Polymeric Drug Delivery Techniques

POLYMERIC
DRUG DELIVERY
TECHNIQUES
Translating Polymer Science
for Drug Delivery
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Preface Solubility Enhancement

Low drug solubility and stability often reduce the effectiveness of an
Rapid advances in medicine and otherwise promising therapeutic candidate. Drug delivery systems
biotechnology have driven the field of drug can be formulated to improve the in vivo solubility of lipophilic and
discovery and led to the development of hydrophobic drugs by encapsulation in a drug delivery carrier or by
many new highly potent and target-specific conjugation with a polymer.
drug candidates. Despite the fast pace of
research and early-stage discovery, many drug
candidates fail during preclinical evaluation Selecting a Polymeric Drug Delivery System
due to poor efficacy, limited bioavailability,
There are three main categories of polymeric drug delivery systems;
and other challenges associated with effective Nicolynn Davis, Ph.D.
colloidal carriers (micro, nanoparticles, micelles, micro/nanogels),
drug delivery. Small molecule drugs can Aldrich Materials Science
implantable networks or hydrogels, and polymer drug conjugates.
suffer from low solubility, poor stability, short Sigma-Aldrich, Milwaukee, WI USA
Email: nicolynn.davis@sial.com Unfortunately, there is no “silver bullet” for effective delivery of
circulation time, and non-specific toxicity
broad classes of therapeutics. Rather, selection of a drug delivery
limiting their therapeutic efficacy. Biopharmaceuticals such as nucleic
system must be driven by the nature of the drug and the inherent
acids, peptides, and proteins are often limited by poor stability and
properties of the drug delivery system (Figure 1). Drug properties,
rapid clearance from the body. These challenges, coupled with the
including chemistry, solubility, potency, site of action, and clearance
complexity and diversity of new pharmaceuticals, are fueling the
rate, each impact the proper selection of a drug delivery system that
evolution of novel drug delivery systems that overcome bioavailability
can achieve the desired outcomes. In addition, the choice of drug
and delivery obstacles. However, despite the growing importance of
delivery system determines the drug loading capacity, longevity of
polymer drug delivery methodologies, the materials and methods of
release, and the route best suited for administration. Furthermore,
drug delivery are not widely available to those outside the polymer
characteristics of the drug delivery system (size, surface charge and
synthesis field.
hydrophobicity, shape, flexibility, inclusion of targeting moieties) will
The objective of effective drug delivery is improving the affect performance and distribution in the body. Each drug delivery
pharmacokinetics and pharmacodynamics of each therapeutic to system has inherent advantages and limitations (Table 1). It should be
enable drug delivery to the right place, at the right time and in the noted that drug release from any carrier is determined by a complex
right amount. Delivery systems apply three main strategies to enable interaction between the drug properties, polymer characteristics, and
improved drug efficacy. environmental/in vivo conditions.
Controlled Release Drug

Drug efficacy can be enhanced by maintaining the concentration
• Drug properties
within the therapeutic window (effective dose). Polymer carriers loaded (solubility, stability)
with therapeutics enable controlled temporal and spatial release • Desired site of Drug Delivery System Formulation
action
of a drug by controlling drug diffusion, the rate of dissolution, or • Desired release rate
• Loading capacity
degradation of the carrier. • Delivery challenge
• Route of
administration Polymer Selection
associated with
drug • Compatibility with
Targeted Delivery drug
Drug efficacy can be enhanced and toxicity minimized by localization • Desired release
kinetics, including
at the organ, tissue, cellular, or organelle level. Targeting can be degradation rate
achieved by coating or conjugating the carrier with affinity reagents
such as nucleic acids, peptides, antibodies, or others that bind specific
cell receptor proteins, nucleic acids, or polysaccharides. Figure 1. Drug delivery formulation selection process.
Table 1. Advantages and limitations of drug delivery systems.

Drug Delivery System and
Polymer Types Advantages Limitations
Microparticles
• Biodegradable polymers • Encapsulate a variety of drugs • Burst release possible, may lead to local toxicity
• Natural polymers • Sustained release can be achieved
Nanoparticles
• Biodegradable polymers • Stable delivery system • Non-specific uptake in RES
• Natural polymers • Small size enables enhanced retention and permeation into tissue and tumor
Micelles
• Amphiphilic block copolymers • Enhanced solubility for hydrophobic drugs • Less stable, may require additional crosslinking
• Facile synthesis
Drug Conjugates
• Hydrophilic polymers • E xtended circulation half-life, reduced clearance due to increased drug • Activity of drug can decrease due to conjugation
• Dendrimers hydrodynamic radius • Approach provides sustained but not controlled release
• Decreased drug immunogenicity and degradation • Low loading capacity of drug
Hydrogels or Implants
• Hydrophilic polymers • Broad range of release timeframes (weeks to months) • Drug solubility may limit utility
• Biodegradable polymers • Useful for localized delivery • Limitation to route of delivery achievable
• Natural polymers • Improved patient compliance due to infrequent dosing • Delivery may require incision or larger gauge needle
• Risk of local dose dumping
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Drug Delivery Material Choices Table of Contents

Polymer selection greatly influences the performance of the drug
delivery system. Careful polymer selection is essential to control the Articles
encapsulation efficiency, release rate, and duration of release. Many
polymers can be formulated into various drug delivery systems to Colloidal Carriers for Drug Delivery
address the three key drug delivery strategies to enable improved Polymer Micelles for Drug Delivery 3
drug efficacy (Table 2). The diversity of polymer building blocks Alice Du, Martina Stenzel
can further complicate formulation decisions. As discussed by Du
and Stenzel (in this publication), the most critical factor in polymer Biodegradable Colloidal Carriers in Drug Delivery Applications 8
selection is considering the interaction of the drug and polymer. Bin Wu, Theresa Logan
Polymer selection will determine the mechanism for drug release (bulk Lipid-polymer Hybrid Nanoparticles for Drug Delivery Applications 14
erosion, system degradation), and the choice of polymer properties Sangeetha Krishnamurthy, Juliana M. Chan
(molecular weight, surface charge) will influence release rate and
impact pharmacokinetics. Further fine-tuning of release from drug Crosslinked Chitosan Nanoparticles and Chemical Modifications for 18
delivery systems can be achieved by using multiple types of polymers Drug Delivery Applications
or including additives. Shady Farah, Joshua Doloff, Daniel Anderson, Robert Langer
Table 2. Polymer categories and the drug delivery strategies they enable. Poly(N-isopropylacrylamide)-based Stimuli-responsive Materials 22
Ganga Panambur, Nicolynn Davis
Controlled
Release
Targeted
Delivery
Solubility
Enhancement
Shape Change Poly(N-isopropylacrylamide) Microstructures 28
for Drug Delivery
Tanvi Shroff, ChangKyu Yoon, David H. Gracias
Biodegradable nanoparticles ✔ ✔
Biodegradable micelles ✔ Hydrogels for Drug Delivery

Responsive polymers ✔ ✔ Formulation of Poly(ethylene glycol) Hydrogels for Drug Delivery 31
Polymeric hydrogels ✔ ✔ Tyler Lieberthal, W. John Kao
PEG conjugation ✔
Polyoxazoline polymers ✔
Drug Conjugates
Dendrimers ✔ ✔ Protein PEGylation 36
Steve Brocchini
About This Guide Poly(2-oxazoline)s for Drug Delivery

Rainer Jordan, Robert Luxenhofer, Alexander V. Kabanov
42
This guide is intended to provide an overview of polymeric drug Polyoxazolines: An Alternative to Polyethylene Glycol 46
delivery systems as well as provide the corresponding example Nicolynn Davis
formulation protocols and product information required to utilize these
techniques in the laboratory. The publication has been developed Dendritic Polyester Scaffolds: Functional and Biocompatible Precision 47
to enable those without a polymer chemistry background to use Polymers for Drug Delivery Applications
polymers to solve their drug delivery research challenges; but, we have Sandra García-Gallego, Michael Malkoch
also kept the expert in mind by including a number of cutting-edge
methodologies. This guide is arranged according to drug delivery
RAFT Polymeric Carriers for Antibody Drug Conjugates of Biologic Drugs 52
Patrick S. Stayton, Anthony Convertine, Geoffrey Berguig
strategies, and these strategies are noted within each method. We
hope this publication will enable chemists, engineers, pharmaceutical Linear and Branched PEIs as Nonviral Vectors for Gene Delivery 57
scientists, and biologists to explore different drug delivery techniques Philip Dimitrov, Nicolynn Davis
to facilitate translational research.
Featured Products
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Poly(lactide-co-glycolide) Copolymers 11
End-functionalized Poly(l-lactide)s 12
PNIPAM and End-functionalized PNIPAM 26
Bifunctional and Multi-arm PEGs 33
Poly(oxazoline)s 45
bis-MPA Dendrimers and Hyperbranched PEG Dendrimers 50
Indexes
Method 60
Trademark 60
For questions, product data, or new product suggestions, contact us at matsci@sial.com. 1

NANOMATERIALS FOR
DRUG DELIVERY AND
THERANOSTICS
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Polymer Micelles for Drug Delivery
Controlled Targeted Solubility
Release Delivery Enhancement
POLYMER MICELLES FOR DRUG DELIVERY

Since many drugs have a strong tendency to crystallize, theoretical
models of the polymer–drug interactions treat this like a solution
where the presence of the homogenous mixture is determined by the
miscibility curve of its phase diagram on the molecular level. Moreover,
the models discussing the thermodynamic stability of a binary system
are based on a fast equilibrium. This may not always be the case since
polymers with high Tg values may trap the drug in the matrix, resulting
in a kinetically stable system. Readers who are interested in the
Alice Du, Martina Stenzel* underpinning thermodynamic principles are referred to an excellent
Centre for Advanced Macromolecular Design review article.8
School of Chemistry
University of New South Wales, Australia How, then, can one choose the right polymer for the right drug to
*
Email: M.Stenzel@unsw.edu.au
achieve good loading and high stability? The assumption “like dissolves
like” is a good starting point. This rule of thumb is based on the Flory–
Introduction Huggins parameter χ in Equation 1:
Polymeric micelles obtained from the self-assembly of amphiphilic ( − )2
= (1)
block copolymers are probably one of the most common drug
delivery carriers among polymeric nanoparticles.1–4 The rise of highly
where δs and δp are the Scatchard–Hildebrand solubility parameter
controlled polymerization techniques, especially processes such as
of the solute and the polymer, respectively.7 In short, polymers that
ATRP5 and RAFT,6 has led to an extraordinary surge of new types of
are chemically similar to the drug should enable the highest loading
block copolymers fit for biomedical applications. Facile control over
capacity. A good example is doxorubixin conjugated to a polymer.9
the polymer structure has also meant access to a large array of self-
While the drug attached to the polymer was found to be inactive,
assembled morphologies including micelles, cylindrical micelles, and
polymer micelles constructed with the polymer-drug conjugate
polymersomes. Micelles in particular are at the center of attention
created an environment that had the highest compatibility possible
as potential drug carriers due to a core-shell structure that is highly
with free doxorubicin leading to an increased loading capacity.
water soluble while still maintaining a hydrophobic core suitable for
Decorating the polymer with the same drug to be loaded is an
hydrophobic drugs. This is crucial for many drugs since they are often
effective but cost-prohibitive option. Alternatively, subtle changes
rendered insoluble in water, and loading them into drug carriers can
to the interior polymeric structure by altering the substitution of the
increase their solubility by several orders of magnitude.4,7
polymer can maximize loading. For example, a PEO-b-PCL polymer
was modified with benzyl, carboxyl, stearyl, palmitoyl, and cholesteryl
Choice of Block Copolymers functional groups with the aim of varying the hydrophobicity to tailor
the polymer matrix toward the highest possible loading capacity of the
The choice of drug carriers can be daunting. In addition to a range chosen drug.10
of commercially available block copolymers, there is basically no However, not every lab has synthetic chemists capable of carefully
limit to the design of amphiphilic structures thanks to advances in tailoring a drug carrier to the drug. A tool is needed to help predict
polymer design. Block copolymers can be further complemented the best possible polymer structure for the drug. This is not easy, but
by other amphiphilic polymers (such as miktoarm starpolymers, an initial estimate can be obtained using the group contribution
multiblock copolymers, and star polymers) to enable the formation method to determine approximate partial solubility parameters. In
of compartmentalized micelles. Whatever architecture is chosen, the this approach, the polymer and drug are essentially dissected into
primary consideration should be the compatibility between the drug their different functional groups, which then are used to determine
and the polymers.7 The polymer–drug interaction plays an important dispersion forces, dipole-dipole interactions, and hydrogen bonding of
role in the drug-loading capacity of a carrier and the stability of the polymer and drug.11 This approach frequently has been employed to
drug in the matrix, which ultimately affects the shelf-life of the carrier. predict the most suitable polymeric drug carrier,12–13 but one also needs
The miscibility of a drug with the polymeric matrix can be described to exercise caution since many aspects are not taken into account
by the Flory–Huggins theory. This contains both entropy and enthalpy resulting in unsuitable predictions. More refined approaches are based
components, expressed by the Flory–Huggins interaction parameter χ, on molecular dynamics simulation,14 which can reveal the critical role of
that describe the interaction between the polymer and the drug. H-bonding, an interaction that is often more crucial than hydrophobic
In other words, the Flory–Huggins parameter χ is a measure of forces to achieve high drug loading.15 Further theories, based on a free
compatibility between polymer and drug.

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energy model, describe the solubilization of low molecular weight Solvent Evaporation
compounds in micelles. Based on this information, one can derive In the solvent evaporation technique, polymer and drug are dissolved
conclusions on the distribution of the drug in the micelle. aggregation in an organic solvent with a low boiling point, followed by evaporation
number, the size of the micelle, the effect on micelle stability, and and subsequent dehydration.19 The chosen organic solvent is selective
the maximum extent of solubilization.16 A summary of various toward one block, which results in the formation of micelles in non-
computational approaches can be found in a review article by Allen aqueous solutions. The outcome is usually determined by the type
and co-workers.17 of solvent, the concentration of polymer and drug, and the rate of
evaporation. The limitation of this approach lies in the limited choice of
Methods: Micelle Drug Loading solvents, and there is no guarantee the resulting particles will be well-
defined core-shell particles that can be easily redissolved in water.
Once a suitable polymer has been identified, the drug must be loaded
into the micelles.7,18 Direct mixing of the hydrophobic drug and the Example Method
micelle in water, although suitable for some selected systems, is rarely 1. Dissolve 2 mg of drug and 20 mg of polymer in methanol (or any
capable of dissolving both the drug and polymer. Therefore, other other low-boiling solvent that can dissolve both components).
techniques must be used to ensure solubilization of both the drug 2. Evaporate solvent under vacuum.
and the polymer. A common solvent is capable of fully dissolving 3. Add distilled water, incubate at 40 °C for 10 min, and vortex to
both the drug and the block copolymer into the unimeric state (single obtain a clear solution.
block copolymers). A clear solution can serve as an initial indication
the polymer has dissolved, but it is advisable to test for the absence Co-solvent Evaporation
of micelles or other aggregates using light scattering techniques to Co-solvent evaporation proceeds by adding water directly to the
ensure full solvation. Examples of commons drug-loading techniques organic solvent to cause the self-assembly of the micelle and
are described in Figure 1. encapsulation of the drug. The outcome is controlled by the type of
solvent, the ratio between organic solvent and water, the concentration
Solvent Evaporation of water and drug, rate of solvent evaporation, and the order and
rate of mixing.20 This approach is limited by the choice of solvent, but
usually results in higher drug encapsulation efficiencies.
Example Method
1. Dissolve 20 mg of polymer and 2 mg of drug in 1 mL of acetone,
Drug and ABC in Evaporate organic Redissolve in water THF, or acetonitrile.
organic solvent solvent under vacuum 2. Add 2 mL of water dropwise to the organic solvent (or vice versa).
3. Mix for 4 h, followed by the evaporation of the organic solvent.
Co-solvent Evaporation
Dialysis
Dialysis is probably the most versatile and most common technique
used for drug encapsulation since it allows the use of high-boiling
solvents such as DMSO, which is removed by dialysis and replaced
with water. Although this approach is applicable to many solvent
Drug and ABC in Addition of organic Evaporation of systems, the drug loading efficiency is usually lower than the
organic solvent phase into water organic solvent co-solvent evaporation and the technique can be time-consuming.
phase or vice versa
The slow process can aid the formation of thermodynamically stable
Dialysis morphologies. A final dialysis step to remove solvent and free drug
is often crucial to obtain a product free of organic solvent while
maintaining maximum drug loading. However, while extensive dialysis
can assist in the thorough purification of the product, it also can cause
the release of the already encapsulated drug and result in low drug
encapsulation efficiencies.
Slow addition of water to drug Removal of organic
and ABC in organic solvent solvent by dialysis Example Method
1. Dissolve 20 mg of polymer and 2 mg of drug in 1 mL of DMF.
Flash Nanoprecipitation 2. Add 5 mL of water slowly, with the help of a syringe pump, if
possible, to control the rate of water addition.
H2O
3. Dialyze against water using a tubular cellulose membrane (Sigma
Fast mixing Prod. No. Z726176).
of both
phases
using
confined
impinging Removal of organic
jet mixer solvent by dialysis
Figure 1. Techniques for drug loading of micelles
4 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

Flash Nanoprecipitation producing a family of relatively uniform spheroids without the use of
Flash nanoprecipitation is a relatively new technique that offers a specialized equipment.
more rapid solution than other time-consuming methods. Fast mixing
and precipitation into a non-solvent for the drug and one polymer
block results in a kinetically trapped structure. Although the resulting Method: Characterization of Toxicity
structures do not have well-defined internal phase boundaries,
as would be the case in thermodynamically stable structures, the with Spheroids
approach provides an alternative to achieve a fast throughput.21 Typical steps for culturing spheroids in this manner include:
Example Method 1. Pre-determine the seeding number of cells required for each
1. Prepare a solution of 40 mg of polymer and 20 mg of drug in spheroid (usually 1,000–2,000 cells/spheroid).
1 mL of THF. 2. Prepare a cell suspension with a per mL concentration 100× that of
2. Use a confined impinging jet mixer to mix the solution with the initial seeding cell number where the suspension is prepared in
1 mL of water. the growth medium used to culture the cells.
3. Introduce the exit stream into 8 mL of water:THF (9:1 v/v%). 3. Place 10 µL of the well-mixed cell suspension on the inside surface
4. Dialyze against water using a tubular cellulose membrane. of the lid of a petri dish.
4. Repeat until the required number of seeding droplets are placed.
Characterization 5. Flip the petri dish lid upside down so that the seeding droplets
are “hanging” and place on top of a petri dish filled with 15 mL of
Independent of the technique, the characterization of drug-loaded sterile PBS.
micelles is similar to other nanoparticles. Parameters of interest are the 6. Incubate at 37 °C and 5% CO2 for a minimum of 3 days without
drug encapsulation efficiency (EE) and the drug-loading capacity (LC) disturbing the petri dish.
(see Equation 2): 7. Check the spheroids daily until the desired size has been reached
(usually 300–400 µm in diameter).
%= 8. Once culturing is complete, move the spheroids into a 96-well plate
(2) filled with 200 µL of medium/well.
%= 9. Culture the spheroids for one more day at 37 °C and 5% CO2 with
slow rotation of the plate on a 4-way mixer during that time.
where WL is the weight of loaded drug, W0 the quantity of drug initially Once the spheroids have been cultured and are of the desired shape
added, and WN the weight of the nanoparticle. and size, the micelle formulation can be loaded for testing:
The International Organization for Standardization (ISO) published a 1. Remove 170 µL of medium from each well in the 96-well plate
catalog of properties for the full characterization of nanoparticles. The containing the spheroids.
Guidance on Physico-chemical Characterization of Engineered Nanoscale 2. Add 100 µL of twice-concentrated culture medium and 100 µL of
Materials for Toxicologic Assessment (ISO/TR 13014:2012) includes the twice-concentrated micellar solution to each well.
dimensions, shape, specific surface area, surface charge, composition,
3. Incubate for the desired amount of time at 37 °C and 5% CO2.
and purity, among others. While most of these points apply to micelles
4. At the desired time points, move the spheroids to be tested into a
as well, the evaluation of the stability of the micelle can be considered
new plate and wash with PBS.
a crucial aspect when trying to evaluate micelles for drug delivery
purposes. It has been shown the dynamic behavior of micelles can 5. Measure the viability of the spheroids via several methods,
affect their cellular uptake22 and their rate of exocytosis.23 The physico- including determination of DNA amount or measuring acid
chemical characterization of nanoparticles is then complemented by phosphatase activity.32
the Compilation and Description of Toxicological Screening Methods for Additional tips for culturing spheroids include:
Manufactured Nanomaterials (ISO/TR 16197:2014), which contains a list
of recommended experiments to understand toxicity, accumulation, 1. Use the surface tension of the droplet to naturally maintain its
and other factors. The reader is referred to comprehensive shape when initially placing it on the petri dish lid.
publications on this topic, which describe background and also give 2. Place droplets at least 1 cm apart to give room for the droplets to
practical advice.24,25 spread slightly upon culturing.
3. Prepare at least 25% more spheroids than required in case of
Multicelluar spheroids are an established drug discovery technique that undesired spheroid shape or size.
is making its way into the nanomedicine area, including the testing
Examples of spheroids are provided in Figure 2. Confocal microscopy
of drug-loaded micelles.26–28 Interestingly, while some drug-loaded
can be used to visualize micelle penetration if fluorescence is
micelles can have poor performance in monolayer cell models, the
incorporated into the polymeric structure.
results in a three-dimensional (3D) cell culture experiment can differ
noticeably.26 The key to this behavior is the differences in penetration
of drugs and micelles into the multicellular spheroid. While this aspect
only plays a minor role in two-dimensional (2D) models, it becomes
one of the main parameters in understanding enhanced delivery in
a 3D environment.29 These 3D models can be further combined with
2D models to create sophisticated systems that mimic the tumor
microenvironment to simulate the behavior of micelles in vivo.30
A typical procedure to test the toxicity of drug-loaded micelles is
outlined below. Although the researcher can choose from a range Figure 2. A) Penetration of uncrosslinked and fluorescent micelles into prostate cancer
(LNCaP) spheroids as visualized by confocal microscopy at a depth of 90 µm (yellow scale
of ways to culture spheroids,31 only the “hanging drop” method is bar = 100 µm); B) LNCaP spheroids after 4 days culture (black scale bar = 300 µm); C) LNCaP
discussed here. The hanging drop method is a convenient method of spheroids after 14 days treatment with the uncrosslinked micelles which have been
conjugated with paclitaxel.

aldrich.com/matsci
Conclusions
(10) Falamarzian, A.; Lavasanifar, A. Macromol. Biosci. 2010, 10, 648–656.
(11) J. Liu, Y. X., C. Allen. J. Pharm. Sci 2004, 93, 132–143.
(12) Kim, Y.; Liemmawa, E. D.; Pourgholami, M. H.; Morris, D. L.; Stenzel, M. H. Macromolecules
The delivery of drugs using polymeric micelles has now matured into 2012, 45, 5451–5462.
(13) Sharma, A.; Soliman, G. M.; Al-Hajaj, N.; Sharma, R.; Maysinger, D.; Kakkar, A.
a well-established field. Compatibility between the drug and polymer Biomacromolecules 2012, 13, 239–252.
is the key to success in obtaining maximum loading efficiency. The (14) Patel, S. K.; Lavasanifar, A.; Choi, P. Biomacromolecules 2009, 10, 2584–2591.
scientist can choose from a range of tools to load the drug. Although (15) Schulz, A.; Jaksch, S.; Schubel, R.; Wegener, E.; Di, Z.; Han, Y.; Meister, A.; Kressler, J.; Kabanov,
many drugs can be loaded using the above techniques, it should be A. V.; Luxenhofer, R.; Papadakis, C. M.; Jordan, R. ACS Nano 2014, 8, 2686–2696.
(16) Nagarajan, R.; Ganesh, K. Macromolecules 1989, 22, 4312–4325.
noted that a range of drugs—such as drugs that have a low drug- (17) Huynh, L.; Neale, C.; Pomès, R.; Allen, C. Nanomedicine: Nanotechnology, Biology and
loading efficiency—are best delivered by conjugating them to the Medicine 2012, 8, 20–36.
block copolymer directly, instead of relying on physical attraction alone. (18) Gaucher, G.; Dufresne, M.-H.; Sant, V. P.; Kang, N.; Maysinger, D.; Leroux, J.-C. J. Controlled
Release 2005, 109, 169–188.
It must be noted that this article has not touched upon the benefits
(19) Lavasanifar, A.; Samuel, J.; Kwon, G. S. J. Controlled Release 2001, 77, 155–160.
of crosslinking micelles.2,33 As briefly discussed here, the dynamic (20) Aliabadi, H. M.; Elhasi, S.; Mahmud, A.; Gulamhusein, R.; Mahdipoor, P.; Lavasanifar, A. Inter.
properties of the micelles and the potential disassembly may affect J. Pharm. 2007, 329, 158–165.
the interaction with biological media, and crosslinking may circumvent (21) York, A. W.; Zablocki, K. R.; Lewis, D. R.; Gu, L.; Uhrich, K. E.; Prud’homme, R. K.; Moghe, P. V.
Adv. Mater. 2012, 24, 733–739.
the issue as it may enhance characteristics such as cellular uptake,22,23 (22) Kim, Y.; Pourgholami, M. H.; Morris, D. L.; Stenzel, M. H. Biomacromolecules 2012, 13,
movement in multicellular tumors,29 and in vivo circulation.33 In 814–825.
summary, polymeric micelles provide limitless avenues of modification (23) Kim, Y.; Pourgholami, M. H.; Morris, D. L.; Lu, H.; Stenzel, M. H. Biomater Sci 2013, 1, 265–275.
possibilities and represent a versatile method of delivering a wide (24) Hall, J. B.; Dobrovolskaia, M. A.; Patri, A. K.; McNeil, S. E. Nanomedicine (Lond) 2007, 2,
789–803.
range of drugs. (25) McNeil, S. Characterization of Nanoparticles Intended for Drug Delivery. Humana Press:
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(4) Lu, Y.; Park, K. Int. J. Pharm. 2013, 453, 198–214. (30) Gao, H.; Yang, Z.; Zhang, S.; Pang, Z.; Liu, Q.; Jiang, X. Acta Biomater 2014, 10, 858–67.
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(6) Gregory, A.; Stenzel, M. H. Prog. Polym. Sci. 2012, 37, 38–105.
(32) Friedrich, J.; Seidel, C.; Ebner, R.; Kunz-Schughart, L. A. Nature Protocols 2009, 4, 309–324.
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(9) Yokoyama, M.; Fukushima, S.; Uehara, R.; Okamoto, K.; Kataoka*, K.; Sakurai, Y.; Okano, T. J.
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Diblock Copolymers
For more information on these products, visit aldrich.com/block.
Name Structure Molecular Weight/Viscosity Degradation Time Prod. No.
Poly(L-lactide-block-acrylic acid) OH PAA average Mn 18,000 - 805718-1G
O CH3 PLA average Mn 4,500
S H 3C CN
O H PAA average Mn 18,000 - 799246-250MG
H3C(C10H20)H2C S y O
S x PLA average Mn 10,000
O
Poly(D,L-lactide-block-acrylic acid) OH PAA average Mn 18,000 - 798126-1G

O H3C
S CH3 PLA average Mn 5,000
CN
CH3(CH2)10CH2 O H
S S O
n m
O
Poly(DL-lactide-co-caprolactone) O inherent viscosity 0.7-0.9 dL/g in - 457639-5G
chloroform, DL 86%
O
O inherent viscosity 0.7-0.9 dL/g in - 457647-5G
CH3 O chloroform, DL 40%
x y
Poly(L-lactide)-block-poly(ethylene O H3C PEG average Mn 5,000 - 570281-250MG

glycol)methyl ether O O PLA average Mn 5,000 570281-1G
H3C O OH
m CH3 O n
Poly(ethylene glycol)-block- O PEG average Mn 350 - 659665-1G

polylactide methyl ether O OH PLA average Mn 1,000
H3C O
PEG average Mn 750 - 659657-1G
x H3C x
PLA average Mn 1,000
Poly(ethylene glycol) methyl ether- O PEG average Mn 2,000 2-4 weeks 764779-1G
block-poly(D,L lactide) O O PLA average Mn 2,200
H O CH3 average Mn 4,000 (total)
n
H3C m


Poly(ethylene glycol) methyl ether- PEG Mn 2,000 1-4 weeks 764825-1G
block-poly(lactide-co-glycolide) CH3 O PLGA Mn 4,000
H O O average Mn 6,000 (total)
O O CH3
O n PEG average Mn 5,000 1-4 weeks 765139-1G
x y PLGA Mn 10,000
m average Mn 15,000 (total)
PEG average Mn 2,000 1-4 weeks 764760-1G
PLGA average Mn 15,000
average Mn 17,000 (total)
PEG average Mn 5,000 1-4 weeks 764752-1G
PLGA Mn 55,000
Poly(ethylene glycol) methyl ether- PEG average Mn 2,000 2-5 weeks 764736-1G
O
block-poly(D,L lactide)-block-decane PLA average Mn 2,000
O O CH2(CH2)8CH3 average Mn 4,000 (total)
H3C O
n
H3C O
m
Poly(ethylene glycol)-block-poly(ε− O PCL average Mn 5,000 >12 months 570303-250MG

caprolactone) methyl ether O OH PEG average Mn 5,000 570303-1G
H3C O average Mn 10,000 (total)
m n
PCL average Mn 13,000 >12 months 570311-250MG
PEG average Mn 5,000 570311-1G
PCL average Mn 32,000 >12 months 570338-250MG
PEG average Mn 5,000 570338-1G
Triblock Copolymers
For more information on these products, visit aldrich.com/block.
Polylactide-block-poly(ethylene CH3 O PEG average Mn 900 <12 months 659630-1G
glycol)-block-polylactide PLA average Mn 1,500
O O
HO O H average Mn 1,500-900-1,500
O y
CH3 PEG average Mn 10,000 <12 months 659649-1G
x z
PLA average Mn 1,000
average Mn 1,000-10,000-1,000
Polyglycolide-block-poly(ethylene O O PEG average Mn 400 - 790230-1G
glycol)-block-polyglycolide O PEG:Gly 8:92
O O PEG average Mn 400 - 790222-1G
m n m
PEG:Gly 12:88
Poly(lactide-co-glycolide)-block- PEG average Mn 1,000 1-2 weeks 764787-1G
poly(ethylene glycol)-block- CH3 O CH3 O PLGA average Mn 2000
poly(lactide-co-glycolide) H O O O average Mn 1,000-1,000-1,000
O O O H
O
n PEG average Mn 1,000 2-3 weeks 764817-1G
x y O x y PLGA average Mn 2,200
m m average Mn 1100-1000-1100
Poly(D,L-lactide-co-glycolide)- PEG average Mn 400 <4 months 790257-1G
block-poly(ethylene glycol)-block- O O CH3 O CH3 O average Mn 6,000‑12,000 790257-5G
poly(D,L-lactide-co-glycolide) based R O O O PEG average Mn 400 <4 months 790249-5G
poly(ether ester urethane) N N O y O O y
H H l average Mn 6,000-15,000 790249-1G
O O
x x
m m PEG average Mn 400 - 790265-1G
n average Mn 8,000-20,000 790265-5G
O O
R
R= O PLGA PEG PLGA O N N
H H
n
Poly(lactide-co-caprolactone)- PEG average Mn 5,000 1-2 months 764833-1G

block-poly(ethylene glycol)-block- O O CH3 PLCL average Mn 5,700
poly(lactide-co-caprolactone) H O O O average Mn 1,000-10,000-1,000
O O O H
y x x y
H3C O O
m n m

aldrich.com/matsci

BIODEGRADABLE COLLOIDAL CARRIERS

IN DRUG DELIVERY APPLICATIONS
acetate microspheres used for the treatment of prostate cancer and
endometriosis, can be subcutaneously administered at 1-month,
3-month, or 6-month intervals. When drug-loaded PLGA microspheres
are administered, the PLGA polymer starts to degrade in vivo, and
as it degrades the drug molecules are gradually released from the
microspheres. The drug release rate can be modulated by the selection
of the type of PLGA polymer and by adjusting the encapsulation
Bin Wu, Theresa Logan
process. For example, the following parameters can affect the drug
Phosphorex, Inc. release profile:
Hopkinton, MA USA
bin.wu@phosphorex.com and tlogan@phosphorex.com yy The ratio of lactide to glycolide (L/G ratio) in the PLGA polymers;
e.g., PLGA with an L/G ratio of 50:50 have the fastest drug release.
Introduction yy The molecular weight or inherent viscosity of the PLGA polymer,

where higher molecular weight provides slower drug release.
Colloidal carriers are particles or vesicles of nanometer to micron
size that facilitate drug delivery. Common colloidal carrier systems yy The terminal group of the PLGA polymer, where carboxyl-
include liposomes, polymeric microspheres and nanoparticles, terminated PLGA polymers offer faster drug release compared to
nanocrystals, and microemulsions. Colloidal carriers can be used to ester-terminated PLGA.
improve the therapeutic index of APIs by transporting loaded drugs
to the target site and modifying their distribution within the body.
Furthermore, colloidal carriers can alter the pharmacokinetics of drug
molecules, increase efficacy, reduce toxicity, and provide controlled and
sustained release.
Polymeric Microspheres
Polymeric microsphere drug carriers are spherical particles in the size
range of several to hundreds of microns that can protect unstable Figure 1. Image of API-loaded PLGA microspheres.
drugs pre- and post-administration. Microspheres have the ability to
release a drug continuously over time,1 thereby providing a prolonged
therapeutic effect and reducing the dosing frequency. In addition to Polymeric Nanoparticles
controlled release, microspheres allow for the targeted drug delivery of
Polymeric nanoparticles (NP), either plain or drug loaded, are
potent drugs at reduced concentrations, thereby minimizing systemic
typically less than 1 micron in size. The use of API-loaded polymeric
exposure and adverse side effects. Finally, polymeric microspheres
nanoparticles for intravenous administration is a promising approach
facilitate manipulation of in vivo behavior, pharmacokinetic profile,
for achieving the controlled release and site-specific delivery of
tissue distribution, and cellular interaction of the drug.2
drugs. The nanoparticle delivery system can be designed to maintain
Microspheres are typically comprised of biodegradable polymers such appropriate therapeutic concentration in the bloodstream (controlled
as poly(lactide-co-glycolide) (PLGA), polylactic acid (PAA), polylactide release) or to target a specific cell type (e.g., bone marrow, blood cells).
(PLA), and polycaprolactone (PCL). These polymers degrade in vivo Various types of APIs, including small molecule drugs and biologic
by hydrolysis of their ester backbone into non-toxic products, which compounds, can be incorporated into PLGA polymer nanoparticles by
are excreted by the kidneys or eliminated as CO2 and water through either microencapsulation or surface conjugation. Nanoencapsulation
biochemical pathways. PLGA microspheres have been widely used can protect the API from early degradation, facilitate cell entry, and
to encapsulate drug molecules and have been used as long-acting, increase solubility and bioavailability.
sustained-release pharmaceutical formulations. There are several
The surface properties of intravenously injected particles are important
drug-loaded PLGA microspheres approved by the FDA and marketed
factors determining in vivo organ distribution and fate. Furthermore,
for clinical use. For example, depot products, such as luprolide
surface modification can be an effective approach to targeting specific

Biodegradable Colloidal Carriers in Drug Delivery Applications
tissues. Surface modification of nanoparticles with polyethylene Natural polymers such as chitosan and alginate have been studied
glycol (PEG) can be used to prolong the in vivo circulation lifetime extensively for the preparation of hydrogel nanoparticles. Hydrogel
of drug-loaded nanoparticles. PEGylation of the nanoparticle can nanoparticles based on synthetic polymers including poly (vinyl
be accomplished by adding a copolymer containing PEG chains alcohol) (PVA), PEG, poly (ethyleneimine) (PEI), poly vinyl pyrrolidone,
during the nanoparticle fabrication process. For example, the and poly-N-isopropylacrylamide have also been used for drug delivery.
addition of an ethylene glycol monomer during lactide and glycolide
Hydrogel systems have various applications including oral, transdermal,
copolymerization can lead to a PEGylated PLGA polymer. PEGylation
nasal, rectal, and ocular drug delivery. Hydrogel membranes facilitate
can increase nanoparticle hydrophilicity and improve degradation rate
transdermal drug delivery through the skin at a predetermined and
and crystallization.3 In addition to being biocompatible, PEG is resistant
controlled rate. They are advantageous as they prevent the first pass
to immunological recognition. PEG units on the NP surface prevent
metabolism effect.12 Additionally, hydrogel delivery systems can
opsonin-NP binding, thus preventing the nanoparticles from being
increase the bioavailability of ophthalmic drugs by increasing the
recognized by monocytes and macrophages and, therefore, increasing
contact time of the drugs with cornea.13
circulation time in the body.4 In some circumstances nanoparticles
have been shown to remain in circulation 40× longer when coated Embolization therapy is another application of hydrogel microspheres.
with PEG compared to uncoated nanoparticles.5 Other advantages of It is typically used to prevent the growth of solid tumors by blocking
PEGylated nanoparticles include increased drug loading of hydrophilic the blood supply to the feeding artery. For example, trans-arterial
drugs, reduced initial burst and improved bioavailability.6 PEGylated chemical embolization is an application where anticancer drug loaded
nanoparticles have been used as carriers for vaccine and protein APIs particles are injected into the feeding artery of cancer tumors. In
and are particularly useful in both sustained/controlled release and addition to blocking the blood supply to the tumor, they release the
targeted drug delivery systems. Currently, there are more than 35 U.S. anticancer drugs at high concentrations inside the tumor.14
FDA-approved products utilizing PEG in their biomedical applications.4
Nanoparticles can also facilitate the crossing of the blood brain barrier
(BBB). Surfactants such as Polysorbate 80 and Poloxamer 188 have been
Methods: Colloidal Carrier Fabrication
shown to facilitate the BBB crossing of drug molecules encapsulated in Colloidal carriers can be fabricated using a variety of techniques. The
polybutyl cyanoacrylate nanoparticles or solid lipid nanoparticles.7 method for synthesis should be selected based on the type and nature
In some nanoparticle formulations, the API is attached to the surface of the drug to be encapsulated as well as the desired particle size,
instead of being encapsulated inside the particle. For example, a delivery route, and release characteristics for the final formulation.
peptide for ocular delivery (POD) and a human immunodeficiency virus
transactivator were conjugated to the surface of PLGA nanoparticles, Nanoprecipitation
and the conjugate was found to improve ocular drug bioavailability.8 Nanoprecipitation is a facile and low energy process for the preparation
The conjugation can be done by reacting the terminal functional group of polymeric nanoparticles. It is based on interfacial deposition due
(e.g., terminal COOH) on the PLGA molecule with a reactive group to the displacement of a solvent with the non-solvent. Miscibility
(e.g., amino) on the peptide, API or protein. Finally, in some cases, of the solvents and the dilute polymer solutions are required for
PLGA nanoparticles themselves can have therapeutic effects against nanopreciptation.13 For drug delivery applications, nanoprecipitation
certain diseases.2 is often used for small-scale preparation of nanoparticles of polylactic
acid, PLGA, and polycaprolactone. It is well-suited for hydrophobic APIs.
Microgels and Nanogels In a typical nanoprecipitation process, a polymer (e.g., PLGA)

(and hydrophobic drug, if desired) is dissolved in a solvent that is
Hydrogel particles, including microgels and nanogels, consist of miscible with water (e.g., acetone). The polymer/drug solution is
crosslinked networks of hydrophilic polymer chains that form added dropwise to an aqueous solution under continuous stirring.
colloidal gels. Hydrogel particles are made from natural or synthetic Surfactants or polymer stabilizers may be added to the aqueous
polymeric networks, are highly absorbent, and can contain over 90% solution to stabilize the nanoparticles during formation. Common
water. Crosslinking between polymer chains is either chemically or surfactants and stabilizers include TWEEN® 20, sodium dodecyl sulfate
physically induced and prevents the dissolution of these networks (SDS), PVA, hydroxymethylcellulose (HPMC), and Pluronic® F-68. The
in water.11 The release of the loaded API from the hydrogel particles organic solvent is removed by evaporation or repetitive washing.
may occur through diffusion, hydrogel matrix swelling, or chemical Additional washing steps may be performed to remove surfactant
reactivity of the drug/matrix. The physical properties of microgels and and unincorporated API. The purified nanoparticles can be lyophilized
nanogels (i.e., swelling, permeation, mechanical strength, and surface for storage.
characteristics) can be optimized by structural modification.11 Many The following is a typical protocol for preparing PLGA nanoparticles, of
hydrogel particles are also environmentally sensitive and have the approximately 200 nm, using a one-step nanoprecipitation.
ability to respond to changes of pH, temperature, or the concentration
of metabolites, and release their load as a response to these stimuli for 1. PLGA polymer with an L/G ratio of 50/50, COOH terminated, and
controlled drug release. inherent viscosity of 0.55–0.75 dL/g (additional PLGA polymers may
be substituted, such as Aldrich Prod. No. 719900) is dissolved in
In drug delivery, microgels and nanogels fill a unique niche because acetone (Aldrich Prod. No. 179124).
they can encapsulate water-soluble, small molecule APIs that are 2. Prepare a polyvinyl alcohol (PVA) solution by dissolving the
difficult to encapsulate using traditional biodegradable polymeric appropriate amount of PVA (Mw 85,000–124,000, 87–89%
particles comprised of PLGA and PCL. As a result, these gels are hydrolyzed, Aldrich Prod. No. 363081) in DI water. A typical
particularly suitable for the sustained release of water-soluble drugs concentration is 1%.
or proteins. Other advantages of hydrogel particles include their high 3. Transfer 100 mL of the 1% PVA solution prepared in Step 2 to a
drug loading and activity, biocompatibility, and biodegradability. 500 mL beaker equipped with a magnetic stir bar. Stir the PVA
Additionally, the manufacturing of colloidal gels does not require solution at 400 rpm.
organic solvents, eliminating toxicity risks and the potential for
protein denaturation.

aldrich.com/matsci
4. Using a disposable pipette, slowly and dropwise add 10 mL of the In double emulsions, achieving high drug loading of hydrophilic
PLGA solution into the stirring PVA solution. The nanoparticles form APIs can be challenging since the drug partitions away from the
on contact when the PLGA solution is added. hydrophobic polymer solution into the aqueous surfactant solution.
5. After the PLGA solution is completely added to the PVA solution, Macromolecular drugs such as proteins and antibodies have all been
continue stirring in a fume hood for 3 h to allow acetone encapsulated into polymeric microspheres and nanoparticles using a
evaporation. double-emulsion processes.
6. Wash the nanoparticles three times using refrigerated However, protein aggregation or denaturing may occur during
centrifugation and follow with lyophilization. formation. During the microencapsulation process, proteins are
7. Store lyophilized PLGA nanoparticles dry at –20 °C. constantly exposed to cavitation, heat, solvents, and high shear force,
which could lead to aggregation and denaturation.17,18–20 Alternative
Emulsification Process techniques have been pursued to encapsulate protein drugs into
Emulsification can be used to prepare plain or drug-loaded polymeric microspheres and nanoparticles and are reviewed elsewhere.19–29
microspheres and nanoparticles. Depending on the type of drug to be
loaded, either a single emulsion or double emulsion may be used. The following is a typical protocol for preparing API-loaded PLGA
nanoparticles using a double-emulsion process:
Single Emulsion 1. Prepare a 1% bovine serum albumin (BSA) solution by dissolving
In a single emulsion, the polymer (e.g., PLGA, PCL) is dissolved in a BSA (lyophilized powder, Sigma Prod. No. 05470) in DI water.
solvent that is not miscible with water. If a drug is to be encapsulated, 2. Prepare a 5% PLGA solution by dissolving PLGA polymer with an
it is preferably hydrophobic and solvent soluble. The hydrophobic L/G ratio of 50/50, COOH terminated, and inherent viscosity of
drug is dissolved in the same solution as the polymer. The polymer/ 0.55–0.75 dL/g (additional PLGA polymers may be substituted,
drug solution is emulsified in an aqueous solution containing a such as Aldrich Prod. No. 719900) in methylene chloride. Prepare
surfactant or a polymeric stabilizer (as noted earlier). Emulsification a 1% PVA (Mw 85,000–124,000, 87–89% hydrolyzed, Aldrich Prod.
can be completed by sonication, magnetic or mechanical stirrer, rotor No. 363081) solution in DI water.
stator, high-pressure homogenizer, or microfluidizer. After the oil-in-
3. Mix 0.5 mL of the BSA solution prepared in Step 1 and 5 mL of the
water emulsion is formed, the solvent is removed by evaporation or
5% PLGA solution prepared in Step 3 in a 15 mL glass vial.
extraction. The particles can be washed to remove the surfactant and
possible unincorporated drug molecules, and then lyophilized. 4. Homogenize the PLGA and BSA solution using an IKA Ultra Turrax
High Speed Homogenizer at 20,000 RPM for 25 seconds.
The single emulsion process can also be used to prepare nanocrystals 5. Mix the resulting emulsion with 100 mL of the 1% PVA solution
of poorly soluble compounds. For example, a single emulsion process prepared in Step 3 in a 500 mL beaker.
was used to prepare albumin bound paclitaxel nanoparticles.14–16 6. Homogenize the mixture of the first emulsion and the PVA solution
The following is a typical protocol for preparing PLGA nanoparticles using an IKA Ultra Turrax High Speed Homogenizer generator at
using a single-emulsion process: 8,000 rpm for 2 min.
7. Stir the resulting double emulsion on a stir plate at 400 rpm in a
1. Prepare a 5% PLGA solution by dissolving PLGA polymer with an
fume hood for 3 h to allow the methylene chloride to evaporate.
L/G ratio of 50/50, COOH terminated, and inherent viscosity of
0.55–0.75 dL/g (additional PLGA polymers may be substituted, such 8. Wash the nanoparticles three times using refrigerated
as Aldrich Prod. No. 719900) in methylene chloride (Sigma Prod. centrifugation. Follow with nanoparticle lyophilization.
No. 443484). 9. Store lyophilized BSA-PLGA nanoparticles dry at –20 °C.
2. Prepare a 1% solution of polyvinyl alcohol (PVA) (Mw 85,000–124,000, Spray Drying
87–89% hydrolyzed, Aldrich Prod. No. 363081) solution in DI water.
In spray drying, the feed (a solution, emulsion, or suspension
3. Mix 5 mL of the 5% PLGA solution prepared in Step 1 with 100 mL of containing the API and the matrix material) is atomized into hot
the 1% PVA solution prepared in Step 2 in a 500 mL beaker. nitrogen, leading to rapid drying and particle formation. The particles
4. Homogenize the mixture of the PLGA and PVA solutions by using an are then separated in a cyclone and/or filter bag.
IKA Ultra Turrax High Speed Homogenizer at 18,000 rpm for 2 min.
5. Stir the resulting emulsion at 400 rpm on a stir plate in a fume hood Spray drying has been used extensively by the pharmaceutical industry
for 3 h to allow the methylene chloride to evaporate. to formulate solid dispersions to overcome solubility limitations.
Crystalline APIs can be encapsulated within polymer microspheres. For
6. Wash the nanoparticles three times using refrigerated
example, enteric polymers can be used to protect a drug from harsh
centrifugation and lyophilize.
gastric conditions or for enhanced delivery to the site of maximum
7. Store lyophilized PLGA nanoparticles dry at –20 °C. absorption. Similar approaches can be used for taste masking and
Double Emulsion protecting the drug from physical environments such as light or
For hydrophilic API encapsulation, a double-emulsion technique is moisture. These drug-loaded particles are normally in the micron to
necessary to prepare the polymeric microspheres and nanoparticles. millimeter range, although the spray-drying process is capable of
The double emulsion is predominately a water-in-oil-in-water producing sub-micron or nanometer sized particles.
emulsion, although in some cases it can be a reverse double emulsion, In addition to enhancing the oral bioavailability of poorly water-soluble
or an oil-in-water-in-oil. In a typical double-emulsion process, the compounds, spray drying offers the ability to control particle size,
hydrophilic API is dissolved in an aqueous media and emulsified in the morphology, and other properties with direct effect in Fine Particle
polymer solution to form the first emulsion. The first emulsion is again Fraction (FPF) and lung deposition. It also allows for a reproducible,
emulsified in an aqueous solution containing appropriate surfactants or controllable, and scalable manufacturing process. Spray drying is the
polymer stabilizers to form the second emulsion, or double emulsion. method of choice where abrasion or shearing needs to be avoided,
The solvent is then removed by evaporation or extraction processes. as in the case of biologic compounds. Inhaled insulin formulations
The equipment used in single-emulsion processes can be used to of Affrezza (MannKind) and Exubera (Pfizer) are manufactured by
generate double emulsions. spray drying.

Biodegradable Colloidal Carriers in Drug Delivery Applications
The following is a typical spray-drying protocol for the preparation (10) Giri, T. K.; Thakur, A.; Alexander, A.; Ajazuddin, H.; Badwaik, H.; Tripathi, D. K.; Acta
Pharmaceutica Sinica B 2012, 2, 439.
of chitosan microspheres. This method uses a Mini Spray Dryer B-290 (11) Genta, I.; Conti, B.; Perugini, P.; Pavanetto, F.; Spadaro, A.; Puglisi, G. J. Pharm. Pharmacol.
(BUCHI Corporation, New Castle, DE, USA) with a 0.7 mm standard 1997, 49, 737–742.
nozzle. (12) Saralidze, K.; Koole, L. H.; Knetsch, M. L. W. Materials 2010, 3 (6), 3537–3564.
(13) Hornig, S.; Heinze, T.; Becer, C. R.; Schubert, U. S. J. Mater.Chem. 2009, 3838–3840.
1. Dissolve an appropriate amount of chitosan (medium Mw, (14) Green, M. R.; Manikhas, G. M.; Orlov, S.; Afanasyev, B.; Makhson, A. M.; Bhar, P.; Hawkins, M. J.
Aldrich Prod. No. 448877) in 1.0% v/v acetic acid (Aldrich Prod. Annals of Oncology 2006, 17 (8), 1263–1268.
(15) Miele, E.; Spinelli, G. P.; Miele, E.; Tomao, F.; Tomao, S. Int. J Nanomed 2009, 4, 99–105.
No. 695092) solution to prepare a 2.5% (w/v) chitosan solution in
(16) Stinchcombe, T. E. Nanomedicine 2007, 2 (4), 415–423.
a 500 mL Erlenmeyer flask. (17) Cleland, J.; Jones, A. Pharm Res 1996, 13, 1464–1475.
2. Use the suction mode as the method of operation. Set the flow rate (18) Morlocka, M.; Kollb, H.; Winterb, G.; Kissel, T. Eur J Pharm Biopharm 1997, 43, 29–36.
of the compressed air at 600 L/h, the inlet temperature at 160 °C, (19) Pérez, C.; Castellanos, I. J.; Costantino, H. R.; Al-Azzam, W.; Griebenow. K. J Pharm Pharmacol
2002, 54, 301–303.
and sample flow rate at 700 mL/h. (20) Boury, F.; Ivanova, T.; Panaieotov, I.; Proust, J. E.; Bois, A.; Richou, J. Langmuir 1995, 11,
3. Start the operation. The chitosan solution is fed to the spray dryer, 1636–1644.
atomized by the force of the compressed air, and blown by heated (21) Carrasquilloa, K. G.; Stanleya, A. M.; Aponte-Carroa, J. C.; Jésusa, P. D.; Costantinob, H. R.;
Bosquesc, C. J.; Griebenow, K. J. Controlled Release 2001 76, 199–208.
air to the drying chamber. (22) Sáncheza, A.; Villamayora, B.; Guob, Y.; McIverb, J.; Alonso, M. J. Int J Pharm 1999, 185,
4. Collect the dried microspheres. The mean residence time is 255–266.
1–1.5 seconds. (23) Sturesson, C.; Carlfors, J. J. Controlled Release 2000, 67, 171–178.
(24) Sah, H. J. Controlled Release 1999, 58, 143–151.
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1999, 16, 1294–1299.
(1) Sahil, K.; Akanksha, M.; Premjeet, S.; Bilandi, A.; Kapoor, B. Int. J. Res.Pharm. Chem. 2011, 1,
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(2) Ramteke, K.; Jadhav, V. B.; Dhole, S. N. IOSR J. of Pharm. 2012, 2, 44.
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(9) Ahmed, E. M. J Adv. Res. 2015, 7, 105.
Poly(lactide-co-glycolide) Copolymers
For more information on these products, visit aldrich.com/biodegradable.
Name Feed Ratio End Group Molecular Weight Degradation Time Prod. No.
Resomer® RG 502 H, Poly(D,L-lactide-co-glycolide) lactide:glycolide 50:50 acid terminated Mw 7,000‑17,000 <3 months 719897-1G
719897-5G
719870-5G
Resomer® RG 504 H, Poly(D,L-lactide-co-gylcolide) lactide:glycolide 50:50 acid terminated Mw 38,000‑54,000 <3 months 719900-1G
719900-5G
Resomer® RG 502, Poly(D,L-Lactide-Co-Glycolide) lactide:glycolide 50:50 ester terminated Mw 7,000‑17,000 <3 months 719889-1G
719889-5G
Resomer® RG 503, Poly(D,L-lactide-co-glycolide) lactide:glycolide 50:50 ester terminated Mw 24,000‑38,000 <3 months 739952-1G
739952-5G
739944-5G
739960-5G
719862-5G
719919-5G
Resomer® RG 756 S, Poly(D,L-lactide-co-glycolide) lactide:glycolide 75:25 ester terminated Mw 76,000‑115,000 <6 months 719927-1G
719927-5G
Poly(D,L-lactide-co-glycolide) lactide:glycolide 85:15 ester terminated Mw 50,000‑75,000 <6 months 430471-1G
430471-5G
Resomer® RG 858 S, Poly(D,L-lactide-co-glycolide) lactide:glycolide 85:15 ester terminated Mw 190,000‑240,000 <9 months 739979-1G
739979-5G

aldrich.com/matsci
Low PDI Polylactides

Name Structure Molecular Weight PDI Degradation Time Prod. No.
Poly(L-lactide) O average Mn 5,000 ≤ 1.2 PDI >3 years 764590-5G
O CH3 average Mn 10,000 ≤ 1.1 PDI >3 years 765112-5G
H O
CH3 average Mn 20,000 ≤ 1.1 PDI >3 years 764698-5G
n
Poly(D,L-lactide) O average Mn 5,000 ≤ 1.1 PDI <6 months 764612-5G

O CH3 average Mn 10,000 ≤ 1.1 PDI <6 months 764620-5G
H O
CH3 average Mn 20,000 ≤ 1.3 PDI <6 months 767344-5G
n
End-functionalized Low PDI Poly(l-lactide)s

Name Structure Molecular Weight PDI Prod. No.
Poly(L-lactide), acrylate terminated O average Mn 2,500 ≤ 1.2 PDI 775991-1G
O O average Mn 5,500 ≤ 1.2 PDI 775983-1G
H O CH2
CH3 n
O
Poly(L-lactide), amine terminated O average Mn 2,500 ≤ 1.3 PDI 776378-1G

776378-5G
O
H O NH2 average Mn 4,000 ≤ 1.2 PDI 776386-1G
CH3 n 776386-5G
Poly(L-lactide), azide terminated O average Mn 5,000 < 1.2 PDI 774146-1G

O
H O N3
CH3 n
Poly(L-lactide) N-2-hydroxyethylmaleimide O O average Mn 5000 < 1.2 PDI 746517-1G

terminated 746517-5G
N O
O O H average Mn 2,000 ≤ 1.2 PDI 746797-1G
CH3 n 746797-5G
Poly(L-lactide) 2-hydroxyethyl, methacrylate CH3 O average Mn 2,000 ≤ 1.1 PDI 771473-1G

H O CH2
O O average Mn 5,500 ≤ 1.2 PDI 766577-1G
O CH3 766577-5G
n
Poly(L-lactide), propargyl terminated average Mn 2,000 ≤ 1.1 PDI 774162-1G

O
O CH average Mn 5,000 ≤ 1.1 PDI 774154-1G
H O
CH3
n
Poly(L-lactide), thiol terminated CH3 average Mn 2,500 ≤ 1.2 PDI 747386-1G

H O 747386-5G
O SH
average Mn 5,000 ≤ 1.2 PDI 747394-1G
O n 747394-5G
Polylactide Block Copoymers

Name Structure Molecular Weight Prod. No.
Poly(L-lactide-co-5-methyl-5- CH3 O average Mn 5,000 795259-1G
allyloxycarbonyl-1,3-dioxan- 2-one) CH3
H 3C H average Mn 10,000 795267-1G
O O O
O n m average Mn 40,000 792039-1G
CH2
O O

BIODEGRADABLE PLGA
MICROSPHERES AND
NANOPARTICLES
Aldrich® Materials Science offers a selection of preformed
Degradex® poly(lactic-co-glycolic acid) (PLGA) microspheres
and nanoparticles for drug delivery applications, such as:
Drug-carrier compatibility testing before active
yy
pharmaceutical ingredient (API)loading
Surface-conjugated drug carriers, covalent attachment of
yy
proteins, peptides, antibodies, and antigens
Fluorescent Degradex® particles available for imaging and
yy
diagnostic applications, including cell tracking, phagocytosis
studies, fluorescent microscopy, and drug discovery
Size standards
yy
Particle Size
Name (Average diameter) Prod. No.
PLGA nanoparticles 100 nm 805092
500 nm 805149
PLGA microspheres 2 µm 805130
50 µm 805122
Green fluorescent PLGA nanoparticles 100 nm 805157
500 nm 805300
Green fluorescent PLGA microspheres 2 µm 805181
50 µm 805165
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Degradex® particles are products of Phosphorex Inc.

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LIPID-POLYMER HYBRID NANOPARTICLES

FOR DRUG DELIVERY APPLICATIONS
One-step Synthesis of LPNs
Previously, researchers used various two-step synthesis methods
(Figure 1), that require the lipid vesicles and polymeric nanoparticle to
be separately synthesized before being fused together.8 This approach
gives rise to LPNs with bilayer or multilayer lipid shells. Techniques
used to fuse the liposomal shell and the polymeric nanoparticle core
Sangeetha Krishnamurthy,1 Juliana M. Chan2 together include extrusion, sonication, direct hydration, vortexing, and
School of Chemical and Biomedical Engineering1,2 and Lee Kong Chian School of Medicine2 high-pressure homogenization.
Nanyang Technological University, Singapore
Email: sangeetha.k@ntu.edu.sg and julianachan@ntu.edu.sg As first demonstrated by Zhang et al., a more convenient one-step
synthesis method uses nanoprecipitation and the spontaneous self-
Introduction assembly of lipid and polymer components (Figure 1A), yielding LPNs
coated with a lipid monolayer shell.9 In this method, the polymer
Since the last decade, there has been a burgeoning interest in the and cargo are dissolved in the organic phase (water-miscible organic
use of nanoparticle-based platforms for drug delivery applications. solvent) and the lipids are dissolved in the aqueous phase. The organic
Nanoparticle-based delivery offers a number of advantages over phase is added dropwise to the aqueous phase under continuous
traditional drug delivery platforms, including the ability to load multiple stirring, followed by self-assembly at room temperature. To the best
drugs, attach targeting ligands, enhance drug circulation time, and of our knowledge, this is the simplest method of synthesizing LPNs
reduce non-specific drug toxicity. Nanoparticle formulations such as currently available.
polymeric nanoparticles, liposomes, dendrimers, gold nanoparticles,
Alternatively, LPNs can be synthesized using an emulsification
carbon nanotubes and quantum dots have been widely researched,
technique where the polymer is dissolved in the organic phase (water-
but only a handful of them have ever reached clinical use.1
immiscible organic solvent) and the lipids are dissolved in the aqueous
The inherent advantages of liposomes and polymeric nanoparticles phase. The solutions are mixed and sonicated to disperse the polymer
make them the most commonly studied among available drug delivery into droplets and coat the polymers with a layer of lipid. The organic
platforms. For example, liposomes offer excellent biocompatibility,2 solvent is slowly evaporated under gentle stirring, and the LPNs are
while polymeric nanoparticles possess excellent stability and drug then purified for further use.
loading capacity.3 Although the majority of polymeric nanoparticles
are still years away from clinical application, researchers have sought A)
to combine the advantages of the two platforms—biocompatibility
and high drug loading—by designing hybrids, known as lipid-polymer Drug
Lipid
hybrid nanoparticles (LPNs).4
A typical LPN has a core-shell structure, consisting of a polymeric core
Polymer
for loading the cargo, such as small molecule drugs and/or diagnostic
molecules, surrounded by a lipid shell for enhanced biocompatibility. Lipid-coated Polymeric Nanoparticle
Lipid-PEG
The most widely used polymer in the core is poly(lactic-co-glycolic
acid) (PLGA) due to its biocompatibility, biodegradability and general B)
drug loading versatility.5,6 Several lipids, including phosphatidylcholine
(PC); 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC); 1,2-distearoyl- Polymer
sn-glycero-3-phosphoethanolamine (DSPE); cholesterol; myristic acid;
stearic acid; and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)
have been used in the shell, in addition to poly(ethylene glycol) (PEG)
lipid conjugates.5,7 Drug Lipid-coated Polymeric Nanoparticle
Figure 1. Schematic showing one- and two-step LPN synthesis. A) One-step synthesis method.
B) Two-step synthesis method.

Lipid-polymer Hybrid Nanoparticles for Drug Delivery Applications
The basic LPN formulation can be modified using targeting moieties 2. The organic solution is prepared by adding the following to a water-
to enable site-specific cargo delivery. In many cases, the lipid shell is miscible organic solvent such as acetonitrile:
functionalized using simple conjugation chemistry such as EDC-NHS or yy Poly(d,l-lactide-co-glycolide) (PLGA) with a 50:50 monomer
thio-maleimide chemistry. For example, LPNs developed by Aravind et ratio, ester-terminated, and viscosity of 0.72–0.92 dl/g
al. include AS1411 anti-nucleolin aptamers conjugated to the lipid shell (Durect Corporation, Pelham, AL). Varying the monomer
to specifically target cancer cells over-expressing nucleolin receptors.10 ratio and viscosity will give rise to LPNs with different sizes,
In another example, Clawson et al. developed stimuli-responsive LPNs biodegradation rates, and drug-loading and release properties.
using a pH-sensitive, lipid-succinate-mPEG coating.11 In a low-pH tumor
yy Small molecule drug such as docetaxel.
microenvironment, disassembly of the PEG layer is triggered causing
the internalization of LPNs by cell membrane fusion. The initial drug weight must not exceed 10–30% of the polymer
weight for the drugs to be properly encapsulated by the polymer.
While it is assumed the polymeric core can hold a variety of
The lipid/polymer weight ratio can range from 15%–20%.
cargo (sometimes more than one type at once), the lipid shell
can also be used to load cargo. Sengupta et al. loaded an anti- 3. The aqueous solution is heated to 65 °C on a hotplate stirrer under
angiogenic agent combrestatin-A4 in a lipid shell containing gentle stirring conditions for 3–5 min.
DSPE-PEG, phosphatidylcholine, and cholesterol. At the tumor site, 4. Once the reaction temperature is reached, the organic solution
combrestatin-A4 is first released, which shuts down the tumor is added dropwise to the aqueous solution under gentle stirring
vasculature. Doxorubicin is later released from the polymeric core to conditions, followed immediately by vigorous vortexing for 3 min.
cause cancer cell cytotoxicity.12 5. The mixture is returned to gentle stirring conditions and the LPNs
are allowed to self-assemble for 2 h at room temperature.
Summary 6. The LPNs are washed three times using an Amicon Ultra-4
centrifugal filter (Millipore, Billerica, MA) with a molecular weight
LPNs have been used to deliver drugs such as docetaxel,13 paclitaxel,14 cut-off of 10 kDa. The washed LPNs are re-suspended in water or
curcumin,15 and doxorubicin,16 in addition to diagnostic molecules for buffer at a final desired concentration.
various disease indications. These nanoparticles have characteristics 7. The LPNs are used immediately, stored at 4 °C overnight, or
that make them ideal candidates for drug delivery applications, lyophilized for extended storage at –80 °C.
including the ability to load multiple drugs, precisely control drug
References
loading and drug release, and functionalize with targeting moieties. (1) Zhang, L.; Gu, F. X.; Chan, J. M.; Wang, A. Z.; Langer, R. S.; Farokhzad, O. C. Clin. Pharmacol.
Ther. 2007, 83 (5), 761–769.
Although LPNs have been previously formulated using two-step (2) Sharma, A.; Sharma, U. S. Int. J. Pharm. 1997, 154 (2), 123–140.
synthesis methods, here we describe a one-step synthesis method that (3) Liechty, W.; Kryscio, D.; Slaughter, B.; Peppas, N. Annu. Rev. Chem. Biomol. Eng. 2010, 1, 149.
is convenient and reproducible. This method gives rise to LPNs that are (4) Krishnamurthy, S.; Vaiyapuri, R.; Zhangb, L.; Chan, J. Biomater. Sci. 2015, DOI: 10.1039/
less polydisperse in size and whose physicochemical properties can be C4BM00427B
(5) Hasan, W.; Chu, K.; Gullapalli, A.; Dunn, S.; Enlow, E.; Luft, J. C.; Tian, S.; Napier, M., Pohlhaus,
precisely controlled.17 P.; Rolland, J.; DeSimone, J. Nano Lett. 2012, 12 (1), 287–292.
(6) Zheng, Y.; Yu, B.; Weecharangsan, W.; Piao, L.; Darby, M.; Mao, Y.; Koynova, R.; Yang, X.; Li, H.;
Method: One-step Synthesis of LPNs

Xu, S.; Lee, L. J.; Sugimoto, Y.; Brueggemeier, R.; Lee, R. Int. J. Pharm. 2010, 390 (2), 234–241.
(7) Gao, L. Y.; Liu, X. Y.; Chen, C. J.; Wang, J. C.; Feng, Q.; Yu, M. Z.; Ma, X. F.; Pei, X. W.; Niu, Y. J.;
Qiu, C.; Pang, W. H.; Zhang, Q. Biomaterials 2014, 35 (6), 2066–2078.
(8) Thevenot, J.; Troutier, A. L.; David, L.; Delair, T.; Ladavière, C. Biomacromolecules 2007, 8 (11),
The following procedure describes a one-step synthesis method 3651–3660.
as performed by Prof. Juliana Chan’s research group at Nanyang (9) Zhang, L.; Chan, J.; Gu, F. X.; Rhee, J-W.; Wang, A.; Radovic-Moreno, A.; Alexis, F.; Langer, R.;
Technological University. Farokhzad, O. ACS Nano 2008, 2 (8), 1696–1702.
(10) Aravind, A.; Jeyamohan, P.; Nair, R.; Veeranarayanan, S.; Nagaoka, Y.; Yoshida, Y.; Maekawa, T.;
LPNs are synthesized from soybean lecithin, DSPE-PEG, and PLGA using Kumar, S. Biotechnol. Bioeng. 2012, 109 (11), 2920–2931.
a one-step nanoprecipitation method combined with self-assembly. (11) Clawson, C.; Ton, L.; Aryal, S.; Fu, V.; Esener, S.; Zhang, L. Langmuir 2011, 27 (17), 10556–
10561.
1. The aqueous solution is prepared by adding the following to a 4% (12) Sengupta, S.; Eavarone, D.; Capila, I.; Zhao, G.; Watson, N.; Kiziltepe, T.; Sasisekharan, R.
Nature 2005, 436 (7050), 568–572.
ethanol aqueous solution in a glass vial (ethanol, Sigma-Aldrich
(13) Liu, Y.; Li, K.; Pan, J.; Liu, B.; Feng, S-S. Biomaterials 2010, 31 (2), 330–338.
Prod. No. E7023): (14) Wang, H.; Zhao, Y.; Wu, Y.; Hu, Y. L.; Nan, K.; Nie, G.; Chen, H. Biomaterials 2011, 32 (32),
yy Soybean lecithin consisting of 90–95% phosphatidylcholine (MP 8281–8290.
Biomedicals, Solon, OH) (15) Kumar, S. S. D.; Mahesh, A.; Mahadevan, S.; Mandal, A. B. Biochim. Biophys. Acta (BBA) -
General Subjects 2014, 1840 (6), 1913–1922.
yy DSPE–PEG2000–COOH (1,2-distearoyl-sn-glycero-3-phospho (16) Prasad, P.; Shuhendler, A.; Cai, P.; Rauth, A.; Wu, X. Y. Cancer Lett. 2013, 334 (2), 263–273.
(17) Chan, J. M.; Zhang, L.; Yuet, K. P.; Liao, G.; Rhee, J. W.; Langer, R.; Farokhzad, O. C. Biomaterials
ethanolamine-N-carboxy(polyethylene glycol)2000) (Avanti, 2009, 30 (8), 1627–1634.
Alabaster, AL).
The soybean lecithin/DSPE–PEG molar ratio can range from
7:3 to 8.5/1.5.

aldrich.com/matsci
Lipids for Drug Delivery

For more information on these products, visit sigma.com/lipids.
Product Description Structure Purity Prod. No.
1,2-Didodecanoyl-sn-glycero-3-phosphocholine, O O CH3 ≥99% P1263-25MG
synthetic O P1263-100MG
P N+
CH3(CH2)9CH2 O O CH3 P1263-500MG
O– H3C
O O
CH2(CH2)9CH3
1,2-Dilinoleoyl-sn-glycero-3-phosphocholine O ≥99%, TLC P0537-5MG

P0537-25MG
CH3 O O (CH2)7 CH3
(CH2)4
H3C N O P O O
CH3 O CH3
O (CH2)7 (CH2)4
1,2-Dioleoyl-sn-glycero-3-phosphocholine CH2(CH2)6CH3 - P6354-25MG

O P6354-100MG
O CH3
P6354-1G
CH2(CH2)5CH2 O O P O N CH3
O O CH3
CH2(CH2)5CH2
O
CH2(CH2)6CH3
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, O CH3 ≥99% P0763-50MG

semisynthetic O P0763-100MG
N CH3
CH3(CH2)13CH2 O O P O P0763-250MG
CH3
CH3(CH2)13CH2 O O P0763-1G
P0763-5G
1,2-Dipalmitoyl-sn-glycero-3-phosphocholine O ≥99%, TLC P4329-25MG
P4329-100MG
P4329-500MG
P4329-1G
1,2-Dipalmitoyl-rac-glycero-3-phosphocholine ~99% P5911-100MG
P5911-250MG
P5911-1G
1,2-Distearoyl-sn-glycero-3-phosphoethanolamine O O ≥99% P3531-100MG

O P3531-500MG
P NH2
H3C(H2C)16 O O
OH
O O
(CH2)16CH3
DOTAP chloride O ≥98%, TLC D6182-50MG

D6182-250MG
CH3(CH2)6 (CH2)6 C O CH2
CH3(CH2)6 (CH2)6 C O CH
O CH2 Cl
CH3 N CH3
CH3
Isopropyl myristate O CH3 ≥90%, GC M0757-250ML

M0757-1L
CH3(CH2)11CH2 O CH3
L-α-Lysophosphatidylcholine - ≥99% L4129-25MG

L4129-100MG
L4129-500MG
L4129-1G
- ≥99% L0906-25MG
L0906-100MG
L0906-500MG
Methoxypolyethylene glycol maleimide O ≥90%, NMR 63187-1G
63187-5G
O N
H3C O
n O

Lipid-polymer Hybrid Nanoparticles for Drug Delivery Applications
Product Description Structure Purity Prod. No.

Methyl 12-hydroxystearate O ≥99%, GC H7002-1G
H3C OCH3
OH
Methyl myristate O ≥99%, GC M3378-1G
M3378-25G
CH3(CH2)11CH2 OCH3
Methyl stearate O ~99%, GC S5376-1G

S5376-5G
CH3(CH2)15CH2 OCH3 S5376-10G
S5376-50G
Myristic acid O ≥99% M3128-10G
M3128-100G
CH3(CH2)11CH2 OH M3128-500G
≥98.0%, GC 70082-50G
70082-250G
70082-1KG
1-Oleoyl-sn-glycero-3-phosphocholine, synthetic O ≥99% L1881-5MG
O CH3 L1881-25MG
CH2(CH2)5CH2 O O P O N CH3 L1881-100MG
OH O CH3
CH2(CH2)6CH3
1-Palmitoyl-sn-glycero-3-phosphocholine, synthetic O O CH3 ≥99% L5254-25MG

O L5254-50MG
P N+
H3C(H2C)14 O O CH3 L5254-100MG
O– H3C
OH L5254-250MG
L5254-1G
L-α-Phosphatidylcholine, egg yolk O ≥99%, TLC P3556-25MG
O CH3 P3556-100MG
R O O P O N+ CH3 P3556-500MG
O O O– CH3 P3556-1G
L-α-Phosphatidylcholine, from egg yolk ~60%, TLC 61755-25G
R' 61755-100G
L-α-Phosphatidylcholine, dried egg yolk R, R' = fatty acid residues ≥40%, enzymatic P5394-10G
P5394-25G
P5394-100G
P5394-500G
L-α-Phosphatidylcholine, egg yolk ≥99% P2772-100MG
P2772-250MG
P2772-500MG
P2772-1G
≥99%, TLC P4279-100MG
P4279-250MG
P4279-1G
L-α-Phosphatidylcholine, soybean O ≥99%, TLC P7443-100MG
O CH3 P7443-500MG
R O O P O N+ CH3 P7443-1G
O O O– CH3 ≥30%, enzymatic P3644-25G
P3644-100G
R' P3644-500G
R, R' = fatty acid residues P3644-1KG
14‑23% choline basis P5638-500G
P5638-1KG
L-α-Phosphatidylcholine, hydrogenated - ≥99% P4139-100MG
P4139-1G
Sodium myristate O ≥99% M8005-10G
M8005-25G
CH3(CH2)11CH2 ONa
Sodium stearate O ≥99% S3381-1G

S3381-5G
CH3(CH2)15CH2 ONa S3381-25G
Stearic acid O ≥98.5%, capillary GC S4751-1G
S4751-5G
CH3(CH2)15CH2 OH S4751-10G
S4751-25G
S4751-100G

aldrich.com/matsci
CROSSLINKED CHITOSAN NANOPARTICLES

AND CHEMICAL MODIFICATIONS FOR DRUG DELIVERY APPLICATIONS
Nanoparticle drug delivery systems, including nanospheres,
nanocapsules, nanomicelles, nanoliposomes, etc., are nanometric
carriers used to deliver drugs or biomolecules by trapping active agents
in their interior structures and/or adsorbing them onto their exterior
surfaces.1,10–11 Presently, nanoparticles (NPs) have been widely used
to deliver drugs, polypeptides, proteins, vaccines, genes, and nucleic
acids. Recently, there has been increased interest in the use of NPs
Shady Farah,1–3 Joshua C. Doloff,1–3Daniel G. Anderson,1–5 Robert Langer1–5*
containing natural polysaccharides for drug delivery applications.12–13
1
David H Koch Institute for Integrative Cancer Research A large number of studies have been conducted on polysaccharides
Massachusetts Institute of Technology, Cambridge, MA, USA and their derivatives for their potential application as NP drug
2
Department of Chemical Engineering delivery systems, and chitosan has been identified among the most
Massachusetts Institute of Technology, Cambridge, MA, USA
promising candidates.14–16
3
Department of Anesthesiology, Boston Children’s Hospital, Boston, MA, USA
4
Institute for Medical Engineering and Science The following sections focus on the leading techniques for preparation
Massachusetts Institute of Technology, Cambridge, MA, USA
and application of chitosan NPs, as well as chemical modification
5
Harvard-MIT Division of Health Science and Technology
Massachusetts Institute of Technology, Cambridge, MA, USA methods for self-assembly structures including nanoparticles
Email: rlanger@mit.edu (*Corresponding Author) and nanomicelles.
Introduction Chitosan NP Preparation Methods

Particulate chitosan structures are 3D crosslinked networks where
As a polysaccharide, chitosan can have a large density of reactive polymeric chains are interconnected by crosslinkers. The main
groups and a wide range of molecular weights (Mw). Chitosan is a parameter, which determines the properties of a crosslinked NP,
linear heteropolymer of N-acetyl-d-glucosamine and d-glucosamine such as drug release and mechanical strength, is the crosslinking
linked by β-(1–4)glycosidic bonds (Figure 1). It is obtained by partial density.17 Depending on the desired chitosan structural characteristics,
deacetylation of chitin, the second largest and most abundant nanoparticles are prepared mainly by four mechanisms:
polysaccharide in nature after cellulose. The degree of acetylation (DA)
is an essential characteristic of chitin and chitosan. It represents the 1. Covalent crosslinking
fraction of N-acetyl-d-glucosamine relative to the total number of units. 2. Ionic crosslinking
3. Polyelectrolyte complexation
OH OH 4. Self-assembly of hydrophobically modified polysaccharide
O O
O O
HO HO
In general, for mechanisms 1–3, chitosan NP preparation starts with
NH2 NH the dropwise addition of the desired crosslinker to the chitosan
O
n solution with continuous stirring for 1–24 h, with or without slight
CH3
heating depending on the crosslinking chemistry. However, the fourth
Figure 1. Chemical structure of chitosan mechanism of NP preparation, in addition to hydrophobic moiety
chemistry, depends on two parameters: the percentage of hydrophobic
For many years chitosan was considered useful as a bioadhesive
substitution (Degree of Substitutions—DS%) and the final modified
material because of its ability to form non-covalent bonds with
chitosan concentration (lower for nanoparticles and nanomicelles;
biological tissues, mainly epithelia and mucous membranes.
higher within hydrogels). Table 1 summarizes and compares these
Bioadhesions formed using natural polymers have unique properties
four mechanisms as well as current delivery applications. Figure 2
as a carrier because they can prolong residence time and, therefore,
represents illustrative photos on the crosslinked chitosan 3D structure
increase the absorbance of loaded drugs.1 Chitosan is hydrophilic
formation highlighting internal interactions. Hydrophobic groups on
and soluble in acidic solutions through the protonation of its amine
chitosan confer new physicochemical properties, including the ability
groups. Modified and unmodified chitosan has been widely used,
to self-associate in water or under sonication,18–19 to form different
albeit with different molecular weights and chemical modifications,
types of drug delivery systems (Figure 2).
in biomedical,2,3 pharmaceutical,4 metal chelation,5,6 food additive,7
and other industrial applications.8 Chitosan is biocompatible and
can be biodegraded by enzymes such as lysozymes, some lipases,
and proteases.9 These properties, as well as its positive charge in
physiological conditions, endow chitosan with a promising future as
a biomaterial.

Crosslinked Chitosan Nanoparticles and Chemical Modifications for Drug Delivery Applications
Table 1. Crosslinked chitosan NP synthesis mechanisms and drug delivery applications—comparison study.
Chitosan NP Formulation Mechanisms

Self-assembly of Hydrophobically
Mechanism Parameters Covalent Crosslinking Ionic Crosslinking Polyelectrolyte Complexation Modified Chitosan
Interaction Irreversible chemical linkers Reversible ionic crosslinking Multi-ionic crosslinking Intramolecular and/or intermolecular associations in
water. Hydrophobic moieties interactions
Crosslinker/Moiety Two reactive functional groups Small ionic molecules Polyelectrolyte or larger ionic molecules Hydrophobic nature
Characteristics (at least)
Main Interaction Covalent bond interactions Electrostatic interactions Multiple electrostatic interactions Hydrophobic interactions
Secondary Interactions Hydrogen bonds and hydrophobic Hydrogen bonds Hydrogen bonds Hydrogen bonds
interactions
Classical Crosslinkers/ Dialdehydes: Tripolyphosphate (TPP) Alginate, Heparin, Peptide, Nucleic Linoleic acid, Stearic acid, 5β-Cholanic acid, Deoxy-
Hydrophobic Moiety Glutaraldehyde acid, Poly(acrylic acid), Carboxymethyl cholic, Oleoyl, Cholesterol
Di/tricarboxylic acids Cellulose
Structures NPs NPs NPs NPs and Nanomicelles
Biocompatibility and Toxicity Aldehydes are highly toxic, while Enhanced biocompatibility since the Enhanced biocompatibility since the Related to modification moiety, particulate structure,
natural carboxylic-based crosslinkers preparation of NPs are by reversible preparation of NPs are by reversible ionic and DS%
are considered biocompatible ionic crosslinking, without use of crosslinking, without use of aldehyde or
alternatives aldehyde or toxic crosslinkers toxic crosslinkers
Particles Size 200–400 nm 100–350 nm 200–900 nm 80–1,400 nm
Drugs and Drug Delivery Toothpaste applications Nanoparticles as carriers for the Great potential in gene delivery: plasmid Drug delivery to brain tumor or general anticancer
Applications anticancer drug doxorubicin DNA as well as insulin and bovine serum drug delivery: methotrexate, paclitaxel, doxorubicin,
albumin epirubicin
References 20–22 20 17,20,23–25 26–28
Notes Rigid network structures Mild-rigid network structures pH-responsive drug release network NP size is controlled by adjusting the length of the
structures hydrophobic moiety and chitosan Mw.17
Core hydrophobic—optimal for hydrophobic
drugs delivery.
Prolonged circulation and thermodynamic stability.29
Hydrogel could form at high concentration and
DS% levels.
(1) Covalent Crosslinking

O NH
H3C O
NH H3CHN
H2N
H2N
H NH2 NH2
E HN (2) Ionic Crosslinking
H N N
H3C O 2
HN O NH E NH
H2N
NH CH3 NH
CHH C E O NH2
NH NH3 NH3
O NH2O 33
HN O
CH3
H3CHN E NH2
O HN H3C O NH O NH
H2N
H3CHN NH2HN NH2 HN
ENH NHH3CHN O
N NH2O NH
RRN 2 HN OCHNH
H H N NH E H3C H CH3H2N
H3C N 3
NH2 E NH2
HN O H RR OCH NH2 O N
NH E NH
NH2 NH3
NH
O HCH
H2N NH2 R R H 2N NH2
3 O CH2 3
NH2
O
2N
3
H2N NH
H3CHN
E HNH
N E NH NH
NH3
NH HN
NH O NO 3C H O H2N
2
O NH H3C N H N CH2 NH2
NH2
CHH33C OH2N H CH3 O
NH
H2N
NHCH
3 O
CH3
HN CHH2 N O NH2 NH3
NH 2
NH2 O CH3 NH3
NH2 (4) Self-assembly of E E H3N NH

CHO3 O
NH
Hydrophobically O
H3C H
N
NH 3 CH3
H3C
Nanomicelles formation Modified Chitosan NH2 NH2 HN
O
R NH NH2 NH2
H3C
O
H3C
O
Hydrophobic H3C
O
O
CH3 NH2 NH2 HN
NH2 NH2 HN
HN NH2 NH moities Protonation
modifications H3C Acidic pH O
NH NH2 NH2
HN NH2 NH NH NH2 NH2 O CH3
DS%> X O
CH3
O
CH3
NH2 NH2 HN
(3) Polyelectrolyte Complexation
C%> Y R NH NH2 NH2
Sonication> T O
CH3 NH2
NH3 NH3
Chitosan O
N NH2O NH
H2N
H3C H CH3H3N
NH
Nanoparticles formation NH3 NH3
O HCH 3
2N
NH NH3
NH2 NHH2N H3N
NH2 O
CH2 NH2
HHN
2N NH NHCH CH3
O H3 N HO
R
3
O HN
N NH2O NH CHH3 N O NH2 NH2
RR
2
CH3H2NH
N
H3C H
R R NH3
RR R NH3 NH
NH OH CH3
NH
2N E E Covalent Crosslinker E Electrophile groups
R Hydrophobic H3N NH
CHO3 O
NH
NH
3 CH3
HHN
2N HN O N
NH HNH2N NH
NH moiety H3 C H
NHH2N NH 3
R 3
NH RNH
CH3
RR
N CH RR
NH
CH3 Ionic Crosslinker Negatively charged groups DS% - Degree of substitution
RR
O 3 O C% - Modified Chitosan concentration
CHH3 N O NH2 NH2
RRR
2 NH
NH33
NH HN X,Y,T - Values related specific moiety
H2N HN NH NH Negatively charged polyelectrolyte
NH O
CHO3 NH 2 CH3 Hydrophobic Interactions
O N
H3C H
Figure 2. Mechanisms of chitosan nanoparticle formation and hydrophobically modified chitosan self-assembly nanomicelles.
Identification of the most appropriate mechanism for chitosan of the crosslinker itself. In covalent crosslinking, for example, the most
nanoparticle formation can be complicated. Many factors must be crucial factor is the crosslinker size/length; in ionic crosslinking, the
considered that may affect the rational design of nanoparticulate global charge of both the crosslinker and the polysaccharide are
delivery systems, including drug nature and loading capacity, dominant. Additionally, unlike covalently crosslinked NPs, ionically
delivery duration, chitosan Mw, NP shape and size, targeting site, and crosslinked particles are generally pH sensitive, a desired trait for drug
biocompatibility. On the other hand, several factors also influence the delivery purposes.
crosslinking reaction such as the characteristics and chemical structure

aldrich.com/matsci
Chitosan Modifications: NPs and Nanomicelles

In addition to Mw diversity, chitosan has a variety of reactive groups,
Concluding Remarks
including hydroxyl and amino groups (Figure 1), which allow for the This short review summarizes the recent research on chitosan and its
possibility of chemical modification to obtain amphiphilic properties.1 amphiphilic derivatives, including experimental chemical modifications,
This gives it a new or improved property. Chitosan, chemically modified the mechanisms of nano-structure fabrication, and their drug delivery
through the grafting of hydrophobic groups, undergoes intra and/ system applications. Special attention is increasingly focused on
or inter-molecular hydrophobic interactions. Amphiphilic properties modified chitosan with amphiphilic properties because of unique
allow it to self-associate in aqueous solution, leading to different properties such as excellent biocompatibility and biodegradability,
kinds of drug delivery systems such as nanomicelles, nanoparticles, non-toxicity, and bioadhesive properties. Self-assembly of amphiphilic
microspheres,30 liposomes,31 and hydrogels (Figure 2. Mechanism 4). chitosan provides further promise for drug delivery systems since
Modified chitosans are of great interest for the development of various several properties such as size, surface charge, loading efficiency,
controlled-release systems. stability, and biodistribution can be altered for a particular application.
Chitosan-based nanomicelle systems have been found to improve
Since the amine group of chitosan is more reactive than the hydroxyl
delivery of hydrophobic drugs and proteins, increase stability, and
groups, all the research describing the formation of amphiphilic
exhibit controllable drug release properties, biocompatibility, and
chitosan have been based on the chemical grafting of hydrophobic
improved targeting for specific applications—all of which are of
groups on the amine functional group by N-acylation reactions.32 The
increasing interest for clinical application.
following section reviews experimental procedures for the chemical
modification of chitosan based on N-acylation and current applications
References
in drug delivery systems, as listed in Table 1 (self-assembly part) (1) Liu, Z. et al., Adv. Drug Deliv. Rev. 2008, 60, 1650–1662.
and Table 2. (2) Langer, R.; Tirrell, D. A. Nature 2004, 428, 487–492.
(3) Alvarez, N. M.; Mano, J. F. Int. J. Biol. Macromol. 2008, 43, 401–414.
Delivery of hydrophobic molecules and proteins has always been (4) Pedro, A. S. et al. Carbohydr. Polym. 2009, 76, 501–508.
an issue due to poor bioavailability after administration. Micelle (5) Guibal, E. et al. Int. J. Biol. Macromol. 2001, 28, 401–408.
carrier systems can improve drug solubility and stability as well as (6) Schmuhl, R. et al. Water SA 2001, 27, 1–7.
help overcome toxicity and immunogenicity problems. It is well (7) Shahidi, F. et al. Trends Food Sci. Technol. 1999, 10, 37–51.
(8) Majeti, N. V.; Kumar, R. React. Funct. Polym. 2000, 46, 1–27.
known the hydrophobic core of the micelles provides a reservoir for (9) Bardot, P.M.; et al. Presses universitaires de Franche-Comte´ mars 2009. 308,
loading water-insoluble drugs. By grafting hydrophobic moieties to ISBN: 2-84867-249-8.
the polysaccharide backbone, self-assembled micelles can be readily (10) Jung, T. et al. Eur. J. Pharm. Biopharm. 2000, 50, 147–160.
(11) Eliaz, R. E.; Szoka, F.C. Cancer Res. 2001, 61, 2592–2601.
formed in aqueous solution. Aggregation of amphiphilic polymers is
(12) Coviello, T. et al. J. Controlled Release 2007, 119, 5–24.
controlled by the balance between the interaction of the hydrophobic (13) Vauthier, C.; Bouchemal, K. Pharm. Res. 2009, 26, 1025–1058.
groups and the hydrophilic chains. The critical aggregation (14) Sinha, V. R.; Kumria, R. Int. J. Pharm. 2001, 224, 19–38.
concentration (CAC) is the concentration at which the polymer (15) Vandamme, T. F. et al. Carbohydr. Polym. 2002, 48, 219–231.
(16) Lemarchand, C. et al. Eur. J. Pharm. Biopharm. 2004, 58, 327–341.
aggregation starts.
(17) Mizrahy, S.; Peer, D. Chem. Soc. Rev. 2012, 41, 2623–2640.
It was found that a chitosan EDC-mediated modification with stearic (18) Liu, C. et al. J. Ocean Univ. China 2005, 4, 234–239.
(19) Liu, C. et al. Curr. Appl. Phys. 2007, 7, 125–129.
acid, linoleic acid, deoxycholic acid, and 5β-cholanic acid tends (20) Berger, J. et al. Eur. J. Pharm. Biopharm. 2004, 57, 19–34.
to self-aggregate to form nanomicelles at very low CAC (0.01– (21) Liu, H. et al. J. Appl. Polym. Sci. 2007, 106, 4248–4256.
0.06 mg/mL).33–36 Amphiphilic chitosan-based micelles were used (22) Bodnar, M. et al. Biomacromolecules 2005, 6, 2521–2527.
to encapsulate doxorubicin,37 paclitaxel,27,34,38 ibuprofen,39 and the (23) Hamman, J. H. Mar. Drugs 2010, 8, 1305–1322.
(24) Park, J. H. et al. Adv. Drug Delivery Rev. 2010, 62, 28–41.
amphiphilic adriamycin.40 Furthermore, hydrophilic peptides, proteins, (25) Sajeesh, S.; Sharma, C.P. J. Biomed. Mater. Res. 2006, 76B, 298–305.
and nucleic acids41,42 can be adsorbed onto chitosan-based micelles. (26) Yang, X. D. et al. Colloids Surf., B 2008, 61, 125–131.
(27) Hu, F. Q. et al. Colloids Surf., B 2006, 50, 97–103.
Table 2. Chitosan modifications—experimental reactions and applications
Modification Chitosan N-acylation Modifications via:

Parameters N-acetyl Chloride N-acetyl Carboxylic Acid N-carboxyacyl
Chemical Reaction OH OH OH
O O O
(Amino Repeating Unit O
O O O O
in Chitosan) HO
R Cl
HO R
HO OH
O
NH2 OR NH2 OH NH2 O
O O OH OH O
O (EDC) O HO
R O R O O R NH
O
HO HO O
NH NH O R
O O O
R OH
R
Hydrophobic Moiety Oleoyl chloride 28

Stearic acid (C18H36O2) 27,45
Cyclic acid anhydride (R): acetic, propionic, n-butyric,
Linoleic acid (C18H32O2)46 n-valeric, hexanoic, octanoic, lauric, palmitic, and stearic
Oleic acid (C18H34O2)47
Deoxycholic acid35,41
Reaction Conditions In mixture (pyridine/chloroform) with oleoyl chloride at 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Reactions performed in presence of dimethyl sulfoxide.48–49
room temperature for 2 h, followed by reflux for 10 h.
Purification: Crude product was poured into methanol,
filtered and dried under vacuum for 24 h.43
Notes DS calculation by infrared spectroscopy, ratio of NP size increased with the amount of active ingredient
absorbance at 1,655 cm-1 (amide I band) and the encapsulated.
hydroxyl band at 3,450 cm-1.44
Drug Delivery NPs as carriers for anticancer drug doxorubicin. For linoleic acid: magnetic resonance imaging of the liver. NP carriers for anticancer drugs
Applications NP carriers for anticancer drugs: paclitaxel and doxorubicin.

Crosslinked Chitosan Nanoparticles and Chemical Modifications for Drug Delivery Applications
(28) Zhang, J. et al. Nanomed-Nanotechnol. 2007, 3, 258–265. (39) Jiang, G. B. et al. Mol. Pharm. 2006, 3, 152–160.
(29) Letchford, K.; Burt, H. Eur. J. Pharm. Biopharm. 2007, 65, 259–269. (40) Lee, K. Y. et al. Colloid. Polym. Sci. 2000, 278, 1216–1219.
(30) Mi, F. L. et al. Carbohydr. Polym. 2005, 60, 219–227. (41) Lee, K. Y. et al. J. Controlled Release 1998, 51, 213–220.
(31) Liang, G. et al. J. Pharm. Pharmacol. 2007, 59, 661–667. (42) Lee, K. Y. et al. Polymer 2005, 46, 8107–8112.
(32) Hassani, L. N. et al. Drug Discovery Today 2012, 17, Num. 11/12. (43) Li, Y. Y. et al. J. Appl. Polym. Sci. 2006, 102, 1968–1973.
(33) Kwon, S. et al. Langmuir 2003, 19, 10188–10193. (44) Le Tien, C. et al. J. Controlled Release 2003, 93, 1–13.
(34) Kim, J. H. et al. (Reprinted from J. Controlled Release 2005, 109, 1), (45) Hu, F. Q. et al. Biomaterials 2009, 30, 6955–6963.
J. Controlled Release 2006, 111, 228–234. (46) Liu, C. G. et al. Carbohydr. Polym. 2005, 62, 293–298.
(35) Lee, K. Y. et al. Macromolecules 1998, 31, 378–383. (47) Huang, L. et al. Front. Biol. China 2009, 4, 321–327.
(36) Kim, K. et al. Macromol. Res. 2005, 13, 167-175. (48) Lee, M. Y. et al. Int. J. Biol. Macromol. 2005, 36, 152–158.
(37) Ye, Y. Q. et al. Int. J. Pharm. 2008, 352, 294–301. (49) Mourya, V. K.; Inamdar, N. N. React. Funct. Polym. 2008, 68, 1013–1051.
(38) Zhang, Y. et al. Carbohydr. Polym. 2009, 77, 231–238.
TECHNICAL SPOTLIGHT
Chitosan Nanoparticles by Covalent or Ionic Crosslinking

Chitosan nanoparticles can be formed by a variety of methods including emulsion, ionic gelation, reverse micellar
methods, or self-assembly. The example method given here uses the simplest approach to form either chemical or ionic
crosslinked chitosan nanoparticles. This method utilizes dropwise addition of a crosslinker to a solution of chitosan under
constant stirring.
1. Prepare chitosan solution: Dissolve chitosan at 0.1–1 wt% in
1–3% acetic acid, once dissolved adjust pH to 4.7.
2. Prepare crosslinker solution:
a) For Ionic gelation: dissolve crosslinker (Tripolyphosphate
(TPP), dextran sulphate, etc.) in deionized water at 0.1 wt%
Solution of Chitosan Add Crosslinker Dropwise, Drug-loaded
to 0.5 wt%. with Drug with Constant Agitation Chitosan Nanoparticles
b) For covalent crosslinking: adjust crosslinker concentration
(example: 1–3% dialdehyde or di/tricarboxylic acid).
3. Slowly add crosslinker solution (1 mL) dropwise into Chitosan
solution (3 mL) at room temperature with constant stirring.
4. Allow nanoparticles to stabilize by a 30 min incubation at
room temperature.
5. Collect nanoparticles by centrifugation at 13,000 × g at 10 °C
for 30 min, and resuspend nanoparticles in aqueous solution.
6. To incorporate drug, add drug to crosslinker aqueous solution
prior to addition to the chitosan solution.
Note: Chitosan and crosslinker concentration need to be
optimized for desired particle size and encapsulation efficiency.
Natural Polymers
For more information on these products, visit aldrich.com/natural.
Name Inherent Viscosity (cP) Degree of Deacetylation Prod. No.
Chitosan 20-300 (1 wt. % in 1% acetic acid Brookfield) 75-85% deacetylated 448869-50G
448869-250G
200-800 (1 wt. % in 1% acetic acid Brookfield) 75-85% deacetylated 448877-50G
448877-250G
800-2000 (1 wt. % in 1% acetic acid Brookfield) >75% deacetylated 419419-50G
419419-250G
Alginic acid 15-20 1 % in H2O - 180947-100G
180947-250G
180947-500G

aldrich.com/matsci
POLY(N-ISOPROPYLACRYLAMIDE)-BASED
STIMULI-RESPONSIVE MATERIALS
sites.9 Stimuli-responsive drug delivery platforms often are prepared
by formulating or functionalizing the system with thermo- and/or
pH-sensitive polymeric materials.10 These materials have polymer–
polymer and polymer–solvent interactions that change abruptly with a
small change in pH and/or temperature, which translates to a polymer
chain transition between extended and compacted coil states. In drug
delivery, this chain configuration disrupts the integrity of the delivery
vehicle and triggers release of the drug.
Ganga Panambur, Nicolynn Davis
Aldrich Materials Science
Poly(N-isopropylacrylamide) (PNIPAM) (Figure 1) is a unique hydrophilic
Sigma-Aldrich, Milwaukee, WI USA polymer with a stimuli-responsive transition temperature close to
Email: gangadhar.panambur@sial.com physiological temperature.
Introduction
Controlled delivery of therapeutic agents has generated significant n
interest1–3 because it can improve drug efficacy by preventing NH
O
premature degradation, enhance uptake, reduce side effects, and help
to maintain appropriate therapeutic concentration in the bloodstream.
Significant research effort has been devoted to the development of
systems that can deliver defined quantities of a therapeutic payload Figure 1. Structure of PNIPAM.
in a site-specific and/or time-controlled fashion. Devices made with In aqueous solution, PNIPAM exhibits a thermo-responsive phase
stimuli-responsive materials have attracted considerable interest for transition at 32 °C. This transition temperature is called the lower
use in controlled delivery.4–6 Stimuli-responsive or “smart” materials critical solution temperature (LCST). Below the LCST, PNIPAM is water-
undergo dramatic property changes in response to small changes in soluble and hydrophilic, with an extended chain conformation. At LCST,
the environment and can be used as programmable, responsive, and PNIPAM undergoes a phase transition to a hydrophobic aggregate state
adaptive materials. The response may be produced through artificial becoming water insoluble above the LCST. This phase transition occurs
stimuli such as thermal, light-irradiation, magnetic, ultrasonic, and within a remarkably narrow temperature range and is reversible. The
electric, as well as natural stimuli like changes in pH, ionic strength, macroscopic manifestation of this thermal change depends on the
redox gradients, and enzymatic stimuli.4 The use of stimuli-responsive chain configuration (Figure 2).11
delivery devices offers an interesting opportunity for controlled delivery
where the delivery system becomes an active participant rather than a A)
passive vehicle in the optimization of a therapy.7 The benefit of stimuli
responsive nano-carriers is especially important when the stimuli are
unique to disease pathology, allowing the nano-carrier to respond
specifically to the pathological “triggers” such as pH, temperature, and S
B) T
redox microenvironment.
I
M
Poly(N-isopropylacrylamide): U
L
U
A Temperature-sensitive Polymer C)
S
Among the stimuli-responsive materials, thermo- and pH-sensitive

materials are the most frequently used for controlled drug delivery
because of the different thermal and pH conditions within various
Figure 2. Classification of stimuli-responsive polymers by their physical form and their
tissue and cellular compartments along the endocytic pathway.8,9 response to external stimuli. A) Linear free chains in solution will undergo a reversible collapse
For example, tumor tissues have slightly higher temperature and after stimulus is applied. B) Covalently crosslinked reversible gels where swelling or shrinking
lower pH than healthy tissues. These variations in temperature and of the gels can be triggered by environmental change. C) Chain adsorbed or surface-grafted
form, where the polymer reversibly swells or collapses on the surface once an external
pH can be exploited for the targeted release of payloads at specific parameter is changed.11

Poly(N-isopropylacrylamide)-based Stimuli-responsive Materials
The solubility of PNIPAMs below the LCST and lack of functional groups PNIPAMs have been developed with a range of functional groups
for further modification can limit the utility of the unmodified polymer including amine,36–38 carboxylic,39–41 NHS ester,42,43 and thiol.44,45 Recent
in many drug delivery applications.12 Therefore, PNIPAM is often developments in controlled radical polymerization, such as RAFT,
modified to tune the LCST through the synthesis of copolymers using provide a very simple tool to prepare end-functionalized PNIPAM with
hydrophilic or hydrophobic monomers. For example, when NIPAAm well-defined structures46–48 that can be used to make nanocarriers
is copolymerized with the hydrophilic monomers acrylamide (AAm), amiable to bioconjugation.49–51 Hoffman et al. prepared amine-
the LCST increases to 45 °C with an AAm content of 18%. Conversely, terminated PNIPAMs using the amine for protein conjugation.52 Meyer
copolymers can be synthesized with an LCST of 10 °C when 40% of et al.39 demonstrated enhanced and targeted delivery of therapeutics
the hydrophobic N-tert-butylacrylamide (N-tBAAm) monomer is added to solid tumors with an amine-terminated poly(NIPA-co-AAm)/
to the polymer.12–14 Copolymers with both thermal and pH sensitivity rhodamine conjugate. Furthermore, carboxyl-terminated PNIPAM has
have been synthesized with acidic monomers such as acrylic acid or been used for bioconjugation.53–55
methacrylic acid.16–21 In addition, protein and peptide conjugates of
PNIPAM and copolymers of poly(NIPAM-co-acrylic acid) have been
explored to further modify the LCST or stimuli responsiveness.22–24 PNIPAM-coated Liposomes
PNIPAM can also be incorporated into liposomes for drug delivery by
PNIPAM Micelles modifying the PNIPAM polymer structure. The incorporation of long
hydrocarbon chains such as C-18 (either on the end or randomly
Amphiphilic copolymers containing PNIPAM have been used to form along the PNIPAM backbone) enables polymer anchoring into the
nano-assembled aggregates for use as drug delivery carriers.25–27 liposome bilayer.56–58 The resulting PNIPAM coated liposomes act as
To do this, block copolymers consisting of a PNIPAM segment stimuli-sensitive delivery devices for tumor delivery.59,60 Moreover,
and a hydrophobic segment are used to form core–shell micellar liposomes were surface modified with PNIPAM copolymers, such as
structures below the PNIPAM LCST. The inner hydrophobic core is then poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and PEG.61
loaded with water-insoluble drugs; the PNIPAM outer shell imparts The release of DOX from these PNIPAM-AAM/PEG-modified liposomes
temperature responsiveness and aqueous solubilization. Upon reaching increases near the polymer transition temperature. In addition, these
the LCST, nano-assemblies of PNIPAM-based micelles loaded with modified liposomes were found to be stable in serum compared
active ingredients collapse and release the payload (Figure 3).28,29 with unmodified liposomes, indicating promise for use in targeted
Chung et al. demonstrated enhanced drug release in response to drug delivery.
temperature fluctuations and improved cytotoxicity of Doxorubicin to
cancer cells when Poly(N-isopropylacrylamide-b-butylmethacrylate)
micelles were used as a drug carrier.30 Summary
Stimuli-responsive polymers, like PNIPAM, play an important role in
PNIPAM Drug Conjugates designing advanced active drug delivery devices. Responsive polymers
enable targeted and controlled drug delivery, resulting in superior
Stimuli-responsive polymers have also been covalently pharmacokinetics. With advances in polymerization techniques, an
linked to biologics for controlled and targeted drug delivery. abundance of PNIPAM copolymer variants can be made to produce
Polymer bioconjugates can improve aqueous stability, reduce materials with a wide range of physical and chemical properties and
immunogenenicity, minimize toxicity, and increase in vivo circulation stimuli-responsive sensitivities. To date, numerous publications illustrate
times of biological drugs.31–33 Site-specific delivery may be obtained the potential of stimuli-responsive nanocarriers in the future arsenal of
by tailoring the conjugates as an inactive prodrug and designing pharmaceutical formulations.
polymer drug linkages susceptible to cleavage by specific enzymes or
pH.34,35 Bioconjugation is generally accomplished using polymers with
chemically reactive end-functional groups.
Polymeric Micelle-drug Complex Drug Release and Cellular Uptake
Below LCST Above LCST
Figure 3. Schematic showing PNIPAM-based micelles. The drug is released at temperatures above the LCST.

aldrich.com/matsci
Methods: PNIPAM Drug Delivery Systems Preparation of PNIPAM-copolymer

Coated Liposomes
PNIPAM Micelles Liposomes can be coated with a thermo- and pH-sensitive PNIPAM
Temperature-responsive drug-loaded micelles can be formed from polymer to create stimuli-responsive drug delivery carriers.59 For
amphiphilic polymers containing PNIPAM blocks.46 The easiest method, example, Poly(N-isopropylacrylamide-co-methacrylic acid-co-
dialysis, can be accomplished by first dissolving the copolymer at a octadecyl acrylate) (Aldrich Prod. No. 724475), shown in Figure 5,
dilute concentration in organic solvent (for example: 30 mg of PNIPAM contains hydrophobic octadecyl side chains randomly distributed
copolymer in 5 mL of DMF or THF, where the organic solvent is selected along the polymer chain; the acid group imparts pH sensitivity to the
based on the copolymer composition and solubility). polymer and octadecyl chain and helps anchor it to the lipid bilayer of
the liposome.
For drug encapsulation, the drug is added to the organic solvent prior
to dialysis. The solution is dialyzed with a molecular weight cut-off
membrane of 3,500 Da against ddH2O for up to 5 days, depending on
the frequency of water change. x y z
PNIPAM copolymer micelles can be formed by additional techniques O NH O OH O OCH2(CH2)16CH3

(highlighted by Du and Stenzel within this guide).
PNIPAM Protein Conjugation

As an example, amine-terminated PNIPAM (Aldrich Prod. No. 724823) Figure 5. Structure of Poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecyl acrylate).
can be used for protein conjugation or further modified to have
a maleimide functional group for thiol conjugation.52 For thiol As a general method, liposomes are first prepared by lipid hydration
conjugation, PNIPAM-amine is converted to PNIPAM-Mal, followed followed by extrusion, and the PNIPAM copolymer is added during the
by conjugation to the protein therapeutic (Figure 4). rehydration step. Liposome preparation involves three steps: vesicle
formation, vesicle size reduction, and purification. The most common
1. Synthesis of PNIPAM-Mal method for liposome preparation is lipid hydration and organic solvent
The amino end group is reacted with succinimidyl4-(N-maleimido exchange by reverse-phase evaporation or organic-solvent injection.
methyl)cyclohexane-1-carboxylate (Sigma-Aldrich Prod.
No. M5525) in dichloromethane at a 1:1.5 ratio, resulting in PNIPAM-
Mal. After the insolubles are filtered, the product can be isolated
and purified by precipitation into 15× excess of diethyl ether.
2. Protein Conjugation
a) Prepare protein solution: Dissolve the protein in a suitable
aqueous buffer (for example, 1 mM protein in 50 mM phosphate,
1 mM EDTA buffer, pH 8.0), and reduce the thiol by using 1 mM
dithiothreitol (DTT, Sigma-Aldrich Prod. No. D9779) for 10 min
at 4 °C. Remove DTT by spin filtration or by passing the solution
over a Sephadex® G-25 gel filtration column equilibrated with the
starting buffer.
b) For protein conjugation: mix protein solution from Step 2a
with purified PNIPAM-Mal from Step 1 using a molar excess
of PNIPAM-Mal (10× is recommended). Allow the reaction to
proceed at room temperature for 4 h with gentle shaking.
The resulting protein-PNIPAM conjugate can be purified by
precipitation with 10% (v/v) saturated (NH4)2SO4 and heating
the solution to 37 °C. The precipitate is then separated by
centrifugation at 10,000 g.
Note: purification can also be completed by additional techniques,
such as column chromatography, depending on the nature of
the protein.
O
O O
O O O O S
S N S n N N
NH2 O N N Protein SH H S Protein
n N n
O
O H O
O O NH
O NH O NH
Figure 4. Scheme for PNIPAM maleimide conjugation to a protein-free thiol.

Preparation of Liposome References

(1) Zhang, Y.; Chan, H. F.; Kam W.; Leong, K. W. Adv Drug Deliv Rev. 2013, 65, 104.
1. Dissolve 20 mg the lipid mixture of 1-Palmitoyl-2-oleoyl-sn-glycero- (2) Rokstad, A. M. A.; Lacík, I.; de Vos, P.; Strand, B. L. Adv Drug Deliv Rev. 2014, 67–68, 111.
3-phosphocholine (POPC) (Sigma-Aldrich Prod. No. 42773) and (3) Reddy, L. H.; Bazile, D. Adv Drug Deliv Rev. 2014, 71, 34.
cholesterol (3:2 mol/mol) (Sigma-Aldrich Prod. No. C8667) in 10 mL (4) Fleige, E.; Quadir, M. A.; Haag, R. Adv Drug Deliv Rev. 2012, 64, 866.
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remove chloroform at reduced pressure at 35 °C using rotary (7) Ganta, S.; Devalapally,H.; Shahiwala, A.; Amiji, M. J. Controlled Release 2008, 126, 187.
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(27) Hang, C, Y.; You, Y. Z.; Pan, C. Y. J. Polym. Sci., Part A: Polym. Chem. 2004, 42, 4873.
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aqueous solution (e.g., 150 mM NaCl, 0.02% w/w NaN3 or (29) Rijcken, C. J. F.; Soga, O.; Hennink, W. E.; Nostrum, C. F. V. J. Controlled Release 2007, 120, 131.
(30) Chung, J. E.; Yokoyama, M.; Yamato, M.; Aoyagi, T.; Sakurai, Y.; Okano, T. J. Controlled Release
another suitable buffer) using a 5 mL syringe. 1999, 62, 115.
vi. Seal the flask and sonicate for 3 min to form a lipid–drug (31) Duncan, R. www.nature.com/reviews/cancer; 2006, 6, 688.
(32) Vicent, M. J.; Dieudonné, L.; Carbajo, R. J.; Pineda-Lucena, A. Expert Opin. Drug Deliv. 2008,
emulsion. 5, 593.
vii. Remove the organic solvents by rotary evaporation under (33) Meyer, O.; Papahadjopoulos, D.; Leroux, J. C. FEBS Lett. 1998, 421, 61.
(34) Hoffman, A. S.; Stayton, P. S. Prog. Polym. Sci. 2007, 32, 922.
vacuum at 37 °C until a viscous gel layer is formed.
(35) Lutz, J. F.; Börner, H. G. Prog. Polym. Sci 2008, 33, 1.
viii. Agitate the gel vigorously using a vortex mixer to convert (36) Meyer, D. E.; Shin, B. C.; Kong, G. A.; Dewhirst, M. W.; Chilkoti, A. J. Controlled Release 2001,
into a suspension of large unilamellar vesicles (LUVs). 74, 213.
(37) Wei, H.; Zhang, X. Z.; Cheng, H.; Chen, W. Q.; Cheng, S. X.; Zhuo, R. X. J. Controlled Release
3. Size reduction: liposome size can be decreased by using an 2006, 116, 266.
extrusion system (LiposoFast) with a 0.2 µm pore size polycarbonate (38) Postma, A.; Davis, T. P.; Li, G.; Moad, G.; O’Shea, M. S. Macromolecules 2006, 39, 5307.
(39) Konak, C.; Reschel, T.; Oupicky, D.; Ulbrich, K. Langmuir 2002, 18, 8217.
membrane. Membranes of different pore sizes may be used to (40) Smithenry, D. W.; Kang, M. S.; Gupta, V. K. Macromolecules 2001, 34, 8503.
obtain liposomes of the desired size range. After extrusion, remove (41) Yasui, M.; Shiroya, T.; Fujimoto, K.; Kawaguchi, H. Colloids and Surfaces B: Biointerfaces 1997,
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G-100 gel column equilibrated with the aqueous buffer used in the (42) Chen, X.; Ding, X.; Zheng, Z.; Peng, Y. New J. Chem. 2006, 30, 577–58.
(43) Yamazaki, A.; Song, J. M.; Winnik, F. M.; Brash, J. L. Macromolecules 1998, 31, 109.
liposome preparation stage above) or by spin-column filtration. (44) Scales, C. W.; Convertine, A. J.; McCormick, C. L. Biomacromolecules 2006, 7, 1389.
4. To create PNIPAM-coated liposomes: (45) Choi, S.; Choi, B. C.; Xue, C.; Leckband, D. Biomacromolecules 2013, 14, 92.
a) Prepare a 6 mg/mL PNIPAM stock solution of Aldrich Prod. (46) Smith, A. E.; Xu, X.; McCormick, C. L. Prog. Polym. Sci. 2010, 35, 45.
(47) Henry, C. M.; Convertine, A. J.; Benoit, D. W.; Hoffman, A. S.; Stayton, P. S. Bioconjugate
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suitable aqueous buffer for liposome rehydration). (48) York, A. W.; Kirkland, S. E.; McCormick, C. L. Adv Drug Deliv Rev. 2008, 60, 1018.
b) Add PNIPAM solution to drug-loaded liposome solution from (49) Zhu, J. L.; Zhang, X. Z.; Cheng, H.; Li, Y. Y.; Cheng. S. X.; Zhuo. R. X. J Polym Sci Polym Chem.
2007, 45, 5354.
Step 3. (50) Yan, J. J.; Ji, W. X.; Chen, E. Q.; Li, Z. C.; Liang, D. H. Macromolecules 2008, 41, 4908.
c) PNIPAM-coated liposomes can be stored in aqueous buffer at (51) You, Y. Z.; Oupicky, D. Biomacromolecules 2007, 8, 98.
4 °C or lyophilized. (52) Chilkoti, A.; Chen, G.; Stayton, P. S.; Hoffman, A. S. Bioconjugate Chem. 1994, 5, 504.
(53) Chen, G.; Hoffman, A. S. Bioconjugate Chem. 1993, 4, 509.
(54) Chen, J-P.; Chu, D-H.; Sun Y-M. J Chem Tech Biotechnol. 1997, 69, 421.
(55) Takei, Y. G.; Aoki, T.; Sanui, K.; Ogata, N.; Okano, T.; Sakurai, Y. Bioconjugate Chem. 1993, 3, 42.
(56) Konoa, K.; Hayashib, H.; Takagishi, T. J. Controlled Release 1994, 30, 69.
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Biomembranes 1993, 1153, 335.
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72, 71.
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Poly(N-isopropylacrylamide) (PNIPAM)
For more information on these products, visit aldrich.com/polynipam.
Poly(N-isopropylacrylamide) H3C CH3 Mn 20,000‑40,000 535311-10G
O NH
End-functionalized PNIPAM
Poly(N-isopropyl acrylamide), amine terminated average Mn 2000 799564-1G
H2N n H
average Mn 5,000 802107-1G
HN O
H3C CH3
Poly(N-isopropylacrylamide), amine terminated H3C CH3 Mn 2000‑3000 724823-1G

724823-5G
O NH
average Mn 5,500 724831-1G
H2N 724831-5G
S
n
Poly(N-isopropylacrylamide), azide terminated H3C CH3 average Mn 15,000 747068-1G

747068-5G
H3C O NH
H3C S
N3 O
S SCH2(CH2)10CH3
n
O
Poly(N-isopropylacrylamide), carboxylic acid H3C CH3 average Mn 2,000 724815-1G
O NH
O average Mn 5,000 724807-1G
724807-5G
S OH
average Mn 7,000 724866-1G
n 724866-5G
average Mn 10,000 724459-5G
Poly(N-isopropylacrylamide), N-hydroxysuccinimide H 3C CH3 average Mn 2,000 725668-1G
(NHS) ester terminated 725668-5G
O NH O
O
N
S O
n
O
Poly(N-isopropylacrylamide), maleimide terminated H3C CH3 average Mn 2,000 731048-1G
731048-5G
O NH
O average Mn 5500 728632-1G
H
N 728632-5G
S N
n
O
O
Poly(N-isopropylacrylamide) triethoxysilane O CH3 average Mn 2,750 760978-1G
Si O CH3
S
n O CH3
O NH
H3C CH3

PolyNIPAM Copolymers
Name Structure Molecular Weight/Viscosity Prod. No.
Poly(N-isopropylacrylamide-co-acrylamide) H3C CH3 average Mn 20,000 738727-5G
O NH O NH2
x y
Poly(N-isopropylacrylamide-co-acrylic acid) H3C CH3 viscosity 7500-12500 5 % in H2O 741930-5G
O NH O OH
x y
Poly(N-isopropylacrylamide-co-butylacrylate) H3C CH3 average Mn 30,000 762857-5G
O NH O O CH3
x y
Poly(N-isopropylacrylamide-co-methacrylic acid) H3C CH3 average Mn 8,000‑10,000 750166-5G

Mn 30,000‑50,000 724467-5G
O NH O OH
CH3
x y
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SHAPE CHANGE POLY(N‑ISOPROPYLACRYLAMIDE)
MICROSTRUCTURES FOR DRUG DELIVERY
Shape Change Polymer Microstructures

Shape memory polymers and hydrogels are perhaps the most
important types of all responsive polymers.4–6 These systems often
are composed of two kinds of molecular moieties, including rigid
and flexible chains or hydrophobic and hydrophilic structural
units. While the use of shape memory polymers in drug delivery is
Tanvi Shroff,1 ChangKyu Yoon,2 David H. Gracias1,2*
reviewed elsewhere,7 we elaborate here on multilayer and patterned
1
Department of Chemical and Biomolecular Engineering microstructures composed of at least one hydrogel component. This
2
Department of Materials Science and Engineering paradigm represents an attractive concept for dynamic DDSs for the
The Johns Hopkins University, Baltimore, MD, USA
*Email: dgracias@jhu.edu
following reasons:
yy Hydrogels have mechanical properties, such as moduli, that are
Introduction well matched with those of human tissue and organs. Many
are biocompatible and capable of swelling by several orders
Therapeutic drugs have evolved from single component formulations of magnitude in volume in response to a range of different
such as powders to multi-component drug delivery systems (DDSs) environmental stimuli.8,9 In aqueous biological systems, swelling or
such as capsules, anisotropic particles, or microfabricated needle collapse can occur due to absorption or expulsion of water.
patches. An effective DDS must be both smart and multifunctional;
yy Hydrogel swelling can be achieved in response to various stimuli
it must release a drug at a specified anatomical location within the
like pH,10 temperature,11 electric field,12 or biomolecules13–17 and can
therapeutic range for a specified period of time and with minimal
be programmed to be responsive to more than one stimulus at a
side effects. DDSs can target cell receptors using antibodies, ligands,
time.18–20 In addition, gelation can also be triggered in vivo.21
or aptamers for more effective cancer therapeutics1 or dissolve and
release their cargo in specific anatomical areas such as the stomach, yy Due to large changes in volume, swelling and de-swelling cause
which is highly acidic.2 Thus far, while polymeric DDSs can bind to large mechanical deformations that can be used to enable
specific cells or be broken apart by biochemical reactions, they are actuation without the need for any other external sources of energy
inherently static and do not reconfigure or change shape dynamically. such as batteries or wires.
Shape change is an emerging concept in DDSs which is inspired by yy Multilayers22 using combinations of more and less swelling
robotics and the tunable shapes of cells or pathogens. Shape change hydrogels or gradient crosslinked hydrogels can be designed so
offers the possibility for autonomous, environmentally responsive multi- that differential volume change during swelling can be converted to
state functionality. There are a number of dynamic polymeric DDSs spontaneous curving and folding to form a wide range of three-
that range in size from nanometer-sized biomolecular constructs to dimensional (3D) shapes and structures.23–25
centimeter-sized implants. These new materials are in different stages
of development, ranging from laboratory curiosity to clinical trial. yy With advances in bio-MEMS, a number of techniques have
Nanometer-sized dynamic shape change structures for use in DDSs are been developed to pattern and structure hydrogels in a wide
composed of smart biomolecules, such as DNA, and can be assembled range of shapes and sizes using mass-producible methods
into shapes such as cubical containers with controllable lids.3 In this such as photolithography, printing, molding, or layer by layer
article, we restrict our discussion to larger, micro or mesoscale systems assembly methods.
and focus on the use of shape change polymer microstructures to
create dynamic DDSs based on reversible swelling.

Shape Change Poly(N‑isopropylacrylamide) Microstructures for Drug Delivery
An Example: Theragrippers A) B)
Self-folding DDSs, referred to as theragrippers for chemomechanical

controlled drug release,26 are hand-shaped DDSs that contain multiple
digits that can open and close in response to a change of temperature
or pH. The shape change is actuated by Poly(N-isopropylacrylamide)
(pNIPAM), a thermoresponsive hydrogel27 that has been studied
extensively for use in drug delivery and for the creation of stiffer
scaffold materials for surgical applications. The swelling of pNIPAM is
driven by the transition from a hydrophilic water-absorbing state to a
hydrophobic water-expelling state as the temperature increases above
physiological temperature. pNIPAM is traditionally formulated using
photolithography, micromolding, emulsion polymerization or radical
polymerization techniques.28 Shah et al. has reviewed methods to
create monodispersed pNIPAM microgels using microfluidic devices as Figure 2. Loading and delivery of drugs using theragrippers. A) Absorption of drugs on porous
hydrogel. B) Free-standing soft grippers for drug delivery.
well as manipulation of the conditions required to induce gelation.29
Crosslinked pNIPAM is typically a relatively soft material with a modulus
of approximately 150 KPa. To increase rigidity of the theragrippers
for gripping applications, the highly swelling gel is paired with a
Outlook and Challenges
stiff non-swelling polypropylene fumarate (PPF) with a significantly Shape change self-folding hydrogel microstructures offer many
higher modulus of 16 MPa to form a bilayer. In addition, NIPAM was advantages as DDSs. In the future, we envision applications such as
co-polymerized with acrylic acid (AAc) to endow pH sensitivity. This self-attaching devices for sustained drug release, self-coiling devices
bilayer was patterned using photolithography in the shape of hands in for blocking aneurisms, self-expanding drug eluting stents, and self-
a mass-producible manner (Figure 1), on a sacrificial layer of polyvinyl propelled and miniaturized locomotive devices.
alcohol using a protocol discussed below. Several schemes were Since DDSs are envisioned for in vivo applications, stringent
used to load drugs such as mesalamine and doxorubicin into the requirements must be met to ensure safety and minimize toxicity,
theragrippers. These included: (a) soaking the fabricated theragrippers which vary depending on the anatomical position and duration of
overnight in the drug solution when the drug soaked primarily into the drug release. In the previous theragrippers example, there is the
porous pNIPAM hydrogel layer; (b) adding the drug to a microporous somewhat questionable biosafety of pNIPAM in the body. On the
PPF layer which was created by leaching salt that was earlier mixed one hand, pNIPAM is widely used in cell culture dishes where no
into the PPF layer; and (c) incorporating the dry drug powder into the significant cellular toxicity has been observed; also, it can be created
PPF mixture before photocrosslinking, which allowed a more uniform with biodegradable crosslinkers offering the possibility for clearance
distribution. Gripping of cells was verified in vitro, and drug release from the body.31–33 Additionally, ophthalmic formulations of pNIPAM
was verified in vivo in the stomach of a live pig (Figure 2).26 In addition, have been reported with no in vitro cytotoxicity.34 On the other hand,
such responsive polymer microstructures can be loaded with magnetic the NIPAM monomers are potentially toxic in vivo.35 Consequently,
nanoparticles for manipulation from afar using magnetic fields, we emphasize that this example of a pNIPAM theragripper is being
allowing for the possibility for remote guidance.30 used for illustrative purposes and more research needs to be done to
evaluate its safety. Additional research is needed, as well, on developing
A) B) C)
such shape change microstructures using alternate bio-friendly soft-
material compositions such as chitosan,36 gelatin,37 cellulose,38 alginate
and hyaluronic acid,39 PLGA,40 PVA,41 PEO, and PEO-PPO-PEO block
copolymers like Pluronic® block copolymers or poloxamers.42,43
Due to significant advances in nanotechnology and biofabrication,
future DDSs are expected to embody more characteristics of living
systems such as self-assembly and disassembly, energy dissipation and
Figure 1. Microfabricated theragrippers. Mass production of hand-shaped bilayer adaptation. They will be dynamic and exquisitely structured at a variety
microstructures fabricated by photopatterning, A) a hand-shaped stimuli-responsive pNIPAM of length scales. Much like a complex macroengineered robotic device
hydrogel, and B) a segmented layer of a stiff polymer PPF on a substrate followed by C) release
from the substrate.
they would likely incorporate feedback, control, logic, and possibly
memory that is manifested through a combination of molecular
binding, chemical reaction and electronic circuits. As highlighted here,
the reversible swelling of hydrogels and their engineering into curved
and folded 3D microstructures, in the absence of any external power
sources, could be used to enable a variety of autonomous shape
changes and, consequently, smart behaviors that are a small step to
enabling the grand vision of the ideal therapeutic.

aldrich.com/matsci
Method: Fabrication of Theragrippers 4. Loading drugs onto the theragrippers26

a) Method 1: Upon release, soak the theragrippers overnight in a
This is the protocol that is used for fabricating PPF/pNIPAM grippers in chemical solution. The hydrophilicity and swelling capacity of
the Gracias Laboratory.26,44,45 the pNIPAM hydrogel layer will cause it to soak up more of the
chemical solution than the PPF layer.
1. Preparation of the stock solutions b) Method 2: Add 5% NaCl solution to PPF, and spin-coated as in
a) PVA stock solution: prepare a 30% PVA (9,000 Da Mw, 80% Step 3. Upon releasing the grippers using DI water, the water
hydrolyzed, Aldrich Prod. No. 360627) solution by weight in will also leach the salt out of the PPF layer to create porosity
deionized water. within the PPF layer. Soak in chemical solution to allow the drug
b) pNIPAM-AAc stock solution44: NIPAM monomer 3 g, (Aldrich to enter both layers of the theragrippers.
Prod. No. 415324) is mixed with pNIPAM 0.4 g, (Aldrich Prod. c) Method 3: Dry load drug by mixing the dry drug powder into
No. 535311) and Bis-acrylamide (0.18 g, Sigma-Aldrich Prod. the PPF layer, and then crosslinking the polymer, to incorporate
No. 146072) in 7.5 mL of n-butanol (Sigma-Aldrich Prod. the drug into the polymer mesh of the PPF layer. Drug release is
No. 34867) as a solvent. This solution is stirred overnight and the controlled both in vitro and in vivo.
undissolved crystals are separated prior to use by decantation.
Before decanting, mix 0.31 mL of acrylic acid (Aldrich Prod. References
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(44) Bassik, N.; Abebe, B. T.; Laflin, K. E.; Gracias, D. H. Polymer 2010, 51, 6093–6098.
(45) Kasper, F. K.; Tanahashi, K.; Fisher, J. P.; Mikos, A. G. Nat. Protoc. 2009, 4, 518–525.

Formulation of Poly(ethylene glycol) Hydrogels for Drug Delivery
FORMULATION OF POLY(ETHYLENE GLYCOL)

HYDROGELS FOR DRUG DELIVERY
PEG-hydrogel Drug Delivery Applications
Drug delivery from a hydrogel involves the diffusion of an encapsulated
pharmaceutical agent through the bulk of the gel into the immediate
microenvironment surrounding the delivery site. Depending on the
porosity, hydrophilicity, and other physicochemical properties of the
hydrogel and the drug, the loaded drug can elute slowly over time
Tyler Lieberthal, W. John Kao* in a pharmacokinetically controlled manner that prolongs circulation
Biomedical Engineering, Surgery, and Pharmaceutical Sciences time.5 Many different agents have been used in PEG-containing
University of Wisconsin-Madison hydrogel drug delivery applications, including small molecules,6
Madison, WI 53705 USA
*Email: wjkao@wisc.edu macromolecules,7 and nano/microparticles.8–9 Such formulations have
been applied to cutaneous, ocular, and cardiac tissue, among others.
Introduction Although described as a “bioinert” drug delivery platform, PEG

hydrogels can also incorporate bioactive materials, such as extracellular
Hydrogels are an attractive vehicle for localized administration of matrix amino acid motifs or macromolecules such as collagen.10
pharmaceutical agents such as proteins and small molecules that Incorporation of biological macromolecules facilitates integration
can be released in a temporally and spatially controlled manner. By into the native tissue environment due to cell-integrin recognition
maintaining a high local concentration of a drug, hydrogels bypass of extracellular matrix components such as laminin, fibronectin,
the need for systemic drug administration and provide a reservoir of collagen, and hyaluronic acid. This may be important for drug delivery
the drug to be released slowly over time. Poly(ethylene glycol) (PEG) is applications where degradation of the hydrogel, and therefore drug
a hydrophilic polymer that, when crosslinked and swollen with water, release, should be mediated by the host microenvironment. Control of
produces a three-dimensional (3D) hydrogel capable of encapsulating degradation is, therefore, critically dependent on the composition of
drugs and cells. Therefore, PEG hydrogels have been studied extensively the hydrogel and the sensitivity of the degradable sequences.
as a platform for drug delivery and tissue engineering. PEG hydrogels
may be an ideal drug delivery vehicle due to favorable host-material
interactions: they are non-toxic, biocompatible, resist biofouling, and
are non-biodegradable.1 The mechanical properties of PEG hydrogels
Interpenetrating Networks of PEG and
are also well-defined and can be tuned for application, including
the drug delivery site and drug diffusion kinetics, by modifying PEG
Biological Materials
molecular weight and crosslink density.2 Functionalization of PEG Due to the poor mechanical stability of biologically derived hydrogels
allows for further robust modification of the chemical and mechanical such as collagen, many have incorporated PEG into the polymer
properties of PEG hydrogels. But perhaps the simplest synthetic matrix to enhance the mechanical strength and longevity in vivo. In
addition is acrylation, which allows crosslinking via free radical general, interpenetrating networks (IPNs) consist of a binary system
additions (Figure 1). of two polymers that are chosen to better control the physical and
biological properties of the hydrogel. Fusion of synthetic and biological
PEG can be end-functionalized with a variety of other groups, including materials can be via physical entanglement or covalent crosslinking.
methoxy, hydroxy, maleimide, thiol, and azide moieties, all of which are Several groups have incorporated hyaluronic acid into degradable
commercially available in homobifunctional and heterobifunctional PEG hydrogels in an IPN to increase cell proliferation and activity for
forms. The diversity of functional groups allows unique crosslinkers with cartilage, cutaneous, vocal folds and other soft tissue.11–12 The PEG in
different crosslinking chemistry, such as Michael-type additions, thiol- these formulations is typically modified with poly(lactic acid) or similar
ene and Diels-Alder click reactions, enzymatic reactions, and carbonyl copolymer to facilitate degradation. Another formulation uses chitosan,
additions.3 These reactions can be triggered with ultraviolet light, a natural polysaccharide derived from crustacean shells, often used in
changes in pH, temperature, electromagnetic fields, or the addition of wound healing applications.13
other chemical compounds.4
A robust and versatile IPN platform that consists of cysteine-conjugated
gelatin processed from collagen (Gel-PEG-Cys), and PEG-diacrylates
1) DCM, TEA O O
H OH 2) Acryloyl chloride O Photoinitiator O

O n O n UV O n
O O
Figure 1. Synthesis of a PEG-diacrylate hydrogel.

aldrich.com/matsci
(PEGdA) has been developed (Figure 2).14–15 Both components of

the IPN, gelatin and PEGdA, are crosslinked together via a thiol-ene Method: Formulation of PEG-gelatin IPN
reaction with or without UV light in situ. In comparison to a physically
entangled network of gelatin and crosslinked PEGdA, the IPN shows Hydrogel Without UV Polymerization*
improved mechanical properties: depending on the ratio of gelatin
This formulation does not require a photoinitiator or UV exposure to
to PEG, the elastic modulus can range from 1 to 360 MPa, whereas
crosslink the network and is ideal for loading bioactives that are known
fixed gelatin alone is ~0.1 MPa.16 In vivo degradation can be further
to be UV-sensitive. Crosslinking proceeds via a Michael-type addition
modified by adjusting a number of parameters, including the gelatin:
between the thiol and acrylate moieties upon a shift to a basic pH.22
PEGdA ratio, gelatin strength bloom, concentration and percent thiol
modification, PEGdA concentration and molecular weight, and initiator 1. Add optional soluble factors (growth factors, small molecules, other
weight percent.17 This IPN biomatrix has been used to encapsulate macromolecules) at desired concentration to sterile water.
various cell types such as fibroblasts, keratinocytes, and mesenchymal 2. Warm the water solution to 37 °C.
stromal/stem cells as well as small molecular weight drugs such as 3. Allow the PEGdA (Aldrich Prod. No. 806757*) and Gel-PEG-Thiol
minocycline, silver sulfadiazine, bupivicaine, and biologics such as (Aldrich Prod. No. 806749*) vials to reach room temperature.
peptides and growth factors.14,18–21 Further, this IPN can be used as a 4. Pipette 2.5 mL of the water solution into the Gel-PEG-Thiol vial and
platform that can be paired with other drugs or drug combinations for 2.5 mL of the water solution into the PEGdA vial. Warm solutions to
site-specific delivery. 37 °C. Mix gently by pipetting up and down.
Cysteine
5. Add the PEGdA solution to the Gel-PEG-Thiol solution and mix gently
PEG Gelatin by pipetting up and down.
Photoinitiator
UV 6. Titrate to pH 8.5 with 1 N NaOH.
+ 7. Quickly pipette the hydrogel solution into desired mold or location.
Gel-PEG-Cys PEGdA 8. The PEGdA/Gel-PEG-Thiol gelatin hydrogel will polymerize within 3 min.
*Note: PEGdA and Gel-PEG-Thiol are available as kit components of
Acrylate-acrylate Aldrich Prod. No. 799629.
Acrylate-thiol
Table 1. Materials
Figure 2. Interpenetrating network composed of cysteine-conjugated gelatin and
PEG-diacrylate. Item Prod. No.
PHSRN (fibronectin domain) 161044K
Optional Bioactives
Method: Formulation of PEG-gelatin IPN Dexamethasone sodium phosphate

Chlorhexidine
D0720000
282227
Hydrogel Using UV Polymerization* Parvalbumin

Basic human fibroblast growth factor
P6393
F0291
The following procedure creates 5 mL of IPN hydrogel containing Methylprednisolone acetate M1755000
poly(ethylene glycol) diacrylate and cysteine-conjugated gelatin. If Keratinocyte growth factor K1757
additional components are added to the IPN, the solubility of those Silver sulfadiazine 481181
components should be determined at the appropriate concentration Bupivacaine hydrochloride 1078507
prior to incorporation into the IPN. A wide range of bioactive Sulfadiazine sodium S6387
compounds was tested and verified using this formulation, as listed in Bovine serum albumin 05470
Table 1. The viability of cells, if incorporated into the hydrogel, should
also be assessed. Hydrogel composition can be varied by mixing References
different volumes of the PEG-diacrylate (PEGdA) and modified gelatin- (1) Hoffman, A. S. Adv Drug Deliv Rev 2002, 54 (1), 3–12.
Cys (Gel-PEG-Cys) solutions (Step 5). (2) Browning, M. B.; Wilems, T.; Hahn, M.; Cosgriff-Hernandez, E. J Biomed Mater Res A 2011, 98
(2), 268–273.
1. Add 5.0 mL H2O to the vial containing 5 mg of photoinitiator (Aldrich (3) Hennink, W. E.; van Nostrum, C. F. Adv Drug Deliv Rev 2002, 54 (1), 13–36.
Prod. No. 410896) to create a 0.1% photoinitiator solution. (4) Peppas, N. A.; Bures, P.; Leobandung, W.; Ichikawa, H. Eur J Pharm Biopharm 2000, 50 (1),
27–46.
2. Warm the photoinitiator solution to 60 °C and vortex occasionally until (5) Hoare, T. R.; Kohane, D. S. Polymer 2008, 49 (8), 1993–2007.
dissolved; then allow the photoinitiator solution to return to 37 °C. (6) Peppas, N. A.; Keys, K. B.; Torres-Lugo, M.; Lowman, A. M. J. Controlled Release 1999, 62 (1-2),
81–87.
3. Add optional soluble factors (growth factors, small molecules, other (7) Saito, N.; Okada, T.; Horiuchi, H.; Murakami, N.; Takahashi, J.; Nawata, M.; Ota, H.; Nozaki, K.;
macromolecules) at desired concentration to photoinitiator solution Takaoka, K. Nature Biotechnology 2001, 19 (4), 332–335.
(see Table 1 for a list of materials). (8) Hamidi, M.; Azadi, A.; Rafiei, P. Adv Drug Deliv Rev 2008, 60 (15), 1638–1649.
(9) Holland, T. A.; Tabata, Y.; Mikos, A. G., J. Controlled Release 2003, 91 (3), 299–313.
4. Allow the PEGdA (Aldrich Prod. No. 806757*) and Gel-PEG-Cys (Aldrich
(10) Zhu, J. M., Biomaterials 2010, 31 (17), 4639–4656.
Prod No. 806730*) vials to reach room temperature. (11) Kutty, J. K.; Cho, E.; Soo Lee, J.; Vyavahare, N. R.; Webb, K. Biomaterials 2007, 28 (33),
5. Pipette 2.5 mL of the photoinitiator solution into the Gel-PEG-Cys vial 4928–4938.
and 2.5 mL of the photoinitiator solution into the PEGdA vial. Warm (12) Skaalure, S. C.; Dimson, S. O.; Pennington, A. M.; Bryant, S. J. Acta Biomater 2014, 10 (8),
3409–3420.
solutions to 37 °C and mix gently by pipetting up and down. (13) Lee, S. J.; Kim, S. S.; Lee, Y. M. Carbohydrate Polymers 2000, 41 (2), 197–205.
6. Add the PEGdA solution to the Gel-PEG-Cys solution and mix gently by (14) Fu, Y.; Xu, K.; Zheng, X.; Giacomin, A. J.; Mix, A. W.; Kao, W. J. Biomaterials 2012, 33 (1), 48–58.
pipetting up and down. (15) Xu, K.; Fu, Y.; Chung, W.; Zheng, X.; Cui, Y.; Hsu, I. C.; Kao, W. J. Acta Biomater 2012, 8 (7),
2504–2516.
7. Pipette the hydrogel solution into desired mold or location. (16) Burmania, J. A.; Martinez-Diaz, G. J.; Kao, W. J. J Biomed Mater Res A 2003, 67 (1), 224–234.
8. Polymerize hydrogel for about 3 minutes under a long-wavelength (17) Martinez-Diaz, G. J.; Nelson, D.; Crone, W. C.; Kao, W. Y. J. Macromolecular Chemistry and
Physics 2003, 204 (15), 1898–1908.
UV lamp (CF1,000 LED, λmax = 365 nm; Clearstone Technologies,
(18) Guerra, A. D.; Cantu, D. A.; Vecchi, J. T.; Rose, W. E.; Hematti, P.; Kao, W. J. AAPS J 2015.
Minneapolis, MN or equivalent). (19) Kleinbeck, K. R.; Bader, R. A.; Kao, W. J. J Burn Care Res 2009, 30 (1), 98–104.
*Note: PEGdA and Gel-PEG-Cys are available as kit components of (20) Cantu, D. A.; Hematti, P.; Kao, W. J. Stem Cells Transl Med 2012, 1 (10), 740–749.
(21) Waldeck, H.; Chung, A. S.; Kao, W. J. J Biomed Mater Res A 2007, 82 (4), 861–871.
Aldrich Prod. No. 799610. (22) Xu, K.; Cantu, D. A.; Fu, Y.; Kim, J.; Zheng, X.; Hematti, P.; Kao, W. J. Acta Biomater 2013, 9 (11),
8802–8814.

Formulation of Poly(ethylene glycol) Hydrogels for Drug Delivery
PRODUCT HIGHLIGHT
Localize Your Drug Delivery

Aldrich® Materials Science offers two new ready-to-use hydrogel kits for the encapsulation and delivery of drugs, biomolecules, and
cells. The hydrogels fuse the durability of synthetic polymers and the biorecognition of a natural protein to create a biocompatible
delivery platform. The biodegradable matrix allows for temporally and spatially controlled delivery.
APPLICATIONS
• Localized delivery of small molecules, proteins, nucleic acids,
and cellular-based therapeutics
• Tissue engineering
• Regenerative medicine
KEY BENEFITS
• Allows tailored delivery profile Description Prod. No.
• Amenable to delivery drugs and cells in a single system Photo-crosslinkable sIPN hydrogel kit 799610-1KT
• Photo or chemical crosslinking Chemically crosslinkable sIPN hydrogel kit 799629-1KT
Learn more about our IPN kits and their applications, at

aldrich.com/ipn
Homobifunctional PEGs
For more information on these products, visit aldrich.com/peg.
α- and ω-ends Structure Molecular Weight Prod. No.
Acrylate O average Mn 2,000 701971-1G
O CH2 average Mn 6,000 701963-1G
H2C O
O n average Mn 10,000 729094-1G
average Mn 20,000 767549-1G
Amine average Mn 2,000 753084-1G
O 753084-5G
H2N NH2
n
average Mn 6,000 752444-1G
752444-5G
average Mn 10,000 752460-1G
752460-5G
Azide O average Mn 10,000 767530-1G
N3 N3
n average Mn 20,000 756601-1G
Methacrylate O CH3 average Mn 6,000 687537-1G
H2C O average Mn 10,000 725684-1G
O CH2
CH3 n O average Mn 20,000 725692-1G
SH average Mn 1,000 717142-1G

HS SH
O average Mn 3,400 704539-1G
n
average Mn 8,000 705004-1G

aldrich.com/matsci
Multi-arm PEGs
Name Molecular Weight Prod. No.
Poly(ethylene oxide), 4-arm, amine terminated average Mn 10,000 565733-250MG
565733-1G
Poly(ethylene oxide), 4-arm, carboxylic acid terminated average Mn 10,000 565717-500MG
565717-1G
Poly(ethylene oxide), 4-arm, hydroxy terminated average Mn 10,000 565709-1G
565709-5G
Poly(ethylene oxide), 4-arm, succinimidyl glutarate terminated average Mn 10,000 565768-250MG
565768-1G
Poly(ethylene oxide), 4-arm, succinimidyl succinate terminated average Mn 10,000 565741-250MG
565741-1G
Poly(ethylene oxide), 4-arm, thiol terminated average Mn 10,000 565725-1G
Poly(ethylene oxide), 6-arm, hydroxy terminated average Mn 17,000 570273-250MG
570273-1G
Multi-arm Block Copolymers

Name Molecular Weight Prod. No.
Poly(ethylene oxide)-block-polylactide, 4-arm poly(ethylene oxide) Mn 2,500 570354-250MG
polylactide average Mn 3,500 570354-1G
Poly(ethylene oxide)-block-polycaprolactone, 4-arm PCL average Mn 2,500 570346-1G
PEG average Mn 2,500

HIGH-PURITY COLLAGEN
Collagen is biocompatible, biodegradable, and contains
biological recognition sequences that promote cell migration,
attachment, and proliferation. These can be exploited for
tissue regeneration and site-specific drug delivery. We offer
a selection of high purity, biocompatible, and biodegradable
collagen to enable biomedical research.
Features Applications
yyType I, Bovine Corium 3D scaffolds
yy
yyPepsin (atelo) and acid Regenerative medicine
yy
(telo) solubilized available Medical devices and
yy
yyHigh purity implants
yyLow endotoxin Drug delivery
yy
In vitro diagnostics
yy
High-purity Collagen Products*

Name Prod. No.
Type I Bovine Collagen Solution, Pepsin soluble 804592
3 mg/mL, ≥95% purity
Type I Bovine Collagen Solution, Pepsin soluble 804622
Type I Bovine Collagen Solution, Acid Soluble telocollagen 804614
Products of Collagen Solutions Plc

*
Learn more about natural polymers and applications,

including our complete product offering, at
aldrich.com/natural
aldrich.com/matsci

PROTEIN PEGYLATION
Many strategies2 have been developed and studied to optimize the
pharmacokinetic properties of proteins, including hyperglycosylation,3
fusion to an antibody Fc or to albumin, non-covalent association
to albumin, and encapsulation or association with particulates.1a,1b,4
PEGylation has been the most clinically successful strategy and
involves the covalent conjugation of poly(ethylene glycol) (PEG) to
the biopharmaceutical of interest (Figure 1). Many different proteins,
peptides, and oligonucleotides have been PEGylated for use in a wide
Steve Brocchini range of medical indications.5
School of Pharmacy
University College London, UK Protein PEGylation was first described by Frank Davis et al. in 1977.6 The
Email: steve.brocchini@ucl.ac.uk
first therapeutic PEGylated products (PEG-enzymes) appeared in the
early 1990s and PEGylated cytokines were registered for clinical use by
Introduction the early 2000s. The field developed rapidly thereafter. Currently, there
are at least 12 innovative PEGylated biopharmaceuticals registered
Therapeutic proteins and other biopharmaceuticals, such as peptides for clinical use, with many more in development. The most recent
and oligonucleotides, are often potent drugs that comprise an of these is Plegridy® (2014).7 A number of PEGylated products have
established and fast growing segment of the pharmaceuticals market. been developed from essentially the same parent protein, and several
Unfortunately, proteins are prone to aggregation and misfolding, PEGylated proteins including PEGylated interferon-a2 (Pegasys® and
making them difficult to formulate and use. In addition, most proteins PegIntron®) and PEGylated GCSF (Neulasta® and Lonquex™) have been
are cleared rapidly from the bloodstream upon administration. developed as first-line treatments. PEGylation is also now a viable
Although therapeutic protein metabolism and pharmacokinetics (PK) strategy for the life-cycle management of unmodified proteins in order
are complex,1 rapid clearance can result in dose dumping, the lack of a to develop improved or biobetter versions of existing therapeutics.
therapeutic dose between each administration and the need for higher With the expiration of patents on the first PEGylated products about to
cumulative and more frequent dosing. Frequent dosing can result in occur, biosimilar versions are rapidly being developed, along with the
increased occurrence of side-effects, increased risk for immunogenicity, required development of international standards8 to govern this new
and suboptimal efficacy. Often there is the need to extend the half- type of generic therapeutic. The PEGylation of many other molecules
life of biopharmaceuticals in the blood to maximize clinical safety (peptides, oligonucleotides, and low molecular-weight chemical
and efficacy. entities) is also an active area of research.5b,9
Protein Reactive
Group
• NH2
• NH
• OH
• SH
• COOH
PEG
• Linear
Biomolecule • Branched
• Protein, peptide • Variable size
• Oligonucleotide • Symmetric
• Small molecule • Bifunctional
Linker
• Permanent
• Releasable
• Site specific
Figure 1. Schematic of a PEGylated biopharmaceutical. (Image courtesy of Dr. Karolina Peciak.)

Protein PEGylation
Protein Modification regarding the impact of PEGylation on clinical efficacy and potential
immune response. For this reason, it has become increasingly
Comprehensive reviews have been published describing the broad important to develop standardized assays for anti-PEG antibodies to
spectrum of conjugation reactions used for protein PEGylation5b,10 better monitor PEG-specific immune responses.13d,18a,22
and conjugation in general. PEGylation is also used for a wide range While PEG remains useful for protein modification, alternative
of other applications such as PEGylating the surface of particulates.11 natural and synthetic polymers23 are also being examined (e.g.,
Recent advances in PEGylation conjugation also are being adapted to polyoxazolines,24 phosphorylcholine methacrylates,25 heparosan
make complex, multi-functional therapeutic proteins like antibody drug polymer,26 hydroxyethyl starch27). Like PEG, these polymers will need
conjugates (ADCs).12 to be thoroughly evaluated from a regulatory perspective, and the
The PEGylation of a protein extends its retention in the body because polymeric conjugation reagents need to be made reproducibly at scale
the hydrophilic PEG polymer increases the hydrodynamic size of the with narrow polydispersity and defined chemical functionality.
protein in solution, slowing renal clearance of the conjugate. Moreover,
PEG sterically shields the protein to decrease proteolytic degradation,
opsonization, and uptake by the mononuclear phagocyte system. PEG PEGylation Reagents
steric shielding can also help reduce immunogenicity by shielding Figure 2 shows various linear PEGylation reagents that are typically
potential antigenic sites on the protein5a,13 and minimize the propensity functionalized at one terminus (monofunctional) for conjugation to a
for aggregation.14 Depending on the site of conjugation, PEG steric protein (Figure 2-1). PEGylation reagents have been described using
shielding can slow the rate of association of the modified protein with both established and recently developed conjugation functional
its target,15 thereby decreasing potency. Disassociation rates for the groups10c to undergo reaction with many different amino acid
PEGylated protein are thought to remain the same as the unmodified residues on proteins,5b,10a,10b including non-native amino acid side-
protein.15 Hence, once a PEGylated protein is bound to its target, chains containing aldehyde and alkyne groups.10d–f Of the many
its properties are essentially the same as the unmodified protein.16 PEGylation reagents described, those designed to undergo reductive
Reduced association due to PEG shielding means in vitro evaluation amination with the amine of the N-terminal amino acid (PEG-
of PEGylated proteins and macromolecular drugs generally do not aldehyde Figure 2-2), acylation of the lysine side-chain amine (PEG-
correlate with in vivo efficacy.17 While a log reduction in protein in vitro N-hydroxysuccinimide (PEG-NHS) Figure 2-3), and Michael addition
activity after PEGylation can be observed, most therapeutic proteins of the cysteine thiol (PEG-maleimide Figure 2-4; PEG-bis-sulfone
are very potent and remain so after PEGylation, thus achieving clinical Figure 2-5) are most common. Linear PEGylation reagents are generally
benefit which is better than the unmodified protein. in the range of 20–30 kDa because the synthesis of narrow molecular
In spite of the widespread clinical use of PEGylated proteins, there weight distribution methoxy-PEGs is more feasible than at higher
is still debate about the clinical effects of PEG accumulation and molecular weights. This has led to the development of “branched”
immunotoxicity.18 In many cases, this can be attributed to other factors. PEGylation reagents (Figure 2-6 for example)5b,10a,10b and other reagents
For example, animal-based accumulation studies often use much with a wide spectrum of PEG morphologies and configurations
higher bolus and cumulative doses than that given to humans.19 Most (e.g., polyPEG).28
therapeutic proteins20 display immunogenic effects in some proportion Narrowly defined reaction conditions with different variants of PEG-
of patients in the clinic, so the metabolism, toxicity, and clearance aldehyde (Figure 3-2) used in stoichiometric excess sometimes can be
of PEGylated products can be dependent on the protein.13b,13c If the found to undergo reaction predominantly with the N-terminal amine
unmodified protein is safe, then the PEGylated variant is expected to on a protein to give an intermediate imine (Figure 3-7). A borohydride
be safe.21 Products conjugated with many PEG molecules (liposomes, reagent (e.g., NaBH4, NaCNBH3, or Na(AcO)3BH) then needs be used to
biomaterials, non-human enzymes) may raise additional questions reduce the imine to give the PEGylated protein (Figure 3-8). Known
O
Conjugation H O O
H 3C
O
O
Moiety O
O
N
H H 3C
O
O O
N
H 3C
n n O n O
1 2 3
O H
O H 3C O N O
O NH O O
O N H 3C O SO 2
H 3C O n n O N
O O
n O O
H 3C O NH
SO 2 O
n O
4 5 6
Figure 2. Linear PEGylation reagents 1 general functionalization for protein conjugation, 2 PEG-aldehyde, 3 PEG-N-hydroxysuccinimide, 4 PEG-maleimide, 5 PEG-bis-sulfone, and 6 branched PEG-
N-hydroxysuccinimide PEGylation reagent.
N-terminal amine H H
H2 N N N
H3C O O N H3C O O N
H O n n
O O H
N
H3C O O H
n
O
2 + imine formation 7 Reduction to amine 8
Protein
Figure 3. N-terminal PEGylation by reductive amination with 2 PEG-aldehyde results in an 7 intermediate imine, that can be reduced by borohydride to give the 8 N-terminal PEGylated protein.

aldrich.com/matsci
as reductive amination, this strategy for PEGylation can achieve

varying degrees of site-specificity at the amine of the N-terminal Thiol-selective Reagents
amino acid residue. Although the N-terminal amine can be targeted To address cost and regulatory issues, current research efforts are
by reductive amination using a PEG-aldehyde reagent (Figure 3-2), underway to ensure PEGylation is more robust, efficient, and site-
specific conditions are narrow and conjugation efficiency can be low. specific. Many site-specific approaches have been described,10c,10d,10f but
Other strategies designed to be more efficient and selective for the only thiol alkylation at an α,β-unsaturated carbonyl (Michael addition
N-terminus (or C-terminus) of proteins have been described.29 reaction) will be described here. Alkylation at an α,β-unsaturated
Amine PEGylation with PEG-NHS esters (Figure 2-3) primarily undergo carbonyl can be rapid in mild conditions that are conducive to
non-selective acylation with lysine amines, the most accessible maintaining the tertiary structure of a protein and where protein
nucleophilic groups on the protein. However, additional amines and amine nucleophiles tend to be protonated and not reactive (typically
nucleophilic residues (terminal amine, tyrosine, serine) may also react, at pH 6–7). The free cysteine thiol is rare in a protein and often not
leading to non-specific PEGylation. Reaction of PEG-NHS with the available for conjugation because in the majority of proteins, cysteines
histidine imidazole side-chain results in the formation of a hydrolytically are paired to form disulfides.
labile carbonyl imidazolide. PEG-NHS esters are also susceptible to For thiol conjugation, PEG-maleimide (Figure 4A-4) has been widely
competitive hydrolysis which requires this reagent to be used in used, but maleimide-derived PEG-conjugates (Figure 4A-9) are
considerable excess. susceptible to deconjugation via a retro-Michael reaction and undergo
Amine reactive reagents generally tend to be used in considerable thiol exchange reactions.31 Efforts to stabilize maleimide conjugates
stoichiometric excess to the target protein. Since each protein has have focused on hydrolysis of the maleimide ring after conjugation.32
several available nucleophilic groups, non-selective PEG conjugation Alternative thiol-specific reagents have also been described.33 One
results in PEG-protein positional isomers and multiple PEGylated example is the PEG-mono-sulfone (Figure 4B-10)34 which is equally
products. Achieving both good purification and high yield can be reactive with PEG-maleimide. This reagent (Figure 4B-10) undergoes
challenging when excess PEGylation reagent is used to overcome elimination in situ to give the reactive enone (Figure 4B-11) that
non-selective conjugation.30 The cost of the PEG reagent can exceed undergoes conjugation. The initially formed PEG conjugate
that of the unmodified protein, making conjugation efficiency during (Figure 4B-13) can be readily treated with a borohydride reagent
clinical development important. Conjugation of many PEG molecules, (NaBH4, NaCNBH3, or Na(AcO)3BH) to give a conjugate (Figure 4B-10)
or hyper-PEGylation, has been shown to be useful for therapeutic that is not susceptible to retro-Michael and other exchange reactions.
enzymes of non-human origin that are selective for a low molecular The scarcity of free cysteine thiols in therapeutic proteins has resulted
weight substrate (e.g., Adagen®, Oncaspar®, and Krystexxa®). Hyper- in many attempts to recombinantly add a free cysteine to a protein
PEGylation is readily achieved using amine acylating reagents such as a site for conjugation. This strategy is viable for proteins without
as PEG-NHS esters. Often these PEGylated products are described as disulfides;35 but for proteins with disulfides, the presence of an unpaired
being immunogenic, even though the non-PEGylated version would cysteine often results in disulfide scrambling, protein misfolding, and
have no clinical utility. Non-selective mono-PEGylation with amine aggregation. Since many therapeutic proteins have disulfides, another
reactive reagents (like PEG-aldehyde Figure 2-2 or PEG-NHS Figure 2-3) way to exploit the selectivity and efficiency of thiol conjugation is by
have been used to develop several very successful clinical products bis-alkylation to re-bridge disulfides using reagents such as PEG-bis-
that bind to cell-surface receptors (e.g., Pegasys®, PegIntron®, Micera™, sulfone (Figure 5-5).36 Elimination of the sulfinic acid leaving group
and Neulasta®).
A)
C C O
O CH3
SH O pH~7 S N O
O CH3 n
N O
+ n O
O
4 9
Protein
B)
O O
SO 2 pH~7 SO2H
NH O NH O
O CH 3 O CH 3
O n O n
+
10 11 Protein 12
C OH C O
Hydride
S S
NH O NH O
O CH 3 O CH 3
O n O n
14 13
Figure 4. A4) PEG-maleimide, A9) maleimide-derived PEG-conjugate, B10) PEG-mono-sulfone, B11) elimination in situ to give the reactive enone, B12) sulfinic acid leaving group,
B13) PEG conjugate, B14) stable PEG conjugated, treated with borohydride

Protein PEGylation
(Figure 5-12) to give the PEG-mono-sulfone enone (Figure 5-15)

allows the PEGylation reaction to proceed by what is thought to be Methods: Protein PEGylation
an addition-elimination mechanism. Disulfide bridging PEGylation
can be accomplished during protein refolding or after mild partial PEGylation of a Protein With a Single Cysteine34
reduction of a disulfide in the purified protein. Disulfide reduction can 1. The protein first can be treated with dithiothreitol (DTT) (Sigma
also be readily accomplished in situ37 without the need to remove Prod. No. 43815) to reduce any intermolecular disulfides
the reducing agent prior to PEGylation. Although not necessary, that may be present. Other disulfide exchange reagents (e.g.,
the conjugate (Figure 5-16) then can be reduced without any loss mercaptoethylamine) can be used as well as phosphine derived
of protein activity using a borohydride reagent (NaBH4, NaCNBH3, reagents, e.g., tris(2-carboxyethyl)phosphine) (TCEP) (Sigma
or Na(AcO)3BH). In practice, this is not required as the hydrophobic Prod. No. 68957). A lyophilized protein or antibody must first be
interactions of the protein and the flexibility of the 3-carbon bridge resuspended in a reaction buffer such as 50 mM sodium phosphate,
prevent de-conjugation. Additionally, two thiols can be conjugated pH 7.4, containing 150 mM NaCl and 10 mM EDTA (0.5 mL). It is
using a bis-alkylating maleimide linker,38 which results in a rigid best to use ultrapure water (18.2 MΩcm) to make solutions used for
vinylic 2-carbon bridge between the thiol cysteines. As with other PEGylation. DTT (1.0 mM stock solution) is added to the resulting
maleimides, hydrolysis is required to prevent de-conjugation and solution to give a final DTT concentration of 20 mM. The resulting
exchange reactions.39 solution is mixed gently and then allowed to incubate at room
The bis-sulfone PEGylation reagents (Figure 5-5) are also selective for temperature for 1 h.
the histidine tag (His-Tag) in proteins,29b which can be placed at either 2. The DTT must be removed by buffer exchange prior to the
terminus of the protein. His-Tag PEGylation can be accomplished with PEGylation reaction. The buffer of the reduced protein is exchanged
greater efficiency than PEGylation by amine acylation and reductive with fresh reaction buffer using an Illustra NAP-5 column. The
amination.29b The difference in reactivity of cysteine thiols and histidine reduced protein concentration can be determined by measuring UV
imidazoles for the PEG-bis-sulfone reagents (Figure 5-5) means it is absorbance at 280 nm and the concentration should be adjusted to
possible to conduct a disulfide bridging conjugation in the presence 0.1 mg/mL.
of a His-Tag. Then this can be followed by subsequent site-specific 3. The PEGylation reagent (either PEG-maleimide Figure 4-A4 (Aldrich
modification at the His-Tag using the same conjugation moiety. Prod. No. 712469), or PEG-mono-sulfone Figure 4-B10; 1 equiv.,
1 mg/mL) is added to the protein solution. The PEGylation reaction
solutions are mixed gently and incubated at ambient temperature
Summary (18–25 °C) for approximately 4 h. Reaction mixtures can be analyzed
by SDS-PAGE (InstantBlue™ stain), and then the conjugate is purified
Drugs that utilize the properties of macromolecules can provide by ion-exchange chromatography.
significant clinical benefit.40 Protein PEGylation is a clinically proven
approach where first the protein is made recombinantly, then Disulfide Bridging PEGylation15b,37,43
modified by a conjugation reaction using a PEGylation reagent. Protein The procedure to PEGylate the two cysteine thiols43 on a protein with
engineering and PEGylation are being utilized to modify10d-f,29b existing PEG-bis-sulfone Figure 5-5 is essentially the same as the PEGylation of
proteins and design non-endogeneous proteins like scaffolds35,41 to a protein that has a single cysteine thiol available for conjugation. The
achieve more efficient PEGylation site-selectivity. These efforts are key difference is the disulfide in the protein to be conjugated must
focused to ensure there will be greater structural homogeneity, activity, first be reduced, typically using DTT or TCEP. It is often possible to use
and stability42 in the final PEGylated protein product. The combination TCEP in stoichiometric equivalence to the protein to avoid the need for
of protein engineering and chemical synthesis strategies provides a buffer exchange to remove the reducing agent prior to the addition
huge range of opportunities for the continued modification of proteins of the PEGylation reagent.37 It is also possible to use immobilized DTT
with PEG and other molecules. or TCEP.
O O NH O SO2 H
O NH H 3C O SO 2
H 3C O SO2
n
O
n
O +
5 SO2 15 12
Native
SO2 R
SO 2R
disulfide C C O C C C
O- O
S SH SH S- S O
PEG
S S- 15 S PEG S PEG S PEG
C C C C C
Protein 16
Open disulfide Addition Elimination Addition Bridged conjugation
Figure 5. Conjugation by bis-alkylation to re-bridge disulfides using an addition-elimination mechanism. 5 PEG-bis-sulfone reagent, 12 sulfinic acid leaving group, 15 PEG-mono-sulfone enone,
16 final bis-alkylated conjugate

aldrich.com/matsci
PEGylation Using a PEG-aldehyde Reagent (16) Gonnelli, M.; Strambini, G. B. Biochim. Biophys. Acta, Proteins Proteomics 2009, (1794),
569–576.
Reductive amination is considered only to be broadly site-specific (17) Duncan, R.; Gaspar, R. Mol. Pharmaceutics 2011, 8 (6), 2101–2141.
for the N-terminal amino group of a protein. Optimization of the (18) (a) Schellekens, H.; Hennink, W. E.; Brinks, V. Pharm. Res. 2013, 30 (7), 1729–1734; (b) Garay,
R. P.; El-Gewely, R.; Armstrong, J. K.; Garratty, G.; Richette, P. Expert Opin. Drug Delivery 2012,
reaction conditions (e.g., pH, reagent stoichiometry, concentration of 9 (11), 1319–1323.
protein and reagent) is required for each protein in an effort to exploit (19) (a) Rudmann, D. G.; Alston, J. T.; Hanson, J. C.; Heidel, S. Toxicol. Pathol. 2013, 41 (7),
the difference in the pKa of the amine of the N-terminal amino acid 970–983; (b) Bendele, A.; Seely, J.; Richey, C.; Sennello, G.; Shopp, G. Toxicol. Sci. 1998, 42
(2), 152–157.
compared to other amines often present in a protein to achieve high- (20) Schellekens, H. Clin. Thera. 2002, 24 (11), 1720–1740.
yielding and homogeneous imine formation in an aqueous medium. (21) Webster, R.; Didier, E.; Harris, P.; Siegel, N.; Stadler, J.; Tilbury, L.; Smith, D. Drug Metabol.
Disposition 2007, 35 (1), 9–16.
1. A stoichiometric excess 5–10 equivalents of a PEG-aldehyde reagent (22) Gorovits, B.; Wakshull, E.; Pillutla, R.; Xu, Y.; Manning, M. S.; Goyal, J. J. Immunol. Methods
Figure 2-2 is usually used. As an example,28 to N-terminally PEGylate 2014, 408 (C), 1–12.
a protein, dissolve the protein in solution (1.7 mg/mL, 20 mM (23) Pasut, G. Polymers 2014, 6 (1), 160–178.
(24) Luxenhofer, R.; Han, Y.; Schulz, A.; Tong, J.; He, Z.; Kabanov, A. V.; Jordan, R. Macromol. Rapid
sodium acetate, pH 4.5, or a suitable buffer based on stability and Commun. 2012, 33 (19), 1613–1631.
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(5–10 equiv.) and NaCNBH3 (40 mM) (Aldrich Prod. No. 156159). 2144–2155.
Then incubate the mixture for 16–24 h at ambient temperature (26) DeAngelis, P. L. Expert Opin. Drug Delivery 2015, 12 (3), 349-352.
(27) Liebner, R.; Mathaes, R.; Meyer, M.; Hey, T.; Winter, G.; Besheer, A. Eur. J. Pharm. Biopharm.
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(29) (a) Soundrarajan, N.; Sokalingam, S.; Raghunathan, G.; Budisa, N.; Paik, H.-J.; Yoo, T. H.;
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the purest fractions then pooled. Laurine, E.; Tommasi, R.; Singh, R.; Dubey, S.; Peciak, K.; Bird, M.; Sivasankar, A.; Swierkosz, J.;
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2262–2277.

Protein PEGylation
Monofunctional mPEGs
For a complete list of available materials, visit aldrich.com/peg.
ω-end Structure Molecular Weight Prod. No.
Acetylene O average Mn 2,000 699802-500MG
O C CH
H3C O
n
Acrylate O average Mn 2,000 730270-1G

O CH2 average Mn 5,000 730289-1G
H3C O
n
Azide H3C O average Mn 1,000 733407-1G

O N3
n average Mn 2,000 689807-250MG
689807-1G
average Mn 5,000 689475-250MG
689475-1G
average Mn 10,000 726168-250MG
average Mn 20,000 726176-250MG
NHS ester O average Mn 5,000 85973-1G
O O
H3C O N
n O
O
Maleimide O average Mn 750 712558-250MG
average Mn 5,000 63187-1G
O N 63187-5G
H3C O
n O average Mn 10,000 712469-250MG
O average Mn 2,000 731765-1G

O 731765-5G
H3C O N
O O
n
O
SH H3C O average Mn 800 729108-1G
O SH 729108-5G
n
average Mn 2,000 729140-1G
729140-5G
average Mn 6,000 729159-1G
729159-5G

aldrich.com/matsci

POLY(2-OXAZOLINE)S FOR DRUG DELIVERY

polymers of low dispersity (PDI <1.05–1.3), controllable compositions
(random, gradient, and block copolymers) and with end groups that
can be quantitatively defined by the initiation and termination reaction.
Among other factors, the solvent polarity and nature of the counter
ion determine the equilibrium and thus, the “livingness” of the LCROP. A
general reaction scheme is given in Figure 1.
Initiation
Rainer Jordan,1* Robert Luxenhofer,2 Alexander V. Kabanov2
1
Chair of Macromolecular Chemistry, School of Science electrophile
Technische Universität Dresden, Dresden, Germany ionic species covalent species
*Email: Rainer.Jordan@tu-dresden.de (initiator salt)
2
Professur für Polymere Funktionswerkstoffe Propagation
Fakultät für Chemie und Pharmazie
random or gradient copolymers
Universität Würzburg, Würzburg, Germany
3
Center for Nanotechnology in Drug Delivery and Division of Molecular Pharmaceutics
Eshelman School of Pharmacy
University of North Carolina at Chapel Hill, Chapel Hill, NC USA
Introduction 2nd block 3rd block

Poly(2-oxazoline)s (POx) were discovered in 19661,2 but have only
recently gained substantial attention as a biomaterial, especially R3 block copolymers
for the development of drug delivery systems (DDS) and polymer Termination
therapeutics.3 One reason for the late recognition of POx in the
NuH
biomedical field was a lack of commercially available monomers and N
N O
N
Nu chain ends AND every monomer unit
- Nu n can be functionalized
well-defined, functionalized polymers, while other suitable polymer R O
n-1 R
R O
platforms such as poly(ethylene glycol) (PEG) and Pluronic® block

copolymers were readily available from commercial sources. As a Figure 1. General reaction scheme of the living ring-opening polymerization of 2-oxazolines.
consequence, PEG became the most widely used polymer therapeutic
and is classified as “Generally Recognized as Safe” (GRAS) by the FDA.
However, recent reports on an “accelerated blood clearance” (ABC) Biocompatibility of POx
effect of PEGylated therapeutics,4 as well as the rather limited chemical
The current interest in POx as an alternative polymer platform to PEG is
variability of the polymer, has helped make the case for developing
largely based on recent reports on its exceptional behavior in complex
suitable alternative polymer platforms. POx has the potential to
biological environments. Zalipsky et al.6 first discovered the so-called
provide such an alternative in the case of the failure of a PEG-based
“stealth behavior” of hydrophilic POx (poly(2-methyl- and 2-ethyl-2-
system. Moreover, POx has the potential to offer more than merely a
oxazoline) (PMeOx, PEtOx)) that leads to long blood circulation times in
replacement to PEG because it provides a more versatile chemistry
animals. Later, Luxenhofer et al.7 found that upon injection in rodents,
and the potential to tune the “hydrophilic lipophilic balance” (HLB) of
hydrophilic POx disperses rapidly throughout the entire organism
amphiphilic POx copolymers.3,5
(except the brain) and shows very little unspecific organ deposition and
rapid renal excretion. Amphiphilic POx of various compositions display
Chemistry no cytotoxicity, low complement activation in vitro8,9 and animal testing
indicates no adverse properties from neutral hydrophilic or amphiphilic
POx is synthesized by living cationic ring-opening polymerization POx. Moreover, depending on the amphiphilic contrast of POx, the cell-
(LCROP) of 2-oxazolines, typically with alkyl tosylates or triflates as uptake can be surprisingly fast even at low polymer concentrations.8
initiators and various nucleophiles (amines, carboxylates, etc.) as
terminating agents. The LCROP of 2-oxazolines is relatively slow but
proceeds reasonably well at T >40 °C to produce well-defined linear

Poly(2-oxazoline)s for Drug Delivery
POx Conjugates amphiphilic

monomer unit
(polysoap)
One advantage of POx chemistry is that it allows for the definition
of the physical and chemical properties of the final polymer over a
very broad range simply by the variation of the monomer side chain
(2-substitution). This enables the introduction of various chemical
functional groups for later polymer ligation. Among the first functional PMeOx PEtOx PiPOx PnPOx PBuOx PNOx PPhOx
groups introduced directly onto POx was the alkyne moiety for the hydrophilic Tcp≈70 ˚C Tcp≈40 ˚C Tcp≈25 ˚C hydrophobic
subsequent polymer analog CuAAC click chemistry.10–12 Recently, other Figure 2. Series of hydrophilic to hydrophobic poly(2-oxazoline)s from the pure hydrophilic
“click reactions” such as the “thiol-ene click” have been successfully PMeOx to PEtOx (slightly amphiphilic and similar to PEG) over thermo-sensitive POx (having a
added to functional POx;13–16 next-generation biomaterials will critical solution temperature as indicated), to the first water-insoluble PBuOx. Because of the
amide group linking the side to the main chain, even water-insoluble POx are amphiphilic
include POx conjugates that incorporate small molar mass drugs and (non-ionic polysoap).
proteins.17–21 Modification of proteins with amphiphilic POx block
copolymers imparts these proteins not only with stealth properties and Short (<C4) pendant chains result in water-soluble polymers while
long circulation, but also with the ability to cross biological barriers; for longer aliphatic or aromatic side chains result in hydrophobic polymers
example, increase protein uptake in cells or transport to the brain.18 For (Figure 2). Similar to other water-soluble polymers, aqueous POx
a more exhaustive list of reported end- and side-functionalized POx, solutions exhibit reversible phase-transitions (C2 and C3 side chains)
there are several recent reviews.3,22–25 which renders POx as an ideal candidate for the design of “smart
polymer materials.”29–32 POx displays a very narrow phase transition
behavior of only 1–2 K and low hysteresis.
POx Polyplexes In principle, all combinations of hydrophilic and hydrophobic monomer
POx has long been used for the preparation of polyplexes for gene units are possible in random, gradient, and multiblock copolymers. This
delivery because linear poly(ethylene imine) (PEI) is derived from linear results in a series of polymeric amphiphiles similar to Pluronic® block
POx (poly(2-ethyl-2-oxazoline)) by hydrolysis. Recently, cationic POx copolymers. In water the POx amphiphiles assemble into polymeric
prepared from protected amine-functionalized 2-oxazolines26 or via micelles of distinct shape and typically small size, with a very low
thiol-ene click reactions27 was investigated as a nonviral vector for critical micellar concentration (CMC ≈ 10–5 M). Thus, amphiphilic POx
use in gene therapy.27,28 Schubert et al.27 generated a library of various is an ideal system for the solubilization, formulation, and delivery of
cationic POx and identified one variant that had similar transfection strongly hydrophobic drugs.
efficiency to linear poly(ethyleneimine) in HEK cells. Kabanov et al.28 The formulation of paclitaxel (PTX) in a P(MeOx-BuOx-MeOx) triblock
reported transfection of very-hard-to-transfect cells using POx-based copolymer is one outstanding example of amphiphilic POx in micellar
polyplexes in combination with Pluronic® block copolymers as the drug delivery. PTX is one of the most frequently used anticancer drugs
transfection enhancer. Interestingly, in the latter report an intriguing that suppresses cell division by association to microtubules and leads
amount of low plasma protein binding with polyplexes was observed, to apoptosis. Like many other small-molecule drugs, PTX suffers from
which highlights the stealth properties of hydrophilic POx and is of very low water solubility, only 1 µg/mL. In currently used commercial
particular interest for targeted gene delivery. Additionally, various other PTX formulations, the ratio of excipient to PTX is around 100:1 (Taxol®)
polycations were combined with hydrophilic POx to create nonviral or 10:1 (Abraxane®). However, the triblock POx formulation P(MeOx-
vectors, taking advantage of the stealth properties of the hydrophilic BuOx-MeOx) was found to solubilize PTX at a ratio of POx:PTX of 1:1
POx shell around the polyplex core.3 (50 wt% drug loading instead of 1 or 10 wt%, respectively). Moreover,
drug concentrations as high as 50 mg/mL were stable in the injectable
Amphiphilic POx as Micellar DDS triblock formulation, increasing drug solubility by a factor of 50,000.9,33,34
This unparalleled high drug loading capacity initiated a series of
As mentioned previously, the physical properties of POx can be studies to solubilize various other drugs in a variety of amphiphilic
fine-tuned through the 2-substitution of the monomer. This can be POx.33,35–38 PBuOx containing block copolymers were the most effective
used to vary the solubility of POx in water over a wide range and, in for drug solubilization, while the use of a more hydrophobic PNOx
combination with (block) copolymerization, results in highly defined block resulted in acceptable but not extraordinary drug loadings. The
amphiphilic POx. In contrast to Pluronic® block copolymers, the high drug loading of PBuOx copolymers can be associated with the
amphiphilic motif is not only found along the polymer backbone but a hydrophobic and polar interactions between the polymer excipient
second amphiphilic motif is located within each monomer unit. Thus, and the drug. Flexible composition of the polymer carrier and
hydrophobic POx is actually a polysoap with a hydrophobic pendant optimization of its interaction with the drug are both critical to efficient
group linked via a polar, hydrophilic amide group to the polymer drug solubilization and formulation. While maintaining high drug
chain (Figure 2). loading capacity is important, it is also critical to maintain simplicity of
the formulation procedure.

aldrich.com/matsci
Typically, the drug and the polymer are solubilized in a common Preparation of Drug-loaded POx Micelles by
solvent, and the solvent is evaporated until a thin film remains and
then water or buffer is added. For most drug-POx combinations, this Thin Film Method/Co-solvent Evaporation
“thin film method” results in clear and stable micellar solutions with 1. POx and drug are dissolved in a minimum amount of a common
different maximum drug loadings depending on the formulation. The solvent (e.g., ethanol).
formulations can typically be reconstructed multiple times without 2. The solvent is slowly evaporated under reduced pressure and/or
noticeable differences in the micellar characteristics. The simple mild heat until a thin, clear film is obtained.
procedure is outlined in Figure 3. 3. Water, buffer, or other aqueous solvent is added dropwise
(possibly mild heat, 50–60 °C, can be applied) until a clear solution
Hydrophobic drug is obtained.
4. Drug loading capacity is determined by HPLC.
Water References
+ (1) Tomalia, D. A.; Sheetz, D. P. Journal of Polymer Science Part A-1: Polymer Chemistry 1966, 4,
2253–2265.
(2) Aoi, K.; Okada, M. Prog. Polym. Sci. 1996, 21, 151–208.
(3) Luxenhofer, R.; Han, Y.; Schulz, A.; Tong, J.; He, Z.; Kabanov, A. V.; Jordan, R. Macromol. Rapid
Commun. 2012, 33, 1613–1631.
Amphiphilic POx (4) Yang, Q.; Lai, S. K. WIRE-Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2015, 7 (5) 655–77
Drug-loaded (5) Sedlacek, O.; Monnery, B. D.; Filippov, S. K.; Hoogenboom, R.; Hruby, M. Macromol. Rapid
Commun. 2012, 33, 1648–1662.
polymeric micelle
(6) Woodle, M. C.; Engbers, C. M.; Zalipsky, S. Bioconjugate Chem. 1994, 5, 493–496.
(7) Gaertner, F. C.; Luxenhofer, R.; Blechert, B.; Jordan, R.; Essler, M. J. Controlled Release 2007,
Figure 3. Preparation of drug loaded polymeric micelles with an amphiphilic triblock POx as
119, 291–300.
the only excipient. (For the general procedure, see A. Du, M. Stenzel in this publication.)
(8) Luxenhofer, R.; Sahay, G.; Schulz, A.; Alakhova, D.; Bronich, T. K.; Jordan, R.; Kabanov, A. V. J.
Controlled Release 2011, 153, 73–82.
(9) Luxenhofer, R.; Schulz, A.; Roques, C.; Li, S.; Bronich, T. K.; Batrakova, E. V.; Jordan, R.;
Conclusions Kabanov, A. V. Biomaterials 2010, 31, 4972–4979.
(10) Luxenhofer, R.; Jordan, R. Macromolecules 2006, 39, 3509–3516.
(11) Manzenrieder, F.; Luxenhofer, R.; Retzlaff, M.; Jordan, R.; Finn, M. G. Angew. Chem. Int. Ed.
Poly(2-oxazoline)s (POx) have recently emerged as a popular candidate 2011, 50, 2601–2605.
for the development of new polymer therapeutics due largely to its (12) Fijten, M. W. M.; Haensch, C.; van Lankvelt, B. M.; Hoogenboom, R.; Schubert, U. S.
biocompatibility and versatility in forming well-defined POx-drug Macromol. Chem. Phys. 2008, 209, 1887–1895.
and POx-protein conjugates, polyplexes, and polymeric micelles (13) Cortez, M. A.; Grayson, S. M. Macromolecules 2010, 43, 4081–4090.
(14) Kempe, K.; Hoogenboom, R.; Jaeger, M.; Schubert, U. S. Macromolecules 2011, 44,
from amphiphilic POx. With preclinical data recently completed, the 6424–6432.
translation of POx-based polymer therapeutics into clinical trials is soon (15) Dargaville, T. R.; Forster, R.; Farrugia, B. L.; Kempe, K.; Voorhaar, L.; Schubert, U. S.;
expected. Hoogenboom, R. Macromol. Rapid Commun. 2012, 33, 1695–1700.
(16) Schubert, U. S.; Hartlieb, M.; Kempe, K. Journal of Materials Chemistry B 2014, 3, 526–538.
(17) Tong, J.; Yi, X.; Luxenhofer, R.; Banks, W. A.; Jordan, R.; Zimmermann, M. C.; Kabanov, A. V.
Method: Block Copolymerization Mol. Pharmaceutics 2013, 10, 360–377.

(18) Tong, J.; Luxenhofer, R.; Yi, X.; Jordan, R.; Kabanov, A. V. Mol. Pharmaceutics 2010, 7,
984–992.
of 2-Oxazolines (19) Mero, A.; Fang, Z.; Pasut, G.; Veronese, F. M.; Viegas, T. X. J. Controlled Release 2012, 159,
353–361.
(20) Harris, J. M.; Bentley, M. D.; Viegas, T. X. 8th Intern. Symp. on Polymer Therapeutics: From
The polymerization is carried out under dry inert conditions Laboratory to Clinical Practice 2010, 19.
with freshly distilled and degassed solvents and monomers (21) Viegas, T. X.; Bentley, M. D.; Harris, J. M.; Fang, Z.; Yoon, K.; Dizman, B.; Weimer, R.; Mero, A.;
Pasut, G.; Veronese, F. M. Bioconjug. Chem. 2011, 22, 976–986.
(Schlenk conditions).
(22) Lava, K.; Verbraeken, B.; Hoogenboom, R. Eur. Polym. J. 2015, 65, 98–111.
(23) Rossegger, E.; Schenk, V.; Wiesbrock, F. Polymers 2013, 5, 956–1011.
Synthesis of P(MeOx27-BuOx12-MeOx27) (24) Luxenhofer, R.; Jordan, R. Materials Matter 2013, 8, 70–73.
(25) de la Rosa, V. R. J. Mater. Sci. Mater. Med. 2014, 25, 1211–1225.
1. At room temperature, 32.2 mg (0.2 mmol, 1 eq) of methyltriflate
(26) Cesana, S.; Auernheimer, J.; Jordan, R.; Kessler, H.; Nuyken, O. Macromol. Chem. Phys. 2006,
(MeOTf ) (Aldrich Prod. No. 164283) and 440 mg (5.17 mmol, 26 eq) 207, 183–192.
of 2-methyl-2-oxazoline (MeOx) (Aldrich Prod. No. 137448) are (27) Rinkenauer, A. C.; Tauhardt, L.; Wendler, F.; Kempe, K.; Gottschaldt, M.; Traeger, A.;
dissolved in 3 mL acetonitrile. Schubert, U. S. Macromol. Biosci. 2015, 15 (3), 414–425
(28) He, Z.; Miao, L.; Jordan, R.; S-Manickam, D.; Luxenhofer, R.; Kabanov, A. V. Macromol. Biosci.
2. The mixture is heated to 130 °C (microwave, 150 W) for 15 min. After 2015, 15, in print.
equilibration to room temperature, the monomer for the second (29) Huber, S.; Jordan, R. Colloid Polym. Sci. 2008, 286, 395–402.
block, 2-butyl-2-oxazoline (256 mg, 2.01 mmol, 10 eq) (Aldrich (30) Park, J. S.; Kataoka, K. Macromolecules 2007, 40, 3599–3609.
(31) Hoogenboom, R.; Thijs, H. M. L.; Jochems, M. J. H. C.; van Lankvelt, B. M.; Fijten, M. W. M.;
Prod. No. 799637) is added under dry nitrogen and heated again Schubert, U. S. Chem. Commun. 2008, 41, 5758–5759
for 15 min. (32) Weber, C.; Hoogenboom, R.; Schubert, U. S. Prog. Polym. Sci. 2012, 37, 686–714.
3. For the third block, the procedure is repeated with 442 mg MeOx (33) Schulz, A.; Jaksch, S.; Schubel, R.; Wegener, E.; Di, Z.; Han, Y.; Meister, A.; Kressler, J.;
Kabanov, A. V.; Luxenhofer, R.; Papadakis, C. M.; Jordan, R. ACS Nano 2014, 8, 2686–2696.
(5.19 mmol, 26 eq). The polymerization is terminated at room
(34) Kabanov, A.; Jordan, R.; Luxenhofer, R., PCT Appl. EP2009/004655, US 61/133,154, US
temperature by the addition of 0.1 mL piperidine (1.01 mmol, 5 eq) 61/134,209, RU 2523714, JP 5671457.
(Sigma-Aldrich Prod. No. 104094) and stirring overnight. (35) Tong, J.; Zimmermann, M. C.; Li, S.; Yi, X.; Luxenhofer, R.; Jordan, R.; Kabanov, A. V.
Biomaterials 2011, 32, 3654–3665.
4. Finally, an access of K2CO3 is added and the mixture is allowed to stir
(36) Han, Y.; He, Z.; Schulz, A.; Bronich, T. K.; Jordan, R.; Luxenhofer, R.; Kabanov, A. V. Mol.
for several hours. Pharmaceutics 2012, 9, 2302–2313.
5. After filtration, the solvent is removed and the polymer precipitated (37) He, Z.; Schulz, A.; Wan, X.; Seitz, J.; Bludau, H.; Darr, D. B.; Perou, C. M.; Jordan, R.; Ojima, I.;
Kabanov, A. V.; Luxenhofer, R. J. Controlled Release 2015, 208, 67–75.
(chloroform to diethylether, 1:10) collected and freeze-dried.
(38) Seo, Y.; Schulz, A.; Han, Y.; He, Z.; Bludau, H.; Wan, X.; Tong, J.; Bronich, T. K.; Luxenhofer, R.;
Expected yield: ≥90%, PDI ≤1.2. Jordan, R.; Kabanov, A. V. Polym. Adv. Technol. 2015, 26, 837–850.

Poly(2-oxazoline)s for Drug Delivery
Low PDI Poly(oxazoline)s

For more information on these products, visit aldrich.com/pox.
Poly(2-methyl-2-oxazoline), hydroxy terminated average Mn 2,000 < 1.2 PDI 795275-5G
OH
N
O n
Poly(2-ethyl-2-oxazoline) average Mn 5,000 ≤ 1.2 PDI 740713-5G

H3C OH
N n average Mn 25,000 ≤ 1.4 PDI 741884-5G
CH3 average Mn 10,000 < 1.3 PDI 741906-5G
O
Poly(2-ethyl-2-oxazoline) α-methyl, ω-2- H average Mn 2,000 ≤ 1.2 PDI 773360-1G

hydroxyethylamine terminated H3C N
N OH average Mn 5,000 < 1.3 PDI 773379-1G
n average Mn 10,000 < 1.3 PDI 773387-1G
O
Poly(2-ethyl-2-oxazoline), alkyne terminated average Mn 2,000 ≤ 1.1 PDI 747262-1G

HO CH
N n average Mn 5,000 ≤ 1.2 PDI 778338-1G
H3C
O
Poly(2-ethyl-2-oxazoline)
For more information on these products, visit aldrich.com/pox.
Poly(2-ethyl-2-oxazoline) average Mw 50,000 372846-100G
N 372846-500G
CH3 average Mw 200,000 372854-100G
O
n
average Mw 500,000 373974-100G
373974-500G

aldrich.com/matsci
POLYOXAZOLINES: AN ALTERNATIVE TO
POLYETHYLENE GLYCOL
Table 1. Property comparison between PEG and Poly(2-oxazoline)s*
Poly(2-oxazoline) PEG
Polymerization
Easily synthesized with commercially available Challenging polymerization
materials
No peroxide formation Forms peroxides
No diol impurities May have up to 6% diol content as an impurity
Polymer Properties
Low viscosity High viscosity at high concentrations
Nicolynn Davis
Aldrich Materials Science Stable at room temperature Stable at <–20 °C
Sigma-Aldrich, Milwaukee, WI USA Non-hygroscopic –
Email: nicolynn.davis@sial.com Drug Delivery Applications
Not approved by the FDA yet FDA approved
Water-soluble, biocompatible polymers are important building High drug loading Low drug loading
blocks in drug delivery formulations, from micelles to drug-polymer Pendant functional groups allow for active Active targeting is reserved for conjugation to
targeting by conjugation techniques end-functional group only
conjugates. Polyethylene glycol (PEG) is often considered the gold
Readily cleared from the body May accumulate in vivo
standard for biocompatible and inert polymers used in drug delivery
– May be immunogenic for a subset of patients
formulations. However, the growing demand for suitable polymer
– Vacuole formation observed
candidates with a range of material properties to fit the increasing
diversity of drugs has generated a need for new biocompatible *adapted from Viegas et al.15
multifunctional polymers.
References
Poly(2-ethyl-2-oxazoline)s and poly(2-methyl-2-oxazoline)s are as (1) Chapman, R. G.; Ostuni, E.; Takayama, S.; Holmlin, R. E.; Yan, L.; Whitesides, G. M. J. Am.
extensively studied as poly(ethylene glycol) substitutes in a variety Chem. Soc. 2000, 122, 8303.
of biomedical applications since they share many of the desirable (2) Konradi, R.; Pidhatika, B.; Muhlebach, A.; Textort, M. Langmuir 2008, 24, 613.
(3) Zalipsky, S., Hansen, C. B.; Oaks, J. M.; Allen, T. M. J. Pharm. Sci. 1996, 85, 133.
properties of PEG1–4 but avoid some of their limitations (Table 1). PEG
(4) Pidhatika, B.; Rodenstein, M.; Chen, Y.; Rakhmatullina, E.; Muhlebach, A.; Acikgoz, C.; Textor,
has been widely used in biomedical applications because it imparts M.; Konradi, R. Biointerphases 2012, 7, 1.
“stealth” properties to the drug delivery system. PEGylation, the (5) Sherman, M. R.; Saifer, M. G.; Perez-Ruiz, F. Adv. Drug Delivery Rev. 2008, 6, 59–68.
conjugation of PEG to a biopharmaceutical, has helped generate both (6) Armstrong, J. K.; Hempel, G.; Koling, S.; Chan, L. S.; Fisher, T.; Meiselman, H. J.; Garratty, G.
Cancer 2007, 110, 103–11.
clinical and market success for a number of drugs. Although PEG has (7) Leger, R. M.; Arndt, P.; Garratty, G.; Armstrong, J. K.; Meiselman, H. J.; Fisher, T. C. Transfusion
traditionally been considered bio-inert, recently, serum antibodies 2001, 41, 29S.
against PEG have been detected in patients treated with PEG-uricase5 (8) Armstrong, J. K.; Leger, R.; Wenby, R. B.; Meiselman, H. J.; Garratty, G.; Fisher, T. C. Blood
2003, 102, 556A.
and PEG-asparaginase.6 Moreover, in a pre-treatment screening, over
(9) Bendele, A.; Seely, J.; Richey, C.; Sennello, G.; Shopp, G. Toxicol. Sci. 1998, 42, 152–157.
25% of patients had pre-existing anti-PEG antibodies even though (10) Conover, C.; Lejeune, L.; Linberg, R.; Shum, K.; Shorr, R. G. L. Immob. Biotechnol. 1996, 24,
they never received prior treatment with PEGylated drugs.7,8 This may 599–611.
be due to the abundance of PEG used in everything from beauty (11) Goddard, P.; Hutchinson, L. E.; Brown, J.; Brookman, L. J. J. Controlled Release 1989, 10, 5–16.
(12) Gaertner, F. C.; Luxenhofer, R.; Blechert, B.; Jordan, R.; Essler, M. J. Controlled Release 2007,
products to food. Furthermore, PEGylated conjugates can cause kidney 119, 291–300.
cell vacuoluation in animals following repeated administration of (13) Kronek, J.; Kronekova, Z.; Luston, J.; Paulovicova, E.; Paulovicova, L.; Mendrek, B. J. Mater Sci
PEGylated therapeutics.9,10 Mater Med. 2011, 22, 1725–34.
(14) Luxenhofer, R.; Sahay, G.; Schulz, A.; Alakhova, D.; Bronich, T. K.; Jordan, R.; Kabanov, A. V.
Poly(2-oxazolines) may be a suitable alternative to PEG due to their J. Controlled Release 2011, 53, 73–82.
biocompatible properties. For example, radiolabeling of polyoxazoline (15) Viegas, T.; Bentley, M.; Harris, J.; Fang, Z.; Yoon, K.; Dizman, B.; Weimer, R.; Mero, A.; Paut, G.;
Veronese, F. Bioconjugate Chem. 2011, 22, 976–986.
has shown that the polymer is rapidly excreted by the kidneys and (16) Mero, A.; Pasut, G.; Dalla, Via L.; Fijten, M. W.; Schubert, U. S.; Hoogenboom, R.; Veronese, F.
shows no accumulation in the body.11,12 In addition, in vitro studies M. J. Controlled Release 2008, 2, 87–95.
with poly(2-ethyl-2-oxazoline) and poly(2-ethyl-2-oxazoline) block (17) Wang, C. H.; Hsiue, G. H. J. Controlled Release 2005, 108, 140–149.
copolymers were reported to be biocompatibile even at higher (18) Zalipsky, S.; Hansen, C. B.; Oaks, J. M.; Allen, T. M. J. Pharm. Sci. 1996, 85, 133–137.
concentrations than PEG.13,14 For these reasons, poly(2-oxazolines)

are being investigated in a variety of pharmaceutical and medical
applications, e.g., drug-polymer conjugates (POZylation),15,16 micelles,17
or grafting onto liposomal bilayers.18

Dendritic Polyester Scaffolds: Functional and Biocompatible Precision Polymers for Drug Delivery Applications
DENDRITIC POLYESTER SCAFFOLDS:

FUNCTIONAL AND BIOCOMPATIBLE PRECISION POLYMERS
FOR DRUG DELIVERY APPLICATIONS
yy They can be tuned to present optimal properties such as
biodegradability, low toxicity, and water solubility.
yy They provide an increased loading capacity, achieved by
encapsulation or direct conjugation of drugs with different
hydrophilic/hydrophobic properties.
yy The drug release profile can selectively be tuned and controlled.
Sandra García-Gallego,1 Michael Malkoch2*

yy They are intrinsically highly functional and can be designed to
1
Post-doctoral Researcher, Division of Coating Technology, Fibre and Polymer Technology, present multipurpose actions (e.g., therapeutic, diagnosis, targeting).
KTH Royal Institute of Technology
2
Associate Professor, Division of Coating Technology, Fibre and Polymer Technology,
KTH Royal Institute of Technology, and Chief Executive Officer, Polymer Factory, Sweden AB
*Email: malkoch@kth.se Non-toxic Biodegradable bis-MPA
Introduction Dendritic Scaffolds
A promising family of dendritic macromolecules is based on the
Dendrimers and dendrons are highly branched polymer structures with
2,2-bis(methylol)propionic acid (bis-MPA) building block.3–4 These
a precise distribution of functional groups. In contrast to their linear
scaffolds exhibit highly valuable properties for biomedical applications
analogs, they are monodisperse in nature and are extremely consistent
including low or no in vitro toxicity, no specific in vivo organ
from batch-to-batch. A broad range of sophisticated applications have
accumulation, no immunogenic profile, and biodegradability under
capitalized on these dendritic materials including catalysis, optics, and
physiological conditions.5–6 Moreover, the use of both the bis-MPA
nanomedicine.1 In nanomedicine, great efforts have been devoted
monomer and dendritic macromolecules has increased due to the
toward the design of selective drug delivery systems and efficient
commercial availability of a wide range of hyperbranched polymers,
imaging probes.2 Thus, dendrimers present significant advantages over
dendrimers, and dendrons (Figure 1). Herein, the most advanced
linear polymers, including the following:
applications of bis-MPA based dendritic materials as drug delivery
yy They are precision nanomedicines carrying an exact number agents will be summarized.
of drugs, all arising from their controlled synthesis and
monodisperse nature.
yy Their properties can be directly correlated to their structure and
present predictable and repeatable pharmacokinetics.
OH OH
OH
OH
HO HO O OH
HO OH
HO HO O O O
HO O O OH
O OO O OH
HO O O O O OH O
OH OH OH OH O HO O O
HO OH OH OH HO O
O OH
OH HO OH O O
HO OH O O O
HO O O O OH OH HO OH HO O O O O OH
O O O
HO O O O O O
O
OH
O
OH O O
O OH
OH O HO O HO O O O O
HO O O OH OH O
OH O HO O O O O OH
HO O O O O OH O O
O O O
OH O O O O O OH O
OH
O O OO HO HO HO OH
HO O O OH O O O O OH HO HO OH OH
O O O OH O O O
O O O O HO O O O HO O O OH
HO O O O O OH OH O O O O O
O O O O O O O O O
OH O O O O O O O O O
HO O O O O O O O OH HO O OH O O O O
O O O O O O O O O O
OO OO OH R1 O O O O O
HO O OO O O OH HO O OH HO O O OO O OH
O O O O O O O O O O
O O OH O O O n O O O
HO O O OH HO O OH HO O O OH
O O O O O O O O O
O O O OH O O O O O
HO HO OH
HO O O O O O OH O O O O OH O O
O O OH O O O O O O
O O O O OH HO O O O OH HO O O OH
HO O O O O OH
O OH O O O O O O
O O HO O
HO O O O O OH O OH O O OH HO O O O OH O O O OH
O O OH HO O OH O
HO O O OH O O HO O OH OH O
O HO O
O OO
HO O O O OO O OH OH HO OH HO O O
O OH
HO OH OH HO OH HO O OH O O OH
HO OH O
HO O O O O
OH OHOH OH OH O
O
pseudoG4 HOHO O O O OH
traditional G4 dendrimer G4 dendron dendritic-linear-dendritic hyperbranched

O O
OH
OH
hybrid polymer HO
HO OH OH
OH
monodisperse polydisperse
Figure 1. Examples of bis-MPA based dendritic structures of generation 4.

aldrich.com/matsci
Novel bis-MPA Dendritic Scaffolds for Facile Formulation Protocols

Targeted Drug Delivery With the wide range of polyester dendrons and dendrimers based on
bis-MPA currently available on the market, straightforward reactions
The dendritic family of bis-MPA scaffolds has proven to be an efficient like one-step functionalization are aiding in the development of many
tool for delivery of both physically and covalently attached drugs. final sophisticated bioactive materials. The recent development of
Fluoride-Promoted Esterification (FPE) through imidazolide-activated
Passive Encapsulators compounds acts as a powerful tool for the modification of highly
Bis-MPA dendrimers have been reported as highly effective functional polymers under benign and green chemistry conditions.22
formulation systems for drugs physically encapsulated in the interior, Through this protocol, the hydroxyl4 or carboxyl23 periphery of
based on hydrophobic, ionic, or charged-based interactions. They the dendritic scaffolds can be easily functionalized with a large
have demonstrated enhancement of the solubility of different amount of moieties (drugs, targeting moieties, dyes, etc.). A more
chemotherapeutics (e.g., such as doxorubicin,7–8 camptothecin,9–10 and advanced process called Click chemistry was introduced in 2001 by
paclitaxel11) and stabilize colloidal silver particles with antimicrobial Prof. K. B. Sharpless.24 Click chemistry is based on a set of highly reliable
properties.12 The dendritic components act as controlled drug delivery and synthetically simple chemical reactions known to proceed in a
carriers to encapsulate a large amount of the hydrophobic drugs wide range of solvents, including organic and aqueous conditions
that are released from the nanocarrier to its surroundings upon with high conversions in short reaction times. In the case of bis-MPA
external stimuli such as changes in pH, temperature, or enzymes. dendritic scaffolds, Click reactions have been used for the successful
The hybridization of bis-MPA dendritic units with linear polymers conjugation of bioactive moieties such as PEG,14 saccharides,18–19
such as polyethylene glycol (PEG) imparts stealth properties, peptides, and antibiotics.19
increases circulation time, and can endow novel properties such as The combination of these one-step functionalization protocols allows
self-assembly into micelles to improve drug encapsulation.13–15 One the straightforward development of advanced dendritic materials. The
of the most remarkable examples is the theranostic nanoparticles presence of common “clickable” groups in the core or periphery of the
described by Nyström et al.16 in which star-shaped dendritic-linear dendritic molecule such as azides (-N3), acetylenes (-CΞCH), thiols (-SH),
hybrids self-assembled into carriers capable of efficiently delivering and alkenes (-C=CH2) allows the simple substitution at this position via
doxorubicin and acting as in vivo contrast agents for 19F-MRI. Analogous Click reactions. The multiple hydroxyl or carboxyl groups in the surface
hyperbranched polymers also have been widely used as passive are susceptible to modification with a broad range of compounds
encapsulators due to their straightforward preparation and similar via the FPE protocol. Typical reaction conditions for the modification
behavior to their monodisperse counterparts.14 of the core and the periphery of the dendritic scaffolds are depicted
in Table 1.
Unimolecular Carriers
Table 1. Typical reaction conditions for the peripheral and core functionalization of
Today many pharmaceutically active drugs are intrinsically commercially available bis-MPA based dendritic structures.
hydrophobic. Researchers are continually proposing new platforms
Reaction ——— Conditions ———
of biocompatible and unimolecular drug delivery systems that Periphery -OH FPE22–23 RCOOH, CDI 1.5 eq./OH
can carry a large cargo of covalently conjugated drugs, along with -COOH CsF 0.2 eq./OH
providing appropriate aqueous solubility and allowing controlled EtOAc, 50 °C
release upon reaching the targeted cells and tissues. Among the many -Ξ CuAAC25 R-N3 1.1 eq./ck
proposed drug delivery systems, monodisperse and highly functional CuSO4∙5H2O 0.1 eq./ck
NaAsc 0.3 eq./ck
dendritic frameworks based on bis-MPA are the most intriguing. This THF:H2O, r.t.
is due to their structural flawlessness coupled with biocompatibility,
Core -OH FPE22 RCOOH, CDI 1.5 eq./OH
biodegradability, and lack of toxicity. Furthermore, careful selection of -COOH CsF 0.2 eq./OH
the conjugation chemistry between the bis-MPA dendritic carrier and EtOAc, 50 °C
the desired cargo enables controlled release of pharmaceutically active -N3 CuAAC25 R-N3 or R-Ξ 1.1 eq./ck
drugs. Such controlled release is typically triggered by external stimuli -Ξ CuSO4∙5H2O 0.1 eq./ck
NaAsc 0.3 eq./ck
such as variations in pH, intracellular glutathione levels, or the reductive
THF:H2O, r.t.
potential in the cell. One of the most advanced studies is based on
-= TEC25 R-SH or R-= 2.0 eq./ene
a bow-tie bis-MPA dendrimer containing degradable linkages of -SH DMPA 0.2 eq./ene
doxorubicin on one side and PEG chains on the other side. This scaffold THF, r.t. hν (365 nm)
produced a 9-fold higher specific tumor uptake compared to the free
drug in a colorectal xenograft tumor animal model.17 The attachment Considering these one-step functionalizations, the preparation of
of certain active components, such as sugar or peptide moieties,18–20 advanced homofunctional and heterofunctional bis-MPA dendrimers
leads to unimolecular therapeutic agents that take advantage of is currently an easily achievable task. As detailed in Figure 2, in a two-
the multivalency of the scaffold. One clear example, described by step process the most advanced heterofunctional dendrimers can be
Hawker et al., is the bifunctional bis-MPA dendrimers containing both realized. Nevertheless, the suggested reactions are only guidelines that
bioactive mannose and fluorescent coumarin units.21 The dendritic may need to be adapted based on the functionalization of interest.
macromolecules proved to be highly efficient recognition/detection
agents for the inhibition of hemagglutination with a 240-fold greater
potency than monomeric mannose.

Dendritic Polyester Scaffolds: Functional and Biocompatible Precision Polymers for Drug Delivery Applications
Commerical
[Ck]G4(OH)16 dendrons OH
OH
Commercially available OH
O OH Examples of Commercially Available Functional Moieties (F)
clickable dendrons (Ck) O
O O OH Reactive Optical Bioactive
O Allyl O O OH
O O
OH O
O Thiol O O O Alkene HO Rhodamine B O
SH O
O O O OH HO O HO Ibuprofen
Ck (No. 245925) Cl
O Acetylene O O O OH N O N (No. I4883)
O O O O
OH (No. R6626)
O N3 Azide O O HO Alkyne
O O OH HO O OH O
O O OH (No. 232211) S
Fluorescein HO
O Biotin
O OH O O H O HN NH
OH HO NHBoc Amine N OH (No. B4501) O
OH COOH O
COOH O
Step 1 OH
O
(No. 15420) (No. 46935)
O
Functionalization N N N N N N N N
of the periphery “in situ”
CsF
FPE protocol
Step 2
Coupling of O O O O
O O O
O
O O O O O O
O
dendrons O O
O
O O
O O O O O O O O O O
O O O O O O O O
O O O O O O O O O O
O O O O O O O O OO
O O O O OO
O O O O O O
OO O O
OO
O
O O
O
O O O O O O O O O O
O O
O
O O
O O O
O O
O O O O Click-reaction O
O O
O O
O O
O O O OO
O O O O O O OO O
Ck Ck O O O O O O O OO
O O O O OO
O O O O O O O O O O O O
O O O O O O O O O O
O O O O O O
O O O O OO OO
OO O O O O O
O O O O O O O O O O O O O
O O O O O O O O O O
O O O O O O O O
O O O O O O O O O O
O O O O O O O
O O O O O O O O O O
O O O
G4(F1)16(F2)16
[Ck]G4(F)16
heterofunctional
dendrons
dendrimer
Figure 2. Example of a two-step approach toward bis-MPA based heterofunctional dendrimers.
Summary Methods: Dendritic Carriers

Dendritic polymers, including monodisperse dendrimers and dendrons, The following procedures describe two general protocols for the
are by definition the ultimate precision polymers. They are defined as preparation of dendritic-based drug delivery systems. The suggested
highly branched scaffolds with a large functional group density that reactions are only guidelines that may need to be adapted based on
can carry a large payload of active drugs. Due to their structurally the molecules of interest.
flawless nature, dendrimers and dendrons belong to a group of
the most advanced drug delivery carriers in which batch-to-batch Drug Passive Encapsulators
consistency plays a crucial role in future therapeutic breakthroughs. Drug loading varies significantly depending upon both the drug
Among the many available dendritic platforms, bis-MPA based and the dendrimer structure, making a universal protocol difficult to
polyester scaffolds have attracted the most research interest for establish. However, the following is an example that can be used to
biomedical applications. This is mainly due to their structural diversity establish an initial protocol.8
and simple functionalization protocols along with being biocompatible,
biodegradable, and non-toxic. Their commercial availability coupled 1. The dendritic component is dissolved in phosphate buffer
with the rise of facile post-functionalization protocols such as Click saline (PBS).
chemistry and FPE reactions gives non-dendrimer specialists the 2. Subsequently, a chloroform solution of the drug is added dropwise
necessary toolbox to custom-build next-generation sophisticated drug under stirring.
delivery systems. 3. After evaporation of the chloroform, the free drug is removed by
spin filtration.

aldrich.com/matsci
Drug Unimolecular Carrier References

(1) Tomalia, D. A.; Christensen, J. B.; Boas, U. Dendrimers, dendrons and dendritic polymers,
As detailed in Figure 2, an advanced heterofunctional dendritic carrier Cambridge University Press, 2012.
can be easily obtained in a two-step process. (2) Caminade, A.-M.; Turrin, C.-O. J. Mater. Chem. B 2014, 2, 4055–4066.
(3) Carlmark, A.; Malmström, E.; Malkoch, M. Chem. Soc. Rev. 2013, 42, 5858–5879.
1. Surface Modification via FPE Protocol. The molecule of interest (4) García-Gallego, S.; Nyström, A.; Malkoch, M. Prog. Polym. Sci. 2015, DOI 10.1016/j.
(drug, dye, etc.) containing a carboxylic group F-COOH is activated progpolymsci.2015.04.006.
with 1,1´-carbonyldiimidazole (CDI, Aldrich Prod. No. 115533) (5) Feliu, N.; Walter, M. V.; Montañez, M. I.; Kunzmann, A.; Hult, A.; Nyström, A.; Malkoch, M.;
Fadeel, B. Biomaterials 2012, 33, 1970–1981.
in EtOAc at 50 ºC for 1 h. The in situ reaction with the hydroxyl- (6) Parrott, M. C.; Benhabbour, S. R.; Saab, C.; Lemon, J. A.; Parker, S.; Valliant, J. F.; Adronov, A. J.
functional dendron at 50 ºC and CsF (Aldrich Prod. No. 20989), and Am. Chem. Soc. 2009, 131, 2906–2916.
subsequent quenching and washing with NaHCO3 10%, provides (7) Zeng, X.; Zhang, Y.; Wu, Z.; Lundberg, P.; Malkoch, M.; Nyström, A. M. J. Polym. Sci., Part A:
Polym. Chem. 2012, 50, 280–288.
the bifunctional dendron [Ck]Gn(F)m. (8) Wu, Z.; Zeng, X.; Zhang, Y.; Feliu, N.; Lundberg, P.; Fadeel, B.; Malkoch, M.; Nyström, A. M.
2. Coupling of Dendrons via Click Reactions. The covalent J. Polym. Sci., Part A: Polym. Chem. 2012, 50, 217–226.
attachment of two dendrons containing complementary clickable (9) Morgan, M. T.; Carnahan, M. A.; Immoos, C. E.; Ribeiro, A. A.; Finkelstein, S.; Lee, S. J.;
Grinstaff, M. W. J. Am. Chem. Soc. 2003, 125, 15485–15489.
moieties can be achieved through different Click reactions. The (10) Morgan, M. T.; Nakanishi, Y.; Kroll, D. J.; Griset, A. P.; Carnahan, M. A.; Wathier, M.; Oberlies, N.
most common protocols are as follows: H.; Manikumar, G.; Wani, M. C.; Grinstaff, M. W. Cancer Res. 2006, 66, 11913–11921.
yy Click Reaction Between Acetylene and Azide (CuAAC). To a (11) Ooya, T.; Lee, J.; Park, K. J. Control. Release 2003, 93, 121–127.
(12) Mahltig, B.; Cheval, N.; Astachov, V.; Malkoch, M.; Montañez, M. I.; Haase, H.; Fahmi, A.
THF solution of the dendrons, freshly prepared solutions Colloid. Polym. Sci. 2012, 290, 1413–1421.
of CuSO4∙5H2O (Aldrich Prod. No. 209198) and sodium (13) Gillies, E. R.; Fréchet, J. M. J. Bioconjugate Chem. 2005, 16, 361–368.
ascorbate (Sigma Prod. No. A7631) are added. The reaction (14) Hed, Y.; Zhang, Y.; Andrén, O. C. J.; Zeng, X.; Nyström, A. M.; Malkoch, M. J. Polym. Sci., Part A:
proceeds at room temperature until the total disappearance Polym. Chem. 2013, 51, 3992–3996.
(15) Lundberg, P.; Walter, M. V.; Montañez, M. I.; Hult, D.; Hult, A.; Nyström, A.; Malkoch, M. Polym.
of the clickable groups’ signals. The same reactions also can Chem. 2011, 2, 394–402.
be accomplished in organic solvents using combinations (16) Porsch, C.; Zhang, Y.; Östlund, Å.; Damberg, P.; Ducani, C.; Malmström, E.; Nyström, A. M.
of CuBr (Aldrich Prod. No. 212865) and stabilizing ligands Part. Part. Syst. Charact. 2013, 30, 381–390.
(17) Lee, C. C.; Gillies, E. R.; Fox, M. E.; Guillaudeu, S. J.; Fréchet, J. M. J.; Dy, E. E.; Szoka, F. C. Proc.
such as tris-(benzyltriazolylmethyl)amine (Aldrich Prod. Natl. Acad. Sci. U.S.A. 2006, 103, 16649–16654.
No. 678937), or organic copper complexes such as (18) Lo Conte, M.; Robb, M. J.; Hed, Y.; Marra, A.; Malkoch, M.; Hawker, C. J.; Dondoni, A. J. Polym.
bromotris(triphenylphosphine) copper(I) (Aldrich Prod. Sci., Part A: Polym. Chem. 2011, 49, 4468–4475.
No. 572144). (19) Ghirardello, M.; Öberg, K.; Staderini, S.; Renaudet, O.; Berthet, N.; Dumy, P.; Hed, Y.; Marra, A.;
Malkoch, M.; Dondoni, A. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 2422–2433.
yy Click Reaction Between Thiol and Alkene (UV-initiated Thiol- (20) Montañez, M. I.; Hed, Y.; Utsel, S.; Ropponen, J.; Malmström, E.; Wagberg, L.; Hult, A.;
Malkoch, M. Biomacromolecules 2011, 12, 2114–2125.
ene). To a deoxygenated MeOH solution of the dendrons, a (21) Wu, P.; Malkoch, M.; Hunt, J. N.; Vestberg, R.; Kaltgrad, E.; Finn, M. G.; Fokin, V. V.; Sharpless, K.
photoinitiator such as 2,2-dimethoxy-2-phenylacetophenone B.; Hawker, C. J. Chem. Commun. 2005, 5775–5777.
(Aldrich Prod. No. 196118) is added and the mixture is irradiated (22) García-Gallego, S.; Hult, D.; Olsson, J. V.; Malkoch, M. Angew. Chem. Int. Ed. 2015, 54,
2416–2419.
with UV light at 365 nm until the completion of the reaction.
(23) Mongkhontreerat, S.; Andrén, O. C. J.; Boujemaoui, A.; Malkoch, M. J. Polym. Sci. A Polym.
Both protocols lead to a bifunctional dendrimer Gn[F1]m[F2]n. Chem. 2015, DOI 10.1002/pola.27750.
(24) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. 2001, 40, 2004–2021.
(25) Antoni, P.; Robb, M. J.; Campos, L.; Montañez, M.; Hult, A.; Malmström, E.; Malkoch, M.;
Hawker, C. J. Macromolecules (Washington, DC, U.S.) 2010, 43, 6625–6631.
Dendrimers
For more information on these products, visit aldrich.com/dendron.
bis-MPA Dendrimers, TMP Core Hyperbranched PEG OH Dendrimers

No. of Pseudo No. of
Surface Group Generation Surface Groups Prod. No. Name Generation Surface Groups Prod. No.
Hydroxyl 1 6 805920-250MG G4-PEG6k-OH 4 32 806218-5G
2 12 805939-250MG G5-PEG6k-OH 5 64 806226-5G
3 24 805947-250MG G6-PEG6k-OH 6 128 806234-5G
4 48 805955-250MG G2-PEG10k-OH 2 8 806242-5G
Carboxyl 1 6 806099-250MG G3-PEG10k-OH 3 16 806250-5G
2 12 806064-250MG G4-PEG10k-OH 4 32 806269-5G
3 24 806080-250MG G5-PEG10k-OH 5 64 806277-5G
4 48 806072-100MG G6--PEG10k-OH 6 128 806285-5G
Acetylene 1 6 806145-250MG G2-PEG20k-OH 2 8 806293-5G
3 24 806153-250MG G3-PEG20k-OH 3 16 806307-5G
5 96 806161-100MG G4-PEG20k-OH 4 32 806323-5G
G5-PEG20k-OH 5 64 806315-5G
G6-PEG20k-OH 6 128 806331-5G

CONTROLLED RADICAL
POLYMERIZATION GUIDE
Enabling Highly Controlled Polymer Synthesis
Procedures written by experts in Controlled Radical

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Authors include:
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-- Atom Transfer Radical Polymerization (ATRP) ligands
and monomers
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RAFT POLYMERIC CARRIERS

FOR ANTIBODY‑DRUG CONJUGATES OF BIOLOGIC DRUGS
endosomal membrane. The key functional property of these proteins
is a membrane-destabilizing activity that is closely coupled to a
pH-sensing mechanism. At physiological pH (7.4), the proteins are in
a “stealth” conformation until they are brought into the intracellular
endosomes via receptor-mediated endocytosis. As the pH of these
compartments drops during endosomal development to values of 5.5
or lower, a conformational change is triggered to expose a membrane-
destabilizing domain in viral proteins such as hemagluttinin, or
Patrick S. Stayton, Anthony Convertine, Geoffrey Berguig pathogenic proteins such as diphtheria toxin.1
Molecular Engineering & Sciences Institute
Department of Bioengineering, University of Washington, Seattle, WA USA We have developed polymeric carriers based on this biological design,
Email stayton@uw.edu incorporating pH-sensing moieties that trigger membrane destabilizing
activity at a defined lower pH value typical of endosomes.2–7 These
Introduction polymers are prepared using the Reversible Addition-Fragmentation
chain Transfer (RAFT) polymerization technique.8–10 Initial designs
Advances in genomics and proteomics have delivered an amazing were directed toward gene (plasmid), antisense oligonucleotide, and
collection of new biological information and new therapeutic targets RNA delivery. A representative design exploited a polyampholyte
for the biopharmaceutical industry. Biologic drugs based on proteins, segment composed of dimethylaminoethyl methacrylate (DMAEMA),
RNA, DNA, and cells have the broad target range necessary to propylacrylic acid (PAA), and a hydrophobizing monomer butyl
translate the potential of the genome era into effective therapeutics methacrylate (BMA).7 The pH-responsive DMAEMA and PAA were
and individualized medicines. Biologic drugs currently comprise incorporated at equimolar ratios, leading to a charge-neutralized
approximately 20–30% of clinically utilized drugs, and they represent and hydrophobized block segment. At neutral pH, this block is core-
the fastest growing segment since they address needs for safety, forming when combined with a hydrophilic block segment, but it
specificity and individualized medicine. The biologics category is transitions into the endosomal pH range as the DMAEMA is protonated
dominated by monoclonal antibodies for the treatment of oncology to become more positively charged and the PAA is protonated to
and inflammatory diseases and by vaccines for the treatment of neutrality. The corresponding micelle destabilization leads to exposure
infectious diseases. However, the number of nucleic acid and protein of the core segments and the carriers become sharply membrane-
therapeutic biologics is still limited, and their larger potential impact destabilizing at endosomal pH values. We characterized the structure–
still remains unrealized due to limitations in drug delivery. The crossing activity relationships that connected intrinsic membrane-destabilizing
of biological barriers at the circulatory, tissue, and cellular level remains activity with siRNA delivery activity. A series of seven diblock
the key to ultimate efficacy and one that, in broad terms, continues to copolymers were synthesized where the BMA hydrophobizing content
stymie the field of biologic drug delivery. was varied from 0–50% in the second block. It was shown that as BMA
content increased, while DMAEMA and PAA were kept at equimolar but
Pathogens display the highest level of success now known for
decreasing mole fraction, membrane-destabilizing activity and gene
the delivery of DNA, RNA, and proteins. Viruses and pathogenic
and protein knockdown increased (Figures 1A–B). This work provided
organisms such as Diphtheria have evolved highly effective systems
initial confirmation of carrier activity and guided further development
for delivery of nucleic acids and proteins to intracellular locations
of active compositional designs.4–6
and targets.1 These organisms feature remarkable molecular
machines that enhance transport of DNA, RNA, or proteins across the

RAFT Polymeric Carriers for Antibody‑drug Conjugates of Biologic Drugs
A) revolutionizing a broad range of disciplines from traditional polymer

90 science to biology. The versatility of RAFT lies in the elegant simplicity
pH 7.4 Increasing
pH 6.6 of the technique, broad chemical compatibility, and ease of use. This
80 BMA %
pH 5.8 technique was first reported by Rizzardo et al. at the Commonwealth
70 Scientific and Industrial Research Organization.8 Since that time, RAFT
has become one of the premier methods to prepare sophisticated
% Hemolysis
60
polymers for biotechnology applications. RAFT employs a thiocarbonyl
50 thio compound as a degenerate chain transfer agent (CTA), which
pDMAEMA-b-p(DMAEMA/PAA/BMA)
40 is most commonly a dithioester or trithiocarbonate. By simple
manipulation of the initial monomer, CTA, and radical initiator
30
stoichiometry, it is possible to prepare low dispersity materials over
20 a range of predefined molecular weights. Because this methodology
10
does not require the use of any toxic metal catalysts, it is particularly
well-suited for use in biotechnology applications. Most typical
0 commercially available RAFT CTAs, which contain carboxylic acid
P1 P2 P3 P4 P5 P6 P7
Polymer functionality, are modified using standard esterification/amidation
reactions. Because the functional groups are introduced as part of the
B) polymerization process, this strategy eliminates the need for costly
120 Increasing and often ill-defined post-polymerization modification reactions.
BMA % Following polymerization of a given monomer or monomers, the
100 Protein Level
resultant macroCTAs can be isolated for use in subsequent block (co)
Relative GAPDH Level
polymerization steps. RAFT polymerizations are also well-controlled in

80 water, assuming appropriate pH conditions are maintained, making
mRNA Level
them ideal for many bioconjugation applications.9
60
Module 1 Module 2
40 protein conjugation & shielding endosomal escape
siRNA [25 nM] [(PEGMA)-c-(bioHEMA)-c-(PyrSMA)]-b-[(DEAEMA)-c-(BMA)]
20 4:1 Charge
Ratio
O O O O O
0 O O O O O
P1 P2 P3 P4 P5 P6 P7
Polymer
O O O N
Figure 1. A) Structure–activity of diblock carrier series (pDMAEMA-b-(DMAEMA/PAA/BMA) O O
in pH-dependent, membrane-destabilization hemolysis assay. P1 (0% BMA), P2 (1% BMA), 6,19
P3 (12% BMA), P4 (19% BMA), P5 (24% BMA), P6 (27% BMA), and P7 (48% BMA). B) Structure- O
activity of carrier series in siRNA delivery and GAPDH knockdown assay (HeLa cells). O
HN
N
O S S S
Antibody-drug Conjugates for Intracellular N

H
S
Delivery of Proteins and Peptides S
Recently, we have adapted these carrier designs to the challenge

of intracellular protein and peptide delivery through an antibody
BIM
drug conjugate (ADC) approach.11,12 The introduction of antibody
and antibody-drug conjugate (ADC) therapeutics has had a major
CD22/SA
impact in the cancer field, demonstrating that anti-tumor efficacy can
be enhanced and toxicity diminished by increasing tumor delivery
selectivity.13,14 Current ADCs exploit small molecule drugs. These
small molecule drugs are typically hydrophobic enough to transport
broadly inside cells to reach their intracellular targets. Additional Figure 2. CD22-targeted, pH-responsive micelles loaded with proapoptotic BIM peptide.
Multifunctional diblock copolymers were synthesized by RAFT polymerization with
selectivity and new therapeutic approaches could be realized by two modules. The first module is composed of PEGMA for steric shielding, bioHEMA for
opening antibody-targeted drugs to new classes of biologic drugs complexing anti-CD22/streptavidin (CD22/SA), and PyrSMA for conjugating thiol-containing
that more specifically target the proteins dysregulated or abnormally BIM peptides. The second module is a block composed of DEAEMA and BMA to drive micelle
assembly, endosomal escape, and cytosolic release of BIM peptides.
produced in transformed tumor cells. The driving goal of developing
new intracellular polymeric carriers is to develop ADC-like delivery Previous work has demonstrated the copolymerization of
systems for biologic drugs—specifically, proteins/peptides that work diethylaminoethyl methacrylate (DEAEMA) with butylmethacrylate
against intracellular targets. These protein drugs could greatly expand (BMA) by RAFT can be tuned for endosomal escape and intracellular
the repertoire of disease-specific targets that are currently undruggable delivery of plasmid DNA.3 A polyethylene glycol methacrylate
with small molecules. (PEGMA) monomer can be incorporated to add steric stabilization
RAFT polymer synthesis allows for the development of well-tuned with “stealth-like” properties, increasing blood circulation of peptides
heterotelechelic diblock copolymers with endosomal-releasing and proteins.15–17 The incorporation of biotinylated monomers
capabilities (Figure 2). RAFT represents one of the most significant in the corona-forming block segment facilitates the integration
recent advances in synthetic chemistry, and its application is of antibody targeting agents and a pyridyl disulfide containing
monomer provides reversible peptide/protein conjugation sites into

aldrich.com/matsci
the carrier systems. Thus, the final hydrophilic first block is composed Synthesis of bioHEMA Monomer
of three different monomers: polyethylene glycol methacrylate, Biotin (2.0 g, 8.19 mmol, 1 eqv., Sigma-Aldrich Prod. No. B4501) and
pyridyldisulfide methacrylate (PyrSMA), and biotinhydroxyethyl 20 mL DMSO are added to a 50 mL round bottom flask. The biotin is
methacrylate (bioHEMA). then allowed to dissolve overnight in the dark.
In two recent papers, we demonstrated this generalized carrier system DMAP (4.0 g, 33.7 mmol, 4 eqv., Aldrich Prod. No. 107700) and
can be tailored for use with both peptide and protein therapeutics. HEMA (4.2 g, 33.7 mmol, 4 eqv, Aldrich Prod. No. 128635) are added
Two segment lengths of PEG 300 and PEG 95018 were studied and the to this solution.
longer-chain PEG-methacrylate monomer was found to show better
PK and tumor biodistribution properties. A proapoptotic BIM peptide19 After the DMAP completely dissolves, DIC (5.07 mL, 33.7 mmol, 4 eqv.)
was delivered via anti-CD22 monoclonal antibody targeting of human is added. The solution is then septa-sealed and allowed to react for 18 h
B-cell lymphoma in a xenograft tumor model.11 A related carrier system in the dark. At this time, the solution is filtered and then precipitated
was also developed for a de novo protein therapeutic that attacks (1 to 20) into cold (3 °C) 150 mM HEPES buffer pH 8.4. The filtrate
an Epstein–Barr (EB) viral target in an EB-human B-cell lymphoma is then washed thoroughly with deionized water and dried under
xenograft tumor model.12 This carrier exploited anti-CD19 monoclonal high vacuum.
antibody targeting, demonstrating the modular nature of the carrier
design. Both in vitro and in vivo studies showed the carrier system could Example
deliver active peptide drug and protein drug to tumor cells from an 500 mHz 1H NMR in DMSO D6: δ ppm 1.25–1.68 (SCHCH2CH2CH2, m,
infusion route of administration, enabling their therapeutic activities 6H), 1.88 (CH3, s, 3H), 2.31 (CH2CO2, t, 2H), 2.55 (SCH2, d, 1H), 2.82 (SCH2,
and survival benefits. dd, 1H), 3.08 (CHCH(CH2)S, m, 1H), 4.13 (CHCH(CH2)S, m, 1H), 4.29
(OCH2CH2O and NHC(H)CH(CH2)S, s/m, 5H), 5.7 (CH2CCH3 trans, s, 1H),
6.03 (CH2CCH3 cis, s, 1H), 6.35 and 6.43 (CONHCH, s, 1H).
Methods: Diblock Copolymer Biologic RAFT Synthesis of macroCTAs
Drug Carriers Two copolymers are synthesized via RAFT using Azobis(4-cyano
pentanoic acid) (ABCVA) (Aldrich Prod. No. 118168) as the initiator,
Synthesis of PyrSMA Monomer 4-cyanopentanoic acid dithiobenzoate (CTP) (Aldrich Prod.
In a 500 mL round-bottom flask, mono-2-(methacryloxy)ethyl No. 722995) as the chain transfer agent (CTA), with 20 wt% monomer
succinate)(SMA) (8.9 g, 0.0387 mol, Aldrich Prod. No. 454974) is in dimethysulfoxide (Scheme 1A).
dissolved in chloroform (300 mL). To this solution N-hydroxysuccinimide The initial monomer ([M]o) to CTA ([CTA]o) to initiator ([I]o) ratios for the
(NHS), (4.89 g, 0.0425 mol, Aldrich Prod. No. 130672) is added and two polymers is 25:1:0.1. One reaction solution contained a feed ratio
stirred for 30 min under N2. This solution is cooled to 0 °C, and the of 80% polyethylene glycol methacrylate 950 Da, 10% bioHEMA, and
reaction mixture is further stirred for 30 min. 10% PyrSMA. The second reaction solution contained a feed ratio of
N,N’-dicyclohexyl carbodimide (DCC), (9.57 g, 0.0464 mol, Aldrich Prod. 85% polyethylene glycol methacrylate 300 Da, 5% bioHEMA, and 10%
No. 379115) and a catalytic amount of 4-(dimethylamino)pyridine PyrSMA (Scheme 1A).
(66 mg, Aldrich Prod. No. 107700) are added, and the solution is Individual polymerization solutions are vortexed in 20 mL scintillation
stirred for 1 h at 0 °C. Reaction is allowed to continue for 22 h at room vials, transferred to septa-sealed 10 mL round bottom flasks, purged
temperature under N2. After the reaction is complete, precipitated under N2 for 30 min, and transferred to a preheated water bath at
dicyclohexylurea is filtered twice. 70 °C for 14 h. The resultant polymers are isolated by precipitation in
A solution of 2-(pyridyldithiol)-ethylamine hydrochloride (1.0 g, diethyl ether.
0.0045 mol) and Et3N (1.13 g, 0.0112 mol, Sigma-Aldrich Prod. The precipitated polymers are then dissolved in acetone and
No. T0886) is cooled to 0 °C and stirred for 30 min. re-precipitated into diethyl ether (×6). The dried polymers are analyzed
The previously synthesized NHS-activated SMA solution (2.2 g, by 1H NMR to assess purity. Gel-permeation chromatography (GPC)
0.0067 mol) in chloroform (65 mL) is then added dropwise for 1 h. can be used to determine number average (Mn) molecular weight and
The reaction mixture is stirred overnight (16 h) at room temperature polydispersity (PDI) of the polymers.
and then transferred to a separating funnel and washed with H2O (3 ×
Example GPC Protocol
150 mL). Organic extracts are then washed with brine and dried over
Na2SO4, filtered, and concentrated in vacuo to afford a crude oil which is Use Tosoh SEC TSK GEL α-3000 and α-4000 columns (Tosoh Bioscience,
purified by column chromatography [SiO2, EtOAc/hexane 3:1] to obtain Montgomeryville, PA) connected in series to a 1200 Series liquid
1.62 g pure product (yield=60.5%). chromatography system (Agilent, Santa Clara, CA) and a miniDAWN
TREOS three-angle light scattering instrument with an Optilab TrEX
Example refractive index detector (Wyatt Technology, Santa Barbara, CA). HPLC-
500 mhz 1H NMR in acetone D6: δ ppm 5.81 (CH2CCH3 trans, s, 1H), grade DMF containing 0.1 wt% LiBr at 60 °C is used as the mobile
6.11 (CH2CCH3 cis, s, 1H), 1.92 (CCCH3, s, 3H), 4.35 (OCH2CH2O), 3.50 phase at a flow rate of 1 mL/min. Reverse-phase high performance
(NHCH2, t, 2H), 2.99 (NHCH2CH2, t, 2H), 2.50 (OCH2CH2COCH2, t, 2H), liquid chromatography (RP-HPLC) can be used to measure monomer
2.61 (OCH2CH2COCH2, t, 2H), 8.48 (NCH, d, 1H), 7.81 (NCHCHCH, m, 2H), conversions for each macroCTA using aliquots collected at To and Tx.
7.47 (NH, b, 1H), 7.23 (CCH, d, 1H).

RAFT Polymeric Carriers for Antibody‑drug Conjugates of Biologic Drugs
A) Formulation of Antibody-Polymer-Peptide
R Z
Conjugates
PEGMA bioHEMA PyrSMA MacroCTA [1]
S Z
R
O O O HO
S
x y z Polymeric micelles are conjugated to peptides via disulfide exchange.
O O O O O O
O HN O O O
First, polymers are dissolved as unimers in ethanol (100 mg/mL),
O O O ABCVA DMSO O O O
then diluted 10× into DPBS, pH 7.4 to spontaneously form micelles.
O O
5 or 19 70 °C 14 h
5 or 19
O O Ethanol content is reduced to less than 0.01% using Amicon-4 Ultra
3k MWCO spin columns (Aldrich Prod. No. Z740186) and the polymer
O
HN
HN
O concentration is verified by absorbance at 290 ηm.
S
S
HN S
The number of pyridyl disulfde (PDS) groups per polymer chain is
HN
NH S
NH S
S
quantified by reducing PDS groups on the polymeric micelles with an
O N O N excess of Bond Breaker TCEP solution (Pierce). Conversion of PDS to
2-mercaptopyridine results in a colorimetric change that is quantified
by a 343 nm absorbance with an extinction coefficient of 8,080 M-1cm-1.
B) Next, BIM or BIM/LE peptide is dissolved in anhydrous dimethyl
DEAMA BMA R Z
sulfoxide (30 mM, Sigma-Aldrich Prod. No. 276855), added to the
S
R y
b Z polymer solutions and mixed vigorously by vortexing, followed by an
O O [1] HO x z a b
O O
S O O O O O overnight reaction at room temperature. In the final reaction mixture,
O HN O O O O O
the polymer concentration is 500 μM with a 1.25 molar excess of
N
ABCVA Dioxane O O O N
peptide, which results in one peptide bound per polymer chain.
70 °C 8 h
5 or 19
O O Reduction of the disulfide bond in the presence of glutathione can be
confirmed by HPLC purification.
O
HN The formation of micelles and the number average diameter before
S
and after peptide loading can be measured by dynamic light scattering
HN
NH S
S
following filtration through a 0.45 μm syringe filter with a polymer
O N concentration of 0.5 mg/mL. The ability of the biotin-containing
polymer micelles to complex with streptavidin and antibody/
Scheme 1. A)The diblock copolymer poly[(PEGMA-PyrSMA-bioHEMA)-(DEAEMABMA)] (O950) streptavidin conjugates can be quantified using a HABA assay.
was synthesized by RAFT with a multifunctional hydrophilic first block containing PEGMA950
for in vivo biocompatibility, a biotin-containing monomer (bioHEMA) for antibody/streptavidin
complexation, and a PDS-containing monomer (PyrSMA) for disulfide conjugation to a
cysteine containing peptide. B) The second block contains the DEAEMA monomer which
undergoes a pH-dependent phase transition and the BMA monomer for hydrophobic
Summary
interactions with membrane disruption. These RAFT polymeric carriers provide a straightforward route
to making functionally sophisticated drug delivery systems. In
RAFT Synthesis of Diblock Copolymers addition to intracellular delivery activities, additional functionalities
can be synthetically incorporated. This includes the incorporation
The two polymers isolated from the previous synthesis are used
of polyethylene glycol (PEG) or alternative chemistries to optimize
as the macroCTAs for RAFT copolymerization with ABCVA (Aldrich
circulatory and biodistribution properties. Drug conjugation and
Prod. No. 118168) as the initiator in 50 wt% monomer in 1,4-dioxane
targeting ligand chemistries can be incorporated via functionalized
(Scheme 1B). The [M]o:[mCTA]o:[I]o ratios for the polymers containing
monomers or through functionalized chain transfer agents that are
PEGMA 950 Da (Aldrich Prod. No. 447951) and PEGMA 300 Da (Aldrich
built into the polymer ends. Finally, the compositions can be optimized
Prod. No. 447935) are 200:1:0.2 and 125:1:0.2, respectively.
to solubilize drugs and protect more fragile drugs from enzymatic
The polymer solutions contain a relative feed of 60% diethylaminoethyl degradation in the blood and other tissues.
methacrylate (DEAEMA, Aldrich Prod. No. 234907) and 40% butyl
methacrylate (BMA, Aldrich Prod. No. 235865). References
(1) Hughson, F. M. Curr. Biol. 1995, 5, 265.
Individual polymerization solutions are vortexed in 20 mL scintillation (2) Bulmus, V., et al. J. Controlled Release 2003, 93,105.
vials, transferred to septa-sealed 5 mL round bottom flasks, purged (3) Manganiello, M. J. et al. Biomaterials 2012, 33, 2301–2309.
under N2 for 20 min, and transferred to a preheated water bath at (4) Duvall, C. L. et al. Molecular Pharmaceutics 2010, 7, 468.
70 °C for 8 h. The resultant polymers can be isolated by precipitation in (5) Palanca-Wessels, M. C. et al. Molecular Therapy 2011, 19, 1529.
(6) Wilson, J. T. et al. ACS Nano 2013.
petroleum ether. (7) Convertine, A. J. et al. J. Controlled Release 2009, 133, 221.
(8) Chiefari, J. et al. Macromolecules 1998, 31, 16, 5559.
The precipitated polymers are then dissolved in acetone and
(9) Lowe, A. B.; McCormick, C. L. Australian Journal of Chemistry 2002, 55, 367.
re-precipitated into petroleum ether (×5). The dried diblock copolymers (10) Boyer, C. et al. Chemical Reviews 2009, 109, 5402.
are analyzed by 1H NMR, GPC, and RP-HPLC as described above and in (11) Berguig, G. et al. Cell 2014, 157, 1644.
recent publications. (12) Berguig, G. et al. Molecular Therapy 2015, 23, 907.
(13) Polson, A. G. et al. Cancer Research 2009, 69, 2358–2364.
(14) Senter, P. D. Current Opinion in Chemical Biology 2009, 13, 235.
(15) Brown, A. A. et al. European Polymer Journal 2005, 41, 1757.
(16) Otsuka, H. et al. Advanced Drug Delivery Reviews 2012, 64, 246.
(17) Little, S. R.; Kohane, D. S. Journal of Materials Chemistry 2008, 18, 832.
(18) Roy, D. et al. Polym. Chem. 2014, 5, 1791.
(19) Guillaume, L. et al. Nature Reviews Drug Discovery 2008, 7, 989.

aldrich.com/matsci
PEG MacroCTAs
For more information on these products, visit aldrich.com/raftagent.
Poly(ethylene glycol) methyl ether (4-cyano-4-pentanoate O average Mn 1,400 ≤ 1.1 PDI 752487-1G
dodecyl trithiocarbonate) O S SCH2(CH2)10CH3 752487-5G
H3C O
average Mn 2,400 ≤ 1.1 PDI 751634-1G
n H3C CN S
751634-5G
average Mn 5,400 ≤ 1.1 PDI 751626-1G
751626-5G
Poly(ethylene glycol) methyl ether 4-cyano-4- O average Mn 10,000 ≤ 1.1 PDI 753033-1G
[(dodecylsulfanylthiocarbonyl)sulfanyl]pentanoate O S SCH2(CH2)10CH3
H3C O
n
H3C CN S
Poly(ethylene glycol) methyl ether O average Mn 1,100 ≤ 1.1 PDI 740705-1G

2-(dodecylthiocarbonothioylthio)-2-methylpropionate O S S average Mn 5,000 ≤ 1.1 PDI 736325-1G
H3C O n C12H25
CH3 CH3 S
Poly(ethylene glycol) methyl ether (2-methyl-2-propionic O average Mn 10,000 ≤ 1.1 PDI 752495-1G
acid dodecyl trithiocarbonate) O S SCH2(CH2)10CH3
H3C O
n
H3C CH3 S
Poly(ethylene glycol) 4-cyano-4-(phenylcarbonothioylthio) O average Mn 2,000 ≤ 1.1 PDI 764914-1G

pentanoate
O S
H3C O
n H3C CN S
Polylactide RAFT and ATRP Polymers

Poly(D,L-lactide), 4-cyano-4-[(dodecylsulfanylthiocarbonyl) average Mn 5000 792020-1G
O
sulfanyl]pentonate terminated average Mn 20,000 797790-1G
CH3(CH2)10CH2S S O
O H
S H3C CN CH3
n
Poly(L-lactide), 2-bromoisobutyryl terminated average Mn 3,000 773247-1G

O Br CH3
O O
H O CH3
CH3 O
n
Macro-RAFT
Poly(tert-butyl acrylate), DDMAT terminated O average Mn 7,000 < 1.2 PDI 772550-1G
S S
HO n
C12H25
H3C CH3 S
t-Bu
O O
Poly(tert-butyl acrylate), DDMAT terminated, azide terminated O average Mn 8,500 ≤ 1.2 PDI 776424-1G
S S
N3 O C12H25
n
H3C CH S
3
O O
H3C CH3
CH3
Poly(N,N-dimethylacrylamide), DDMAT terminated O average Mn 10,000 ≤ 1.1 PDI 773638-1G

S S
HO n
C12H25
H3C CH3 S
CH3
O N
CH3

Linear and Branched Polyethylenimines as Nonviral Vectors for Gene Delivery
LINEAR AND BRANCHED POLYETHYLENIMINES

AS NONVIRAL VECTORS FOR GENE DELIVERY
toxicity.1 The cytotoxicity of PEIs is the result of the high positive charge
density within the polymer chains that can lead to destabilization of
the cell wall and cellular necrosis. The overall cytotoxicity of the PEI
vectors can be modulated by adjusting the nitrogen-to-phosphate
(N/P) ratio of the nucleotide/PEI complex.6 The preferred molecular
weight for PEI for gene delivery is estimated to be in the range of
5,000–25,000 Da, whereas lower molecular weight PEI transfection
efficiency can outperform the higher molecular weight counterparts
Philip Dimitrov, Nicolynn Davis only if higher N/P ratios are used.7
Aldrich Materials Science R&D
6000 North Teutonia Avenue, Milwaukee, WI 53209 USA Branched PEIs possess higher charge density and carry approximately
Email: philip.dimitrov@sial.com
equal amounts of primary, secondary and tertiary amino groups. Linear
PEI contains only secondary amino groups and therefore linear PEIs are
Introduction somewhat less cytotoxic.8 In general, primary amines condense DNA
better than other amines due to their higher protonation. In addition,
Gene therapy, the delivery of DNA or RNA into cells, has gained
binding capacity is correlated to the number of primary amines, and
momentum over the past few decades as a potent tool for treatment
complex stability increases with increasing primary amine content,
of genetic disorders and cancer. A significant obstacle to gene therapy
leading to higher transfection efficiencies. Linear PEI is a particularly
is developing effective carriers that protect the DNA or RNA from
effective gene transfer agent. It has lower complexation capability
serum nuclease degradation, facilitate cellular uptake, and enable
than branched PEI toward nucleotides, but at the same time linear
transfer of the cargo into the nucleus or cytoplasm. Nonviral vector
PEI is more efficient than branched PEI due to its topology and lower
systems, based on cationic lipids, dendrimers, peptides, and polymers,1
cytotoxicity as demonstrated in a number of in vivo studies.9
are currently preferred for gene delivery because they are much
safer than viral systems,2 which are burdened by immunogenic or The design of the PEI polymeric carrier can be challenging since
inflammatory responses. transfection efficiency and cytotoxicity depend on the physiochemcial
properties of the polymer. In addition, PEI can be used for different
Polyethylenimine (PEI) is a cationic polymer widely adopted in
gene therapy applications as a carrier for plasmids, oligonucleotides, or
nonviral gene delivery systems both in vitro and in vivo due to its high
siRNA. In general, molecular weight and branching need to be adjusted
transfection efficiency.3 PEI is capable of condensing plasmid DNA
depending on application to design stable complexes with the desired
and RNA through electrostatic interaction to form complexes that are
release rates.
internalized into cells through endocytosis. This ability is attributed to
the “proton sponge” characteristic of PEI, in which the single nitrogen
per monomer subunit in PEI forms an ionic interaction with the
phosphate backbone of nucleic acids. Thus, DNA or RNA complexes
Methods: PEI Solutions and Complexes
(or polyplexes) are readily formed through mixing with PEI.4 These PEI
complexes result in increased transfection efficiency due to the large Linear PEI
buffering capacity of PEI, leading to changes in endosomal osmolarity, Linear PEI is not directly soluble in water at ambient temperature. To
and resulting in lysis and release (endosomal escape). PEI-based vectors make a PEI solution based on monomer units (7.5 ×10–3 M), linear PEI
have been used to deliver oligonucleotides, plasmid DNA, as well as (0.323 g/L) is added to endotoxin-free de-ionized H2O and stirred for
RNA and intact ribozymes.5 approximately 1 h on low heat until all of the particles are in solution.
The volume is then adjusted with de-ionized H2O to achieve the
The molecular weight, structure, and branching of PEI can each desired concentration. Allow the solution to cool to room temperature,
influence the condensation behavior, complex size, and transfection neutralize to the desired pH, and sterile filter (0.22 µM). A large PEI stock
efficiency of the vector. For example, the transfection efficiency of PEI solution can be prepared and stored frozen at −20 °C. Aliquots of linear
increases by increasing the molecular weight of the polymer. Higher PEI can be thawed and stored at 4 °C while in use. Aliquots of linear PEI
molecular weight PEI generally results in a decreased complex size. may be adjusted to pH 7.0, 8.0, and 9.0 for testing the effect of the pH
However, higher molecular weight PEI polymers also lead to higher of the PEI solution on the transfection.10

aldrich.com/matsci
Branched PEI Solutions References

(1) Mintzer, M. A.; Simanek, E. E. Chem. Rev. 2009, 109, 259–302.
Branched PEI is an extremely viscous liquid and cannot be pipetted; (2) Karmali, P. P.; Chaudhuri, A. Medicinal Research Reviews 2007, 27 (5) 696–722.
therefore, stock solutions of branched PEI should be made in water, first (3) Gebhart, C. L.; Kabanov, A. V. J. Controlled Release 2001, 73, 401–416.
at high concentrations (100 g/L). Once the solution is homogeneous, (4) Jager, M.; Schubert, S.; Ochrimenko, S.; Fischer, D.; Schubert, U. Chem. Soc. Rev. 2012, 41,
4755–4767
it is further diluted to 1 mg/mL, the pH is adjusted to 7 with HCl, then (5) Aigner, A.; Fischer, D.; Merdan, T.; Brus, C.; Kissel, T.; Czubayko, F. Gene Ther. 2002, 9,
filter sterilized (0.22 µm filter) and aliquoted. Aliquots are stored frozen 1700–1707.
at –20 °C, and working solutions can be kept at 4 °C for long periods of (6) Erbacher, P.; Bettinger, T.; Brion, E.; Coll, J.-L.; Plank, C.; Behr, J.-P.; Remy, J.-S. Journal of Drug
time (months) without any loss in transfection efficiency.11 Targeting 2004, 12 (4), 223–236.
(7) Kunath, K.; von Harpe, A.; Fischer, D.; Petersen, H.; Bickel, U.; Voigt, K.; Kissel, T. J. Controlled
Release 2003, 89, 113–125.
Formation of PEI/Polynucleotide Complexes (8) von Harpe, A.; Petersen, H.; Li, Y.; Kissel, T. J. Controlled Release 2000, 69, 309–322.
(9) Lungwitz, U.; Breunig, M.; Blunk, T.; Gopferich, A. European Journal of Pharmaceutics and
The size of the complexes depends on the PEI-to-DNA ratio, often Biopharmaceutics, 2005, 60, 247–266.
referred to as the N/P ratio. The dimensions of the polyplexes can (10) Reed, S. E.; Staley, E. M.; Mayginnes, J. P.; Pintel, D. J.; Tullis, G. E. Journal of Virological
range from 50 to >1,000 nm depending on protocol conditions (i.e., Methods 2006, 138, 85–98.
N/P ratios) and whether the PEI is branched or linear.12,13 In general, N/P (11) Aricescu, A. R.; Lu, W.; Jones, E. Y. Acta Cryst. 2006, D62, 1243–1250.
(12) Goula, D.; Benoist, C.; Mantero, S.; Merlo, G.; Levi, G.; Demeneix, B. A. Gene Ther. 1998 5,
ratios of 2:1 to 20:1 are used to achieve stable complexes using linear or 1291–1295.
branched PEI. Note the sequence of addition (adding PEI to DNA) can (13) Wightman, L.; Kircheis, R.; Rossler, V.; Carotta, S.; Ruzicka, R.; Kursa, M.; Wagner, E. J. Gene
influence polyplex size and transfection efficiency. Med. 2001, 3, 362–372.
1. Allow stock reagents to warm to room temperature.

2. Ensure cells for transfection are at the desired confluency (70–90%)
to improve transfection efficiency. Replace growth medium with
low serum media (<2% serum).
3. Prepare diluted PEI and plasmid separately to the desired
concentrations (in endotoxin-free water, 150 mM saline, or phenol-
free growth medium), vortex and leave at room temperature for
10–15 min.
For a standard 15 cm tissue culture dish (approximate surface area
of 176 cm2), 50 µg of plasmid DNA are required (quantities need to
be scaled accordingly for the different surface areas of plates/flasks/
bottles). The DNA is added to 5 mL serum-free medium, and mixing
is followed by the addition of 75 µL PEI stock (1 µg/mL) and brief
vortexing.
4. Slowly add diluted PEI (1 µg/mL) to the DNA solution to achieve
the desired N/P ratio, vortex, and incubate at room temperature for
another 15 min to allow DNA/PEI complex formation.
5. Add DNA/PEI mixture to cells and briefly rotate the dish to allow
mixing. Allow transfection to occur in an incubator for 3–24 h.
6. Remove transfection complexes by washing cells with PBS. Add
growth medium and assay transfection efficiency.

Linear and Branched Polyethylenimines as Nonviral Vectors for Gene Delivery
Linear PEI
For more information on these products, visit aldrich.com/pei.
Polyethylenimine, linear H3C OH average Mn 2,500 < 1.2 PDI 764604-1G
N
H average Mn 5,000 < 1.2 PDI 764582-1G
n
average Mn 10,000 ≤ 1.2 PDI 765090-1G
Polyethylenimine hydrochloride average Mn 4,000 ≤ 1.1 PDI 764892-1G
H3C OH • xHCl 764892-5G
N
H
n average Mn 8,000 ≤ 1.1 PDI 764647-1G
average Mn 20,000 ≤ 1.2 PDI 764965-1G
Branched PEI
Polyethylenimine, ethylenediamine branched NH2 average Mn ~600 by GPC 408719-100ML
NH2 N 408719-250ML
H H
N N N 408719-1L
H2N N N NH2
Polyethylenimine, branched H average Mn ~10,000 by GPC 408727-100ML
408727-250ML
N 408727-1L
H 2N NH2
n
Polyphosphoesters for Gene Delivery

Name Structure Prod. No.
Poly[1,4-bis(hydroxyethyl)terephthalate-alt- O O 659738-1G
ethyloxyphosphate]-co-1,4-bis(hydroxyethyl) O
terephthalate-co-terephthalate O O P O O
O H3C O O
O O
O O
x
O
y
Poly[(lactide-co-ethylene glycol)-co- O CH3 O CH3 659606-1G

ethyloxyphosphate] O
O O O
O O O P
CH3 O H3C O O
x y
CH3
n
Poly[1,4-bis(hydroxyethyl)terephthalate-alt- O O 659614-1G
ethyloxyphosphate] O
O O P O
O H3C O O
O OH
O O
n

aldrich.com/matsci
METHOD INDEX
Micelle Drug Loading 4 Formulation of PEG-gelatin IPN Hydrogel
Characterization of Toxicity with Spheroids 5 Without UV Polymerization 32
Colloidal Carrier Fabrication 9 Protein PEGylation 39
One-step Synthesis of LPNs 15 Block Copolymerization of 2-Oxazolines 44
PNIPAM Drug Delivery Systems 24 Dendritic Carriers 49
Fabrication of Theragrippers 30 Diblock Copolymer Biologic Drug Carriers 54
Formulation of PEG-gelatin IPN Hydrogel PEI Solutions and Complexes 57
Using UV Polymerization 32
TRADEMARK INDEX
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Amgen Inc. — Neulasta® Hoffmann-La Roche Inc. — Pegasys®
BASF — Pluronic® Merck Sharp & Dohme Corp — PegIntron®
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Bristol-Myers Squibb Co. — Taxol® Roche — Micera™
Celgene Corporation — Abraxane® Sigma-Aldrich Co. LLC — Aldrich®, Material Matters™, Sigma®,
Crealta Pharmaceuticals LLC — Krystexxa® Sigma-Aldrich®
Croda International PLC — TWEEN® Sigma-Tau Rare Disease Ltd — Adagen®, Oncaspar®
Evonik Industries AG — RESOMER® Teva Pharmaceutical Industries Ltd. — Lonquex™
Expedeon Protein Solutions — InstantBlue™

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POLYMER SYNTHESIS
Aldrich® Materials Science has established a
Center of Excellence in Polymer Science with
expertise in the research and development of
custom polymers and monomers.
We can serve you from research through
development to commercial-scale materials
manufacturing. Polymers for biomedical
applications include:
Monomers, crosslinkers, and functional
yy
polymers for drug delivery
Polymers with controlled biomedical
yy
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Biocompatible polymers for multilayer films
yy
on medical device surfaces
Monomers and polymers for dental or
yy
ophthalmic applications
For more information on capabilities

or to request a quote, contact us at
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TRANSLATE DISCOVERIES INTO
THERAPEUTICS
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your drug discovery and drug delivery research
• Biodegradable Polymers: polylactide and polyglycolide TRANSLATIONAL RESEARCH SOLUTIONS

(PLA, PGA, PLGA), RESOMER®, and chitosan
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POLYMERIC

Preface Solubility Enhancement

Controlled Release Drug

Table 1. Advantages and limitations of drug delivery systems.

TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

Drug Delivery Material Choices Table of Contents

Biodegradable micelles ✔ Hydrogels for Drug Delivery

About This Guide Poly(2-oxazoline)s for Drug Delivery

For questions, product data, or new product suggestions, contact us at matsci@sial.com. 1

To access the complete portfolio, visit

POLYMER MICELLES FOR DRUG DELIVERY

For questions, product data, or new product suggestions, contact us at matsci@sial.com. 3

Figure 1. Techniques for drug loading of micelles

4 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

For questions, product data, or new product suggestions, contact us at matsci@sial.com. 5

Poly(D,L-lactide-block-acrylic acid) OH PAA average Mn 18,000 - 798126-1G

Poly(L-lactide)-block-poly(ethylene O H3C PEG average Mn 5,000 - 570281-250MG

Poly(ethylene glycol)-block- O PEG average Mn 350 - 659665-1G

6 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

Name Structure Molecular Weight/Viscosity Degradation Time Prod. No.

Poly(ethylene glycol)-block-poly(ε− O PCL average Mn 5,000 >12 months 570303-250MG

Poly(lactide-co-caprolactone)- PEG average Mn 5,000 1-2 months 764833-1G

For questions, product data, or new product suggestions, contact us at matsci@sial.com. 7

Controlled Targeted Solubility

BIODEGRADABLE COLLOIDAL CARRIERS

Introduction yy The molecular weight or inherent viscosity of the PLGA polymer,

8 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

Microgels and Nanogels In a typical nanoprecipitation process, a polymer (e.g., PLGA)

For questions, product data, or new product suggestions, contact us at matsci@sial.com. 9

10 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

For questions, product data, or new product suggestions, contact us at matsci@sial.com. 11

Low PDI Polylactides

Poly(D,L-lactide) O average Mn 5,000 ≤ 1.1 PDI <6 months 764612-5G

End-functionalized Low PDI Poly(l-lactide)s

Poly(L-lactide), amine terminated O average Mn 2,500 ≤ 1.3 PDI 776378-1G

Poly(L-lactide), azide terminated O average Mn 5,000 < 1.2 PDI 774146-1G

Poly(L-lactide) N-2-hydroxyethylmaleimide O O average Mn 5000 < 1.2 PDI 746517-1G

Poly(L-lactide) 2-hydroxyethyl, methacrylate CH3 O average Mn 2,000 ≤ 1.1 PDI 771473-1G

Poly(L-lactide), propargyl terminated average Mn 2,000 ≤ 1.1 PDI 774162-1G

Poly(L-lactide), thiol terminated CH3 average Mn 2,500 ≤ 1.2 PDI 747386-1G

Polylactide Block Copoymers

12 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

Learn more about preformed particles and applications,

Degradex® particles are products of Phosphorex Inc.

Controlled Targeted Solubility

LIPID-POLYMER HYBRID NANOPARTICLES

14 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.

Method: One-step Synthesis of LPNs

For questions, product data, or new product suggestions, contact us at matsci@sial.com. 15

Lipids for Drug Delivery

1,2-Dilinoleoyl-sn-glycero-3-phosphocholine O ≥99%, TLC P0537-5MG

1,2-Dioleoyl-sn-glycero-3-phosphocholine CH2(CH2)6CH3 - P6354-25MG

1,2-Dipalmitoyl-sn-glycero-3-phosphocholine, O CH3 ≥99% P0763-50MG

1,2-Distearoyl-sn-glycero-3-phosphoethanolamine O O ≥99% P3531-100MG

DOTAP chloride O ≥98%, TLC D6182-50MG

Isopropyl myristate O CH3 ≥90%, GC M0757-250ML

L-α-Lysophosphatidylcholine - ≥99% L4129-25MG

16 TO ORDER: Contact your local Sigma-Aldrich office or visit aldrich.com/matsci.