CA2445125A1 - Microemulsions as precursors to solid nanoparticles - Google Patents

Abstract

The preparation of novel microemulsions to be used as precursors for solid nanoparticles is described. The microemulsion precursors consist of either alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions. The formed solid nanoparticles have diameters below 200 nanometers and can be made to entrap various materials including drugs, magnets, and sensors. The solid nanoparticles can be made to target different cells in the body by the inclusion of a cell-specific targeting ligand. Methods of preparing the nove l microemulsion precursors and methods to cure solid nanoparticles are provide d.

Description

DESCRIPTION
MICROEMULSIONS AS PRECURSORS TO SOLID NANOPARTICLES
BACKGROUND OF THE INVENTION
Field of the Invention The invention relates to making targeted nanoparticle delivery systems for drugs, magnets, 1 o and sensors. The invention relates to the preparation of microemulsion precursors whereby the dispersed droplets are templates for the curing of solid nanoparticles. The invention also relates to making solid nanoparticles from microemulsion precursors without the use of light, electricity, free radicals, or y-rays to make nanoparticles. The invention also relates to a nanoparticulate delivery system for delivering a molecule of interest to the body.
Brief Description of the Related Art Nanotechnology is becoming increasingly more important in the pharmaceutical, chemical and engineering fields. This is primarily due to the fact that particles made at the nanoscale have much different physical, chemical, and biological properties than larger particles. For example, in 2 o the pharmaceutical field, nanoparticles have been used to more efficiently deliver drugs, genes, diagnostics, and vaccines (Douglas et al., 1987; MacLaughlin, et al., 1998;
Kreuter 1995). Due to their small size, nanoparticles can aid in the direct entry of entrapped molecules into cells (either non-specifically or specifically via cell targeting ligands). Cellular uptake of drug molecules is often desirable and even necessary if the mechanism of action of the drug requires it to be in the cell as in the case of larger biologically-based molecules such as antisense oligonucleotides, ribozymes, and plasmid DNA. Further, the benefit of being able to deliver a vaccine antigen intxacellularly to achieve a cellular-based immune response has been established (for a review see Mumper et al., 1999). However, getting these larger molecules efficiently into cells is difficult.
Unlike small drugs, which may efficiently enter cells by diffusion and/or transport mechaiusms, large molecules often require a carrier system to achieve sufficiently high intracellular concentrations. Nanoparticles may provide a way of increasing the cellular uptake of larger molecules if these molecules can be efficiently packaged into pharmaceutically acceptable Garners using a cost-effective method.
Gene therapy has emerged as a promising approach for the treatment of a munber of genetic s and acquired diseases. Non-viral gene therapy involves the delivery of genetic material (plasmid DNA) into cells of the body to produce therapeutic proteins endogenously by exploiting the cell's transcriptional and translational machinery. Most non-viral gene delivery strategies employ polyelectrolyte complexation using cationic lipids, peptides, or polymers to complex and condense negatively charged plasmid DNA into particles having diameters in the 100-1000 nm range. The Z o complexation strategy is fraught with problems since: i) the cationic molecules are relatively. toxic materials and are not approved by the FDA in any maxketed medical product, ii) the complexes are prone to aggregation at or near charge neutrality, iii) stable particles having diameters below 100 nm are very difficult to engineer, iv) scale-up of these complexes is complicated and expensive since very controlled mixing systems are needed to introduce the ions in solution, and v) the .
15 complexes tend to aggregate or dissociate when injected in the body.
Although a few reports in the literature have demonstrated proof of concept in animals, ;
attempts to specifically target polyelectrolyte complexes to cells in the body using cell-specific ligands have been largely unsuccessful. Also, no such technology has advanced to clinical testing in humans. Contributing factors to this lack of success may be that these ligands (i.e., monoclonal 2o antibodies, carbohydrates, etc.) are attached to biologically unstable particles and/or that the.these particles cannot be made small enough to be efficiently taken up by cells by receptor-mediated endocytosis.
As an alternative to polyelectrolyte complexation, researchers have also attempted to encapsulate plasmid DNA into conventional solid nanoparticles based on biodegradable polymers 25 such as polylactic acid-co-glycolic acid (Ando et al., 1999; Wang et al., 1999), gelatin (Truong-Le, et al., 1998), and other polymers (Mumper and Klakamp, 1999). However, these techniques and systems have disadvantages such as: a) the relatively high cost of these carrier materials, b) the unknown safety of some of these materials, c) the use of rigorous processes typically used to make the nanoparticles (i.e., interfacial polymerization andlor high-torque mechanical mixing that may be damaging to biologically-based drugs and expensive to scale-up and manufacture), d) the inability to produce nanoparticles below 50 nm, and e) the low encapsulation efficiency of plasmid DNA.
Yet another alternative to polyelectrolyte complexation is to incorporate plasmid DNA into microemulsions. A microemulsion is a stable biphasic mixture of two immiscible liquids stabilized by a surfactant and usually a co-surfactant. Microemulsions are thermodynamically stable, isotropically clear, form without excessive mixing, and have dispersed droplets in the range of 5 nm to 100 nm diameter. Microemulsions have been proposed as drug delivery systems to enhance the absorption of drugs across biological membranes (Bhargava et al. 1987; Ho et al. 1996;
Constantinides, 1995). Although microemulsions have advantages as delivery systems, they do 1 o have important limitations. For example, the dispersed droplets are a liquid and are not stable in biological fluids. Thus, microemulsions are not effective in delivering drugs intracellularly or targeting drugs to different cells in the body.
A significant advancement in the field of non-viral gene delivery would be made if one could avoid the problems associated with polyelectrolyte complexation and instead combine the unique advantages of solid nanoparticles and microemulsions into one pharmaceutically engineered gene delivery system.
Finally, there have been a handful of reports pertaining to the use of microemulsions to make nanoparticles (Li et al., 1999; Cavalli et al., 1999; Bocca et al., 1998;
Tojo et al., 1998;
Nlunshi et al., 1997; Ruys et al., 1999). These reports have primarily dealt with the preparation of 2 o water-in-oil (hydrocarbon) microemulsions (Lade et al., 2000; Song et al., 2000; Porta et al., 1999) whereby nanoparticles are formed in the water phase by the use of photochemistry (Agostiano et al., 2000), y-rays (Niangling et al., 1999), or electrochemistry (Tang et al., 2000) to induce crosslinking, polymerization (Fang et al., 2000; Capek, 1999; Meier, 1999) and/or complexation of the appropriate agents in the water phase. The great majority of these reports do not use 2 5 pharmaceutically acceptable materials or methods of preparation that would be suitable for scale-up and preparation of nanoparticles containing drugs, magnets, or sensors that are intended for use in humans.
U.S. Patent No. 4,826,689 to Violanto, discloses methods of making uniformly sized particles of less than 10 microns from water-insoluble drugs by precipitation.
Although Violanto 3 o discloses a method of making drug particles by precipitation, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions as precursors to prepare solid nanoparticles containing drug.
U.S. Patent No. 4,997,599 to Steiner, discloses the preparation of cellulose acetate microspheres having a size of less than 1 micron to a maximum of 1000 microns by spraying a polymer solution through a nozzle. Although Steiner discloses the use of a film-forming cellulose polymer, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions as precursors to prepare solid nanoparticles containing drug.
to U.S. Patent No. 5,049,322 to Devissaguet discloses a process of preparing a colloidal system containing nanocapsules of less than about 500 nanometers. The patent reports that the colloidal system of nanocapsules forms practically instantaneously with gentle agitation. The wall of the nanoparticles is reported to be preferably formed of a ftlm forming polymer, e.g., cellulose, and the core may be a biologically active substance. Although the patent describes 15 nano-sized products, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions as precursors to prepare solid nanoparticles containing drug.
U.S. Patent S,S00,224 to Vranckx describes pharmaceutical compositions containing nanocapsules. The nanocapsules are prepared by adding an aqueous solution containing an 20 active ingredient to an oil to form a water-in-oil emulsion and removing the nanocapsules having a size of less than 500 nanometers. Although the patent describes nano-sized products, the patent does not disclose alcohol-in-fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions as precursors to prepare solid nanoparticles containing drug.
25 U.S. Patent No. 5,733,526 to Trevino discloses hydrocarbon oillfluorochemical preparations which may be used for the administration of bioactive agents. It is reported that the hydrocarbon oil, e.g., paraffin or vegetable oil, is preferably dispersed in a continuous fluorochemical phase. In an embodiment, the patent discloses a hydrocarbon oil-fluorochemical disperse phase in a continuous polar liquid. Trevino does not appear to disclose alcohol-in-3o fluorocarbon microemulsions, liquid hydrocarbon-in-fluorocarbon microemulsions, or liquid hydrocarbon-in-water microemulsions wherein the dispersed alcohol or liquid hydrocarbon phases contain a film-forming substance dissolved or dispersed therein.
Further, the patent does not disclose the use of such microemulsions to prepare solid nanoparticles containing drug.
United States Patent 5,250,236 by Gasco describes the use of solid lipid microspheres that are formed by diluting one volume of the mixture of molten lipid substance, water, surfactant and possibly a co-surfactant to 100 volumes of cold water. Gasco teaches the preparation of rnicrospheres smaller than one micrometer and in particular between 50-800 nanometers, and preferably between 100 and 400 nanometers. Gasco also teaches the preparation of microspheres wherein said solid lipid microspheres may contain a 1o pharmacologically active substance, such as a drug. Gasco does not teach the use of nanoparticles made from oil-in-water 'microemulsion precursors wherein said nanoparticles containing drugs are formed directly by cooling the oil-in-water microemulsion with no dilution of the most useful system.
United States Patent 5,510,118 by Bosch teaches a process of preparing nanoparticulate drug substances comprising the steps of: preparing a premix of the drug substance and a surface modifier, and subjecting the premix to mechanical means such as shear, impact, and attrition to reduce the particle size of the dmg substance. Bosch does not teach the method of preparing nanoparticles made without mechanical means or the formation of microemulsion precL~xsors wherein said nanoparticles are formed directly by cooling the microemulsion with no dilution of the most useful system.
United States Patent 6,177,103 by Pace describes a process to stabilize microparticulate suspensions (having diameters up to 2000 nm) with surface modifier molecules by rapid expansion into an aqueous medium from a compressed solution of the compound and surface rnodif ers in a liquefied gas and optionally homogenizing the aqueous suspension thus formed with a high pressiu-e homogenizes. Bosch does not teach the method of preparing nanoparticles less than 300 nanometers made by the formation of microemulsion precursors wherein said nanoparticles are formed directly by cooling the microemulsion with no dilution of the most useful system.
United States Patent 6,238,694 by Gasco teaches a method to produce nanoparticles less 3o than 1 micron by heating a lipidic substance at a temperature at least equal to its melting point, heating a mixture comprising water, a surfactant and a co-surfactant at a temperature at least equal to the melting point of the lipidic substance and combining with the lipidic substance, obtaining a microemulsion, and then dispersing the microemulsion in 1 to 10 volumes of cold water to form solid nanoparticles. Gasco does not teach the use of nanoparticles made from oil-s in-water microemulsion precursors wherein said nanoparticles are formed directly by cooling the oil-in-water microemulsion with no dilution of the most useful system.
Conventional microemulsions are water-in-oiI type, and use various methods of curing the nanoparticles (i.e., crosslinking, polymerization, radiation, and so on).
There is a need in the art to provide a non water-in-oil type microemulsions using curing methods specific to those non water-in-oil microemulsions, such as by cooling and evaporation or by the addition of a solvent, to prepare solid nanoparticles containing drug or other molecules of interest. An additional advantage of this invention over prior art is that the described nanoparticle systems can be engineered rapidly;
reproducibly, and cost-effectively from the microemulsion precursors in a one-step process and contained in one manufacturing vessel, vial, or container.
l5 SUlYIMARY OF THE INVENTION
The present invention has met the hereinbefore described need.
The present application discloses a method for incorporating a molecule of interest into microemulsion precursors and subsequently engineering stable solid nanoparticles (about 5-300 2o nm) containing the molecule of interest from the microemulsion precursor.
The molecule of interest may be a drug molecule (such as plasmid DNA, a peptide, a protein, a small drug molecule, a food, a magnet or a sensor molecule). The molecule of interest may be physically contained in the nanoparticle or adsorbed onto the surface of the nanoparticle. .The microemulsion precursors may be either an ethanol-in-fluorocarbon microemulsion, a liquid hydrocarbon-in-fluorocarbon 25 microemulsion, or a liquid hydrocarbon-in-water microemulsion wherein filin-forming substance is initially contained in the dispersed phase (ethanol or liquid hydrocarbon).
Solid nanoparticles with the entrapped molecule of interest are made from the microemulsion precursors by a simple curing process. For all processes, the film-forming substance cures to form solid nanoparticles containing the molecule of interest. The present application also discloses characterizing the solid 3 o nanoparticles (i.e., size, surface charge and porosity, drug release and stability) and demonstrates that the solid nanoparticles are stable in biological fluids. The present application also discloses incorporating a~ targeting ligand such as asialofetuin or mannan onto the surface of the solid nanoparticles for targeting of the solid nanoparticles to specific cells of the body such as liver hepatocytes, macrophages, or dendritic cells. The present application also discloses methods to use solid nanoparticles to delivery macromolecules such as plasmid DNA more efficiently in-vivo to result in more robust immune responses.
These and other objects of the invention will be more fully understood from the following description of the invention, the referenced drawings attached hereto and the claims appended hereto.
$RIEF DESCRIPTION OF THE DRAWINGS
The present invention will become more fully understood from the detailed description given herein below, and the accompanying drawings which are given by way of illustration only, and thus are not limitative of the present invention:
Figure 1 A diagram of a method to engineer solid nanoparticles from microemulsion precursors.
Figvire 2 Solubilized ethanol in ethanol-in-fluorocarbon (E/F) microemulsions as a function 2 0 of fluorosurfactant (PDFOA, pentadecafluorooctanoic acid) content.
Figure 3 Particle size of cured emulsifying wax nanoparticles (2 mg/mL) made from oil-in-water microemulsion precursors as a function of final surfactant (Brij 78, polyoxyl 20 stearyl ether) concentration in the microemulsion precursors.
Figure 4 Stability of cured emulsifying wax nanoparticles (2 mg/mL) as a function of final surfactant (Brij 78, polyoxyl 20 stearyl ether) concentration in the microemulsion precursors.
Figure S The effect of three different curing methods of oil-in-water microemulsion precursors on the resulting particle size of Brij 72 nanoparticles made with Tween 80 as the surfactant. Method A) cooling of the undiluted oil-in-water microemulsion at 55°C to room temperature while stirring, Method B) cooling of the oil-in-water microemulsion at 55°C by placing undiluted in a refrigerator at 4°C, and Method C) diluting (1/10) the oil-in-wafer microemulsion at 55°C with water at 4°C.
Figm-e 6 The effect of three different curing methods of oil-in-water microemulsion precursors on the resulting particle size of emulsifying wax nanoparticles made to with Brij 78 as the surfactant. Method A) cooling of the undiluted oil-in-water microemulsion at 55°C to room temperature while stim-ing, Method B) cooling of the oil-in-water microemulsion at 55°C by placing undiluted in a refrigerator at ~.°C, and Method C) diluting (1/10) the oil-in-water microemulsion at 55°C with water at 4°C.
l5 Figure 7 The stability of Brij 72 nanoparticles challenged with different media at 37°C:
(~) water, (~) 10% FBS (~) 10 mM PBS, (O) 150 mM NaCl, (o) 10% lactose.
Figure 8 Particle size of cured emulsifying wax nanoparticles coated with different 20 amoLxnts of a hepatocyte-specific ligand, asialofetuin-palmitate (ASF-pal).
Figure 9 Entrapment of Gadolinium Acetylactetonate (GdAcAc) in both emulsifying wax and Brij 72 nanoparticles.
25 Figure 10 Entrapment efficiency of Gadolinium Acetylactetonate (GdAcAc) in emulsifying wax nanoparticles as determined by gel permeation chromatography (GPC) elution profiles. Nanoparticles were detected by laser light scattering (counts per second, CPS) and entrapped GdAcAc was detected by absorbance at 288 nm.
_g_ Figure 11 Particle size analysis of warm o/w microemulsion precursors at 55°C and cured (cooled) emulsifying wax nanoparticles at 25°C. The cationic nanoparticles were made using hexadecyltrimethylammonium bromide (CTAB) as the surfactant.
Figure 12 Antigen-specific IgG levels in sera to expressed (3-galactosidase 28 days after the administration of pDNA-coated nanoparticles ("nano") and 'naked' pDNA to Balb/C mice by three routes (intramuscular, "i.m."; subcutaneous, "s.c."., and topically to skin, "skin"). See Example 14 for more details. Mice (n= 5/group) were immunized with 40 p,g pDNA on day 0, 7, and 14. IgG titers are the mean +
standard deviation of all mice.
Figure 13 Antigen-specific IgG levels in sera to expressed ~3-galactosidase 28 days after the administration of either net positively-charged pDNA-coated nanoparticles ("+"), net negatively-charged pDNA-coated nanoparticles ("-"), or 'naked' pDNA to Balb/C mice by three routes (intramuscular, "i.m."; subcutaneous, "s.c.", and topically to skin, "skin"). See Example 15 for more details. Mice (n=4/group) were immunized with 4 ~.g pDNA on day 0, 7, and 14. Results are expressed as the individual titer for each mouse.
2o Figure 14 Secretion of Interleukin-2 (IL-2) from isolated splenocytes (1 x I06 cells) from immunized BalblC mice after ih-vitro exposure to (3-galactosidase protein for hours at a concentration of either 10 ~,g/mL or 100 p,g/mL. Mice were immunized with either net positively-charged pDNA-coated nanoparticles ("+"), net negatively-charged pDNA-coated nanoparticles ("-"), or 'naked' DNA to Balb/C
by three routes (intramuscular, "i.m."; subcutaneous, "s.c."., and topically to skin, "skin"). See Example 15 for more details. Mice were immunized with 4 ~.g pDNA on day 0, 7, and 14. Results are expressed as the mean IL-2 levels from pooled splenocytes harvested on day 28.
_g_ Figure 15 Secretion of Interferon-y (INF-y) from isolated splenocytes (1 x 10g cells) from immunized Balb/C mice after in-vitro exposure to (3-galactosidase protein for hours at a concentration of either 10 p,g/mL or 100 p,g/mL. Mice were immunized with either net positively-charged pDNA-coated nanoparticles ("+"), net wegatively-charged pDNA-coated nanoparticles ("-"), or 'naked' DNA to Balb/C .
by three routes (intramuscular, "i.m."; subcutaneous, "s.c."., and topically to skin, "skin"). See Example 15 for more details. Mice were immunized with 4 ~g pDNA on day 0, 7, and 14. Results are expressed as the mean INF-y levels from pooled splenocytes harvested on day 28.
to Figure 16 Secretion of Interleukin-4 (IL-4) from isolated splenocytes (1 x 106 cells) from , immunized Balb/C mice after in-vitro exposure to (3-galactosidase protein for 60 , hours at a concentration of either 10 p.g/mL or 100 p.g/mL. Mice were immunized ' with either net positively-charged pDNA-coated nanopaxticles (''+"), net negatively-charged pDNA-coated nanoparticles ("-"), or 'naked' DNA to Balb/C
by three routes (intramuscular, "i.m."; subcutaneous, "s.c."., and topically to skin, "skin"). See Example 15 for more details. Mice were immunized with 4 yg pDNA on day 0, 7, and 14. Results are expressed as the mean IL-~. levels from pooled splenocytes harvested on day 28.
Figure 17 Verification of Ligand-Coated Nanoparticles. Nanoparticles were coated with cholesterol-mannan as described in Example 16. Mannan-coated nanoparticles were purified by GPC to remove unincorporated or free chol-mannan. Various samples were added to Concanavalin A (Con-A; 1 mg/mL) and the increase in turbidity at 360 nm was measured for 200 seconds. A "mannan negative control"
Was taken from the same fraction that nanoparticles normally elute from the GPC
column (fraction 2-4). This confirmed that the positive agglutination results from mannan-coated emulsifying wax nanoparticles were not caused by co-elution of nanoparticles with unincorporated chol-mannan.
-lo-Figure 18 . Antigen-specific IgG levels in sera to expressed (3-galactosidase 28 days after subcutaneous administration of 'naked' pDNA, mannan-coated nanoparticles with pDNA, , nanoparticles with pDNA, or mannan with free pDNA. Mice were immunized with 10 ~g pDNA on day 0, 7, and 14. See Example 17 for more details: Mice were immunized with 4' ~,g pDNA on day 0, 7, and 14. Results are expressed as the individual titer for each mouse. All groups had 5 mice except for pDNA which had 4 mice.
Figure 19 Secretion of Interleukin-2 (IL-2) from isolated splenocytes (1 x 106 cells) from immunized Balb/C mice after in-vitro exposure to (3-galactosidase protein for hours at a concentration of either 10 yg/mL or 100 ~g/mL. Mice were immunized with 10 p.g pDNA on day 0, 7, and 14 by subcutaneous administration of 'naked' pDNA, mannan-coated nanoparticles with pDNA, nanoparticles with pDNA, or ~ mannan with free pDNA. Results are expressed as the mean IL-2 levels from pooled splenocytes harvested on day 28.
Figure 20 Secretion of Interferon-y (INF-y) from isolated splenocytes (1 x 106 cells) from immmuzed Balb/C mice after in-vitro exposure to (3-galactosidase protein for 2 o hours at a concentration of either 10 ~g/mL or 100 ~ug/mL. Mice were immunized with 10 ~,g pDNA on day 0, 7, and 14 by subcutaneous administration of 'naked' pDNA, mannan-coated nanoparticles with pDNA, nanoparticles with pDNA, or mannan with free pDNA. Results are expressed as the mean INF-y levels from pooled splenocytes harvested on day 28.
-l1-Figure 21 Secretion of Interleukin-4 (IL-4) from isolated splenocytes (1 x 10~
cells) from inununized Balb/C mice after in-vitro exposure to (3-galactosidase protein for hours at a concentration of either 10 ~,g/mL or 100 ~,ghnL. Mice were immunized with 10 ~g pDNA on day 0, 7, and 14 by subcutaneous administration of 'naked' pDNA, mannan-coated nanoparticles with pDNA, nanoparticles with pDNA, or mannan with free pDNA. Results are expressed as the mean IL-4 levels from pooled splenocytes harvested on day 28.
Figure 22 Gel Permeation Chromatography (GPC) elution of cured emulsifying wax Z o nanoparticles containing fluorescein-labelled plasmid DNA. See Example 18 for more details.
Figl.~re 23 Gel Permeation Chromatography (GPC) elution of cured emulsifying wax nanoparticles containing fluorescein-labelled plasmid DNA before and after DNase I nuclease treatment. See. Example 18 for more details.
Figure 24 The particle size of the pDNA-coated nanoparticles (net negatively-charged) in various media at 37°C immediately after dilution (0 min) and after 30 min.
2 o Figure 25 Ira-vitro transfection of liver HepG2 cells with pDNA-coated nanopaxticles.
(50,000 cells) (n=3) in thepresence of 10% FBS after 52 hr with: pDNA (Neg), Lipofectin~ (LPFN), nanoparticles (N) with 5% (w/w) DOPE (N+D), and pullulan-coated nanoparticles (PN). For PN-2 and PN-3, 50 ~,g and 250 ~g free Cholesterol-pullulan was added 30 min prior to pullulan-coated nanoparticles to block the glucose receptors. The pDNA dose was 2.5 ~,g for all samples.
Figure 26 Antigen-specific total IgG titer in serum to expressed (3-galactosidase 28 days after subcutaneous administration. One hundred microliters (100 p,L) containing five micrograms (5 p.g) of pDNA was administered to anesthetized Balb/C mice -l2-on day 0, day 7, and day 14. Groups: (1) pDNA-coated nanopanticles; (2) mannan-coated pDNA-nanoparticles; (3) mannan-coated pDNA-nanoparticles with DOPE (5% w/w); (4) 'naked' pDNA. (5) 10 ~g of (3-galactosidase protein adjuvanted with 15 ~g'Alum'.
Figure 27 The effect of final pDNA concentration on .the particle size (white bars) and zeta potential (~) of mannan-coated,nanoparticles with pDNA at a final concentration of either 100 ~,g/mL ("Man-NP/100") or 150 p,g/mL ("Man-NP/150") 1 o Figure 28 Ira-vity-o proliferation of isolated splenocytes 28 days after topical immunization with (1) pDNA-coated nanoparticles; (2) mannan-coated pDNA-nanoparticles; (3) mannan-coated pDNA-nanoparticles with DOPE (5% w/w); (4) 'naked' pDNA; (5) ~cg of (3-galactosidase antigen adjuvanted with 15 l.cg 'Alum'. One hundred ~,L
(100 L) of the formulations containing 5 ~,g of pDNA -was applied to anesthetized Balb/C mice on day 0, day 7, and day 14 to shaved skin. The pDNA-nanoparticles were prepared with emulsifying wax (2 mg/mL) and CTAB (15 mM).
Figure 29 Specific total IgG titer and IgA titer in serui~n to expressed j3-galactosidase antigen 28 days after intranasal administration to mice. Twenty five microliters (25 ~,L =
1.25 ~,g) of pDNA (either formulated as 'naked' pDNA or pDNA-coated nanoparticles) was administered to Balb/C mice (n=4-5) on day 0, day 7, and day 14.
Figure 30 Antigen-specific total IgG titer in serum to (3-galactosidase 28 days after subcutaneous administration of protein-coated anionic nanoparticles. One hundred microliters (100 ~,L) of the formulations containing 10 ~,g of (3-galactosidase were injected in anesthetized Balb/C mice on day 0, day 7, and day 14. The groups were:
(white bars) nGal, 10 ~,g of (3-galactosidase protein adjuvanted with 15 ~g 'Alum';
(gray bars) cGal, 10 ~.g of (3-galactosidase protein; (black bars) cGal-NPs, 10 ~,g of cationized (3-galactosidase protein coated on the surface of GPC purified, sterilized anionic nanoparticles.
Figure 31 Particle size of Gadolinium Hexanedione (GdH) nanoparticles engineered from microemulsion templates.
Figure 32 Particle size of Coenzyme Q10 nanoparticles engineered from microemulsion templates.
to Figure 33 Phospholipid nanoparticles were engineered from microemulsion precursors comprised of various phosphatidylcholine (lecithin)/emulsifying wax mixtures or phospholipid alone.
figure 34 Entrapment of a hydrophilic macromolecule in nanoparticles using a water-in-oil microemulsion precursor:
Figw-e 35 Adsorption of a HIV Tat peptide on anionic nanoparticles.
DETAILED DESCRIPTION OF THE INVENTION
2 o The growing interest in nanotechnology has also resulted in the use of microemulsions as precursors or templates to form nanoparticles within the small dispersed droplets (L,i et al., 1999;
Cavalli et al., 1999; Bocca et al., 1998; Tojo et al., 1998; Munshi et al., 1996; Ruys et al., 1999).
These reports have primarily dealt with the preparation of water-in-oil microemulsions (Lade et al.
2000; Song et al., 2000; Porta et al., 1999) whereby nanoparticles are formed in the water phase by the use of photochemistry (Agostiano et al., 2000), y-rays (Xiangling et al., 1999), or electrochemistry (Tang et al., 2000) to induce crosslinking, polymerization (Fang et al., 2000;
Capek, 1999; Meier; 1999) and/or complexation of the appropriate agents in the water phase.
This invention relates to microemulsions to be used as precursors for solid nanoparticles.
The microemulsion precursors consist of either alcohol-in-fluorocarbon microemulsions, liquid 3 o hydrocarbon-in-fluorocarbon microcmulsions, or liquid hydrocarbon-in-water microemulsions. The formed solid nanoparticles have diameters below 300 manometers and can be made to contain various materials including drugs, magnets, and sensors. The solid nanoparticles can be made to target different cells in the body by the inclusion of a cell-specific targeting ligand. Methods of preparing the microemulsion precursors, and methods to cure solid nanoparticles axe provided.
Methods to administer useful nanoparticles to the human body are also described.
As used herein, certain terms may have the following defined meanings.
As used in the specification and claims, the singular form a, aaz, and tdae include plural references unless the context clearly dictates otherwise. For example, the term a nanoparticle may refer to one or more nanoparticles for use in the presently disclosed systems.
to As used.herein, the term "solubility" refers to the extent to which a solute is dissolved in a solvent. Solubility can be described in terms such as described in REMINGTON'S
PHARMACEUTICAL SCIENCES ranging from very soluble (less than 1 part of solvent per 1 part of solute) to insoluble (more than 10,000 parts of solvent for 1 part of solute). The term "water-insoluble" refers to a substance or solute where more than 10,000 parts of water are a. 5 needed to dissolve 1 part of solute.
The term "nanoparticle" refers to particles that have diameters below 1 micrometer in diameter that are comprised of primarily one solid phase. "Stable nanoparticles" remain largely unaffected by environmental factors such as temperature, pH, body fluids, or body tissues..
However, solid nanoparticles may be designed to respond to these environmental factors in a 2 o controlled and predictable manner. The solid nanoparticles may contain, or have adsorbed to, many different materials for various pharniaceutical and. engineering applications such as plasmid DNA for gene therapy and genetic vaccines, peptides and proteins or small drug molecules, magnetic substances for use as nanomagnets, lubricants, or chemical, thermal, or biological sensors. It is preferred that the nanoparticles have a diameter of less than about 300 25 manometers and are present in the system at a concentration froml about 0.1-30 mg/mL, even more preferably that the nanoparticles have a diameter of less than about 200 manometers and axe present in the system at a concentration from about 0.1-10 mg/mL.
As used herein, a "microemulsion" is a stable biphasic mixture of tyvo immiscible liquids stabilized by a surfactant and usually a co-surfactant. Microemulsions are thermodynamically 30 stable, isotropically clear, form spontaneously without excessive mixing, and have dispersed droplets in the range of about 5 nm to 140 nm. In contrast, emulsions are opaque mixtures of two immiscible liquids. Emulsions are thermodynamically unstable systems, and usually require the application of high-torque mechanical mixing or homogenization to produce dispersed droplets in the range of about 0.2 to 25 Vim. Both microemulsions and emulsions can be made as water-in-oil or oil-in-water systems. Whether water-in-oil or oil-in-water systems will form is largely influenced by the properties of the surfactant. The use of surfactants that have hydrophilic-lipophilic balances (HLB) of about 3-6 tend to promote the formation of water-in-oil microemulsions while those with HLB values of about 8-18 tend to promote the formation of oil-in-water microemulsions.
Microemulsions were first described by Hoar and Schulman in 1943 after they observed that a medium chain alcohol could be added to an emulsion to produce a clear system within a defined 'window', now referred to as a microemulsion window. A unique physical aspect of microemulsions is the very low interfacial surface tension (y) between the dispersed and continuous phases. In a microemulsion, the small size of the dispersed droplets present a very large interface. A thermodynamically stable microemulsion can only be made if the interfacial surface tension is low enough so that the positive interfacial energy (yA, where A equals the interfacial area) can be balanced by the negative free energy of mixing (~Gm).
The limiting y value needed to produce a stable microemulsion with a dispersed droplet of 10 nm, for example, can be calculated as follows: ~G", _ -TOSm (where T is the temperature and the entropy of mixing OSn, is of the order of the Boltzman constant KB). Thus, KBT= 4~rzy and the limiting y value is calculated to be KBT/4~crz or 0.03 mN W '. Often, a co-surfactant is required in addition to the surfactant to achieve this limiting interfacial surface tension.
Tn addition to their unique properties as mentioned above, microemulsions have several key advantages for use as delivery systems intended for use in marketed pharmaceutical products, namely; i) increased solubility and stability of drugs incorporated into the dispersed phase, ii) increased absorption of drugs across biological membranes, iii) ease and economy of scale-up (since expensive mixing equipment is often not needed), and iv) rapid assessment of the physical stability of the microemulsion (due to the inherent clarity of the system). For example, oil-in-water microemulsions have been used to increase the solubility of lipophilic drugs into formulations that are primarily aqueous-based (Constantinides, 1995). Both oil-in-water and water-in-oil microemulsions have been also been shown to enhance the oral bioavailability of drugs including peptides (Bhargava et al. 1987; Ho et al. 1996;
Constantinides, 1995).
Although microemulsions have many potential advantages they do have potential limitations, namely; a) they are complex systems and often require more development time, b) a large number of the proposed surfactants/co-surfactants are not pharmaceutically acceptable (Constantinides, 1995), c) the microemulsions are not stable in biological fluids due to phase W version. Thus, the microemulsions themselves are not effective in delivering drugs intracellularly or targeting drugs to different cells in the body. The development of a microeinulsion involves the very careful selection and titration of the dispersed phase, the continuous phase, the surfactant and the co-surfactant. Time consuming pseudo-phase ternary diagrams involving the preparation of a large number of samples must be generated to find the existence of the 'rnicroemulsion window', if any (Attwood, ,1994). In general, . a water-in-oil microemulsion is typically much easier to prepare than an oil-in-water microemulsion. The , former system is useful for formulating water-soluble peptides and proteins to increase their stability and absorption while the latter system is preferred for formulating drugs with little or no aqueous solubility.
As used herein, a "surfactant" refers to a surface-active agent, including substances commonly referred to as wetting agents, detergents, dispersing agents, or emulsifying agents.
2 o For the purposes of this invention, it is preferred that the surfactant has an HLB value of about 6 20, and most preferred that the surfactant has an HLB value of about 8-18. It is preferred, but not required, that the surfactant, either non-ionic, anionic, or cationic, is selected from the following groups; polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, or fatty alcohols or their derivatives, hexadecyltrimethylammonium bromide, or combinations thereof. A "co-surfactant" refers to a surface-active agent, including substances commonly referred to as wetting agents, detergents, dispersing agents, or emulsifying agents. It is preferred, but not required, that the co-surfactant is selected from the following groups; polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, or fatty alcohols or their derivatives, hexadecyltrimethylammonium bromide, or combinations thereof. It is also preferred that the total concentration of surfactant andlor co-surfactant present in both the oil-in-water microemulsion precursor and the cured nanoparticles system is in the range of about 1-5000 mM, more preferably in the range of about 1-1000 mM, and most preferably in the range of about I-300 mM.
As used herein, an "ethanol-in-fluorocarbon microemulsion" is a stable biphasic mixture of ethanol dispersed in a fluorocarbon wherein the ethanol droplets have diameters ranging from about 5 rim to~about 500 nrn, preferably from about 5 nm to about 250 nm, and most preferably from about 5 nm to about IOU nm.
As used herein, a "liquid hydrocarbon-in-fluorocarbon microernulsion" is a stable biphasic mixture of a liquid (melted) hydrocarbon dispersed in a fluorocarbon wherein the liquid 1o hydrocarbon droplets have diameters ranging from 5 nm to 500 nm, preferably from 5 nm to 250 nm, and most preferably from 5 nm to 100 nm. A "liquid hydrocarbon" is any material that is a solid below body temperature (35-38°C), but a liquid at temperatures greater than body temperature.
As used herein, a "liquid hydrocarbon-in-water microemulsion" is a stable biphasic mixture Of a liquid (melted) hydrocarbon dispersed in water wherein the liquid hydrocarbon droplets have diameters ranging from about 5 nm to about 500 nm, preferably from about 5 nm to about 250 mn, and most preferably from about 5 nm to about 100 nm.
As used herein, a "film-forming substance" may be any pharmaceutical material that is soluble or dispersible in the dispersed phase, or actually be the dispersed phase when melted to a 2 0 liquid, and that can be cured by a curing process to form a solid membrane suitable for the delivery of drugs by different routes of administration.
As used herein, the term "nanopaz-ticle matrix material" refers to those materials that can form both the shell and majority of the weight composition of the said nanoparticle. Two types of matrix materials are envisioned, both serving as the oil-phase in the oil-in-water microemulsion precursor. The first matrix materials are those materials that are amphipathic in nature (having both hydrophilic and hydrophobic moieties), are primarily water-insoluble, and that melt above room temperature in the range of about 35-100°C, more preferably in the range of about 35-80°C, and most preferably in the range of about 35°C-65°C. It is envisioned that these materials can be any substance meeting the above criteria and that are a wax, lipid, polymeric surfactant, or 3o combinations thereof. It is most preferred, but not absolutely required, that these materials are -l8-selected from the following: emulsifying wax, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene stearates, phospholipids, fatty acids or fatty alcohols or their derivatives, or combinations thereof. It is preferred that the nanoparticle matrix material is present in both the microemulsion precursor and cured nanoparticles at a concentration of about 0.1-30 mg/mL, and more preferably at a concentration of about 0.1-10 mg/mL. It is also envisioned that the nanoparticle matrix material may be a pharmacologically active substance such as a drug.
As used herein, a "curing process" is the process whereby the film-forming substance.
residing in the dispersed phase of the stable microemulsion precursor is cured to form solid nanoparticles containing entrapped drug molecules. In particular, the curing process may consist of evaporating the dispersed liquid to precipitate the film-fornling substance, adding water or some combination of water to precipitate the film-forming substance, or by cooling the liquid (melted) dispersed phase to solidify the film-forming substance.
As used herein, a "solid nanoparticles" are particles below 1 micron in diameter that are comprised of primarily one solid phase. "Stable solid nanoparticles" remain largely vmaffected by environmental factors such as temperature, pH, body fluids, or body tissues.
However, solid nanoparticles may be designed to respond to these environmental factors in a controlled and predictable manner to deliver their contents in a controlled and predictable manner. The solid nanoparticulate systems may contain (by entrapment or adsorption) many different materials for various pharmaceutical and engineering applications such as plasmid DNA for gene therapy and genetic vaccines, peptides and proteins or small drug molecules, magnetic substances for use as nanomagnets, or chemical, thermal, or biological sensors fox use as nanosensors.
As used herein, the term "permanently suspended" refers to nanoparticles, engineered.
from said microemulsion precursors, that remain suspended in aqueous media such as water or buffer for at least one month at room temperature and that cannot be settled by ultracentrifugation treatment at about 50,000 cpm for about S minutes.
As used herein, the term "ligand" refers to those substances that can be recognized and bind to a specific molecule, a cell-receptor, an antibody, an anti-antibody, or combinations thereof. It is preferred that the ligand be comprised of carbohydrates or amino acids or combinations thereof. It 3o is most preferred that the ligand be a monoclonal or polyclonal antibody.
The ligand may be attached onto said nanoparticles by any number of processes including, but not limited to, covalent attachment, ionic interaction, hydrophobic interaction, and hydrogen bonding.
It is also envisioned that the ligand may be .chemically modified to enhance the attachment of said ligand to said nanoparticle to either increase the efficiency of detection or selectively detecting one or more radioactive molecules from other molecules. Most preferred. in the invention are those ligands specific for, 1) tumor cells, such as folate and antibodies, 2) hepatocytes, such as asialofetuin and other galactose containing ligands, and 3) macrophages and dendritic cells, such as mannan, mannose, or synthetic or natural peptides, and 4) brain or the blood-brain-barrier (BBB), such as choline, and or derivatives of choline Microemulsion Precursors: To overcome the problems associated with polyelectrolyte complexation and conventional solid' nanoparticles and to exploit the benefits of microemulsions, we have developed a process to pharmaceutically engineer solid nanoparticles containing drugs that do not require the use of rigorous processes. The strategy involves the spontaneous formation of a microemulsion precursors wherein the microemulsion is subsequently treated to cure solid 25 . nanoparticles between about 5-300 nm. Curing may be completed either by adding water to the microemulsion, applying heat to remove the solvent, or most preferably, by simple cooling of the microemulsion. For the purposes of this invention, three microemulsion precursors are preferred, 1) an ethanol-in-fluorocarbon microemulsion, also referred to as E/F, 2) a liquid hydrocarbon-in fluorocarbon microemulsion, also referred to as O/F, and 3) a liquid hydrocarbon-in water 2 o microemulsion, also referred to as O/W.
Ethanol-in-fluorocarbon microemulsion containing film-forming polymers: Ethyl celluose is an inert, biocompatible polymer available in a variety of molecular weights. Ethyl cellulose polymers, dissolved in ethanol, instantaneously precipitate when exposed to water. When ethanol solutions containing ethyl cellulose and a drug are applied to the skin or mucosal surfaces, the ethyl 25 cellulose very quickly forms a strong film. This film functions as a reservoir for the controlled-release of drug. We hypothesized that if this ethanolic phase could be dispersed into a fluorocarbon phase and stabilized to form an E/F microemulsion, the dispersed ethanol droplets (about 5-140 nm) containing ethyl cellulose may constitute a "template" for solid nanoparticles when the E/F
microemulsions were exposed to aqueous solutions or if the ethanol was removed by evaporation.
-ao-If plasmid DNA was dissolved in the ethanol phase then it could be entrapped in the solid naiioparticles.
Fluorocarbons are carbon-based molecules with some or all of the hydrogen atoms replaced with fluorine. They have unique properties including chemical and biological inertness, Iow surface tension, high density, unique hydrophobicity, and he ability to dissolve large amounts of gases (Riess and I~rafft, 1997). In the present application, perfluorooctyl bromide (CF3(CF~)6CFZBr;
perflubron) is exemplified as the fluorocarbon because of its well-documented safety profile and its growing use in (micro)emulsion-based delivery systems for drugs and oxygen (Riess and Krafft, 1997; Lattes et al., 1997; Cornelus et al., 1994; Gauger et al., 1996).
Importantly, for forming a s o microemulsion, ethanol is not miscible with perflubron but may be dispersed in perflubron using an appropriate .surfactant. Thus, the use of E/F microemulsions and specifically the use of perflubron, have a niunber 'of advantages. The engineering of solid ethyl cellulose nanoparticles containing plas~°nid DNA following the rationale and concepts described above are not possible using traditional water-in-oil (W/O) or oil-in-water (0/W' microemulsions.
15 In addition to the ethyl cellulose and a molecule such as plasmid DNA, the ethanol droplets in the E/F microemulsion may contain a number of excipients including a small amount of water (only up to 20% v/v), pore-forming polymers to control the release of the plasmid DNA, endosomolytic agents to disrupt endosomal membranes, nuclear targeting agents to target plasmid to the nuclear membrane, and other excipients as needed. This E/F
microemulsion precursor 2 o strategy has advantages since; i) all ingredients axe potentially biocompatible and those that may not be are removed when the solid nanoparticles are cured and isolated, ii) perflubron and the surfactant and co-surfactant may be recycled, iii) well-defined and uniform solid nanoparticles (about 5-300 um) may be reproducibly made without the use of high-torque mechanical mixing, microfluidization, or homogenization, iv)'the formed solid nanoparticles may have superior in-vivo 25 stability, and v) cell-specific targeting ligands can easily be incorporated into the system (during or after the engineering process). These solid nanoparticles may have applications in the areas of non-viral gene delivery. For example, due to their small size and stability, these nanoparticles may have greater access to tissues and cells than larger, Iess stable plasmid DNA
polyelectrolyte complexes (i.e., liver hepatocytes, tumor cells, or antigen-presenting cells, etc.).
_21_ Cell-Specific Targeting to the Liver. Since the discovery of the hepatic asialoglycoprotein receptor in the early 1970s there have been a plethora of attempts to target molecules, such as genes, specifically to hepatocytes. The asialoglycoprotein receptor on hepatocytes functions, in part, to remove circulating glycoproteins from the blood. This aspect of the receptor has led to the design and testing of a number of targeted systems primarily using different natural, synthetic, or semi-synthetic glycosylated proteins, polymers, or lipids (Meijer et al.
1995). The carrier systems described are comprised of conventional nanoparticles, liposomes, conjugated soluble polymers, or viruses. It is generally thought that the targeted systems must have three properties to specifically target hepatocytes. First, the systems must be less than about I00 nm in size, and preferably less zo than about 50 nm. Particles with diameters greater than about I00 nm cannot traverse the fenestrae (pores) in the endothelial lining to gain access to the hepatocytes (Schlepper-Schafer et al. 1986;
Mandeville et al., 1997). Second, the systems must be stable in the blood.
Unstable particles that aggregate will become too large to diffuse through the fenestrae. For example, although hepatocyte-targeted polyelectrolyte complexes of poly-L-lysine and plasmid DNA
have been 15 produced in the size range of about 50-100 nm, these complexes aggregate even in physiological saline alone (Wu and Wu, 1988; Kwoh et al. 1999; Plank et al., 1992). Third, the systems generally have to employ a clustering of the glycosylated ligands. For example, tri-antennary asialoglycoproteins have much greater affinity for the receptor than do bi-and mono-antennary .
asialoglycoproteins (Wadhwa and Rice, 1995).
20 In this application, a unique system comprising stable ethyl cellulose nanoparticles (about 5-50 nm) containing a molecule of interest, such as plasmid DNA and asialofetuin as the hepatocyte-specific ligand. Asialofetuin (45,450 g/mol) is a natural glycopeptide having three major glycosylation sites comprised of about two-thirds tri-antennary and one-third bi-antennary functionalities. Asialofetuin has been used previously to increase delivery of substances to the liver 25 including, liposomes (Sliedregt et al. 1999; Wu et al. 1998), contrast agents (Mandeville et al.
1997), plasmid DNA (Plank et al. 1992), gold particles (Schlepper-Schafer et al. 1986), and several other drugs (Wadhwa and Rice, 1995; Meijer et al. 1995). Importantly, we are not limited to the use of ethyl cellulose as the film-forming material. In fact, any water insoluble material including lipids, peptides, or other polymers that have solubility in ethanolic solutions may be utilized as the 3 o film-forming material.

It has been demonstrated that stable ethanol-in-perflubron microemulsions could be formed using at least four different fluorosurfactants. A microemulsion using pentadecafluorooctanoic acid (PDFOA) in a ratio of 70:30 (w/w) perflubron/PDFOA was able to solubilize up to 37.2% ethanol (w/w). In this same microemulsion, a film-forming polymer could be incorporated into the system by adding ethyl cellulose dissolved in ethanol without altering the existence of the microemulsion window. For E/F microemulsions made without a co-surfactant or ethyl cellulose, the average droplet size of ethanol was 34.9 nm. The actual amount of PDFOA needed to coat the surface of all ethanol droplets in this microemulsion was within 3% of the theoretical amount needed and supported the formation of an E/F microemulsion. E/F microemulsions inco~orating about 5%
Zo ethyl cellulose in the ethanol phase had an unexpectedly small droplet size of only about 8 nm suggesting unknown effects of the ethyl cellulose.
Cell-Specific Targeting to the Dendritic Cells. Positive results with the delivery of (genetic) vaccines and ex vavo loading of dendritic cells with antigen has strengthened the movement towards directly targeting antigen presenting cells as a means to amplify, control and 15 mediate the immunological consequences of prophylactic and/or therapeutic vaccines {Tang et al.
1992; Liu et at. 1995; Ulmer et al., 1996; Mumper et al. 1999; Cui and Mumper 2001) Of particular interest is targeting antigens to the lymph nodes via the skin or subcutaneous routes wherein a rich population of dendritic cells are located (Steinman et al.
x997; Banchereau and Steinman, 1998). Moreover, specifically targeting dendritic cells in the skin (Langerhan's cells) or 2 o draining lymph node would theoretically have advantages in eliciting stronger T cell-mediated immunity. Human dendritic cells have been shown to express a mannose-receptor (MR) and this receptor has been exploited to deliver antigens resulting in more robust Th1 and CTL responses (Jordens et al. 1999; Engering et al. 1997; Tan et al. 1997; Gu et al. 1998;
McKenzie et al. 1998;
Loflhouse et al. 1997; Ohishi et al. 1997). For example, Tan et al. have demonstrated that 25 mannosylation of antigens has been shown to enhance the stimulation of HLA
class-II restricted peptide specific T cell clones by 200-10,000-fold compared to non-mannosylated peptides (Tan et al. 1997). Further, the use of mannan to deliver antigens to dendritic cells has been shown by several groups to elicit strong Thl-type immune responses that subsequently provided protection in several different viral and W rnor challenge models (Gu et al. 1998; McKenzie et al. 1998;
3o Lofthouse et al. 1997; Ohishi et al. 1997). It is also envisioned that other types of dendritic cell-specific ligands can be used including, but not limited to, synthetic peptides and/or peptides/proteins isolated from phage-display techniques.
Targeting to the Brain: Pharmacologic intervention in brain disease is often limited by poor drug transport into the central nervous system. Attempts have been made to increase drug delivery to brain across the blood-brain barrier (BBB) either by increasing a drug's lipid solubility, and therefore it's passive permeability, or by causing a temporary "opening" of the BBB (Greig, 1989).
Additional methods employed to augment brain drug delivery include direct injection into brain or cerebrospinal fluid, intracarotid infusion to maximize brain arterial concentrations, and inhibition of active removal from brain or blocking drug metabolism (Smith, 1993). Several investigators have l o ' also explored the use of nanoparticles to deliver dnigs through the BBB;
however, these particles were quite large (i.e., > 200 nm) and the 'efficiency of delivery was usually less than 1% (Alyautdin et al., 1997; Olivier et al., 1999). Fox example, Troster et al. (1990) observed that about 1% of the total dose of poly(methylinethacrylate) nanoparticles coated with polysorbate-80 was delivered to the brain of rats after intravenous injection. Similarly, Gulyaev et al.
(1999) observed that about 1-IS 2% of the total dose of poly(butyl cyanoacrylate) nanoparticles coated with polysorbate-80 was-delivered to the brain of rats after intravenous injection. Krueter (2001 ) hypothesized that the most likely rnecharusm of brain uptake of polysorbate 80 coated nanoparticles was via endocytosis by the endothelial cells lining the brain blood capillaries. Transport to'the brain was dependent on the coating of the nanoparticles by surfactants, especially polysorbates. Further, polysorbate-80 coated nanoparticles were shown to lead to the adsorption of apolipoprotein E from blood plasma thereby creating particles that mimic low density lipoprotein (LDL) particles that could lead to their uptake by the endothelial cells on the BBB. A plausible alternative approach is to design very small nanoparticles (<100 nm) that have at their surface a ligand molecule that is normally transported into the brain by saturable nutrient carriers of the BBB. Transporters for the BBB have been 2 5 successfully utilized to deliver several amino acid drugs to the brain, such as D,L-NAM for the treatment of brain tumors (Takada et al., 1991; Takada et aL, 1992) and L-DOPA
for the treatment of Parkinson's disease. Both of these drugs are taken up into brain by the BBB
large neutral amino acid transporter. Baclofen, melphalan, acivicin, 6-diazo-5-oxo-norleucine, azaserine, buthionine sulfoximine and alpha-methyl DOPA are transported by the large neutral amino acid transporter as 3o well (Smith, 1993). One such BBB transporter investigated has been the choline transporter.

Choline enters the brain via a comparable BBB transport system as the ones described above (Diamond 1971; Oldendorf and Braun, 1976; Cornford et al.~ 1978; Metting et aL, 1998; Allen and Smith, 1999). Thus, the BBB choline transporter may have utility in delivering drugs and even very small nanoparticles to the central nervous system. Indeed, lymphoblast choline transporters have .
been shown to transport nitrogen mustard alkylating agents (Goldenberg and Begleiter, 1979) suggesting choline transporters deliver drugs across cell membranes.
Targeting to Solid Tumors: As compared to normal .tissues, solid tumors have a relatively - large interstitial space or microvasculature (Jain et al., 1994; Brown and Giaccia, 1998; Jain 1997). The therapeutic efficacy of FDA-approved liposomal anticancer drugs has been . demonstrated to be due, in part, to the increased microvascular permeability of 100-200 nm liposomes in tumors. This increased permeability results in enhanced transport of particles and macromolecules into the tumor capillaries and interstitial space. Studies by several groups using liposomes and other macromolecules have shown that the typical pore size of vascularized tumors is about 200 nm to 500 nm with some fenestrations as small as 50 nm to 60 nm (Wu et al., 1993;
~.5 Yuan et al., 1995; Hobbs et al., 1998; Bally et al., 1994; Nagayasu et al., 1999; Kong et al., 2000).
Despite the fact that tumors tend to be more "leaky" than normal tissues, the transport of larger particles into some regions of the tumor (i.e., central necrotic regions) is fuz~ther hindered by the high hydrostatic pressure which tends to forces these particles out of tumors.
Taken as a whole, these studies highlight the importance of using smaller particles to deliver cancer therapies. Tumor 2 0 therapies' have also included active targeting modalities to increase tumor deposition, including antibody, carbohydrate, peptide, or vitamins such as folic acid (Vyas et al., 2001; Thorpe and Derbyshire, 1997; Wang and Low, 1998). The folic acid receptor is overexpressed in a number of human honors including cancer of the lung, colon, brain, testis, uterus, ovary, kidney, and some blood cells (Wang and Low, 1998; Weitman et al., 1992). In contrast, normal tissues have ony 25 rarely been found to express the folic acid receptor. Folic acid binds to the cell surface folate receptors with very high affinity (Kd = 10-'° M) and is internalized by receptor-mediated endocytosis (Kamen et al., 1986; Lee et al., 1996). These properties have been exploited by several groups to deliver toxins, imaging agents, antisense oligonucleotides, genes, and liposomes to tumor cells (see review by Wang and Low, 1998). Two folate-based radioactive cancer diagnostic agents 30 (folate-Indium-111 and folate-Technetium-99m) are now in human clinical trials. The use of folic acid has several advantages over monoclonal antibodies including low cost, high chemical and biological stability, non-irmnunogenicity, and small size. It is hypothesized that engineered folate-targeted nanoparticles, having diameters less than I00 nm or even 50 nm, may have excellent potential for enhanced tumor targeting, transport, and therapy.
' Liquid hydrocarbon-in-water microemulsions: In another aspect of the invention, liquid matrix microemulsions are made. The basic concept of liquid matrix microemulsion precursors is shown in Figure 2. The concept avoids the use of an ethanol dispersed phase to solubilize the film-forming polymer. Instead, the nanoparticle matrix material alone is melted and then dispersed in a heated continuous phase with an appropriate surfactant and/or co-surfactant to form a heated 1 o microemulsion precursor at the same temperature. The heated microemulsion precursor is then simply cooled to room temperature to cure solid nanoparticles. The nanoparticles are then isolated and purified as described in Figure 2.
The liquid matrix microemulsion precursor method may have a number of advantages over the E/F microemulsion method namely, 1) no additional materials such as water have to be added to the formed microemulsion to cure the solid nanoparticles, the microemulsion precursor is simply cooled, 2) high entrapment efficiencies may be achieved since the dispersed droplets are composed entirely of the matrix material. 3) the dispersed phase is not limited only to the use of ethanol, which is used in the E/F microemulsions, but to any matrix material meeting the criteria and from .
which a stable microemulsion precursor can be made, and 4) no organic solvents are needed to 2 o form the microemulsion precursors.
Gadolinium and Neutron Capture Therapy of Tumors: Gadolinium, a rare-earth metal, has been proposed as an alternative to boron for neutron capture therapy of tumors. In contrast to Boron-10, which emits short-range alpha-particles when exposed to thermal neutron irradiation, Gadolinium-157 emits gamma rays and Auger electrons. Consequently, Gadolinium-157 neutron 25 capture therapy may increase the probability of hitting a larger number of tumor cells with long-range photons (> 100 pm) and high-energy electrons. Gadolinium-I57 also has a very large neutron capture cross section of 255,000 barns which is almost 70-fold greater than Boron-10. Thus, much shorter neutron irradiation times are needed for Gadolinium-157 neutron capture therapy than for Boron-10 neutron capture therapy. The intratumoral administration of delivery systems for 3o gadolinium such as gadoliniumlchitosan complexes, and emulsions and microspheres containing gadolinium have recently been reported. (Tokumitsu et al. 1999; Tokumitsu et al. 2000; Miyamoto et al. 1999; Jono et aI..1999). In most cases, these particulate delivery systems for gadolinium were quite large (i.e:, 400 nn to several microns in size). For targeting solid honors, it preferred that the nanoparticles containing Gadolinium, or its derivatives or complexes thereof, have a particle size below about 300 nn, and more preferably below about 200 nn, and most preferably below about 100 nn.
HIV-l and Tat: HIV-1 Tat protein is an RNA binding transcriptional regulatory protein expressed early in- HIV-1 infection, and necessary for high level expression of viral proteins (Garber et al. 1999). .Tat is a small, 86 to 102 as protein that is encoded by two exons. Tat is 1 o released from cells at relatively high levels and can be detected in the serum of HIV infected.
individuals. Tat shows very little variability and is highly conserved in the first exon among the different subtypes, with the exception of the O subtype (Korber et al. 1995).
Neutralization of Tat might prevent the trans-activation of other infected cells, and also prevent Tats immunosuppressive arid neurotoxic properties. Tat is also expressed early in HIV infected cells, and elimination of Tat expressing cells by CTL would prevent the production of infectious virus:
CTL responses have been repeatedly detected in HIV infected individuals (van Baalen et al. 2997;
Venet et al. 1992; Froebel et al. 1994; Ogg et al. 1998), and studies have shown that the presence of anti-Tat CTL in the initial phase of infection correlates inversely with progression to AIDS.
Antibodies to Tat may neutralize extracellular Tat and have protective effects in controlling disease 2 o progression (Re et al. 1995). Several groups have advanced the idea that Tat would make a logical vaccine candidate. For example, In 1999, Cafaro et al. reported that cynamolgus monkeys immunized with Tat (1 prime with 8 boosts) using either the R.IBI or Alum adjuvant developed high antibody titers and Tat neutralizing activity; however the CTL activities were low (~I O% cell lysis) and were detected in only two of five monkeys (Cafaro et al. 1999).
Further, the same group 25 reported in 2001 that intramuscular injection of 'naked' pDNA expressing Tat to cynomolgus monkeys (1 prime with 8 boosts) induced anti-Tat CTLs which provided protection against viral challenge by blocking virus replication at the early stage (Cafaro et al.
2001). However, antibody titers to Tat were very low and no detectable neutralizing antibodies were found in any immunized monkeys. These studies provide a compelling argument for the further development of Tat as a 3o vaccine as well as the development of a heterologous prime-boost regimen using both adjuvanted _2~_ protein and plasmid DNA to generate high neutralizing antibodies and CTL.
Heterologous immunization regimens have been reported to induce 10- to 100-fold higher frequencies of T cells than priming and boosting with protein, plasmid DNA, or virus alone (Amara et al. 2001; Robinson et al. 1999).
s Genetic Immunization: Genetic immunization has emerged as one of the most promising applications of non-viral gene therapy (Liu et al. 1995, Ulmer et al. 1996, Levine et al. 1997).
The potential advantages of DNA vaccines over conventional vaccines include, i) the high stability of plasmid DNA, ii) low W anufacturing costs, iii) lack of infection risk associated with attenuated viral vaccines, iv) the capacity to target multiple antigens on one plasmid, and v) the Zo ability to elicit both humoral and cellular immune responses (Mumper et al.
2001). The ability of genetic vaccines to elicit cellular immunity is of great importance to those working in the vaccine field. Vaccines that generate cellular immunity mediated by the generation of cytotoxic' T lymphocyte (CTL) responses have been called the "the immunologist's grail"
(Liu 1997).
These vaccines may be of prime importance for protection from intracellular viral infections and 15 as immunotherapies for cancer. Immunization with 'naked' plasmid DNA has been found to induce strong T helper cell type 1 (THl) immune responses (Robinson and Torres, 1997) as evidenced by the protection of cytokines such as interleukin-2 (IL-2) and interferon-'y (INF-y). In contrast, subunit, or protein-based, vaccines tend to induce T helper cell type 2 (TH2) immune responses as evidenced by the protection of cytokines such as interleukin-4 (IL-4) and 2o interleukin-10 (IL-10). Importantly, TF.,1 cells aid in the regulation of cellular immunity. In contrast, TH2 cells aid in the production of antibodies such as IgA and IgE.
Improved immunization methods to induce cellular immunity and TH1 type immune responses are needed in the field.
Until recently, intramuscular (i.m.) injection was the primary route of administration .for 25 DNA vaccines. The i.m. route has been shown to elicit protective and therapeutic immune responses in many animal models. However, the low bioavailability of plasmid DNA in the muscle coupled with the redundant nature of antigen transfer by muscle cells clearly raised the issue about the rationale of this route (Ulmer et al. 1996, Corr et al. 1996, Huang et al. 1994, Doe et al. 1996, Corr et al. 1999, Torres et al. 1997). Also, there has been no conclusive clinical data 3o suggesting that the i.m. route is viable in humans. As an alternative to intramuscular _2s_ administration of plasmid DNA, researchers have investigated targeting plasmid DNA to the skin using intradermal needle injection, needle-free jet injection devices, or the gene gun. Intradermal needle injections of plasmid DNA into the skin has been shown to be more effective than intramuscular injection in several animal species in eliciting immune responses (Braun et al.
1998, Gerdts et al. 1997, Van Rooij et al. 1998, Van Drunen et al., 1998).
Further, several preclinical animals studies have reported on the use of needle-free jet injection devices and the gene gun to administer plasmid DNA (Tang et al. 1992, Fynan et al. 1993, Degano et al. 1998, Pertmer et al. 1995, Yoshida et al. 2000). Recent clinical trials using the gene gun to administer plasmid DNA-based vaccines to the skin epidermis in humans showed that this technology may 1 o be an effective clinical vaccine modality for the treatment or prevention of hepatitis B or malaria. .
Although these preclinical and clinical results are promising, it is not clear whether these technologies will translate into safe, commercially available and affordable vaccines.
The growing body of evidence that pointed to the significance of the role of Langerhan's .
cells in the epidermis prompted researchers to consider alternatives to gene gun and jet injection ~.5 to target plasrnid to this site. Topical delivery of formulated plasmid in the form of a patch, cream, or gel may provide many advantages in terms of cost and patient compliance (Shi et al. .
1999, Fan et al. 1999, Tang et al. 1997). Shi et al. demonstrated the feasibility of topical genetic immunization in mice by applying plasmid DNA complexed to cationic liposomes to chemically., (Hair)-treated skin for 18 h (Shi et al. 1999). Antigen expression at the site of administration was 2o extremely low and virtually unquantifiable in the skin 18 h after topical administration and the immune responses Were 100-fold lower than those for mice immunized with 50 p,g of 'naked' plasmid injected into the muscle. Fan et al. demonstrated that the immune response to expressed J3-galactosidase in mice were comparable at fom weeks after both topical and intramuscular administration of 100 ~g 'naked' plasmid and I00 ~,g plasmid complexed with cationic 25 liposomes (Fan et al. 1999). It was further demonstrated, by skin graft transplantation studies, that the presence of normal hair follicles was required to elicit a humoral immune response to expressed antigen. Taken together, the studies by Shi et al. and Fan et al.
demonstrated the feasibility of topical genetic immunization. However, these studies also indicated the need for more effective topical delivery systems that would allow for much lower doses of plasmid DNA
3 o to skin not pre-treated with chemicals.

Singh et czl. have demonstrated enhanced immune responses in mice over 'naked' plasmid DNA after intramuscular injection of pDNA-coated cationic polylactic acid-co-glycolic acid (PLGA) microspheres (Singh et al. 2000). These PLGA microspheres were made cationic by the inclusion of cationic surfactants such as hexadecyltrimethylammonimn bromide (CTAB). Singh et czl. also investigated immune responses to p55 Gag protein using pDNA-coated on different sizes of cationic PLGA microspheres (300 nm, 1 Vim, 30 ~,m). Singh et al.
found a direct correlation between microsphere size and immune response wherein pDNA-coated PLGA/CTAB
microspheres with a size of 300 nm led to the highest immune response after intramuscular injections (2 x 1 ~.~g pDNA) of the formulations. Although these results were encouraging, the z o difficulty in preparing PLGA microspheres below 300 nm, the use of solvents such as methylene chloride, and the inclusion of cationic surfactants may be problematic for future clinical investigation. As a result, we sought to investigate the more facile preparation of smaller nanoparticles engineered from microemulsion precursors in a single vial that could then serve as a more pharmaceutically-acceptable template to coat or entrap pDNA.
15 Immunization via the nasal route or other mucosal sites: It is generally accepted that many' new vaccines will require the induction of mucosal immunity since almost all viral, bacterial, and .
parasitic pathogens causing common infectious diseases of the intestinal, respiratory and genital tract enter or infect through the body via mucosal surfaces (McGhee & Kiyono 1992; Mestecky et al 1997). Systemic administration of vaccines generally fails to induce mucosal immunity.
2 o However, with appropriate systems, it is possible to induce both mucosal and systemic immune responses by mucosal immunization (McGhee et al 1992, 1999). The nasal mucosa is an important arm of the mucosal system since it is often the first. point of contact for inhaled antigens. As a consequence, intranasal immunization has proven to be an effective method for stimulating both upper and lower respiratory immunity (Davis 200,1; Partidos 2000). Moreover, because of the 25 properties of the common mucosal immune system; nasal immunization has the ability to induce both local and distal mucosal immunities in the nasal and upper respiratory mucosa and the draining lymph node, as well as the intestinal and vaginal mucosa (Mestecky and McGhee 1987;
Klavinskis et al 1999). Thus, nasal immunization may have important role in the prophylaxis of both (upper) respiratory infections or infections at distant mucosal sites.
Additionally, intranasal 3 o immunization, rnay require less vaccine presumably due to the minimization of vaccine loss andlor degradation after oral administration (Wu et al 2001). Intranasal immunization requires no needles and therefore it may be amenable for wide-spread immunization of large populations. A nasal vaccine (Nasal Flu~) is now commercially available, and the United States FDA
is currently reviewing an application to market an additional influenza vaccine (Flmnist~) (Illum et al. 200I).
Traditionally, vaccines have been comprised of (subunit) proteins, live attenuated viruses, or killed bacteria. However, much attention has recently been focused on pDNA
vaccines. One important advantage of pDNA vaccine is that it is able to induce in animals both humoral and cellular immune responses including T helper type-I (Thl) and cytotoxic lymphocytes (CTL) responses (Liu 1997). Moreover, the poor safety issues associated with the attenuated virus or 1 o bacterial vaccines may be avoided (LTlmer et al 1996). Plasmid DNA vaccine is relatively stable, cost-effective for manufacture and storage. It is also possible to express multiple antigens on' one plasmid. ,However, until recently, intramuscular injection has been the primary route for administration of pDNA vaccines. Although pDNA immunization by intramuscular route has proven to be very potent in small animal models, the potency in primates and humans has been disappointing (lVlumper and Ledebur 2001). There exists a clear need to improve the potency of pDNA vaccines and/or to search for alternative routes) to administer pDNA
vaccines.
Methods used to administer and evaluate genetic vaccines: For topical application, the hair covering the back of the mouse was shaved with clippers. The skin was wiped with an alcohol..
swab, allowed to air dry, and 100 p,1 of each formulation was dripped and subsequently rubbed with 2o pipette tips onto the skin covering an area of about 2 cmz. For intramuscular injection, the hair covering the back of the gastrocnemious muscle on both legs .was shaved, wiped with an alcohol swab, and allowed to air dry. Filly microliters of each formulation was injected into the gastrocnemious muscles on both legs. A typical immunization schedule is to dose the formulation at day 0, 7, and 14. At day 28, alI mice were anesthetized using pentobarbital (i.p.) and blood was collected by cardiac puncture. The blood was transferred into a Vacutainer Collection Tube (Becton Dickinson). Senurz was separated by centrifugation and stored. at -20°C
until analyzed. The ~3-galactosidase specific sera IgG level were quantified by ELISA. Briefly, Costar high binding 96-well assay plates were coated with 8 ~,g/ml of (3-galactosidase antigen overnight at -4.°C. The plates were then blocked with 100 pl/well of 4% BSA/4% Normal Goat Serum (NGS) solution made in 10 mM PBS/Tween 20 (Scytei> Laboratories; Logan, UT) for 1 hr at 37°C.
Mouse serum samples (50 pL/well; starting dilution of 20:100 in 4% BSA/4% NGS/PBS/Tween 20) were serially diluted and then incubated for 2 hr at 37°C. Afterward samples Were washed with 10 mM PBS/Tween 20 buffer three times, and Anti-mouse IgG HRP F(ab')z fragment from sheep (diluted 1:2,000 in 1%
BSA) was added (50 p,L/well) and incubated for 1 hr at 37°C. Plates were washed three additional times with 10 mM PBS/Tween 20 buffer. One hundred microliters of tetramethybenzidine (TMB) solution reagent was added to each well and incubated at room temperature for 10 min followed by the addition of 50 X10.2 M of HZSOa. The O.D. of each sample was measured by using Universal Microplate Reader (Bio-Tek Instruments, Inc., Winooski, VM) at 450 nm.
Purified monoclonal anti-(3-galactosidase was used for the standard curve.
1o Secretion of various cytokines (IL-2, INF-y, and IL-4) from splenocytes of immunized Balb/C mice was determined by isolating splenoctyes (1 x 106 cells) and exposing to ~3-galactosidase protein for 60 hours at a concentration of either 10 ~g/mL or 100 ~.g/mL. Mouse cytokine kits were purchased from Endogen, Inc. (Woburn, MA) and used as directed.
Splenocyte proliferation assay: A CellTiter 96~ Aqueous non-radioactive cell proliferation assay kit was used to determine the isolated splenocyte proliferation. Spleens from each group of mice were pooled together and placed into 5 mL, of HBSS (Hank's Balanced Salt Solution) (1X) in a Stomacher Bag 400 from Fisher Scientific (Pittsburgh, PA). The spleens were homogenized at high speed for 60 s using a Stomacker Homogenizer. Cell suspensions were then transferred into 15 mL Falcon tube and filled to 15 mL with 1X ACi~ buffer (156 mM of NH4Cl, 10 mM
of KHC03, 2 o and 100 ~.IVI of EDTA) for red blood cell lysis. After 5-8 min at room temperature, the suspension was spun down at 1,500 rpm for 7 min at 4°C. After pouring off the supernatant, the cell pellet was re-suspended in 15 mL HBSS. The suspension was then spun down at 1,500 rpm for 7 min at 4°C.
After washing with 15 mL of RPMI-1640 (BioWhittaker, Walkersville, MD) supplemented with 10% fetal bovine serum (FBS) (Sigma, St. Louis, 1VI0) and 0.05 mg/mL of gentamycin (Gibco BRL), the cells were re-suspended in RPMI 1640 media (4-5 mL, or 1 mL for each spleen in the group pool). Isolated splenocytes (5 X 106/well) with three replicates (n=3) were seeded into a 48-well plate (Costar), and stimulated with 0 or 3.3 yg/well of (3-galactosidase (Spectrum). After incubation at 37°C with 5% CO, for 94 horns, 60 pL of the combined MTS/PMS solution (Promega) was pipetted into each well (20 pL/100 pL of cells in medimn). After an additional three hours of incubation at 37°C with 5% COZ, the absorbance at 490 nm was measured using a Universal Microplate Reader. The cell proliferation was reported as the %
increase of the OD~9° of the stimulated cells (3.3 ~.g/well) over the OD~9° of un-stimulated cells (0 pg/well) (i.e., 100 X
(OD4905h°,a,~,ea - OD490""_S~;""~ate~~OD490""-Snn,u,a'e~. Statistical analyses were completed using a two-sample t-test assuming unequal variances. A p-value of < 0.05 was considered to be statistically significant.
The following examples are offered by way illustration of the present invention, and not by way of limitation.
F.Y A MP7 .~'. 1 Plasmid DNA Solubility in Ethanol. For all studies, we utilized plasmid DNA
that contains the cytomegalovirus (CMV) enhancer and ~ promoter and the luciferase gene ligated into a pBluescupt KS-derived backbone modified to contain the kanarnycin resistance gene, Tn5 (derived from pNEO, Pharmacia, Piscataway, NJ) and with the deletion of the fl origin of replication. The plasmid DNA was obtained from GeneMedicine, Inc. (now Valentis, Inc.).
Solubility and stability of plasmid DNA in the ethanol phase is imperative in order to encapsulate large amounts of plasmid DNA in the solid ethyl cellulose nanoparticles. Fortunately, the exposure of plasmid DNA to ethanol/salt mixtures is a common technique to precipitate and 2 o purify plasmid DNA. A simple solubility experiment was performed to demonstrate that plasmid DNA remained soluble when aqueous solutions of plasmid DNA were diluted with ~95% ethanol.
To stirring plasmid DNA (1 mg in 1 mL of water) in a glass vial, ethanol was added in 25 p,L
aliquots (25 ~.L/minute) until the final solution was 95% ethanol (i.e., 19 mL
ethanol plus 1 mL
water). Our observation showed that plasmid DNA remained soluble in 95%
ethanol. At this point, 15 p,L of 5 M NaCI was added to the plasmid DNA in 95% ethanol. Plasmid DNA
immediately precipitated into a large stringy precipitate. This experiment demonstrated that plasmid DNA may be solubilized in the ethanol dispersed phase for subsequent encapsulation in the ethyl cellulose nanopaxticles. Although the final plasmid DNA concentration in this experiment twas only 50 ~g/mL, it is likely that the concentration can be increased considerably.

Ti .X A MPT .F. 7.
Selection of Fluorosurfactants. For initial testing as possible surfactants, we selected or synthesized a series of fluorinated surfactants as shown in Table 1. These fluorosurfactants have chemical moieties allowing for both association or solubility with the perflubron continuous phase (highly fluorinated chains) and association or solubility with the ethanol dispersed phase (polar head-groups).
Table 1: Fluorosurfactants Used in Preliminary Studies Fluorosurfactant Structure FSN-100 (Zonyl~) F(CFZCFZ),_9CHZCH20(CHZCHzO)_zsF~

FSO-I00 (Zonyl~) F(CFZCFZ),_~CHzCHZO(CHZCH20)_,5H

Pentadecafluorooctanoic CF3(CF2)6COOH
acid D2 Proprietary structure Tridecatluoro-1-octanol CF3(CF2)5(CHZ)~OH
(D3) D4 Proprietary structure Tridecafluoroheptanoic acidCF3(CF~)SCOOH
(DS) Perfluorotetradecanoic acidCF3(CF,),~COOH
(D6) Perfluorododecanoic acid CF3(CFZ),COOH
(D7) PentadecafluoromethyloctanoateCF3(CFJ6COOCH3 (D8) Octanoic acid (Control) CH3(CH~)SCHZCOOH
~

Pseudo-Phase Diagrams: Fluorosurfactant Screen. Studies focused on E/F
microemulsions that allowed for maximum solubilization of ethanol. E/F microemulsion systems having higher ethanol content allow for increased concentration of plasmid DNA in the systems and ultimately s5 the cured solid nanoparticles. To achieve maximum solubilization of ethanol, we constructed classical pseudo-phase diagrams for microemulsions (Bhargava et al., 1987) using a matrix screening approach for each fluorosurfactant candidate. Briefly, a small amount of ethanol was added to defined perflubron (X%)/(fluorosurfactant) (100-X%) mixtures in an attempt to solubilize ethanol and define the microemulsion window (i.e., portion of diagram that represents a clear 2o system). A 0.5 g mixture of perflubron (X = 70% to 95% w/w) and fluorosurfactant (S% to 30%
w/w) was prepared in glass vials. While the mixture was stirring, ethanol was added in S ~L (= 4.1 pg ethanol) aliquots. The clarity (transparency) of the systems as a function of the percentages of the three phases was plotted in order to define the microemulsion window, if any. This screen was repeated for each of the fluorosurfactant candidates. The maximum amount of ethanol incorporated into a stable clear microemulsion for selected fluorosurfactants is shown in Table 2.
Table 2: Incorporation of Ethanol in E/F Microemulsions Using Different Fluorosurfactants Fluorosurfactant Final (Ethanol) % w/w Solubilized in Perflubron/Fluorosurfactant (70/30 w/w) FSN-100 10.1 FSO-100 18.8 Pentadecafluorooctanoic acid 37.2 (PDFOA) D4 32.0 Perfluorotetradecanoic acid 66.6 (D6) Perfluorododecanoic acid (D7)59.2 I

As expected, no E/F microemulsion could be formed with the octanoic acid control due to the fact Z o that the molecule is not fluorinated. The microemulsions shown in Table 2 were clear and stable throughout the microemulsion window, except for the system made with D4. The use of D4 produced a slightly opaque system. The use of D6 and D7 resulted in microemulsions that apparently solubilized a high amount of ethanol in perflubron. However, it is very likely that these microemulsions were actually reverse perflubron-in-ethanol systems based on mathematical modeling of surfactant-coated ethanol or perflubron droplets (see examples of similar calculations shown later). Fluorosurfactants D2, D3, D5, and D8 were all soluble in perflubron, and we have not yet determined the microemulsion windows for systems made using these surfactants.
FXAMPT.F 4 Preparation of Stable E/F Microemulsions Containing Filin-Forming Polymers.
After demonstrating that ethanol could be incorporated into E/F microemulsions in a sufficient amount, we then sought to incorporate ethyl cellulose dissolved in ethanol inta the same microemulsion.
Pharmaceutical grade ethyl cellulose with National Farmulary (NF) designation was obtained from Hercules, Inc. (Wilmington, DE). Six different molecular weights, all having 48.0-49.5% ethoxyl content, were obtained (ethyl cellulose N7, N10, N14, N22, N50, and N100). Our previous research in film-forming ethyl cellulose-based gels showed that films made with 1:1 blends of the higher molecular weight ethyl celluloses, N50 and N100, produced the strongest films. We deduced from this that the same blend would produce the most stable solid nanoparticles when exposed to biological fluids. Our intent was to identify an E/F microemulsion that could incorporate as high of a weight percentage of N50 and N100 as possible.
Fortunately, the higher molecular weight ethyl cellulose polymers also precipitate more rapidly than the lower molecular weight polymers when ethanolic solutions of these polymers are exposed to water. More rapid precipitation of ethyl cellulose in the E/F microemulsions may be needed to entrap a greater amount 1G of plasmid DNA. For these experiments, we added ethanol containing various types and concentrations of dissolved ethyl cellulose to perflubron/surfactant (70:30 w/w) mixtures. The mixtures were visually observed for the existence of the expected microemulsion window and/or whether ethyl cellulose remained dissolved in the E/F microemulsion. For all E/F microemulsions, the incorporation of ethyl cellulose had no effect on the formation of the E/F
microernulsions or the width of the microemulsion window. As shown in Table 3, the incorporation of ethyl cellulose into the E!F microemulsion was maximized using the PDFOA surfactant. In an E/F
microemulsion rriade with perflubron (0.508 g), PDFOA (0.214 g), and ethanol (0.228 g) with 5% ethyl cellulose, the stable microemulsion contained 11.4 mg of dissolved ethyl cellulose (NSO/N100 1:1 w/w) 2 o Table 3: Incorporation of Ethyl Cellulose Filin-Forming Polymers into the Ethanol Dispersed Phase of E/F Microernulsions Fluoro Ethyl Cellulose Maximum Conc. Of Ethyl Cellulose surfactantT a In Ethanol in E/F Microernulsions FSN-100 N50/N100 (1:l) < 0.25%

FSN-100 N50 0.25%

FSN-100 N22, or N14 0.50%

FSN-100 N10 1.0%

FSO-100 N50/N100 (1:l) or < 0.25%

PDFOA N50/N100 (1:1) 5%

Modeling of E!F microemulsions: An ethanol-in-fluorocarbon (ElF) microemulsion was made having the following components: perflubron (0.7382 g), PDFOA (0.3195 g), and ethanol (0.3603 g). The weight of ethanol corresponded to 25.4% w/w of the final microernulsion or 65.7%
of the microemulsion window (see Figure 2). The average droplet size of 34.9 nm determined by photon correlation spectroscopy (dynamic light scattering at 11.3° for 300 seconds) agreed very well with the predicted ethanol droplet size based on simple mathematical modeling of surfactant-coatetl ethanol droplets. The total surface area of ethanol droplets that have an average diameter.of 34.9 nm can be calculated in a series of equations as follows:
Volume, of one ethanol droplet (Equation 1) _ ~d3/6 = - 2.226 x 10-z' cm3 ~_ 5 Total number of ethanol droplets (Equation 2) _ (0.3603 g * 0.814 g/cm3)/2.258 x 10-" cm3 = 1.989 x 1016 droplets Total sW face area of each droplet = ~d' = 3.827 x 10'" cm2 (Equation 3) 2 o Total surface area of all ethanol droplets (Equation 4) = 1.989 x 10'6 droplets * 3.827 x 10-" cm'-= 760966 cm'.
If we assume that PDFOA head-group (Mw 414.06 g/mol) occupies a space of 301', then we can calculate the number of PDFOA molecules needed to cover the total surface area of all ethanol 2 5 droplets from Equation 4 as:
Total number of PDFOA molecules needed to cover the (Equation 5) total surface area of all ethanol droplets =760966 cmz/30 x 10-16 cmz = 2.537 x 1 OZ° molecules of PDFOA.
JO

Thus, 2.537 x l OZ° molecules of PDFOA would theoretically be needed to coat all of the surface area provided by all of the ethanol droplets. The ratio of ethanol to PDFOA
molecules 65.7%
through the microemulsion window was 10:1. We showed that if we continued adding ethanol to this E/F microemulsion to reach 100% of the microemulsion window, a total of 833 p,L (or 0.678 g) ethanol could be added until the microemulsion became turbid. Deriving the same equations as shown above, it can be demonstrated that theoretically 0.3282 g PDFOA is needed to form a microemulsion incorporating 0.678 g ethanol (8.864 x 10'' molecules) into droplets having an average droplet size of 34.9 nm. The actual amount of PDFOA used to prepare the microemulsion was 0.3195 g which was within 3% of the predicted amount (0.3195 g/ 0.3282 g =
0.973 or 97.3%) 2 o based on the measured droplet size of ethanol. Thus, this simple modeling supports the formation of a E/F microemulsion as well as the particle sizing method used.
F.xewrpr.F ~
To determine the existence of an oil-in-water microernulsion window for the ~5 microemulsion precursor, exactly two (2) milligrams of emulsifying wax were weighed accurately into ten separate 7-mL glass vials and melted at 50°C on a temperature calibrated magnetic hot plate. Water (0.2 ~,m filtered) was then added (750-1000 ~,L) to . form a homogeneous milky slurry in the stirring water at 50°C. To form the microemulsion precursor, the surfactant polyoxy 20 steer-yl ether (100 mM) in water was added (0-250 ~,L) so that the final 2 o surfactant concentration ranged from 0 mM to 25 mM in the ten vials. The microemulsion precursor was then removed from heat (52-54°C) and allowed to cool to 25°C while stirnng.
When cooled, visual inspection showed that systems with final surfactant concentration less than 2.5 mM were precipitated, systems with final surfactant concentration between 2.5 mM and 10 mM were either very slightly turbid or clear, and 'systems with a final surfactant concentration 25 greater than 10 mM were either very turbid or precipitated. Thus, an apparent microemulsion window was defined. One hundred (100) ~L of each cooled system was taken and diluted with 900 ~L water. The particle size of the diluted solid nanoparticles was determined using a Coulter N4 Plus Sub-Micron Particle Sizer at 20°C by scattering light at 90° for 120 seconds. The particle sizes of the cured solid nanoparticles as a function of surfactant concentration are shown 3 o in Figure 3. The particle sizes of systems with no surfactant addzd could not be determined since the systems contained precipitates that were greater than 3000 nm in diameter.
In general, the particle size results agreed with the visual observations and suggested the following; 1) solid nanoparticles less than 100 nm could be engineered from the liquid matrix oil-in-water microemulsion precursor, and 2) the resulting clarity and particle size were related to the final concentration of the surfactant used. The droplet size of the oil phase in the microemulsion nanotemplates made with a final surfactant concentration of 10 mM was measured at 55°C and was found to be 11 + 3 nm demonstrating that oil-in-water microemulsion precursor could be made. To determine if the measured droplet sizes in either the microemulsion nanotemplate or the cured solid nanoparticles were due to the presence of surfactant micelles, samples were made Zo as described above with no emulsifying wax and with final surfactant concentrations ranging from 0 mM to 100 mM in water. Interestingly, no published critical micellar concentration (CMC) value could be found for polyoxyethylene 20 stearyl ether. It is likely that the relatively heterogeneous nature of the polymeric surfactant makes the determination of its CMC difficult using conventional techniques. Photon correlation spectroscopy, using a Coulter N4 Plus Submicron Particle Sizer, was used to determine the existence and the size of the surfactant micelles. The results indicated that the surfactant does begin to form micelles (5-20 nm) between .
a concentration of 0.5 mM to 1 mM in water. However, these micelles are clearly absent in the cured solid nanoparticles indicating that the 50-100 nm nanoparticles could be engineered directly from the microemulsion precursors.
ao F.X A MPT .F. 7 Stability of cured emulsifying wax nanoparticles over time: Emulsifying wax nanoparticles (2 mg/mL) were prepared as described in Example 6 using final concentrations of Brij 78 surfactant between 3 mu'VI and 15 mM. The particle size of cured nanoparticles was determined both at 10 minutes and 24 hours after curing. For particle size analysis, one hundred microliters (100 ~,L) of each preparation was taken and diluted with 900 ~,L
distilled water. As shown in Figure 4, although cured nanoparticles made with' higher concentrations of Brij 78 surfactant were initially smaller than those made with lower concentrations of Brij 78, the nanoparticle made with higher concentrations of Brij 78 agglomerated to larger particles over 24 3o hours. Ideally, a final surfactant concentration of 3 mM produced stable nanoparticles. These findings were unexpected and the reason for this phenomena is still unknown.
It is clear, however, that a non-obvious and optimal amount of surfactant is needed to both engineer stable microemulsion precursors as well as stable cured nanoparticles from these precursors.

Preparation of Brij 72 nanoparticles. Three separate samples of Brij 72 nanoparticles were engineered using the following process. Brij 72 (2 mg) was melted at 50-55°C and dispersed in 970 microliters of water at the same temperature. Thirty microliters of solution of Tween 80 (10% v/v in water) was added to produce a clear oil-in-water microemulsion at approximately 55°C. The oil droplet size of liquid Brij 72 was measured by photon correlation spectroscopy to be 22.2 + 1.8 nanometers at approximately 55°C. Brij 72 nanoparticles were cured by three different methods as follows: Method A) cooling of the undiluted oil-in-water microemulsion at 55°C to room temperature while stirring, Method B) cooling of the oil-in-water microemulsion at 55°C by placing undiluted in a refrigerator at 4°C, and Method C) diluting 15 (1/10) the oil-in-water microemulsion at 55°C with water at 4°C. The results as shown in Figure demonstrate that the method of curing had no effect on the size of nanoparticles formed.
Further,~Method A illustrated a key advantage of simply allowing the oil-in-water microemulsion to cool to room temperature to form useful solid nanoparticles. This method allows for rapid, reproducible, and cost-effective method to engineer useful nanoparticles.
~F'.XAMPT.F 9 Preparation of emulsifying wax nanoparticles. Three separate samples of emulsifying wax nanoparticles were engineered using the following process. Emulsifying wax (2 mg) was melted at 50-55°C and dispersed in 970 microliters of water at the same temperature. Thirty microliters 100 mM Brij 78 were added to produce a clear oil-in-water microemulsion at approximately 55°C. The oil droplet size of liquid emulsifying wax was measured by photon correlation spectroscopy to be 24.5 + 0.4 nanometers at approximately 55°C. Emulsifying nanoparticles were cured by three different methods as follows: Method A) cooling of the undiluted oil-in-water microemulsion at SS°C to room temperature while stirring, Method B) 3o cooling of the oil-in-water microemulsion at 55°C by placing undiluted in a refrigerator -at 4°C, and Method C) diluting (1/10) the oil-in-water microemulsion at 55°C
with water at 4°C. The results as shown in Figure 6 demonstrate that the method of curing had no effect on the size of nanoparticles formed. Further, Method A illustrated a key advantage of simply allowing the oil-in-water microemulsion to cool to room temperature to .form useful solid nanoparticles. This method allows for rapid, reproducible, and cost-effective method to engineer useful nanoparticles.
Further, the solid nanoparticles made from Method A were subjected to ultracentrifugation at 50,000 rpm for 30 minutes. Photon correlation spectroscopy analysis showed that these ultracentrifugation conditions had no effect on the intensity of light scattering or particle size indicating a very stable colloidal suspension.
EXAlVIPLE 10 Stability of nanoparticles in biological conditions: To assess the potential stability of nanoparticles in biological media, Brij 72 nanoparticles (2 mg/mL) were diluted 1:10 with 10%
fetal bovine serum (FBS), 10 mM phosphate buffered saline (pH 7.4), IO%
lactose, or I50 mM
ufaCl. The particle size of nanoparticles in each media Was monitored for 60 minutes at 37°C. As shown in Figi.~re 7, cured Brij 72 nanoparticles challenged with various biological media at 37°C
were found to be stable over 60 minutes wider all conditions.

Incorporation of cell-specific ligand on nanoparticles: To determine the feasibility of adding a hydrophobized cell-specific targeting ligand to the cured solid nanoparticles, asialofeW in-palmitate (ASF pal) was synthesized and purified. Asialofetuin was derivatized with about 12 palmitate 'arms' per molecule as measured by a colorimetric hydroxamic acid reaction assay. ASF-pal (1-100 ~,L; 13.4 ~,g/mL water) was added to cured solid nanoparticles in water so that the final concentration of nanoparticles was 200 ~,g nanoparticles per 1 mL. Stirring was continued at 25°C
for a total of 1'hour to ensure complete adsorption/insertion of the palinitate arm of ASF-pal into the nanoparticles. The results as shown in Figure 8 demonstrate that even very high concentrations of ASF-pal could be added to the nanoparticles with only a small effect on the particle size. As -4l-controls, the particle size of ASF-pal alone in water at a concentration of either 67 ~,g/mL or 1340 ~g/xnL were measured. The results showed that ASF-paI formed micelles (3-15 nm) at 67 p,g/mL
At a concentration of 1340 ~.g/mL, ASF-pal formed a mixture of micelles (3-10 mn) as well as .
larger aggregates (40-300 nm). It was apparent from these results that a hydrophobized cell-specific targeting ligand could be added to cured nanopartieles.

It was discovered that the solubility of Gadolinium acetylacetonate (GdAcAc), a potential agent for neutron capture therapy of tumors, in water could' effectively be increased :by at least Z o 4000-fold using the methods described in this invention. Specifically, the solubility of GdAcAc is only 1 mg per 2000 mL water However, utilizing the said methods described in this invention to entrap GdAcAc in stable nanoparticles having diameters of about 50 nanometers, only 1 milliliter of water is required to solubilize 2 mg GdAcAc. Various amounts (0.1 mg to 1 mg) of gadoliniuri2 acetylacetonate were entrapped in both emulsifying wax and Brij 72 nanoparticles (2 15 mg/mL). As shown in Figure 9, the entrapment of GdAcAc had little or no effect on the resulting particle sizes of the cured nanoparticles. Entrapment efficiencies of GdAcAc in nanoparticles were determined using gel permeation chromatography (Sephadex G75; 30 cm x 0.5 cm cohunn) with water as the mobile phase. One hundred microliters (100 ELI,) of nanoparticles (2 mg/mL) containing GdAcAc (0.5 mg/mL) was eluted down the column. Each fraction (1m1) was 2 o monitored acing light both scattering (counts per second) and LTV
absorption of GdAcAc (at 288 nm). As shown in Figure 10, GPC analysis confirmed that GdAcAc co-eluted with nanoparticles .
and that the apparent entrapment efficiency of GdAcAc in nanoparticles was approximately 100%.

Preparation of cationic nanoparticles made from microemulsion~orecursors:
Exactly 2 mg of emulsifying wax was placed into six 7-ml glass scintillation vials.
After melting at 50-55°
C, water was added to form a homogenous milky slurry. Different volumes of a hexadecyltrimethylammonium bromide (CTAB) stock solution (50 mM in water) was added 3o while stirring to obtain a final CTAB concentration of 5 ~0 30 xnM. After 3-5 min, the milky slurry turned clear or stayed cloudy, depending on the amount of CTAB used.
The droplet size of the microemulsion was measured at 55°C using a Coulter N4 Plus Submicron Particle Sizer (Coulter Corporation, Miami, FL) at a 90° angle for 90 seconds. These microemulsions were then cooled down (cured) to room temperature while stirring to form nanoparticles.
The nanoparticle suspension was diluted 10 times with water (0.22 mm filtered) and the particle size was measured as above. When the required volume of CTAB (50 mM) solution was added into the milky slurry wax in warm suspension, the suspension turned clear within seconds if the final CTAB concentration was greater than 10 mM. For the samples with final CTAB
concentration of S mM or below, the samples turned slightly turbid. As shown in Figure I l, the droplet sizes of Zo the warm microemulsions (at 55°C) were in the range of 30-70 nm and cured nanoparticles (at 25°C) were in the range of 60-120 nm.

Preparation of cationic nanoparticles with adsorbed plasmid DNA for genetic 15 immunization: Cationic nanoparticles comprised of 6 mg emulsifying wax per 1 mL water containing a final concentration of 15 mM CTAB were prepared as described in Example 13. Free CTAB was separated from the cured nanoparticles using a Sephadex G-75 column (14 x 230 mm) and using 10% lactose as the mobile phase. Two milliliters of the cured nanoparticle suspension.
was applied to the column. The particle size and zeta potential of the purified cationic 2o nanoparticles was measured and found to be 99 ~ 27 ntn and 35.8 + 2.3 mV, respectively. Plasmid DNA (CMV-~3-galactosidase) was coated on the surface of the nanoparticle by gently mixing the required amomt of pDNA and nanoparticle suspension to obtain a final pDNA
concentration of 400 ~.g/mL. After the addition of pDNA to the cationic nanoparticles, the particle size and zeta potential of the pDNA-coated nanoparticles was 245 + 25 nm and -4.7.7 ~ 1.2 mV, respectively.
25 The change in particle size and zeta-potential demonstrated that pDNA was successfully coated on the cationic nanoparticles. pDNA-coated nanoparticles and 'naked' DNA were administered to Balb/C mice (10-I2 weeks old) by three different routes (intramuscular injection, i.m.;
subcutaneous injection, s.c., or by topical application to skin) on day 0, 7, and 14. The pDNA dose on each day was 40 p.g. On day 28, the IgG titers in sera were determined and are plotted in Figure 30 12. Sera IgG titers at day 28 resulting from immunization by pDNA-coated nanoparticles and 'naked' DNA after intramuscular and subcutaneous administration were comparable. However, a surprising finding was observed after topical administration of formulations to slcin. Mice immunized with pDNA-coated nanoparticles had an approximately 10-.fold increase in IgG titers over mice immunized with 'naked' pDNA.

Cationic emulsifying wax nanoparticles containing two different concentrations of CTAB
were prepared as described in Example 13 above. Plasmid DNA (CMV-(3-galactosidase) was coated on the surface of the nanoparticle by gentle mixing to form, 1) a pDNA-coated nanoparticle 'having a net positive charge with pDNA at a final concentration of 40 ~g/mL, and 2) a pDNA-coated nanoparticle having a net negative charge with pDNA at a final concentration of 40 p,g/mL.
pDNA-coated nanoparticles (negatively-charged and positively-charged) and 'naked' DNA were administered to Ba.lb/C mice (10-12 weeks old) by three different routes (intramuscuhr injection, i.m.; subcutaneous inj ection, s.c., or by topical application to skin) on day 0, 7, and 14. The pDNA
:i 5 dose on each day was 4 ,ug. The IgG titers in sera were determined and are plotted in Figure 13.
The results were similar to those obtained and reported in Example 14 above.
Sera IgG titers at day 2~ resulting from immunization by pDNA-coated nanoparticle were lower than 'naked' pDNA for the 4 pg doses after both intramuscular and subcutaneous administration.
However, mice immunized with pDNA-coated nanoparticles by topical application to skin showed up to 1-2 log 2 o increases in TgG titers as compared to mice immunized with 'naked' pDNA.
As shown in Figure 14, mice immunized with pDNA-coated nanoparticles induced greater IL-2 production hom stimulated splenocytes by all three routes of administration. For example, IL-2 production from stimulated splenocytes was approximately 3-fold higher after immunization with pDNA-coated nanoparticles by both intramuscular and subcutaneous injection as compared to 'naked' pDNA. As 2 5 shown in Figure 15, TFN-y production from stimulated splenocytes was comparable after immunization with pDNA-coated nanoparticles and 'naked' pDNA by all three routes. As shown in Figure 16, IL-4 production from stimulated splenocytes was comparable after immunization with pDNA-coated nanoparticles and 'naked' pDNA by all three routes, except for subcutaneous administration of pDNA-coated nanoparticles that were net negatively-charged.
Immunization with these pDNA-coated nanoparticles resulted in an approximately 2.4-fold increase in . IL-4 production from stimulated splenocytes over 'naked' pDNA given by the same route.

Confirmation of cell-specific ligand on the surface of cured emulsifying wax nanoparticles:
Cholesterol-marman (Chol-mannan) was purchased from Dojindo (Gaithersburg, MD). Various amounts of ~ Chol-mannan were incorporated into cured nanoparticles either during the preparation of the o/w microemulsion precursor or by adsorbing it on the surface of cured nanoparticles as described in Example 11. Mannan-coated nanoparticles were purified by GPC to remove Z o . unincorporated or free, chol-mannan. An in-vitro agglutination was be used to verify that mannan was on the surface of GPC purified nanoparticles. Con-A is tetrameric protein with four binding sites specific for terminal glucosyl or ma~~nosyl residues. Binding to the mannan will cause agglutination (or aggregation) of the complex in solution resulting in an increase in turbidity. This assay was performed at room temperaW re by adding various samples to Con-A (1 mg/mL) in 25 phosphate buffered saline, pH 7.4 with 5 mM calcium chloride and ~ mM
magnesium chloride and monitoring the increase in turbidity at 360 nm for 200 seconds. As shown in Figure 17, as expected, nanoparticles alone (uncoated) or pDNA alone resulted in no detectable agglutination of , Con-A over 200 seconds. In comparison, mannan-coated nanoparticles and mannan-coated nanoparticles containing pDNA (at a concentration of 100 p,g/mL or 150 p,g/mL) caused significant 2o agglutination of Con-A over 200 seconds as confirmed by the increase in absorbance. It was also shown that a "mannan negative control" also produced no agglutination of Con-A. This negative control was taken from the same fraction that nanoparticles normally elute from the GPC column (fraction 2-4). Tlus confirmed that the positive agglutination results Were not caused by co-elution of nanoparticles with unincorporated chol-mannan.

Emulsifying wax nanoparticles made with CTAB as the surfactant were prepared.
Chol-mannan and/or pDNA (CMV-(3-galactosidase) were coated on the surface of the nanoparticles as described in Example 16 to prepare the following formulations, 1) pDNA alone, 2) mannan-coated 3 o nanoparticles with pDNA, 3) pDNA-coated nanoparticles, and 4) mannan plus free pDNA as a control. These formulations were administered to Balb/C mice (10-12 weeks old) by subcutaneous injection on day 0, 7, and 14. The pDNA dose on each day was 10 p,g. The IgG
titers in sera were determined on day 28 and are plotted in Figure 18. Results are expressed as the individual titer for each mouse. All groups had 5 mice except for pDNA which had 4 mice. As shown in Figure 18, the mean sera IgG titers at day 28 for all formulations were comparable. Two mice immunized with 'naked' pDNA had IgG titers that were clearly higher than any other mice, however, one mouse immunized with 'naked' pDNA could be considered a non-responder. As shown in Figure 19, mice immunized with pDNA-coated nanoparticles or mannan-coated nanoparticles showed up to 2-fold greater IL-2 production from stimulated splenocytes as compared to 'naked' pDNA. As shown in Figure 20, IFN-y production from stimulated splenocytes (100 ~Cg ~-gal/mL) was comparable after immunization with pDNA-coated nanoparticles or mannan-coated nanoparticles and 'naked' pDNA. However, IF'N-y production from stimulated splenocytes (10 p.g [3-gal/mL) .
was up to 4-fold greater with pDNA-coated nanoparticles or mannan-coated nanoparticles as compared to 'naked' pDNA. Finally, as shown in Figm-e 21, IL-4 production from stimulated splenocytes was significantly higher for all groups as compared to 'naked' pDNA ,which showed IL-4 levels near or at background.
E~~AMPLE 18 Entrapment of plasmid DNA into nanoparticles engineered from microemulsion precursors:
2 o A significant challenge to the use of the new O/W microemulsion precursors is that plasmid DNA, a highly negatively-charged hydrophilic molecule, would have to be contained in the oil phase if it were to be entrapped in the solid nanoparticles. To this end, a series of positively charged s~.~rfactants or lipids were screened as potential agents to complex and 'hydrophobize' plasmid DNA as described previously (tiara et al., 1997; Yi et al., 2000; Liu et al., 1996; tiara et al., 1997).
Among the several hydrophobizing candidates investigated was DOTAP (1,2-dioleoyl-sn-glycero-3-trimethyl-ammonium-propane), an ester-linked biodegradable Lipid. A
DOTAP/plasmid DNA
complex (1.5:1 -/+ verify) with plasmid DNA at a final concentration of 40 pg/mL was added to the following 1 mL total volume formulation: emulsifying wax (2 mg), Brij 78 (10 mM), and 50 p.L, of Tween 20 (1:3 w/w diluted with water). The formulation was briefly heated to 52°C to form an 3o O/W microemulsion and then cooled to 25°C to form nanoparticles containing plasmid DNA. The nanoparticle size was measured as 58.5 + 26.8 rm. To verify that plasmid DNA
was entrapped in the nanoparticles, an identical formulation was made using fluorescein labeled plasmid DNA (final 20 yg/mL). Formed nanoparticles were purified via Sephadex-G75 gel permeation chromatography and the fractions were detected using photon correlation spectroscopy (to obtained light scattering result, cps) or fluorescence spectroscopy (to obtained plasmid DNA.concentration). The results as shown in Figtue 22 show that fluorescein labeled plasmid DNA did elute in the same fractions as those of solid nanoparticles. To further confirm that the plasmid DNA is entrapped in the solid nanoparticles and not adsorbed onto the nanoparticles, the preparation was treated with DNase I, a nuclease that rapidly degrades plasmid DNA. The results as shown in Figure 23 demonstrate that to nuclease treatment for 15 minutes at 37°C failed to degrade plasmid DNA and suggest that plasmid DNA was entrapped in the cured 58 nm nanoparticles.

Preparation of liquid hydrocarbon-in-fluorocarbon microemulsion precursors: To ~5 demonstrate that liquid matrix in perflubron microemulsions can be formed, the following experiment was completed. PDFOA (94.7 mg) was added to 500 yI~ perflubron in a scintillation vial under gentle magnetic stirring. PDFOA did not dissolve in perflubron at 25°C. Wlute beeswax, USP (33.4 mg) was added as a solid to the PDFOA suspended in the stirring perflubron.
The vial was heated to 60°C and the mixture became a clear homogenous solutian. The mixture 20 was removed from heat and within 1 minute the mixture slowly turned opaque.
After 5 minutes, the precipitated wax agglomerated into a clump. As a control, the experiment was completed without the PDFOA. Melted wax and perflubron were not miscible at 60°C
indicating that the fluorost~rfactant was needed. The results demonstrated that liquid matrix O/F
microemulsion was possible, but that a more suitable nanoparticle matrix material (that did not agglomerate) was z 5 needed.

The stability of pDNA-coated nanoparticles in various media at 37°C:
The particle size of the pDNA-coated nanoparticles (net negatively-charged) was measured in various media at 30 37°C immediately after dilution (0 min) and after 30 min. Plusmid DNA was added to a suspension of GPC purified and filter sterilized cationic emulsifying wax nanoparticles so that the final pDNA concentration was 75 ~ig/mL and the f nal zeta potential of the pDNA-coated nanoparticles was -22 + 1 mV. For particle size measurements, 100 1.~L of each sample was diluted with 900 p,L of 150 mM NaCl, 10% FBS1150 mM NaCI, or 10% lactose. The results of the stability study, as shown in Figure 24, demonstrated that the nanoparticles with pDNA were stable over 30 minutes at 37°C in normal saline, 10% FBS/150 mM NaCl, and 10% lactose.

I3Z-VLLT"O transfection of liver HepG2 cells with pDNA-coated nanoparticles:
Hep G2 cells Zo were obtained from American Type Culture Collection (ATCC, Rockville, MD) and were maintained in Eagle's Minimum Essential Medium (EMEM) (Gibco, BRL) media containing 10% fetal bovine serum (Gibco, BRL) and 1% penicillin-streptomycin (Gibco, BRL).
Transfections were performed with cells that were approximately 80% confluent.
Cells were plated in 48-well plates at a cell density of 5 X 10' cells/well and incubated overnight. The cells were incubated with the formulations having a plasmid dose of 2.5 p,g/well.
Cells were harvested at 52 h by removing the media, washing with 1X PBS buffer, and then adding 200 1.~L 1X Lysis buffer (Promega, Madison, WI) for 5-10 min and then freeze-thawing three times. Luciferase activity was assayed as described previously. Net positively-charged pDNA-nanopaxticles were prepared from pre-formed cationic nanoparticles comprised of emulsifying wax (6 mg/mL) and 15 mM CTAB. The pDNA-coated nanoparticles had a particle size and zeta potential of 221 ~ 5 nm and 44 + 2 niV, respectively. The particle size and zeta potential of the other modified positively-charged nanoparticles used in the transfection studies were comparable. The net negatively-charged nanoparticles were prepared from pre-formed cationic -nanoparticles comprised of emulsifying wax (2 mg/mL) and 15 mM CTAB and had a particle size and zeta ~5 potential of 220 + 4 nm and -23 ~ 2 mV, respectively. HepG2 cells (50,000 cells/well) were transfected in the presence of 10% FBS after 52 hr with: pDNA (Neg), Lipofectin~ (LPFN), nanoparticles (I~ with 5% (w/w) DOPE (N+D), and pullulan-coated nanoparticles (PN). For PN-2 and PN-3, 50 p,g and 250 p,g free Cholesterol-pullulan was added 30 min prior to pullulan-coated nanoparticles to block the glucose receptors. The pDNA dose was 2.5 p.g for all samples.

As recommended by Dynatech, luciferase expression data were reported as the ratio of the .full integral of the samples to that of the negative control (Neg), divided by the total amount of protein in the 20 ~,l of samples assayed. The results, as shown in Figure 25, demonstrate that coating of pDNA ,on the surface of the cationic nanoparticles significantly enhanced the in-vitYo cell transfection, compared to that of'naked' pDNA. As expected, the incorporation of DOPE, an endosomolytic lipid, increased the transfection ability of the nanoparticles by over 5-fold (N+D
vs. N) (p = 0.000. Surface coating of a HepG2 cell-targeting ligand, pullulan, a polysaccharide of glucose, increased the transfection ability of the pDNA-coated nanoparticles by over 40-fold .
(PN vs. N) (p = 0.03) to levels that were comparable to that of the Lipofectin°. The enhanced cell 1 o transfection ability of the pDNA-coated nanoparticles with pullulan can be at least partially attributed to receptor-mediated endocytosis since pre-incubation of the HepG2 cells with free cholesterol-pullulan for 30 min significantly reduced the transfection efficiency of the pullulan-coated pDNA-nanoparticles, in proportion to the amount of the free cholesterol-pullulan added.
1 s EXAMPLE 22 Genetic immunization via the subcutaneous route: Plasmid DNA (CMV-(3-galactosidase) was coated on the surface of the cationic nanoparticles to obtain a final pDNA
concentration of 5 ,ug/100, p,L. Three different pDNA-coated nanoparlicle formulations, 'naked' pDNA,~ and (3ga1 protein (10 pg) adjuvanted with 'Alum' (15 p,g) were administered to mice by subcutaneous 20 injection (100 ~.L) to BaIbJC mice on day 0, 7, and 14. The IgG titer in serum of mice immunized with NPs was enhanced by 3-fold over that of 'naked' pDNA, although the result was not statistically significant (Figure 26). Mannan coating enhanced the immune response by 10-fold over that of'naked' pDNA. Again, this enhancement was not statistically significant (p=0.065) since one of the S mice in the NP group was a very low responder. However, a combination of both DOPE
2 5 and cholesterol-mannan on the NPs (Man-NPs/DOPE) enhanced the IgG titer by more than 16-fold over that of 'naked' pDNA (p=0.005). Both fecal IgA and serum IgA (by OD 450 nm) were enhanced with NP groups over 'naked' pDNA (data not shown). In-vitf°o cytokine release from isolated splenocytes after stimulation with (3-gal was also investigated (Table 4). hnmunization with pDNA-coated NPs resulted in a strong Thl biased cytokine release as demonstrated by the significant increase (up to 300%) of Thl-type cytokines IL=2 and IFN-y. As expected, 'Alum' adjuvanted (3-gal protein resulted in very low levels of IL-2 and IFN-y. The incorporation of DOPE
in the NPs'as well as the coating of the NPs with mannan significantly enhanced Thl-type cytokine release over that of the DOPE-free and mannan-free NPs. In contrast, 'Alum' adjuvanted ~i-gal protein resulted in high levels of IL-4 release, whereas all nanoparticle groups and 'naked' pDNA' resulted in significantly lower, although still positive, IL-4 release.
~. o Table 4: In-Vitro Cytokine Release from Isolated Splenocytes Thl type Th2 type IL-2 IFN-~y~ IL-4 ~PgJmL~) lPgimL) ~PgimL) Native 546 + 54 570 + 28 < 1 pDNA-IVPs ~ 2300 + 167 15098 + 360 8.6 + 2.4 lVIannan-pDNA-NPs 4540 + 346b 24902 + 1619' 25.4 + 5.5e Mannan-pDNA-NPs with DOPE 4044 + 479b 28006 + 1597°'d 57.7 +
4.3e°f 'naked' pDNA 1761 + 164a 19971 + 393 54.4 + 4.5e>c R_gal + 'Alum' 171 + 13 678 + 80 164.4 + 14.5 In-vitro cytokine release from isolated splenocytes (5 x 106 cells) exposed to ~3-galactosidase protein for 60 lir. l~Iice were immunized with 5 P.g pDNA or 10 qg (3Ga1 (with 15 P,g 'Alum') on day 0, 7, and 14 by subcutaneous injection. On day 28, the spleens were removed and pooled for each group.
Isolated splenocytes (5 X 106/well) with three replicates were stimulated with (3-galactosidase protein (3.3 ~,g/well) for 60 hours at 37°C. Cytokine release was quantified ELISA kits (Endogen).

ap<0.05 over all nanoparticle groups; by<0.05 over pDNA-NPs; °p<0.05 over pDNA-NPs; dp<0.05 over Matman-pDNA-NPs; ep<0.05 over pDNA-NPs; fp<0.05 over Mannan-pDNA-NPS;
°°p<0.05 over all other groups. The reported data are the mean + standard deviation Statistical analysis was completed using a two-sample t-test assuming unequal variances. A result of p<
0.05 was considered statistically significant.

The effect of nanoparticle matrix material concentration on the microemulsion droplet size and the cured cationic nanoparticle size: The effect of emulsifying wax concentration on the z o microemulsion droplet size at 55°C (black bars), and both the cm-ed cationic nanoparticle size (white bars) and zeta potential (~) at 25°C. Microemulsion precursors (1 mL) were prepared at 55°C with emulsifying wax at either 2, 4, 6, or 8 mg/mL in water using CTAB (15 mM) 'as the cationic surfactant. Emulsifying wax nanoparticles having a final concentration from 2-8 mg/mL
were engineered by simple cooling of the microemulsion precursors to room temperature. The data reported are the mean + standard deviation for three replicates (n=3). As shown in Figure 27 emulsifying wax nanoparticles could be engineered with a final concentration of 6 mg/mL while.
still retaining a particle size below 100 nm. In contrast, nanoparticles engineered with a final emulsifying wax concentration of 8 rng/mL had a particle size of 137 + 9 mn.
For all samples, the microemulsion droplet sizes at 55°C were in the range of 51-58 nm demonstrating that cationic 2 o nanoparticles could be ' engineered directly from O/W microemulsion precursors. The zeta potentials of cationic nanoparticles having final emulsifying wax concentrations from 2-8 mg/mL
ranged from +58 mV to +65 mV. Cationic nanoparticles having final concentrations of 2 mg/mL
and 6 mg/mL were used for subsequent pDNA coating experiments since these systems had sufficiently small size (94 + 5 nm and 100 ~ 8 nm, respectively) and positive zeta-potentials (+60 +
4 mV and +61 + 2 mV, respectively).

Genetic immunization via the topical route: Plasmid DNA (CMV-(3-galactosidase) was coated on the surface of the cationic nanoparticles to obtain a final pDNA
concentration of 5 ~,g/100 ~,L. Three different pDNA-coated nanoparticle formulations, 'naked' pDNA, and [3gal protein (10 fig) adjuvanted with 'Aliun' (1S yg) were administered to shaved Balb/Cmice by topical application (100 ~,L) to Balb/C mice on day 0, 7, and 14. Plasmid DNA
(CMV-(3-galactosidase) was coated on the sl~rface of the cationic nanoparticles to obtain a final pDNA
concentration of S ~,g/100 pL. Three different pDNA-coated nanoparticle formulations, 'naked' pDNA, and (3ga1 protein (10 p,g) adjuvanted with 'Alum' (1S fig) were administered to mice by topical application (100 yL) to shaved skin on day 0, 7, and 14. As shown in Table S, the pDNA-coated nanoparticles (NPs) led to over a 6-fold enhancement in total antigen-specific IgG titer over 'naked' pDNA on day 2&. However, due to the fact that two of f ve mice in each group were very low responders, the enhancement only trended toward significance (p=0.066).
Surface deposition of the marman ligand on the nanoparticles (Man-NPs) enhanced the total antigen-specific IgG titer about 13-fold over 'naked' DNA, but again the enhancement only trended toward significance (p=U.OS4) due to a few low responders. In contrast, mice immunized with pDNA-nanoparticles with both DOPE and the mannan ligand (Man-NPs/DOPE) resulted in an approximately 16-fold increase in amtigen-specific IgG titer over 'naked' with statistical significance (p=0.002). Both fecal IgA and serum IgA (by OD 4S0 nm) were enhanced with NP groups over 'naked' pDNA
(data not shown). As shown in Figure ?8, splenocytes from 'naked' pDNA
immunized mice did not proliferate after stimulation with J3-gal antigen. In contrast, all the splenocytes isolated from mice immunized with pDNA-coated nanoparticles strongly proliferated in the presence of antigen (p<O.OS for all NP groups). Finally, splenocytes isolated from mice immunized with (3-gal,protein 2o adjuvanttdwith'Ahun' also Table S: Antigen-specific IgG titer in serum 28 days after topical application of formulations to mice IgG titer for individual mice p_ value Group I II III IV V Mean ~ SD vs.
pDNA
(Group 4) 1 NPs 2560 10 1280 10 2560 1284 1275 0.066 2 Man-NPs 5120 5120 10 2560 10 2564 2555 0.054 3 Man-NPs/DOPE 2560 5120 2560 2560 2560 3072 1144 0.002*

4 pDNA 320 10 320 10 320 196 169 N/A

(3-gal+~Aluia'320 320 160 160 320 256 87 0.254 (1) "NPs" is pDNA-coated nanoparticles; (2) "Man-NPs" is mannan-coated pDNA-nanoparticles;
(3) "Man-NPs/DOPE" is mannan-coated~pDNA-nanoparticles with DOPE (5% w/w); (4) "pDNA"
is 'naked' pDNA; (5) "(3-gal + Alum" is 10 pg of J3-galactosidase antigen adjuvanted with 15 ~g~
'Alum'. One hundred yL (100~,L) of each formulation (corresponding to a pDNA
dose of 5 ~,g) was applied to anesthetized Balb/C mice on day 0, day 7, and day 14 to shaved skin. * indicates that the .
result for group 3 was significantly different from that for group 4.
Statistical analysis was completed using a two-sample t-test assuming unequal variances.

Genetic immunization via the intranasal route: Ten to 12 week old female Balb/C mice from Harlan Sprague-Dawley Laboratories were used for aII animal studies. 1VIH
guidelines for.
the care and use of laboratory animal were observed. Twenty five ~,L ofpDIVA
(CMV-(3-gal, 'SO
~.g/mL) coated nanoparticle suspensions in 10% lactose or pDNA alone (50 ~,g/mL) in 155 mM
saline was administered intranasally into mice (n=4-5) on day 0, day 7, and day 14. Mice were held upright, and the formulations were carefully dripped on one nostril with a pipette tip for the mouse to inhale. On day 28, the mice were anesthetized and then bled by cardiac puncture. The blood was transferred into a Vacutainer Brand Blood Collection Tube (Becton Dickinson and Company, Franklin Lakes, NJ). Serum was separated and isolated by centrifugation, and frozen at -20°C. After blood collection, the spleens of the mice were removed and placed into 5 mL of 2o Hank's Balanced Salt Solution (HBSS) (1X) (Gibco BRL) for splenocyte preparation. The two different pDNA-coated nanoparticle suspensions administered to the mice were;
1) Brij 78 containing nanoparticle ("Brij-NP/DNA") and, 2) Brij 78-free nanoparticles ("CTAB-NP/DNA"). CTAB-NPs were engineered from microemulsions containing emulsifying wax (2 mg/mL) and CTAB (15 mM). The Brij-NPs were engineered from microemulsions containing emulsifying wax (2 mg/mL), CTAB (2.5 mM), and Brij 78 (6 mM). Five % w/w DOPE
was also ' incorporated into both nanoparticles. As shown in Figure 29, after intranasal administration, both of the pDNA-coated the nanoparticles led to significantly enhanced antigen-specific IgG
level in serum, compared to the 'naked' pDNA alone (i.e., 18-fold IgG titer using Brij-NP/DNA
and 28-fold using CTAB-NP/DNA). Moreover, the antigen-specific IgA titers in the pDNA
coated nanoparticles immunized mice were 25-30-fold higher than that in the 'naked' pDNA
immunized mice, strongly indicating an enhancement of mucosal immunity caused by these pDNA-coated nanoparticles. Although it would be more persuasive if the local specific IgA
levels, such as in nasal, vaginal, and genital .fluids, were measured in these present studies, such a to ~ strong enhancement of the antigen-specific IgA Level in serum strongly suggested the local specific IgA level should also be enhanced.

immunization with protein-coated nanoparticles via the subcutaneous route: (3-gal s5 protein was cationized as previously described with some modification.
Briefly, twenty (20) mg of (3-galactosidase (Spectrum) was dissolved into 1 mL of PBS buffer (10 mM, pH 7.4). Then, 2 mL of HMD (2 M, pH 6.8) and 54 mg of EDC were added into the protein solution.
After the pH
was adjusted to 6.8 with concentrated HCI, the mixture was stirred at room temperature for 3 h.
The reaction was stopped by adding I. mL of glycine solution (2 M) and stirred for another 1 h.
20 Cationized proteins were purified with a Sephadex G-25 column (Pre-packed PD-10, Amersham Phaimacia) and concentrated with ultra-centrifugation filter tubes (100 kDa cut-off) from Eppendorf Scientific, Inc. (Westbury, NY).
Verification of cationization was completed by electrophoresis. The pI of native (3-gal (nGal) was reported to be 4.6 and moved toward the cathode whereas cationized [3-gal (cGal) 25 moved toward the anode confirming that the pI of the cGal was greater than 7.4 (since the TAE
buffer used in the gel electrophoresis method had a pH of 7.4). Anionic nanoparticles were prepared as follows. Microemulsion precursors (1 mL) were prepared at 55°C with emulsifying wax (2 mg/mL) in water using SDS (1-30 mM) as the surfactant. Nanoparticles were engineered by simple cooling of the microemulsion precursor to room temperature. The data reported in 3o Table 6 for microemulsion droplet size, and nanoparticle size are mean +
S.D. (polydispersity index); the zeta potential data are the mean + S.D. (n=3). * indicates that the results are statistically the same for all values (p = 0.07; ANOVA). ** indicates that the results are statistically different for all values marked by * (p < 0.05; ANOVA).
Table 6: The physical properties of anionic nanoparticles engineered from microemulsion precursors using sodium dodecyl sulfate (5D5) as an anionic surfactant.
Final [5D5] Microemulsion size Nanoparticle size Zeta potential mM (nm) (nm) (m~
1 N/A 425 + 184 (0.724)-84 + 1 5 N/A 298 + 116 (0.386)-73 + 6 59 + 19 (0.185)96 + 36 (0.296)-62 + 2 48 + 17 (0.247)90+ 25 (0.117)-49 + 6 50 + 19 (0.322)99 + 22 (0.061)-61 + 4 *

52 + 21 (0.348)97 + 35 (0.275)-60 + 4 *

70 + 18 (0.084)113 + 46 (0.503)-67 + 2 **

n o For mouse immunization studies, nanoparticles were made with a final SDS
concentration of 15 mM, and purified to remove free SDS by GPC purification. Cationized (3-galactosidase'(cGal) was coated on the surface of the GPC purified anionic nanoparticles by gently mixing the required amount o.f cGal with 1 mL of purified nanoparticles in suspension to obtain a final cGal concentration of 100 ~.g/mL. At least 30 min at room temperature was allowed for complete 15 adsorption of cGal to the surface of the nanoparticles before particle sizing and TEM were performed. Mice (n=4-5/group) were immunized by subcutaneous injection on day 0, 7, and day 14 with either nGal (10 p.g) adjuvanted with 'Alum' (15 pg), cGal (10 ~,g) alone, or cGal (10 ~.g) coated on anionic nanoparticles. As shown in Figure 30, coating of cGal on the surface of anionic nanoparticles resulted in significantly enhanced and more reproducible antigen-specific IgG titer 20 ovzr both the nGal with 'Alum' (p = 0.03) and cGal alone (p = 0.008).
Immunization with nGal with 'Alum' produced relatively low levels of Thl cytokines whereas mice immunized with cGal alone or cGal-coated NPs produced very high levels of Thl cytokines. The IL-4 level from splenocytes isolated from mice immmuzed with the cGal-coated NPs was 3-4-fold greater than that from the nGal or cGal immunized mice, strongly suggesting an enhanced Th2-type immune response. Interestingly, cGal-coated NPs also enhanced the Th1-type cytokine release. (IL-2 and IFN-y), compared to mice immunized with nGal with 'Alum'. A more balanced enhancement of both Thl-type and Th2-type immune responses as observed with the cGal-coated NPs was somewhat surprising since it is generally thought that immunization methods that promote strong Th2-type responses (high antibody and strong Th2-type cytokines) tend to product weak Thl-type so responses, and vice-versa.
Table 7: In-vitro cytokine release from isolated splenocytes after stimulation with (3-galactosidase for 60 hours '1'h1 type 'i'h2 type I -2 (p mL) IFN-~, (pg/mI,) ~~(Pg/mL) Naive 545 + 54 460 + 30 undetectable n~al 171 ~ 13 4,340 ~ 170 164 ~ 15 cGal 3,184~ 83a 32,690 -~ 5,900a 121 ~- 16 coal-NPs 2,288 ~ 298b 10,520 ~ 3,600b 408 ~ 24°
On day 28, the spleens were removed and pooled for each group. Isolated splenocytes (5 x Z 5 1 O~lwell) with three replicates were stimulated with (3-galactosidase protein (3.3 pg/well) for 60 hours at 37°C. Cytokine release was quantified using ELISA kits (Endogen). Groups are: nGal, 10 ~.g of (3-galactosidase protein adjuvanted with 15. p.g 'Alum'; cGal, 10 p.g of (3-galactosidase protein; cGal-NPs, 10 ~.g of (3-galactosidase protein coated on the surface of GPC purified, sterilized anionic nanoparticles. aindicates that the results for cGal were significantly different from 2 o that of both nGal and cGal-NPs. bindicates that the results for cGal-NPs were significantly -different from that of both nGal and cGal. °indicates that the results for cGal-NPs were significantly different from that of both nGal and cGal. The reported data are the mean + S.D.
Statistical analysis was completed using a two-sample t-test assuming tmequal variances. A result of p<
0.05 was considered statistically significant.

Preparation of nanoparticles containing increased concentrations of Gadolinium: Since the concentration of Gd is critical to the performance of Gd-NCT, we have therefore studied an approach of increasing the amount gadolinium in the nanoparticle formulations, by eliminatiilg the use of emulsifying wax or other matrix matezzals altogether. To achieve this objective, a gadolinium complex (gadolinium hexanedione) was synthesized. The suitability of ~gadoliniurn Z o hexanedione (GdH) over GdAcAc is due to the lower melting point of GdH
(55°C) and increased lipoplulicity that make it more amenable for iilcorporation into O/W
microemulsions. Gadolinium hexanedione (GdH) was synthesized by using the modified method previously described by Mumper and. Jay (1992). Briefly, ~46 ml of an aqueous solution (pH 2.5) containing approximately 5 g of Gd chloride and approximately 6 ml of the complexing agent (2,4 hexanedione) was titrated with standard solution of sodium hydroxide (1.038 I~
to raise the pH to 7.34. At this pH, GdH precipitated from the solution. GdH was collected on a preweighed filter paper and washed with distilled and deionized water to remove excess complexing agent. Various templates were prepared by mixing the matrix material (2.5 mg GdH alone or in combination with 0.5 mg of either emulsifying wax or PEG 400 monostearate, surfactant (Brij 78 (polyoxyl 20 2 o stearyl ether) 0-10 mM) and water to make a final volume of 1 mL. The ingredients were mixed together and heated to 65°C while stirring. The formation of transparent oil-in-water microemulsions was verified by the clarity of the mixture and by photon correlation spectroscopy (PCS) using Coulter N4 Plus Submicron Particle Sizer. Table 8 contains the final compositions of selected formulations.
Table 8: Components of three different GdH nanoparticles formulations (A,B, and C). Each formulation was made to final volume of 1 ml with filtered water Components A B C

Gd hexanedione (mg) 2.5 2.5 2.5 Emulsifying Wax (mg) 0 0.5 0 PEG 400 monostearate 0 0 0.5 (mg) Brij 78 (mM) 6.0 4.0 6.0 As shown in Figure 31, stable nanoparticles could be engineered containing up to 2.5 mg/ml of GdH, indicating an increase in the overall gadolinium concentration in all rianoparticle formulations. Further, all of the nanoparticle formulations were stable when "challenged" with various biologically relevant media for 60 minutes at 37°C such as 10%
fetal bovine serum (FBS), 10 mM phosphate buffered saline (pH 7.4), 10% lactose, or 150 mM NaCl.
. .

Entrapment of Coenzyme Q10 in nanoparticles: Coenzyme Q10 is an endogenously synthesized provitamin, and an essential component of the mitochondrial respiratory chain as a cofactor in electron-transport chain and the synthesis of ATP. Coenzyme Q10 has very low solubility, . and hence, very poor and variable bioavailability when given orally. It is commercially available in tablets, or oil-based capsules. Enhanced solubility of Coenzyme Q10 via nanoparticles may lead to enhanced oral bioavailability. Coenzyme Q10 was entrapped in emulsifying wax nanoparticles in water using a microemulsion precursor comprised of emulsifying wax (2 mg/mL), 6 mM Brij 78, and final concentrations of Tween 20 of 0, 2, 4, or 6 mM. Either 1 mg or 2 mg of Coenzyme QIO was dissolved in the liquid dispersed phase. As shown in Figure 32, nanoparticles containing Coenzyme Q10 (either 1 mg/mL or 2 mg/mL) could be engineered having particle sizes of around 50 run.

Engineering of phospholipid nanoparticles from microemulsion precursors:
Phospholipid nanoparticles were engineered from microemulsion precursors comprised of various phosphatidylcholine (lecithin)/emulsifying wax mixtures or phospholipid alone.
Various ratios of nanoparticle matrix materials (2 mg total) emulsifying wax and lecithin (phosphatidyl choline; egg hydrogenated from Avanti Polar Lipids) were mixed with water and a final Brij 78 concentration of mM and stirred at 52°C until clear microemulsions formed. As shown in Figure 33, nanoparticles comprised of mixtures of phosphatidylcholine and emulsifying wax could be engineered using Brij 78 as the surfactant. As the ratio of phosphatidylcholine increased in the 5 matrix, the particle size of nanoparticles actually decreased. Nanoparticles engineered with phosphatidylcholine alone had particle sizes in the range of 5-10 nm.

Entrapment of a hydrophilic macromolecule in nmoparticles using a water-in-oil to microemulsion precursor: A water-in-oil microemulsion precursor was prepared by adding 7%
(v/iv) of FITC-dextran (FD-4; fluorescein-labeled dextran) solution (Mw 4000;
10 mg/ml in lOmM
PBS) to the melted mixture of oil and surfactants (emulsifying wax 44.2%, Capmul-GMS 4.4%, Capmul MCM 22.2%, Tween 80 22.2%). FD-4 was used as model peptide or protein.
When the system turned clear, an external water phase with Brij 78 was added to form a water-in-oil-water microemulsion with final Brij 78 concentration of ~.6 mM. The final concentration of nanoparticles containing FD-4 in the .suspension was 2 mg/ml. A control sample was prepared the . same way using 0.2 p,m-filtered water as internal water phase instead of FD-4 solution.
The nanoparticles suspension was spiked with FITC-dextran (sample was left under stirring conditions for about 10 min) so that the final concentration was 1.4 p,g/ml (as calculated for nanoparticles containing FD-4). One hundred microliters (100 p1) bf each preparation was eluted down a GPC
column (Sephadex G-7j, 25 cm by 1 cm) using 10 mM PBS as mobile phase. Twenty-three (23) one ml fractions were collected; each fraction was monitored for the presence of nanoparticles (6y light scattering intensity) and fluorescence intensity (by EX 494 nm, EM 521 nm) as shown in Figure 34.
Based on the calculation of area under the curve the entrapment efficiency of FD-4 in the 2 5 nanoparticles was calculated to be about 20%.
Example 31 Adsorption of a HIV Tat peptide on anionic nanoparticles: A stock solution of the HIV
Tat peptide in water was added to cured GFC-purified anionic nanoparticles made using a final 3 o emulsifying wax and SDS concentration of 2 mg/mL and 15 mM, respectively.
As shown in Figure 35, the overall zeta potential of the anionic nanoparticles in suspension increased (i.e., became less negative) as the final concentration of Tat increased from 0 to 130 pg/mL indicating that the Tat peptide adsorbed to the surface of the nanoparticles.
Example 32 Ethanol-in-Perflubron Microemulsions for topical genetic immunization:
Traditionally, vaccines have been adminstered by needle injection. Topical immunization through the intact skin with either protein- or DNA-based vaccines has attracted much attention recently. We sought to enhance the immune responses induced by DNA-based vaccines after topical application by developing novel ethanol-in-perflubron (E/P) microemulsion systems to aid in the delivery of plasmid DNA (pDNA). Ten different fluorosurfactants were selected and/or synthesized, and screened using pseudo-phase diagram construction for their feasibility to form E/P microemulsions.
Plasmid DNA (pDNA) was successfully incorporated into E/P microemulsions using several different fluorosurfactants. For several reasons, Zonyl° FSN-100 (ethoxylated nonionic n 5 fluorosurfactant) was selected for further studies. pDNA incorporated into E/P microemulsions using FSN-100 was found to be stable. For the preparation of the ethanol-in-perflubron (E/P) microemulsion using FSN-I00 as a fluorosurfactant, exactly 350 mg of perflubron and 100 mg FSN-100 were weighed and mixed in a glass vial to make a 70:30 w/w system.
While stirnng, a combination of 35 ~,L ethanol and 5-6 p,L of pDNA solution (4 p,g/pL in water) was added into the 2 o mixture. The system was stirred and turned clear within 2-3. minutes. The final pDNA
concentration was about 50 ~,g/mL. The pDNA was Found to be stable (i.e., retained supercoiled form) during 24 hours of storage at room temperature in the E/P microemulsion.
The E/P
microemulsion containing pDNA, pDNA in saline, and pDNA in ethanol was applied topically to shaved Balb/C mice on day 0, 7, and 14. The pDNA (CMV-(3-gal) dose on each day was 5 ~g per 25 100 ~L applied topically. As a control the same dose of pDNA in saline was injected into the gastrocnemious muscle of mice. On day 28, the [3-galactosidase specific IgG, IgA, IgGl, and IgG2a levels in sera were quantified by ELISA. The antigen-specific IgG in sera with the E/P
microemulsion (titer ~ 6000) was 45-fold greater than those obtained using pDNA in saline or ethanol (p = 0.02). Further, the titer with the E/P microemulsion was only 2.3-fold lower than those obtained using pDNA in saline by the intramuscular route. The 2.3-fold difference was not statistically significant (p=0.08). IgA titer with the E/P microemulsions (titer ~ 1600) were significantly greater than all other groups - IgA titers were undetectable in mice immunized topically with pDNA in saline or ethanol and the IgA titer in mice immunized using pDNA ill saline by the intramuscular route were also very low (i.e., only one of five mice had positive sera IgA).
-6l-UTILITY
The application relates to new and improved methods to engineer useful nanoparticulate systems that may solve many of the hurdles associated with conventional technologies and provide unique research opportunities across many different fields. The invention involves the use of microemulsion precursors (ethanol-in-fluorocarbon, liquid hydrocarbon-in-fluorocarbon, or liquid hydrocarbon-in-water) to engineer useful nanoparticles. The engineered nanoparticulate systems may contain many different materials for various medical and engineering applications such as plasmid DNA for gene therapy and genetic vaccines, magnetic substances for use as nanomagnets, Zo or chemical, thermal, or biological sensors for use as nanosensors. An additional advantage of this invention over prior art is that the described nanoparticle systems can be engineered rapidly, reproducibly, and cost-effectively from the microemulsion precursors in a one-step process and contained in one manufacturing vessel, vial, or container.
All of the references cited in the application are incorporated herein by reference in their entiret<,~.
* * *
Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention specifically 2 o described herein. Such equivalents are intended to be encompassed in the scope of the .following claims.

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Claims (56)

1. A stable alcohol-in-fluorocarbon microemulsion composition comprising:
an alcohol dispersed phase, a fluorocarbon continuous phase, a molecule of interest dissolved or dispersed in alcohol, a film-forming substance dissolved or dispersed in the alcohol, a surfactant or co-surfactant or a mixture thereof, and a cell-targeting ligand.

2. The microemulsion according to claim 1, wherein the molecule of interest comprises a drug molecule, a food, a magnet, or a sensor molecule.

3. The microemulsion according to claim 1, wherein the fluorocarbon comprises perflubron.

4. The microemulsion according to claim 1, wherein the film-forming substance comprises ethyl cellulose.

5. The microemulsion according to claim 1, wherein the surfactant comprises a fluorosurfactant.

6. The microemulsion according to claim 1, wherein the alcohol comprises ethanol.

7. The microemulsion according to claim 2, wherein the drug molecule comprises plasmid DNA, oligonucleotide, peptide, protein, antibody, small drug molecule, or a rare-earth molecule.

8. The microemulsion according to claim 2, where the sensor molecule responds in a controlled and predictable manner to changes in temperature, pH, pressure, or the presence of another substance.

9. The microemulsion according to claim 1, wherein the cell-targeting ligand comprises asialofetuin, mannan, mannose, folate, a saccharide, or an antibody.

10. The microemulsion according to claim 1, wherein the alcohol is removed from the microemulsion by evaporation or by dilution with a suitable solvent to cause the film-forming substance to precipitate into solid nanoparticles having a diameter less than about 300 nanometers.

11. A method for purifying or characterizing a solid nanoparticle comprising removing alcohol from the microemulsion according to claim 1 by evaporating or diluting with a suitable solvent thereby curing the nanoparticle in a continuous phase, and subjecting the cured nanoparticle to gel permeation or ultracentrifugation, and obtaining a solid nanoparticle.

12. A stable liquid hydrocarbon-in-fluorocarbon microemulsion prepared at a temperature of between about 35-100°C and having a composition comprising:
a liquid hydrocarbon dispersed phase, a fluorocarbon continuous phase, a molecule of interest dissolved or dispersed in the liquid hydrocarbon, a surfactant or co-surfactant or a mixture thereof, and a cell-targeting ligand.

13. The microemulsion according to claim 12, wherein the fluorocarbon comprises perflubron.

14. The microemulsion according to claim 12, wherein the liquid hydrocarbon comprises a solid at about 25°C, has a melting point of between about 35-100°C, is water-insoluble, and is amphipathic having both hydrophobic and hydrophilic moieties.

15. The microemulsion according to claim 12, wherein the surfactant comprises a fluorosurfactant.

16. The microemulsion according to claim 12, wherein the molecule of interest comprises a drug molecule.

17. The microemulsion according to claim 12, where the molecule of interest comprises a sensor molecule that responds in a controlled and predictable manner to changes in temperature, pH, pressure, or the presence of another substance.

18. The microemulsion according to claim 12, wherein the cell-targeting ligand comprises asialofetuin, mannan, mannose, folate, a saccharide, or an antibody.

19. The microemulsion according to claim 12, wherein the microemulsion is cooled to cause the hydrocarbon to solidify into solid nanoparticles having a diameter less than about 300 nanometers.

20. A method for purifying or characterizing a solid nanoparticle, comprising cooling the microemulsion according to claim 12, wherein the hydrocarbon is solidified into solid nanoparticles containing the molecule of interest thereby curing the nanoparticle in a continuous phase, and subjecting the cured nanoparticle to gel permeation or ultracentrifugation, and obtaining a solid nanoparticle.

21. A nanoparticle comprising:
at least one liquid nanoparticle matrix material, at least one surfactant or co-surfactant or a mixture thereof, and at least one molecule of interest, wherein the nanoparticle is made from an oil-in-water microemulsion precursor.

22. The nanoparticle according to claim 21, wherein in the oil-in-water microemulsion precursor, an oil phase comprised of at least one nanoparticle matrix material and at least one molecule of interest is dispersed in an aqueous continuous phase to form a surfactant stabilized microemulsion between about 35°C and about 100°C, wherein the micromulsion is cooled to room temperature while stirring to form solid stable nanoparticles containing at least one molecule of interest either entrapped in or adsorbed to the nanoparticles having a diameter of less than about 300 nanometers.

23. The nanoparticle according to claim 22, wherein the nanoparticle matrix material comprises one or more of the following materials: emulsifying wax, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene stearates, phospholipids, fatty acids or fatty alcohols or their derivatives, or combinations thereof.

24. The nanoparticle according to claim 21, wherein the liquid nanoparticle matrix material is present in the microemulsion at a concentration from about 0.1 to about 30 mg/mL.

25. The nanoparticle according to claim 22, wherein the oil phase is present as liquid droplets having a diameter of less than about 100 manometers.

26. The nanoparticle according to claim 22, wherein the continuous phase comprises water or an aqueous buffer present at a concentration of greater than about 95% w/w

27. The nanoparticle according to claim 21, wherein the surfactant or co-surfactant comprises polyoxyethylene alkyl ethers, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, hexadecyltrimethylammonium bromide, fatty alcohol and their derivatives, or combinations, thereof.

28. The nanoparticle according to claim 21, wherein the surfactant is present at a total concentration of about 1-5000 mM.

29. The nanoparticle according to claim 21, wherein the molecule of interest is present at a total concentration in the range of about 20 µg/mL to about 5 mg/mL.

30. The nanoparticle according to claim 21, wherein the molecule of interest comprises a drug molecule, a food, a magnet, or a sensor molecule.

31. The nanoparticle according to claim 21, wherein the molecule of interest comprises plasmid DNA.

32. The nanoparticle according to claim 21, wherein the molecule of interest comprises Gadoliniums its derivatives or complexes thereof.

33. The nanoparticle according to claim 21, wherein the nanoparticle is coated with a cell-specific ligand comprising an antibody, carbohydrate, peptide, protein, or derivatives or combinations thereof.

34. The nanoparticle according to claim 21, wherein the nanoparticle is coated with a cell-specific ligand comprising a mannan or peptide for targeting dendritic cells, a protein including asialofetuin, a polysaccharide including pullulan for targeting hepatocytes, folate and thiamine for targeting tumors, or choline or its derivatives for targeting a brain.

35. The nanoparticle according to claim 21, wherein the molecule of interest comprises Tat peptide from HIV, nerve-growth factor, or calcitonin.

36. The nanoparticle according to claim 21, wherein the nanoparticle is anionic.

37. The nanoparticle according to claim 36, wherein the nanoparticle further comprises a positively-charged drug or antigen coating.

38. The nanoparticle according to claim 37, wherein the coating comprises Tat peptide from HIV or nerve-growth factor.

39. The nanoparticle according to claim 21, wherein the nanoparticle is cationic.

40. The nanoparticle according to claim 39, wherein the nanoparticle further comprises a negatively-charged drug or antigen coating.

41. The nanoparticle according to claim 40, wherein the coating comprises DNA.

42. A method for delivering a nanoparticle, comprising:
obtaining a nanoparticle according to claim 21; and administering the nanoparticle to a subject.

43. The method of claim 42, wherein administering comprises administering topically, intranasally, subcutaneously, intramuscularly, intravenously, or orally.

44. A method of making a solid stable nanoparticle, comprising:
obtaining a nanoparticle matrix material;
melting the nanoparticle matrix material at a temperature between about 35°C and about 100°C to form a liquid dispersed phase;
dispersing a molecule of interest into the liquid dispersed phase;
dispersing the liquid dispersed phase, including the molecule of interest, in an aqueous continuous phase to form a surfactant stabilized microemulsion between about 35°C and about 100°C; and cooling the microemulsion while stirring to form a solid stable nanoparticle having a diameter of less than about 300 nanometers, which includes the molecule of interest either entrapped in or adsorbed to the nanoparticle.

45. The method according to claim 44, wherein the cooling comprises cooling with no dilution in water.

46. The method according to claim 44, wherein the nanoparticle matrix material comprises one or more of the following materials: emulsifying wax, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, polyoxyethylene stearates, phospholipids, fatty acids or fatty alcohols or their derivatives, or combinations thereof.

47. The method according to claim 44, wherein the nanoparticle matrix material is present in the microemulsion at a concentration from about 0.1 to about 30 mg/mL.

48. The method according to claim 44, wherein the aqueous continuous phase comprises water or an aqueous buffer present at a concentration of greater than about 95% w/w.

49. The method according to claim 44, wherein the molecule of interest is present at a total concentration in the range of about 20 µg/mL to about 5 mg/mL.

50. The method according to claim 44, wherein the molecule of interest comprises a drug molecule, a food, a magnet, or a sensor molecule.

51. The method according to claim 44, wherein the molecule of interest comprises plasmid DNA.

52. The method according to claim 44, wherein the molecule of interest comprises Gadolinium, its derivatives, or complexes thereof, Tat peptide from HIV, nerve-growth factor, or calcitonin.

53. The method according to claim 44, further comprising coating the nanoparticle with a cell-specific ligand.

54. The method according to claim 53, wherein the cell-specific ligand comprises an antibody, carbohydrate, mannan, peptide, polysaccharide, folate, thiamine, choline, protein, or derivatives or combinations thereof.

55. The method according to claim 44, further comprising isolating the nanoparticle by gel permeation or ultracentrifugation.

56. The method according to claim 55, further comprising treating the nanoparticle with a buffer.