JP4841066B2 - Nitric oxide-forming hydrogel materials - Google Patents

Abstract

Hydrogels releasing or producing NO, most preferably polymerizable biodegradable hydrogels capable of releasing physiological amounts of NO for prolonged periods of time, are applied to sites on or in a patient in need of treatment thereof for disorders such as restenosis, thrombosis, asthma, wound healing, arthritis, penile erectile dysfunction or other conditions where NO plays a significant role. The polymeric materials can be formed into films, coatings, or microparticles for application to medical devices, such as stents, vascular grafts and catheters. The polymeric materials can also be applied directly to biological tissues and can be polymerized in situ. The hydrogels are formed of macromers, which preferably include biodegradable regions, and have bound thereto groups that are released in situ to elevate or otherwise modulate NO levels at the site where treatment is needed. The macromers can form a homo or hetero-dispersion or solution, which is polymerized to form a hydrogel material, that in the latter case can be a semi-interpenetrating network or interpenetrating network. Compounds to be released can be physically entrapped, covalently or ionically bound to macromer, or actually form a part of the polymeric material. The hydrogel can be formed by ionic and/or covalent crosslinking. Other active agents, including therapeutic, prophylactic, or diagnostic agents, can also be included within the polymeric material.

Description

[0001]
Biocompatible polymeric substances that release or produce physiological amounts of NO over time can include restenosis, thrombosis, asthma, wound healing, arthritis, erectile dysfunction, or other disorders where NO plays an important role It is applied outside or inside the body of a patient in need of treatment such as a condition. The polymeric material can also be formed as a film, coating or particulate. Polymers are generally formed from macromers, which contain biodegradable regions with attached groups that are released in situ to increase or otherwise adjust the NO concentration at the site that requires treatment. To do. Macromers can form homo- or hetero-dispersions or solutions that are polymerized to form a polymeric material. In the latter case, it may be a semi-interpenetrating network structure or an interpenetrating network structure. The released compound is either physically trapped, covalently or ionically bound to the macromer, or actually forms part of the polymer material. Hydrogels can be formed by ionic and / or covalent crosslinking. Other active agents, including therapeutic, prophylactic or diagnostic agents can also be included in the polymeric material.
[0002]
I. Polymeric material for NO release
The polymeric material is biocompatible and releases or produces NO. In various preferred embodiments, the polymer is also biocompatible, forms a hydrogel, polymerizes in situ, and is tissue adhesive. These properties are imparted not only by selection of the macromer component, but also by addition to various groups of components.
[0003]
The term “polymerizable” refers to a region that creates an additional covalent bond that results in a macromer linkage of an acrylate-type molecule, eg, a carbon-carbon double bond. Such polymerization is characterized by the free radical formation that results from photon adsorption of certain dyes and chemical compounds that ultimately generate free radicals. However, free radicals can also be obtained using other methods and reagents known to those skilled in the art.
[0004]
A. Polymer material
The polymeric material must be biocompatible, i.e., must not induce a significant or unacceptable toxicity or immune response after administration or implantation in a patient.
[0005]
Many biocompatible polymeric materials are known, including both natural and synthetic polymers. Examples include proteins (from the same origin as the recipient), polysaccharides such as chondroitin sulfate, and hyaluronic acid, polyurethanes, polyesters, polyamides, and acrylates. The polymer may be degradable or non-degradable.
[0006]
Most polymer materials are selected based on a combination of properties imparted by various components. These include water-soluble regions such as PEG or PVA, biodegradable regions such as regions that degrade by hydrolysis, groups that can be used to polymerize macromers in situ, and the like.
[0007]
(Water-soluble and / or tissue adhesion area)
Various water-soluble substances can be mixed into the polymer. The term “at least substantially water soluble” indicates an aqueous solution having a solubility of at least about 5 g / 100 ml. In a preferred embodiment, the core water-soluble region is poly (ethylene glycol), poly (ethylene oxide), poly (vinyl acetate), poly (vinyl alcohol), poly (vinyl pyrrolidone), poly (ethyl oxazoline), Poly (ethylene oxide) -poly (propylene oxide) block copolymers, polysaccharides or carbohydrates such as polysaccharose, hyaluronic acid, dextran, heparin sulfate, chondroitin sulfate, heparin or alginate, gelatin, collagen, albumin or ovalbumin, etc. It can be composed of
[0008]
The hydrophilic (ie, water soluble) region is generally tissue adhesive. Both hydrophobic and hydrophilic polymers containing large amounts of exposed carboxylic acid groups are tissue or bioadhesive. Ligands such as RGD peptides and lectins that bind to cellular carbohydrate molecules can also adhere to polymers, improving tissue adhesion.
[0009]
(Degradable region)
Polyesters of α-hydroxy acids (ie, lactic acid, glycolic acid) (Holland et al., 1986 Controlled Release, 4: 155-180) are available from a closure device (suture and clasp) to a drug delivery system (US Pat. No. 4 to Smith et al.). 741,337; Spilizewski et al., 1985 J. Control. Rel. 2: 197-203), the most widely used biodegradable material. Domb et al., 1989 Macromolecules, 22: 3200; Heller et al., 1990 Biodegradable Polymers as Drug Delivery Systems, Chasin, M .; And Langer, R .; Hen, Dekker, New York, 121-161, in addition to poly (hydroxy acids), other polymers including polyanhydrides and polyorthoesters that utilize labile backbone bonds are also available. It is known to be biodegradable. As reported by Miyake et al., 1974, polyamino acids have also been synthesized since it is desirable to include a polymer that degrades into the natural form material for in vivo use.
[0010]
The time required for polymer degradation can be adjusted by selecting an appropriate monomer. The difference in crystallinity also changes the decomposition rate. Since these polymers are relatively hydrophobic, the actual mass loss only begins when the oligomeric fragments are so small that they are soluble in water. Thus, the initial polymer molecular weight affects the degradation rate.
[0011]
Preferably, the biodegradable region can be hydrolyzed under in vivo conditions. Hydrolyzable groups are glycolide, lactide, ε-caprolactone, other α-hydroxy acid polymers and oligomers, and other biodegradable polymers that are non-toxic or exist as normal metabolites in the body. Preferred poly (α-hydroxy acids) are poly (glycolic acid), poly (DL-lactic acid) and poly (L-lactic acid). Other useful materials are poly (amino acids), poly (anhydrides), poly (orthoesters), and poly (phosphate esters). For example, polylactones such as poly (ε-caprolactone), poly (ε-caprolactone), poly (δ-valerolactone), and poly (γ-butyrolactone) are also useful.
[0012]
Biodegradable regions can also be composed of polymers or monomers with linkages that undergo enzymatic biodegradation, such as ester, peptide, anhydride, orthoester and phosphate ester linkages. For example, biodegradable substances such as crosslinked gelatin are known. Hyaluronic acid is crosslinked and used in biomedical applications as a degradable swelling polymer (U.S. Pat. No. 4,987,744 to Della Valley et al., U.S. Pat. No. 4,957,744 to Della Valle et al.). (1991) Polym. Mater. Sci. Eng., 62: 731-735).
[0013]
(Biodegradable hydrogel)
Many polymers have been described that contain both water-soluble and biodegradable regions. Sawhney et al. (1990) J. MoI. Biomed. Mater. Res. 24: 1397-1411 copolymerized lactide and ε-caprolactone with PEG to improve hydrophilicity and degradation rate. US Pat. No. 4,716,203 to Casey et al. (1987) synthesized PGA-PEG-PGA block copolymers containing PEG in the range of 5-25% by weight. US Pat. No. 4,716,203 to Casey et al. (1987) also reports the synthesis of PGA-PEG diblock copolymers containing PEG in the range of 5-25% by weight again. U.S. Pat. No. 4,526,938 (1985) to Churchill et al. Describes non-crosslinked materials with molecular weights greater than 5,000 based on PEG based on similar compositions. However, these materials are not water soluble. Cohn et al. Biomed. Mater. Res. 22: 993-1009 described PLA-PEG copolymers that swell to 60% in water. These polymers are also not water-soluble and are not crosslinked. A common feature of these materials is that they use both water-soluble and degradable polymers, are insoluble in water, and swell up to a total of 60%.
[0014]
U.S. Pat. No. 5,410,016 issued to Hubbell et al. On April 25, 1995 describes a short polylactide extension that imparts biodegradability and observability due to high biocompatibility and antithrombotic properties Materials based on polyethylene glycol (PEG) with acrylic acid ends that enable rapid photopolymerization without heat generation are described. These materials are easily modified to produce hydrogels that release or produce NO.
[0015]
The polymerizable moieties are separated by at least one degradable region to facilitate uniform degradation in vivo. There are several variations of these polymers. For example, as long as the polymerizable region is separated by the degradable region, it can be bonded directly to the degradable extension or indirectly through a water-soluble non-degradable portion. For example, if the macromer composition includes a simple water-soluble region bonded to a degradable region, one polymerizable region binds to the water-soluble region and another polymerizable region binds to the degradable extension or region. In another embodiment, the water soluble region forms the core of the macromer composition and has at least two degradable regions attached to the core. Since at least two polymerizable regions are bonded to the degradable region, the polymerizable region is separated upon decomposition, particularly in the form of a polymer gel. Conversely, if the core of the macromer composition is formed by a degradable region, at least two water-soluble regions can be bound to the core, and a polymerizable region can be bound to each water-soluble region. The final result will be the same as after gel formation and exposure to in vivo degradation conditions.
[0016]
In another embodiment, the macromer composition has a water soluble backbone region and a degradable region attached to the macromer backbone. Since at least two polymerizable regions are bound to the degradable region, they are separated during degradation, resulting in dissolution of the gel product. In a further embodiment, the macromer backbone is formed of a non-degradable backbone with a water soluble region as a branch or graft attached to the degradable backbone. Two or more polymerizable regions are attached to a water-soluble branch or graft. In another variation, the backbone is star-shaped and may contain water-soluble regions, biodegradable regions, or water-soluble regions that are also biodegradable. In this general embodiment, the star region comprises either a water-soluble or biodegradable branch or graft to which a polymerizable region is attached. Again, the polymerizable region is separated at some point by the degradable region.
[0017]
(Polymerizable group)
The polymerizable group is most preferably polymerizable by photoinitiation by free radical generation upon visible or long wavelength UV irradiation. Preferred polymerizable regions are acrylates, diacrylates, oligoacrylates, dimethacrylates, oligomethacrylates or other biologically acceptable photopolymerizable groups. A preferred tertiary amine is triethanolamine.
[0018]
Usable photoinitiators are photoinitiators that can be used for initiation by free radical-generating polymerization of macromers within a short time frame of no more than a few minutes, most preferably a few seconds, without cytotoxicity. Preferred dyes selected as initiators for LWUV initiation are ethyl eosin, 2,2-dimethoxy-2-phenylacetophenone, other acetophenone derivatives, and camphorquinone. In all cases, crosslinking and polymerization is carried out, for example, 2,2-dimethoxy-2-phenylacetophenone or ethyl eosin (10 ^-4 -10 ^-2 Initiated within the copolymer by a photoactivatable free radical polymerization initiator, such as a combination of milliM) and triethanolamine (0.001-0.1 M).
[0019]
The choice of photoinitiator is highly dependent on the photopolymerizable region. For example, if the macromer contains at least one carbon-carbon double bond, light absorption by the dye causes the dye to take the triplet state, which then reacts with the amine to form a free radical that initiates polymerization. To do. Preferred dyes for use with these materials include eosin dyes and initiators such as 2,2-dimethyl-2-phenylacetophenone, 2-methoxy-2-phenylacetophenone, and camphorquinone. With such an initiator, the copolymer is polymerized in situ by, for example, long wavelength ultraviolet light or laser light of about 514 nm.
[0020]
Polymerization is initiated by irradiation with light of a wavelength of about 200-700 nm, most preferably light of 320 nm or longer in the long wavelength ultraviolet range or visible range, most preferably about 514 nm or 365 nm.
[0021]
Multiple photooxidation and photoreduction dyes can be used to initiate the polymerization. These include, for example, acridine dyes such as acribraline; thiazine dyes such as thionine; xanthine dyes such as rose bengal; and phenazine dyes such as methylene blue. These include, for example, amines such as triethanolamine; RSO ₂ R ₁ Used together with cocatalysts such as sulfur compounds such as: heterocycles such as imidazole; enolates; organometallics; and other compounds such as N-phenylglycine. Other initiators include camphorquinone and acetophenone derivatives.
[0022]
Thermal polymerization initiator systems can also be used. Such systems that are unstable at 37 ° C. and initiate free radical polymerization at physiological temperatures include, for example, potassium persulfate in the presence or absence of tetramethylethylenediamine; excess in the presence or absence of triethanolamine. There is benzoyl oxide; and ammonium persulfate with sodium bisulfite present.
[0023]
In addition to photoinitiation, other initiation chemistries can be used. These include, for example, water and amine initiated systems with isocyanates or isothiocyanates containing macromers used as the polymerizable region.
[0024]
(Preferred embodiment)
In a preferred embodiment, the polymeric material is a biodegradable, polymerizable, at least substantially water soluble macromer composition. The first macromer includes at least one water soluble region, at least one NO retention region and at least one free radical polymerizable region. The second macromer includes at least one water soluble region and at least two free radical polymerizable regions. The region may be water soluble and biodegradable in certain embodiments. The macromer composition is polymerized by exposing the polymerizable region to free radicals generated, for example, by photosensitive chemicals and dyes.
[0025]
Examples of these macromers are PVA or PEG-oligoglycolyl-acrylate. Choosing the appropriate end cap speeds polymerization and gelation. Acrylates are preferred because they can be polymerized using a plurality of initiating systems, such as eosin dyes, by brief exposure to ultraviolet or visible light. Poly (ethylene glycol) or PEG central structural unit (core) is preferred based on its high hydrophilicity and water solubility with excellent biocompatibility. Short oligos or poly (α-hydroxy acids) such as polyglycolic acid are selected as preferred chain extensions because they degrade rapidly upon hydrolysis into glycolic acid, an innocuous metabolite of the ester bond. Highly crystalline polyglycolic acid is insoluble in water and most common organic solvents, but the entire macromer composition is water soluble and rapidly gels in a biodegradable network while in contact with aqueous tissue fluids. The Such a network can be used to capture and uniformly disperse water-soluble drugs and enzymes and deliver them at a controlled rate. Further, the mesh is used to capture a particle suspension of a water-soluble drug. Other preferred chain extensions are polylactic acid, polycaprolactone, polyorthoesters, and polyanhydrides. Polypeptides can also be used. It should be understood that such “polymer” blocks include dimer, trimer and oligomer blocks.
[0026]
PVA contains many pendant hydroxyl groups. These hydroxyl groups readily react to form side chains such as various crosslinkers and nitric oxide donors. PVA is water soluble and has excellent biocompatibility. When PVA is modified to bind to a methacrylate group via a diacetal bond with pendant hydroxyl groups and an appropriate photoinitiator is added, PVA is photopolymerized by long wavelength ultraviolet light to form a hydrogel. In another preferred embodiment, U.S. Pat. Nos. 5,508,317, 5,665,840, 5,849,841, 5,932,674, 6,011,077, , 939,489 and 5,807,927, hydrogels are produced from modified polyvinyl alcohol (PVA) macromers. For example, the macromer disclosed in US Pat. No. 5,508,317 is a PVA prepolymer modified with pendant crosslinkable groups such as acrylamide groups containing crosslinkable olefinically unsaturated groups. These macromers can be polymerized, for example, by photopolymerization or redox free radical polymerization. The starting polymer is in particular a derivative of polyvinyl alcohol or a copolymer of vinyl alcohol comprising, for example, a 1,3-diol framework. The crosslinkable group or further modifier can be modified in various ways, for example by modifying a few percent of 1,3-diol units to 1,3-dioxane containing a crosslinkable radical, or in the 2-position. Can be attached to the framework of the starting polymer. Another possibility is to esterify a few percent of the hydroxyl groups of the starting polymer with unsaturated organic acids, and these ester bond radicals contain crosslinkable groups. The hydrophobicity of these macromers can be improved by replacing some pendant hydroxyl groups with more hydrophobic substituents. Macromer properties such as hydrophobicity can be modified by incorporating comonomers into the macromer backbone. Macromers with pendant groups that can be cross-linked by other means can also be formed.
[0027]
B. NO groups or modulating compounds
A number of molecules that produce NO under physiological conditions (No donors) have been identified and evaluated both in vitro and in vivo, including S-nitrosothiols, organic nitrates and NO complexes with nucleophilic atoms. Since L-arginine is a substrate for NO synthase, it is a NO donor, and therefore administration of L-arginine improves endogenous NO production, and in most cases, a reaction similar to that produced by a NO donor is observed. Be triggered. Other NO donors include molsidin, CAS754, SPM-5185 and SIN-1. Other compounds that can generate and / or donate NO may be used. These include organic nitrates, nitrosylated compounds, nitrosoesters and L-arginine.
[0028]
Molecules that generate NO or that release or generate NO are preferably bound to a region containing a nucleophilic atom capable of forming a complex with NO and / or a thiol such as S-nitrosothiol.
[0029]
C. Prophylactic, therapeutic and diagnostic agents
The polymeric material can be loaded with systemically released drugs, but can also be used for drug delivery, preferably local release of prophylactic, therapeutic and diagnostic agents at the site where the material is needed. These agents include proteins or peptides, polysaccharides, nucleic acid molecules, and simple natural and synthetic molecules. Representative agents are involved in antibiotics, antiviral and antibacterial agents, anti-inflammatory agents (steroidal or nonsteroidal), hormones, growth factors, cytokines, neurostimulants, vasoconstrictors, and cardiovascular responses Other molecules, enzymes, antitumor agents, local anesthetics, antiangiogenic agents, antibodies, drugs that affect the reproductive organs, and oligonucleotides such as antisense oligonucleotides. The diagnostic agent is radioactive and is detected by ultrasound, x-ray, MRI or other standard imaging methods, binding to or cleaving the chromogenic substrate.
[0030]
These agents can be mixed with the prepolymerized macromer, or applied in or on the polymer, so that it can be released by either degradation or diffusion (by enzyme or hydrolysis) at the site where the polymer is applied. It can be covalently or ionically bound to the macromer before or during the polymerization.
[0031]
II. how to use
A. Coating; Film; Fine particle
Although described with respect to in vivo therapy, the polymeric materials described herein can be used for coating cell cultures, cell culture substrates, or medical implants or devices such as stents or catheters, using standard techniques, Clearly, it can be formed into microparticles or other types of formulations that are used or administered to a patient.
[0032]
B. Therapeutic use
Polymeric substances that can release physiological amounts of NO over time can be applied to the site or patient in need of such treatment. Representative disorders or symptoms that can be treated with NO include restenosis, thrombosis, asthma, wound treatment, arthritis, penis or erectile dysfunction in women. The material is usually applied as a macromer solution and can be polymerized before application, but is polymerized in situ.
[0033]
(Wound treatment)
The formulations are particularly useful for the treatment of all types of wounds, including burns, surgical wounds, open leg and foot wounds. There are generally three types of open leg wounds called ulcers. Venous congestive ulcers that are common in older people who are always sitting and when blood flow to the legs is stagnant; the most common position in bedridden people who are bedridden and often unable to roll over Ulcers: Diabetic foot ulcers caused by stagnant blood circulation to the feet. As the aging population increases, there will be an increasing demand for effective and user-friendly ulcer treatments in the near future.
[0034]
As used herein, the term “wound” refers to any type of injury, including those caused by surgery and trauma, including burns, as well as damage from chronic or acute medical conditions such as atherosclerosis or diabetes. Refers to tissue damage.
[0035]
(Treatment of restenosis)
A preferred application is a method of reducing the effects of restenosis in patients after surgery. The method is to coat the intra-arterial surface with an aqueous solution of a photosensitive free radical polymerizable initiator and a number of macromers. The coated artery is irradiated with a Xenon arc laser that induces macromer polymerization. When a newly polymerized polymer composition is produced, NO release is induced by physiological conditions in the artery. This release is strictly localized over a long period of time.
[0036]
(Prevention of adhesion due to surgery)
A preferred application is a method of reducing adhesion formation after a patient's surgery. In certain embodiments, the method comprises coating a patient's damaged tissue surface with an aqueous solution of the photosensitive free radical polymerizable initiator and an aqueous macromer solution described above. The coated tissue surface is exposed to sufficient light for macromer polymerization. The photosensitive free radical polymerization initiator may be a single compound (eg 2,2-dimethoxy-2-phenylacetophenone) or a combination of a dye and a cocatalyst (eg ethyl eosin and triethanolamine).
[0037]
(Tissue adhesion)
Another application of the polymer is a method of adhering patient tissue surfaces. In certain embodiments, the macromer is mixed with a photoinitiator or photoinitiator / cocatalyst mixture to form an aqueous mixture and the mixture is applied to the surface of the tissue where tissue adhesion is desired. The tissue surface is in contact with tissue where adhesion is desired and creates a tissue bond. The tissue bond is then irradiated until the macromer polymerizes.
[0038]
(Tissue coating)
In particularly preferred applications of these macromers, the ultrathin coating is applied to the surface of the tissue, most preferably to the lumen of a tissue such as a blood vessel. One application for such coatings is in the treatment or prevention of restenosis, sudden reclosure, vasoconstriction after vascular intervention, and the like. When an initiator is applied to the tissue surface and allowed to react, absorb or bind to the tissue, unbound initiator is removed by dilution or rinsing, and the macromer solution is applied and polymerized. This method can produce a uniform polymer coating with a thickness of 1 to 500 microns, most preferably about 20 microns, and does not cause thrombus formation or local inflammation.
[0039]
(Tissue support)
The polymeric material can also be used to generate a tissue support by forming a molded product to provide mechanical functions within the body. Examples of such a support include a sealant for a bleeding organ, a sealant for a bone defect, and a filler for an aneurysm. In addition, such supports can include stenosis to hold organs, blood vessels, and tubes in a special position for management time.
[0040]
(Controlled drug delivery)
As described above, the polymer substance can also be used as a carrier for physiologically active substances as therapeutic, prophylactic and diagnostic agents, including hormones, enzymes, antibiotics, antitumor agents, and cell suspensions. The polymeric material can be used for long-term controlled release of the drug for local tissue or systemic circulation as well as for temporary maintenance of the functional properties of the released drug.
[0041]
In a variation of the method for controlled drug delivery in which the drug is mixed into the macromer solution and then polymerized in situ, the macromer is polymerized with the bioactive substance to form microspheres or nanoparticles containing the bioactive substance. The macromer, photoinitiator and encapsulated drug are mixed in an aqueous mixture. For example, standard techniques by mixing in oil to form an emulsion, using a nozzle to form droplets in oil, or using a nozzle to form droplets in air Used to form particles of the mixture. The suspension or droplets are irradiated with light suitable for macromer photopolymerization.
[0042]
These materials are particularly useful for controlled drug delivery of hydrophilic materials because the water-soluble regions of the polymer allow water to reach the material trapped within the polymer. Furthermore, it is possible to polymerize a macromer composition containing a substance without exposing the substance to an organic solvent. Release occurs by diffusion of the material from the polymer prior to degradation and / or diffusion of the material from the polymer upon degradation, due to the characteristic pore size within the polymer controlled by the molecular weight between crosslinks and the crosslink density. Gel immobilization as well as protective effects reduce capture material inactivation and avoid catastrophic burst effects associated with other controlled release systems. If the capture material is an enzyme, the enzyme is exposed to the substrate while being captured if the gel ratio is selected so that the substrate can permeate the gel. Degradation of the polymer by gradual hydrolysis of the terminal ester bond facilitates final controlled release of free macromolecules in vivo.
[0043]
III. Example
As shown in Example 1-3, three types of NO-producing, PEG-based polymers were synthesized and their release rates were determined in vitro under organized conditions. Physiological responses to appropriate substances were assessed in vitro using cultured smooth muscle cells and endothelial cells and in vivo using a rat carotid artery injury model resembling human restenosis. The substance is then NaNO ₂ Polyethylene glycol (A) and polycysteine (B) BAB block copolymer that reacts with NO to produce nucleophile / NO complex that reacts with NO gas to produce S-nitrosothiol Polyethylene glycol (A) and polylysine which react with BAB block copolymer of ("PEG") (A) and diethylenetriamine ("DETA") (B) and then NO gas to form a nucleophile / NO complex The BAB block copolymer (B) is included. All polymers were further terminated with active acrylate groups for rapid photopolymerization in situ.
[0044]
If a water-soluble, biocompatible polymer, such as PEG, contains a large amount of the substance and the molecular weight is sufficiently high, such a substance is well biocompatible and has two ester linkages and two amides in each polymer chain. Slow biodegradation is expected due to the presence of binding. These three substances were chosen because they were expected to have very different release kinetics: nucleophiles / NO complexes have been shown to release NO over 5 weeks (Smith et al., (1996). ) J. Med. Chem., 39: 1148-56), S-nitrosocysteine has a half-life of 0.023 hours (Mathews et al., (1993) J. Pharmacol. Exp. Therap., 267: 1529-37). ). The amount of NO produced by these copolymers may be adjusted by changing the ratio of polyethylene glycol (PEG) to cysteine or lysine.
[0045]
The advantage of these macromer compositions is that they can be polymerized rapidly in an aqueous environment. Thus, semi-permeable, biodegradable films or membranes can be formed that fit exactly in situ on the tissue, serving as biodegradable barriers, live cells or bioactive agent carriers, surgical adhesives. The polymer exhibits excellent biocompatibility as can be seen from the minimal fiber overgrowth on the implant sample. Model hydrogels gel in situ from water soluble precursors upon brief exposure to long wavelength ultraviolet (LWUV) light, resulting in an interpenetrating network of hydrogels containing tissue protein and glycosaminoglycan components Formed.
[0046]
As shown in Examples 4 and 5, the following three PVA hydrogels were made to show the release of NO and the containing drug (bFGF). PVA-NH ₂ -NO hydrogel; PVA-Cys-NO hydrogel; PVA-NO-bFGF hydrogel. The results are similar to PEG based hydrogels.
[0047]
Example 1: Synthesis of PEG-Cys-NO macromers and hydrogels
As shown in FIG. 1, acryloyl PEG-Cys-NO polymer is first made of polyethylene glycol N-hydroxysuccinimide monoacrylate (ACRL-PEG-NHS, molecular weight 3400, Shearwater) in 50 mM sodium bicarbonate buffer (pH 8.5). It was formed by reacting Huntington, AL) with L-cysteine at a molar ratio of 1: 2 for 2 hours. The product is then diH ₂ Dialyzed in cellulose with a cellulose ester membrane (molecular weight cut-off 500, Spectrum Labs, Laguna Hills, Calif.) And lyophilized. Analysis of the acryloyl PEG-Cys-NO copolymer was performed using gel permeation chromatography (GPC) (Polymer Laboratories, Amherst, Mass.) Equipped with a light scattering detector and a 260 nm UV detector. Successful synthesis of acryloyl-PEG-Cys was determined by shifting the peak position of the evaporative light scattering detector. Next, the molar amount of NaNO interrogating the copolymer ₂ And pH 2 and 37 ° C. for 20 minutes to form S-nitrosocysteine. Conversion of the thiol group to S-nitrosothiol was measured using an Ellman assay (Hermanson, (1995) Bioconjugate Technologies, San Diego, CA, Academic Press; 88-90). After adjusting the pH of the solution to 7.4, the acryloyl-PEG-Cys-NO polymer is PEG-diacrylate in an aqueous solution containing 1500 ppm 2,2-dimethoxy-2-phenylacetophenone as a long wavelength UV initiator. (Molecular weight 3400) was mixed at a molar ratio of 1:10 and mixed in a photopolymerizable hydrogel. 0.15% N-vinylpyrrolidone contained in this mixture was used as the solvent for the photoinitiator. UV light (365 nm, 10 mW / cm ² ) Was converted to a hydrogel when exposed to exposure to (Sawney et al., (1993) Macromol 26: 581-7).
[0048]
Example 2: PEG-Lys ₅ -Synthesis of NO macromers and hydrogels
As shown in FIG. 2, acryloyl-PEG-Lys ₅ For NO hydrogels, ACRL-PEG-NHS (molecular weight 3400, Shearwater Polymers) and poly-L-lysine (DP = 5) were reacted in an equimolar ratio in 50 mM sodium bicarbonate buffer (pH 8.5). A copolymer was synthesized. After the obtained copolymer was analyzed by GPC, it was dissolved in water and reacted with NO gas in a vacuum vessel to produce a NO-nucleophile complex having an amine group in the lysine side chain group. The degree of conversion of the amine group to the NO-nucleophile complex was measured using a ninhydrin assay and a hydrogel as described in Example 1 was formed.
[0049]
Example 3: Synthesis of PEG-DETA-NO macromers and hydrogels
Diethylenetriamine (DETA, Aldrich, Milwaukee, WI), poly-L-lysine (DP = 5), etc. with ACRL-PEG-NHS (molecular weight 3400, Shearwater Polymers) in 50 mM sodium bicarbonate buffer (pH 8.5) etc. Reacted at a molar ratio, lyophilized and analyzed by GPC as described above. The copolymer is then dissolved in water and exposed to NO gas to produce PEG-Lys. ₅ A NO-nucleophile complex was formed as described for -NO and analyzed for amine content using a ninhydrin assay. PEG-DETA-NO was lyophilized and then photopolymerized as described above to form a hydrogel, as shown in FIG.
[0050]
Example 4: PVA-NH ₂ -Synthesis of NO macromers and hydrogels
Poly (vinyl alcohol) (Hoechst, Mowiol 4-88) is diH ₂ Dissolved in O and warmed to 95 ° C. in a round bottom flask with constant stirring. After 1 hour, the solution was cooled to room temperature and methacrylamide acetaldehyde dimethyl acetal (NAAADA), a crosslinkable acetal group, was added. Amine acetal and gamma-butyraldehyde diethyl acetal were also added and the mixture was acidified using glacial acetic acid and 37% hydrochloric acid. After the mixture was stirred at room temperature for 9 hours, the pH was adjusted to 3.6 using triethylamine. In order to purify the polymer, the solution is then used with a cellulose membrane with a molecular weight of 3000 and a volume of diH of 6 times the polymer. ₂ Diafiltered in O. The polymer concentration was adjusted to 22% w / v using diafiltration and the pH adjusted to 7.4 with 1N NaOH. The amine concentration of the polymer was determined using the ninhydrin assay.
[0051]
PVA-NH ₂ The neutralized amine-modified polymer was placed in a round bottom flask with a stopcock to make a NO donor bound to. The flask was evacuated and filled with nitric oxide gas until the desired conversion of the amine to the NO nucleophile complex occurred. The photocrosslinking hydrogel is PVA-NH ₂ -NO with 0.1% IRGACURE ^TM 2959 (Ciba-Geigy) photoinitiator (based on the total volume of the solution) was added, followed by UV light (2 mW / cm ² And 365 nm) for 30 seconds. When the photoinitiator is added, the final polymer concentration is 20% w / v.
[0052]
Example 5: Synthesis of PVA-Cys-NO macromers and hydrogels
PVA-NH ₂ Was synthesized as described above. The amine terminus of cysteine is acetylated using acetic anhydride, and the carboxyl terminus of cysteine is PVA-NH using water soluble EDAC chemistry. ₂ Bound to. The resulting PVA-Cys was then purified using diafiltration to a concentration of 22% w / v. For PVA-Cys-NO, an equimolar amount of sodium nitrite was added to the cysteine residue, the pH was adjusted to 2, and the mixture was incubated at 37 ° C. for 15 minutes. The extent of reaction of cysteine to Cys-NO was analyzed using both Ellman and Griess reactions. The photoinitiator 2,2-methyl-2-phenylacetophenone was dissolved in N-vinylpyrrolidone at a concentration of 600 mg / ml and added to the polymer solution (0.1% based on the total volume of the solution). The polymer was then crosslinked by exposure to UV light for 30 seconds and placed in HEPES buffered saline at pH 7.4, 37 ° C.
[0053]
Example 6: Synthesis of PVA-NO-bFGF hydrogel
In the case of PVA-NO-bFGF hydrogel, the above procedure was used to make PVA-NO polymer. Just prior to exposure to UV light, 25 μg / ml bFGF was added to the polymer solution and mixed well. The gel was cross-linked as described above and stored in HEPES buffered saline at pH 7.4 and 37 ° C.
[0054]
Example 7: NO release from hydrogel
After preparation of the NO releasing material and photopolymerization as described above, the hydrogel was weighed and stored in HEPES buffered saline at pH 7.4, 37 ° C. A portion of the buffer was removed at each time point and fresh buffer was added. Samples taken at each time point were then analyzed for nitrite content using a colorimetric assay based on the Griess reaction.
[0055]
In order to explore possible storage conditions for the hydrogel, the NO release kinetics of the hydrogel stored in various pH level buffers were also investigated. NO release from the hydrogel was significantly suppressed at acidic pH levels.
[0056]
Acryloyl-PEG-Lys ₅ -NO release from the NO hydrogel is shown in FIG.
[0057]
NO release from acryloyl-PEG-DETA-NO hydrogel is shown in FIG.
[0058]
NO release from acryloyl-PEG-Cys-NO hydrogel is shown in FIG.
[0059]
Example 8: Release of NO and bFGF from PVA-NO-bFGF hydrogel
NO release from PVA-NO-bFGF hydrogel prepared as described in Example 6 was determined in the same manner as Example 7 and is shown in FIG. 7A. bFGF release was quantified using the BCA assay (Pierce Chemicals) and is shown in FIG. 7B. The release of NO continues significantly beyond 12 hours, but the growth factor is completely released within the first 5 hours.
[0060]
Example 9: Effect of NO releasing macromer on cultured smooth muscle cells: proliferation and viability
In order to evaluate the potential of substances that reduce smooth muscle cell (SMC) proliferation after vascular injury, cultured smooth muscle cells were cultured in the presence of NO-releasing substances and the effects of these substances on the cells were evaluated. Minimal smooth muscle cells isolated from Wistar-Kyoto rats (passage 11-15, provided by T. Scott-Burden) plus 10% FBS, 2 mM L-glutamine, 500 units penicillin, 100 mg / l streptomycin CO at 37 ° C using essential medium ₂ Cultured in the environment. The cells were transferred to a 24-well tissue culture plate (Becton Dickinson, Franklin Lakes, NJ) at 10,000 cells / cm. ² Seeded at a density of One day after seeding, either the dissolved or hydrogel form of the NO donor was added to the medium in the wells. On the fourth day of culture, a single cell suspension of trypsin was prepared and 3 samples in each group were counted using a Coulter counter (Multisizer number 0646, Coulter Electronics, Hialeah, FL) to determine the number of cells. Were determined.
[0061]
The effect of NO donors in solution on SMC growth was examined by first performing a NO dose response curve. There, cells were cultured at a range of NO donor concentrations (1 μM to 10 mM) to confirm the appropriate dose for hydrogel studies. NO-nucleophile complexes (Lys-NO and DETA-NO) were made by reacting either L-lysine or DETA with NO gas for 24 hours in water. Soluble Cys-NO converts equimolar amounts of L-cysteine to NaNO at pH 2 and 37 ° C. ₂ And synthesized for 20 minutes. All NO donor solutions were adjusted to pH 7.4 before being added to the cell culture.
[0062]
The optimal dose of NO determined above was then used to examine smooth muscle cell proliferation in the presence of NO production and control hydrogel. Acryloyl-PEG-Lys ₅ -Hydrogels containing NO, acryloyl-PEG-DETA-NO, acryloyl-PEG-Cys-NO were prepared by subjecting the gel solution to 0.2 μm syringe filter (Gelman Sciences, Ann) before adding 2,2-dimethoxy-2-phenylacetophenone. Formed as described above, except that it was sterile filtered through Arbor, MI). A PEG-diacrylate hydrogel containing no NO donor was used as a control. The hydrogel was photopolymerized in a cell culture insert (8 μm pore size, Becton Dickinson, Franklin Lakes, NJ) and placed in the medium on the cultured cells.
[0063]
All three hydrogel NO donors showed significant SMC growth (p <0.0001). The number of smooth muscle cells remained close to the number of seeding densities and ranged from 10-15% of the final control cell number in all experiments.
[0064]
Acryloyl-PEG-Lys ₅ Inhibition of SMC proliferation by -NO hydrogel is shown in FIG. 8A compared to the macromer solution control shown in FIG. 8B. Both significantly inhibited SMC proliferation.
[0065]
Inhibition of SMC proliferation by acryloyl-PEG-DETA-NO-nucleophile complex hydrogel is shown in FIG. 9A compared to the macromer solution control shown in FIG. 9B. Both significantly inhibited SMC proliferation.
[0066]
Inhibition of SMC proliferation by acryloyl-PEG-Cys-NO hydrogel is shown in FIG. 10A compared to the macromer solution control shown in FIG. 10B. Both significantly inhibited SMC proliferation.
[0067]
Inhibition of SMC proliferation by acryloyl-PEG-Cys-NO hydrogel, acryloyl-PEG-DETA-NO hydrogel, acryloyl-PEG-Lys-NO hydrogel was compared to the FIG. 11 control hydrogel. All NO hydrogels significantly inhibited SMC growth.
[0068]
Example 10: Effect of NO releasing macromer on platelet adhesion in vitro
To evaluate the potential of these substances for preventing thrombosis, the effect of NO-releasing macromers on platelet adhesion was examined. Blood was collected from healthy volunteers by venipuncture and anticoagulated with 10 U / ml heparin. Platelets and white blood cells were fluorescently labeled with mepacrine at a concentration of 10 μM. 3% glacial acetic acid diH ₂ A 2.5 mg / ml collagen I solution with O was prepared and applied to a glass slide and placed in a humidified environment at room temperature for 45 minutes. Acryloyl-PEG-Cys-NO and PEG-diacrylate hydrogels were prepared as described above and incubated with labeled whole blood at 37 ° C. for 30 minutes. The hydrogel was removed and then the blood was incubated with collagen-coated glass slides for 20 minutes at 37 ° C. (2 per group) and then rinsed with HBS. Four areas were randomly selected per slide, and the number of platelets per field at 40x was counted under a fluorescence microscope (Zeiss Axiovert 135, Thornwood, NY).
[0069]
Photographs of platelets exposed to control PEG-diacrylate or acryloyl-PEG-Cys-NO hydrogels show that exposure to NO-releasing hydrogels inhibits platelets from adhering to the clot-forming surface. A glass slide coated with collagen was used as a thrombus-forming surface to which platelets normally adhere. When blood was incubated with a control PEG-diacrylate hydrogel, 69.25 ± 4.46 (mean ± standard deviation) adherent platelets per field were seen. When blood was previously exposed to acryloyl-PEG-Cys-NO hydrogel, this number decreased to 7.65 ± 6.16 per field (p <0.0001).
[0070]
Modifications or variations of the methods and materials described herein will become apparent to those skilled in the art from the foregoing detailed description and accompanying drawings. These methods and materials are intended to be included in the claims.
[Brief description of the drawings]
FIG. 1 is a schematic representation of the synthesis of S-nitrosocysteine hydrogel (acryloyl-PEG-Cys-NO).
FIG. 2 shows acryloyl-PEG-lysine ₅ 1 is a schematic diagram of the synthesis of —NO—nucleophile complex hydrogel. FIG.
FIG. 3 is a schematic representation of the synthesis of acryloyl-PEG-DETA-NO-nucleophile complex hydrogel.
FIG. 4 shows acryloyl-PEG-Lys at pH 7.4 (circle) and pH 3.0 (square). ₅ -NO is a graph showing the time release of NO from hydrogel (% NO release with respect to days).
FIG. 5 is a graph showing the time release of NO from acryloyl-PEG-DETA-NO hydrogel at pH 7.4 (circle) and pH 2.0 (square) (% NO release vs. days).
FIG. 6 is a graph showing time release of NO from acryloyl-PEG-Cys-NO hydrogel (% NO release over days) at pH 7.4 (circles) and pH 2.0 (squares).
7A and 7B are graphs showing the time release of NO from PVA-NO-bFGF hydrogel at pH 7.4 and 37 ° C. (NO mol released per gram of polymer over time). FIG. 7B is a graph showing the time release of NO from the PVA-NO-bFGF hydrogel at pH 7.4, 37 ° C. (theoretical release bFGF% per gram polymer over time).
FIGS. 8A and 8B are graphs showing that acryloyl-PEG-lysine-NO hydrogel inhibits smooth muscle cell proliferation. FIG. 8A,% control cell number, hydrogel formulation. FIG. 8B,% control cell number, soluble polymer.
FIGS. 9A and 9B are graphs showing inhibition of SMC proliferation as a percentage of control by NO released from acryloyl-PEG-DETA-NO hydrogel (FIG. 9A) and soluble polymer (FIG. 9B). It is.
FIGS. 10A and 10B show the inhibition of SMC proliferation by NO released from acryloyl-PEG-CYSNO, Lys-NO hydrogel (FIG. 10A) and soluble polymer (FIG. 10B) as a percentage of the control. It is a graph to show.
FIG. 11 shows smooth muscle cells with NO release from hydrogels: acryloyl-PEG-Lys-NO, acryloyl-PEG-DETA-NO and acryloyl-PEG-Cys-NO compared to a control hydrogel containing NO. It is a graph which compares the degree of inhibition of growth. The percent inhibition of smooth muscle cell growth is determined relative to the cell growth of each NO-releasing hydrogel compared to the control PEG-diacrylate hydrogel.
(Background of the Invention)
Endothelial cells, usually present as a monolayer in the intimal layer of the arterial wall, are thought to play an important role in regulating smooth muscle cell (SMC) proliferation in vivo. Endothelial cells are severely inhibited by most forms of vascular injury, including by percutaneous transluminal coronary angioplasty and similar procedures. Approximately 35-50% of patients undergoing percutaneous transluminal coronary angioplasty experience clinical arterial severe restenosis, or restenosis, within 6 months of initial treatment. Restenosis is due, at least in part, to the migration and proliferation of smooth muscle cells within the arterial wall, along with increased secretion of matrix proteins to create an occlusive neointimal layer within the arterial wall. Similar problems limit the performance of vascular grafts. The process of regulating arterial wound healing after vascular injury, such as caused by angioplasty, is not yet well understood, but appears to involve a complex cascade of blood and vessel wall-derived factors.
A number of factors that promote intimal thickening and restenosis have been identified by the administration of exogenous proteins, cellular genetic alterations, or the blocking of certain signals using antibodies or other specific growth factor inhibitors. These smooth muscle cell mitogenic factors and chemoattractants are derived from both blood or thrombus formation and the vessel wall itself. Endothelial cells produce many substances including heparin sulfate, prostacyclin (PG12) and NO, which are known to down regulate smooth muscle cell proliferation.
NO is a target molecule derived from endothelial cells useful for preventing restenosis. Because, in addition to restricting smooth muscle cell proliferation (Garg et al., (1989) J. Clin. Invest., 83: 1774-7), platelet aggregation is reduced (de Graaf et al., (1992) Circulation, 85: 2284-90; Radomski et al., (1987) Br. J. Pharmacol., 92: 181-7) and increased endothelial cell proliferation (Ziche et al., (1993) Biochem. Biophys. Res. Comm., 192. 1198-1203) to reduce leukocyte adhesion (Lefer et al., (1993) Circulation, 88: 2337-50), all of which are highly desirable for reducing intimal thickening and restenosis (Loscalzo, (1996). ) Clin. Appl. mb.Hemostas., 2: 7-10). Because the restenosis process is complex, an approach that works with multiple targets appears to be most successful.
The mechanism by which NO affects these multiple reactions is not yet fully understood, but NO binds to the hemo moiety of soluble guanylate cyclase and is a second messenger between cells that has multiple cellular effects. It is known to activate guanylate cyclase by increasing the level of cyclic guanosine monophosphate (cGMP) (Moro et al., (1996) Proc. Natl. Acad. Sci. USA, 93: 1480-5). The effect of NO can often be mimicked by administration of cGMP or a more stable derivative of cGMP (Garg et al. (1989) J. Clin. Invest., 83: 1774-7). Furthermore, NO converts ribonucleotides into deoxyribonucleotides and thus significantly contributes to DNA synthesis, as is the case with a plurality of enzymes involved in cell respiration (Stuhr et al., (1989) J. Exp. Med., 169: 1543-55). Ribonucleotide, an enzyme that affects (Lepoivre et al., (1991) Biochem. Biophys. Res. Comm., 179: 442-8; Kwon et al. (1991) J. Exp. Med., 174: 761-7) It has been shown to inhibit lactase.
A number of molecules (NO donors) that produce NO under physiological conditions have been identified and evaluated both in vitro and in vivo. NO donor molecules exert a physiological effect that mimics NO, and S-nitrosothiols (Diodati et al., (1993) Thromb. Haem., 70: 654-8; Lefer et al., (1993) Circulation, 88: 2337-50. DeMeyer et al., (1995) J. Cardiovasc. Pharmacol., 26: 272-9), organic nitrates (Ignaro et al., (1981) J. Pharmacol. Exp. Ther., 218: 739-49) and NO and nucleophilicity. Complexes of reagents (Diodati et al., (1993) Thromb. Haem., 70: 654-8; Diadati et al., (1993) J. Cardiovasc. Pharmacol., 22: 287-92; Maragos et al., (1993) Cancer Res., 53: 564-8). Many of these are low molecular weight molecules that are administered systemically and have a short half-life under physiological conditions, thus exerting activity on many tissue types for a short period of time. Furthermore, since L-arginine is often considered as a NO donor and L-arginine is a substrate for NO synthase, administration of L-arginine improves endogenous NO production and, in most cases, NO. It induces a reaction similar to that caused by the donor (Cook et al., (1992) J. Clin. Invest., 90: 1168-72).
The development of NO releasing polymers containing NO / nucleophile complexes is described in Smith et al. (1996) J. MoI. Med. Chem. 39: 1148-56. These substances are capable of releasing NO in vitro for 5 weeks, limiting smooth muscle cell proliferation in culture and preventing platelet adhesion to vascular graft material in an arterio-venous shunt model. It was possible to reduce. These materials are promising for many clinical applications where local NO production is desired, such as anti-thrombogenic coating materials for catheters, but these materials suffer from a number of drawbacks, so that in vivo tissue Probably not useful for direct treatment. Although these polymers can be made as films, powders or microparticles, they cannot be formed in situ in direct contact with cells and tissues, making it difficult to strictly localize NO treatment for certain tissues, Problems with keeping the polymer in place can occur. Formulation issues also make local administration difficult or impossible during laparoscopic or catheter procedures. Furthermore, the biocompatibility of the base polymer is a significant problem for implantable NO-releasing polymers, especially for long-term use, as an inflammation or thrombotic reaction can occur after cessation of NO release.
If these compounds can be administered alone at the site where treatment is required, or in some cases, the side effects of systemic administration of the drug can be reduced and eliminated, it is even more effective, especially over the long term.
Accordingly, it is an object of the present invention to provide an agent for controlled release at a specific site of a compound that modulates NO and / or NO levels after local or topical administration.
It is a further object of the present invention to provide a method for the treatment of conditions including inflammatory reactions by providing a hydrogel material that releases a compound that modulates NO levels at the site of application.

Claims (20)

A biocompatible and polymerizable macromer composition for controlled release of NO or NO donor , comprising at least one NO retention region or NO donor;
NO or NO donor is released from the macromer composition under physiological conditions after polymerization on tissue ,
The macromer composition is a water soluble region, a region selected from the group consisting of tissue adhesion area, and a polymerizable end group regions, proteins, carbohydrates, nucleic acids, organic molecules, inorganic molecules, bioactive molecules, cells, tissues, and I saw including at least one therapeutic or diagnostic agent is selected from the group consisting of tissue aggregates,
The macromer composition comprises acryloyl-PEG-Cys-NO macromer, acryloyl-PEG-Lys ₅ -NO macromer, acryloyl-PEG-DETA-NO macromer, PVA-NH ₂ -NO macromer, PVA-Cys-NO macromer, and Comprising a macromer selected from the group consisting of PVA-NO-bFGF macromers,
Macromer composition . The macromer composition of claim 1, wherein the macromer composition further comprises a macromer that does not release NO after polymerization. The macromer composition of claim 1, wherein the macromer further comprises crosslinkable side groups. The macromer composition of claim 1, wherein the macromer composition comprises at least one degradable region. The macromer composition of claim 1, wherein the macromer composition is water soluble. The macromer composition of claim 1, wherein the macromer composition adheres to tissue. 2. The macromer composition of claim 1, comprising a water soluble region that binds to the degradable region, at least one polymerizable region that binds to the water soluble region, and at least one polymerizable region that binds to the degradable region. Macromer composition . 2. The macromer composition of claim 1 , wherein the degradable region is a central nucleus, at least two water soluble regions are attached to the nucleus, and at least one polymerizable region is attached to each water soluble region. 2. The macromer of claim 1, wherein the macromer composition comprises a water soluble region that forms a central nucleus, at least two degradable regions that bind to the nucleus, and at least two polymerizable regions that bind to the degradable region. Composition . 2. The macromer composition of claim 1, wherein the macromer composition comprises at least one water soluble region, at least one NO retention region, and at least one free radical polymerizable region. The macromer composition of claim 10 , further comprising at least one degradable region. 2. The macromer composition of claim 1, comprising or releasably bound to a NO donor that produces NO under physiological conditions. The macromer composition of claim 1, which releases NO under physiological conditions. Use of a macromer in the manufacture of a biocompatible and polymerizable macromer composition for the controlled release of NO or a NO donor ,
The macromer composition comprises at least one NO retention region or NO donor , wherein the NO or NO donor is released from the macromer composition under physiological conditions after polymerization on tissue;
The macromer composition is a water soluble region, a region selected from the group consisting of tissue adhesion area, and a polymerizable end group regions, proteins, carbohydrates, nucleic acids, organic molecules, inorganic molecules, bioactive molecules, cells, tissues, And at least one therapeutic or diagnostic agent selected from the group consisting of tissue aggregates ,
The macromer composition comprises acryloyl-PEG-Cys-NO macromer, acryloyl-PEG-Lys ₅ -NO macromer, acryloyl-PEG-DETA-NO macromer, PVA-NH ₂ -NO macromer, PVA-Cys-NO macromer, and Comprising a macromer selected from the group consisting of PVA-NO-bFGF macromers,
Use . At a site macromer composition solution is polymerized, the polymerization initiator is first applied, use according to claim 14. Polymerization initiator is bound to tissue, and unbound initiator prior to applying the macromer composition solution is removed, use according to claim 15. Use of a biocompatible polymerizable macromer composition comprising at least one NO retention region or NO donor in the manufacture of a medicament for treating a disorder or condition with NO comprising :
NO or NO donor is released from the macromer composition under physiological conditions after polymerization on the tissue ,
The macromer composition comprises a region selected from the group consisting of a water soluble region, a tissue adhesion region, and a polymerizable end group region ;
The macromer composition comprises acryloyl-PEG-Cys-NO macromer, acryloyl-PEG-Lys ₅ -NO macromer, acryloyl-PEG-DETA-NO macromer, PVA-NH ₂ -NO macromer, PVA-Cys-NO macromer, and Comprising a macromer selected from the group consisting of PVA-NO-bFGF macromers,
Use . The use according to claim 17 , wherein the macromer composition further comprises a degradable region. 18. Use according to claim 17 , wherein the disorder or condition is selected from the group consisting of wound treatment, restenosis, thrombus, asthma, arthritis, and erectile dysfunction. By macromer composition to adhere to tissue, or to prevent surgical adhesions, or to adhere the tissue, or providing support to the tissue, or coating of tissue, use of claim 17.

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