Synthesis and characterization of novel thiol-reactive poly(ethylene glycol) cross-linkers for extracellular-matrix-mimetic biomaterials

Synthetic extracellular matrix hydrogels can be used for three-dimensional cell culture, wound repair, and tissue engineering. Using the bifunctional electrophile poly(ethylene glycol) diacrylate (PEGDA), thiol-modified glycosaminoglycans and polypeptides can be cross-linked into biocompatible materials in the presence of cells or tissues. However, the rate of in situ cross-linking with PEGDA under physiological conditions may occur too slowly for clinical applications requiring a fast-curing preparation. To explore a wider range of cross-linking time courses, five homo-bifunctional PEG derivatives were synthesized and examined as cross-linking agents for thiol-modified derivatives of hyaluronan (HA). Thiol reaction rate constants were measured over a pH range of 7.4 to 8.6. The order of reactivity for the functional groups used was determined to be maleimide > iodoacetate > bromoacetate > iodoacetamide > acrylate > bromoacetamide, with rates increasing exponentially with increasing pH. The range of gelation times at physiological pH varied from less than 1 min to over 2 h. Addition of the cross-linkers to cell culture medium showed minimal cytotoxicity toward primary human dermal fibroblasts at concentrations anticipated during in situ cross-linking. Moreover, hydrogels prepared from thiol-modified gelatin and thiol-modified HA were biocompatible and supported attachment and proliferation of fibroblasts and hepatocytes.     

Engineering a clinically-useful matrix for cell therapy

The design criteria for matrices for encapsulation of cells for cell therapy include chemical, biological, engineering, marketing, regulatory, and financial constraints. What is required is a biocompatible material for culture of cells in three-dimensions (3-D) that offers ease of use, experimental flexibility to alter composition and compliance, and a composition that would permit a seamless transition from in vitro to in vivo use. The challenge is to replicate the complexity of the native extracellular matrix (ECM) environment with the minimum number of components necessary to allow cells to rebuild a given tissue. Our approach is to deconstruct the ECM to a few modular components that can be reassembled into biomimetic materials that meet these criteria. These semi-synthetic ECMs (sECMs) employ thiol-modified derivatives of hyaluronic acid (HA) that can form covalently crosslinked, biodegradable hydrogels. These sECMs are “living” biopolymers, meaning that they can be crosslinked in the presence of cells or tissues to enable cell therapy and tissue engineering. Moreover, the sECMs allow inclusion of the appropriate biological cues needed to simulate the complexity of the ECM of a given tissue. Taken together, the sECM technology offers a manufacturable, highly reproducible, flexible, FDA-approvable, and affordable vehicle for cell expansion and differentiation in 3-D.     

Engineering a clinically-useful matrix for cell therapy

The design criteria for matrices for encapsulation of cells for cell therapy include chemical, biological, engineering, marketing, regulatory, and financial constraints. What is required is a biocompatible material for culture of cells in three-dimensions (3-D) that offers ease of use, experimental flexibility to alter composition and compliance, and a composition that would permit a seamless transition from in vitro to in vivo use. The challenge is to replicate the complexity of the native extracellular matrix (ECM) environment with the minimum number of components necessary to allow cells to rebuild a given tissue. Our approach is to deconstruct the ECM to a few modular components that can be reassembled into biomimetic materials that meet these criteria. These semi-synthetic ECMs (sECMs) employ thiol-modified derivatives of hyaluronic acid (HA) that can form covalently crosslinked, biodegradable hydrogels. These sECMs are “living” biopolymers, meaning that they can be crosslinked in the presence of cells or tissues to enable cell therapy and tissue engineering. Moreover, the sECMs allow inclusion of the appropriate biological cues needed to simulate the complexity of the ECM of a given tissue. Taken together, the sECM technology offers a manufacturable, highly reproducible, flexible, FDA-approvable, and affordable vehicle for cell expansion and differentiation in 3-D.Key words: hyaluronan, 3-D cell culture, tissue engineering, extracellular matrix, crosslinked hydrogel, design criteria, commercial utility, stem cells, regenerative medicine     

Disulfide cross-linked hyaluronan hydrogels

A new disulfide cross-linking strategy was developed to prepare hyaluronic acid (HA) hydrogel from thiol-modified HA. First, dithiobis(propanoic dihydrazide) (DTP) and dithiobis(butyric dihydrazide) (DTB) were synthesized and then coupled to HA with carbodiimide chemistry. Next, disulfide bonds of the initially formed gel were reduced using dithiothreitol (DTT) to give, after exhaustive dialysis, the corresponding thiol-modified macromolecular derivatives HA-DTPH and HA-DTBH. The degree of substitution of HA-DTPH and HA-DTBH could be controlled from 20% to 70% of available glucuronate carboxylic acid groups. The pK(a) values of the HA-thiol derivatives were determined spectrophotometrically to be pK(a) = 8.87 (HA-DTPH) and pK(a) = 9.01 (HA-DTBH). The thiol groups could be oxidized in air to reform disulfide linkages, which resulted in HA-DTPH and HA-DTBH hydrogel films. Further oxidation of these hydrogels with dilute H(2)O(2) created additional cross-links and afforded poorly swellable films. The disulfide cross-linking was reversible, and films could be again reduced to sols with DTT. Release of blue dextran from cross-linked films was used as a model for drug release. The rapid gelation of the HA-DTPH solution under physiological conditions was also achieved, which demonstrated the capacity for in situ cell encapsulation. Thus, L-929 murine fibroblasts were encapsulated in HA-DTPH hydrogel; these cells remained viable and proliferated during 3 days of culture in vitro.     

Aminooxy pluronics: synthesis and preparation of glycosaminoglycan adducts

Novel biomaterials have been prepared in which glycosaminoglycans (GAGs) are chemically modified to create amphiphilic multiblock copolymers that are able to adhere to hydrophobic surfaces and can self-assemble into cross-linker-free hydrogels. First, the triblock poly(ethylene oxide)-polypropylene oxide copolymers (Pluronics) were converted into the previously unknown aminooxy (AO) derivatives. Both mono-AO and bis-AO Pluronics (AOPs) were synthesized and fully characterized in order to prepare tetrablock and pentablock copolymers, respectively. Second, the AOPs were coupled to the uronic acid carboxylates of heparin (HP) and hyaluronic acid (HA) using carbodiimide chemistry in order to give the previously undescribed amidooxy GAG derivatives. The coupling chemistry was confirmed using a newly prepared fluorescent AO reagent. Third, AOP-heparin and AOP-fluorescently labeled heparin were shown to adsorb efficiently to polystyrene surfaces, as determined by IL-8 based ELISA and fluorescence measurements, respectively. Fourth, AOP-linked fluorescently labeled HA was shown to adsorb efficiently to plastic surfaces. Finally, three different AOPs were evaluated for self-assembling hydrogel formation by AOP-HA pentablock polymers. In short, AOP-GAG adducts are semisynthetic amphiphilic biomacromolecules that offer a range of valuable practical opportunities for surface modification, preparation of cross-linker-free hydrogels, and formation of self-assembling mimics of the extracellular matrix.     

Synthesis and biological evaluation of a cross-linked hyaluronan-mitomycin C hydrogel

A cross-linked hyaluronan (HA) hydrogel that contained a covalently bound derivative of the anti-proliferative drug mitomycin C (MMC) was synthesized and evaluated in vitro and in vivo. The HA-MMC hydrogel was prepared by coupling MMC-aziridinyl-N-acrylate with thiol-modified HA followed by cross-linking with poly(ethylene glycol) diacrylate (PEGDA). MMC was released from 0.5% and 2.0% MMC films by hydrolysis in proportion to the MMC loading. When incubated in vitro with human T31 tracheal scar fibroblasts, 0.5% MMC films inhibited proliferation, whereas 2.0% MMC films were cytotoxic. When implanted in vivo into a rat peritoneal cavity, neither 0.5% nor 2.0% HA-MMC films elicited a severe peritoneal fluid leukocyte response. Importantly, MMC reduced the thickness of fibrous tissue formed surrounding the implanted films. Thus, cross-linked HA-MMC films have strong potential as anti-fibrotic barriers for the prevention of post-surgical adhesions.     

Rheological properties of cross-linked hyaluronan-gelatin hydrogels for tissue engineering

Hydrogels that mimic the natural extracellular matrix (ECM) are used in three-dimensional cell culture, cell therapy, and tissue engineering. A semi-synthetic ECM based on cross-linked hyaluronana offers experimental control of both composition and gel stiffness. The mechanical properties of the ECM in part determine the ultimate cell phenotype. We now describe a rheological study of synthetic ECM hydrogels with storage shear moduli that span three orders of magnitude, from 11 to 3 500 Pa, a range important for engineering of soft tissues. The concentration of the chemically modified HA and the cross-linking density were the main determinants of gel stiffness. Increase in the ratio of thiol-modified gelatin reduced gel stiffness by diluting the effective concentration of the HA component.     

Anti-calcification of bovine pericardium for bioprosthetic heart valves after surface modification with hyaluronic acid derivatives

Surface modification of glutaraldehyde fixed bovine pericardium (GFBP) was successfully carried out with hyaluronic acid (HA) derivatives. At first, HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional group into the carboxyl group of HA backbone. Then, GFBP was surface modified by grafting HA-ADH to the free aldehyde groups on the tissue and the subsequent HA-ADH hydrogel coating. HA-ADH hydrogels could be prepared through selective crosslinking at low pH between hydrazide groups of HA-ADH and crosslinkers containing succinimmidyl moieties with minimized protein denaturation. When HA-ADH hydrogels were prepared at low pH of 4.8 in the presence of erythropoietin (EPO) as a model protein, EPO release was continued up to 85% of total amount of loaded EPO for 4 days. To the contrary, only 30% of EPO was released from HA-ADH hydrogels prepared at pH=7.4, which might be due to the denaturation of EPO during the crosslinking reaction. Because the carboxyl groups on the glucuronic acid residues are recognition sites for HA degradation by hyaluronidase, the HA-ADH hydrogels degraded more slowly than HA hydrogels prepared by the crosslinking reaction of divinyl sulfone with hydroxyl groups of HA. Following a two-week subcutaneous implantation in osteopontin-null mice, clinically significant levels of calcification were observed for the positive controls without any surface modification. However, the calcification of surface modified GFBP with HA-ADH and HA-ADH hydrogels was drastically reduced by more than 85% of the positive controls. The anti-calcification effect of HA surface modification was also confirmed by microscopic analysis of explan ted tissue after staining with Alizarin Red S for calcium, which followed the trend as observed with calcium quantification.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.     

Modular extracellular matrices: solutions for the puzzle

The common technique of growing cells in two-dimensions (2-D) is gradually being replaced by culturing cells on matrices with more appropriate composition and stiffness, or by encapsulation of cells in three-dimensions (3-D). The universal acceptance of the new 3-D paradigm has been constrained by the absence of a commercially available, biocompatible material that offers ease of use, experimental flexibility, and a seamless transition from in vitro to in vivo applications. The challenge-the puzzle that needs a solution-is to replicate the complexity of the native extracellular matrix (ECM) environment with the minimum number of components necessary to allow cells to rebuild and replicate a given tissue. For use in drug discovery, toxicology, cell banking, and ultimately in reparative medicine, the ideal matrix would therefore need to be highly reproducible, manufacturable, approvable, and affordable. Herein we describe the development of a set of modular components that can be assembled into biomimetic materials that meet these requirements. These semi-synthetic ECMs, or sECMs, are based on hyaluronan derivatives that form covalently crosslinked, biodegradable hydrogels suitable for 3-D culture of primary and stem cells in vitro, and for tissue formation in vivo. The sECMs can be engineered to provide appropriate biological cues needed to recapitulate the complexity of a given ECM environment. Specific applications for different sECM compositions include stem cell expansion with control of differentiation, scar-free wound healing, growth factor delivery, cell delivery for osteochondral defect and liver repair, and development of vascularized tumor xenografts for personalized chemotherapy.     

Synthesis and degradation test of hyaluronic acid hydrogels

Hyaluronic acid (HA) hydrogels prepared with three different crosslinking reagents were assessed by in vitro and in vivo degradation tests for various tissue engineering applications. Adipic acid dihydrazide grafted HA (HA-ADH) was synthesized and used for the preparation of methacrylated HA (HA-MA) with methacrylic anhydride and thiolated HA (HA-SH) with Traut's reagent (imminothiolane). (1)H NMR analysis showed that the degrees of HA-ADH, HA-MA, and HA-SH modification were 69, 29, and 56 mol%, respectively. HA-ADH hydrogel was prepared by the crosslinking with bis(sulfosuccinimidyl) suberate (BS(3)), HA-MA hydrogel with dithiothreitol (DTT) by Michael addition, and HA-SH hydrogel with sodium tetrathionate by disulfide bond formation. According to in vitro degradation tests, HA-SH hydrogel was degraded very fast, compared to HA-ADH and HA-MA hydrogels. HA-ADH hydrogel was degraded slightly faster than HA-MA hydrogel. Based on these results, HA-MA hydrogels and HA-SH hydrogels were implanted in the back of SD rats and their degradation was assessed according to the pre-determined time schedule. As expected from the in vitro degradation test results, HA-SH hydrogel was in vivo degraded completely only in 2 weeks, whereas HA-MA hydrogels were degraded only partially even in 29 days. The degradation rate of HA hydrogels were thought to be controlled by changing the crosslinking reagents and the functional group of HA derivatives. In addition, the state of HA hydrogel was another factor in controlling the degradation rate. Dried HA hydrogel at 37 degrees C for a day resulted in relatively slow degradation compared to the bulk HA hydrogel. There was no adverse effect during the in vivo tests.     

Polymers containing isonitrile functional groups as supports for the covalent fixation of biologically active molecules

1) Isonitrile derivatives of synthetic polyamides, polyesters and polyalcohols, of polysaccharides such as cellulose, cross-linked dextran and agarose, linear dextran and of cross-linked and linear polyacrylamide were prepared. 2) Proteins and low-molecular-weight ligands were bound covalently to these materials by four-component reactions involving the -NC function on the polymer, amino or carboxyl groups deriving from the ligand, aldehyde and the complementary fourth component (-COOH or - NH2) being added to the aqueous reaction medium. 3) Supports of modified surface properties could be prepared by grafting of water-soluble macromolecules containing -NC groups onto preformed polymeric structures. 4) The isonitrile functional groups on polymers could be transformed into other types of functional groups by simple one-step reactions.     

Diels-Alder Click cross-linked hyaluronic acid hydrogels for tissue engineering

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step, aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder "click" chemistry. Furan-modified HA derivatives were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.     

Biotinylated biodegradable nanotemplated hydrogel networks for cell interactive applications

We describe the synthesis of a novel biotinylated nanotextured degradable hydrogel that can be rapidly surface engineered with a diverse range of biotinylated moieties. The hydrogel is synthesized by reacting methacrylated biotin-PEG with dimethacrylated P LA-b- PEG-b-P LA (LPLDMA, PEG = poly(ethylene glycol), PLA = poly(lactic acid)),or dimethacrylated PEG-b-P LA-b- PEG (PLPDMA). Methacrylated biotin-PEG is prepared by reacting biotin-PEG-OH with methacrylic anhydride. Biotin-PEG-OH is prepared by reacting alpha-hydroxy-omega-amine PEG with N-hydroxysuccinimide-biotin. Confirmation of the final product is determined using (1)H NMR and Fourier transform infrared spectroscopy (FTIR). The integrity and surface presentation of the biotin units is observed spectrophotometrically using the HABA/avidin assay. To produce nanostructured polymer topography, a self-assembling lyotropic liquid crystalline mesophase is used as a polymerization template, generating biotinylated hydrogels with highly organized lamellar matrix geometry. Traditionally processed isotropic hydrogels are used for comparison. Scanning electron microscopy shows that isotropic hydrogels have a smooth glassy appearance while lamellar templated hydrogels have defined surface topographical features that enhance preosteoblast human palatal mesenchymal cell (HEPM) attachment. Engineering the surfaces of the hydrogels with cell adhesive Arg-Gly-Asp (RGD) peptide sequences using the biotin-avidin interaction significantly enhances cell attachment. Surface engineering of cell adhesive peptides in conjunction with the lamellar template induced surface topography generates additive enhancements in cell attachment.     

Enzymatic degradation of hyaluronan hydrogels with different capacity for in situ bio‐mineralization

In situ cross‐linked hyaluronan (HA) hydrogels with different capacities for biomineralization were prepared and their enzymatic degradation was monitored. Covalent incorporation of bisphosphonates (BPs) into HA hydrogel results in the increased stiffness of the hydrogel in comparison with the unmodified HA hydrogel of the same cross‐linking density. The rate of enzymatic degradation of HABP hydrogel was significantly lower than the rate of degradation of control HA hydrogel in vitro. This effect is observed only in the presence of calcium ions that strongly bind to the matrix‐anchored BP groups and promote further mineralization of the matrix. The degradation of the hydrogels was followed by noninvasive fluorescence measurements enabled after mild and chemoselective labeling of cross‐linkable HA derivatives with a fluorescent tag.     

Reductive alkylation of hyaluronic acid for the synthesis of biocompatible hydrogels by click chemistry

Hyaluronan (HA) based hydrogels have been synthesized combining chemical modification of the polysaccharide by partial oxidation, reductive amination and 'click chemistry'. HA was oxidized by 4-acetamido-TEMPO-mediated reaction, using sodium hypochlorite as primary oxidant and NaBr in buffered pH, so that the produced aldehyde moieties (hemiacetals) were trapped in situ by adding primary amines containing azide or alkyne-terminal groups. The structure of the reaction products, oxidized-HA and primary amines bonded to HA, was elucidated using 2D NMR spectroscopy. SEC-MALLS analysis of the modified substrates showed a negligible degradation of the polysaccharide using this procedure. Furthermore, azido- and alkynyl derivatives underwent cross-linking by click chemistry into hydrogels, which were characterized by NMR, FT-IR, swelling degree and mechanical properties. Possible application of the material as scaffold for tissue engineering was tested by seeding and proliferation of chondrocytes for up to 15 days.     

Investigation of Overrun-Processed Porous Hyaluronic Acid Carriers in Corneal Endothelial Tissue Engineering

Hyaluronic acid (HA) is a linear polysaccharide naturally found in the eye and therefore is one of the most promising biomaterials for corneal endothelial regenerative medicine. This study reports, for the first time, the development of overrun-processed porous HA hydrogels for corneal endothelial cell (CEC) sheet transplantation and tissue engineering applications. The hydrogel carriers were characterized to examine their structures and functions. Evaluations of carbodiimide cross-linked air-dried and freeze-dried HA samples were conducted simultaneously for comparison. The results indicated that during the fabrication of freeze-dried HA discs, a technique of introducing gas bubbles in the aqueous biopolymer solutions can be used to enlarge pore structure and prevent dense surface skin formation. Among all the groups studied, the overrun-processed porous HA carriers show the greatest biological stability, the highest freezable water content and glucose permeability, and the minimized adverse effects on ionic pump function of rabbit CECs. After transfer and attachment of bioengineered CEC sheets to the overrun-processed HA hydrogel carriers, the therapeutic efficacy of cell/biopolymer constructs was tested using a rabbit model with corneal endothelial dysfunction. Clinical observations including slit-lamp biomicroscopy, specular microscopy, and corneal thickness measurements showed that the construct implants can regenerate corneal endothelium and restore corneal transparency at 4 weeks postoperatively. Our findings suggest that cell sheet transplantation using overrun-processed porous HA hydrogels offers a new way to reconstruct the posterior corneal surface and improve endothelial tissue function.     

Rapid cross-linking of elastin-like polypeptides with (hydroxymethyl)phosphines in aqueous solution

In situ gelation of injectable polypeptide-based materials is attractive for minimally invasive in vivo implantation of biomaterials and tissue engineering scaffolds. We demonstrate that chemically cross-linked elastin-like polypeptide (ELP) hydrogels can be rapidly formed in aqueous solution by reacting lysine-containing ELPs with an organophosphorous cross-linker, beta-[tris(hydroxymethyl)phosphino]propionic acid (THPP) under physiological conditions. The mechanical properties of the cross-linked ELP hydrogels were largely modulated by the molar concentration of lysine residues in the ELP and the pH at which the cross-linking reaction was carried out. Fibroblasts embedded in ELP hydrogels survived the cross-linking process and were viable after in vitro culture for 3 days. DNA quantification of ELP hydrogels with encapsulated fibroblasts indicated that there was no significant difference in DNA content between day 0 and day 3 when ELP hydrogels were formed with an equimolar ratio of THPP and lysine residues of the ELPs. These results suggest that THPP cross-linking may be a biocompatible strategy for the in situ formation of cross-linked hydrogels.     

Hyaluronic acid-based microgels and microgel networks for vocal fold regeneration

Vocal fold scarring disrupts the viscoelastic properties of the lamina propria that are critical for normal phonation. There is a clinical need for the development of advanced biomaterials that approximate the mechanical properties of the lamina propria for in vivo vocal fold regeneration. We have developed hyaluronic acid (HA)-based microgels and cross-linked microgel networks with tunable degradation and mechanical properties. HA microgels were prepared by cross-linking HA derivatives carrying hydrazide (HAADH) and aldehyde (HAALD) functionalities within the inverse emulsion droplets. Alternatively, poly(ethylene glycol) dialdehyde (PEGDiALD) was employed in place of HAALD. Microgels based on HAADH/HAALD are more resistant to enzymatic degradation than those generated from HAADH/PEGDiALD. In vitro cytotoxicity studies using vocal fold fibroblasts indicate that microgels synthesized from HAADH/HAALD are essentially nontoxic, whereas microgels derived from HAADH/PEGDiALD exhibit certain adverse effects on the cultured cells at high concentration (> or =2 mg/mL). These microgels exhibit residual functional groups that can be used as reactive handles for covalent conjugation of therapeutic molecules. The presence of residual functional groups also allows for subsequent cross-linking of the microgels with other reactive polymers, giving rise to doubly cross-linked networks (DXNs) with tunable viscoelasticity. Mechanical measurements using a torsional wave apparatus indicate that HA-based DXNs exhibit elastic moduli that are similar to those of vocal fold lamina propria at frequencies close to the range of human phonation. These HA-based microgel systems are promising candidates for the treatment of vocal fold scarring, not just as biocompatible filler materials, but as smart entities that can repair focal defects, smooth the vocal fold margin, and potentially soften and dissolve scar tissue.     

Synthesis and NMR characterization of new hyaluronan-based NO donors

Nitric oxide (NO) and hyaluronic acid (HA), two species widely different in terms of molecular complexity and biological competence, are both known to play an important role in the wound healing process. To combine the properties of HA and NO, we synthesized new NO-donors based on hyaluronic acid derivatives exhibiting a controlled NO-release under physiological conditions (in vitro tests). Since two molecules of NO can form a covalent bond with secondary amines to yield structures, named NONO-ates, able to release NO in solution, we used spermidine bound to HA as the NO-linker. The HA-spermidine derivative was obtained by controlled HA amidation in aqueous media, activating the biopolymer carboxylate groups with a water soluble carbodiimide. The resulting derivative, soluble in water, was fully characterized by high field 1H and 13C NMR spectroscopy. The amount of grafting of spermidine on HA was determined by integration of suitable 1H NMR signals. In addition, cross-linked derivatives of HA were synthesized by the Ugi's four-component reaction using formaldehyde, cyclohexylisocyanide, and spermidine. The HA-spermidine networks were characterized by 13C CP-MAS NMR spectroscopy. The degree of cross-linking of the networks was also determined. Finally, the release of NO from the swollen hydrogels freshly saturated with NO, in contact with aqueous media, was monitored by means of UV spectrophotometric measurements.