Synthesis and characterization of radiopaque iodine-containing degradable PVA hydrogels

Poly (vinyl alcohol) (PVA) hydrogels are highly attractive for biomedical applications, especially for controlled release of drugs and proteins. Recently, degradable PVA hydrogels have been described, having the advantage that the material disappears over time from the implantation site. Herein, we report the synthesis of radiopaque degradable PVA, which gives a further advantage that the position of the hydrogel can precisely be determined by X-ray fluoroscopy. Radiopacity has been introduced by replacing 0.5% of the pendent alcohol groups on the PVA with 4-iodobenzoylchloride. This level of substitution rendered the polymer adequately radiopaque. The subsequent modification of 0.8% of the pendent hydroxyl groups with an ester acrylate functional group allowed for cross-linking of the macromers. The radiopaque hydrogels degraded over a time span of 140 days. Rheology data suggested that the macromer solutions were appropriate for injection.     

Viscoelastic characterization and modeling of gelation kinetics of injectable in situ cross-linkable poly(lactide-co-ethylene oxide-co-fumarate) hydrogels

Cell transplantation by injection of biodegradable hydrogels is a recently developed strategy for the treatment of degenerated tissues. A cell carrier should be cytocompatible, have suitable working time and rheological properties for injection, and harden in situ to attain dimensional stability and the desired mechanical strength. Hydrophilic macromer/cross-linker polymerizing systems, due to the relatively high molecular weight of the macromer and its inability to cross the cell membrane, are very attractive as injectable cell carriers. The objective of this research was to determine the effects of cross-linker, initiator, and accelerator concentrations on the gelation kinetics and ultimate modulus of a biodegradable, in situ cross-linkable poly(lactide-co-ethylene oxide-co-fumarate) (PLEOF) macromer. The in situ polymerizing mixture consisted of PLEOF macromer, methylene bisacrylamide cross-linker, and a neutral redox initiation system of ammonium persulfate initiator and tetramethylethylenediamine accelerator. Measurement of the time evolution of the viscoelastic properties of the network during the sol-gel transition showed the important influence of each component on the gel time and stiffness of the hydrogels. A kinetic model was developed to predict the modulus as a function of composition. Model predictions were consistent with most of the experimental findings. The values of the storage and loss moduli at the gel point were found to be approximately equal for samples with equal PLEOF concentrations, resulting in a simple method to predict the gelation time based on the Winter--Chambon criterion, with the use of the proposed kinetic model. The results of this study can be coupled with component cytocompatibility measurements to predict the effect of composition on the viability of the cells encapsulated in the hydrogel matrix.     

Characterization of protein release from hydrolytically degradable poly(ethylene glycol) hydrogels

We present a novel fully hydrophilic, hydrolytically degradable poly(ethylene glycol) (PEG) hydrogel suitable for soft tissue engineering and delivery of protein drugs. The gels were designed to overcome drawbacks associated with current PEG hydrogels (i.e., reaction mechanisms or degradation products that compromise protein stability): the highly selective and mild cross‐linking reaction allowed for encapsulating proteins prior to gelation without altering their secondary structure as shown by circular dichroism experiments. Further, hydrogel degradation and structure, represented by mesh size, were correlated to protein release. It was determined that polymer density had the most profound effect on protein diffusivity, followed by the polymer molecular weight, and finally by the specific chemical structure of the cross‐linker. By examining the diffusion of several model proteins, we confirmed that the protein diffusivity was dependent on protein size as smaller proteins (e.g., lysozyme) diffused faster than larger proteins (e.g., Ig). Furthermore, we demonstrated that the protein physical state was preserved upon encapsulation and subsequent release from the PEG hydrogels and contained negligible aggregation or protein–polymer adducts. These initial studies indicate that the developed PEG hydrogels are suitable for release of stable proteins in drug delivery and tissue engineering applications. Biotechnol. Bioeng. 2011; 108:197–206. © 2010 Wiley Periodicals, Inc.     

Synthesis and characterization of poly(dimer acid-brassylic acid) copolymer and poly(dimer acid-pentadecandioic acid) copolymer

Poly(dimer acid-brassylic acid) [P(DA-BA)] copolymers and poly(dimer acid-pentadecandioic acid) [P(DA-PA)] copolymers were prepared by melt polycondensation of the corresponding mixed anhydride prepolymers. The copolymers were characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle x-ray powder-diffraction, and thermal gravimetric analysis (TGA). In vitro studies show that all the copolymers are degradable in phosphate buffer at 37 degrees C, and leaving an oily dimer acid residue after hydrolysis for the copolymer with high content of dimer acid. The release profiles of hydrophilic model drug, ciprofloxcin hydrochloride, from the copolymers, follow first-order release kinetics. All the preliminary results suggested that the copolymer might be potentially used as drug delivery devices.     

Synthesis and properties of carboxymethylchitosan hydrogels modified with poly(ester-urethane)

Preparation and properties of carboxymethylchitosan (CMC) modified with polyurethane (PU) containing poly(ethylene adipate) (PEA) as a soft segment is described. Urethane prepolymer was first synthesized by the reaction of PEA with an excess of 1,6-hexamethylene diisocyanate (HDI) to terminate its ends with isocyanate functional groups, followed by chain extension reaction using ethylene glycol as a chain extender. Its chemical structure was characterized by ¹H NMR and FTIR, molecular weight by GPC, and thermal behavior by DSC. To prepare PU-modified CMC (CMC-PU), 1–60 wt% of PU were introduced into the CMC solution of THF:H₂O mixture (50:50 v/v) in the presence of 10 wt% of hexamethylene-1,6-di-(aminocarboxysulfonate) (HDA) to increase network density. Formation of the network structure was confirmed by investigating percent crosslinking and water swelling properties of CMC-PU compared to CMC network without PU. When percent of PU increased from 1 to 60 wt%, percent crosslinking of CMC-PU gradually increased up to 82%, whereas equilibrium water content (EWC) dropped and retained at 1000%. SEM showed microphase separation of PU (10–50 μm) thoroughly dispersed in CMC surface and in the bulk. In addition, CMC-PU exhibited a slight enhancement in toughness properties. Cytotoxicity and biocompatibility tests indicated that CMC-PU was non-toxic.     

Synthesis and characterization of thermally and chemically gelling injectable hydrogels for tissue engineering

Novel, injectable hydrogels were developed that solidify through a physical and chemical dual-gelation mechanism upon preparation and elevation of temperature to 37 °C. A thermogelling, poly(N-isopropylacrylamide)-based macromer with pendant epoxy rings and a hydrolytically degradable polyamidoamine-based diamine cross-linker were synthesized, characterized, and combined to produce in situ forming hydrogel constructs. Network formation through the epoxy-amine reaction was shown to be rapid and facile, and the progressive incorporation of the hydrophilic polyamidoamine cross-linker into the hydrogel was shown to mitigate the often problematic tendency of thermogelling materials to undergo significant postformation gel syneresis. The results suggest that this novel class of injectable hydrogels may be attractive substrates for tissue engineering applications due to the synthetic versatility of the component materials and beneficial hydrogel gelation kinetics and stability.     

Transdermal photopolymerization of poly(ethylene oxide)-based injectable hydrogels for tissue-engineered cartilage

Transdermal photopolymerization, a minimally invasive method for implantation, was used to subcutaneously place a mixture of polymer and isolated chondrocytes to regenerate cartilage tissue in vivo. Semi-interpenetrating networks of varying proportions of poly(ethylene oxide)-dimethacrylate and poly(ethylene oxide) and primary bovine articular chondrocytes were implanted in athymic mice. Four mice (12 implants) were harvested at 2, 4, and 7 weeks. Chondrocytes survived implantation and photopolymerization and formed neocartilage containing 1.5 to 2.9% wet weight collagen and 4 to 7% glycosaminoglycan. Thirty-five percent of the total collagen was type II collagen. Histologic analysis exhibited tissue structure resembling neocartilage, and safranin O staining demonstrated glycosaminoglycan distribution throughout the hydrogels. This study demonstrates the potential use of transdermal photopolymerization for minimally invasive subcutaneous implantation of hydrogels and chondrocytes for in vivo cartilage regeneration.     

Hydrolytically degradable hyaluronic acid hydrogels with controlled temporal structures

Polysaccharides are being processed into biomaterials for numerous biological applications due to their native source in numerous tissues and biological functions. For instance, hyaluronic acid (HA) is found abundantly in the body, interacts with cells through surface receptors, and can regulate cellular behavior (e.g., proliferation, migration). HA was previously modified with reactive groups to form hydrogels that are degraded by hyaluronidases, either added exogenously or produced by cells. However, these hydrogels may be inhibitory and their applications are limited if the appropriate enzymes are not present. Here, for the first time, we synthesized HA macromers and hydrogels that are both hydrolytically (via ester group hydrolysis) and enzymatically degradable. The hydrogel degradation and growth factor release was tailored through the hydrogel cross-linking density (i.e., macromer concentration) and copolymerization with purely enzymatically degradable macromers. When mesenchymal stem cells (MSCs) were encapsulated in the hydrogels, cellular organization and tissue distribution was influenced by the copolymer concentration. Importantly, the distribution of released extracellular matrix molecules (e.g., chondroitin sulfate) was improved with increasing amounts of the hydrolytically degradable component. Overall, this new macromer allows for enhanced control over the structural evolution of the HA hydrogels toward applications as biomaterials.     

Synthesis and characterization of photo-cross-linked hydrogels based on biodegradable polyphosphoesters and poly(ethylene glycol) copolymers

Novel biodegradable hydrogels by photo-cross-linking macromers based on polyphosphoesters and poly(ethylene glycol) (PEG) are reported. Photo-cross-linkable macromers were synthesized by ring-opening polymerization of the cyclic phosphoester monomer 2-(2-oxo-1,3,2-dioxaphospholoyloxy) ethyl methacrylate (OPEMA) using PEG as the initiator and stannous octoate as the catalyst. The macromers were characterized by 1H NMR, Fourier transform infrared spectroscopy, and gel permeation chromatography measurements. The content of polyphosphoester in the macromer was controlled by varying the feed ratio of OPEMA to PEG. Hydrogels were fabricated by exposing aqueous solutions of macromers with 0.05% (w/w) photoinitiator to UV light irradiation, and their swelling kinetics as well as degradation behaviors were evaluated. The results demonstrated that cross-linking density and pH values strongly affected the degradation rates. The macromers was compatible to osteoblast cells, not exhibiting significant cytotoxicity up to 0.5 mg/mL. "Live/dead" cell staining assay also demonstrated that a large majority of the osteoblast cells remained viable after encapsulation into the hydrogel constructs, showing their potential as tissue engineering scaffolds.     

Synthesis and gelation of DOPA-modified poly(ethylene glycol) hydrogels

3,4-Dihydroxyphenylalanine (DOPA) residues are known for their ability to impart adhesive and curing properties to mussel adhesive proteins. In this paper, we report the preparation of linear and branched DOPA-modified poly(ethylene glycol)s (PEG-DOPAs) containing one to four DOPA endgroups. Gel permeation chromatography-multiple-angle laser light scattering analysis of methoxy-PEG-DOPA in the presence of oxidizing reagents (sodium periodate, horseradish peroxidase, and mushroom tyrosinase) revealed the formation of oligomers of methoxy-PEG-DOPA, presumably resulting from oxidative polymerization of DOPA endgroups. In the case of PEG-DOPAs containing two or more DOPA endgroups, oxidative polymerization resulted in polymer network formation and rapid gelation. The amount of time required for gelation of aqueous PEG-DOPA solutions was found to be as little as 1 min and was dependent on the polymer architecture as well as the type and concentration of oxidizing reagent used. Analysis of reaction mixtures by UV-vis spectroscopy allowed the identification of reaction intermediates and the elucidation of reaction pathways. On the basis of the observed reaction intermediates, oxidation of the catechol side chain of DOPA resulted in the formation of highly reactive DOPA-quinone, which further reacted to form cross-linked products via one of several pathways, depending on the presence or absence of N-terminal protecting groups on the PEG-DOPA. N-Boc protected PEG-DOPA cross-linked via phenol coupling and quinone methide tanning pathways, whereas PEG-DOPA containing a free amino group cross-linked via a pathway that resembled melanogenesis. Similar differences were observed for the rate of gel formation as well as the molecular weight between cross-links ((-)M(c)), calculated using equilibrium swelling and the Flory-Rehner equation.     

Synthesis and characterization of injectable, thermally and chemically gelable, amphiphilic poly(N-isopropylacrylamide)-based macromers

In this study, we synthesized and characterized a series of macromers based on poly( N-isopropylacrylamide) that undergo thermally induced physical gelation and, following chemical modification, can be chemically cross-linked. Macromers with number average molecular weights typically ranging from 2000-3500 Da were synthesized via free radical polymerization from, in addition to N-isopropylacrylamide, pentaerythritol diacrylate monostearate, a bifunctional monomer containing a long hydrophobic chain, acrylamide, a hydrophilic monomer, and hydroxyethyl acrylate, a hydrophilic monomer used to provide hydroxyl groups for further chemical modification. Results indicated that the hydrophobic-hydrophilic balance achieved by varying the relative concentrations of comonomers used during synthesis was an important parameter in controlling the transition temperature of the macromers in solution and stability of the resultant gels. Storage moduli of the macromers increased over 4 orders of magnitude once gelation occurred above the transition temperature. Furthermore, chemical cross-linking of these macromers resulted in gels with increased stability compared to uncross-linked controls. These results demonstrate the feasibility of synthesizing poly( N-isopropylacrylamide)-based macromers that undergo tandem gelation and establish key criteria relating to the transition temperature and stability of these materials. The data suggest that these materials may be attractive substrates for tissue engineering and cellular delivery applications as the combination of mechanistically independent gelation techniques used in tandem may offer superior materials with regard to gelation kinetics and stability.     

Synthesis and physicochemical characterization of end-linked poly(ethylene glycol)-co-peptide hydrogels formed by Michael-type addition

The synthesis of novel hybrid hydrogels by stepwise copolymerization of multiarm vinyl sulfone-terminated poly(ethylene glycol) macromers and alpha-omega cysteine oligopeptides via Michael-type additions is described. Cross-linking kinetics, studied by in situ rheometry, can be controlled by pH and the presence of charged amino acid residues in close proximity to the Cys, which modulates the pK(a) of the thiol group. These end-linked networks were characterized by their equilibrium swelling in water, by their viscoelastic properties in the swollen state, and by their soluble fraction. It was demonstrated that structure and properties are very sensitive to the preparation state including stoichiometry and precursor concentration and less sensitive to the pH during cross-linking. For each network the concentration of elastically active chains (nu) was calculated from experimentally determined sol fractions using Miller-Macosko theory and compared to values obtained from swelling and rheometry studies and by calculation from Flory's classical network models. Hydrogels were also prepared with varying macromer structures, and their properties were shown to respond to both macromer functionality and molecular weight.     

Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP-ribose) synthetase

Calf thymus poly(ADP-ribose) synthetase (Mr = 120,000) is cleaved with papain into two fragments of M(r) = 74,000 and 46,000 and also split with chymotrypsin into two fragments of M(r) = 66,000 and 54,000. Each fragment purified to homogeneity is enzymatically inactive, but combined incubation of the 74,000 and 46,000 fragments in the presence of DNA restored 20% of the enzyme activity. In contrast, combined incubation of the 66,000 and 54,000 fragments does not restore any enzyme activity. In the former incubation, autopoly(ADP-ribosyl)ation reaction occurs exclusively on the 74,000 fragment. When each fragment is incubated with [adenine-U-14C]NAD in the presence of DNA and a catalytic amount of the native enzyme, poly(ADP-ribosyl)action occurs in the overlapped portion (22,000) of the 66,000 fragment and the 74,000 fragment. Nevertheless, the purified 22,000 fragment is a poor acceptor for poly(ADP-ribosyl)ation. The degree of poly(ADP-ribosyl)ation of the proteolytic fragments is significantly reduced by increasing NaCl concentration, probably due to the lack of the interaction between the enzyme fragments and DNA. These results, taken together, indicate that DNA is indispensable for the reconstitution of the catalytic activity as well as the poly(ADP-ribosyl)ation of the fragmented enzyme.     

Synthesis and characterization of stereoregular poly(D-methionyl-L-methionine) and poly(L-methionyl-D-methionyl-L-methionine)

By use of a polycondensation procedure free of racemization, stereoregular polymethionines have been synthesized from C-activated D -methionyl-L -methionine and L -methionyl-D -methionyl-L -methionine. The poly(D -methionyl-L -methionine) and poly(L -methionyl-D -methionyl-L -methionine) so prepared are soluble in chloroform and can be purified through dissolution in this solvent and precipitation by ligroin. Poly(D -Met-L -Met)which is obtained in a 25% yield, is about 5000 in average molecular weight. It has no discernible optical activity when examined between 400 and 600 nm in a trifluoroacetic acid solution. Poly(L -Met-D -Met-L -Met) (40% yield, M. W. = 10,000) is an optically active polymer. [α]₄₃₆²⁴ ≈ + 170° for a chloroformic solution (c = 0.2 CHCl₃).     

Synthesis and characterization of biodegradable cationic poly(propylene fumarate-co-ethylene glycol) copolymer hydrogels modified with agmatine for enhanced cell adhesion

We synthesized positively charged biodegradable hydrogels by cross-linking of agmatine-modified poly(ethylene glycol)-tethered fumarate (Agm-PEGF) and poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) to investigate the effect of the guanidino groups of the agmatine on hydrogel swelling behavior and smooth muscle cell adhesion to the hydrogels. The weight swelling ratio of these hydrogels at pH 7.0 increased from 279 +/- 4 to 306 +/- 7% as the initial Agm-PEGF content increased from 0 to 200 mg/g of P(PF-co-EG), respectively. The diffusional exponents, n, during the initial phase of water uptake were independent of the initial Agm-PEGF content and were determined to be 0.66 +/- 0.08, 0.71 +/- 0.07, and 0.60 +/- 0.05 for respective initial Agm-PEGF contents of 0, 100, and 200 mg/g. The heat of fusion of water present in the hydrogels increased from 214 +/- 11 to 254 +/- 4 J/g as the initial Agm-PEGF content increased from 0 to 200 mg/g. The number of adherent smooth muscle cells increased dose-dependently from 15 +/- 6 to 75 +/- 7% of the initial seeding density as the initial Agm-PEGF content increased from 0 to 200 mg/g. These results suggest that the incorporation of the guanidino groups of agmatine into P(PF-co-EG) hydrogels increases the hydrogel free water content and the total water content of the hydrogels and also enhances cell adhesion to the hydrogels.     

Synthesis and properties of thermo- and pH-sensitive poly(N-isopropylacrylamide)/polyaspartic acid IPN hydrogels

Various interpenetrating polymer network (IPN) hydrogels with sensitivity to temperature and pH were prepared by introducing the pH-sensitive polymer polyaspartic acid (PASP) hydrogel, into the poly(N-isopropylacrylamide) (PNIPAAm) hydrogel system for the purpose of improving its response rate to temperature. The morphologies and thermal behavior of the prepared IPN hydrogels were studied by both scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The IPN hydrogels showed a large and uneven porous network structure, without showing the common PNIPAAm hydrogel structure. The paper moreover studied their swelling properties, such as temperature dependence of equilibrium swelling ratio, shrinking kinetics, re-swelling kinetics and oscillatory swelling behavior in water. The swelling experiment results revealed that IPN hydrogels exhibited much faster shrinking and re-swelling in function of the composition ratio of the two network components. These fast responsive hydrogels foster potential applications in biomedical and biotechnology fields.     

Synthesis and characterization of poly(LysAla3)

The synthesis and characterization of poly(LysAla₃) are described. The polytetrapeptide is a model for short sequences found in proelastin, and is presumably involved in desmosine or isodesmosine cross-link formation in the native protein. Poly(LysAla₃) is found to possess a mixture of conformations in aqueous solution dependent on molecular weight and pH. Low-molecular-weight (ca. 3000) material appears to be a mixture of random and extended helix at neutral pH. However, as the molecular weight is increased an increasing amount of α-helix is observed rising to >50% for mol wt = 21,000. The α-helical chain segments are thermally stable, melting to a mixture of extended and random forms at T_m = 25°C. High pH (10.5) promotes further α-helix formation but at pH >11.0 the polypeptide becomes insoluble. The inference is that short chain segments of the peptide in elastin are unlikely to be α-helical in the equilibrium state but may fluctuate through such a conformation.     

Synthesis and decoloring properties of sodium humate/poly (N-isopropylacrylamide) hydrogels

A series of novel sodium humate/poly(N-isopropylacrylamide) (SH/PNIPA) hydrogels were synthesized by solution polymerization. The swelling and decoloring properties of SH/PNIPA hydrogels were also examined. Experiment results show that there exist hydrogen-bonding interactions between SH and PNIPA in the SH/PNIPA hydrogels network, which are not strong enough to disrupt the aggregation of dehydrated PNIPA chains at phase transition temperature, leading to the same volume phase transition temperature as pure PNIPA hydrogel. The adsorption and desorption of methylene blue (MB) for the hydrogels were influenced by temperature, initial MB concentration and SH amount. Low temperature favors the adsorption and desorption of MB. Appropriate SH amount of the hydrogels is crucial for the adsorption and desorption of MB. The maximum adsorption capacity was 10.8 mg MB per gram of SH/PNIPA gel.     

Bio-based hydrogels prepared by cross-linking of microbial poly(gamma-glutamic acid) with various saccharides

Novel bio-based hydrogels were prepared by cross-linking of microbial poly(gamma-glutamic acid) (PGA) with saccharides such as glucose, maltotriose, and cyclodextrin (CD) in the presence of water-soluble carbodiimide in dimethyl sulfoxide (DMSO) by one-pot synthesis at 25 degrees C for 24 h. The degradation of the gels in alkaline solution (pH 9) at 37 degrees C was also investigated. The PGA gels cross-linked with various neutral saccharides were obtained in relatively high recovery yields by use of a base like 4,4-(dimethylamino)pyridine. The PGA gel cross-linked by glucose showed the highest water absorption of 3000 g/g. The PGA gels cross-linked by CDs showed higher water absorption than those cross-linked by the corresponding linear saccharides. It was revealed that the water absorption of the PGA gel was affected by the cross-linker content and also the structure of cross-linkers as they had an effect on the cross-linking density of the PGA gel. The PGA gels were hydrolyzed under alkaline condition (pH 9) at 37 degrees C. The degradation rate was higher when the cross-linker content of the gel was lower.     

Synthesis and characterization of a cationic poly(beta-hydroxyalkanoate)

Poly(beta-hydroxyalkanoates) (PHAs) are biodegradable polyesters produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the carbon source composition in the feed stock, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials. Here, we report the synthesis of the first cationic PHA, poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Pseudomonas putida Gpo1 was used to produce poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-10-undecenoate) (PHOU), whose vinyl-terminated side chains were first converted to terminal epoxides and then modified with diethanolamine. The modification of PHOU was examined using (1)H, COSY, and HSQC NMR and GPC and resulted in a loss of molecular weight due to aminolysis and also in the introduction of side chains terminated with tertiary amine groups, which are protonated at physiological pH. The polycationic PHA is soluble in polar solvents such as DMSO, DMF, and water. The new biodegradable cationic polymers are envisioned as nucleic acid delivery systems.