New dextrin-vinylacrylate hydrogel: Studies on protein diffusion and release

New dextrin hydrogels with degrees of substitution (DS) from ca. 10% (DS 10) to 70% (DS 70) were prepared by radical polymerization of aqueous solutions of vinylacrylate (VA)-derivatized dextrin. A preliminary analysis on the potential of these hydrogels for the controlled release of bioactive molecules was carried out. The protein (bovine serum albumin) diffusion coefficients on the hydrogels were calculated using the lag-time analysis. Values in range 10^?7 cm²/s were obtained for DS 20 and DS 40 and a smaller value of 10^?8 cm²/s arised upon DS increasing to 70%, revealing the dependence of the diffusivity on the crosslinking density. The release of BSA from dextrin-VA hydrogels, in the presence of amyloglucosidase was shown to be mainly dependent on the diffusion and, to a smaller extent, on the degradation kinetics. The protein release can be tailored from days to months by varying the DS.     

Photocrosslinked bovine serum albumin hydrogels with partial retention of esterase activity

Novel biohybrid hydrogels based on bovine serum albumin (BSA) were synthesized by one-pot photopolymerization of chemically modified protein in the presence of N,N'-methylenebisacrylamide (MBA) as cross-linking agent under mild conditions. Two batches of methacrylated albumins were prepared by treating the protein with different amounts of methacrylic anhydride (MAN) and the degree of substitution (DS) of primary amines was quantified via trinitrobenzesulfonic acid (TNBS) colorimetric assay. Hydrogels readily formed when a diluted buffered solution of the modified protein and MBA was exposed to LW-UV in the presence of 1-[4-(2-hydroxyethoxy)phenyl]-2-hydroxy-2-methyl-1-propan-1-one (Irgacure 2959) as the radical initiator. In contrast, no hydrogel was obtained in the absence of a polymerizable BSA, nor when the cross-linker, the radical initiator or UV light exposure was excluded from the reaction, suggesting the critical importance of the combined conditions for hydrogel formation. Hydrogels were characterized via scanning electron microscopy (SEM) and the swelling ratios were monitored at different pHs. The esterolytic activity of the novel biohybrid materials was quantitatively investigated via UV-vis spectroscopy by measuring the release of para-nitrophenol upon incubation with para-nitrophenyl acetate (p-NPA) substrate. The effect of the addition of acrylic acid co-monomer and of the monomer concentration in the catalytic activity and in the swelling behavior was also examined. Finally, the reusability of the materials following one round of catalysis was evaluated.     

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.     

β-Cyclodextrin grafted polyethyleneimine hydrogel immobilizing hydrophobically modified glucose oxidase

Hydrogels which release their contents in response to glucose concentration were prepared by immobilizing glucose oxidase (GOD) into β-cyclodextrin grafted polyethyleneimine hydrogels (PEI-βCD hydrogel). For the tight immobilization, hydrophobically modified GOD (HmGOD) was prepared by reacting GOD with palmitic acid-N-hydroxysuccinimide ester (PA-NHS) in the molar ratio of 1:40. According to trinitrobenzene sulfonic acid (TNBS) assay, five palmitic acids were covalently attached to one GOD molecule. The activity of HmGOD was about 76% of native enzyme. The swelling ratios of HmGOD loaded hydrogels increased from about 960% to 1190% in 24h, when glucose concentration was varied from 0 to 100mg/dl. The % release in 48 h of fluorescein isothiocyanate dextran increased from about 53% to 89%, when glucose concentration was varied in the same range. Gluconic acid, produced by the enzymatic reaction, would protonate and swell the PEI-βCD hydrogel, leading to a higher release.     

Dermatan sulfate as a stabilizer for protein stability in poly(lactide-co-glycolide) depot

Dermatan sulfate (DS), a glycosaminoglycan family, was investigated as a additive to enhance the stability of therapeutic protein with low p/ value loaded in poly(lactide-co-glycolide) (PLGA) microspheres prepared by water-in-oil-in-water (W₁/O/W₂) method. DS maintains negative charge below pH 3.0 because of its sulfate groups, while most anionic polymer with carboxyl groups becomes neutral charge at that pH. Thus, at pH 3.0 DS can form a polyelectrolyte complex with a protein with lower p/ such as exendin-4, insulin, and human growth hormone. In order to complex with DS, bovine serum albumin (BSA) was employed as a model protein, which has low p/value (p/= 4.8). The complex prepared at pH 3.0 showed a nano-size in the range of 100∼200 nm with a mono distribution. During the preparation of PLGA depot, DS concentration in water phase increases with decreasing the formation of non-covalent BSA aggregates and enhancing BSA loading efficiency. It means that DS/BSA complex system enabled to keep a stability of BSA at the water/organic interface. In an in vitro BSA release test, PLGA depot with DS exhibited a lower initial burst kinetic than only PLGA depot and continuous BSA release in almost 100% for 23 days. From the results, it was concluded that DS as an additive in PLGA depot, has a potential for the long-term delivery of therapeutic proteins with lower p/ value.     

Synthesis and characterization of thermo-sensitive semi-IPN hydrogels based on poly(ethylene glycol)-co-poly(ε-caprolactone) macromer, N-isopropylacrylamide, and sodium alginate

Thermo-sensitive semi-IPN hydrogels were prepared via in situ copolymerization of N-isopropylacrylamide (NIPAAm) with poly(ethylene glycol)-co-poly(ε-caprolactone) (PEG-co-PCL) macromer in the presence of sodium alginate by UV irradiation technology. The effects of the sodium alginate content, temperature, and salt on the swelling behavior of the as-obtained hydrogels were studied. The results showed that the swelling ratio of the hydrogels increased with the increasing sodium alginate content at the same temperature, and decreased with the increase in temperature. The salt sensitivity of the semi-IPN hydrogels was dependent on the content of sodium alginate introduced in the hydrogels. The mechanical rheology of the hydrogels and in vitro release behavior of bovine serum albumin (BSA) in situ encapsulated within the hydrogels were also investigated. It was found that the introduction of sodium alginate with semi-IPN structure improved mechanical strength of the hydrogels and the cumulative release percentage of BSA from the hydrogels. Such double-sensitive semi-IPN hydrogel materials could be exploited as potential candidates for drug delivery carriers.     

Microspheres of chitosan/carboxymethyl cashew gum (CH/CMCG): Effect of chitosan molar mass and CMCG degree of substitution on the swelling and BSA release

Chitosan/carboxymethyl cashew gum microspheres (CH/CMCG) were prepared with carboxymethyl cashew gum with two different degrees of substitution (DS) and loaded with bovine serum albumin (BSA). In water, for microspheres formed using low molar mass chitosan (LCH) sample swelling was observed for both CMCG samples and CMCG sample with higher DS showed greater swelling. Using high molar mass chitosan (HCH) sample swelling was observed only for microsphere with high DS of CMCG (DS = 0.44). At pH 7.4, the HCH sample led to a lower degree of swelling. The diffusion coefficients D_v were higher for the higher DS of CMCG in both media and the HCH sample had a lower D_v than LCH one. Faster BSA release rates were observed for beads prepared with the higher DS, whereas those prepared with DS = 0.16 took twice the time to reach similar release profiles. All microsphere systems investigated had a non-Fickian BSA release mechanism.     

IPNs based on chitosan with NVP and NVP/HEMA synthesised through photoinitiator-free photopolymerisation technique for biomedical applications

Biocompatible interpenetration polymeric network (IPN) hydrogels based on chitosan with N-vinylpyrrolidinone (NVP) as well as its copolymer with 2-hydroxymethyl methacrylate (HEMA) were synthesised using the photopolymerisation technique without the inclusion of any photoinitiator or crosslinking agent. These hydrogels were characterised using the Fourier-transform infrared spectroscopy (FTIR) technique. Equilibrium swelling of these hydrogels was performed in Milli-Q water and drug release studies were carried out using theophylline as the model drug. These tests showed that the IPN comprised of chitosan and NVP with a very small amount of N-hydroxymethyl maleimide (HMMI) included exhibited higher swelling abilities and fast drug release rates than the IPN which contained chitosan, NVP and HEMA. Kinetic studies of water diffusion into these hydrogels and drug release revealed that with the exception of the IPN with HEMA incorporated, the other hydrogels did not adhere to the Fickian diffusion model. These hydrogels were tested for their biocompatibility with human epidermal keratinocyte cells (HaCaT). A positive cell growth as evidenced by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) cell proliferation assay indicated that these hydrogels are non-toxic to human keratinocytes and can be potentially used as biomaterials for biomedical applications.     

Poly(vinyl alcohol) nanofibers by electrospinning as a protein delivery system and the retardation of enzyme release by additional polymer coatings

Protein-loaded (bovine serum albumin (BSA) or luciferase) poly(vinyl alcohol) (PVA) nanofibers were obtained by electrospinning. Poly(p-xylylene) (PPX, also coined as parylene) coated PVA/BSA nanofibers were prepared by chemical vapor deposition (CVD). The release of BSA from PVA nanofibers under physiological conditions was monitored by absorption spectroscopy. Burst release of BSA was noted with uncoated PVA nanofibers. In contrast, PPX-coated nanofibers exhibited a significantly retarded release of BSA depending on the coating thickness of PPX (ranging from 40 to 300 nm). Luciferase was used here as model enzyme, which after electrospinning retained its enzyme activity. This preservation of enzyme activity and the continuous release of the intact enzyme from the immersed fibers meets a fundamental prerequisite for the application of enzymes or other sensitive agents released from electrospun nanofibers under physiological conditions.     

Preparation and characterization of a novel pH-sensitive chitosan-g-poly (acrylic acid)/attapulgite/sodium alginate composite hydrogel bead for controlled release of diclofenac sodium

A series of pH-sensitive composite hydrogel beads composed of chitosan-g-poly (acrylic acid)/attapulgite/sodium alginate (CTS-g-PAA/APT/SA) was prepared as drug delivery matrices crosslinked by Ca²⁺ owing to the ionic gelation of SA. The structure and surface morphology of the composite hydrogel beads were characterized by FTIR and SEM, respectively. pH-sensitivity of these composite hydrogels beads and the release behaviors of drug from them were investigated. The results showed that the composite hydrogel beads had good pH-sensitivity. The cumulative release ratios of diclofenac sodium (DS) from the composite hydrogel beads were 3.76% in pH 2.1 solution and 100% in pH 6.8 solutions within 24 h, respectively. However, the cumulative release ratio of DS in pH 7.4 solution reached 100% within 2 h. The DS cumulative release ratio reduced with increasing APT content from 0 to 50 wt%. The drug release was swelling-controlled at pH 6.8.     

Chitosan cross-linked with poly(ethylene glycol)dialdehyde via reductive amination as effective controlled release carriers for oral protein drug delivery

The covalently cross-linked chitosan-poly(ethylene glycol)₁₅₄₀ derivatives have been developed as a controlled release system with potential for the delivery of protein drug. The swelling characteristics of the hydrogels based on these derivatives as the function of different PEG content and the release profiles of a model protein (bovine serum albumin, BSA) from the hydrogels were evaluated in simulated gastric fluid with or without enzyme in order to simulate the gastrointestinal tract conditions. The derivatives cross-linked with difunctional PEG₁₅₄₀-dialdehyde via reductive amination can swell in alkaline pH and remain insoluble in acidic medium. The cumulative release amount of BSA was relatively low in the initial 2 h and increased significantly at pH 7.4 with intestinal lysozyme for additional 12 h. The results proved that the release-and-hold behavior of the cross-linked CS–PEG₁₅₄₀H-CS hydrogel provided a swell and intestinal enzyme controlled release carrier system, which is suitable for oral protein drug delivery.     

Biocompatible glucose sensor prepared by modifying protein and vinylferrocene monomer composite membrane

This paper proposes a very simple procedure for preparing a biocompatible sensor based on a protein (bovine serum albumin, BSA), enzyme and vinylferrocene (VF) composite membrane modified electrode. The membrane was prepared simply by first casting vinylferrocene and then coating it with BSA and glucose oxidase immobilised with glutaraldehyde. The sensor response was independent of dissolved oxygen concentration from 3 to 10 ppm and showed good stability for serum sample measurement, unlike the commonly used BSA/enzyme modified electrode. The sensor response was almost unchanged over the measurement time (>10 h) whereas the responses of a BSA and glucose oxidase modified platinum electrode and an osmium-polyvinylpyridine wired horseradish peroxidase modified electrode (Ohara et al., 1993) fell to 68% of their initial value in a serum sample containing 10mM glucose.     

One-step synthesis of interpenetrating network hydrogels: Environment sensitivities and drug delivery properties

A novel interpenetrating network hydrogel for drug controlled release, composed of modified poly(aspartic acid) (KPAsp) and carboxymethyl chitosan (CMCTS), was prepared in aqueous system. The surface morphology and composition of hydrogels were characterized by SEM and FTIR. The swelling properties of KPAsp, KPAsp/CMCTS semi-IPN and KPAsp/CMCTS IPN hydrogels were investigated and the swelling dynamics of the hydrogels was analyzed based on the Fickian equation. The pH, temperature and salt sensitivities of hydrogels were further studied, and the prepared hydrogels showed extremely sensitive properties to pH, temperature, the ionic salts kinds and concentration. The results of controlled drug release behaviors of the hydrogels revealed that the introduction of IPN observably improved the drug release properties of hydrogels, the release rate of drug from hydrogels can be controlled by the structure of the hydrogels and pH value of the external environment, a relative large amount of drug released was preferred under simulated intestinal fluid. These results illustrated high potential of the KPAsp/CMCTS IPN hydrogels for application as drug carriers.     

芽孢杆菌M50产生β—甘露聚糖酶的条件研究

从土壤中分离到９株产生β－甘露聚糖酶的芽孢杆菌（Ｂａｃｉｌｌｕｓ ｓｐ．）。Ｂａｃｉｌｌｕｓ ｓｐ．Ｍ５０２５０ｍＬ三角瓶摇瓶培养试验,以４％的魔芋粉为碳源,１．０％（ＮＨ４）２ＳＯ４为氮源,０．３５％Ｎａ２ＣＯ３,３０～３４℃培养６０ｈ产酶达到高峰。酶活力为１８０～２２０ｕ／ｍＬ。１００Ｌ罐发酵,在３０～３２℃,１：０．７５ｖｖｍ通气量,２００ｒ／ｍｉｎ条件下,发酵液酶活力高达３３０ｕ／ｍＬ。     

Effect of protein-hydroxyethylmethacrylate hydrogels on cultured endothelial cells

The use of protein hydroxy ethylmethacrylate (HEMA) hydrogels to control cell morphology and growth, as well as the synthesis of extracellular matrix components, is described in this communication. HEMA hydrogels prepared with collagen support growth of embryonic lung fibroblasts (IMR-90), as well as bovine aortic and pulmonary artery endothelial cells at a level comparable to the respective cells grown on tissue culture surfaces. On the other hand, HEMA hydrogels prepared with solubilized elastin inhibit the fibroblast growth and prevent both types of endothelial cell cultures from achieving their normal morphology. These morphologically altered endothelial cells resume a normal cobblestone-like appearance when subcultivated from the elastin-HEMA hydrogels to tissue culture plastic. When pulsed with [14C]proline, the procollagens synthesized by the endothelial cells on the different surfaces vary, as shown by immunoprecipitation and polyacrylamide gel electrophoresis. On the standard tissue culture plastic, the confluent cells produce mainly type III procollagen in the medium, whereas those endothelial cells grown on collagen and elastin-HEMA hydrogels synthesize primarily type I procollagen (much like sprouting cells on tissue culture plastic), regardless of their morphology.     

Biocomposite hydrogels with carboxymethylated, nanofibrillated cellulose powder for replacement of the nucleus pulposus

Biocomposite hydrogels with carboxymethylated, nanofibrillated cellulose (c-NFC) powder were prepared by UV polymerization of N-vinyl-2-pyrrolidone with Tween 20 trimethacrylate as a cross-linking agent for replacement of the native, human nucleus pulposus (NP) in intervertebral disks. The swelling ratios and the moduli of elasticity in compression of neat and biocomposite hydrogels were evaluated in dependence of c-NFC concentration (ranging from 0 to 1.6% v/v) and degree of substitution (DS, ranging from 0 to 0.23). The viscoelastic properties in shear and the material relaxation behavior in compression were measured for neat and biocomposite hydrogels containing 0.4% v/v of fibrils (DS ranging from 0 to 0.23), and their morphologies were characterized by cryo-scanning electron microscopy (cryo-SEM). The obtained results show that the biocomposite hydrogels can successfully mimic the mechanical and swelling behavior of the NP. In addition, the presence of the c-NFC shows lower strain values after cyclic compression tests and consequently creates improved material relaxation properties compared with neat hydrogels. Among the tested samples, the biocomposite hydrogel containing 0.4% v/v of c-NFC with a DS of 0.17 shows the closest behavior to native NP. Further investigation should focus on evaluation and improvement of the long-term relaxation behavior.     

Designing chitosan-dextran sulfate nanoparticles using charge ratios

The purpose of this study was to examine the effect of charge ratio on the formation and properties of the chitosan (CS)-dextran sulfate (DS) nanoparticles developed for the delivery of water-soluble small and large molecules, including proteins. Rhodamine 6G (R6G) and bovine serum albumin (BSA) were chosen as model molecules. CS-DS nanoparticles were formulated by a complex coacervation process under mild conditions. The influence of formulation and process variables, including the charge ratio of the 2 ionic polymers, on particle size, zeta potential, and nanoparticle entrapment of R6G and BSA was studied. The in vitro release of R6G and BSA was also evaluated, and the integrity of BSA in the release fraction was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Depending on the concentration and charge ratio of DS and CS, nanoparticles with varied size (>or=244 nm) and zeta potential (-47.1-60 mV) were obtained. High entrapment efficiency (98%) was achieved for both R6G and BSA when the charge ratio of the 2 ionic polymers was greater than 1.12. The release of both R6G and BSA from nanoparticles was based on the ion-exchange mechanism. BSA showed much slower continuous release for up to 7 days while still maintaining its integrity for an extended period. The CS-DS nanoparticles developed based on the modulation of charge ratio show promise as a system for controlled delivery of both small and large molecules, including proteins.     

Rapidly in situ-forming degradable hydrogels from dextran thiols through Michael addition

Thiol-functionalized dextrans (dex-SH) (M(n,dextran) = 14K or 31K) with degrees of substitution (DS) ranging from 12 to 25 were synthesized and investigated for in situ hydrogel formation via Michael type addition using poly(ethylene glycol) tetra-acrylate (PEG-4-Acr) or a dextran vinyl sulfone conjugate with DS 10 (dex-VS DS 10). Dex-SH was prepared by activation of the hydroxyl groups of dextran with 4-nitrophenyl chloroformate and subsequent reaction with cysteamine. Hydrogels were rapidly formed in situ under physiological conditions upon mixing aqueous solutions of dex-SH and either PEG-4-Acr or dex-VS DS 10 at polymer concentrations of 10 to 20 w/v%. Rheological studies showed that these hydrogels are highly elastic. By varying the DS, concentration, dextran molecular weight, and type of cross-linker, hydrogels with a broad range of storage moduli of 9 to 100 kPa could be obtained. Varying the ratio of thiol to vinyl sulfone groups from 0.9 to 1.1 did not alter the storage modulus of the hydrogels, whereas larger deviations from equimolarity (thiol to vinyl sulfone ratios of 0.75 and 1.5) considerably decreased the storage modulus. The plateau value of hydrogel storage modulus was reached much faster at pH 7.4 compared to pH 7, due to a higher concentration of the thiolate anion at higher pH. These hydrogels were degradable under physiological conditions. Degradation times were 3 to 7 weeks for dex-SH/dex-VS DS 10 hydrogels and 7 to over 21 weeks for dex-SH/PEG-4-Acr hydrogels, depending on the DS, concentration, and dextran molecular weight.     

Novel method using a temperature-sensitive polymer (methylcellulose) to thermally gel aqueous alginate as a pH-sensitive hydrogel

A novel method using a temperature-sensitive polymer (methylcellulose) to thermally gel aqueous alginate blended with distinct salts (CaCl2, Na2HPO4, or NaCl), as a pH-sensitive hydrogel was developed for protein drug delivery. It was noted that the salts blended in hydrogels may affect the structures of an entangled network of methylcellulose and alginate and have an effect on their swelling characteristics. The methylcellulose/alginate hydrogel blended with 0.7 M NaCl (with a gelation temperature of 32 degrees C) demonstrated excellent pH sensitivity and was selected for the study of release profiles of a model protein drug (bovine serum albumin, BSA). In the preparation of drug-loaded hydrogels, BSA was well-mixed to the dissolved aqueous methylcellulose/alginate blended with salts at 4 degrees C and then gelled by elevating the temperature to 37 degrees C. This drug-loading procedure in aqueous environment at low temperature may minimize degradation of the protein drug while achieving a high loading efficiency (95-98%). The amount of BSA released from test hydrogels was a function of the amount of alginate used in the hydrogels. The amount of BSA released at pH 1.2 from the test hydrogel with 2.5% alginate was relatively low (20%), while that released at pH 7.4 increased significantly (86%). In conclusion, the methylcellulose/alginate hydrogel blended with NaCl could be a suitable carrier for site-specific protein drug delivery in the intestine.     

Macroscopic hydrogels by self-assembly of oligolactate-grafted dextran microspheres

A novel approach is presented to create self-assembling hydrogels. Microspheres based on cross-linked dextran were chemically modified with L- or D-oligolactate chains. Successful grafting of the particles was confirmed by Fourier transform infrared (FT-IR) and Raman and X-ray photoelectron spectroscopy (XPS). Rheological analysis of aqueous dispersions of oligolactate-grafted microspheres demonstrated that hydrophobic interactions between oligolactate chains on the surface of various microspheres resulted in the formation of an almost fully elastic gel. A mixture of microspheres substituted with L- or D-oligolactates of opposite chirality resulted in gels with highest strength, likely due to stereocomplexation between the enantiomers. The network properties could be modulated by varying the solid content of the gel, the DS (i.e., number of lactate grafts per 100 glucopyranose units) and the DP (i.e., degree of polymerization) of the oligolactate grafts. Protein loading of the hydrogels could be achieved by simply mixing the microspheres with protein solution. Release experiments showed a continuous release of the entrapped lysozyme, with 50% released after 5 days and full preservation of its enzymatic activity. The biocompatible nature of the material, the protein-friendly self-assembly of the hydrogel and the possibility to tailor the gel properties, makes this hydrogel system an attractive candidate for pharmaceutical and biomedical applications.