Nanogel engineering and chaperone engineering

Chaperone-like activity (to trap proteins in biomaterials without their aggregations and to control release of proteins in a native form) is important to design protein delivery systems as well as protein engineering. In general, irreversible adsorption of proteins is unavoidable in trapping them in hydrogel biomaterials because it is difficult to control the mesh size of the hydrogel matrix. We suggested that physically cross-linked nanogels with a size comparable to that of proteins are useful for these purposes. Tailor-made functional nanogels and hydrogels were designed by self-assembly of functional associating polysaccharides such as cholesterol-bearing pullulans. The nanogels can trap hydrophobic molecules, proteins and nucleic acids. They are useful as artificial molecular chaperones and also polymeric nanocarriers in DDS.     

A review of hydrogel use in fracture healing and bone regeneration

This review explores the application of hydrogels in orthopaedic clinical situations which may benefit from enhanced growth factor delivery and improved osteogenesis of bone graft material. Hydrogels are defined, and in vivo evidence supporting their application in these clinical areas is explored. Our focus is on clinically pertinent properties, such as the chemistry of formation, biocompatibility, efficacy of cell and growth factor delivery, ability to withstand mechanical loading and potential to be delivered via an injection. Naturally derived hydrogels, such as gelatin, hyaluronic acid and fibroin, together with a number of synthetic polyethylene glycol‐based gels combined with protease‐sensitive domains, have shown excellent biocompatibility. There is significant literature evidence supporting the ability of hydrogels to facilitate growth factor and cell delivery. Burst release of the selected growth factor remains a consistent challenge, which has been overcome in some studies with chemical modifications of the hydrogel. Interestingly, a number of studies detail percutaneous delivery with hydrogels combined with calcium‐based minerals to enhance osteogenicity, with mixed results. Few of the studies explored the biomechanical properties of the materials, and none of the studies reviewed demonstrated the ability of a hydrogel/graft material to withstand mechanical loading in a clinically relevant segmental bone defect model. Copyright © 2014 John Wiley & Sons, Ltd.     

Core‐shell silk hydrogels with spatially tuned conformations as drug‐delivery system

Hydrogels of spatially controlled physicochemical properties are appealing platforms for tissue engineering and drug delivery. In this study, core‐shell silk fibroin (SF) hydrogels of spatially controlled conformation were developed. The core‐shell structure in the hydrogels was formed by means of soaking the preformed (enzymatically crosslinked) random coil SF hydrogels in methanol. When increasing the methanol treatment time from 1 to 10 min, the thickness of the shell layer can be tuned from about 200 to about 850 μm as measured in wet status. After lyophilization of the rehydrated core‐shell hydrogels, the shell layer displayed compact morphology and the core layer presented porous structure, when observed by scanning electron microscopy. The conformation of the hydrogels was evaluated by Fourier transform infrared spectroscopy in wet status. The results revealed that the shell layer possessed dominant β‐sheet conformation and the core layer maintained mainly random coil conformation. Enzymatic degradation data showed that the shell layers presented superior stability to the core layer. The mechanical analysis displayed that the compressive modulus of the core‐shell hydrogels ranged from about 25 kPa to about 1.1 MPa by increasing the immersion time in methanol. When incorporated with albumin, the core‐shell SF hydrogels demonstrated slower and more controllable release profiles compared with the non‐treated hydrogel. These core‐shell SF hydrogels of highly tuned properties are useful systems as drug‐delivery system and may be applied as cartilage substitute. Copyright © 2016 John Wiley & Sons, Ltd.     

Genetically engineered polymers: status and prospects for controlled release.

Genetic engineering methodology has enabled the synthesis of protein-based polymers with precisely controlled structures. Protein-based polymers have well-defined molecular weights, monomer compositions, sequences and stereochemistries. The incorporation of tailor-made motifs at specified locations by recombinant techniques allows the formation of hydrogels, sensitivity to environmental stimuli, complexation with drugs and nucleic acids, biorecognition and biodegradation. Accordingly, a special interest has emerged for the use of protein-based polymers for controlled drug and gene delivery, tissue engineering and other biomedical applications. This article is a review of genetically engineered polymers, their physicochemical characteristics, synthetic strategies used to produce them and their biomedical applications with emphasis on controlled release.     

Hyaluronic acid and fibrin hydrogels with concentrated DNA/PEI polyplexes for local gene delivery

Local delivery of DNA through a hydrogel scaffold would increase the applicability of gene therapy in tissue regeneration and cancer therapy. However, the delivery of DNA/cationic polymer nanoparticles (polyplexes) using hydrogels is challenging due to the aggregation and inactivation of polyplexes during their incorporation into hydrogel scaffolds. We developed a novel process (termed caged nanoparticle encapsulation or CnE) to load concentrated and unaggregated non-viral gene delivery nanoparticles into various hydrogels. Previously, we showed that PEG hydrogels loaded with DNA/PEI polyplexes through this process were able to deliver genes both in vitro and in vivo. In this study, we found that hyaluronic acid and fibrin hydrogels with concentrated and unaggregated polyplexes loaded through CnE were able to deliver genes in vivo as well, demonstrating the universality of the process.     

Reversible adsorption by a pH- and temperature-sensitive acrylic hydrogel

Thermo- and pH-sensitive hydrogels were synthesized using N-isopropylacrylamide (NIPA) and N-aminopropylmethacrylamide, cross-linked with N,N′-methylenebis(acrylamide). The dependence of the degree of swelling on the cross-linking density was analyzed according to the Flory-Huggins theory and a master curve obtained. To optimize the efficiency of these hydrogels in controlled release, we studied the loading and release of a divalent molecule (naphthalenedisulfonic acid, NS-2) in media of different ionic strengths and pH. The uptake process followed the Langmuir adsorption isotherm model. The highest loading occurred when the amino groups in the gel were protonated (acidic pH) and could come close each other to form a binding site for the two sulfonic groups of NS-2, i.e. low degree of cross-linking and collapsed state. Below the phase transition temperature (33 °C), NS-2 loaded hydrogels quickly released a significant amount of adsorbate until a new equilibrium between free NS-2 and adsorbed NS-2 was achieved. Above that temperature, hydrogels not only stopped the release but were even able to take free NS-2 up again from the medium, showing that the loading/release process was reversible and reproducible after several temperature cycles. At 37 °C, the release rate was independent of the degree of cross-linking (NIPA caused the hydrogel to collapse), but was strongly affected by the pH and salt concentrations of the medium, which condition the strength of the interaction between the hydrogel amino groups and the NS-2 sulfonic groups. In an acidic medium, the protonated amino groups bind NS-2 strongly and the amount released is small. In contrast, at pH 7.4 or in the presence of a high salt concentration, the hydrogel loses its affinity for NS-2 and the release rate increases, giving pH- or salt-sensitive delivery systems. Additionally, since the hydrogel is collapsed, the release can be prolonged for a long period of time.     

Physicochemical behavior and cytotoxic effects of p(methacrylic acid–g-ethylene glycol) nanospheres for oral delivery of proteins

The challenges faced to orally deliver therapeutic agents with unfavorable physicochemical properties, such as proteins, have been the primary motivation for the design and development of novel oral delivery systems that could circumvent biological barriers. In this work, we examined complexation-sensitive hydrogel nanospheres composed of poly[methacrylic acid–grafted-poly(ethylene glycol)] (P(MAA–g-EG)), on a model biological environment. For this purpose, a gastrointestinal cell culture model, the Caco-2 cell line, was employed to investigate the cytotoxic effects of the polymeric carrier and its effects on the cell monolayer integrity. The determination of the cytotoxic effects of the polymer network on the cell monolayer was performed by a colorimetric assay and by the counting of viable cells using the trypan blue exclusion method. Electrophysiological measurements were performed to measure the transepithelial electrical resistance changes in the monolayers in the presence and absence of the nanosphere suspension. The examination of the physicochemical interactions of the P(MAA–g-EG) nanosphere system with Caco-2 cell monolayers revealed that these systems possessed low cytotoxicity and were capable of opening the tight junctions between epithelial cells, therefore significantly reducing the transepithelial electrical resistance.     

Manipulation of hydrogel assembly and growth factor delivery via the use of peptide-polysaccharide interactions.

The design of materials in which assembly, mechanical response, and biological properties are controlled by protein-polysaccharide interactions could provide materials that mimic the biological environment and find use in the delivery of growth factors. In the investigations reported here, a heparin-binding, coiled-coil peptide, PF4 ZIP, was employed to mediate the assembly of heparinized polymers. The heparin-binding affinity of this peptide was compared with that of other heparin-binding peptides (HBP) via heparin-sepharose chromatography and surface plasmon resonance (SPR) experiments. Results from these experiments indicate that PF4 ZIP demonstrates a higher heparin-binding affinity and heparin association rate when compared to the heparin-binding domains of antithrombin III (ATIII) and heparin-interacting protein (HIP). Viscoelastic hydrogels were formed upon the association of PF4 ZIP-functionalized star poly(ethylene glycol) (PEG-PF4 ZIP) with low-molecular-weight heparin-functionalized star PEG (PEG-LMWH). The viscoelastic properties of the hydrogels can be altered via variations in the ratio of LMWH to PF4 ZIP. bFGF release from these gels have also been investigated. Comparison of the bFGF release profiles with the hydrogel erosion profiles indicates that bFGF delivery from this class of hydrogels is mainly an erosion-controlled process and the rates of bFGF release can be modulated via choice of HBP or via variations in the mechanical properties of the hydrogels. Manipulation of hydrogel physical properties and erosion profiles will provide novel materials for controlled growth factor delivery and other biomedical applications.     

Recent advances in delivery of drug–nucleic acid combinations for cancer treatment

Cancer treatment that uses a combination of approaches with the ability to affect multiple disease pathways has been proven highly effective in the treatment of many cancers. Combination therapy can include multiple chemotherapeutics or combinations of chemotherapeutics with other treatment modalities like surgery or radiation. However, despite the widespread clinical use of combination therapies, relatively little attention has been given to the potential of modern nanocarrier delivery methods, like liposomes, micelles, and nanoparticles, to enhance the efficacy of combination treatments. This lack of knowledge is particularly notable in the limited success of vectors for the delivery of combinations of nucleic acids with traditional small molecule drugs. The delivery of drug–nucleic acid combinations is particularly challenging due to differences in the physicochemical properties of the two types of agents. This review discusses recent advances in the development of delivery methods using combinations of small molecule drugs and nucleic acid therapeutics to treat cancer. This review primarily focuses on the rationale used for selecting appropriate drug–nucleic acid combinations as well as progress in the development of nanocarriers suitable for simultaneous delivery of drug–nucleic acid combinations.     

Protein and peptide parenteral controlled delivery

Protein and peptide delivery has been a challenge due to their limited stability during preparation of formulation, storage and in vitro and in vivo release. These biopolymers have traditionally been administered via intramuscular or subcutaneous routes. Recent efforts have been made to develop formulations for non-invasive routes of administration, including oral, intranasal, transdermal and transmucosal delivery. Despite these efforts, invasive delivery remains the main method of administering peptide and protein drugs. This review focuses on recent developments in injectable, polymeric controlled-release formulations, with an emphasis on hydrogels and particulate systems.     

Poloxamine/fibrin hybrid hydrogels for non‐viral gene delivery

Hydrogels have been widely investigated for localized, sustained gene delivery because of the similarity of their physical properties to native extracellular matrix and their ability to be formed under mild conditions amenable to the incorporation of bioactive molecules. The objective of this study was to develop bioactive hydrogels composed of macromolecules capable of enhancing the efficiency of non‐viral vectors. Hybrid hydrogels were prepared by simultaneous enzymatic and Michael‐type addition crosslinking of reduced fibrinogen and an acrylated amphiphilic block copolymer, Tetronic T904, in the presence of dithiothreitol (DTT) and thrombin. T904/fibrin hydrogels degraded by surface erosion in the presence of plasmin and provided sustained release of polyplex vectors up to an order of magnitude longer than pure fibrin gel control. In addition, the rate of gel degradation and time‐course of polyplex vector release were readily controlled by varying the T904/fibrinogen ratio in the gel composition. When added to transfected neuroblastoma (N2A) cells, both native T904 itself and hydrogel degradation products significantly increased polyplex transfection efficiency with minimal effect on cell viability. To evaluate gel‐based transfection, N2A cells encapsulated in small fibrin clusters were covered by or suspended within polyplex‐loaded hydrogels. Cells progressively degraded and invaded the hybrid hydrogels, exhibiting increasing gene expression over 2 weeks and then diminishing but persistent gene expression for over 1 month. In conclusion, these results demonstrate that T904/fibrin hybrid hydrogels can be promising tissue engineering scaffolds that provide local, controlled release of non‐viral vectors in combination with the generation of bioactive gel degradation products that actively enhance vector efficiency. Copyright © 2014 John Wiley & Sons, Ltd.     

Dynamic crosslinked and injectable biohydrogels as extracellular matrix mimics for the delivery of antibiotics and 3D cell culture

Antibiotics are widely used in clinical medicine. As an important member, vancomycin often plays an irreplaceable role in some serious infections but for its use, there is still a lack of suitable carriers and effective formulations. To find a vancomycin carrier with potential for clinical applications, a new class of poly(γ-glutamic acid)/dextran-based injectable hydrogels have been constructed through dynamic covalent hydrazone linkages. Adipic dihydrazide (ADH)-grafted poly(γ-glutamic acid) (PGAADH) and sodium periodate-oxidized dextran (OD) precursors were synthesized; then, the hydrogels were formed by blending PGAADH and OD buffer solutions without any additives under physiological conditions. The newly formed precursor structures, mechanical properties, morphologies, hydrogel degradation profiles, and the interaction between the drug and precursors were investigated with FTIR spectroscopy, ¹H NMR spectroscopy, rheological experiments, compression tests, SEM, and isothermal titration calorimetric (ITC) measurements. The resulting hydrogels exhibited excellent antibacterial ability and ideal variable performances. Moreover, the hydrogels exhibited different drug release kinetics and mechanisms and were applied effectively towards the controlled release of vancomycin. Significantly, benefitting from the reversibly cross-linked systems and the excellent biocompatibility, the hydrogels can work as the ideal material for HeLa cell culture, leading to encapsulated cells with higher viability and capacity that is proliferative. Therefore, the injectable PGAADH/OD hydrogels demonstrated attractive properties for future applications in pharmaceutics and tissue engineering.

Polysaccharides-polypeptide derived biohydrogels were formed using hydrazone chemistry as crosslinking strategy, which have controllable drug release rate and many other potential applications, especially in sustained drug delivery and cell scaffold.     

Non-viral vector delivery from PEG-hyaluronic acid hydrogels

Hydrogels have been widely used in tissue engineering as a support for tissue formation or to deliver non-viral gene therapy vectors locally. Hydrogels that combine these functionalities can provide a fundamental tool to promote specific cellular processes leading to tissue formation. This report investigates controlled release of gene therapy vectors from hydrogels as a function of the physical properties for both the hydrogel and the vector. Hydrogels were formed by photocrosslinking acryl-modified hyaluronic acid (HA) with a 4-arm poly(ethylene glycol) (PEG) acryl. The polymer content, and relative composition of HA and PEG modulated the swelling ratio, water content, and degradation, which can influence transport of the vector through the hydrogel. All hydrogels had a water content of 94% or higher, though the water content and swelling ratio increased with a decrease in the PEG:HA ratio. Plasmids were stably incorporated into the hydrogel, with a majority of the release occurring during the initial 2 days. For incubation in buffer, the cumulative release increased with a decreasing PEG or increasing HA content, with approximately 20% to 80% released during the first week depending on the hydrogel composition. Hydrogels incubated in hyaluronidase, an enzyme that degrades HA, significantly increased plasmid release for hydrogels containing 4% PEG and 4% HA-Acryl. The encapsulation of plasmid complexed with polyethylenimine had less than 14% of the complexes released from the hydrogel both in the presence and absence of hyaluronidase. The limited release of the complexes likely results from the complex size and interactions between the vector and hydrogel. These studies demonstrate the dependence of non-viral vector release on the physical properties of the hydrogel and the vector, suggesting vector and hydrogel designs for maximizing localized delivery of non-viral vectors.     

Cellulose-based self-healing hydrogel through boronic ester bonds with excellent biocompatibility and conductivity

Self-healing hydrogels based on degradable resources have developed rapidly in the past decade due to their extensive bioapplications with biosecurity. In this research, a new kind of cellulose-based self-healing hydrogel with bio-degradability is constructed through boronic ester linkage. The carboxyethyl cellulose-graft-phenylboronic acid (CMC–B(OH)₂) was synthesized through condensation reaction conveniently and then hydrogels were prepared with dynamic boronic ester cross-linking. The chemical structures, microscopic morphologies, mechanical and self-healing properties of the hydrogels were investigated intensively through Fourier transform infrared (FT-IR) spectroscopy, rheological, SEM and tensile testing. The hydrogels formed instantly without any additional catalyst and exhibit excellent self-healing ability with good mechanical properties. Moreover, the hydrogels were applied for controlled release of doxorubicin (DOX·HCl) and showed a successive slow release profile. Importantly, the hydrogel exhibited excellent biocompatibility and show potential applications in controlled drug delivery, 3D cell culture and tissue engineering.

Self-healing hydrogel with excellent biocompatibility and conductivity fabricated from cellulose through boronic ester bond.     

Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching.

In this paper, the release of proteins from a novel self-gelling hydrogel based on biodegradable dextran microspheres is investigated. The protein-loaded macroscopic gels are obtained by hydration of mixtures of oppositely charged hydroxyethyl methacrylate-derivatized dextran microspheres with a protein solution. In media of low ionic strength (100 mM Hepes pH 7.0) it was found that the release of the entrapped model proteins (lysozyme, BSA and IgG) was slower than in saline (150 mM NaCl, 100 mM Hepes pH 7.0). The reason behind this observation is that substantial adsorption of the proteins onto the microspheres' surface and/or absorption in the microspheres takes place. Confocal images showed that independent of their crosslink density the microspheres are impermeable for BSA and IgG. BSA, bearing a negative charge at neutral pH, was adsorbed onto the surface of positively charged microspheres. Lysozyme, which is positively charged at neutral pH, was able to penetrate into the negatively charged microspheres. In saline, the gels showed continuous release of the different proteins for 25 to 60 days. Importantly, lysozyme was quantitatively and with full preservation of its enzymatic activity released in about 25 days. This emphasizes the protein friendly technology to prepare the protein-loaded gels. Mathematical modeling revealed that protein release followed Fick's second law, indicating that the systems are primarily diffusion controlled. These results show that these hydrogels are very suitable as injectable matrix for diffusion-controlled delivery of pharmaceutically active proteins.     

Gelatin‐based hydrogel for vascular endothelial growth factor release in peripheral nerve tissue engineering

Hydrogels are promising materials in regenerative medicine applications, due to their hydrophilicity, biocompatibility and capacity to release drugs and growth factors in a controlled manner. In this study, biocompatible and biodegradable hydrogels based on blends of natural polymers were used in in vitro and ex vivo experiments as a tool for VEGF‐controlled release to accelerate the nerve regeneration process. Among different candidates, the angiogenic factor VEGF was selected, since angiogenesis has been long recognized as an important and necessary step during tissue repair. Recent studies have pointed out that VEGF has a beneficial effect on motor neuron survival and Schwann cell vitality and proliferation. Moreover, VEGF administration can sustain and enhance the growth of regenerating peripheral nerve fibres. The hydrogel preparation process was optimized to allow functional incorporation of VEGF, while preventing its degradation and denaturation. VEGF release was quantified through ELISA assay, whereas released VEGF bioactivity was validated in human umbilical vein endothelial cells (HUVECs) and in a Schwann cell line (RT4‐D6P2T) by assessing VEGFR‐2 and downstream effectors Akt and Erk1/2 phosphorylation. Moreover, dorsal root ganglia explants cultured on VEGF‐releasing hydrogels displayed increased neurite outgrowth, providing confirmation that released VEGF maintained its effect, as also confirmed in a tubulogenesis assay. In conclusion, a gelatin‐based hydrogel system for bioactive VEGF delivery was developed and characterized for its applicability in neural tissue engineering. Copyright © 2014 John Wiley & Sons, Ltd.     

Development and characterization of novel agar and gelatin injectable hydrogel as filler for peripheral nerve guidance channels

Injectable hydrogels are becoming of increasing interest in the field of tissue engineering thanks to their versatile properties and to the possibility of being injected into tissues or devices during surgery. In peripheral nerve tissue engineering, injectable hydrogels having shear‐thinning properties are advantageous as filler of nerve guidance channels (NGCs) to improve the regeneration process. In the present work, gelatin‐based hydrogels were developed and specifically designed for the insertion into the lumen of hollow NGCs through a syringe during surgery. Injectable hydrogels were obtained using an agar–gelatin 20:80 weight ratio, (wt/wt) blend crosslinked by the addition of genipin (A/GL_GP). The physicochemical properties of the A/GL_GP hydrogels were analysed, including their injectability, rheological, swelling and dissolution behaviour, and their mechanical properties under compression. The hydrogel developed showed shear‐thinning properties and was applied as filler of NGCs. The A/GL_GP hydrogel was tested in vitro using different cell lines, among them Schwann cells which have been used because they have an important role in peripheral nerve regeneration. Viability assays demonstrated the lack of cytotoxicity. In vitro experiments showed that the hydrogel is able to promote cell adhesion and proliferation. Two‐ and three‐dimensional migration assays confirmed the capability of the cells to migrate both on the surface and within the internal framework of the hydrogel. These data show that A/GL_GP hydrogel has characteristics that make it a promising scaffold material for tissue engineering and nerve regeneration. Copyright © 2014 John Wiley & Sons, Ltd.     

Affinity-based growth factor delivery using biodegradable, photocrosslinked heparin-alginate hydrogels

Photocrosslinkable biomaterials are promising for tissue engineering applications due to their capacity to be injected and form hydrogels in situ in a minimally invasive manner. Our group recently reported on the development of photocrosslinked alginate hydrogels with controlled biodegradation rates, mechanical properties, and cell adhesive properties. In this study, we present an affinity-based growth factor delivery system by incorporating heparin into photocrosslinkable alginate hydrogels (HP-ALG), which allows for controlled, prolonged release of therapeutic proteins. Heparin modification had minimal effect on the biodegradation profiles, swelling ratios, and elastic moduli of the hydrogels in media. The release profiles of growth factors from this affinity-based platform were sustained for 3 weeks with no initial burst release, and the released growth factors retained their biological activity. Implantation of bone morphogenetic protein-2 (BMP-2)-loaded photocrosslinked alginate hydrogels induced moderate bone formation around the implant periphery. Importantly, BMP-2-loaded photocrosslinked HP-ALG hydrogels induced significantly more osteogenesis than BMP-2-loaded photocrosslinked unmodified alginate hydrogels, with 1.9-fold greater peripheral bone formation and 1.3-fold greater calcium content in the BMP-2-loaded photocrosslinked HP-ALG hydrogels compared to the BMP-2-loaded photocrosslinked unmodified alginate hydrogels after 8 weeks implantation. This sustained and controllable growth factor delivery system, with independently controllable physical and cell adhesive properties, may provide a powerful modality for a variety of therapeutic applications.     

Delivery of osteoinductive growth factors from degradable PEG hydrogels influences osteoblast differentiation and mineralization.

Degradable poly(ethylene glycol) (PEG) hydrogels with varying mass loss profiles were investigated to assess their applicability as delivery vehicles for osteoinductive growth factors in bone tissue engineering. Protein release is readily controlled by changes in both the structure (i.e., macromer concentration) and chemistry (i.e., number of degradable units) of the starting macromer. In vitro studies indicate an increase in total protein levels, alkaline phosphatase, and mineralization by osteoblasts cultured in the presence of osteoinductive growth factors compared to cells cultured with standard media. When growth factors are delivered from a 25 wt% hydrogel, a significant increase in both alkaline phosphatase and mineralization was seen after 3 weeks of culture over growth factor delivery in a bolus fashion. Additionally, gene expression levels of both osteocalcin and type I collagen were higher at early timepoints when growth factors were released from hydrogels. These results indicate that growth factors remain active after photoencapsulation, that the sustained delivery of growth factors alters various markers of osteoblastic differentiation, and that these networks could be useful as delivery vehicles for growth factors in bone tissue engineering. Finally, ectopic bone formation was present in subcutaneous rat tissue after implantation of hydrogel networks loaded with osteoinductive growth factors.     

pH-sensitive freeze-dried chitosan-polyvinyl pyrrolidone hydrogels as controlled release system for antibiotic delivery.

The aim of this study was to develop a pH-sensitive chitosan/polyvinyl pyrrolidone (PVP) based controlled drug release system for antibiotic delivery. The hydrogels were synthesised by crosslinking chitosan and PVP blend with glutaraldehyde to form a semi-interpenetrating polymer network (semi-IPN). The semi-IPN formation was confirmed by Fourier transform infrared spectroscopic (FTIR) analysis. Semi-IPNs, viz, air-dried and freeze-dried, were compared for their surface morphology, wettability, swelling properties and pH-dependent swelling. Air- and freeze-dried membranes were also incorporated with amoxicillin and antibiotic release was studied. Porous freeze-dried hydrogels (pore diameter, 39.20+/-2.66 microm) exhibited superior pH-dependent swelling properties over non-porous air-dried hydrogels. A high octane contact angle (144.20+/-0.580) of hydrogel was indicative of its hydrophilic nature. Increased swelling of hydrogels, under acidic conditions, was due to the protonation of a primary amino group on chitosan, as confirmed by FTIR analysis. Freeze-dried membranes released around 73% of the amoxicillin (33% by air-dried) in 3 h at pH 1.0 and, thus, had superior drug-release properties to air-dried hydrogels. Freeze-dried membranes could serve as potent candidates for antibiotic delivery in an acidic environment.