Genipin-crosslinked Chitosan Research Articles

Suture pullout strength and in vitro fibroblast and RAW 264.7 monocyte biocompatibility of genipin crosslinked nanofibrous chitosan mats for guided tissue regeneration

Guided tissue regeneration (GTR) is a surgical technique used to direct the formation of bone in the graft space by protecting it with a barrier membrane used to exclude soft tissues during healing. Chitosan has been advocated for GTR applications because of its biocompatibility, degradability, wound healing, and osteogenic properties. In this study, electrospun chitosan membranes, crosslinked with 5 mM or 10 mM geinipin, a natural crosslinker extracted from the gardenia plant, were evaluated for suture pullout strength, crystallinity, and cytocompatibility with normal human dermal fibroblast and TIB 71™ RAW 264.7 monocyte cells. Ultimate suture pullout strength was significantly lower (51-67%) than that of commercially available collagen membranes. Crystallinity of the electrospun chitosan mats decreased upon crosslinking by 14-17% (p = 0.013). The molecular weight of the chitosan polymer was decreased by 75% during the electrospinning process. Uncrosslinked and genipin-crosslinked chitosan mats were cytocompatible and supported fibroblast cell proliferation for 9 days. Uncrosslinked and genipin-crosslinked membranes did not activate monocytes to produce nitric oxide (NO) in vitro in the absence of lipopolysaccharide (LPS). Finally, chitosan membranes inhibited LPS-induced NO production of RAW 264.7 cells by 59-67% as compared to tissue culture plastic and collagen membrane. Improvements are needed in the tear strength of electrospun chitosan membranes for clinical application.

Intra-articular delivery of genipin cross-linked chitosan microspheres of flurbiprofen: Preparation, characterization, in vitro and in vivo studies

In this study, genipin cross-linked chitosan microspheres of flurbiprofen for intra-articular (i.a.) delivery were prepared. Emulsion-cross-linking method was used to prepare the microspheres using different concentrations of genipin and drug-to-polymer ratios. The mean particle size was found to be in the range of 5.18–9.74μm with good % drug entrapment up to 80.97%. SEM indicated the spherical shape with smooth surface of drug-loaded cross-linked microspheres. FTIR confirmed cross-linking of genipin with chitosan and the absence of chemical interactions between drug, polymer, and cross-linker, which was further confirmed by TGA showing unaltered melting point of entrapped drug. DSC and XRD revealed the molecular dispersion of drug within microspheres. The optimized microspheres were able to release the drug for more than 108h. The biocompatibility of the microspheres in the rat (Sprague-Dawley) knee joints was confirmed by histopathology. The results of pharmacokinetic study, decreased Ka and Tmax, lower Cmax and AUC(0–24) and delayed MRT, suggested the significant extended release of flurbiprofen from microspheres in comparison with its solution at P<0.05. The recovery of flurbiprofen as the percent of administered dose followed by 24h after i.a. injection of microspheres was found to be 8.7 folds higher than its solution.

Characterization and antibacterial properties of genipin-crosslinked chitosan/poly(ethylene glycol)/ZnO/Ag nanocomposites

Novel nanocomposites consisting of genipin-crosslinked chitosan (GC), poly(ethylene glycol) (PEG), zinc oxide (ZnO), and silver (Ag) nanoparticles were prepared for biomedical applications as the wound-healing materials. Various amounts of ZnO and Ag nanoparticles were dispersed in the GC/PEG hydrogel matrix without severe aggregation. The effects of composition and ZnO nanoparticles on the physico-chemical properties of nanocomposite samples were evaluated by infrared analysis, X-ray diffraction, and scanning electron microscopy. GC/PEG/ZnO/Ag nanocomposite showed the pH-sensitive swelling behavior and the improved mechanical properties. The antibacterial activities of nanocomposite films were tested toward the bacterial species Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis. GC/PEG/ZnO/Ag composite films had higher antibacterial activities than GC/PEG and GC/PEG/ZnO nanocomposite films. GC/PEG/ZnO/Ag composite films have potential application as wound and burn dressings.

Genipin-crosslinked chitosan scaffolds and its efficacy in releasing anti-inflammatory medicine

Controlled release carriers are often made into microspheres or tablets. Systematic and quantitative characterization of porous tissue engineered scaffolds as release carriers have not been done. Chitosan and chitosan crosslinked with various concentrations of genipin were made into porous tissue engineered scaffolds. Their thermal and enzymatic stabilities, hydrophobicities, porous structures, swelling and release properties, and compressional moduli were measured. The effects of scaffolds loaded with pentoxifylline (PTX) in suppressing inflammatory reactions in vitro were quantified.Fourier Transform Infrared spectra showed new bond formation after crosslinking chitosan with genipin. As genipin increased from 0.01% to 0.1%, the crosslinked chitosan scaffolds swelled 0.5% to 1.8% less, had 1.9-5% decrease in PTX release efficiencies, became less wettable, were less favorable for initial cell attachment, had 4-20% increase in Young's modulus and were more resistant to enzymatic degradation. In vitro tests showed that when PTX was released more slowly from crosslinked scaffolds, PTX became more effective in suppressing macrophage cells from releasing IL-6 and TNF-α.

Genipin-crosslinked N-carboxymethyl chitosan microcapsules prepared by electrostatic spray method

Genipin-crosslinked N-carboxymethyl chitosan microcapsules prepared by electrostatic spray method

Mechanical and Antimicrobial Properties of Genipin-Crosslinked Chitosan/Poly(Ethylene Glycol) IPN

Chitosan/poly(ethylene glycol) (PEG) interpenetrating networks (IPN) were prepared using a solution casting technique. The natural and nontoxic cross-linking agent, genipin, was used to form genipin-crosslinked chitosan (GC). Morphology and cross-linking of GC/PEG IPN were investigated as a function of the composition with scanning electron microscope and Fourier transform infrared spectroscopy, respectively. GC/PEG (80/20) IPN showed the most satisfactory mechanical properties for antibacterial packaging films. The surface properties of GC/PEG IPN films were also examined by contact angle measurements. GC/PEG IPN films became more hydrophilic with increasing the content of PEG. The antimicrobial properties of GC/PEG IPN films were evaluated against Escherichia coli. Chitosan contributed a large share to the antimicrobial properties of GC/PEG IPN films.

In Vitro Response of Retinal Pigment Epithelial Cells Exposed to Chitosan Materials Prepared with Different Cross-Linkers

The interaction between cells and biopolymers is the evaluation indicator of the biocompatibility of materials. The purpose of this work was to examine the responses of retinal pigment epithelial (RPE) cells to genipin (GP) or glutaraldehyde (GTA) cross-linked chitosan by means of cell viability assays, cytokine expression analyses, and apoptosis assays. Evaluations of non-cross-linked chitosan were conducted simultaneously for comparison. Both GP and GTA treated samples with the same extent of cross-linking (around 80%) were prepared by varying cross-linking time. Our results showed that GP cross-linking was carried out by either radical polymerization of the monomers or SN2 nucleophilic substitution reaction involving the replacement of the ester group on the monomer with a secondary amide linkage. On the other hand, GTA could react with free amino groups of chitosan, leading to the formation of either the Schiff bases or the Michael-type adducts with terminal aldehydes. The biocompatibility of non-cross-linked chitosan membranes was demonstrated by the absence of any signs of toxicity or inflammation reaction. The present study showed that the ARPE-19 cells exposed to GTA cross-linked chitosan membranes had significantly higher cytotoxicity, interleukin-6 levels, and number of TUNEL-positive nuclei than did those exposed to GP treated samples. In addition, the materials modified with GTA trigger apoptosis at an early stage and may induce toxicity in the RPE cells later. The findings suggest that while the chitosan molecules bridged by GP are satisfactorily cytocompatible, the counterparts treated by GTA do not seem to be tolerated. In terms of material safety, the GP cross-linked chitosan may be compatible with human RPE cells and may have a potential application as delivery carriers in the treatment of posterior segment diseases.

Antibacterial activity of pH-sensitive genipin cross-linked chitosan/poly(ethylene glycol)/silver nanocomposites

Novel nanocomposites consisting of genipin cross-linked chitosan (GC), poly(ethylene glycol) (PEG), and silver nanoparticles were prepared for such biomedical applications as the wound-healing materials. Various amounts of silver nanoparticles were dispersed in the GC/PEG hydrogel matrix without severe aggregation. The effects of composition and silver nanoparticles on the physico-chemical properties of samples were evaluated by infrared analysis, contact angle measurements, and swelling tests. The GC/PEG/Ag nanocomposite showed a pH-sensitive swelling behavior. The surface hydrophilicity of GC/PEG/Ag nanocomposites was improved with the increase of silver nanoparticle content. L929 cell attachment was improved in the presence of silver nanoparticles. The antimicrobial function was assessed for the GC/PEG/Ag nanocomposites containing the silver content over 100 ppm. The silver nanoparticles had the dual functions of reinforcing structural stability and enhancing antimicrobial activity of GC/PEG/Ag nanocomposites. Copyright © 2010 John Wiley & Sons, Ltd.

Genipin-crosslinked hydrophobical chitosan microspheres and their interactions with bovine serum albumin

In this work chitosan microspheres have been prepared by ion precipitation using sodium sulfate as precipitant, and crosslinked with natural crosslinker genipin. Then the microspheres were hydrophobically modified by covalent and ion interaction grafting of stearic acid and sodium stearate respectively. Results indicated that the there were still some amount of –NH 2 groups on the genipin-crosslinked chitosan microspheres. And the contact angles for hydrophobical graft microspheres were increased greatly from 27° to 104°, which indicates stearic chains have been grafted on the surface of the microspheres even only by a simple graft process based on ion interaction. The interactions of protein with the hydrophobically modified chitosan microspheres were investigated by surface absorption method using bovine serum albumin as a model protein. And results indicated that adsorption efficiency for bovine serum albumin of the fatty chain grafted chitosan microspheres was higher than that of the prestine one distinctly, due to the additional hydrophobic interaction. And the adsorbed protein could well-off be desorbed under a general condition.

A novel wound dressing composed of nonwoven fabric coated with chitosan and herbal extract membrane for wound healing

AbstractThis study presents a novel design of an easily stripped bilayer composite that consists of an upper layer of soybean protein nonwoven fabric coated with a lower layer, genipin‐crosslinked chitosan and Bletilla striata herbal extract (GCB) film as a wound dressing material (GCB/NWF). Preliminary tests investigated the in vitro characteristics of the genipin‐crosslinked chitosan (GC) film. Experimental results showed that the dark bluish color of the GC film may have resulted from the reaction of genipin with the amino acid group in the chitosan solution. Additionally, adding more wt% genipin caused the bluish color of the GC films to be darker. This new wound dressing material also exhibited good mechanical properties. Genipin and chitosan were released from the soaked GC film. Cytotoxic test revealed that 20 ppm of the genipin in the culture medium could be criteria, over which cytotoxicity to L929 fibroblasts could be produced. However, the chitosan and B. striata herbal extract not only were not toxic but also promoted the viability and growth of L929 fibroblasts. Additionally, the GCB film more effectively supported cell attachment and growth. The in vivo histological assessment results revealed that epithelialization and reconstruction of the wound were achieved by covering the wound with the GCB/NWF wound dressing material, and it would be easily stripped from the wound surface without damaging newly regenerated tissue. POLYM. COMPOS., 2010. © 2009 Society of Plastics Engineers

Physicochemical and biological activity study of genipin-crosslinked chitosan scaffolds prepared by using supercritical carbon dioxide for tissue engineering applications

The present study deals with the preparation of porous chitosan scaffolds by using a greener technique i.e., supercritical carbon dioxide (sc.CO 2). 0.2 M chitosan (CS) solution in aqueous acetic acid was treated with 4% (w/v) genipin solution; the resulting hydrogels were subjected to solvent exchange prior to the final treatment procedures. Their morphology, pore structure, and physical properties were characterized by thermal analysis, X-ray diffractogram (XRD), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and the specific surface areas and porosimetry of scaffolds were determined by using N 2 adsorption. The biological activity of scaffolds was investigated by immersing them into 1.5× simulated body fluid (SBF) and cellular attachment study was assessed using Alamar Blue assay by seeding MG63 osteoblastic cells onto the scaffolds. The sc.CO 2 assisted chitosan scaffold prepared by using green chemistry approach is highly pure and bioactive and can be served as a potential material for tissue engineering applications.

Mechanical properties, drug eluting characteristics and in vivo performance of a genipin-crosslinked chitosan polymeric stent

A limitation with the use of polymers as stent matrices is their inherent mechanical weakness. In this study, a polymeric stent, made from chitosan-based films fixed by genipin which has a cyclic molecular structure, was developed (the genipin stent). The mechanical properties of the genipin stent were investigated; its counterpart fixed by a linear epoxy compound (the epoxy stent) and a commercially available metallic stent were used as controls. The results indicated that the cyclic crosslinking structures formed within the genipin stent matrix were beneficiary to the improvement of its mechanical property. Additionally, the tolerable compression load of the genipin stent was superior to that of the control metallic stent. The cytotoxicity of the genipin stent was significantly lower than the epoxy stent. The deployment of the genipin stent in rabbit infrarenal abdominal aortas was performed using a French sheath. At 3 months postoperatively, the retrieved arteries remained patent; no thrombosis was observed. A nearly intact layer of endothelial cells was seen on the stent-implanted vessel wall. To evaluate its possibility as a drug delivery vehicle, sirolimus (an anti-proliferative drug) was loaded in the genipin stent. It was found that the genipin stent with heparin coating exhibited a linear sustained-release profile and the released sirolimus still possessed its original activity in inhibiting smooth muscle cell proliferation. These findings suggest that the genipin stent with enhanced mechanical strength can be used as an attractive stent platform for local drug delivery.

Genipin Cross-Linked Polymeric Alginate-Chitosan Microcapsules for Oral Delivery: In-Vitro Analysis

We have previously reported the preparation of the genipin cross-linked alginate-chitosan (GCAC) microcapsules composed of an alginate core with a genipin cross-linked chitosan membrane. This paper is the further investigation on their structural and physical characteristics. Results showed that the GCAC microcapsules had a smooth and dense surface and a networked interior. Cross-linking by genipin substantially reduced swelling and physical disintegration of microcapsules induced by nongelling ions and calcium sequestrants. Strong resistance to mechanical shear forces and enzymatic degradation was observed. Furthermore, the GCAC membranes were permeable to bovine serum albumin and maintained a molecular weight cutoff at 70 KD, analogous to the widely studied alginate-chitosan, and alginate-poly-L-lysine-alginate microcapsules. The release features and the tolerance of the GCAC microcapsules in the stimulated gastrointestinal environment were also investigated. This GCAC microcapsule formulation offers significant potential as a delivery vehicle for many biomedical applications.

Nanocomposites of Genipin‐Crosslinked Chitosan/Silver Nanoparticles ‐ Structural Reinforcement and Antimicrobial Properties

This study investigates the feasibility of a novel nanocomposite (GC/Ag) of a genipin-crosslinked chitosan (GC) film in which was embedded various amounts of Ag nanoparticles for wound-dressing applications. In situ UV-vis results revealed that adding chitosan solution did not affect the characteristics of Ag nanoparticles. The water uptake ratios and surface hydrophilicity of the GC/Ag nanocomposite were better and the degradation rates slightly lower than those of the pure GC film. The presence of Ag nanoparticles enhanced L929 cell attachment and growth. Its function as an anti-microbial agent in a GC/Ag nanocomposite was assessed for Ag contents of over 100 ppm. In conclusion, silver ions had dual functions--structural reinforcement and provision of antimicrobial properties to a biocompatible polymer.

Evaluation of a Non‐Woven Fabric Coated with a Chitosan Bi‐Layer Composite for Wound Dressing

This study presents a novel design of an easily stripped bi-layer composite that consists of an upper layer of a soybean protein non-woven fabric coated with a lower layer, a genipin-crosslinked chitosan film, as a wound dressing material. This study examines the in vitro properties of the genipin-crosslinked chitosan film and the bi-layer composite. Furthermore, in vivo experiments are conducted to study wounds treated with the composite in a rat model. Experimental results show that the degree of crosslinking and the in vitro degradation rate of the genipin-crosslinked chitosan films can be controlled by varying the genipin contents. In addition, the genipin contents should exceed 0.025 wt.-% of the chitosan-based material if complete crosslinking reactions between genipin and chitosan molecules are required. Water contact angle analysis shows that the genipin-crosslinked chitosan film is not highly hydrophilic; therefore, the genipin-crosslinked chitosan layer is not entangled with the soybean protein non-woven fabric, which forms an easily stripped interface layer between them. Furthermore, this new wound dressing material provides adequate moisture, thereby minimizing the risk of wound dehydration, and exhibits good mechanical properties. The in vivo histological assessment results reveal that epithelialization and reconstruction of the wound are achieved by covering the wound with the composite, and the composite is easily stripped from the wound surface without damaging newly regenerated tissue.

Genipin Cross-Linked Alginate-Chitosan Microcapsules: Membrane Characterization and Optimization of Cross-Linking Reaction

The genipin cross-linked alginate-chitosan (GCAC) microcapsule, composed of an alginate core and a genipin cross-linked chitosan membrane, was recently proposed for live cell encapsulation and other delivery applications. This article for the first time describes the details of the microcapsule membrane characterization using a noninvasive and in situ method without any physical or chemical modifications on the samples. Results showed that the cross-linking reaction generated the fluorescent chitosan-genipin conjugates. The cross-linked chitosan membrane was clearly visualized by confocal laser scanning microscopy (CLSM). A straightforward assessment on the membrane thickness and relative intensity was successfully achieved. CLSM studies showed that the shell-like cross-linked chitosan membranes of approximately 37 microm in thickness were formed surrounding the microcapsule. The reaction variables, including cross-linking temperature and time significantly affected the fluorescence intensity of the membranes. Elevating the cross-linking temperature from 4 to 37 degrees C drastically intensified the membrane fluorescence, suggesting the attainment of a high degree of cross-linking on the chitosan membrane. Extended cross-linking time altered the cross-linked membranes in modulation. Although genipin concentration and cross-linking time had little effects on the membrane thickness, cross-linking at higher temperatures tended to form relatively thinner membranes.

Using NaCl particles as porogen to prepare a highly adsorbent chitosan membranes

Macroporous chitosan membranes were prepared by using NaCl particles porogen and genipin as cross-linking agent. For characterization and sorption behavior comparison, other genipin cross-linked chitosan membranes were prepared by either freeze drying or by using silica particles as porogen. The mean pore diameter, the porosity, the crystallinity index (CrI) as well as the effect of the drying procedures of these chitosan membranes were examined. NaCl reduced the CrI of the chitosan membrane. The oven drying (OD) procedure decreased the mean pore diameter, the porosity, and increased the CrI of the chitosan membranes when compared with the vacuum drying (VD) procedure. The heat treatment of chitosan membrane in aqueous NaOH to attract silica porogen increased the CrI of the membrane. Under the same conditions, the membranes prepared with NaCl had better sorption performance on RR 189 and Cu 2+ than other membranes. The maximum sorption capacity ( q e) reached 1836.17 mg RR 189/g chitosan and 151.98 mg Cu 2+/g chitosan. The pore diameter ( d pore) of the membranes was much larger than the diameter of the adsorbate molecules ( d adsorbate), such that the ratio of d pore/ d adsorbate had little influence on q e. The porosity and the amorphous extent of the membranes were almost the same on q e. When using tyrosinase catalyzing, the hydrocaffeic acid (HCA) grafted on the NaCl treated chitosan membrane was almost 10 times more than on chitosan beads. The chitosan membrane prepared with NaCl can be used as a good adsorbent with high loading capacity for implanting molecules (such as ligands, enzymes, etc.) on.

Swelling and Thermal Characteristics of Genipin Crosslinked Chitosan and Poly(vinyl pyrrolidone) Hydrogels

Chitosan / poly(vinyl pyrrolidone) (PVP) were used to prepare semi-interpenetrating polymeric networks. The hydrogels were crosslinked using genipin, a non-toxic cross-linking agent extracted from the fruits of Gardenia jasminoides Ellis. Swelling properties of these hydrogels were studied in media of different pH’s and temperatures. States of water in the swollen hydrogels at 25°C and pH 7 were determined using Differential Scanning Calorimetry (DSC). The swelling behaviour of the hydrogels was found to be dependent on the temperature and the pH of the swelling medium. The total water content in the hydrogels was found to increase with increasing PVP content.

Characterization of ring-opening polymerization of genipin and pH-dependent cross-linking reactions between chitosan and genipin

In this study, a novel chitosan-based polymeric network was synthesized by crosslinking with a naturally occurring crosslinking agent—genipin. The results showed that the crosslinking reactions were pH-dependent. Under basic conditions, genipin underwent a ring-opening polymerization prior to crosslinking with chitosan. The crosslink bridges consisted of polymerized genipin macromers or oligomers (7 ∼ 88 monomer units). This ring-opening polymerization of genipin was initiated by extracting proton from the hydroxyl groups at C-1 of deoxyloganin aglycone, followed by opening the dihydropyran ring to conduct an aldol condensation. At neutral and acidic conditions, genipin reacted with primary amino groups on chitosan to form heterocyclic amines. The heterocyclic amines were further associated to form crosslinked networks with short chains of dimmer, trimer, and tetramer bridges. An accompanied reaction of nucleophilic substitution of the ester group on genipin by the primary amine group on chitosan would occur in the presence of an acid catalysis. The extent in which chitosan gels crosslinked with genipin was significantly dependent on the crosslinking pH values: 39.9 ± 3.8% at pH 5.0, 96.0 ± 1.9% at pH 7.4, 45.4 ± 1.8% at pH 9.0, and 1.4 ± 1.0% at pH 13.6 (n = 5, p < 0.05). Owing to the different crosslinking extents and different chain lengths of crosslink bridges, the genipin-crosslinked chitosan gels showed significant difference in their swelling capability and their resistance against enzymatic hydrolysis, depending on the pH conditions for crosslinking. These results indicated a direct relationship between the mode of crosslinking reaction, and the swelling and enzymatic hydrolysis properties of the genipin-crosslinked chitosan gels. The ring-opening polymerization of genipin and the pH-dependent crosslinking reactions may provide a novel way for the preparation and exploitation of chitosan-based gels for biomedical applications. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 1985–2000, 2005

In vivo biocompatibility and degradability of a novel injectable-chitosan-based implant

A novel injectable-chitosan-based delivery system with low cytotoxicity was fabricated in the study. The chitosan microspheres with small particle size, low crystallinity and good sphericity were prepared by a spray-drying method followed by treating with a crosslinker. In the study, a naturally occurring crosslinking reagent (genipin), which has been used in herbal medicine and in the production of food dyes, was used to crosslink the chitosan microspheres. The glutaraldehyde-crosslinked counterparts were used as a control. Histological study of the genipin-crosslinked chitosan microspheres injected intramuscularly into the skeletal muscle of a rat model showed a less inflammatory reaction than its glutaraldehyde-crosslinked counterparts. The results of the scanning electron microscopic examination indicated that the glutaraldehyde-crosslinked chitosan microspheres retrieved at 12-week postoperatively were already degraded into a loose and porous structure. However, the degradation of the genipin-crosslinked chitosan microspheres was not significant after 20 weeks of implantation. The results of the study demonstrated that the genipin-crosslinked chitosan microspheres have a superior biocompatibility and a slower degradation rate than the glutaraldehyde-crosslinked chitosan microspheres. Accordingly, the genipin-crosslinked chitosan microspheres may be a suitable polymeric carrier for long-acting injectable drug delivery.