Cross-linked Chitosan Microparticles Research Articles

Crosslinked chitosan microparticles as a safe and efficient DNA carrier for intranasal vaccination against cutaneous leishmaniasis

Intranasal (i.n.) vaccination with adjuvant-free plasmid DNA encoding the leishmanial antigen LACK (LACK DNA) has shown to induce protective immunity against both cutaneous and visceral leishmaniasis in rodents. In the present work, we sought to evaluate the safety and effectiveness of d,l-glyceraldehyde cross-linked chitosan microparticles (CCM) as a LACK DNA non-intumescent mucoadhesive delivery system. CCM with 5 μm of diameter was prepared and adsorbed with a maximum of 2.4 % (w/w) of DNA with no volume alteration. Histological analysis of mouse nostrils instilled with LACK DNA / CCM showed microparticles to be not only mucoadherent but also mucopenetrant, inducing no local inflammation. Systemic safeness was confirmed by the observation that two nasal instillations one week apart did not alter the numbers of bronchoalveolar cells or blood eosinophils; did not alter ALT, AST and creatinine serum levels; and did not induce cutaneous hypersensitivity. When challenged in the footpad with Leishmania amazonensis, mice developed significantly lower parasite loads as compared with animals given naked LACK DNA or CCM alone. That was accompanied by increased stimulation of Th1-biased responses, as seen by the higher T-bet / GATA-3 ratio and IFN-γ levels. Together, these results demonstrate that CCM is a safe and effective mucopenetrating carrier that can increase the efficacy of i.n. LACK DNA vaccination against cutaneous leishmaniasis.

Modified chitosan microparticles to molecular diagnostic of Chagas disease

Background: Chagas disease is caused by the protozoan Trypanosoma cruzi (T.cruzi), is a major public health problem in Latin America and an emerging threat in worldwide. Direct diagnosis is mainly based on the microscopic observation of the parasite but has low sensitivity mainly during the chronic phase; molecular techniques increase the sensitivity, but still limited. Nanobiotechnology is latest advance in biomedicine, recently the innovation of natural polymers is has been explored. The novel use of micro and/or nanoplatforms can be used to increase the sensitivity. The objective of the study was to develop different modified chitosan microparticles with capacity of adsorption of deoxyribonucleic acid (DNA) for the molecular diagnosis of Chagas disease. Methods and materials: Different microparticles of chitosan were synthesized and characterized, the DNA adsorption procedure was optimized evaluating parameters (type, pH of the medium, concentration, time and the desorption method) The limit of detection of plasmid DNA and T. cruzi DNA was determined. The sensitivity and specificity in the detection of kinetoplast DNA (kDNA) and satellite DNA (satDNA) was determined in urine samples of experimentally infected animals obtained at 14 and 45 d post-infection (dpi). Results: The results showed the glutaraldehyde crosslinked chitosan microparticles has the highest DNA adsorption. Using these microparticles the best conditions were obtained with: 1% glutaraldehyde, pH at 6.9, contact time of 60 m and EDTA-SDS as a desorption buffer. The detection limit of plasmid DNA was 101 copies/mL and T.cruzi DNA was 10-1 parasites/mL. Higher sensitivity was obtained with kDNA (71%) compared to satDNA (25%). The sensitivity in urine samples of infected animals at 14 dpi was 42% using ADNsat target and 78% with kDNA target; at 45 dpi with ADNsat target was 7% and with the ADNk target was 64%. High specificity (100%) was obtained for both targets in non-infected animals. Conclusion: Chitosan microparticles is innovative method and could be used to capture and concentrate T.cruzi DNA in urine (Tr-DNA).The detection of Tr-DNA could be used for diagnosis of Chagas disease.

Preparation and characterization of a controlled-release formulation based on carbofuran loaded in ionically cross-linked chitosan microparticles

A controlled-release formulation (CRF) of the nematicide carbofuran (CAB) was prepared using ionically cross-linked chitosan (CHS). Four cross-linker agents were assessed, among which sulfate (SUL) obtained the highest encapsulation efficiency. Several parameters for the encapsulation of the pesticide were optimized, including pH, SUL/CHS molar ratio, stirring time, and stirring speed. The EE and the pesticide loading (PL) were 67% and 24%, respectively, in the optimized process. Scanning electron microscopy (SEM) images for empty microparticles (EMP) showed a rough and porous surface, with particle size of 1023 nm, whereas the CRF surface was found to be smoother, with a particle size of 1127 nm. The release kinetics of CAB from CRF and commercial carbofuran (CC) were evaluated in water under laboratory conditions, and the release data were fitted to the generalized Korsmeyer-Peppas model. The results indicated that the release of CAB was accomplished by a diffusion-controlled process. The CAB release from CRF was slower than that from CC, and required 6 days for 70% release, whereas CC required 5 days for 100% delivery of the pesticide. A diffusion coefficient (n) of 0.603 was determined, meaning that delivery was not governed by Fick’s laws of diffusion, but by an anomalous transport mechanism.

Cross-linked chitosan microparticles preparation by modified three fluid nozzle spray drying approach

Owing to chitosan’s incredible biocompatibility and biodegradability, bioactive molecules loaded delivery systems based on this polymer have been widely studied for improved health benefits. In the present work, quercetin and ferulic acid encapsulated core-shell structure chitosan microparticles crosslinked with tripolyphosphate and sodium alginate have been fabricated using unique three fluid nozzle (3-FN) spray drying technique. Fluorescent molecules incorporated into the microparticles aided observation of core-shell structure formation. Compatibility studies, physicochemical properties, and free radical scavenging activity of microparticles were assessed. Microscopic analysis revealed core-shell structured, spherical shaped microparticles. Thermal and non-thermal method of evaluation confirmed the crosslinking, compatibility and encapsulation of bioactive molecules with the carrier materials. The dual bioactive molecules-loaded microparticles exhibited sustained release pattern (85–88%) for 24 h and had better free radical scavenging potency. Such cross-linked core-shell structured microparticle is a promising dual delivery system for encapsulating bioactive molecules with synergistic antioxidant activity.

Synthesis of dual-responsive Janus nanovehicle via PNIPAm modified SPIONs deposition on crosslinked chitosan microparticles and decrosslinking process in the core

In this study, we developed a novel method to fabricate “Janus” nanoparticles with asymmetric surface chemistry through masking method in three steps which included the covalent deposition of modified magnetic iron oxide nanoparticles on the crosslinked substrate, modification of unprotected site with P(NIPAm-co-NMA) and then removing of the crosslinkages of the substrate. In continuum, the “mixed” nanoparticles with homogeneous surface chemistry were prepared through one-pot reaction of modified iron oxide nanoparticles with chitosan and P(NIPAm-co-NMA). In this process, the two types of polymers were simultaneously reacted with the surface of modified iron oxide nanoparticles. Both nanoparticles were reacted with Doxorubicin via imine reaction. The in vitro release was performed at two temperatures (37 and 40 °C) and pH values (5.8 and 7.4). Results of the comparison between the two formulations revealed that the Janus nanoparticles provided the more controllable release rather than the mixed ones. In addition, rat C6 glioma cells and OLN-93 cells were used to evaluate the cell cytotoxicity at 37 °C, the results demonstrated that Dox-loaded Janus nanoparticles had the desirable performance in inhibition of cancerous cells while they had less toxicity for normal cells rather in comparison to the Dox-loaded mixed ones.

Chemical cross-linking: A feasible approach to prolong doxylamine/pyridoxine release from spray-dried chitosan microspheres

Spray-dried chitosan microparticles have been widely exploited as vehicles for mucosal drug delivery. Despite their advantages as pharmaceutical formulations, one of the major challenges is achieving sustained drug release, which would diminish toxicity and dosage frequency. The aim of this study was to formulate mucoadhesive glutaraldehyde cross-linked chitosan microparticles loaded with doxylamine succinate and pyridoxine hydrochloride as potential nasal drug delivery systems with sustained release. Microparticle models were formulated via spray-drying technique, using glutaraldehyde in different concentrations (0.1–1.0 mg/mL) as a cross-linking agent for chitosan. The obtained particles were with spherical shape, smooth surface and median diameter of 4 μm. The drug entrapment efficiency was high (80.47%–94.25%), indicating a tendency to decrease at higher glutaraldehyde concentrations. FTIR data demonstrated that there were no chemical interactions between glutaraldehyde and the drugs. The in vitro studies showed that the cross-linking process substantially limited particles swelling. The cross-linked particles exhibited sustained drug release characteristics at pH 6.8 over a period of 5 h with an initial burst-effect in the first 30 min. Drug release followed Korsmeyer-Peppas kinetics. Although a decrease of the particles mucoadhesive properties was observed after modification, all cross-linked formulations demonstrated high in vitro adsorption of mucin. The proposed models of mucoadhesive microsphere with sustained drug release are a perspective ground for further development of a novel delivery system for nasal administration of doxylamine and pyridoxine.

Novel Spray Dried Glycerol 2-Phosphate Cross-Linked Chitosan Microparticulate Vaginal Delivery System-Development, Characterization and Cytotoxicity Studies.

Chitosan microparticulate delivery systems containing clotrimazole were prepared by a spray drying technique using glycerol 2-phosphate as an ion cross-linker. The impact of a cross-linking ratio on microparticle characteristics was evaluated. Drug-free and drug-loaded unmodified or ion cross-linked chitosan microparticles were examined for the in vitro cytotoxicity in VK2/E6E7 human vaginal epithelial cells. The presence of glycerol 2-phosphate influenced drug loading and encapsulation efficacy in chitosan microparticles. By increasing the cross-linking ratio, the microparticles with lower diameter, moisture content and smoother surface were observed. Mucoadhesive studies displayed that all formulations possessed mucoadhesive properties. The in vitro release profile of clotrimazole was found to alter considerably by changing the glycerol 2-phosphate/chitosan ratio. Results from cytotoxicity studies showed occurrence of apoptotic cells in the presence of chitosan and ion cross-linked chitosan microparticles, followed by a loss of membrane potential suggesting that cell death might go through the mitochondrial apoptotic pathway.

Injectable chitosan microparticles incorporating bone morphogenetic protein-7 for bone tissue regeneration

This study investigates the influence of the controlled release of bone morphogenetic protein 7 (BMP-7) from cross-linked chitosan microparticles on pre-osteoblasts (OB-6) in vitro. BMP-7 was incorporated into microparticles by encapsulation during the particle preparation and coating after particle preparation. Chitosan microparticles had an average diameter of 700 µm containing ∼10-15 ng of BMP-7. The release study profile indicates that nearly 98% of the BMP-7 coated on the microparticles was released in a period of 18 days while only 36% of the BMP-7 encapsulated in the microparticles was released in the same time period. Cell attachment study indicated that the BMP-7 coated microparticles have many cells adhered on the microparticles in comparison with microparticles without growth factors on day 10. DNA assay indicated a statistical significant increase (p < 0.05) in the amount of DNA obtained from BMP-7 encapsulated and coated microparticles in comparison with microparticles without any growth factors. A real-time RT-PCR experiment was performed to determine the expression of a few osteoblast specific genes-Dlx5, runx2, osterix, osteopontin, osteocalcin, and bone sialoprotein. The results thus suggest that chitosan microparticles obtained by coacervation method are biocompatible and helps in improving the encapsulation efficiency of BMP-7. Also BMP-7 incorporated in the microparticles is being released in a controlled fashion to support attachment, proliferation and differentiation of pre-osteoblasts, thus acting as a good scaffold for bone tissue regeneration.

Effect of cross-linking method on the activity of spray-dried chitosan microparticles with immobilized laccase

The effect of processing method on the properties of cross-linked chitosan microparticles and on the enzymatic activity of laccase immobilized in the particles has been investigated. Chitosan has been cross-linked by tri-polyphosphate (TPP) using two methods – the so called ex situ cross-linking whereby the solutions of chitosan, TPP and the enzyme have been pre-mixed and spray-dried by a standard two-fluid kinetic nozzle, and a novel in situ cross-linking method, whereby the solutions have been contacted at the tip of a three-fluid nozzle and cross-linking occurred within a drying droplet. The influence of the cross-linking method on the particle size and morphology, surface charge, and swelling ratio has been determined. The enzymatic activity of laccase toward the oxidation of a chromophore substrate (ABTS) has been systematically investigated and found to be superior in particles produced by the in situ cross-linking method.

Fabrication and Characterization of Injectable Biomaterials for Biomedical Applications.

The aim of this study is to evaluate the injectable cross-linked chitosan (CS) microparticles (MPs) to apply for biomedical applications specifically for bone regeneration. The CS MPs were fabricated by emulsification method and formed the cross-links between the amide groups in the CS and phosphate groups in the sodium tripolyphosphate (TPP) ionic cross-linking agent. The MPS were analyzed for morphology by Scanning Electron Microscope (SEM). The fabricated CS MPs were in the spherical shape with the size range of 20-100 μm. These CS MPs were analyzed for biodegradation by immersing in phosphate buffered saline (PBS, pH = 7.4) at 37°C for 30 weeks. The biodegradation of CS MPs in PBS was initiated at week 25. Mesenchymal stem cells (MSCs) were harvested from the bone marrow of mice tibia and femurs. The MSC attachment on CS MPs was tested using LIVE/DEAD cell sassy with a Fluorescence Microscope. The murine MSCs attachment onto CS MPs at day 2 was confirmed by visualizing fluorescence images. The CS MPs were also analyzed for the injectability and retainability at the site using a subcutaneous injection in a rat model. The fabricated CS MPs possess injectability, biodegradability and biocompatibility. Therefore, these CS MPs have a great potential to apply for various biomedical applications including bone regeneration by injection.

Ionically cross-linked chitosan/tripolyphosphate microparticles for the controlled delivery of pyrimethamine

Chitosan ionically cross-linked with tripolyphosphate at regulated temperatures (25°C, 40°C, and 50°C) and varying cross-linking times (30 min, 2 h and 4 h respectively) was used to form microparticles employed in the encapsulation of pyrimethamine, an antiprotozoal drug. The yields, equilibrium moisture contents, percentage concentration, swelling characteristics, entrapment efficiency, release properties, infrared spectroscopy, and differential scanning calorimetry of the formulated microparticles were evaluated. The yield of microparticles produced ranged from 0.3515 to 0.7749 g per 100 ml of cross linking solution. The products possessed relatively little amounts of moisture (0.22 − 3.04 % w/v). The entrapment efficiencies ranged from 25.55 to 99 % with the product formed at ambient temperature and cross linking time of 30 min possessing the highest efficiency. The swelling kinetics on the microcapsules revealed that all the products swelled in the various pH media following mainly anomalous sorption mechanism with a few diffusion controlled mechanism. The greatest swellings however occurred at the swelling medium of pH 1 while the least swelling occurred at pH 7. Spectral and differential scanning calorimetric properties of the chitosan used in the study were consistent with those of standard chitosan. The infrared spectroscopy and differential scanning calorimetry of the products confirmed that encapsulation actually occurred with the spectral characters of the products differing from those of the parent constituents (chitosan, tripolyphosphate and pyrimethamine). Based on these factors, tripolyphosphate cross-linked chitosan microparticles present a suitable matrix for the controlled release of pyrimethamine.

Composite magnetic chitosan microspheres: In situ preparation and characterization

A method for preparing magnetic crosslinked chitosan microparticles was developed. The chitosan (CS) encapsulated magnetic particles were produced in alkaline conditions by in situ oxidation of the ferrous ions initially dispersed uniformly within the polysaccharide matrix. The polymer was then crosslinked using glutaraldehyde (GLA). The products were characterized regarding their size distribution and surface charge (by laser diffraction analysis, ζ-potential measurement, and conductometric titration), morphology (TEM), and magnetic properties (magnetic susceptibility analysis). The fact that the particles contain both magnetic iron oxide and chitosan was confirmed by FTIR and thermogravimetric analysis. The synthesis parameters were optimized for obtaining stable magnetic microparticles bearing surface amino groups that can subsequently be used for heavy metal ion complexation. The composite particles obtained by the optimum procedure had an average diameter of 40 μm and a saturation magnetization of 24 emu/g, corresponding to about 47% magnetic iron oxide content.

Biosorption of Cu(II), Zn(II), Ni(II) and Pb(II) ions by cross-linked metal-imprinted chitosans with epichlorohydrin

Cross-linked metal-imprinted chitosan microparticles were prepared from chitosan, using four metals (Cu(II), Zn(II), Ni(II), and Pb(II)) as templates, and epichlorohydrin as the cross-linker. The microparticles were characterized by Fourier transform infrared spectroscopy, solid state 13C nuclear magnetic resonance spectroscopy, and energy-dispersive X-ray spectroscopy. They were used for comparative biosorption of Cu(II), Zn(II), Ni(II) and Pb(II) ions in an aqueous solution. The results showed that the sorption capacities of Cu(II), Zn(II), Ni(II), and Pb(II) on the templated microparticles increased from 25 to 74%, 13 to 46%, 41 to 57%, and 12 to 43%, respectively, as compared to the microparticles without metal ion templates. The dynamic study showed that the sorption process followed the second-order kinetic equation. Three sorption models, Langmuir, Freundlich, and Dubinin–Radushkevich, were applied to the equilibrium isotherm data. The result showed that the Langmuir isotherm equation best fitted for monolayer sorption processes. Furthermore, the microparticles can be regenerated and reused for the metal removal.

Chitosan Microparticles Prepared by the Simple Emulsification-Diffusion Method

Biodegradable chitosan microparticles were prepared by a water-in-oil emulsion solvent diffusion method without any surfactants. Aqueous chitosan solution and ethyl acetate were used as the water and oil phases, respectively. The chitosan microparticles; with spherical, deflated, and hollow shapes, could be fabricated from different chitosan concentrations. Chitosan microparticle matrices were sponge-like. The particle sizes increased with the chitosan concentration and molecular weight. Cross-linked chitosan microparticles obtained from post-glutaraldehyde treatment showed rougher surfaces and denser matrices. The particle shapes did not change after cross-linking. The degrees of cross-linking of chitosan microparticles were in order of hollow > deflated > spherical shapes.

Evaluation of ultrasonic atomization as a new approach to prepare ionically cross-linked chitosan microparticles

Ultrasonic atomization was evaluated as a new approach for the preparation of ionically cross-linked controlled-release chitosan microparticles loaded with theophylline as the model drug, using tripolyphosphate (TPP) as counter-ion. It was possible to nebulize both 2% and 3% (w/v) chitosan solutions as a function of their viscosity, usually not processed by employing the conventional nebulizer. The results of the chitosan molecular characterization using the SEC-MALS analysis revealed that ultrasonic atomization caused a certain depolymerization, probably due to the main chain scission of the 1,4-glycosidic bond; however, Fourier transform-infrared spectroscopy revealed the absence of other chemical modifications. The ultrasonic atomization allowed preparation of TPP cross-linked chitosan microparticles mostly ranging between 50 and 200 mum. As regards manufacturing parameters, the linking time and washing medium were found to affect the properties of the microparticles, while the stirring rate of the TPP solution did not show any influence. The evaluation of the formulation variables revealed that chitosan concentration strongly affected both the feasibility of the ultrasonic atomization and the drug release. All the microparticles showed an encapsulation efficiency of > 50 % and, after an initial burst effect, a controlled release of drug for 48 h. In conclusion, the ultrasonic atomization could be proposed as a robust and innovative single-step procedure with scale-up potential to successfully prepare ionically cross-linked chitosan microparticles.

Effect of chitosan crosslinking on bitterness of artemether using response surface methodology

This work examines the influence of various process parameters on artemether entrapped in crosslinked chitosan microparticles for masking bitterness. A central composite design was used to optimize the experimental conditions for bitterness masking. Critical parameters such as the amounts of artemether, chitosan and crosslinking agent have been studied to evaluate how they affect responses such as incorporation efficiency, particle size and drug release at pH 6.8. The desirability function approach has been used to find the best compromise between the experimental results. The optimized microparticles were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. Bitterness score was evaluated by human gustatory sensation test. Multiple linear regression analysis revealed that the crosslinking of chitosan significantly affects incorporation efficiency, particle size and drug release at pH 6.8. The bitterness score of microparticles was decreased to 0, compared with 3+ for pure artemether. The proposed method completed masked the bitter taste of artemether.

Preparation and characterization of genipin-cross-linked chitosan microparticles by water-in-oil emulsion solvent diffusion method

Chitosan (CS) microparticles with and without cross-linking were prepared by a water-in-oil emulsion solvent diffusion method without any surfactants. Aqueous CS solution and ethyl ace- tate were used as water and oil phases, respectively. Genipin was used as a cross-linker. Influ- ences of genipin ratios and cross-linking times on CS microparticle characteristics were investigated. Non-cross-linked and cross-linked CS microparticles were spherical in shape and rough in surface. Microparticle matrices showed porous structures. Surface roughness, mean par- ticle sizes and bulk density of CS microparticles increased and their dissolutions in acetic acid solution decreased when genipin ratio and cross- linking time increased.

Biosorption of azo dyes from aqueous solution by glutaraldehyde-crosslinked chitosans

The crosslinked chitosan microparticles prepared through homogeneous coupling reaction and microparticle formation using a sodium hydroxide solution showed the largest adsorbed amounts toward the RB5 and 3R dyes than those from the three other methods through heterogeneous coupling reaction and microparticle formation using sodium hydroxide or sodium tripolyphosphate solutions. The dynamical experimental study showed that the dye adsorption accurately followed the second-order adsorption process. The experimental isotherm data were analyzed using three isotherm models, namely, Langmuir, Freundlich, and Dubinin-Radushkevich. The results revealed that the adsorption behavior of the RB5 and 3R dyes on the microparticles fitted well with the Langmuir model. In addition, the mean adsorption energy ( E) from the Dubinin-Radushkevich isotherm and the activation energy ( E a) from Arrhenius equation indicated that the adsorption process might be the dual nature of the process, physisorption and chemisorption, and was predominant on the chemisorption process The competitive adsorption showed that the adsorption of the 3R dye on the microparticles in the mixture solution was much more affected by the existence of the RB5 dye than the other way around. Furthermore, it was also found that the crosslinked chitosan microparticles can be regenerated and reused for dye adsorption.

Influence of Chitosan Crosslinking on Bitterness of Mefloquine Hydrochloride Microparticles Using Central Composite Design

The present work examines the influence of various process and product parameters on mefloquine hydrochloride (MFL) entrapped in crosslinked chitosan microparticles for masking the bitterness. A central composite design (CCD) was employed to investigate the effect of three process and product variables, namely amount of MFL, chitosan and sodium hydroxide (crosslinking agent) on the incorporation efficiency, particle size, drug release at pH 6.8 and bitterness score. The microparticles were prepared by ionotropic gelation method, with a hardening time of 60 min. The optimum condition for process and product variables was evaluated using desirability function. The model is further cross validated for bias. The optimized microparticles were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. Bitterness score was evaluated by human gustatory sensation test. Multiple linear regression analysis revealed that the crosslinking of chitosan significantly affects incorporation efficiency, particle size, drug release and bitterness score. The bitterness score was decreased to zero compared to 3+ of pure MFL. It can be inferred that the proposed methodology can be used to prepare MFL microparticles for bitter taste masking. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:690–703, 2009

Evaluation of cross-linked chitosan microparticles containing acyclovir obtained by spray-drying

The aim of this study was to obtain microparticles containing acyclovir (ACV) and chitosan cross-linked with tripolyphosphate using the spray-drying technique. The resultant system was evaluated through loading efficiency, differential scanning calorimetry (DSC), thermogravimetric analysis (TG), X-ray powder diffraction (XRPD), scanning electron microscopy (SEM), in vitro release and stability studies. The results obtained indicated that the polymer/ACV ratio influenced the final properties of the microparticles, with higher ratios giving the best encapsulation efficiency, dissolution profiles and stability. The DSC and XRPD analyses indicated that the ACV was transformed into amorphous form during the spray-drying process.