Preparation and pharmacodynamics of low-molecular-weight chitosan nanoparticles containing insulin

Low-molecular-weight chitosan (LMWC) was obtained by enzymatic degradation and ultrafiltration separation. LMWC nanoparticles with LMWC having 20 kDa weight average molecular weight (M_w) were then prepared by solvent evaporation method. The resultant nanoparticles were spherical with a narrow particle size distribution. LMWC nanoparticles loaded with insulin as a model drug were prepared. The average entrapment efficiency of insulin could reach up to 95.54%. The in vitro drug release profiles from the nanoparticles showed an initial burst of release in the first 2 h, followed by zero order release kinetics. In vivo pharmacodynamics of chitosan nanoparticles containing insulin showed that the nanoparticles showed some hypoglycemic activity. Compared with an insulin solution, a relative bioavailability of 0.737 was observed for four times the dosage of insulin in the chitosan nanoparticles after pulmonary administration.     

Preparation,Statistical Optimization,and <Emphasis Type="Italic">In vitro</Emphasis> Characterization of Insulin Nanoparticles Composed of Quaternized Aromatic Derivatives of Chitosan

The aim of this study was the preparation, optimization, and in vitro characterization of insulin nanoparticles composed of methylated N-(4-N,N-dimethylaminobenzyl), methylated N-(4-pyridinyl), and methylated N-(benzyl) chitosan. Three types of derivatives were synthesized by the Schiff base reaction followed by quaternization. Nanoparticles were prepared by the polyelectrolyte complexation method. Experimental design D-optimal response surface methodology was used for the optimization of the nanoparticles. Independent variables were pH of polymer solution, concentration ratio of polymer/insulin, and also polymer type. Dependent variables include size, zeta potential, polydispersity index (PdI), and entrapment efficiency (EE%). Optimized nanoparticles were studied morphologically by transmission electron microscopy (TEM), and in vitro release of insulin from nanoparticles were determined under phosphate buffer (pH = 6.8) condition. Although a quadratic model has been chosen to fit the responses for size, PdI, and EE%, the zeta potential of the particles has been best fitted to 2-FI model. The optimized nanoparticles were characterized. The size of the particles were found to be 346, 318, and 289 nm; zeta potentials were 28.5, 27.7, and 22.2 mV; PdI of particles were 0.305, 0.333, and 0.437; and calculated EE% were 70.3%, 84.5%, and 69.2%, for methylated (aminobenzyl), methylated (pyridinyl), and methylated (benzyl) chitosan nanoparticles, respectively. TEM images show separated and non-aggregated nanoparticles with sub-spherical shapes and smooth surfaces. An in vitro release study of the prepared nanoparticles showed that the cumulative percentage of insulin released from the nanoparticles were 47.1%, 38%, and 68.7% for (aminobenzyl), (pyridinyl), and (benzyl) chitosan, respectively, within 300 min.     

Preparation of low molecular weight N-maleated chitosan-graft-PAMAM copolymer for enhanced DNA complexation

Low molecular weight N-maleated chitosan-graft-PAMAM (polyamidoamine) copolymer was prepared through N-maleated chitosan (NMC) by Michael type addition reaction to enhance its solubility in water as well as its cationic character for enhancement of DNA complexation. FTIR, (1)H NMR, XRD and GPC were used to characterize the graft copolymers. The copolymer showed better DNA complexation ability at low N/P ratio than that of chitosan due to increased surface charge density by the incorporation of PAMAM molecule on to chitosan backbone. The copolymer can effectively protect the DNA toward anionic surfactant. In vitro release study showed efficient DNA release occurred at physiological pH (pH 7.4). In vitro cell cytotoxicity test indicated toward less cytotoxicity of NMC-graft-PAMAM copolymers compared to that of 25kDa PEI. Thus, the synthesized NMC-graft-PAMAM copolymers have great potential of finding application in drug and gene delivery.     

Development of pH-sensitive Insulin Nanoparticles using Eudragit L100-55 and Chitosan with Different Molecular Weights

Insulin is a polypeptide hormone and usually administered for treatment of diabetic patients subcutaneously. The aim of this study was to investigate the efficiency of enteric nanoparticles for oral delivery of insulin. Nanoparticles were formed by complex coacervation method using chitosan of various molecular weights. Nanoparticles were characterized by drug loading efficiency determination, particle size analysis, Scanning Electron Microscopy (SEM), Zeta potential and CD spectroscopy (Circular Dichrosim). The in vitro release studies were performed at pH 1.2 and 7.4. The drug loaded nanoparticles showed 3.38% of entrapment, loading efficiency of 30.56% and mean particle size of 199 nm. SEM studies showed that the nanoparticles are non-spherical. Zeta potential increased with increasing molecular weight of chitosan. The CD spectroscopy profiles indicated that the nano-encapsulation process did not significantly disrupt the internal structure of insulin; additionally, pH-sensitivity of nanoparticles was preserved and the insulin release was pH-dependent. These results suggest that the complex coacervation process using chitosan and Eudragit L100-55 polymers may provide a useful approach for entrapment of hydrophilic polypeptides without affecting their conformation.     

A Novel Method for Preparing Surface-Modified Fluocinolone Acetonide Loaded PLGA Nanoparticles for Ocular Use: <Emphasis Type="Italic">In Vitro</Emphasis> and <Emphasis Type="Italic">In Vivo</Emphasis> Evaluations

Our objective was to prepare nanoparticulate system using a simple yet attractive innovated method as an ophthalmic delivery system for fluocinolone acetonide to improve its ocular bioavailability. Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared by adopting thin film hydration method using PLGA/poloxamer 407 in weight ratios of 1:5 and 1:10. PLGA was used in 75/25 and 50/50 copolymer molar ratio of DL-lactide/glycolide. Results revealed that using PLGA with lower glycolic acid monomer ratio exhibited high particle size (PS), zeta potential (ZP) and drug encapsulation efficiency (EE) values with slow drug release pattern. Also, doubling the drug concentration during nanoparticles preparation ameliorated its EE to reach almost 100%. Furthermore, studies for separating the un-entrapped drug in nanoparticles using centrifugation method at 20,000 rpm for 30 min showed that the separated clear supernatant contained nanoparticles encapsulating an important drug amount. Therefore, separation of un-entrapped drug was carried out by filtrating the preparation using 20–25 μm pore size filter paper to avoid drug loss. Aiming to increase the PLGA nanoparticles mucoadhesion ability, surface modification of selected formulation was done using different amount of stearylamine and chitosan HCl. Nanoparticles coated with 0.1% w/v chitosan HCl attained most suitable results of PS, ZP and EE values as well as high drug release properties. Transmission electron microphotographs illustrated the deposition of chitosan molecules on the nanoparticles surfaces. Pharmacokinetic studies on Albino rabbit’s eyes using HPLC indicated that the prepared novel chitosan-coated PLGA nanoparticles subjected to separation by filtration showed rapid and extended drug delivery to the eye.     

Synthesis and property of chitosan graft copolymer by RAFT polymerization with tosylic acid-chitosan complex

pH- and thermo-sensitive (1→4)-2-amino-2-deoxy-β-d-glucan (i.e. chitosan) graft copolymer was prepared by reversible addition fragmentation chain transfer polymerizations of N-isopropylacrylamide with 4-methylbenzenesulfonic acid (i.e. tosylic acid)-chitosan complex. The polymerization was controlled well, and the amino group of chitosan could be deprotected easily and mildly with 15% Tris solution. The model aldehyde vanillin was conjugated with amino group of chitosan-g-PNIPAM via Schiff base bond (Loading efficiency, LE=77.6 mg/g), and the drug release could be controlled with temperature and pH. This property may promote the chitosan graft copolymer to be used in the field of "smart" drug delivery.     

Poly(L-lysine)-graft-chitosan copolymers: synthesis, characterization, and gene transfection effect

Polypeptide/polysaccharide graft copolymers poly(L-lysine)-graft-chitosan (PLL-g-Chi) were prepared by ring-opening polymerization (ROP) of epsilon-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of 6-O-triphenylmethyl chitosan. The PLL-g-Chi copolymers were thoroughly characterized by 1H NMR, 13C NMR, Fourier transform infrared (FT-IR), and gel permeation chromatography (GPC). The number-average degree of polymerization of PLL grafted onto the chitosan backbone could be adjusted by controlling the feed ratio of NCA to 6-O-triphenylmethyl chitosan. The particle size of the complexes formed from the copolymer and calf thymus DNA was measured by dynamic light scattering (DLS). It was found in the range of 120 approximately 340 nm. The gel retardation electrophoresis showed that the PLL-g-Chi copolymers possessed better plasmid DNA-binding ability than chitosan. The gene transfection effect in HEK 293T cells of the copolymers was evaluated, and the results showed that the gene transfection ability of the copolymer was better than that of chitosan and was dependent on the PLL grafting ratio. The PLL-g-Chi copolymers could be used as effective gene delivery vectors.     

Preparation, characterization, and self-assembled properties of biodegradable chitosan-poly(L-lactide) hybrid amphiphiles

Biodegradable chitosan-graft-poly(L-lactide) (CS-g-PLLA) hybrid amphiphiles were prepared through direct grafting of a PLLA precursor to the backbone of CS. The average number of PLLA grafts per CS could be adjusted by the feed ratio of PLLA to CS, and it varied from 1.3 to 16.8. Moreover, both the crystallization rate and degree of crystallization of PLLA grafts with these graft copolymers could be adjusted by the chain length of PLLA and the number of PLLA grafts per CS, respectively. Meanwhile, CS-g-PLLA exhibited good solubility in some nonpolar and polar organic solvents compared with the original CS. Furthermore, self-assembled nanoparticles could be generated by direct injection of these graft copolymer solutions into distilled water, and their critical aggregation concentration decreased with increasing number of PLLA grafts per CS. The average size of the nanoparticles (25-103 nm) could be adjusted through the graft copolymer composition and the graft copolymer concentration, which was very different from the observations in ordinary PLLA-b-poly(ethylene oxide) amphiphiles. Significantly, this will provide a convenient method not only to combine the bioactive functions of CS with the good mechanical properties of biodegradable polymers, but also to generate nanoparticles with adjustable sizes for targeted drug release.     

Preparation, characterization and transfection efficacy of chitosan nanoparticles containing the intestinal trefoil factor gene

Intestinal trefoil factor (ITF) is a novel polypeptide with potential pharmacological value for the prevention and healing of tissue injury; however, poor production capacity limits its clinical application. Chitosan, as a non-viral vehicle, has been successfully used in gene delivery for its intrinsic characteristics. In this context, we prepared chitosan nanoparticles enwrapping ITF cDNA and investigated its size, zeta potential, stability, release profiles, loading efficiency and loading capacity. Gene transfer capability was assessed in HEK293 cells. The data revealed that the chitosan/DNA nanoparticles were successfully prepared with sizes less than 500 nm and positive zeta potentials. The nanoparticles could protect DNA from nuclease degradation, and release profiles of DNA were dependent on N/P ratios. In addition, transfection efficiency of chitosan/DNA nanoparticles was equivalent to Lipofectamine (TM). Collectively, the results suggest that chitosan/DNA nanoparticles could be a promising method for ITF gene therapy.     

Comparative Studies on Chitosan and Polylactic-co-glycolic Acid Incorporated Nanoparticles of Low Molecular Weight Heparin

This study was performed to test the feasibility of chitosan and polylactic-co-glycolic acid (PLGA) incorporated nanoparticles as sustained-release carriers for the delivery of negatively charged low molecular weight heparin (LMWH). Fourier transform infrared (FTIR) spectrometry was used to evaluate the interactions between chitosan and LMWH. The shifts, intensity, and broadening of the characteristic peaks for the functional groups in the FTIR spectra indicated that strong interactions occur between the positively charged chitosans and the negatively charged LMWHs. Three types of LMWH nanoparticles (NP-1, NP-2, and NP-3) were prepared using chitosan with or without PLGA: NP-1 nanoparticles were formed by polyelectrolyte complexation after single mixing, NP-2 nanoparticles were prepared by polyelectrolyte complexation after single emulsion–diffusion–evaporation, and NP-3 nanoparticles were optimized by double emulsion–diffusion–evaporation. NP-3 nanoparticles of LMWH prepared by the emulsion–diffusion–evaporation method showed significant differences in particle morphology, size, zeta potential, and drug release profile compared to NP-1 nanoparticles formed by polyelectrolyte complexation. Another ionic complex of LMWH with chitosan-incorporated PLGA nanoparticles (NP-2) showed lower drug entrapment efficiency than that of NP-1 and NP-3. The drug release rate of NP-3 was slower than the release rates of NP-1 and NP-2, although particle morphology of NP-3 was similar to that of NP-2. Cell viability was not adversely affected when cells were treated with all three types of nanoparticles. The data presented in this study demonstrate that nanoparticles formulated with chitosan–PLGA could be a safe sustained-release carrier for the delivery of LMWH.Key words: chitosan, low molecular weight heparin, nanoparticles, PLGA     

Preparation and characterization of the graft copolymer of chitosan with poly[rosin-(2-acryloyloxy)ethyl ester]

Graft copolymerization of rosin-(2-acryloyloxy)ethyl ester (RAEE) onto chitosan (Cts) was carried out under microwave irradiation using potassium persulfate as an initiator. The structures, morphology, and thermal properties of the Cts graft copolymer (Cts-g-PRAEE) were characterized by means of FT-IR, XRD, SEM, and TG. Also, Cts and Cts-g-PRAEE copolymer were used as carriers of fenoprofen calcium (FC), and their controlled release behavior in artificial intestinal juice were studied. The results show that the rate of release of fenoprofen calcium from the carrier of Cts-g-PRAEE copolymer becomes very slower than that of Cts in artificial intestinal juice.     

Preparation of chitosan derivative with polyethylene glycol side chains for porous structure without specific processing technique

The graft copolymer, chitosan-g-polyethylene glycol (PEG), was prepared through graft polymerization of PEG chains to chitosan due to the esterification reaction between PEG and 6-O-succinate-N-phthaloyl-chitosan (PHCSSA). The graft copolymer with porous structure was observed from scanning electron micrographs. It is a potential method to combine chitosan with the hydrophilic synthetic polymers. The graft reaction was carried out in homogeneous system and yielded copolymers with high grafting content. FTIR, NMR, XRD, DSC, spectrofluorophotometer and SEM were detected to characterize the copolymer.     

Preparation of aspirin and probucol in combination loaded chitosan nanoparticles and in vitro release study

We design and develop chitosan nanoparticles which load two different drugs simultaneously. Aspirin (acetylsalicylic acid, ASA), a hydrophilic drug and probucol (PRO), a hydrophobic drug, are chosen as typical drugs, which are widely used to treat restenosis. The drug loaded chitosan nanoparticles are prepared by gelation of chitosan with tripolyphosphate (TPP) by ionic cross-linking. The physicochemical properties of nanoparticles are investigated by FTIR, transmission electron microscope (TEM), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). The images show that these particles are spherical in shape with ASA being in the amorphous phase, while PRO is crystalline. The properties of chitosan nanoparticles such as encapsulation capacity and controlled release behaviors of ASA and PRO are evaluated. Experimental results indicate that the loading capacity (LC), encapsulation efficiency (EE) and ASA and PRO release behaviors are affected by several factors including pH, concentration of TPP, chitosan molecular weight (MW) and ASA initial concentration as well as PRO. In vitro release shows that the nanoparticles provide a continuous release. Entrapped ASA is released for more than 24 h and PRO lasts longer for 120 h.     

The immobilization of enzymes and cells of Bacillus stearothermophilus onto poly(maleic anhydride/styrene)-Co-polyethylene and poly(maleic anhydride/vinyl acetate)-Co-polyethylene

The graft copolymer, poly(maleic anhydride/styrene)-co-polyethylene was prepared. The copolymer immobilized bovine serum albumin (BSA), but the amount coupled appeared to be effected by the amount of styrene in the graft copolymer, temperature, and pH of the coupling medium. Competition existed between hydrolysis of the grafted anhydride groups and the protein. A graft copolymer with 66% add-on immobilized 4.5 mg/glucose oxidase/g copolymer, 4.6 mg alkaline phosphates/g copolymer and 0.2 mg cell of Bacillus stearothermophilus/g copolymer. A number of copolymers containing poly(maleic anhydride/vinyl acetate)-co-polyethylene were prepared to cover a range of grafting levels. These immobilized larger quantities of BSA, alkaline phosphatase, and cells of B. stearothermophilus than did the styrene graft copolymer. The copolymer was also hydrolyzed to release the hydroxyl group from the poly(vinyl acetate) component of the grafted chains. Using p-benzoquinone as the "activating agent," the copolymer coupled to BSA and to acid phosphatase. Using p-toluene-sulfonyl chloride, the copolymer was very effective in immobilizing trypsin.     

Preparation, characterization, and in vitro drug release behavior of biodegradable chitosan-graft-poly(1, 4-dioxan-2-one) copolymer

A novel copolymer of chitosan-g-poly(p-dioxanone) (CGP) was synthesized in bulk by ring-opening polymerization of p-dioxanone (PDO) initiated by the hydroxyl group or amino group of chitosan using SnOct₂ as catalyst. The chemical structure was determined by ¹H NMR. It was found that the feed ratio of chitosan to PDO had a great effect on the degree of polymerization (DP) and the substitution (DS) of PDO. The thermal stability and crystallization behavior of graft copolymer CGP were closely related to the values of DP and DS. When the resulting copolymer was used as Ibuprofen carrier, the release rate of Ibuprofen decreased compared with that of pure chitosan carrier. The drug release behavior was also influenced by the structure of graft copolymers.     

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.     

Development of Acid-Resistant Alginate/Trimethyl Chitosan Nanoparticles Containing Cationic β-Cyclodextrin Polymers for Insulin Oral Delivery

In this study, the use of trimethylchitosan (TMC), by higher solubility in comparison with chitosan, in alginate/chitosan nanoparticles containing cationic β-cyclodextrin polymers (CPβCDs) has been studied, with the aim of increasing insulin uptake by nanoparticles. Firstly, TMCs were synthesized by iodomethane, and CPβCDs were synthesized within a one-step polycondensation reaction using choline chloride (CC) and epichlorohydrine (EP). Insulin–CβCDPs complex was prepared by mixing 1:1 portion of insulin and CPβCDs solutions. Then, nanoparticles prepared in a three-step procedure based on the iono-tropic pregelation method. Nanoparticles screened using experimental design and Placket Burman methodology to obtain minimum size and polydispercity index (pdI) and the highest entrapment efficiency (EE). CPβCDs and TMC solution concentration and pH and alginate and calcium chloride solution concentrations are found as the significant parameters on size, PdI, and EE. The nanoparticles with proper physicochemical properties were obtained; the size, PdI, and EE% of optimized nanoparticles were reported as 150.82 ± 21 nm, 0.362 ± 0.036, and 93.2% ± 4.1, respectively. The cumulative insulin release in intestinal condition achieved was 50.2% during 6 h. By SEM imaging, separate, spherical, and nonaggregated nanoparticles were found. In the cytotoxicity studies on Caco-2 cell culture, no significant cytotoxicity was observed in 5 h of incubation, but after 24 h of incubation, viability was decreased to 50% in 0.5 mμ of TMC concentration. Permeability studies across Caco-2 cells had been carried out, and permeability achieved in 240 min was 8.41 ± 0.39%, which shows noticeable increase in comparison with chitosan nanoparticles. Thus, according to the results, the optimized nanoparticles can be used as a new insulin oral delivery system.KEY WORDS: alginate, cationic β-cyclodextrin, insulin nanoparticle, oral delivery, trimethyl chitosan     

Polyelectrolyte nanoparticles based on water-soluble chitosan-poly(l-aspartic acid)-polyethylene glycol for controlled protein release

Water-soluble chitosan (WSC)-poly(l-aspartic acid) (PASP)-polyethylene glycol (PEG) nanoparticles (CPP nanoparticles) were prepared spontaneously under quite mild conditions by polyelectrolyte complexation. These nanoparticles were well dispersed and stable in aqueous solution, and their physicochemical properties were characterized by turbidity, FTIR spectroscopy, dynamic light scattering (DLS), transmission electron microscope (TEM), and zeta potential. PEG was chosen to modify WSC-PASP nanoparticles to make a protein-protective agent. Investigation on the encapsulation efficiency and loading capacity of the bovine serum albumin (BSA)-loaded CPP nanoparticles was also conducted. Encapsulation efficiency was obviously decreased with the increase of initial BSA concentration. Furthermore, its in vitro release characteristics were evaluated at pH 1.2, 2.5, and 7.4. In vitro release showed that these nanoparticles provided an initial burst release, followed by a slowly sustained release for more than 24 h. The BSA released from CPP nanoparticles showed no significant conformational change compared with native BSA, which is superior to the BSA released from nanoparticles without PEG. A cell viability study suggested that the nanoparticles had good biocompatibility. This nanoparticle system was considered promising as an advanced drug delivery system for the peptide and protein drug delivery.     

Development of a chitosan-based nanoparticle formulation for delivery of a hydrophilic hexapeptide, dalargin

Nanoparticle based delivery systems can offer opportunities for targeting, controlled release, and enhanced stability of their drug, protein, or gene therapy payload. This study investigated the use of chitosan in combination with the ionic additives sulfobutyl-ether-7-beta-cyclodextrin (SB-CD) or SB-CD/dextran sulfate (SB-CD/DS) mixture in comparison with chitosan: DS in the formulation of nanoparticles incorporating the hexapeptide dalargin. The physical characteristics (particle size, zeta potential), entrapment and loading efficiency, and release of dalargin were quantified. It was demonstrated that anionic cyclodextrin, SB-CD, can be used in complex coacervation with chitosan, with and without the presence of DS, to form nanoparticles. The presence of SB-CD or DS in the nanoparticle formulation and the weight ratio of chitosan to anionic additive(s) influenced the physical properties of the nanoparticles and their ability to carry dalargin. In addition, the particle size of nanoparticles was also affected by the molecular weight of chitosan and DS. The use of either DS or SB-CD/DS mixture produced chitosan nanoparticles with small particle size, high dalargin entrapment efficiency, enhanced peptide stability, and sustained release characteristics.     

Preparation and antibacterial activity of chitosan nanoparticles

Chitosan nanoparticles, such as those prepared in this study, may exhibit potential antibacterial activity as their unique character. The purpose of this study was to evaluate the in vitro antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against various microorganisms. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. Copper ions were adsorbed onto the chitosan nanoparticles mainly by ion-exchange resins and surface chelation to form copper-loaded nanoparticles. The physicochemical properties of the nanoparticles were determined by size and zeta potential analysis, atomic force microscopy (AFM), FTIR analysis, and XRD pattern. The antibacterial activity of chitosan nanoparticles and copper-loaded nanoparticles against E. coli, S. choleraesuis, S. typhimurium, and S. aureus was evaluated by calculation of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Results show that chitosan nanoparticles and copper-loaded nanoparticles could inhibit the growth of various bacteria tested. Their MIC values were less than 0.25 microg/mL, and the MBC values of nanoparticles reached 1 microg/mL. AFM revealed that the exposure of S. choleraesuis to the chitosan nanoparticles led to the disruption of cell membranes and the leakage of cytoplasm.