Adding chitosan nanoparticles of green tea extract in diluent and thawing temperatures ameliorate the post-thawed quality of Boer buck semen

Objective:To improve the quality of post-thawing Boer buck semen for artificial insemination by adding green tea extract chitosan nanoparticles to skimmed egg yolk diluent,and the proper thawing temperature.Methods:The ejaculate of Boer buck was added to skimmed egg yolk diluent without(the control group)and with adding 1μg of chitosan nanoparticles of green tea extract per mL of diluent(the treatment group).Then,the diluted semen was filled in French mini straws containing 60×106 live sperm per straw,frozen in a standard protocol,and stored as frozen semen at−196℃for a week.Six replicates from each group were diluted for 30 s at 37℃or 39℃sterile water to evaluate the semen quality.Results:Post-thawing(at 37℃or 39℃)of live sperm,progressive motility,and plasma membrane integrity were lower compared to those of the pre-freezing stage(P<0.05).Thawing at 37℃resulted in no significant difference in live sperm,progressive motility,and plasma membrane between the control group and the treatment group(P>0.05).The live sperm,progressive motility,and plasma membrane of the treatment group in the pre-freezing stage,and post-thawed at 39℃were higher compared to those of the control group(P<0.05).There was no significant difference in malondialdehyde(MDA)concentration,DNA fragmentation,and catalase concentration of thawing at 37℃compared to those of 39℃in the same group.The MDA concentration and DNA fragmentation in thawing at 37℃and 39℃of the treatment group were significantly lower than those of the control group(P<0.05).However,the catalase concentration in thawing at 37℃and 39℃of the treatment group was not significantly different than the control group(P>0.05).Conclusions:Higher quality post-thawing Boer buck semen is achieved by adding 1μg/mL of chitosan nanoparticles of green tea extract to the skimmed egg yolk diluent and thawing at 39℃.

Preparation of Magnetic Chitosan Nanoparticles and Immobilization of Laccase

The magnetic chitosan nanoparticles were prepared by reversed-phase suspension method using Span-80 as an emulsifier, glutaraldehyde as cross-linking reagent. And the nanoparticles were characterized by TEM, FT-IR and hysteresis loop. The results show that the nanoparticles are spherical and almost superparamagnetic. The laccase was immobilized on nanoparticles by adsorption and subsequently by cross-linking with glutaraldehyde. The immobilization conditions and charac-terizations of the immobilized laccase were investigated. The optimal immobilization conditions were as follows： 10 mL of phosphate buffer （0.1 M, pH 7.0） containing 50 mg of magnetic chitosan nanoparticles, 1.0 mg·mL-1 of laccase and 1% （v/v） glutaraldehyde, immobilization temperature of 4 ℃ and immobilization time of 4 h. The immobilized laccase exhibited an appreciable catalytic capability （480 units·g-1 support） and had good storage stability and operation stability. The Km of immobilized and free laccase for ABTS were 140.6 and 31.1 μM in phosphate buffer （0.1 M, pH 3.0） at 37 ℃, respectively. The immobilized laccase is a good candidate for the research and development of biosensors based on laccase catalysis.

New modular platform based on multi-adjuvanted amphiphilic chitosan nanoparticles for efficient lipopeptide vaccine delivery against group A streptococcus

An effective vaccine against group A streptococcus(GAS)is highly desirable for definitive control of GAS infections.In the present study,two variants of amphiphilic chitosan nanoparticles-based GAS vaccines were developed.The vaccines were primarily composed of encapsulated KLH protein(a source of T helper cell epitopes)and lipidated M-protein derived B cell peptide epitope(lipoJ14)within the amphiphilic structure of nanoparticles.The only difference between themwas one of the nanoparticles vaccines received additional surface coating with poly(I:C).The formulated vaccines exhibited nanosized particles within the range of 220–240 nm.Cellular uptake study showed that nanoparticles vaccine without additional poly(I:C)coating has greater uptake by dendritic cells and macrophages compared to nanoparticles vaccine that was functionalized with poly(I:C).Both vaccines were found to be safe in mice and showed negligible cytotoxicity against HEK293 cells.Upon immunization in mice,both nanoparticle vaccines produced high antigen-specific antibodies titres that were regulated by a balanced Th1 and Th2 response compared to physical mixture.These antibodies elicited high opsonic activity against the tested GAS strains.Overall,our data demonstrated that amphiphilic chitosan nanoparticles platform induced a potent immune response even without additional inclusion of poly(I:C).

Low Releasing Mitomycin C Molecule Encapsulated with Chitosan Nanoparticles for Intravesical Installation

The aim of this investigation is preparation of Mitomycin-C encapsulated with chitosan nanoparticles synthesis using ionic gelation technique for intravesical controlled drug delivery systems. This study was conducted in vitro. Cumulative amount of drug released from the nanoparticles was calculated. Mitomycin-C release studies were examined for different pH values. During the drug loading and release studies, initial amount of drug was changed (i.e., 0.5, 1.25 and 2.5 mg) to get different release profiles and the release studies were repeated (n = 6). The loading efficiencies of Mitomycin-C with three different initial concentrations 0.5mg/ml, 1.25 mg/ml and 2.5 mg/ml into chitosan nanoparticles were 54.5%, 47.1% and 36.4%, respectively. For different pH values, the cumulative releases of Mitomycin-C from chitosan nanoparticles were 47% and 53% for pH 6.0 and 7.4, respectively (p < 0.01). For different drug doses, the cumulative releases of Mitomycin-C (MMC) from Chitosan nanoparticles were 44%, 53% and 65% for 0.5 mg/mL, 1.25 mg/mL and 2.5 mg/mL respectively (p < 0.01). The anticancer activity of Mitomycin-C loaded chitosan nanoparticles was measured in T24 bladder cancer cell line in vitro, and the results revealed that the 2.5 MMC coated Chitosan nanoparticles had better tumor cells decline activity. From this investigation, we conclude that the drug encapsulated synthesized chitosan nanoparticles possess a high ability to be used as pH and dose responsive drug delivery system. This systematic investigation demonstrates a promising future for the intravesical installation in treatment of the superficial bladder cancer.

All-in-one glycol chitosan nanoparticles for co-delivery of doxorubicin and anti-PD-L1 peptide in cancer immunotherapy

Synergistic immunotherapy of immune checkpoint blockade (ICB) and immunogenic cell death (ICD) has shown remarkable therapeutic efficacy in various cancers. However, patients show low response rates and undesirable outcomes to these combination therapies owing to the recycling mechanism of programmed death-ligand 1 (PD-L1) and the systemic toxicity of ICD-inducing chemotherapeutic drugs. Herein, we propose all-in-one glycol chitosan nanoparticles (CNPs) that can deliver anti-PD-L1 peptide (PP) and doxorubicin (DOX) to targeted tumor tissues for a safe and more effective synergistic immunotherapy. The PP-CNPs, which are prepared by conjugating ᴅ-form PP (NYSKPTDRQYHF) to CNPs, form stable nanoparticles that promote multivalent binding with PD-L1 proteins on the targeted tumor cell surface, resulting in effective lysosomal PD-L1 degradation in contrast with anti-PD-L1 antibody, which induces recycling of endocytosed PD-L1. Consequently, PP-CNPs prevent subcellular PD-L1 recycling and eventually destruct immune escape mechanism in CT26 colon tumor-bearing mice. Moreover, the ICD inducer, DOX is loaded into PP-CNPs (DOX-PP-CNPs) for synergistic ICD and ICB therapy, inducing a large number of damage-associated molecular patterns (DAMPs) in targeted tumor tissues with minimal toxicity in normal tissues. When the DOX-PP-CNPs are intravenously injected into CT26 colon tumor-bearing mice, PP and DOX are efficiently delivered to the tumor tissues via nanoparticle-derived passive and active targeting, which eventually induce both lysosomal PD-L1 degradation and substantial ICD, resulting in a high rate of complete tumor regression (CR: 60%) by a strong antitumor immune response. Collectively, this study demonstrates the superior efficacy of synergistic immunotherapy using all-in-one nanoparticles to deliver PP and DOX to targeted tumor tissues.

Drying curve simulation and LF-NMR online monitor of water state in ursolic acid loaded chitosan nanoparticles during microwave freeze drying

The changes in various states of water in ursolic acid(UA)loaded chitosan nanoparticles were assessed using lowfield nuclear magnetic resonance(LF-NMR)during microwave freeze drying(MFD)process,and six thin-layer models were applied to simulate the drying kinetics.UA nanoparticles were dried at different microwave power densities(1 W/g,2 W/g and 4 W/g).The results showed that three water fractions with different transverse relaxation times(T_(2))were detected in fresh UA nanoparticles.The T_(2)relaxation time of water decreased significantly with drying time at different microwave power densities.And the mutual migration and transformation of water in different states during the drying process of chitosan nanoparticles occurred.Furthermore,mathematical model analysis showed that the Page model provided the best description during the process of UA nanoparticle dried by MFD.The Page model can better simulate the drying kinetics of chitosan nanoparticles dried by MFD,and LF-NMR technology can monitor the changes in water status of UA nanoparticles.The results revealed that LF-NMR can monitor the changes of water in UA nanoparticles during the drying process.

Induction of Th1-Type Immune Response by Chitosan Nanoparticles Containing Plasmid DNA Encoding House Dust Mite Allergen Der p2 for Oral Vaccination in Mice

This study was to prepare the chitosan-pDer p 2 nanoparticles and to investigate the effect of chitosan-DNA nanoparticles on immune response in mice by oral delivery of chitosan-DNA nanoparticles. The nanoparticles were synthesized by complexing chitosan with plasmid DNA. The DNA was fully complexed into chitosan-DNA nanoparticles, suggesting a 100% encapsulation efficiency. Chitosan-DNA complex renders a significant protection of the plasmid. No effect on cell viability was observed in both cell types and average cell viability over 100% was obtained. Oral gene delivery with chitosan-DNA nanoparticles can generate a higher level expression of gene in vivo. Oral chitosan-pDer p 2 nanoparticles in BALB/c mice can induce IFN-γ in serum and prevent subsequent sensitization of Th2 cell-regulated specific IgE responses. The data indicate that the oral administration of chitosan-pDer p 2 nanoparticles results in the expression of Der p 2 in the epithelial cells of both stomach and small intestine and the induction of Th1-type immune response. Cellular ＆ Molecular Immunology.

A Novel Chitosan CpG Nanoparticle Regulates Cellular and Humoral Immunity of Mice

Objective To develop a safe and novel immunoadjuvant to enhance the immunity and resistance of animals against E. coli infection. Methods An 88-base immunostimulatory oligodeoxynuleotide containing eleven CpG motifs （CpG ODN） was synthesized and amplified by PCR. The chitosan nanoparticle （CNP） was prepared by ion linking method to entrap the CpG ODN that significantly promotes the proliferation of lymphocytes of pig in vitro. Then the CpG- CNP was inoculated into 21-day old Kunming mice, which were orally challenged with virulent K88/K99 E. Coil 35 days after inoculation. Blood was collected from the tail vein of mice on days 0, 7, 14, 21, 28, 35, 42, and 49 after inoculation to detect the changes and content of immunoglobulins, cytokines and immune cells by ELISA, such as IgG, IgA, IgM, IL-2, IL-4, and IL-6. Results The CpG provoked remarkable proliferation of lymphocytes of pig in vitro in comparison with that of control group （P〈0.05）. The inoculation with CpG-CNP significantly raised the content of IgG, IgM, and IgA in the sera of immunized mice （P〈0.05）. The levels of IL-2, 1L-4, and IL-6 in the mice significantly increased in comparison with those in controls （P〈0.05）, so was the number of white blood cells and lymphocytes in immunized mice. The humoral and cellular immunities were significantly enhanced in immunized mice, which resisted the infection of E coli and survived, while the control mice manifested evident symptoms and lesions of infection. Conclusions CpG-CNP can significantly promote cellular and humoral immunity and resistance of mice against E. coli infection, and can be utilized as an effective adjuvant to improve the immunoprotection and resistance of porcine against infectious disease.

Application of chitosan-based nanoparticles in skin wound healing

The rising prevalence of impaired wound healing and the consequential healthcare burdens have gained increased attention over recent years.This has prompted research into the development of novel wound dressings with augmented wound healing functions.Nanoparticle(NP)-based delivery systems have become attractive candidates in constructing such wound dressings due to their various favourable attributes.The non-toxicity,biocompatibility and bioactivity of chitosan(CS)-based NPs make them ideal candidates for wound applications.This review focusses on the application of CS-based NP systems for use in wound treatment.An overview of the wound healing process was presented,followed by discussion on the properties and suitability of CS and its NPs in wound healing.The wound healing mechanisms exerted by CS-based NPs were then critically analysed and discussed in sections,namely haemostasis,infection prevention,inflammatory response,oxidative stress,angiogenesis,collagen deposition,and wound closure time.The results of the studieswere thoroughly reviewed,and contradicting findings were identified and discussed.Based on the literature,the gap in research and future prospects in this research area were identified and highlighted.Current evidence shows that CS-based NPs possess superior wound healing effects either used on their own,or as drug delivery vehicles to encapsulate wound healing agents.It is concluded that great opportunities and potentials exist surrounding the use of CSNPs in wound healing.

Self-assembled Nanoparticles based on Folic Acid Modifi ed Carboxymethyl Chitosan Conjugated with Targeting Antibody

Nanoparticles conjugated with antibody were designed as active drug delivery system to reduce the toxicity and side effects of drugs for acute myeloid leukemia（AML）.Moreover,methotrexate（MTX）was chosen as modeldrug and encapsulate within folic acid modified carboxymethylchitosan（FACMCS）nanoparticles through self-assembling.The chemicalstructure,morphology,release and targeting of nanoparticles were characterized by routine detection.It is demonstrated that the mean diameter is about 150 nm,the release rate increases with the decreasing of p H,the binding rate of CD33 antibody and FA-CMCS nanoparticles is about 5：2,and nanoparticles can effectively bind onto HL60 cells in vitro.The experimentalresults indicate that the FA-CMCS nanoparticles conjugated with antibody may be used as a potentialp Hsensitive drug delivery system with leukemic targeting properties.

Neuroprotective effect of chitosan nanoparticle gene delivery system grafted with acteoside (ACT) in Parkinson's disease models

Developing new drugs to treat Parkinson's disease efficiently is challenging. Here we report that chitosan nanoparticles(APPDNs) could serve as novel candidates for the design of anti-PD drugs. In this study, we investigated the effects of chitosan poly ethyleneglycol-poly lactic acid(PEG-PLA) nanoparticles conjugated with nerve growth factor(NGF), acteoside(ACT) and plasmid DNA(p DNA) for PD therapy using in vitro and in vivo models. Using PD cell models, we demonstrated that APPDN had good neuroprotective effects. More significantly, experiments using mouse PD models demonstrated that APPDNs could ameliorate the behavioral disorders of sick mice. Immunohistochemical and western blot(WB) analyses demonstrated that APPDNs could significantly reverse dopaminergic(DA) neuron loss in the substantia nigra and striatum of sick mice. This study opens up a novel avenue to develop anti-PD drugs.

Evaluation of anti-metastatic effect of chitosan nanoparticles on esophageal cancer-associated fibroblasts

Aim:Esophageal cancer is one of the major types of cancers,causing death of approximately 5%of all cancer deaths.This is due,in large part,to both relatively ineffectual and unavailable treatment.In order to develop an effective treatment strategy against esophageal cancer,it is important to target metastatic genes.In the present study,we have used a cancer-associated fibroblast(CAF)cell line derived from culturing peripheral blood mononuclear cells from a metastatic esophageal cancer patient to see whether chitosan nanoparticles(Ch-Np)treatment can modulate the metastatic phenotype of CAF cells by using various cellular and molecular markers.Methods:A CAF cell line was developed from peripheral blood mononuclear cells(PBMC)from a metastatic esophageal cancer patient.The cells were treated with 100μg/mL of chitosan nanoparticle in vitro for the morphological and oncogenic characteristic studies,along with the expression of various genes involved in process of tumor development and metastasis.Techniques such as Light and Phase Contrast Microscopy,cell growth rate,Scratch metastatic assay,and molecular profiling were carried out to see changes in CAF cells before and after Ch-Np treatment.Results:It was observed that CAF cells grew in monolayer and had a doubling time of 25±0.38 h.Morphologically,the cells had a fibroblastic appearance.After treatment with 100μg/mL of Ch-Np in vitro,there was an increased doubling time to 30±0.83 h.Similarly,Scratch Assay showed an inhibition in the metastatic property of these cells.These findings were confirmed with gene expression studies.It was also observed that there was complete down-regulation of metastatic genes MMP1 and MMP9 and chemokines such as CXCR-4,CXCR-7,CCR-5,and SDF-1,indicating that Ch-Np inhibited the metastatic characteristic of CAF cells.Conclusion:This study has shown that there was an inhibition of metastatic properties of CAF cells after treatment with Ch-Np,suggesting that Ch-Np can be a delivery system used for targeting cancer cells for treatment of esophageal cancer.

Preparation and Evaluation of Insulin-loaded Nanoparticles based on Hydroxypropyl-β-cyclodextrin Modified Carboxymethyl Chitosan for Oral Delivery

Novel insulin-loaded nanoparticles based on hydroxypropyl-β-cyclodextrin modified carboxymethyl chitosan（CMC-HP-β-CD） were prepared to improve the oral bioavailability of insulin. The CMC-HP-β-CD was characterized by FT-IR spectroscopy and 1H-NMR spectra. The insulin-loaded nanoparticles were prepared through crosslinking with calcium ions, and the morphology and size of the prepared nanoparticles were characterized by transmission electron microscopy（TEM） and dynamic light scattering（DLS）. Cumulative release in vitro study was performed respectively in simulated gastric medium fluid（SGF, p H=1.2）, simulated intestinal fluid（SIF, p H=6.8） and simulated colonic fluid（SCF, p H=7.4）. The encapsulation efficiency of insulin was up to 87.14 ± 4.32% through high-performance liquid chromatography（HPLC）. Statistics indicated that only 15% of the encapsulated insulin was released from the CMC-HP-β-CD nanoparticles in 36 h in SGF, and about 50% of the insulin could be released from the nanoparticles in SIF, whereas more than 80% was released in SCF. In addition, the solution containing insulin nanoparticles could effectively reduce the blood glucose level of diabetic mice. The cytotoxicity test showed that the samples had no cytotoxicity. CMC-HP-β-CD nanoparticles are promising candidates as potential carriers in oral insulin delivery systems.

Polypyrrole Chitosan Cobalt Ferrite Nanoparticles Composite Layer for Measuring the Low Concentration of Fluorene Using Surface Plasmon Resonance

Fluorene is a polycyclic aromatic hydrocarbon, which is a hazardous toxic chemical in the environment. The measurement of low concentrations of fluorene is a subject of intense interest in chemistry and in the environment. Polypyrrole chitosan cobalt ferrite nanoparticles are prepared using the electrochemical method. The prepared layers are characterized using field emission scanning electron microscopy, Fourier transform infrared spectroscopy, and energy dispersive spectroscopy. The layers are used to detect fluorene using the surface plasmon resonance technique at room temperature. The composite layer is evaluated after detection of fluorene using atomic force microscopy. The fluorene is bound on the layer, and the shift of the resonance angle is about 0.0052°, corresponding to the limitation of 0.01 ppm.

Bactericidal activity of hydrogel beads based on N,N,N-trimethyl chitosan/alginate complexes loaded with silver nanoparticles

Polysaccharide-based composite materials（beads） containing silver nanoparticles（AgNPs） were successfully prepared.Hydrogel beads acted as an efficient vehicle for Ag＊ delivery.Beads promoted the AgNPs protection and inhibited their aggregation.Antimicrobial assays showed that the beads/AgNPs concentration can be modulated to deliver an amount of Ag＊ necessary for kill Escherichia coli cells.

On-Chip Fabrication of Carbon Nanoparticle–Chitosan Composite Membrane

The on-chip fabrication of a carbon nanoparticle-chitosan composite membrane （i.e. a sorbent membrane or a mixed matrix membrane） using laminar flow-based interfacial deprotonation technology was presented in this paper. In addition, the effects of carbon nanoparticles and reactant flow rates on membrane formation were investigated. Finally, the permeability and adsorption capacities of the membrane were discussed. During fabrication, an acidic chitosan solution and a basic buffer solution that contained carbon nanoparticles were introduced into a microchannel. At the flow interface, a freestanding composite membrane with embedded carbon nanoparticles was formed due to the deprotonation of the chitosan molecules. The membrane growth gradually stopped with time from upstream to downstream and the thickness of the membrane increased rapidly and then slowly along the reactant flow direction. The formation of the membrane was divided into two stages. The average growth rate in the first stage was significantly larger than the average growth rate in the second stage. Carbon nanoparticles in the basic solution acted as nucleating agents and made the membrane formation much easier. As the flow rate of the chitosan solution increased, the averaged membrane thickness and the membrane hydraulic permeability initially increased and then decreased. Because of the addition of carbon nanoparticles, the formed membrane had adsorption abilities. The carbon nanoparticle-chitosan composite membrane that was fabricated in this study could be employed for simultaneous adsorption and dialysis in microdevices in the future.

A novel antibacterial biomaterial mesh coated by chitosan and tigecycline for pelvic floor repair and its biological performance

The biomaterials composed of mammalian extracellular matrix(ECM)have a great potential in pelvic floor tissue repair and functional reconstruction.However,bacterial infection does cause great damage to the repair function of biomaterials which is the major problem in clinical utilization.Therefore,the development of biological materials with antimicrobial effect is of great clinical significance for pelvic floor repair.Chitosan/tigecycline(CS/TGC)antibacterial biofilm was prepared by coating CS/TGC nanoparticles on mammalian-derived ECM.Infrared spectroscopy,scanning electron microscopy,bacteriostasis circle assay and static dialysis methods were used to characterize the membrane.MTS assay kit and DAPI fluorescence staining were used to evaluate cytotoxicity and cell adhesion.The biocompatibility was assessed by subabdominal implantation model in goats.Subcutaneous antimicrobial test in rabbit back was used to evaluate the antimicrobial and repairing effects on the infected wounds in vivo.Infrared spectroscopy showed that the composite coating had been successfully modified.The antibacterial membrane retained the main structure of ECM multilayer fibers.In vitro release of biomaterials showed sustained release and stability.In vivo studies showed that the antibacterial biological membrane had low cytotoxicity,fast degradation,good compatibility,anti-infection and excellent repair ability.