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Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 μg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies. [ABSTRACT FROM AUTHOR]

Safety and effectiveness are the cornerstone objectives of nanomedicine in developing nanotherapies. It is crucial to understand the biological interactions between nanoparticles and immune cells. This study focuses on the manufacture by the microfluidic technique of N-trimethyl chitosan/protein nanocarriers and their interaction with J774 cells to elucidate the cellular processes involved in absorption and their impact on the immune system, mainly through endocytosis, activation of lysosomes and intracellular degradation. TEM of the manufactured nanoparticles showed spherical morphology with an average diameter ranging from 36 ± 16 nm to 179 ± 92 nm, depending on the concentration of the cargo protein (0, 12, 55 μg/mL). FTIR showed the crosslinking between N-trimethyl chitosan and the sodium tripolyphosphate and the α-helix binding loss of BSA. TGA revealed an increase in the thermal stability of N-trimethyl chitosan/protein nanoparticles compared with the powder. The encapsulation of the cargo protein used was demonstrated using XPS. Their potential to improve cell permeability and use as nanocarriers in future vaccine formulations was demonstrated. The toxicity of the nanoparticles in HaCaT and J774 cells was studied, as well as the importance of evaluating the differentiation status of J774 cells. Thus, possible endocytosis pathways and their impact on the immune response were discussed. This allowed us to conclude that N-trimethyl chitosan nanoparticles show potential as carriers for the immune system. Still, more studies are required to understand their effectiveness and possible use in therapies.

Colon-targeted delivery offers several benefits for oral protein delivery, such as low proteolytic enzyme activity, a natural pH environment, and extended residence time, which improve the bioavailability of the encapsulated protein. Therefore, we hypothesize that developing a novel colonic nanocarrier system, featuring modified chitosan that is soluble at physiological pH and coated with a colon-degradable polymer, will provide an effective delivery system for oral insulin. This study aims to synthesize insulin-loaded pectin-trimethyl chitosan nanoparticles (Ins-P-TMC-NPs) as an oral insulin delivery system and to evaluate its efficacy both in vitro and in vivo. N -trimethyl chitosan (TMC), synthesized via a methylation method, was used to prepare insulin-TMC nanoparticles coated with pectin via the ionic gelation method. The nanoparticles were characterized for their physicochemical properties, cumulative release profile, and surface morphology. The in vitro biological cytotoxicity and cellular uptake of the nanoparticles were evaluated against HT-29 cells. The in vivo blood glucose-lowering effect and histological toxicity were assessed in diabetic male Sprague–Dawley rats. The results showed that Ins-P-TMC-NPs were spherical, with an average size of 379.40 ± 40.26 nm, a polydispersity index of 24.10 ± 1.03 %, a zeta potential of +17.20 ± 0.52 mV, and a loading efficiency of 83.21 ± 1.23 %. Compared to uncoated TMC nanoparticles, Ins-P-TMC-NPs reduced insulin loss in simulated gastrointestinal fluid by approximately 67.23 ± 0.97 % and provided controlled insulin release in simulated colonic fluid. In vitro bioactivity studies revealed that Ins-P-TMC-NPs were non-toxic, with cell viability of 91.12 ± 0.91 % after 24 h of treatment, and exhibited high cellular uptake in the HT-29 cell line with a fluorescence intensity of 37.80 ± 2.40 after 4 h of incubation. Furthermore, the in vivo study demonstrated a sustained reduction in blood glucose levels after oral administration of Ins-P-TMC-NPs, peaking after 8 h with a blood glucose reduction of 87 ± 1.03 %. Histological sections showed no signs of toxicity when compared to those of healthy rats. Overall, the developed colon-targeted oral insulin delivery system exhibits strong potential as a candidate for effective oral insulin administration. [Display omitted] [ABSTRACT FROM AUTHOR]

Qingqing Meng,1,* Aiping Wang,1,2,* Hongchen Hua,1 Ying Jiang,1 Yiyun Wang,1 Hongjie Mu,1 Zimei Wu,1 Kaoxiang Sun1 1School of Pharmacy, Collaborative Innovation Center of Advanced Drug Delivery System and Biotech Drugs in Universities of Shandong, Yantai University, Yantai, People’s Republic of China; 2State Key Laboratory of Long-Acting and Targeting Drug Delivery System, Shandong Luye Pharmaceutical Co., Ltd, Yantai, People’s Republic of China *These authors contributed equally to this work Background: Safe and effective delivery of therapeutic drugs to the brain is important for successful therapy of Alzheimer’s disease (AD).Purpose: To develop Huperzine A (HupA)-loaded, mucoadhesive and targeted polylactide-co-glycoside (PLGA) nanoparticles (NPs) with surface modification by lactoferrin (Lf)-conjugated N-trimethylated chitosan (TMC) (HupA Lf-TMC NPs) for efficient intranasal delivery of HupA to the brain for AD treatment.Methods: HupA Lf-TMC NPs were prepared using the emulsion–solvent evaporation method and optimized using the Box–Behnken design. The particle size, zeta potential, drug entrapment efficiency, adhesion and in vitro release behavior were investigated. The cellular uptake was investigated by fluorescence microscopy and flow cytometry. MTT assay was used to evaluate the cytotoxicity of the NPs. In vivo imaging system was used to investigate brain targeting effect of NPs after intranasal administration. The biodistribution of Hup-A NPs after intranasal administration was determined by liquid chromatography–tandem mass spectrometry.Results: Optimized HupA Lf-TMC NPs had a particle size of 153.2±13.7 nm, polydispersity index of 0.229±0.078, zeta potential of +35.6±5.2 mV, drug entrapment efficiency of 73.8%±5.7%, and sustained release in vitro over a 48 h period. Adsorption of mucin onto Lf-TMC NPs was 86.9%±1.8%, which was significantly higher than that onto PLGA NPs (32.1%±2.5%). HupA Lf-TMC NPs showed lower toxicity in the 16HBE cell line compared with HupA solution. Qualitative and quantitative cellular uptake experiments indicated that accumulation of Lf-TMC NPs was higher than nontargeted analogs in 16HBE and SH-SY5Y cells. In vivo imaging results showed that Lf-TMC NPs exhibited a higher fluorescence intensity in the brain and a longer residence time than nontargeted NPs. After intranasal administration, Lf-TMC NPs facilitated the distribution of HupA in the brain, and the values of the drug targeting index in the mouse olfactory bulb, cerebrum (with hippocampus removal), cerebellum, and hippocampus were about 2.0, 1.6, 1.9, and 1.9, respectively.Conclusion: Lf-TMC NPs have good sustained-release effect, adhesion and targeting ability, and have a broad application prospect as a nasal drug delivery carrier. Keywords: Huperzine A, lactoferrin, N-trimethyl chitosan, nose-to-brain, nanoparticles, Alzheimer’s disease

Here we report the development of tristearin-based nanostructured-lipid carriers (NLCs) which were further surface-modified with mucoadhesive N,N,N-trimethyl chitosan (TMC) to form mucoadhesive nanostructured-lipid carriers (mNLCs). The NLCs were loaded with high partitioning ropinirole-dextran sulphate (ROPI-DS) nanoplex. NLCs were optimised using Box-Behnken experimental design. Obtaining controlled drug release with prolonged residence on the nasal mucosa for treating Parkinson's disease (PD) is a major objective of the present investigation. Enhanced brain targetability and improved therapeutic efficacy are the additional objective of this study. Lyophilised NLCs and mNLCs were characterised in solid state by FTIR, DSC, XRD, and HR-TEM analysis. The bioadhesive strength of mNLCs was observed to be 13.3-folds greater compared to NLCs. Plasma and brain pharmacokinetic studies in mice model demonstrated the potential of mNLCs in nose-to-brain drug delivery via olfactory pathway. If investigated for behavioural and neurotoxicity studies clinically, a commercial formulation for patient looks feasible. [ABSTRACT FROM AUTHOR]

Many water-solubleN-substituted chitosan derivatives have been prepared having the typical -NH-CH2- linkage. Both the Michael reaction and alkylation with monochloroacetic acid at pH 8- 8.5 offer relatively simple ways for their synthesis.

Graphical abstract Highlights • Quaternized trimethyl chitosan (TMC) was functionalized for waste water treatment. • TMC was synthesized via reductive alkylation and methylation. • The reduction in contact angle increases the surface wettability. • The functionalized membranes had shown noticeable antifungal activity. • Highest water flux of 1400 L/m2.h was observed for maximum TMC content. Abstract Chitosan was functionalized to synthesize Quaternized N -trimethyl chitosan (TMC) and incorporated in polyethersulfone (PES) polymer to fabricate membranes for enhanced antifungal activity and water treatment. The TMC was synthesized from chitosan via reductive alkylation and methylation. The effect of concentration of chitosan and TMC on the properties of functionalized PES membranes was investigated. The membrane with the lowest concentration of TMC 5% (w/w) resulted in the largest average pore size than the PES membrane without chitosan or TMC. The surface wettability was enhanced as contact angle was reduced from 90° to 57° by increasing concentration of TMC to 15% (w/w). The resultant membranes exhibited improved water hydrophilicity, permeability and inhibition against fungal species. The functionalized membranes had shown noticeable antifungal activity against Aspergillus niger in the case of 15% TMC with 72% antifungal activity and 15% chitosan with 63% antifungal activity against Fusarium solani. [ABSTRACT FROM AUTHOR]

Lumbrokinase (LK) has strong fibrinolytic and thrombolytic activities, but it has a short half-life, can be easily inactivated, and may cause hemorrhage as a side effect. This study develops a potential thrombolytic therapy by fabricating N,N,N-Trimethyl Chitosan (TMC) nanoparticles modified with the cyclic Arg-Gly-Asp-Phe-Lys peptide (c-RGD) and loaded with LK (i.e. c-RGD-LK-NPs). The binding of c-RGD to platelet membrane GPIIb/IIIa receptors is expected to enable targeted delivery of the c-RGD-conjugated TMC to the thrombus. The synthesized c-RGD-LK-NPs had a mean particle size of 232.0 nm, zeta potential of 19.8 mV, entrapment efficiency of 52.7% ± 2.5%, and loading efficiency of 17.4% ± 0.65%. Transmission electron microscopy showed that they were generally spherical. The c-RGD-LK-NPs gave a cumulative in vitro LK release of 80.6% over 8 h, and the activity of LK was close to 80%, indicating that the nanoparticles protected the activity of LK. In vitro blood clot lysis assays were carried out and in vivo thrombolysis effect was tested in Sprague-Dawley rats carotid artery thrombus model. In all cases, the c-RGD-LK-NPs showed superior performance compared with the free LK and the unmodified TMC nanoparticles loaded with LK. The c-RGD-LK-NPs reagent is expected to be potentially useful in treating thromboembolic diseases. [ABSTRACT FROM AUTHOR]

The unique properties and applications of iron oxide and Au nanoparticles have motivated researchers to synthesize and optimize a combined nanocomposite containing both. By using various polymers such as chitosan, some of the problems of classic core–shell structures (such as reduced saturation magnetization and thick coating) have been overcome. In the present study, chitosan and one of its well-known derivatives, N-trimethylchitosan (TMC), were applied to construct three-layer nanocomposites in an Au/polymer/Fe3O4 system. It was demonstrated that replacement of chitosan with TMC reasonably improved the properties of the final nanocomposites including their size, magnetic behavior and thermal stability. Moreover, the results of the MTT assay showed no significant cytotoxicity effect when the Au/TMC/Fe3O4 nanocomposites were applied in vitro. These TMC-containing magnetic nanoparticles are well-coated by Au nanoparticles and have good biocompatibility and can thus play the role of a platform or a label in various fields of application, especially the biomedical sciences and biosensors.

Context: Cataracts have become the leading cause of blindness around the world, which is mainly mediated by oxidative stress. Objective: N-trimethyl chitosan (TMC)-coated liposomes of cyanidin-3-glycoside (C3G) (C3G-TCL) were prepared to attenuate oxidative stress induced by selenite sodium in rats. Materials and methods: C3G-TCL were prepared by reverse-phase evaporation method and then coated with self-synthesized TMC. The physicochemical properties were determined. A gamma-scintigraphy study was employed to evaluate the precorneal elimination of the radioactive preparations. The transcorneal visualization for fluorescence-labeled samples was determined by confocal laser scanning microscopy (CLSM). Thein vivoanti-oxidative study using C3G-TCL was carried out in rats with selenite-induced cataracts by topical administration. Results: The sphere-like morphological characterization of the vesicles was confirmed by TEM, with a size of 158.3 ± 2.8 nm and a zeta potential of 31.7 mV. The encapsulation efficiency was (53.7 ± 0.2) % as measured by ultrafiltration. C3G-TCL showed a 3.3-fold increment in precorneal residence time when compared with that of the99mTc-solution. A TMC coating enhanced the transepithelial transport of liposomes to a depth of 40-μm in the cornea. Moreover, C3G-TCL could significantly elevate the activity of superoxide dismutase and catalase in lens and also show a considerable reversal of reduced glutathione activity. The lipid peroxidation in lens was strongly prevented when compared with that of groups treated with uncoated C3G-loaded liposomes. Discussion and conclusion: The coating material TMC for liposomes helps improve the anti-oxidative effect of C3Gin vivothrough prolonged residence time on the cornea and improved permeability in the corneal epithelium. [ABSTRACT FROM PUBLISHER]

Purpose: Solid lipid nanoparticles (SLNs) have been proposed as a colloidal carrier system that could enhance the oral bioavailability of curcumin. However, a burst release of the loaded drug, which occurs in acidic environments, has been a main obstacle to the oral delivery of curcumin by using SLNs as a carrier system. We hypothesized that a quarternized chitosan derivative could be used for acid-resistant coating to stabilize the SLNs and circumvent the burst release. Methods: N-trimethyl chitosan (TMC) was synthesized and determined by H-NMR and FT-IR. To investigate the details of chitosan and TMC surface modification on SLCNs composed of palmitic acid, cholesterol, TPGS and curcumin, a number of factors such as optimized SLNs composition, solid state characterization, stability, cell viability, in vitro release in GI conditions, curcumin oral bioavailability and brain distribution studies, were evaluated. Results: The TMC-SLCNs exhibited prolonged stability in room and refrigerated conditions, controlled drug release in simulated intestinal fluid, significantly higher oral bioavailability, and brain distribution of curcumin than free curcumin, chitosan and non-coated SLCNs. Conclusions: These finding suggests that the TMC-SLCNs is a promising nanocarrier system for oral delivery and brain distribution of curcumin. [ABSTRACT FROM AUTHOR]

The influence of DNA vaccine formulations on immune responses in combination with adjuvants was investigated with the aim to increase cell-mediated immunity against plasmid DNA (pDNA) encoding Mycobacterium tuberculosis antigen 85A. Different ratios of pDNA with cationic trimethyl chitosan (TMC) nanoparticles were characterized for their morphology and physicochemical characteristics (size, zeta potential, loading efficiency and pDNA release profile) applied in vitro for cellular uptake studies and in vivo, to determine the dose-dependent effects of pDNA on immune responses. A selected pDNA/TMC nanoparticle formulation was optimized by the incorporation of muramyl dipeptide (MDP) as an immunostimulatory agent. Cellular uptake investigations in vitro showed saturation to a maximum level upon the increase in the pDNA/TMC nanoparticle ratio, correlating with increasing Th1-related antibody responses up to a definite pDNA dose applied. Moreover, TMC nanoparticles induced clear polarization towards a Th1 response, indicated by IgG2c/IgG1 ratios above unity and enhanced numbers of antigen-specific IFN-γ producing T-cells in the spleen. Remarkably, the incorporation of MDP in TMC nanoparticles provoked a significant additional increase in T-cell-mediated responses induced by pDNA. In conclusion, pDNA-loaded TMC nanoparticles are capable of provoking strong Th1-type cellular and humoral immune responses, with the potential to be further optimized by the incorporation of MDP.

Vitexin is a flavonoid which exerted many protective activities. However, the low bioavailability discounts the in vivo effects of vitexin. This study designed vitexin loaded bilayer nanoparticles by the assembly of soybean peptides and the coating of goblet cell targeting peptide CSKSSDYQC (CSK) coupled N -trimethyl chitosan (TMC), to improve the bioavailability of vitexin. The results showed that the bilayer nanoparticles could protect vitexin from being released in stomach and promote sustained release in intestine. Ex vivo experiments confirmed that nanoparticles promoted absorption of vitexin through tight junctions. Further in vivo studies suggested that the embedding of vitexin by nanoparticles increased the bioavailability and antioxidant activity of vitexin. Our results indicated that the nanoparticles could not only promote the absorption of vitexin, but also improve its in vivo antioxidant activity. Therefore, the reported nanoparticles could be effective delivery platforms to improve the bioavailability of vitexin. [Display omitted] [ABSTRACT FROM AUTHOR]

Abstract: N-trimethyl chitosan (TMC) with different degree of quaternization (DQ) as the coating materials, vitamin A palmitate (VAP)-loaded cationic liposomes dispersed in thermo-sensitive in situ gels (ISG) with poloxamer 407 (P407) as the base were prepared in this study. VAP-loaded liposomes (VAPL) were prepared using a film dispersion method followed by TMC-coating with DQ of 20%, 40% and 60% (TMC20, TMC40 and TMC60), respectively, then dispersed in P407 solution to obtain TMC-coated VAPL ISG. The in vitro properties of the system including morphology, size, zeta potential, entrapment efficiency, drug release and ocular retention were investigated. The ocular retention in vivo, eye irritation and pharmacokinetics in aqueous humor of the system were also studied in rabbits. VAPL revealed a spherical surface with mean size below 100nm and negative zeta potential. After TMC-coating, the morphology and entrapment efficiency showed no significant changes, while the mean size was increased, zeta potential was changed into positive, and drug release was further sustained, and above all was influenced by DQ of TMC. TMC-coated VAPL exhibited little effect on the gelation temperature of P407 solution, and at the P407 concentration of 25% (w/v), TMC-coated VAPL ISG had the gelation temperature closest to the eye surface (34°C) after diluted by the artificial tears. Compared with uncoated VAPL ISG and the marketed Oculotect gels, TMC-coated VAPL ISG displayed more retarded drug release and gel corrosion with a good linear relationship between them, and the higher DQ was, the slower drug release and gel corrosion. The ocular retention time in vitro and in vivo of TMC-coated VAPL ISG were both notable prolonged with a positive correlation with DQ of TMC. Compared with the marketed gels, TMC60-coated VAPL ISG showed delayed T max, improved C max and AUC(0–24) in rabbit aqueous humor, suggesting the sustained drug release and better corneal penetration and absorption. The local irritation of TMC-coated VAPL ISG was proved to be negligible. TMC-coated VAPL ISG with the properties of sustained drug release, prolonged ocular retention, improved corneal penetration and promising bio-safety is valuable to be studied further. [Copyright &y& Elsevier]

Abstract: N-trimethyl chitosan-graft-poly(vinyl alcohol) (TMC-g-PVA) copolymers were prepared. The grafting reactions were conducted in water changing the feed ratios of poly(vinyl alcohol)/6-O-succinate-N-trimethyl chitosan (PVA/STMC). The structure of TMC-g-PVA copolymers was characterized through 1H NMR spectroscopy, thermogravimetric analysis (TGA/DTG), wide-angle X-ray scattering (WAXS) and scanning electron microscopy (SEM). The quaternization degree (DQ) and substitution degree (DS) of N-trimethyl chitosan (TMC) and 6-O-succinate-N-trimethyl chitosan (STMC) were determined by 1H NMR, being the spectroscopy 14.0 and 5.5mol-% found, respectively. The viability of HCT-116 cancerous cells was investigated at different concentrations. The effect of PVA/STMC ratios on the cytotoxicity of the TMC-g-PVA copolymers was examined and the CC50 values determined for every case. [Copyright &y& Elsevier]

Highlights: [•] N-trimethyl chitosan of two quaternization degrees (DQ=20 and 80mol%) were synthesized and characterized. [•] Polyelectrolyte complexes of N-trimethyl chitosan/alginate (TMC/ALG) were prepared at different pHs. [•] Using the N-trimethyl chitosan of DQ 20mol%, it was possible to obtain beads of TMC/ALG, at pH 2. [•] In vitro controlled release of curcumin were evaluated and showed the release being dependent of pH. [Copyright &y& Elsevier]

Doxorubicin (DOX)-loaded cationic liposomes (DOXL) coated by N-trimethyl chitosan (TMCs) has been previously shown to enhance DOX uptake by human umbilical vein endothelial cells (HUVECs) in vitro and the tumor inhibition on solid tumor in vivo, and the effects were both enhanced with the degree of quaternization (DQ) increase of TMCs. The aim of the present work is to study the cytotoxicity of the blank cationic liposomes (CLs) coated by TMCs with various DQ on L-929 mouse fibroblasts, by MTT assay, using the relative proliferation rate as the indicator, and the toxicity extent was classified according to the evaluation criteria of United States Pharmacopoeia. Furthermore, the in vivo tumor angiogenesis targeting of DOXL coated by TMC60 was studied. It was found that with the increase of TMCs concentration and DQ, cytotoxicity was increased accordingly. However, the cell proliferation rates of TMCs-coated CLs with TMCs concentrations of 0.02% and 0.05%(w/w) were all above 80%, even the concentration of TMC20 was increased to 0.2%(w/w), the cell proliferation rate was still above 80%, showing noncytotoxicity. The mouse H22 tumor model was established by transplanted tumor experiment. In vivo fluorescence in tumor tissue was investigated through the tail vein injection of fluorescein isothiocyanate conjugated dextran at the 7th day after the administration of different DOX preparations. Compared with DOX solution and uncoated DOXL, the mice given TMC60-coated DOXL showed tumor angiogenesis with good shape, uniform arrangement, and small vascular branches, and the vascular density was decreased, suggesting promising tumor angiogenesis targeting of TMC60-coated DOXL. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013 [ABSTRACT FROM AUTHOR]

Abstract: N-Trimethyl chitosan (TMC) was synthesized and used to prepare lactosyl-norcantharidin TMC nanoparticles (Lac-NCTD-TMC-NPs) using an ionic cross-linkage process. Lac-NCTD-TMC-NPs with an average particle size of 120.6 ± 1.7 nm were obtained, with an entrapment efficiency of 69.29% ± 0.76%, and a drug-loading amount of 9.1% ± 0.07%. The release of Lac-NCTD-TMC-NPs in vitro was investigated through a dialysis method, and its sustained effect was evident. In the human liver cancer cell line HepG2, the half-maximum inhibiting concentration (IC50) of TMC-encapsulated Lac-NCTD (Lac-NCTD-TMC-NPs) was only 24.2% that of free Lac-NCTD at 24 hours. Lac-NCTD induced HepG2 cell death by triggering apoptosis. In vitro cellular uptake and in vivo NIR fluorescence real-time imaging both indicated a high targeting efficacy. In comparison with Lac-NCTD and Lac-NCTD chitosan NPs (Lac-NCTD-CS-NPs ), Lac-NCTD-TMC-NPs had the strongest antitumor activity on the murine hepatocarcinoma 22 subcutaneous model. From the Clinical Editor: In this article the preparation of N-trimethyl chitosan-encapsulated lactosyl-norcantharidin nanoparticles is described that displayed efficient targeting and sustained release in a hepatocarcinoma SC murine model. [Copyright &y& Elsevier]

Abstract: In this study, a novel class of stabilizer, cationic polymer, was introduced into the system of nanocrystals. Taking itraconazole as a poorly soluble model drug, the influence of three cationic polymers—chitosan, N-trimethyl chitosan, and polyethyleneimine—on the properties of the nanocrystals prepared by high-pressure homogenization method was investigated. Physicochemical properties of the nanoparticles were characterized. It was demonstrated that the cationic polymers could act as both an electrostatic and steric stabilizer to facilitate particle size reduction. Factors influencing charge density and stretching state of the cationic polymers influenced the magnitude of the particle. The electrostatic stabilization effect is more prominent than that of the steric stabilizing mechanism. Drug crystalline state was not changed by the addition of cationic polymers. Physical stability of the nanocrystals with cationic polymers was remarkably improved. The in vivo antifungal efficacy of the nanocrystals was dependent on physicochemical properties and pH. From the Clinical Editor: Cationic polymer stabilizers were used to modify the surface of nanocrystals and the resulting stabilizing mechanisms were compared. The electrostatic stabilization effect was found to be stronger than the steric one. Crystallinity of itraconazole was unchanged by the addition of cationic polymers, while drug physical stability remarkably improved. [Copyright &y& Elsevier]

Abstract: N-trimethyl chitosan (TMC) nanoparticles have been shown to increase the immunogenicity of subunit antigens after nasal and intradermal administration. This work describes a second generation of TMC nanoparticles containing ovalbumin as a model antigen (TMC/OVA nanoparticles) and an immunopotentiator (TMC/OVA/immunopotentiator nanoparticles). The selection of immunopotentiators included Toll-like receptor (TLR) ligands lipopolysaccharide (LPS), PAM3CSK4 (PAM), CpG DNA, the NOD-like receptor 2 ligand muramyl dipeptide (MDP) and the GM1 ganglioside receptor ligand, cholera toxin B subunit (CTB). The TMC/OVA/immunopotentiator nanoparticles were characterised physico-chemically and their immunogenicity was assessed by determining the serum IgG, IgG1, IgG2a titres and secretory IgA levels in nasal washes after intradermal and nasal vaccination in mice. After nasal vaccination, TMC/OVA nanoparticles containing LPS or MDP elicited higher IgG, IgG1 and sIgA levels than non-adjuvanted TMC/OVA particles, whereas nanoparticles containing CTB, PAM or CpG did not. After intradermal vaccination, the TMC/OVA/CpG and TMC/OVA/LPS nanoparticles provoked higher IgG titres than plain TMC/OVA particles. Altogether, our results show that co-encapsulation of an additional immunopotentiator with the antigen into TMC nanoparticles can further improve the immunogenicity of the vaccine. However, the strength and quality of the response depends on the immunopotentiator as well as the route of administration. [Copyright &y& Elsevier]

Cationic liposomes (CLs) can accumulate in tumor vascular endothelial cells (VECs) to show high selective targeting ability. Therefore, chemotherapeutic agent-loaded CLs are considered as new therapeutic vehicles to enhance the treatment efficacy. This study investigated the effect of N-trimethyl chitosan (TMC), one of derivatives of chitosan with positive charge determined by its degree of quaternization (DQ), on preparing doxorubicin (DOX)-loaded CLs. TMCs with various DQ, i.e., 20% (TMC20), 40% (TMC40), and 60% (TMC60) were synthesized and characterized by HNMR. DOX-loaded liposomes (DOXL) were prepared by ammonium sulfate gradients followed by TMC-coating to obtain TMC-coated DOXL with various positive surface charges. The morphology, size, ζ-potential and drug release in vitro of TMC-coated DOXL were studied compared with those of DOXL. Human umbilical vein endothelial cells (HUVECs) as cell model, the vascular targeting ability of TMC-coated DOXL was evaluated in vitro. A solid tumor, formed by implantationmurine hepatoma cells (H) into mice, as tumor model, the tumor inhibition rate and tumor histological sections stained by HE of TMC-coated DOXL group were researched compared with those of free DOX and DOXL group. It was found that with the increase of TMC's DQ, the positive surface charge of TMC-coated DOXL was enhanced accordingly, which had little effect on DOX release in vitro while led to the significant increase of DOX uptake by HUVECs in vitro and the treatment effect on solid tumor in vivo. Especially, TMC-coated DOXL showed better targeting ability to the nuclei compared with free DOX and DOXL, which could further enhance the efficacy of DOX in vivo. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011 [ABSTRACT FROM AUTHOR]

Abstract: To provoke an immune response, a transcutaneously administered vaccine has to diffuse into the skin, reach the lymph nodes and be taken up by dendritic cells (DCs). To study these three steps we immunised mice transcutaneously (with microneedles), intradermally and intranodally. The effect of the formulation was investigated by formulating ovalbumin (OVA) in three ways with N-trimethyl chitosan (TMC): TMC+OVA mixtures, TMC–OVA conjugates and TMC/OVA nanoparticles. Both the percentage OVA+ DCs in the lymph node and the resultant immunogenicity (serum IgG titres) were studied. Transcutaneously, the TMC–OVA conjugates induced the highest IgG levels and resulted in more OVA+ DCs in the lymph nodes after 24h than the other TMC formulations. Intradermally, all TMC-adjuvanted OVA formulations increased IgG titres compared to plain OVA. These formulations formed a depot in the skin, prolonging OVA delivery to the lymph nodes. The prolonged delivery of TMC-adjuvanted OVA to lymph node resident DCs was also observed after intranodal immunisation, but in this case the higher uptake did not correspond with elevated antibody titres compared to plain OVA. In conclusion, after transcutaneous administration, TMC–OVA conjugates are most immunogenic among the tested formulations, likely because they penetrate the skin more easily than nanoparticles and consequently are better delivered to DCs, while they show higher uptake by DCs than TMC+OVA mixtures. [Copyright &y& Elsevier]

Abstract: The evaluation of N-trimethyl chitosan (TMC)-coated liposomes containing Coenzyme Q10 as potential ophthalmic drug delivery system was carried out. Firstly, transcorneal permeation studies were conducted at 34°C using a side-by-side diffusion apparatus. The transport process of the fluorescent marker, rhodamine B, across the corneal epithelium was visualized with confocal laser scanning microscopy. Secondly, the human lens epithelial cells (HLECs) were cultured without or with Coenzyme Q10 followed by addition of H2O2. The cell viability and apoptosis were evaluated. The permeability coefficient for rhodamine B with TMC-coated liposomes increased more than two times in comparison with the value obtained for solution as control, from (0.42±0.018)×105 cms−1 to (1.31±0.030)×105 cms−1. Confocal laser scanning microscopy revealed that a TMC coating enhanced the transepithelial transport, dependent on the TMC concentration and contacting time. Coenzyme Q10 elevated the cell viability and reduced the oxidative damage with the decreased percentage of apoptotic cells in a positive concentration-dependent manner. The ATP content of liposome-treated cells was increased about 2-fold compared with that of H2O2-treated cells. Together, our findings demonstrate that with the enhanced permeation effect of the TMC coating, Coenzyme Q10-loaded TMC-coated liposomes appear to be a promising ophthalmic drug delivery carrier with an efficacy in protecting HLECs against H2O2-induced oxidative damage. [ABSTRACT FROM AUTHOR]

Abstract: Nasal vaccination is a promising, but challenging vaccination strategy. Poor absorption by the nasal epithelium and failure to break nasal tolerance are regarded as important reasons for poor efficacy of nasally applied vaccines. Formulation of the antigen into mucoadhesive nanoparticles, made of N-trimethyl chitosan (TMC) crosslinked with tripolyphosphate (TPP), has been shown to overcome these obstacles. However, although nasally administered antigen loaded TMC/TPP nanoparticles induce a strong humoral response, antibody subtyping indicates a Th2 bias. To design a nasal antigen delivery system capable of inducing stronger Th1 type responses, TPP as a crosslinking agent was replaced by unmethylated CpG DNA, a TLR-9 ligand and a potent inducer of Th1 responses, to prepare ovalbumin (OVA) loaded TMC nanoparticles (TMC/CpG/OVA). Several physicochemical characteristics of TMC/CpG/OVA (size, zetapotential, loading efficiency and antigen release profile) were assessed and compared to TMC nanoparticles prepared by crosslinking with TPP (TMC/TPP/OVA). Mice were nasally administered TMC/TPP/OVA and TMC/CpG/OVA after which antibody responses in serum and nasal washes were assessed and T-cell activation in the spleens determined. TMC/CpG/OVA showed similar physical properties as TMC/TPP/OVA in terms of particle size (380nm), zetapotential (+21mV) and antigen release characteristics. Nasal administration of TMC/CpG/OVA and TMC/TPP/OVA to mice resulted in comparable serum IgG levels (ca. 1000 fold higher than those induced by unadjuvanted OVA) and local secretory IgA levels. Moreover, TMC/CpG/OVA induced a 10 fold higher IgG2a response than TMC/TPP/OVA and enhanced the number of OVA specific IFN-gamma-producing T-cells in the spleen. In conclusion, OVA loaded TMC nanoparticles, containing CpG as adjuvant and crosslinker, are capable of provoking strong humoral as well as Th1 type cellular immune responses after nasal vaccination. [ABSTRACT FROM AUTHOR]

Abstract: The efficacy of chitosan (CS) and its derivatives used as transdermal penetration enhancers has been confirmed in our previous research. This study investigated the mechanisms of penetration enhancement by CS and its derivatives, i.e., N-trimethyl chitosan (TMC) with different degree of quaternization (DQ) and mono-N-carboxylmethyl chitosan (MCC). After treatment with CS, TMCs or MCC, the secondary structure changes of keratin in stratum corneum (SC) from mice were examined by an Attenuated Total Reflection-Fourier Transform Infrared (ATR-FTIR) combined with the application of the second-order derivative, deconvolution and curve-fitting. The water content in the SC was also studied by ATR-FTIR. HaCaT cell lines were employed as the cell models in the study. HaCaT cells were first treated with blank D-Hanks solution, CS or its derivatives, and were then fluorescent labeled with DiBAC4 (3). The change of membrane potential was measured by a flow cytometer (FCM). Alternatively, the treated HaCaT cells were labeled with NBD-C6-HPC and the change of membrane fluidity was examined under a Confocal Laser Scanning Microscope (CLSM). It was found that CS, TMCs and MCC could significantly affect the secondary structure of keratin in SC in different ways. Although the amide II absorption peak of keratin moved to a lower wave number following treatment with CS, TMCs, or MCC, the β-turning structure of keratin was converted to β-sheeting and random coiling after treatment with TMCs and was converted to β-sheeting and α-helix following treatment with MCC and CS. At the same time, CS and its derivatives all could increase the water content of SC, decrease HaCaT cells membrane potentials and enhance HaCaT cells membrane fluidity significantly. The effect of TMCs appeared to be independent of their DQ. The results suggest that the mechanisms of transdermal enhancement of CS, TMCs and MCC are closely related to their effects on the secondary structure of keratin and water content in SC, cell membrane potential and fluidity. [Copyright &y& Elsevier]

Abstract: In this study, for the first time, TMC/MCC complex nanoparticles as a delivery system and as an adjuvant were developed and evaluated to obtain systemic and mucosal immune responses against nasally administered tetanus toxoid (TT). Nanoparticles were developed by complexation between the oppositely charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic) and mono-N-carboxymethyl chitosan (MCC, polyampholytic) without using any crosslinker for mucosal vaccination. The cellular viability was found to be higher with TMC/MCC complex compared to that of MCC and TMC alone. Size, zeta potential and morphology of the nanoparticles were investigated as a function of preparation method. Nanoparticles with high loading efficacy (95%) and positively charged surface were obtained with an average particle size of 283±2.5nm. The structural integrity of the TT in the nanoparticles was confirmed by SDS–PAGE electrophoresis analysis. Cellular uptake studies indicated that FITC-BSA loaded nanoparticles were effectively taken up into the mouse Balb/c monocyte macrophages. Mice were nasally immunized with TT loaded TMC/MCC complex nanoparticles and compared to that of TMC and MCC nanoparticles. TMC/MCC complex nanoparticles were shown to induce both the mucosal and systemic immune response indicating that this newly developed system has potential for mucosal administration of vaccines. [Copyright &y& Elsevier]

Background: Multivesicular liposomes (MVLs), uncoated and coated with N-trimethyl chitosan (TMC), have been studied for their potential use for drug delivery by the oral route. Method: Synthesized TMC was characterized by infrared spectroscopy, revealing the presence of trimethyl groups, and by proton nuclear magnetic resonance spectroscopy, allowing the calculation of the degree of substitution quaternization (70.2%). Oxymatrine (OM), a natural quinolizidine alkaloid used clinically for treating hepatitis B, was chosen as a model drug. The surface-modified MVLs and uncoated MVLs were characterized in vitro in terms of their shape, size, zeta potential, entrapment efficiency, coating efficiency, the stability of MVLs in polymer suspension, and the stability in simulated gastric and intestinal fluids. Results: In vivo, the area under the plasma concentration–time curve obtained from the pharmacokinetics study of TMC-coated MVLs was found to be about 3.26- and 1.96-fold higher than that of OM solution and uncoated MVLs, respectively. Conclusion: TMC-coated MVLs can be considered as a potential carrier for oral drug administration. [ABSTRACT FROM AUTHOR]

Abstract: Mucosal application of a vaccine can effectively induce both systemic and mucosal immune responses. In general, mucosal applications of antigens result in poor immune responses. Therefore, adjuvant/delivery systems are required to enhance the immune response. Chitosan is a cationic biopolymer which exerts advantages as a vaccine carrier due to its immune stimulating activity and bioadhesive properties that enhance cellular uptake and permeation as well as antigen protection. Similar effects are also shown by chitosan derivatives. In this study, the nanoparticulate systems were prepared by using differently charged chitosan derivatives, N-trimethyl chitosan (TMC, polycationic), and mono-N-carboxymethyl chitosan (MCC, polyampholytic) for mucosal immunisation. The derivatives were synthesised and characterised in-house. The aqueous dispersions of the derivatives were also prepared for comparison. The cytotoxicity studies (MTT assay) on Chinese hamster ovary (CHO-K1) cell lines showed that cell viability was in the order of MCC, chitosan and TMC. Nanoparticles were prepared using ionic gelation method and loaded with tetanus toxoid (TT). Nanoparticles with high loading efficacy (>90% m/m), particle size within the range of 40–400nm, with a negative surface charge for MCC and positive surface charge for TMC and chitosan were obtained. The structural integrity of the TT in the formulations was confirmed by SDS-PAGE electrophoresis analysis. The effective uptake of the FITC-BSA loaded nanoparticles into the cells was demonstrated by cellular uptake studies using J774A.1 cells. Immune responses induced by the formulations loaded with tetanus toxoid were studied in vivo in Balb/c mice. Enhanced immune responses were obtained with intranasal (i.n.) application of nanoparticle formulations. Chitosan and TMC nanoparticles which have positively charged surfaces induced higher serum IgG titres when compared to those prepared with MCC which are negatively charged and smaller in size. Nanoparticle formulations developed in this study can be used as promising adjuvant/delivery systems for mucosal immunisation. [Copyright &y& Elsevier]

Abstract: The aim of this research was to investigate the effect of degrees of quaternization (DQ) and dimethylation (DD) on physicochemical properties and cytotoxicity of N-trimethyl chitosan (TMC). TMC was synthesized by reductive methylation of chitosan in the presence of a strong base at elevated temperature and polymer characteristics were investigated. The number of methylation process and duration of reaction were demonstrated to affect the DQ and DD. An increased number of reaction steps increased DQ and decreased DD, while an extended duration of reaction increased both DQ and DD. The molecular weight of TMC was in the range of 60–550kDa. From the Mark–Houwink equation, it was found that TMC in 2% acetic acid/0.2M sodium acetate behaved as a spherical structure, approximating a random coil. The highest solubility was found with TMC of an intermediate DQ (40%) regardless of DD and molecular weight. The effect of DD on the physicochemical properties and cytotoxicity was obviously observed when proportion of DD to DQ was higher than 1. TMC with relatively high DD showed reduction in both solubility and mucoadhesion and hence decreased cytotoxicity. However, the influence of DD was insignificant when DQ of TMC was higher than 40% at which physicochemical properties and cytotoxicity were mainly dependent upon DQ. [Copyright &y& Elsevier]

Abstract: The aim of this study was to evaluate the transdermal permeation enhancement of N-trimethyl chitosan (TMC) with different degrees of quaternization (DQ). TMCs with DQ of 40 and 60% (TMC40 and TMC60) were synthesized and characterized by 1H NMR. Testosterone (TS) used as an effective drug, four different gels were prepared without enhancer, with 5% TMC40, 5% TMC60 or 2% Azone, respectively as enhancer. The effect of TMC60 on the stratum corneum was studied by Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR) combined with the technique of deconvolution. The results showed that TMC60 could significantly affect the secondary structure of keratin in stratum corneum. In vitro permeation studies were carried out using Franz-diffusion cells and in vivo studies were performed in rabbits. Both in vitro and in vivo permeation studies suggested the transdermal permeation enhancement of TMCs. Compared to the TS gel without enhancer, TS gels with enhancers all showed significant enhancing effect on transdermal permeation of TS (P <0.05). Meanwhile, compared to 2% Azone, 5% TMC60 had a stronger enhancement (P <0.05) while 5% TMC40 had a similar effect (P >0.05). The results suggested that the enhancement of TMCs increased with the increase of DQ. [Copyright &y& Elsevier]

Abstract: In this study, the effects of alginate modification on absorption properties of FITC-BSA loaded TMC nanoparticles were investigated on an in vitro model of GI epithelium (Caco-2 cells). The feasibility of applying TMC nanoparticles loaded with a model vaccine urease in oral vaccination was also studied. Alginate modified TMC nanoparticles showed higher FITC-BSA permeate efficiency than non-modified TMC nanoparticles. However, alginate modification barely had any effect on TMC nanoparticles’ property of decreasing TEER or enhancing drug paracellular transport. Mice s.c. immunized with urease loaded TMC nanoparticles showed highest systematic immune response (IgG levels) but the lowest mucosal response (secretory IgA levels). In the contrast, mice i.g. immunized with urease loaded TMC nanoparticles showed much higher antibody titers of both IgG and secretory IgA than those with urease solution or urease co-administrated with TMC solution. These results indicated that TMC nanoparticles are potential carriers for oral protein and vaccine delivery. [Copyright &y& Elsevier]

N -trimethyl chitosan (TMC) is a multifunctional polymer that can be used in various nanoparticle forms in the pharmaceutical, nutraceutical and biomedical fields. In this study, TMC was used as a mucoadhesive adjuvant to enhance the oral bioavailability and hence antitumour effects of gemcitabine formulated into nanocomplexes composed of poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) conjugated with d-α-tocopheryl polyethylene glycol 1000 succinate (TPGS). A central composite design was applied to achieve the optimal formulation. Cellular uptake and drug transportation studies revealed the nanocomplexes permeate over the intestinal cells via adsorptive-mediated and caveolae-mediated endocytosis. Pharmacokinetic studies demonstrated the oral drug bioavailability of the nanocomplexes was increased 5.1-fold compared with drug solution. In pharmacodynamic studies, the formulation reduced tumour size 3.1-fold compared with the drug solution. The data demonstrates that TMC modified nanocomplexes can enhance gemcitabine oral bioavailability and promote the anticancer efficacy. [Display omitted] • Synthesized TMC modifies the PLGA-TPGS nanocomplexes to enhance oral absorption. • The central composite design model was applied to obtain the optimal formulation. • The drug release mechanism is governed by anomalous diffusion. • TMC led cell uptake of particles by adsorptive and caveolae-mediated endocytosis. • The carrier greatly enhanced oral drug bioavailability and inhibited tumour growth. [ABSTRACT FROM AUTHOR]