Evaluation of the antifungal properties of nanoliposomes containing rhinacanthin-C isolated from the leaves of Rhinacanthus nasutus.

Fungal infections represent a challenging threat to the human health. Microsporum gypseum and Trichophyton rubrum are pathogenic fungi causing various topical mycoses in humans. The globally emerging issue of resistance to fungi demands the development of novel therapeutic strategies. In this context, the application of nanoliposomes as vehicles for carrying active therapeutic agents can be a suitable alternative. In this study, rhinacanthin-C was isolated from Rhinacanthus naustus and encapsulated in nano-liposomal formulations, which were prepared by the modified ethanol injection method. The two best formulations composed of soybean phosphatidylcholine (SPC), cholesterol (CHL), and tween 80 (T80) in a molar ratio of 1:1:0 (F1) and 1:1:0.5 (F2) were proceeded for experimentation. The physical characteristics and antifungal activities were performed and compared with solutions of rhinacanthin-C. The rhinacanthin-C encapsulating efficiencies in F1 and F2 were 94.69 ± 1.20% and 84.94 ± 1.32%, respectively. The particle sizes were found to be about 221.4 ± 13.76 nm (F1) and 115.8 ± 23.33 nm (F2), and zeta potential values of -38.16 mV (F1) and -40.98 mV (F2). Similarly, the stability studies of rhinacanthin-C in liposomes demonstrated that rhinacanthin-C in both formulations was more stable in mediums with pH of 4.0 and 6.6 than pure rhinacanthin-C when stored at the same conditions. Rhinacanthin-C in F1 was slightly more stable than F2 when stored in mediums with a pH of 10.0 after three months of storage. However, rhinacanthin-C in both formulations was less stable than pure rhinacanthin-C in a basic medium of pH 10.0. The antifungal potential was evaluated against M. gypsum and T. rubrum. The findings revealed a comparatively higher zone of inhibition for F1. In the MIC study, SPC: CHL: T80 showed higher inhibition against M. gypseum and a slightly higher inhibition against T. rubrum compared to free rhinacanthin-C solution. Moreover, rhinacanthin-C showed significant interaction against 14α-demethylase in in silico study. Overall, this study demonstrates that nanoliposomes containing rhinacanthin-C can improve the stability and antifungal potential of rhinacanthin-C with sustained and prolonged duration of action and could be a promising vehicle for delivery of active ingredients for targeting various fungal infections.

Development of pH-Responsive N-benzyl-N-O-succinyl Chitosan Micelles Loaded with a Curcumin Analog (Cyqualone) for Treatment of Colon Cancer.

This work aimed at preparing nanomicelles from N-benzyl-N,O-succinyl chitosan (NBSCh) loaded with a curcumin analog, 2,6-bis((3-methoxy-4-hydroxyphenyl) methylene) cyclohexanone, a.k.a. cyqualone (CL), for antineoplastic colon cancer chemotherapy. The CL-loaded NBSCh micelles were spherical and less than 100 nm in size. The entrapment efficiency of CL in the micelles ranged from 13 to 39%. Drug release from pristine CL was less than 20% in PBS at pH 7.4, whereas the release from CL-NBSCh micelles was significantly higher. The release study of CL-NBSCh revealed that around 40% of CL content was released in simulated gastric fluid at pH 1.2; 79 and 85% in simulated intestinal fluids at pH 5.5 and 6.8, respectively; and 75% in simulated colonic fluid at pH 7.4. CL-NBSCh showed considerably high selective cytotoxicity towards mucosal epithelial human colon cancer (HT-29) cells and lower levels of toxicity towards mouse connective tissue fibroblasts (L929). CL-NBSCh was also more cytotoxic than the free CL. Furthermore, compared to free CL, CL-NBSCh micelles were found to be more efficient at arresting cell growth at the G2/M phase, and induced apoptosis earlier in HT-29 cells. Collectively, these results indicate the high prospective potential of CL-loaded NBSCh micelles as an oral therapeutic intervention for colon cancer.

Enzyme-Immobilized Chitosan Nanoparticles as Environmentally Friendly and Highly Effective Antimicrobial Agents.

Highly effective and minimally toxic antimicrobial agents have been prepared by immobilizing glucose oxidase (GOx) onto biocompatible chitosan nanoparticles (CS-NPs). CS-NPs were prepared via ionotropic gelation and used for the immobilization of GOx via approaches of covalent attachment (CA), enzyme coating (EC), enzyme precipitate coating (EPC), and magnetic nanoparticle-incorporated EPC (Mag-EPC). EPC represents an approach consisting of enzyme covalent attachment, precipitation, and cross-linking, with CA and EC being control samples while Mag-EPC was prepared by mixing magnetic nanoparticles (Mag) with enzymes during the preparation of EPC. The GOx activities of CA, EC, EPC, and Mag-EPC were 8.57, 17.7, 219, and 247 units/mg CS-NPs, respectively, representing 26 and 12 times higher activity of EPC than those of CA and EC, respectively. EPC improved the activity and stability of GOx and led to good dispersion of CS-NPs, while Mag-EPC enabled facile magnetic separation. To demonstrate the expandability of the EPC approach to other enzymes, bovine carbonic anhydrase was also employed to prepare EPC and Mag-EPC samples for their characterizations. In the presence of glucose, EPC of GOx generated H2O2 in situ, which effectively inhibited the proliferation of Staphylococcus aureus in both suspended cultures and biofilms, thereby demonstrating the potential of EPC-GOx as environmentally friendly and highly effective antimicrobial materials.

Effects of silymarin-loaded amphiphilic chitosan polymeric micelles on the renal toxicity and anticancer activity of cisplatin.

This research aimed to evaluate the effects of silymarin (SM)-loaded polymeric micelles (PMs) on the renal toxicity and anticancer activity of cisplatin. Amphiphilic chitosan derivatives were employed to develop SM-loaded PMs. The permeation across an intestinal membrane, cytotoxicity, and renal toxicity of cisplatin during the treatment were evaluated. The SM-loaded PMs had small particle sizes (326-336 nm), negative surface charge, high entrapment efficiency (47-70%), and demonstrated pH-sensitive release. Rapid drug release was obtained at pH 7.4 (81-87% in 4 h). The SM-loaded PMs exhibited higher flux than free SM. Moreover, the pretreatment of SM (50-100 µg/mL)-loaded PMs increased the killing efficacy of cisplatin on the cancer cells. The renoprotective effect was witnessed (p < 0.05) on the cells pretreated with SM-loaded benzyl-functionalized succinyl chitosan (BSC) PMs compared with those treated with only cisplatin, which the % cell viability increased from 29% to 82% and 96% for the PMs with SM concentration of 50 and 100 µg/mL, respectively. Moreover, the reduction in cell apoptosis and necrosis induced by cisplatin has been observed. In conclusion, SM-loaded BSC PMs could improve the bioavailability of SM, enhance the therapeutic effect, and protect renal damage during the treatment with cisplatin.

Gonadotropin-releasing hormone-modified chitosan as a safe and efficient gene delivery vector for spermatogonia cells.

The use of male gonadal tissue as a site for the local delivery of DNA is an interesting concept. Previously, we reported synthesis, physiochemical and biological properties of gonadotropin-releasing hormone (GnRH)-conjugated chitosan as a carrier for DNA delivery to GnRH receptor-overexpressing cells. In this study, the application of modified chitosan as a potential vector for gene delivery to testicular cells was carried out. Transfection efficiency was investigated in mouse-derived spermatogonia cells (GC-1 cells) using green fluorescent protein as a reporter gene. GnRH-conjugated chitosan exhibited higher transfection activity and specificity compared to the unmodified chitosan. Furthermore, the GnRH-modified chitosan showed less cytotoxicity. In conclusion, we have developed and successfully tested the GnRH-modified chitosan for delivery of a transgene of interest to spermatogonia cells in vitro. Such vector could be useful in particular for testis-mediated gene transfer.

Apoptosis Induction and Antimigratory Activity of Andrographolide Analog (3A.1)-Incorporated Self-Assembled Nanoparticles in Cancer Cells.

Andrographolide analog, namely 19-tert-butyldiphenylsilyl-8,17-epoxy andrographolide (or 3A.1) has been reported to be a potential anticancer agent for several types of cancer. Due to its poor aqueous solubility, 3A.1 was incorporated within self-assembly polymeric nanoparticles made of naphthyl-grafted succinyl chitosan (NSC), octyl-grafted succinyl chitosan (OSC), and benzyl-grafted succinyl chitosan (BSC). These 3A.1-loaded nanoparticles were nanosized (< 200 nm) and spherical in shape with a negative surface charge. 3A.1-loaded nanoparticles were produced using a dropping method, which 40% initial drug adding exhibited the highest entrapment efficiency. The release of 3A.1 from the 3A.1-loaded nanoparticles displayed a delayed release pattern. Under acidic conditions (pH 1.2), there was no free drug release. After the pH was adjusted to 6.8, a high cumulative 3A.1 release was obtained which was dependent on the hydrophobic moieties. These 3A.1-loaded pH-sensitive nanoparticles proved to be beneficial for specifically delivering anticancer drugs to the targeted colon cancer sites. In vitro anticancer activity against HT-29 found that the 3A.1-loaded nanoparticles had significantly lower IC50 than that of the free drug and promoted apoptosis. Additionally, in vitro wound-healing migration on HN-22 revealed that free 3A.1 and the 3A.1-loaded nanoparticles inhibited cell motility compared with untreated cells. These pH-sensitive amphiphilic chitosan nanoparticles may be promising nanocarriers for oral anticancer drug delivery to colorectal cancer cells. Graphical abstract ᅟ.

Development of Chitosan-Based pH-Sensitive Polymeric Micelles Containing Curcumin for Colon-Targeted Drug Delivery.

pH-sensitive N-naphthyl-N,O-succinyl chitosan (NSCS) and N-octyl-N,O-succinyl chitosan (OSCS) polymeric micelles carriers have been developed to incorporate curcumin (CUR) for colon-targeted drug delivery. The physical entrapment methods (dialysis, co-solvent evaporation, dropping, and O/W emulsion) were applied. The CUR-loaded micelles prepared by the dialysis method presented the highest loading capacity. Increasing initial amount of CUR from 5 to 40 wt% to polymer resulted in the increase in loading capacity of the polymeric micelles. Among the hydrophobic cores, there were no significant differences in the loading capacity of CUR-loaded micelles. The particle sizes of all CUR-loaded micelles were in the range of 120-338 nm. The morphology of the micelles changed after being contacted with medium with different pH values, confirming the pH-responsive properties of the micelles. The release characteristics of curcumin from all CUR-loaded micelles were pH-dependent. The percent cumulative release of curcumin from all CUR-loaded micelles in simulated gastric fluid (SGF) was limited to about 20%. However, the release amount was significantly increased after contacted with simulated intestinal fluid (SIF) (50-55%) and simulated colonic fluid (SCF) (60-70%). The released amount in SIF and SCF was significantly greater than the release of CUR from CUR powder. CUR-loaded NSCS exhibited the highest anti-cancer activity against HT-29 colorectal cancer cells. The stability studies indicated that all CUR-loaded micelles were stable for at least 90 days. Therefore, the colon targeted, pH-sensitive NSCS micelles may have potential to be a prospective candidate for curcumin delivery to the colon.

Dry Formulations Enhanced Mucoadhesive Properties and Reduced Cold Chain Handing of Influenza Vaccines.

To alleviate concerns in health security, emergency flu vaccine stockpiles are required for ensuring rapid availability of vaccines when needed. Cold chain preservation, at high cost and risk, is necessary to maintain vaccine efficacy. This study aimed to develop a dry, easily storable formula for influenza vaccine preparation. The formulation with mucoadhesive properties is expected to facilitate rapid delivery via nasal administration. Chitosan, a cationic polymer, was used as cryo-protectant and to promote mucoadhesion. Optimal concentrations and molecular weights of chitosan polymers were screened, with short chain chitosan (10 kDa) being most suitable. H1N1 dry powder, in different formulations, was prepared via freeze-drying. A series of cryo-protectants, trehalose (T), chitosan (C), fetal bovine serum (FBS; F), or a combination of these (TCF), were screened for their effects on prolonging vaccine shelf life. Physicochemical monitoring (particle size and zeta potential) of powders complexed with mucin revealed that the order of cryo-protectant mixing during preparation was of critical importance. Results indicated that the TCF formula retains its activity up to 1 year as indicated by TCID50 analysis. This approach was also successful at prolonging the shelf life of H3N2 vaccine, and has the potential for large-scale implementation, especially in developed countries where long-term storage of vaccines is problematic.

Development and evaluation of N-naphthyl-N,O-succinyl chitosan micelles containing clotrimazole for oral candidiasis treatment.

Clotrimazole (CZ)-loaded N-naphthyl-N,O-succinyl chitosan (NSCS) micelles have been developed as an alternative for oral candidiasis treatment. NSCS was synthesized by reductive N-amination and N,O-succinylation. CZ was incorporated into the micelles using various methods, including the dropping method, the dialysis method, and the O/W emulsion method. The size and morphology of the CZ-loaded micelles were characterized using dynamic light scattering measurements (DLS) and a transmission electron microscope (TEM), respectively. The drug entrapment efficiency, loading capacity, release characteristics, and antifungal activity against Candida albicans were also evaluated. The CZ-loaded micelles prepared using different methods differed in the size of micelles. The micelles ranged in size from 120 nm to 173 nm. The micelles prepared via the O/W emulsion method offered the highest percentage entrapment efficiency and loading capacity. The CZ released from the CZ-loaded micelles at much faster rate compared to CZ powder. The CZ-loaded NSCS micelles can significantly hinder the growth of Candida cells after contact. These CZ-loaded NSCS micelles offer great antifungal activity and might be further developed to be a promising candidate for oral candidiasis treatment.

Synthesis and fluorescence properties of N-substituted 1-cyanobenz[f]isoindole chitosan polymers and nanoparticles for live cell imaging.

Highly fluorescent N-substituted 1-cyanobenz[f]isoindole chitosans (CBI-CSs) with various degrees of N-substitution (DS) were synthesized by reacting chitosan (CS) with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide under mild acidic conditions. Introduction of 1-cyanobenz[f]isoindole moieties into the CS backbone resulted in lowering of polymer thermal stability and crystallinity. The fluorescence quantum yield (Φf) of CBI-CS was found to be DS- and molecular-weight-dependent, with Φf decreasing as DS and molecular weight were increased. At similar DS values, CBI-CS exhibited 26 times higher Φf in comparison with fluorescein isothiocyanate-substituted chitosan (FITC-CS). CBI-CS/TPP nanoparticles were fabricated using an ionotropic gelation method in which pentasodium triphosphate (TPP) acted as a cross-linking agent. CS and CBI-CS exhibited low cytotoxicity to normal skin fibroblast cells over a concentration range of 0.1-1000 µg/mL, while an increased cytotoxicity level was evident in CBI-CS/TPP nanoparticles at concentrations greater than 100 µg/mL. In contrast with CBI-CS polymers, the CBI-CS/TPP nanoparticles exhibited lower fluorescence; however, confocal microscopy results showed that living normal skin fibroblast cells became fluorescent on nanoparticle uptake. These results suggest that CBI-CS and fabricated nanoparticles thereof may be promising fluorescence probes for live cell imaging.

Preparation of Self-Assembled, Curcumin-Loaded Nano-Micelles Using Quarternized Chitosan-Vanillin Imine (QCS-Vani Imine) Conjugate and Evaluation of Synergistic Anticancer Effect with Cisplatin.

Nano-micelles are self-assembling colloidal dispersions applied to enhance the anticancer efficacy of chemotherapeutic agents. In this study, the conjugate of quarternized chitosan and vanillin imine (QCS-Vani imine) was synthesized using the reaction of a Schiff base characterized by proton-NMR (1HNMR), UV-Vis spectroscopy, and FT-IR. The critical micelle concentration (CMC), particle size, and zeta potential of the resulting product were determined. The QCS-Vani imine conjugate was used as a carrier for the development of curcumin-loaded nano-micelles, and their entrapment efficiency (%EE), drug-loading capacity (%LC) and in vitro release were investigated using HPLC analysis. Moreover, the nano-micelles containing curcumin were combined with various concentrations of cisplatin and evaluated for a possible anticancer synergistic effect. The anticancer activity was evaluated against lung cancer A549 and mouse fibroblast L929 cell lines. The percent yield (%) of the QCS-Vani imine conjugate was 93.18%. The curcumin-loaded QCS-Vani imine nano-micelles were characterized and found to have a spherical shape (by TEM) with size < 200 nm (by DLS) with high %EE up to 67.61% and %LC up to 6.15 ± 0.41%. The loaded lyophilized powder of the nano-micelles was more stable at 4 °C than at room temperature during 120 days of storage. pH-sensitive release properties were observed to have a higher curcumin release at pH 5.5 (cancer environment) than at pH 7.4 (systemic environment). Curcumin-loaded QCS-Vani imine nano-micelles showed higher cytotoxicity and selectivity toward lung cancer A549 cell lines and exhibited lower toxicity toward the normal cell (H9C2) than pure curcumin. Moreover, the curcumin-loaded QCS-Vani imine nano-micelles exhibited an enhanced property of inducing cell cycle arrest during the S-phase against A549 cells and showed prominently induced apoptosis in lung cancer cells compared to that with curcumin. The co-treatment of cisplatin with curcumin-loaded QCS-Vani imine nano-micelles presented an enhanced anticancer effect, showing 8.66 ± 0.88 µM as the IC50 value, in comparison to the treatment with cisplatin alone (14.22 ± 1.01 µM). These findings suggest that the developed QCS-Vani imine nano-micelle is a potential drug delivery system and could be a promising approach for treating lung cancer in combination with cisplatin.

Validated HPLC method for simultaneous quantitative determination of dimethylcurcumin and resveratrol in pluronic-F127 nanomicelles: Formulation with enhanced anticancer efficacy.

Nano-micelles offer a promising vehicle for the delivery various therapeutically significant biologicals. Development of convenient and efficient chromatographic methods for the quantitative determination of the active pharmaceutical ingredients in such systems is of immense importance. In this study pluronic-F-127 nano-micelles were prepared and loaded with dimethylcurcumin (DMC) and resveratrol (Res). A simple, convenient and effective HPLC method was developed for the quantitative estimation of DMC and Res in the polymeric nano-micelles through a single injection. A reverse-phase ACE® C18 column (250 mm × 4.6 mm) was used with a gradient mobile phase system consisting of 1 % MeOH and 0.1 % H3PO4:100 % acetonitrile at 1 mL/min flow rate with UV detection for Res, and fluorescence detector for DMC. The calibration curves generated for both the compounds were found linear with r2 values of 1.000 over a concentration range of 2-25 µg/mL with low limit of detection (LOD) values of 0.37 and 0.16 µg/mL for DMC and Res respectively and limit of quantification (LOQ) values of 1.23 and 0.55 µg/mL for DMC and Res respectively. Similarly, accuracy was found in a range of 98.80 -102.47 % for DMC and 100.58-101.77 % for Res. Furthermore, the within-run precisions (%RSD) were 0.073 - 0.444% for DMC and 0.159 - 0.917% for Res, while between-run precisions (%RSD) were 0.344 - 1.47 for DMC and 0.458 - 1.651 for Res. Moreover, the DMC with Res co-loaded nanomicelles showed higher activity against MCF-7 and MDA-MB 231 compared to DMC and Res alone. Overall, this study presented a simple, convenient, precise and accurate method for the quantitative determination of DMC and Res in polymeric nano-micelles which have anticancer potential.•A simple HPLC for the quantitative determination of DMC and Res in nanomicelles having anti-cancer potential.•Non complicate with high degree of recoveries of sample preparation process.•This method can be used to determine a mixture of DMC and Res in pharmaceutical formulation in single injection.

Quaternization of high molecular weight chitosan for increasing intestinal drug absorption using Caco-2 cells as an in vitro intestinal model.

Potential use of a quaternized chitosan (MW 600 kDa) with 65% of 3-chloro-2-hydroxypropyltrimethylammonium (600-HPTChC65) as an absorptive enhancer was investigated in Caco-2 monolayers. 600-HPTChC65 (0.005% w/v) quickly reduced transepithelial electrical resistance (TEER) to the maximum level in 40 min with full recovery within 6 h after removal. Its TEER reduction was corresponded to increased FD4 transport across the monolayers and disrupted localization of tight junction proteins ZO-1 and occludin at the cell borders. 600-HPTChC65 was densely localized at the membrane surface and intercellular junctions. This chitosan (0.08-0.32% w/v) reduced the efflux ratio of [3H]-digoxin by 1.7- 2 folds, suggesting an increased [3H]-digoxin transport across the monolayers. Its binding with P-gp on Caco-2 monolayer increased the signal of fluorescence-labeled anti-P-gp (UIC2) reactivity due to conformational change. 600-HPTChC65 (0.32% w/v) had no effect on P-gp expression in the Caco-2 monolayers. These results suggest that 600-HPTChC65 could enhance drug absorption through tight junction opening and decreased P-gp function. Its interaction with the absorptive barrier mainly resulted in disrupting ZO-1 and occludin organization as well as changing in P-gp conformation.

Fouling mitigation in an anaerobic membrane bioreactor via membrane surface modification with tannic acid and copper.

Anaerobic membrane bioreactors (AnMBRs) have recently received a great amount of attention as an alternative anaerobic treatment process due to their superior capability for sludge retention with high effluent quality. Nevertheless, membrane fouling in AnMBRs has been a major concern. In this study, the surfaces of polyvinylidene fluoride (PVDF) ultrafiltration membranes were modified with tannic acid (TA) and Cu(II) at various molar ratios of TA to Cu(II), including 3:1, 2:1, 1:1, 1:2, and 1:3. The hydrophilicity, morphology, chemical structure, elemental composition, and antibacterial properties of the unmodified and modified membranes were analyzed using water contact angle measurements, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), SEM-energy dispersive X-ray spectrometry (SEM-EDX), and the clear zone method, respectively. The modified membrane with a TA-to-Cu(II) molar ratio of 1:3 had high hydrophilicity with certain antibacterial properties; therefore, it was selected to be further tested in an AnMBR along with an unmodified membrane. The chemical oxygen demand (COD) removal efficiencies of the unmodified membrane and modified membrane were 92.2 ± 3.6% and 91.8 ± 4.0%, respectively. The modified membrane had higher permeability after backwashing with less chemical cleaning (CC) than the unmodified membrane. Surface modification with TA and Cu(II) appeared to reduce irreversible fouling on the membranes.

Loading of curcumin in polyelectrolyte multilayers.

Polyelectrolyte multilayer (PEM) thin films prepared using the layer-by-layer technique are proposed as a matrix for the immobilization of 1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-2,5-dione (curcumin), a lipophilic model drug. The PEM assembly was based on the layer-by-layer deposition of cationic poly(diallyldimethyl-ammonium chloride) (PDADMAC) and anionic poly(4-styrene sulfonate, sodium salt) (PSS) onto a quartz slide. Curcumin was loaded by dipping the PEM film into a dilute solution of curcumin dispersed in an 80/20% v/v water/ethanol solution. Within a few minutes, the film turned bright yellow as a result of the curcumin loading. The effect of the solvent composition, curcumin concentration and film thickness on the final concentration of curcumin in the PEM films was measured by UV-vis spectroscopy. The loading of curcumin was driven by its partitioning in the PEM film, and its partitioning coefficient between the 80/20 solvent and the PEM thin film was found to have a value of 2.07 x 10(5). The extinction coefficient of curcumin loaded into PEM was calculated to 64,000 M(-1) cm(-1). Results show that the loading of curcumin into the PEM films increased with the number of deposited layers, implying that curcumin partitioned into the bulk of the thin film. The maximum curcumin dose in the PEM film was measured by exposing films of various thicknesses to a high concentration (0.01% w/v) of curcumin and recording the maximum absorbance after saturation. The films thicknesses were controlled by the number of deposited PDADMAC/PSS layers (10, 20, 30, 40, 50, and 60). Results show that increasing amounts of curcumin could be loaded into the film with an increasing number of layers and up to 8 microg/cm(2) of curcumin could be loaded into a 20-layer film. These results demonstrate that the loading of lipophilic curcumin in PEM thin films is done through a partitioning mechanism and that the PDADMAC/PSS film can be used as a loading matrix for lipophilic drugs.

In vitro permeability enhancement in intestinal epithelial cells (Caco-2) monolayer of water soluble quaternary ammonium chitosan derivatives.

The aim of this study was to investigate the effects of a type of hydrophobic moiety, extent of N-substitution (ES), and degree of quaternization (DQ) of chitosan (CS) on the transepithelial electrical resistance and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4,400 (FD-4) as the model compound for paracellular tight junction transport. CS was substituted with hydrophobic moiety, an aliphatic aldehyde (n-octyl) or aromatic aldehyde (benzyl), for the improved hydrophobic interaction with cell membrane, and they were quaternized with Quat-188 to render CS soluble. The factors affecting the epithelial permeability have been evaluated in the intestinal cell monolayers, Caco-2 cells. Cytotoxicity was evaluated by using the trypan blue and MTT viability assay. The results revealed that at pH 7.4 CSQ appeared to increase cell permeability in dose-dependent manner, and this effect was relatively reversible at the lower doses of 0.05-1.25 mM. The higher DQ and ES caused the higher permeability of FD-4. Cytotoxicity of CSQ was concentration, %DQ, and %ES dependent. Substitution with hydrophobic moiety caused decreasing in permeability of FD-4 and cytotoxicity by benzyl group had more effect than octyl group. These studies demonstrated that these novel modified chitosan derivatives had potential for using as absorption enhancers.

Flexible and Hydrophilic Copolyamide Thin-Film Composites on Hollow Fiber Membranes for Enhanced Nanofiltration Performance.

Flexible and hydrophilic copolyamide (Co-PA) thin-film composite (TFC) membranes were fabricated as a selective layer on the outer surface of the polyvinylidene fluoride hollow fiber membrane substrate. The fabrication process was carried out by the dip-coating method to create three TFC membranes. The first layer is tannic acid and the second layer is (3-aminopropyl)triethoxysilane, which is followed by Co-PA as a final selective layer. The Co-PA TFC membrane was prepared through interfacial polymerization via the combination of various short-chain aliphatic diamines and conventional aromatic diamines with trimesoyl chloride. The influence of coating layers and total diamine concentration on the Co-PA TFC membrane was investigated in terms of the membrane's physicochemical and mechanical properties, morphology, surface thickness and roughness, water contact angle, surface charge, and nanofiltration (NF) performance. The obtained Co-PA TFC membrane system was operated under low pressure (2 bar) with pure water flux in the range of 23.8-83.9 L m-2 h-1 and exhibited better hydrophilicity, flexibility, molecular weight cutoff, and NF performance compared to the conventional PA TFC membrane. The superior properties of Co-PA are due to the increased chain mobilities provided by short-chain aliphatic diamines in its structure. The best Co-PA TFC membranes, which were synthesized using diamines containing four carbon atoms, achieved a significant improvement in NF membrane performance and selectivity (pure water flux = 56.9 L m-2 h-1 and salt and dye rejection in the range of 46.2-99.2%). This Co-PA TFC membrane is a promising membrane for its high flexibility, hydrophilicity, and selectivity of the NF membrane.

Methylated N-(4-N,N-dimethylaminobenzyl) chitosan for novel effective gene carriers.

The objective of this study was to investigate the transfection efficiency of quaternized N-(4-N,N-dimethylaminobenzyl) chitosan, TM(47)-Bz(42)-CS, using the plasmid DNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh7 cells), in comparison to quaternized chitosan (TM(43)-CS) and chitosan (CS). Factors affecting the transfection efficiency, such as the carrier/DNA weight ratio, the pH of the culture medium, and the presence of serum, have been investigated. The results revealed that TM(47)-Bz(42)-CS was able to condense with pDNA. As illustrated by the agarose gel electrophoresis, the complete complexes of TM(47)-Bz(42)-CS/DNA were formed at a weight ratio of above 0.5, whereas those of TM(43)-CS/DNA and CS/DNA were formed at a ratio of above 1. TM(47)-Bz(42)-CS showed superior transfection efficiency to TM(43)-CS and CS at all weight ratios tested. Higher transfection efficiency and gene expression were observed when the carrier/DNA weight ratios increased. The highest transfection efficiency was found at a weight ratio of 8. The results indicated that the improved gene transfection was due to the hydrophobic group (N,N-dimethylaminobenzyl) substitution on CS, which promoted the interaction and condensation with DNA, as well as N-quaternization, which increased the CS water solubility. During cytotoxicity studies, it was found that high concentrations of TM(47)-Bz(42)-CS and TM(43)-CS could decrease the Huh7 cell viability. In conclusion, this novel CS derivative, TM(47)-Bz(42)-CS, shows promising potential as a gene carrier by efficient DNA condensation and a mediated higher level of gene transfection in Huh7 cells.

Methylated N-(4-pyridinylmethyl) chitosan as a novel effective safe gene carrier.

The objective of this study was to study the transfection efficiency of quaternized N-(4-pyridinylmethyl) chitosan; TM-Py-CS, using the pDNA encoding green fluorescent protein (pEGFP-C2) on human hepatoma cell lines (Huh 7 cells). The factors affecting the transfection efficiency, e.g. degree of quaternization (DQ), the extent of N-pyridinylmethyl substitution (ES) and weight ratio, have been investigated. The results revealed that TM-Py-CS was able to condense with pDNA. Illustrated by agarose gel electrophoresis, complete complexes of TM(69)Py(62)CS/DNA were formed at weight ratio above 1.1, whereas those of TM(53)Py(40)CS/DNA and TM(52)Py(13)CS/DNA were above 1.8 and 8, respectively. TM(69)Py(62)CS showed superior transfection efficiency to TM(53)Py(40)CS, TM(52)Py(13)CS, TM(65)CS and TM(43)CS at all weight ratios tested. The highest transfection efficiency of TM(69)Py(62)CS/DNA complexes was found at weight ratio of 4. The results indicated that the improved gene transfection was possibly due to 4-pyridinylmethyl substitution on CS which promoted the interaction and condensation with DNA as well as N-quaternization which increased CS water solubility. In cytotoxicity studies, high concentration of TM-Py-CS and TM-CS could decrease the Huh 7 cell viability. In conclusion, this novel CS derivative, TM(69)Py(62)CS, showed promising potential as a gene carrier by efficient DNA condensation and mediated higher level of gene transfection.

Synthesis and characterization of N-aryl chitosan derivatives.

Selective N-arylation of chitosan was performed via a Schiff bases formed by the reaction between the 2-amino group of glucosamine residue of chitosan with an aromatic aldehyde under acidic condition followed by reduction of the Schiff base intermediate with sodium cyanoborohydride (Borch reduction). Aromatic aldehydes bearing either an electron donating or electron withdrawing substituent were used. The chemical structures and thermal properties of the N-aryl chitosans were characterized by FT-IR, (1)H NMR, (13)C NMR, TGA, and DSC. The extent of N-substitution (ES) was influenced by the molar ratio of the aldehyde to the glucosamine residue of chitosan, the reaction time and the substituent on the aromatic ring. Lower ESs resulted from N-arylation using an aldehyde with an electron donating substituent. A linear relationship between the targeted ES and the ES obtained was observed when aldehydes bearing electron withdrawing substituents were employed.