Theerasak Rojanarata

Lysozyme-loaded, electrospun chitosan-based nanofiber mats for wound healing

In this study, a blend mixture of chitosan-ethylenediaminetetraacetic acid (CS 2 wt%-EDTA) at a weight ratio of 30/70 and polyvinyl alcohol (PVA) solution (10 wt%) was electrospun to produce fibrous mats with lysozyme (10, 20 and 30 wt%) used for wound healing. The morphology and diameter of the electrospun fiber mats with and without lysozyme were analyzed by scanning electron microscopy (SEM). The amount of lysozyme loaded in the nanofiber mats was measured by HPLC. The cell lysis activity of the lysozyme was investigated with Micrococcus lysodeikticus cells as a substrate. The wound healing activity was performed in vivo using male Wistar rats. The SEM images of all lysozyme-loaded fibers show a smooth fiber without beads with an average diameter of 143-209 nm. The amount of lysozyme loaded in the nanofiber mats was slightly decreased when the initial concentration of lysozyme was increased. The rapid lysozyme release from the nanofiber mats was obtained and is dependent on the lysozyme-loading amount. In animal wound healing, lysozyme loaded CS-EDTA nanofiber mats accelerated the rate of wound healing when compared to the controls (gauze). In conclusion, our experiments demonstrated that biomaterials composed of lysozyme loaded CS-EDTA nanofibers have a potential for wound healing.

Characterization and In Vitro Skin Permeation of Meloxicam-Loaded Liposomes versus Transfersomes

The goal of this study was to develop and evaluate the potential use of liposome and transfersome vesicles in the transdermal drug delivery of meloxicam (MX). MX-loaded vesicles were prepared and evaluated for particle size, zeta potential, entrapment efficiency (%EE), loading efficiency, stability, and in vitro skin permeation. The vesicles were spherical in structure, 90 to 140 nm in size, and negatively charged (−23 to −43 mV). The %EE of MX in the vesicles ranged from 40 to 70%. Transfersomes provided a significantly higher skin permeation of MX compared to liposomes. Fourier Transform Infrared Spectroscopy (FT-IR) and Differential Scanning Calorimetry (DSC) analysis indicated that the application of transfersomes significantly disrupted the stratum corneum lipid. Our research suggests that MX-loaded transfersomes can be potentially used as a transdermal drug delivery system.

Development of Meloxicam-Loaded Electrospun Polyvinyl Alcohol Mats as a Transdermal Therapeutic Agent

This study reports on the use of electrospun polyvinyl alcohol (PVA) nanofiber mats loaded with meloxicam (MX) as a transdermal drug delivery system. The amounts of MX loaded in the base PVA solution (10% w/v solution) were 2.5, 5, 10 and 20% weight, based on the dry weight of PVA (% wt). The average diameters of these fibers ranged from 121–185 nm. In all concentrations of MX loaded in spun PVA fiber mats, an amorphous nanodispersion of MX with PVA was obtained. Both the degree of swelling and the weight loss of the electrospun PVA mats were greater than those of the as-cast PVA films. The tensile strength of the as-spun fiber mats was lower than that of the as-cast PVA films, but the strain at the maximum of the as-spun fiber mats was about six times higher than that of the as-cast PVA films. The skin permeation flux of the MX permeated from MX-loaded as-spun PVA were significantly higher than from MX-loaded as-cast PVA films, and increased when the MX content in both MX-loaded as-spun PVA and MX-loaded as-cast PVA films was increased. Our research suggests a potential use for MX-loaded electrospun PVA mats as a transdermal drug delivery system.

Chitosan lactate as a nonviral gene delivery vector in COS-1 cells

The purpose of this research was to evaluate chitosan lactate (CL) of different molecular weights (MWs) as a DNA complexing agent for its efficiency in transfecting COS-1 cells (green monkey fibroblasts) and its effect on cell viability compared with polyethylenimine (PEI), a commercially available cationic polymer. CL and chitosan base dissolved in dilute acetic acid (chitosan acetate, [CA]) of different MWs (20, 45, 200, 460 kDa) and N/P ratios (2∶1, 4∶1, 8∶1, 12∶1, 24∶1) formed complexes with pSV β-galactosidase plasmid DNA. The complexes were characterized by agarose gel electrophoresis and investigated for their ability to transfect COS-1 cells compared with PEI. Additionally, the effect of CL on the viability of COS-1 cells was investigated using 3-(4,5-dimethyliazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The binding of CL/DNA and CA/DNA was dependent on chitosan MWs. The N/P ratio of CL to completely form the complex with the DNA was higher than that of CA. Both CL and CA were comparable in transfection efficiencies at an N/P ratio of 12∶1, but less efficient than PEI (P<.05). The cell viability in the presence of CL and CA at all MWs was over 90%, whereas that of PEI-treated cells was ≈50%. These results suggest the advantage of CL for in vitro gene transfection, with the ease of preparation of polymer/DNA complexes and low cytotoxicity.

Electrospun chitosan-based nanofiber mats loaded with Garcinia mangostana extracts.

The aim of this study was to prepare electrospun chitosan-based nanofiber mats and to incorporate the fruit hull of Garcinia mangostana (GM) extracts into the mats. Chitosan-ethylenediaminetetraacetic acid/polyvinyl alcohol (CS-EDTA/PVA) was selected as the polymers. The GM extracts with 1, 2 and 3 wt% α-mangostin were incorporated into the CS-EDTA/PVA solution and electrospun to obtain nanofibers. The morphology and diameters of the mats were analyzed using scanning electron microscopy (SEM). The mechanical and swelling properties were investigated. The amount of GM extracts was determined using high-performance liquid chromatography (HPLC). The antioxidative activity, antibacterial activity, extract release and stability of the mats were evaluated. In vivo wound healing tests were also performed in Wistar rats. The results indicated that the diameters of the fibers were on the nanoscale and that no crystals of the extract were observed in the mats at any concentration. The mats provided suitable tensile strength and swelling properties. All of the mats exhibited antioxidant and antibacterial activity. During the wound healing test, the mats accelerated the rate of healing when compared to the control (gauze-covered). The mats maintained 90% of their content of α-mangostin for 3 months. In conclusion, the chitosan-based nanofiber mats loaded with GM extracts were successfully prepared using the electrospinning method. These nanofiber mats loaded with GM extracts may provide a good alternative for accelerating wound healing.

Evaluation of Meloxicam-Loaded Cationic Transfersomes as Transdermal Drug Delivery Carriers

The aim of this study is to develop meloxicam (MX)-loaded cationic transfersomes as skin delivery carriers and to investigate the influence of formulation factors such as cholesterol and cationic surfactants on the physicochemical properties of transfersomes (i.e., particle size, size distribution, droplet surface charge and morphology), entrapment efficiency, stability of formulations and in vitro skin permeation of MX. The transfersomes displayed a spherical structure. Their size, charge, and entrapment efficiency depended on the composition of cholesterol and cationic surfactants in the formulation. Transfersomes provided greater MX skin permeation than conventional liposomes and MX suspensions. The penetration-enhancing mechanism of skin permeation by the vesicles prepared in this study may be due to the vesicle adsorption to and/or fusion with the stratum corneum. Our results suggest that cationic transfersomes may be promising dermal delivery carriers of MX.

Neomycin-loaded poly(styrene sulfonic acid-co-maleic acid) (PSSA-MA)/polyvinyl alcohol (PVA) ion exchange nanofibers for wound dressing materials.

In this study, poly(styrene sulfonic acid-co-maleic acid) (PSSA-MA) blended with polyvinyl alcohol (PVA) was electrospun and then subjected to thermal crosslinking to produce PSSA-MA/PVA ion exchange nanofiber mats. The cationic drug neomycin (0.001, 0.01, and 0.1%, w/v) was loaded onto the cationic exchange fibers. The amount of neomycin loaded and released and the cytotoxicity of the fiber mats were analyzed. In vivo wound healing tests were also performed in Wistar rats. The results indicated that the diameters of the fibers were on the nanoscale (250 ± 21 nm). The ion exchange capacity (IEC) value and the percentage of water uptake were 2.19 ± 0.1 mequiv./g-dry fibers and 268 ± 15%, respectively. The loading capacity was increased upon increasing the neomycin concentration. An initial concentration of 0.1% (w/v) neomycin (F3) showed the highest loading capacity (65.7 mg/g-dry fibers). The neomycin-loaded nanofiber mats demonstrated satisfactory antibacterial activity against both Gram-positive and Gram-negative bacteria, and an in vivo wound healing test revealed that these mats performed better than gauze and blank nanofiber mats in decreasing acute wound size during the first week after tissue damage. In conclusion, the antibacterial neomycin-loaded PSSA-MA/PVA cationic exchange nanofiber mats have the potential for use as wound dressing materials.

Electrospun chitosan/polyvinyl alcohol nanofibre mats for wound healing.

Chitosan (CS) aqueous salt blended with polyvinyl alcohol (PVA) nanofibre mats was prepared by electrospinning. CS was dissolved with hydroxybenzotriazole (HOBt), thiamine pyrophosphate (TPP) and ethylenediaminetetraacetic acid (EDTA) in distilled water without the use of toxic or hazardous solvents. The CS aqueous salts were blended with PVA at different weight ratios, and the effect of the solution ratios was investigated. The morphologies and mechanical and swelling properties of the generated fibres were analysed. Indirect cytotoxicity studies indicated that the CS/PVA nanofibre mats were non‐toxic to normal human fibroblast cells. The CS‐HOBt/PVA and CS‐EDTA/PVA nanofibre mats demonstrated satisfactory antibacterial activity against both gram‐positive and gram‐negative bacteria, and an in vivo wound healing test showed that the CS‐EDTA/PVA nanofibre mats performed better than gauze in decreasing acute wound size during the first week after tissue damage. In conclusion, the biodegradable, biocompatible and antibacterial CS‐EDTA/PVA nanofibre mats have potential for use as wound dressing materials.

Fabrication of a novel scaffold of clotrimazole-microemulsion-containing nanofibers using an electrospinning process for oral candidiasis applications.

Clotrimazole (CZ)-loaded microemulsion-containing nanofiber mats were developed as an alternative for oral candidiasis applications. The microemulsion was composed of oleic acid (O), Tween 80 (T80), and a co-surfactant such as benzyl alcohol (BzOH), ethyl alcohol (EtOH) or isopropyl alcohol (IPA). The nanofiber mats were obtained by electrospinning a blended solution of a CZ-loaded microemulsion and a mixed polymer solution of 2% (w/v) chitosan (CS) and 10% (w/v) polyvinyl alcohol (PVA) at a weight ratio of 30:70. The nanofiber mats were characterized using various analytical techniques. The entrapment efficiency, drug release, antifungal activity and cytotoxicity were investigated. The average diameter of the nanofiber mats was in the range of 105.91-125.56 nm. The differential scanning calorimetry (DSC) and powder X-ray diffractometry (PXRD) results revealed the amorphous state of the CZ-loaded microemulsions incorporated into the nanofiber mats. The entrapment efficiency of CZ in the mats was approximately 72.58-98.10%, depended on the microemulsion formulation. The release experiment demonstrated different CZ release characteristics from nanofiber mats prepared using different CZ-loaded microemulsions. The extent of drug release from the fiber mats at 4h was approximately 64.81-74.15%. The release kinetics appeared to follow Higuchis model. In comparison with CZ lozenges (10mg), the nanofiber mats exhibited more rapid killing activity. Moreover, the nanofiber mats demonstrated desirable mucoadhesive properties and were safe for 2h. Therefore, the nanofiber mats have the potential to be promising candidates for oral candidiasis applications.

Effect of Salt Forms and Molecular Weight of Chitosans on In Vitro Permeability Enhancement in Intestinal Epithelial Cells (Caco-2)

The purpose of this study was to investigate the effect of molecular weight (MW) and salt forms of chitosans (aspartate; CS A, glutamate; CS G, lactate; CS L and hydrochloride, CS HCl) on the transepithelial electrical resistance (TEER) and permeability of Caco-2 cells monolayer, using fluorescein isothiocyanate dextran 4000 (FD-4) as the model compound for paracellular tight junction transport. Chitosan salts were prepared by spray-drying method. FTIR and solid-state 13C NMR spectra showed the functional groups of salts in their molecular structures. Salt form, MW of chitosan, and amount of chitosan influenced the permeation-enhancing effects. These studies showed that chitosan salts appeared to increase cell permeability in a dose-dependent manner and caused relatively reversible effects only at the lower doses of 0.001–0.01% w/v. As the MW of chitosan increased from 20 to 460 kDa, the reduction in TEER significantly decreased in the following order: 20 < 45 < 200 < 460 kDa, observed in CS L and CS HCl. In CS A and CS G, the decrease in TEER was not significantly different in all MW because both chitosan salts showed rapid reduction in TEER within 20 min after the start of the experiment. Among chitosan salts, CS A was the most potent absorption enhancer in acidic (pH 6.2) environment. Cytotoxicity of chitosan salts was concentration dependent and varied slightly among the salt forms of chitosan used. CS HCl (MW 45 kDa) was the most toxic having an IC50 of 0.22 ± 0.06 mg/mL. The ranking of chitosan salts cytotoxicity was CS HCl > CS L> CS G > CS A.