Catechol-Functionalized Alginate Nanoparticles as Mucoadhesive Carriers for Intravesical Chemotherapy.

This research aimed to synthesize and evaluate mucoadhesive catechol-functionalized alginate (Cat-Alg) nanoparticles (NPs) for bladder cancer. Cat-Alg was synthesized using coupling chemistry, and the structure was verified using NMR and FT-IR. Cat-Alg NPs were generated by ionic gelation between the synthesized Cat-Alg and calcium chloride. Garcinia mangostana L. extract (GM extract) was entrapped into the NPs during particle formation. The physical characteristics, mucoadhesive properties, drug loading and release, cellular uptake, and anticancer activity of the GM extract-loaded NPs were investigated. The Cat-Alg NPs were spherical with sizes in the range of 155-186 nm. The slightly negative surface charge of the NPs provided them with excellent stability. The Cat-Alg NPs could be retained on a porcine bladder mucosa to a greater extent compared with unmodified Alg NPs. High loading efficiency (71.6%) and loading capacity (292 µg/mg) of GM extract in the NPs were achieved, and a constant release of GM extract was obtained for up to 8 h with zero-order kinetics. Moreover, the GM extract-loaded NPs were deposited in bladder tissue and accumulated in MB49 cells at a higher rate compared with GM extract suspension. In addition, the NPs could kill a mouse urothelial carcinoma cell line with low IC50. Therefore, these NPs have the potential to be a mucoadhesive drug delivery system for bladder cancer treatment. However, additional in vivo investigations are needed for clinical application in cancer treatment. Graphical abstract.

Enabling Combinatorial siRNA Delivery against Apoptosis-Related Proteins with Linoleic Acid and α-Linoleic Acid Substituted Low Molecular Weight Polyethylenimines.

PURPOSE: Short interfering RNA (siRNA) therapy promises a new era in treatment of breast cancers but effective delivery systems are needed for clinical use. Since silencing complementary targets may offer improved efficacy, this study was undertaken to identify non-viral carriers for combinatorial siRNA delivery for more effective therapy. METHODS: A library of lipid-substituted polymers from low molecular weight polyethyleneimine (PEI), linoleic acid (LA) and α-linoleic acid (αLA) with amide or thioester linkages was prepared and investigated for delivering Mcl-1, survivin and STAT5A siRNAs in breast cancer cells. RESULTS: The effective polymers formed 80-190 nm particles with similar zeta-potentials, but the serum stability was greater for complexes formed with amide-linked lipid conjugates. The LA and αLA substitutions, with the low molecular weight PEI (1.2 kDa and 2.0 kDa) were able to deliver siRNA effectively to cells and retarded the growth of breast cancer cells. The amide-linked lipid substituents showed higher cellular delivery of siRNA as compared to thioester linkages. Upon combinational delivery of siRNAs, growth of MCF-7 cells was inhibited to a greater extent with 2.0PEI-LA9 mediated delivery of Mcl-1 combined survivin siRNAs as compared to individual siRNAs. The qRT-PCR analysis confirmed the decrease in mRNA levels of target genes with specific siRNAs and 2.0PEI-LA9 was the most effective polymer for delivering siRNAs (either single or in combination). CONCLUSIONS: This study yielded effective siRNA carriers for combinational delivery of siRNAs. Careful choice of siRNA combinations will be critical since targeting individual genes might alter the expression of other critical mediators.

A paper-based analytical device coupled with electrochemical detection for the determination of dexamethasone and prednisolone in adulterated traditional medicines.

The adulteration of herbal medicines by dexamethasone or prednisolone is regarded as a serious problem in many communities. Herein, a novel platform for the separation and quantification of both target steroids in herbal medicines based on electrochemical paper-based analytical devices (ePADs) has been created. The ePAD was composed of Whatman SG81 chromatography paper, 3D-printed devices and a commercial screen-printed electrode. Whatman SG81 silica-coated paper was used for the separation of dexamethasone and prednisolone based on the difference in their partition coefficients during the flow of the mobile phase. The optimal mobile phase was composed of 60% ethyl acetate in cyclohexane and required 7 min for separation. The separated steroids on the paper were then quantified by electrochemical detection using differential pulse voltammetry, in which the 3D-printed devices facilitated the measurement. Analytical detection ranges of 10-500 µg mL-1 were obtained for both dexamethasone and prednisolone (r2 = 0.988 and 0.994, respectively). The limits of detection for dexamethasone and prednisolone were 3.59 and 11.98 µg mL-1, respectively, whereas the limits of quantification were 6.00 and 20.02 µg mL-1, respectively. The amounts of both target steroids derived from real herbal medicine samples determined by the proposed method were comparable to those obtained with assays using standard high-performance liquid chromatography. In addition, a simple evaporation step can be used to increase the concentration of the samples before analysis. These ePADs are simple, low-cost, rapid, and very promising for on-site quantitative detection.

Fast, affordable and eco-friendly enzyme kinetic method for the assay of α-ketoglutaric acid in medical product and sports supplements.

A novel kinetic method was developed for the quantitation of α-ketoglutaric acid (AKG) in cardioplegic solution and athletic supplements. The assay relied on an enzymatic transamination of AKG and d-4-hydroxyphenylglycine to form 4-hydroxybenzoylformic acid and l-glutamic acid using d-phenylglycine aminotransferase. Since 4-hydroxybenzoylformic acid absorbed UV strongly at 334 nm, the initial rate of the reaction was determined by the increasing absorbance at this wavelength without the need for colorimetric probes or coupling reactions, and this information was used for the construction of a standard curve against AKG concentration. The method showed good linearity (r2 = 0.9994) over an AKG concentration range of 20-160 µM. The limits of detection and quantitation were 4.09 and 13.62 µM respectively. It was simple, inexpensive, accurate and precise, as well as repeatable, and was not interfered with by excipients in the samples. Regarding the environmental friendliness, the method was free from the use of organic solvents or hazardous reagents and required no chemical pre-treatment of samples. The proposed method gave assay results tested in real samples in agreement with the HPLC method and commercial assay kits, therefore being suitable for routine analysis of AKG in quality control laboratories.

Development of Microemulsions and Microemulgels for Enhancing Transdermal Delivery of Kaempferia parviflora Extract.

The purpose of this research was to develop microemulsions (ME) and microemulgels (MG) for enhancing transdermal delivery of Kaempferia parviflora (KP) extract. The methoxyflavones were used as markers. Various formulations of ME and MG containing 10% w/v KP extract were prepared, and the in vitro skin permeation and deposition were investigated. The potential ME system containing oleic acid (5% w/v), Tween 20 (20% w/v), PG (40% w/v), and water (35% w/v) was successfully formulated. ME with 10% w/v limonene (ME-L10%) showed higher methoxyflavones flux than ME-L5%, ME-L1%, ME without limonene, and KP extract in water, respectively. ME-L10% was selected for adding a gelling agent to form microemulgels (MG-L10%). However, the high viscosity of the gel formulation might control the diffusion of the compound from gel layer into the skin. Therefore, the liquid formulation provided potential ME droplets to deliver KP extract through the skin. Limonene also plays an effective role on the skin permeation, in which the histological image of the skin treated with ME-L10% exhibited larger space of each flattened keratinocyte layer in the stratum corneum compared to the skin treated with KP extract in water. Moreover, ME-L10% showed good stability. Therefore, ME-L10% was a potential formulation for improving transdermal delivery of KP extract.

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.

Fabrication and Evaluation of Nanostructured Herbal Oil/Hydroxypropyl-ß-Cyclodextrin/Polyvinylpyrrolidone Mats for Denture Stomatitis Prevention and Treatment.

This work aims to develop the herbal oil-incorporated nanostructure mats with antifungal activity for the prevention and treatment of Candida-associated denture stomatitis. The nanofiber mats loaded with betel oil or clove oil were fabricated via electrospinning process. The morphologies and physicochemical properties of the herbal oil loaded nanofiber mats were examined using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC), and mechanical testing. The release characteristic, antifungal activity, and cytotoxicity were also investigated. The SEM images confirmed the homogeneous and smooth nanoscale fibers. The addition of the herbal oil into the nanofiber mats reduced the fiber diameters. The DSC and FT-IR results confirmed the presence of the oil in the nanofiber mats. The herbal oils can be released from the mats in a very fast manner and inhibit the growth of candida cells within only few minutes after contact. These nanofiber mats may be beneficial for the prevention and treatment of denture stomatitis.

Electrospun cellulose acetate nanofibers as thin layer chromatographic media for eco-friendly screening of steroids adulterated in traditional medicine and nutraceutical products.

Nanofibers fabricated from cheap, naturally derived biopolymer, namely cellulose acetate via facile electrospinning technique were successfully applied for the first time to use as separation media for thin layer chromatography (TLC). From the optimization studies, uniform, bead-free nanofibers with good adherence to the backing plates were obtained by electrospinning 17% (w/v) cellulose acetate solution prepared in acetone/N,N-dimethylacetamide (2:1, v-v), using a feed rate of 0.6 mL/h and an electrostatic field strength of 17.5 kV/15 cm for 4h. The nanofibers exhibited reversed phase characteristics, thereby offering the possibility to use simple, polar and more environmental friendly mixtures of water and alcohols as mobile phase. In this work, the application of the fabricated fibers was illustrated by using them combined with the optimal mobile phase e.g. ethanol/water (40:60, v-v) for the screening of steroids adulterated in traditional medicine and nutraceutical products. Due to the satisfactory separation performance, electrospun cellulose acetate nanofibers were shown to be an efficient alternative for TLC media and could be potentially used for the development of green and facile analytical methods.

One-enzyme catalyzed simultaneous plant cell disruption and conversion of released glycoside to aglycone combined with in situ product separation as green one-pot production of genipin from gardenia fruit.

A direct one-pot production of genipin, an iridoid aglycone, from crude gardenia fruit was developed. The method relied on the use of single cellulase to disrupt plant cells and to cleave off sugar molecules simultaneously, thereby enhancing the release of intracellular iridoids and converting geniposide to genipin. During the biocatalysis, eco-friendly ethyl acetate was used to extract the product, providing the partial purification and the minimization of genipin degradation. By using 10 mg/mL cellulase and 24 h-incubation at 50 °C, pH 4, combined with in situ extraction, genipin with good purity was yielded at 58.83 mg/g, which increased 12.38 and 1.72 times compared with those obtained from the procedures without either the aid of enzyme or in situ extraction, respectively. Therefore, this integrated approach is promising for the production of genipin and should be potentially applied to the preparation of other plant aglycones.

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.

Methylated N-(4-N,N-dimethylaminocinnamyl) chitosan-coated electrospray OVA-loaded microparticles for oral vaccination.

The purpose of this study was to prepare microparticles entrapping ovalbumin (OVA) as a model antigen to induce immune responses in mice following oral vaccination. In this study, calcium-alginate and calcium-yam-alginate microparticles were prepared by crosslinking alginate with calcium chloride solution using an electrospraying technique. 0.1% (w/v) of methylated N-(4-N,N-dimethylaminocinnamyl) chitosan (TM65CM50CS) was used to coat microparticles entrapping an initial OVA of 20% w/w to polymer. The results indicated that the coated microparticles were spherical and had a smooth surface, with an average size of 1-3 µm, and were positively charged. In addition, the particles demonstrated a greater swelling and mucoadhesive properties than did uncoated microparticles. The in vitro release from the microparticles indicated that the coated microparticles resulted in more sustained release than uncoated microparticles. The cytotoxicity results showed that all of the formulations were safe. The in vivo oral administration demonstrated that at the same amount of 250 µg OVA, coated microparticles exhibited the highest in vivo adjuvant activity in both IgG and IgA immunogenicity.

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.

Development and characterization of pectinate micro/nanoparticles for gene delivery.

The aim of this study was to investigate the possibility of using pectinate micro/nanoparticles as gene delivery systems. Pectinate micro/nanoparticles were produced by ionotropic gelation. Various factors were studied for their effects on the preparation of pectinate micro/nanoparticles: the pH of the pectin solution, the ratio of pectin to the cation, the concentration of pectin and the cation, and the type of cation (calcium ions, magnesium ions and manganese ions). After the preparation, the size and charge of the pectin micro/nanoparticles and their DNA incorporation efficiency were evaluated. The results showed that the particle sizes decreased with the decreased concentrations of pectin and cation. The type of cations affected the particle size. Sizes of calcium pectinate particles were larger than those of magnesium pectinate and manganese pectinate particles. The DNA loading efficiency showed that Ca-pectinate nanoparticles could entrap DNA up to 0.05 mg when the weight ratio of pectin:CaCl(2):DNA was 0.2:1:0.05. However, Mg-pectinate could entrap only 0.01 mg DNA when the weight ratio of pectin:MgCl(2):DNA was 1:100:0.01 The transfection efficiency of both Ca-pectinate and Mg-pectinate nanoparticles yielded relatively low levels of green fluorescent protein expression and low cytotoxicity in Huh7 cells. Given the negligible cytotoxic effects, these pectinate micro/nanoparticles can be considered as potential candidates for use as safe gene delivery carriers.