Development of Alginate/Chitosan Microparticles for Dust Mite Allerge

Purpose: To develop chitosan/alginate microparticles for the mucosal delivery of allergen from dust mite (Dermatophagoides pteronyssinus).Methods: Chitosan/alginate microparticles were prepared by ionotropic gelation. The effects of polymer content, crosslinking agent, and preparation method on the physicochemical characteristics of the microparticles as well as their in vitro cytotoxicity were investigated.Results: The microparticles were small (1 - 17 μm) and spherical in shape. The highest allergen content (0.30 ± 0.07 mg/g) was obtained with 2.5 % initial allergen loading in chitosan- triphosphate (CS-TPP) microparticles. Sustained allergen release (approx. 50 % over 24 h) was observed from alginate-coated chitosan microparticles. Allergen incorporation method and initial drug-loading could be varied to obtain optimum particle size with high allergen-loading and sustained release. The cytotoxicity of various microparticle formulations did not differ significantly (p > 0.05 ), as cell viability values were close to 100 %.Conclusion: This study indicates that alginate and alginate-coated chitosan microparticles are safe and can be further developed for mucosal allergen delivery.Keywords: Alginate, Chitosan, Microparticle, Allergen delivery, Dust mite, Dermatophagoides pteronyssinu

In Vitro Antioxidant Activity of Chitosan Aqueous Solution: Effect of Salt Form

Purpose: To investigate the effect of salt form on the antioxidant activities of chitosan aqueous solution.Methods: The antioxidant activities of chitosan acetate (CS-acetate), chitosan hydroxybenzotriazole (CS-HOBt), chitosan thiamine pyrophosphate (CS-TPP) and chitosan ethylenediaminetetraacetic acid (CS-EDTA) solution were determined employing various established in vitro system such as superoxide and hydroxyl radicals scavenging, metal ion chelating and reducing power. Their chemical structures were characterized by nuclear magnetic resonance (NMR) and Fourier transform infraredspectrophotometry (FT-IR).Results: NMR and FT-IR show confirmed formation of chitosan salts. The 50 % inhibition concentration (IC50) of superoxide and hydroxyl radicals was 0.349 – 1.34 and 0.34 – 1.54 mg/mL, respectively. Among the salt forms, CS-acetate (IC50 = 0.349 mg/mL) showed the highest superoxide radical scavenging effect while CS-HOBt (IC50 = 0.34 mg/mL) showed the highest hydroxyl radical scavenging effect. With regard to metal ion chelating activity, CS-EDTA showed the highest chelating activity (approx 100 % at 1 mg/mL) while the others showed 20 % activity at a concentration of 1 mg/mL. The results for reducing power indicate that CS-TPP had the highest reducing power.Conclusion: The results indicate that antioxidant activity varied with the salt form. Thus, CS salts may be used as a source of antioxidants for pharmaceutical applications.Keywords: Chitosan, Antioxidant, Hydroxybenzotriazole, Thiamine pyrophosphate, Ethylenediaminetetraacetic aci

Fabrication of Cationic Exchange Polystyrene Nanofibers for Drug Delivery

Purpose: To prepare polystyrene nanofiber ion exchangers (PSNIE) with surface cation exchange functionality using a new method based on electrospinning and also to optimize crosslinking and sulfonation reactions to obtain PSNIE with maximum ion exchange capacity (IEC).Method: The nanofibers were prepared from 15% w/v polystyrene solution in dimethylacetamide (DMAc) containing 0.025 %w/v tetrabutylammonium bromide (TBAB) using electrospinning technique, followed by crosslinking with sulfuric acid/formaldehyde in a ratio ranging from 100/0 to 50/50 v/v and sulfonation in sulfuric acid. Degree of crosslinking was determined as the amount of fibers that remained in dichloromethane. The morphology and diameter of the fibers were evaluated by scanning electron microscopy (SEM) while IEC of PSNIE was performed by salt splitting titration.Results: PSNIE crosslinked with a sulfuric acid/formaldehyde ratio of 90/10 with 0.1 %w/v silver sulfate for 10 min at 70°C and sulfonated in 98 % sulfuric acid with 0.2 %w/v silver sulfate as the catalyst at 100°C for 30 min showed a maximum IEC of 3.21 meq/g-dry-PSNIE. Increase in  sulfonation temperature caused the IEC of PSNIE to increase due to faster sulfonation. It was observed that the higher the temperature the faster the rate of sulfonation reaction. The diameter of the fibers after sulfonation was 404 ± 42 nm.Conclusion: These results indicate that PSNIE can be successfully prepared by electrospinning. Furthermore, cationic drug can be loaded onto the novel PSNIE for controlled release delivery.Keywords: Polystyrene, Ion exchange capacity, Nanofibers, Ion exchangers, Crosslinking, Sulfonation

Development and Evaluation of Ketoprofen Acrylic Transdermal Patches

Purpose: To fabricate ketoprofen transdermal patches (KTPs) using an acrylic pressure-sensitive adhesive (PSA) polymer.Methods: KTPs were prepared using solvent casting method. The influence of the amount of PSA, drug content, and terpenes as penetration enhancers on the characteristics of the patch, namely, thickness, W/A ratio, and adhesiveness and in vitro skin permeation, were investigated. Scanning electron microscope (SEM) and differential scanning calorimetry (DSC) studies were also performed on the patches. The physical and chemical stability of KTPs after storage at 40 oC, and 75 %RH for 1 month was also evaluated.Results: DSC thermograms demonstrate that the drug was dispersed molecularly in the polymer in all the formulations. Increase in PSA content increased the W/A ratio and adhesiveness of KTPs. Ketoprofen release from the transdermal patches followed the Higuchi diffusion model. Ketoprofen flux increased with increase in the ketoprofen content of the adhesive matrix. Inclusion of terpenes in the patch formulations significantly increased the permeation of ketoprofen through the skin, with enhancement ratio (ER) ranging from 1.4 to 2.6.Conclusion: KTPs formulated with acrylic pressure-sensitive adhesive and incorporating terpenes as permeation enhancers demonstrated suitable characteristics for transdermal delivery of ketoprofen.Keywords: Ketoprofen, Transdermal patch, Skin permeation, Acrylic matrix, Terpenes, Pressuresensitive adhesive

Mechanistic study of decreased skin penetration using a combination of sonophoresis with sodium fluorescein-loaded PEGylated liposomes with D-limonene

Worranan Rangsimawong, Praneet Opanasopit, Theerasak Rojanarata, Tanasait Ngawhirunpat Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand Abstract: The effect of low frequency sonophoresis (SN, 20 kHz) on the skin transport of sodium fluorescein (NaFI)-loaded liposomes was investigated. An in vitro skin penetration study in open and blocked hair follicles was performed, and confocal laser scanning microscopy and scanning electron microscopy were used to visualize the penetration pathways. The results showed that SN significantly increased the flux of NaFI solution, whereas it significantly decreased the flux of NaFI-loaded polyethylene glycol-coated (PEGylated) liposomes with D-limonene (PL-LI). SN did not significantly affect the flux of NaFI-loaded conventional liposomes and PEGylated liposomes. In the blocked follicles, the flux of NaFI-loaded PL-LI both with and without SN decreased, indicating that NaFI-loaded PL-LI penetrated the skin via the transfollicular pathway. A confocal laser scanning microscopy image showed that in the skin without SN, the fluorescence intensity of NaFI-loaded PL-LI was observed in the skin and along the length of hair inside the skin, whereas in the skin with applied SN, the fluorescence intensity was detected only on the top of hair outside the skin. From scanning electron microscopy images, SN dislocated the corneocytes and reduced the deposition of PL-LI around hair follicles. These results revealed that SN may partially plug hair follicle orifices and reduce percutaneous absorption through the follicular pathway. Keywords: sonophoresis, PEGylated liposomes, hydrophilic compound, follicular pathwa

Evaluation of some anionic exchange resins as potential tablet disintegrants

Purpose: To determine the potential of some anionic exchange resins as tablet disintegrants.Methods: Dowex1® x2, x4 and x8 resins (crosslinked copolymers of styrene and divinylbenzene with quaternary methyl amine functionality) were evaluated as disintegrant for dibasic calcium phosphate dihydrate tablets. The best resin providing the fastest disintegration and highest hardness of obtained tablets was selected for further investigation. The effect of resin concentration and compression force on the properties of tablets using the selected resin was investigated. In addition, the disintegrant efficacy of the selected resin in the tablet formulations containing either a basic drug, e.g., dextromethorphan hydrobromide (DMP), or an acidic drug, e.g., diclofenac sodium (DCN), was determined in comparison with sodium starch glycolate (SSG).Results: Dowex1®x2 resin exhibited the fastest disintegration (6.0 s) and the highest hardness (103.6 N) of obtained tablets. These disintegrating and tablet properties depended upon the resin concentration and compression force. For DMP, the resin provided faster disintegration and drug release (8.0 s and 100.4 % at 10 min) as compared with SSG (16.2 s and 98.9 % at 30 min). In contrast, the resin caused the depleted release of DCN (61.6 % at 120 min) in spite of providing the faster tablet disintegration (10.0 s) than SSG (15.5 s) due to the ionic binding of the drug and resin.Conclusion: The Dowex1®x2 resin was shown to be a potential disintegrant for the tablets of basic drugs.Keywords: Anionic exchange resin, Disintegrant, Dextromethorphan hydrobromide, Diclofenac sodium, Calcium phosphat

The development of poly-L-arginine-coated liposomes for gene delivery

Praneet Opanasopit1, Jintana Tragulpakseerojn1, Auayporn Apirakaramwong1, Tanasait Ngawhirunpat1, Theerasak Rojanarata1, Uracha Ruktanonchai21Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand; 2National Nanotechnology Center, Thailand Science Park, Pathumthani, Thailand Abstract: In this study, liposomes coated with cationic polymers, poly-L-arginine (PLA), were assessed as a promising gene transfer system in human cervical carcinoma (HeLa) cells and human hepatoma cell line (Huh7) cells. The liposomes were prepared using egg yolk phosphatidylcholine and sodium oleate in the molar ratio of 10:2 with an ultrasonic generator and then coated with PLA. The PLA-coated liposomes (PCLs) formed complexes with plasmid DNA encoding green fluorescent protein. The complexes were characterized by agarose gel electrophoresis and investigated for their transfection efficiency in HeLa and Huh7 cells. The data were compared with PLA/DNA complexes and the positive control Lipofectamine 2000TM. The results showed that complete PCL/DNA complexes were formed at weight ratios of more than 0.05. Efficient gene transfer by PCLs was dependent on the cell type. The transfection efficiency of PCLs was about two times higher than that of PLA/DNA complexes in both HeLa cells and Huh7 cells. Cytotoxicity was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and showed that 80%-100% of both of the cells were viable after treating PCL/DNA complexes. The present results demonstrate that PCLs are a promising, nonviral gene carrier with low toxicity.Keywords: PLA-coated liposomes, PLA, gene delivery, transfection efficiency&nbsp

A combined approach of hollow microneedles and nanocarriers for skin immunization with plasmid DNA encoding ovalbumin

Boonnada Pamornpathomkul,1 Adisak Wongkajornsilp,2 Wanida Laiwattanapaisal,3 Theerasak Rojanarata,1 Praneet Opanasopit,1 Tanasait Ngawhirunpat1 1Department of Pharmaceutical Technology, Faculty of Pharmacy, Pharmaceutical Development of Green Innovations Group, Silpakorn University, Nakhon Pathom, 2Department of Pharmacology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 3Department of Clinical Chemistry, Faculty of Allied Health Sciences, Chulalongkorn University, Bangkok, Thailand Abstract: The aim of this study was to investigate the use of different types of microneedles (MNs) and nanocarriers for in vitro skin permeation and in vivo immunization of plasmid DNA encoding ovalbumin (pOVA). In vitro skin permeation studies indicated that hollow MNs had a superior enhancing effect on skin permeation compared with solid MN patches, electroporation (EP) patches, the combination of MN and EP patches, and untreated skin. Upon using hollow MNs combined with nanocarriers for pOVA delivery, the skin permeation was higher than for the delivery of naked pOVA, as evidenced by the increased amount of pOVA in Franz diffusion cells and immunoglobulin G (IgG) antibody responses. When the hollow MNs were used for the delivery of nanocarrier:pOVA complexes into the skin of mice, they induced a stronger IgG immune response than conventional subcutaneous (SC) injections. In addition, immunization of mice with the hollow MNs did not induce signs of skin infection or pinpoint bleeding. Accordingly, the hollow MNs combined with a nanocarrier delivery system is a promising approach for delivering pOVA complexes to the skin for promoting successful immunization. Keywords: hollow microneedle, solid microneedle, electroporation, plasmid DNA encoding ovalbumin, skin immunization, nanocarrie

Role of the charge, carbon chain length, and content of surfactant on the skin penetration of meloxicam-loaded liposomes

Sureewan Duangjit,1,2 Boonnada Pamornpathomkul,1 Praneet Opanasopit,1 Theerasak Rojanarata,1 Yasuko Obata,2 Kozo Takayama,2 Tanasait Ngawhirunpat11Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand; 2Department of Pharmaceutics, Hoshi University, Shinagawa-ku, Tokyo, JapanAbstract: The objective of this study was to investigate the influence of surfactant charge, surfactant carbon chain length, and surfactant content on the physicochemical characteristics (ie, vesicle size, zeta potential, elasticity, and entrapment efficiency), morphology, stability, and in vitro skin permeability of meloxicam (MX)-loaded liposome. Moreover, the mechanism for the liposome-enhanced skin permeation of MX was determined by Fourier transform infrared spectroscopy and differential scanning calorimetry. The model formulation used in this study was obtained using a response surface method incorporating multivariate spline interpolation (RSM-S). Liposome formulations with varying surfactant charge (anionic, neutral, and cationic), surfactant carbon chain length (C4, C12, and C16), and surfactant content (10%, 20%, and 29%) were prepared. The formulation comprising 29% cationic surfactant with a C16 chain length was found to be the optimal liposome for the transdermal delivery of MX. The skin permeation flux of the optimal formulation was 2.69-fold higher than that of a conventional liposome formulation. Our study revealed that surfactants affected the physicochemical characteristics, stability, and skin permeability of MX-loaded liposomes. These findings provide important fundamental information for the development of liposomes as transdermal drug delivery systems.Keywords: optimal liposome, optimization, transdermal drug delivery, surfactant charge, surfactant carbon chain length, surfactant conten