Farahidah Mohamed

Microencapsulation of alpha-mangostin into PLGA microspheres and optimization using response surface methodology intended for pulmonary delivery

Abstract Documented to exhibit cytotoxicity and poor oral bioavailability, alpha-mangostin was encapsulated into PLGA microspheres with optimization of formulation using response surface methodology. Mixed levels of four factors Face central composite design was employed to evaluate critical formulation variables. With 30 runs, optimized formula was 1% w/v polyvinyl alcohol, 1:10 ratio of oil to aqueous and sonicated at 2 and 5 min time for primary and secondary emulsion, respectively. Optimized responses for encapsulation efficiency, particle size and polydispersity index were found to be 39.12 ± 0.01%, 2.06 ± 0.017 µm and 0.95 ± 0.009, respectively, which matched values predicted by mathematical models. About 44.4% of the encapsulated alpha-mangostin was released over 4 weeks. Thermal analysis of the microspheres showed physical conversion of alpha-mangostin from crystallinity to amorphous with encapsulated one had lower in vitro cytotoxicity than free alpha-mangostin. Aerodynamic diameter (784.3 ± 7.5 nm) of this alpha-mangostin microsphere suggests suitability for peripheral pulmonary delivery.

Co-encapsulation of Nigella sativa oil and plasmid DNA for enhanced gene therapy of Alzheimer’s disease

Abstract Alzheimer disease involves genetic and non-genetic factors and hence it is rational to be treated with genetic and non-genetic therapeutic agents. Nigella sativa has multiple therapeutic properties including neuroregeneration. Nigella sativa oil (NSO) was encapsulated in PLGA nanoparticles and pDNA was loaded either by adsorption on chitosan-modified particles or encapsulation within PLGA nanoparticles. The particle size and zeta potential of NSO-pDNA-chitosan-PLGA nanoparticles were highly dependent on the medium and exhibited high burst release. Meanwhile, NSO-pDNA-PLGA nanoparticles were more consistent with lower burst release. The fabricated nanoparticles revealed the expected outcomes of both pDNA and NSO. The pDNA transfected N2a cell while the encapsulated NSO promoted neurite outgrowth that is crucial for neuroregeneration. Results from this study suggest that NSO could be added to the gene delivery carrier to enhance treatment benefits for Alzheimer disease.

Fabrication of Fucoxanthin-Loaded Microsphere(F-LM) By Two Steps Double-Emulsion Solvent Evaporation Method and Characterization of Fucoxanthin before and after Microencapsulation

Microencapsulation is a promising approach in drug delivery to protect the drug from degradation and allow controlled release of the drug in the body. Fucoxanthin-loaded microsphere (F-LM) was fabricated by two step w/o/w double emulsion solvent evaporation method with poly (L-lactic-coglycolic acid) (PLGA) as carrier. The effect of four types of surfactants (PVA, Tween-20, Span-20 and SDS), homogenization speed, and concentration of PLGA polymer and surfactant (PVA), respectively, on particle size and morphology of F-LM were investigated. Among the surfactants tested, PVA showed the best results with smallest particle size (9.18 µm) and a smooth spherical surface. Increasing the homogenization speed resulted in a smaller mean F-LM particle size [d(0.50)] from 17.12 to 9.18 µm. Best particle size results and good morphology were attained at homogenization speed of 20 500 rpm. Meanwhile, increased PLGA concentration from 1.5 to 11.0 (% w/v) resulted in increased F-LM particle size. The mean particle size [d(0.5)] of F-LM increased from 3.93 to 11.88 µm. At 6.0 (% w/v) PLGA, F-LM showed the best structure and external morphology. Finally, increasing PVA concentration from 0.5 to 3.5 (% w/v) resulted in decreased particle size from 9.18 to 4.86 µm. Fucoxanthin characterization before and after microencapsulation was carried out to assess the success of the microencapsulation procedure. Thermo gravimetry analysis (TGA), glass transition (Tg) temperature of F-LM and fucoxanthin measured using DSC, ATR-FTIR and XRD indicated that fucoxanthin was successfully encapsulated into the PLGA matrix, while maintaining the structural and chemical integrity of fucoxanthin.

Preparation, characterization and in vitro release study of BSA-loaded double-walled glucose-poly(lactide-co-glycolide) microspheres

Abstract The aim of this study was to prepare a model protein, bovine serum albumin (BSA) loaded double-walled microspheres using a fast degrading glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA) and a moderate-degrading carboxyl-terminated PLGA polymers to reduce the initial burst release and to eliminate the lag phase from the release profile of PLGA microspheres. The double-walled microspheres were prepared using a modified water-in-oil-in-oil-in-water (w/o/o/w) method and single-polymer microspheres were prepared using a conventional water-in-oil-in-water (w/o/w) emulsion solvent evaporation method. The particle size, morphology, encapsulation efficiency, thermal properties, in vitro drug release and structural integrity of BSA were evaluated in this study. Double-walled microspheres prepared with Glu-PLGA and PLGA polymers with a mass ratio of 1:1 were non-porous, smooth-surfaced, and spherical in shape. A significant reduction of initial burst release was achieved for the double-walled microspheres compared to single-polymer microspheres. In addition, microspheres prepared using Glu-PLGA and PLGA polymers in a mass ratio of 1:1 exhibited continuous BSA release after the small initial burst without any lag phase. It can be concluded that the double-walled microspheres made of Glu-PLGA and PLGA polymers in a mass ratio of 1:1 can be a potential delivery system for pharmaceutical proteins.

Development and Validation of Analytical Method by RP-HPLC for Quantification of Alpha-Mangostin Encapsulated in PLGA Microspheres

A simple, rapid, precise and highly accurate RP-HPLC method was developed and validated for determination of alpha-mangostin content extracted from PLGA-microspheres. Method was developed using a silica-based deactivated C-18 column (4.6×100 mm, 3 μm) with a mobile phase of 70-80 % v/v acetonitrile (A) and 0.1% v/v orthophosphoric acid (B), with the following pre-determined timed-gradient program: 70% (A) isocratic for 6 min, 70-75% (A) in 1.2 min, 75-80% (A) in 0.4 min, 80% (A) isocratic for 2.4 min, 80-70% (A) in 0.4 min, finally 70% (A) isocratic for 5 min, with a flow rate of 1 mL/min, detected at 320 nm by a UV detector. Linearity was obtained over the range of 1-200 μg/mL with r 2 =0.9995. The precision was achieved based on repeatability and intermediate precision with RSD of 0.13-0.6% and 0.57-1.2%, respectively. Percent recovery of 100.55% to 103.82% with RSD 0.086 - 0.15 implied high accuracy of the method. Limit of detection and limit of quantitation were 0.038 and 0.121 μg/ml, respectively suggesting good sensitivity of the method. The method is envisaged to be effectively used for routine quality control assay for encapsulated alpha-mangostin in PLGA microspheres.

Cellular uptake of Nigella sativa oil-PLGA microparticle by PC-12 cell line.

Abstract The aim of this study is to investigate the cell uptake of Nigella sativa oil (NSO)-PLGA microparticle by neuron-like PC-12 cells in comparison to surfactants; hydrophilic (Tween 80 & Triton X100) and hydrophobic (Span 80). Solvent evaporation was used to precisely control the size, zeta potential and morphology of the particle. The results revealed varying efficiencies of the cell uptake by PC-12 cells, which may be partially attributed to the surface hydrophobicity of the microparticles. Interestingly, the uptake efficiency of PC-12 cells was higher with the more hydrophilic microparticle. NSO microparticle showed evidence of being preferably internalised by mitotic cells. Tween 80 microparticle showed the highest cell uptake efficiency with a concentration-dependent pattern suggesting its use as uptake enhancer for non-scavenging cells. In conclusion, PC-12 cells can take up NSO-PLGA microparticle which may have potential in the treatment of neurodegenerative disease.

Spectrophotometric Determination of Gentamicin Loaded PLGA Microparticles and Method Validation via Ninhydrin-Gentamicin Complex as a Rapid Quantification Approach

The purpose of this study was to provide a quantification method with rapid, sensitive, reproducible and cost effective for gentamicin in the form of ninhydrin-gentamicin complex. The utilization of spectrophotometric module to validate the method development for gentamicin loaded microparticles was intended to provide alternative quantification method without compromising the sensitivity of the developed method. The wavelength was determined to be at 418 nm with 421 nm and 415 nm were selected for the robustness of the method selectivity. The linearity of 6 known concentrations of ninhydrin-gentamicin complex was obtained with the R2 of 0.9998. The interday and intraday precisions were determined with the acceptance %RSD values of less than 2%. In fact, the highest %RSD value was 1.09% which suggested the method to be acceptably precise. The LOD and LOQ values were recorded to be at 0.016 mg/mL and 0.196 mg/mL respectively. The % recovery of 4 known concentrations indicated the accuracy of the method was high with the recovery range between 98.66% to 101.8%. The parameters analyzed in this study were in accordance with ICH Q2 (R1) guidelines. This quantification method developed by using spectrophotometric module was a promising approach to provide a rapid and cost effective in evaluating gentamicin concentration for in-vitro applications.

Method Development and Validation using UV Spectrophotometry for Nigella sativa Oil Microparticles Quantification

Nigella sativa oil (NSO) has been exploited for medical purposes for many generations. The fabrication of microparticles containing NSO intended for sustained release was done to be used in treating osteomyelitis. Method in quantifying NSO using UV-spectroscopy was developed and validated. Linearity shown a good correlation coefficient with the values higher than 0.995, both for actual and different analysts. The LOD and LOQ values were recorded to be 2.89 µg/mL and 8.75 µg/mL respectively. In addition, the highest %RSD values for the intermediate and repeatability studies were 0.970% and 0.445% which suggested the method was precise. The percentage recovery for 4 known concentrations gave the range between 98.16% to 99.39%, indicating the high accuracy of the method. The parameters analyzed in this study were in accordance with ICH Q2 (R1) guidelines.

Comparative assessment of plasmid DNA delivery by encapsulation within or adsorbed on poly (D, L-lactide-coglycolide) nanoparticles

Purpose : To compare the gene delivery effectiveness of plasmid DNA (pDNA) encapsulated within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with that adsorbed on PLGA nanoparticles. Methods: PLGA nanoparticles were prepared using solvent-evaporation method. To encapsulate pDNA within the particles, it was first complexed with cetyltrimethylammonium bromide (CTAB) and then added to the oil phase during the synthesis. For the adsorption, PLGA nanoparticles were first modified with either CTAB or chitosan and then pDNA was adsorbed on the particle surface by electrostatic interaction. Results : Nanoparticles encapsulating pDNA exhibited better plasmid loading and protection with significantly lower burst release (p < 0.05) compared to that of the nanoparticles with adsorbed plasmid. Cell uptake of chitosan-modified nanoparticles by murine neuroblastoma (N2a) cells was significantly (p < 0.05) higher than that of chitosan-free nanoparticles. Nanoparticles encapsulating pDNA showed higher transfection efficiency (p < 0.05) in N2a cells. Conclusion : Encapsulation of pDNA within PLGA nanoparticles presents a potential strategy for gene delivery that is superior to pDNA adsorbed on the nanoparticle surface. In addition, encapsulation keeps the particle surface free for further modifications such as the addition of targeting ligands. Keywords : Poly (D,L-lactide-co-glycolide), Plasmid DNA, Encapsulation, Adsorption, Cellular uptake, Gene therapy, Targeting ligands

Isolation of the biofilm producing bacteria from osteomyelitis patients at Hospital Tengku Ampuan Afzan (HTAA), Kuantan

Aims: Accurate diagnosis and proper treatments of osteomyelitis are often difficult and ineffective due to several reasons such as less sensitive sample collected and the formation of biofilm following prosthetic use. Thus, our goal of this study is to identify suitable sample for laboratory diagnosis and also microbial species that cause osteomyelitic infection and discriminate between biofilm and non-biofilm producing strains in patients at Hospital Tengku Ampuan Afzan, Kuantan. Methodology and results: Samples of bone, prosthetic material, tissue and swab were collected from patient with suspected osteomyelitis at the hospital. Bacteria were isolated from sample using methods such as homogenization, direct transfer, and sonication. Then, species identification was done by colony characterization, biochemical test and the API identification system. Once species identified, tissue culture plate method was performed to discriminate the biofilm-producing strain from the non-biofilm-producing strain. The total number of 57 samples were collected from 17 cases of suspected osteomyelitis with 34 samples were found positive bacterial growth. Prosthetic samples produced highest positive growth with 81.3%, following by bone samples with 66.7% while swab and tissue samples with 46.2% and 43.8% respectively. We found that 14 from total 16 pathogens identified were biofilm producing-strains. Conclusion, significance, and impact of study: Prosthetic and bone samples produced higher bacterial growth, in contrast to other type of samples. Sonication method improves bacterial detection. Biofilm producing-bacteria were also the most common isolated strains from osteomyelitic infection. These have underscored the need to revise current clinical and laboratory practice as proper identification biofilm bacteria may influences management an outcome.