Storage Stabilisation of Albumin-Loaded Chitosan Nanoparticles by Lyoprotectants

Purpose: To investigate the effect of lyoprotectants on the physical and storage stability of lyophilised bovine serum albumin-loaded chitosan/dextran sulphate (BSA-loaded CS/DS) nanoparticles.Methods: BSA-loaded CS/DS nanoparticles were prepared by ionic-gelation technique. The nanoparticles were harvested by ultra-centrifugation and then various lyoprotectants at different concentrations were added to the nanoparticles prior to lyophilisation at – 40 oC for 24 h. Particle sizeand distribution as well as zeta potential of the nanoparticles were measured by dynamic light scattering method. Entrapment efficiency and BSA retained in the nanoparticles matrices were determined spectrophotometrically at ëmax of 595 nm.Results: The results indicate that 0.5 %w/v trehalose was the most effective lyoprotectant and it essentially maintained the particle size of lyophilised BSA-loaded CS/DS nanoparticles which changed slightly from 188 ± 11 nm to 174 ± 14 nm during lyophilisation. Mannitol was also as effective as trehalose at 0.1 and 1.0 % w/v in stabilising the nanoparticles. The particle size of lyophilized nanoparticles increased moderately from 188 ± 11 nm to 234 ± 12 nm and 287 ± 18 nm at 0.1 and 1.0 % w/v, respectively. In contrast, the other lyoprotectants (inulin and histidine) did not show stabilizing effects. Moreover, trehalose also reduced the degree of particle aggregation from 329 ± 16 to 836 ± 21 nm upon storage for 24 h as compared to CS/DS nanoparticles without trehalose; from 438 ± 14 to1298 ± 18 (p < 0.05). The rate of BSA leakage from the nanoparticles containing trehalose was reduced from 92 to 42 % over a 11-day storage period compared with 99 to 0 % for CS/DS nanoparticles without trehalose.Conclusion: Trehalose (0.5 % w/v) is a promising lyoprotectant for storage stabilisation of BSA-loaded CS/DS nanoparticles.Keywords: Lyoprotectant, Chitosan, Nanoparticles, Trehalose, Bovine serum albumin, Ultracentrifugatio

Controlled release of lysozyme from double-walled poly(Lactide-Co-Glycolide) (PLGA) microspheres

Double-walled microspheres based on poly(lactide-co-glycolide) (PLGA) are potential delivery systems for reducing a very high initial burst release of encapsulated protein and peptide drugs. In this study, double-walled microspheres made of glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA), and carboxyl-terminated PLGA were fabricated using a modified water-in-oil-in-oil-in-water (w1/o/o/w2) emulsion solvent evaporation technique for the controlled release of a model protein, lysozyme. Microspheres size, morphology, encapsulation efficiency, lysozyme in vitro release profiles, bioactivity, and structural integrity, were evaluated. Scanning electron microscopy (SEM) images revealed that double-walled microspheres comprising of Glu-PLGA and PLGA with a mass ratio of 1:1 have a spherical shape and smooth surfaces. A statistically significant increase in the encapsulation efficiency (82.52 ± 3.28%) was achieved when 1% (w/v) polyvinyl alcohol (PVA) and 2.5% (w/v) trehalose were incorporated in the internal and external aqueous phase, respectively, during emulsification. Double-walled microspheres prepared together with excipients (PVA and trehalose) showed a better control release of lysozyme. The released lysozyme was fully bioactive, and its structural integrity was slightly affected during microspheres fabrication and in vitro release studies. Therefore, double-walled microspheres made of Glu-PLGA and PLGA together with excipients (PVA and trehalose) provide a controlled and sustained release for lysozyme

Storage Stabilisation of Albumin-Loaded Chitosan Nanoparticles by Lyoprotectants

Purpose: To investigate the effect of lyoprotectants on the physical and storage stability of lyophilised bovine serum albumin-loaded chitosan/dextran sulphate (BSA-loaded CS/DS) nanoparticles.Methods: BSA-loaded CS/DS nanoparticles were prepared by ionic-gelation technique. The nanoparticles were harvested by ultra-centrifugation and then various lyoprotectants at different concentrations were added to the nanoparticles prior to lyophilisation at – 40 oC for 24 h. Particle sizeand distribution as well as zeta potential of the nanoparticles were measured by dynamic light scattering method. Entrapment efficiency and BSA retained in the nanoparticles matrices were determined spectrophotometrically at ëmax of 595 nm.Results: The results indicate that 0.5 %w/v trehalose was the most effective lyoprotectant and it essentially maintained the particle size of lyophilised BSA-loaded CS/DS nanoparticles which changed slightly from 188 ± 11 nm to 174 ± 14 nm during lyophilisation. Mannitol was also as effective as trehalose at 0.1 and 1.0 % w/v in stabilising the nanoparticles. The particle size of lyophilized nanoparticles increased moderately from 188 ± 11 nm to 234 ± 12 nm and 287 ± 18 nm at 0.1 and 1.0 % w/v, respectively. In contrast, the other lyoprotectants (inulin and histidine) did not show stabilizing effects. Moreover, trehalose also reduced the degree of particle aggregation from 329 ± 16 to 836 ± 21 nm upon storage for 24 h as compared to CS/DS nanoparticles without trehalose; from 438 ± 14 to1298 ± 18 (p < 0.05). The rate of BSA leakage from the nanoparticles containing trehalose was reduced from 92 to 42 % over a 11-day storage period compared with 99 to 0 % for CS/DS nanoparticles without trehalose.Conclusion: Trehalose (0.5 % w/v) is a promising lyoprotectant for storage stabilisation of BSA-loaded CS/DS nanoparticles.Keywords: Lyoprotectant, Chitosan, Nanoparticles, Trehalose, Bovine serum albumin, Ultracentrifugatio

Synergistic effect of pH-responsive folate-functionalized poloxamer 407-TPGS-mixed micelles on targeted delivery of anticancer drugs

Adeel Masood Butt, Mohd Cairul Iqbal Mohd Amin, Haliza Katas Centre for Drug Delivery Research, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia Background: Doxorubicin (DOX), an anthracycline anticancer antibiotic, is used for treating various types of cancers. However, its use is associated with toxicity to normal cells and development of resistance due to overexpression of drug efflux pumps. Poloxamer 407 (P407) and vitamin E TPGS (d-α-tocopheryl polyethylene glycol succinate, TPGS) are widely used polymers as drug delivery carriers and excipients for enhancing the drug retention times and stability. TPGS reduces multidrug resistance, induces apoptosis, and shows selective anticancer activity against tumor cells. Keeping in view the problems, we designed a mixed micelle system encapsulating DOX comprising TPGS for its selective anticancer activity and P407 conjugated with folic acid (FA) for folate-mediated receptor targeting to cancer cells. Methods: FA-functionalized P407 was prepared by carbodiimide crosslinker chemistry. P407-TPGS/FA-P407-TPGS-mixed micelles were prepared by thin-film hydration method. Cytotoxicity of blank micelles, DOX, and DOX-loaded micelles was determined by alamarBlue® assay. Results: The size of micelles was less than 200 nm with encapsulation efficiency of 85% and 73% for P407-TPGS and FA-P407-TPGS micelles, respectively. Intracellular trafficking study using nile red-loaded micelles indicated improved drug uptake and perinuclear drug localization. The micelles show minimal toxicity to normal human cell line WRL-68, enhanced cellular uptake of DOX, reduced drug efflux, increased DOX–DNA binding in SKOV3 and DOX-resistant SKOV3 human ovarian carcinoma cell lines, and enhanced in vitro cytotoxicity as compared to free DOX. Conclusion: FA-P407-TPGS-DOX micelles show potential as a targeted nano-drug delivery system for DOX due to their multiple synergistic factors of selective anticancer activity, inhibition of multidrug resistance, and folate-mediated selective uptake. Keywords: doxorubicin nanocarriers, folate targeting, doxorubicin cytotoxicity, synergistic drug delivery, Pgp-inhibiting micelle

Preparation of polyethyleneimine incorporated poly(D,L-lactide-co-glycolide) nanoparticles by spontaneous emulsion diffusion method for small interfering RNA delivery

Gene therapy based on small interfering RNA (siRNA) has emerged as an exciting new therapeutic approach. However, insufficient cellular uptake and poor stability have limited its usefulness. Polyethyleneimine (PEI) has been extensively studied as a vector for nucleic acids and incorporation of PEI into poly(D,L-lactide-co-glycolide) (PLGA) particles has been shown to be useful in the development of gene delivery. PEI was incorporated into the PLGA particles by spontaneous modified emulsification diffusion method. Incorporation of PEI into PLGA particles with the PLGA to PEI weight ratio 29:1 was found to produce spherical and positively charged nanoparticles where type of polymer, type and concentration of surfactant could affect their physical properties. Particle size of around 100 nm was obtained when 5% (m/v) PVA was used as a stabiliser. PLGA-PEI nanoparticles were able to completely bind siRNA at N/P ratio 20:1 and to provide protection for siRNA against nuclease degradation. In vitro cell culture studies subsequently revealed that PLGA-PEI nanoparticles with adsorbed siRNA could efficiently silence the targeted gene in mammalian cells, better than PEI alone, with acceptable cell viability. PLGA-PEI nanoparticles have been found to be superior to its cationising parent compound; PEI polymer. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved

Preparation, characterization and reduction of burst release of bovine serum albumin (BSA) from biodegradable PLGA microspheres

In this study, model protein bovine serum albumin (BSA) loaded poly(lactide-co-glycolide) (PLGA) microspheres have been prepared by a conventional water-in-oil-in-water (w/o/w) and a modified water-in-oil-in-oil-in-water (w/o/o/w) double emulsion solvent evaporation method. The prepared microspheres were characterized with respect to their morphology, particle size, encapsulation efficiency, production yield, thermal properties and in vitro drug release. By using w/o/o/w method, a significant decrease in mean particle size and a significant increase in encapsulation efficiency were observed when a binary solvent mixture of ethyl acetate and dichloromethane was used. In w/o/o/w double emulsion solvent evaporation method, the optimized formulation of BSA loaded microspheres was nonporous, smooth-surfaced, and spherical shape under field-emission scanning electron microscope (FE-SEM) with a mean particle size of 3.95 µm and encapsulation efficiency of 98.46%. From 72 days In vitro release studies, microspheres prepared by modified (w/o/o/w) method with a combination of hydroxyl and carboxyl terminated PLGA polymers exhibited a significantly lower initial burst release followed by sustained and almost complete release compared to microspheres prepared by conventional w/o/w technique using a single PLGA polymer. It can be concluded that the modified w/o/o/w method can be proposed as a potential delivery system of therapeutic proteins

Effect of preparative variables on small interfering RNA loaded Poly(D,L-lactide-co-glycolide)-chitosan submicron particles prepared by emulsification diffusion method

In this study, poly(D,L-lactide-co-glycolide) (PLGA)-chitosan particles were investigated as an effective delivery system for small interfering RNA (siRNA) by emulsification diffusion method. The type, molecular weight and concentration of chitosan, PLGA type as well as centrifugation and freeze-drying process were amongst the investigated variables. PLGA-chitosan particles obtained were positively charged with particle size between 0.4-1 m depending on type, molecular weight and concentration of chitosan as well as type of PLGA. A better siRNA loading capacity was observed when a higher degree of 'uncapped end groups' were used. The addition of trehalose has also been shown to stabilize these particles from severe aggregation induced by freeze-drying. It was found that physical properties of PLGA-chitosan particles and their siRNA binding capacity were highly influenced by certain preparation parameters. The desired positive charge of submicron size range PLGA-chitosan particles could therefore be obtained by adjusting and optimizing these preparative and formulation parameters

Effect of process variables on the preparation of BSA loaded double-walled poly(lactide-co-glycolide) microspheres

Biodegradable poly(lactide-co-glycolide) (PLGA) microspheres have received much attention over the last twenty-five years for controlled parenteral delivery of therapeutic protein and peptide drugs [1, 2]. In general, for protein drugs delivery, PLGA and PLGA-based single-polymer microspheres system still suffer from two major technical problems associated with their inherent stability problem [3]. Initial burst release followed by very slow and incomplete release is one of the most serious problems in the formulation of PLGA-based protein drugs delivery system. Many strategies have been explored currently to reduce the high initial burst release of protein and peptide drugs from PLGA microspheres [4]. Fabrication of double-walled microspheres in which protein drugs encapsulated in the inner core surrounded by a drug free outer polymer layer offers a promising technique in reducing the high initial burst release. In previous studies, large size double-walled microspheres have been prepared using poly(L-lactic acid) (PLLA) and different co-polymers of PLGA (ester-terminated and carboxyl-terminated) as the core and shell material. Double-walled microspheres consisting PLLA and ester-terminated PLGA as the core or shell material required more time for the complete release of encapsulated drugs due to the slow degrading nature of these polymers