Allan G.A. Coombes

Surface Modification of Poly(lactide-co-glycolide) Nanospheres by Biodegradable Poly(lactide)-Poly(ethylene glycol) Copolymers

The modification of surface properties of biodegradable poly(lactide-co-glycolide) (PLGA) and model polystyrene nanospheres by poly(lactide)-poly(ethlene glycol) (PLA:PEG) copolymers has been assessed using a range of in vitro characterization methods followed by in vivo studies of the nanospheres biodistribution after intravenous injection into rats. Coating polymers with PLA:PEG ratio of 2:5 and 3:4 (PEG chains of 5000 and 2000 Da, respectively) were studied. The results reveal the formation of a PLA: PEG coating layer on the particle surface resulting in an increase in the surface hydrophilicity and decrease in the surface charge of the nanospheres. The effects of addition of electrolyte and changes in pH on stability of the nanosphere dispersions confirm that uncoated particles are electrostatically stabilized, while in the presence of the copolymers, steric repulsions are responsible for the stability. The PLA:PEG coating also prevented albumin adsorption onto the colloid surface. The evidence that this effect was observed for the PLA:PEG 3:4 coated nanospheres may indicate that a poly(ethylene glycol) chain of 2000 Da can provide an effective repulsive barrier to albumin adsorption. The in vivo results reveal that coating of PLGA nanospheres with PLA:PEG copolymers can alter the biodistribution in comparison to uncoated PLGA nanospheres. Coating of the model polystyrene nanospheres with PLA:PEG copolymers resulted in an initial high circulation level, but after 3 hours the organ deposition data showed values similar to uncoated polystyrene spheres. The difference in the biological behaviour of coated PLGA and polystyrene nanospheres may suggest a different stability of the adsorbed layers on these two systems. A similar biodistribution pattern of PLA:PEG 3:4 to PEG 2:5 coated particles may indicate that poly(ethylene glycol) chains in the range of 2000 to 5000 can produce a comparable effect on in vivo behaviour.

Precipitation casting of polycaprolactone for applications in tissue engineering and drug delivery

Microporous materials have been produced by gradual precipitation from solutions of poly(epsilon-caprolactone) (PCL) in acetone induced by solvent extraction across a semi-permeable PCL membrane which is formed in situ at the polymer solution/non-solvent interface. Microparticulates of hydroxyapatite and inulin polysaccharide, respectively, were incorporated in precipitation cast PCL matrices to illustrate potential applications in hard tissue repair and macromolecular drug release. Microporous PCL and HA filled PCL materials were found to provide a favourable surface for attachment and growth of primary human osteoblasts in cell culture. The in vitro degradation characteristics of microporous PCL and inulin/PCL materials in PBS at 37 degrees C were monitored over 45 months. Microporous PCL demonstrated zero weight loss, minor changes in molecular weight characteristics and a fairly constant indentation resistance of around 1 MN/m2. Inulin-loaded PCL materials exhibited a total weight loss of approximately 17% after 12 months in PBS. The indentation resistance decreased by 50% from an initial value of 28 MN/m2 in the first 2 months and then remained stable. Precipitation cast materials based on PCL are expected to be useful for formulating long-term, controlled release devices for bioactive molecules such as growth factors and hormones and extended-residence supports for cell growth and tissue development.

The preparation of sub-200 nm poly(lactide-co-glycolide) microspheres for site-specific drug delivery

Abstract The biodistribution of injected colloidal carriers for targeted delivery will be highly dependent upon their size and surface properties. This paper describes investigations on the preparation of sub-200 nm poly(lactide-co-glycolide) (PLGA) microspheres by the variation of processing parameters using the solvent evaporation technique. Smaller particle sizes were found to be favoured by a two stage emulsification process, a low PLGA concentration and an increased surfactant concentration. In the latter case, it would appear that the viscosity of the continuous phase is a crucial factor. The process of particle size reduction could further be complimented by using a surfactant of lower molecular weight. Employing such procedures, microspheres as low as 90 nm in diameter of low polydispersity were prepared in a reproducible manner.

Protein-loaded poly(dl-lactide-co-glycolide) microparticles for oral administration: formulation, structural and release characteristics

Abstract FITC-labelled bovine serum albumin has been entrapped in sub-5 micron particles of poly( dl -lactide-co-glycolide copolymer) (PLG) using a water-in-oil-in-water (w/o/w) emulsification-solvent evaporation technique. The concentration of PVA stabiliser in the external continuous phase was found to affect not only the particle size, size distribution and protein content but also the release characteristics and internal structure of the microparticles. The importance of primary emulsification was underlined by the finding that the protein content of microparticles with mean size 1 μ m could be increased from about 1% w/w to around 12% w/w by increasing the amount of protein added to the primary emulsion and the homogenisation time in this stage. Under conditions of low stabiliser concentration, multi-nucleate particles formed by polymer precipitation and envelopment of the droplets of the primary w/o emulsion. In this case surface protein loading was of the order of 30% w/w. Under conditions of high PVA stabiliser concentration, disruption of the primary emulsion occurred, resulting in sub-micron particles which were characterised by a high surface protein loading of the order of 70% w/w. A mechanism for protein microencapsulation is presented which is heavily influenced by the shear stresses induced during the process of secondary emulsification. This can explain certain aspects of the relationship between microparticle size and size distribution, protein content and release and the structural characteristics of microparticles produced using the w/o/w emulsification/solvent evaporation technique.

Preparation of sub-100 nm human serum albumin nanospheres using a pH-coacervation method.

Human serum albumin (HSA) nanospheres of about 100 nm diameter were prepared using a pH-coacervation method whereby acetone was added to an HSA solution (pH 9.0). The particles obtained were cross-linked by glutaraldehyde. Increasing the pH of the HSA solution resulted in a gradual rise in the particle size of the resultant nanospheres. A higher cross-linking efficiency was obtained with increased glutaraldehyde concentration and cross-linking time. No significant differences in surface properties, as determined by zeta potential measurements, were recorded between particles prepared from HSA solutions with different pH. The nanospheres were quite stable over 4 days in both phosphate buffer saline (PBS) solution (pH 7.4) and rat serum, but degraded rapidly over 6 hours when incubated in PBS solution containing trypsin.

In vitro cell interaction and in vivo biodistribution of poly(lactide-co-glycolide) nanospheres surface modified by poloxamer and poloxamine copolymers

Abstract Biodegradable poly(lactide-co-glycolide) (PLGA) nanospheres in the size range 80–150 nm have been produced, using an interfacial polymer deposition (nanoprecipitation) method. The nanospheres have been surface modified with poly(ethylene glycol) (PEG) either by adsorption of polypropylene oxide-polyethylene oxide (PPO-PEO) block copolymers of the poloxamer and poloxamine series (poloxamer 407, poloxamine 904 and poloxamine 908) or by incorporation of the same copolymers into the nanospheres during the production. The nanospheres were radiolabelled by incorporation of indium-111-oxine during nanosphere production. The biological behaviour of the PLGA nanosphere systems is discussed in terms of the in vitro cell interaction with isolated non-parenchymal liver cells and the in vivo biodistribution in the rat and rabbit models after intravenous injection. The results are compared to those for model polystyrene nanospheres surface modified in the same manner. It is shown that PLGA nanospheres coated with poloxamer 407 or poloxamine 908 exhibit prolonged blood circulation times accompanied by a combined reduction in liver and spleen accumulation after intravenous injection to the rat. Three hours post intravenous injection, 39% and 28% of the administered dose of poloxamer 407- and poloxamine 908-coated PLGA nanospheres remains in the blood circulation.

The control of protein release from poly(dl-lactide co-glycolide) microparticles by variation of the external aqueous phase surfactant in the water-in oil-in water method

Poly(DL-lactide co-glycolide) microparticles below 5 microm in size and containing ovalbumin (OVA), were prepared using the water-in oil-in water (w/o/w) technique with either polyvinyl alcohol (PVA) or polyvinylpyrrolidone (PVP) as stabilisers in the external aqueous phase. PVP-stabilised microparticles exhibited higher protein loading (8.2%, w/w relative to 4.0% for PVA stabilised microparticles) and increased core loading (encapsulation) of protein (70% vs. 30% for the PVA system). The use of PVP instead of PVA to prepare microparticles also resulted in reduction in the initial burst release of OVA, together with sustained protein release over 28 days and an increase in the protein delivery capacity from 35 to 45 microg/mg particles. The changes in protein loading and delivery characteristics are considered to arise in part from an increase in the viscosity of the droplets of polymer solution, constituting the primary water-in oil emulsion, by diffusion of PVP from the external aqueous phase. Variation of the external aqueous phase surfactant provides a promising approach for improving the loading of therapeutic proteins and vaccine antigens within biodegradable microparticles and for modulating their release pattern.

Single dose, polymeric, microparticle-based vaccines: the influence of formulation conditions on the magnitude and duration of the immune response to a protein antigen

Ovalbumin-loaded poly (D,L-lactide co-glycolide) [OVA-loaded PLG] microparticles, produced by emulsion/solvent evaporation stimulated the production of high serum IgG antibody levels after a single subcutaneous (s.c.) administration in mice and the duration of the immune response paralleled the degradation rate of the carrier. Formulations based on slow resorbing PLG maintained relatively constant peak antibody levels for 26 weeks and high titres for over 1 year at a level approximating the peak response to the faster resorbing, OVA-loaded particles which was of lower duration. Vaccine formulations prepared by simple mixing of blank PLG microparticles and OVA exhibited low primary immune responses which were only elevated by boosting. OVA-loaded PLG microparticles exhibited a substantial surface protein component amounting to ca 40% and 60% of the total protein loading for slow resorbing and fast resorbing PLG, respectively. These findings suggest that sustained presentation of surface protein to the immune system was a major factor in the induction and long-term maintenance of high antibody titres following a single s.c. administration of OVA-loaded microparticles.

Detection and determination of surface levels of poloxamer and PVA surfactant on biodegradable nanospheres using SSIMS and XPS

The surface chemical characterisation of sub-200 nm poly(DL-lactide co-glycolide) nanospheres has been carried out using the complementary analytical techniques of static secondary ion mass spectrometry (SSIMS) and X-ray photoelectron spectroscopy (XPS). The nanospheres, which are of interest for site-specific drug delivery, were prepared using an emulsification-solvent evaporation technique with poly(vinyl alcohol), Poloxamer 407 and Poloxamine 908 respectively as stabilisers. The presence of surfactant molecules on the surface of cleaned biodegradable colloids was confirmed and identified on a qualitative molecular level (SSIMS) and from a quantitative elemental and functional group analysis (XPS) perspective. SSIMS and XPS data were also used in combination with electron microscopy to monitor the effectiveness of cleaning procedures in removing poorly bound surfactant molecules from the surface of nanospheres. The findings are discussed with respect to the development of nanoparticle delivery systems, particularly the composition of the surface for extending blood circulation times and achieving site-specific deposition.

Survivability of probiotics encapsulated in alginate gel microbeads using a novel impinging aerosols method

Encapsulation of probiotic bacteria in cross-linked alginate beads is of major interest for improving the survivability in harsh acid and bile environment and also in food matrices. Alginate micro beads (10-40 μm) containing the probiotics Lactobacillus rhamnosus GG and Lactobacillus acidophilus NCFM were produced by a novel technique based on dual aerosols of alginate solution and CaCl(2) cross linking solution. Extruded macro beads (approximately 2mm diameter) produced by the conventional method and micro beads produced by novel aerosols technique offered comparable protection to L. rhamnosus in high acid and bile environment. Chitosan coating of micro beads resulted in a significant increase in survival time of L. rhamnosus from 40 to 120 min in acid condition and the reduction in cell numbers was confined to 0.94 log over this time. Alginate macro beads are more effective than micro beads in protecting L. acidophilus against high acid and bile. Chitosan coating of micro beads resulted in similar protection to L. acidophilus in macro beads in acid and extended the survival time from 90 to at least 120 min. Viability of this organism in micro beads was 3.5 log after 120 min. The continuous processing capability and scale-up potential of the dual aerosol technique offers potential for an efficient encapsulation of probiotics in very small alginate micro beads below sensorial detection limits while still being able to confer effective protection in acid and bile environment.