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Biomedical subjects

A G Coombes

Publications and source records attributed to A G Coombes.

At least 19 recordsLinked to original sources
PubMed1999

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.

Adsorption↗
PubMed1999

Biodegradable lamellar particles of poly(lactide) induce sustained immune responses to a single dose of adsorbed protein.

The adjuvanticity of lamellar particles of poly(L-lactide) (PLA) towards adsorbed ovalbumin (OVA) was investigated. The aim of vaccine formulation was to maximise the amount of antigen retained on the particles and the time of retention during incubation of the formulations in PBS at 37 degrees C. Unmodified PLA lamellae were capable of adsorbing large quantities of OVA (up to 12.5% w/w) but major and rapid desorption occurred in PBS at 37 degrees C (80% released in 24 h). Retention of OVA on PLA lamellae was improved (25% released in 24 h) by precipitating the particles using aqueous sodium deoxycholate solution (DOC-modified PLA lamellae and lyophilising the lamellae-protein preparation after adsorption. Sustained immune responses were elicited in mice to a single sub-cutaneous injection of OVA adsorbed onto DOC-modified PLA lamellae. The level of antibodies induced and the pattern of response was similar to that induced by an alum-adsorbed OVA formulation. Normally boosting is required to obtain high levels of antibody when OVA is adsorbed on poly(DL-lactide co-glycolide) (PLG) microspheres. The lamellar forms of PLA may function as an efficient immunomodulator by effectively retaining adsorbed antigen and by activating immune cells due to their irregular shape. PLA lamellae have potential to stimulate enhanced immune responses to a variety of adsorbed antigens.

Adjuvants, Immunologic↗
PubMed1999

Induction of cellular immunity to a mycobacterial antigen adsorbed on lamellar particles of lactide polymers.

Microspheres prepared from synthetic, biodegradable poly (L-lactide) [PLA] and copolymers of lactide and glycolide such as poly (DL lactide co-glycolide) [PLG] have been widely investigated for controlled delivery of encapsulated vaccine antigens. In this study we describe novel lamellar microparticles produced from PLA to which protein antigens can be adsorbed. These particles when administered to mice, induced strong Th1-type T cell responses to the adsorbed 38 kDa protein antigen from M. tuberculosis characterised by high levels of Interferon-gamma. In addition to proteins, we were also able to adsorb synthetic peptides resulting in specific T cell proliferation. Induction of strong cellular immunity together with the versatility of antigen adsorption to these particles should make such lamellae a useful tool to deliver protective antigens from intracellular pathogens.

Adsorption↗
PubMed1999

The stability and immunogenicity of a protein antigen encapsulated in biodegradable microparticles based on blends of lactide polymers and polyethylene glycol.

Protein-loaded microparticles were produced from blends of poly(ethylene glycol) (PEG) with poly(L-lactide) (PLA) homopolymer or poly(DL-lactide co-glycolide) copolymers (PLG) using a water-in oil-in oil method. The stability of ovalbumin (OVA) associated with microparticles prepared using PEG and 50:50 PLG, 75:25 PLG and PLA, respectively, was analysed by SDS-PAGE and quantified by scanning densitometry following incubation in PBS at 37 degrees C for up to 1 month. Fragmentation and aggregation of OVA was detected with all 3 formulations. The extent of both processes correlated with the degradation rate of the lactide polymer used and decreased in the order PLA < 75:25 PLG < 50:50 PLG. Extensive degradation of the PLG/PEG microparticles also occurred over 4 weeks whereas the use of PLA/PEG blends resulted in a stable microparticle morphology and much reduced fragmentation and aggregation of the associated protein. Following a single sub-cutaneous immunisation, high levels of specific serum IgG antibody were elicited by OVA associated with the PLA/PEG particles. Injection of OVA associated with the 75:25 PLG/PEG microparticles resulted in very low levels of specific antibody. A higher response was induced by the 50:50 PLG/PEG formulation but there was very large inter-animal variation in this group. Antibody levels elicited by all 3 formulations were significantly higher than those elicited by a single injection of soluble OVA. Analysis of antigen specific IgG1 and IgG2a antibody subtype levels also revealed the greater efficacy of the PLA/PEG microparticles as an adjuvant system. The use of PLA/PEG microparticles shows improved protein loading and delivery capacity while maintaining a high level of stability of the associated protein. These results indicate a strong correlation between the stability of microencapsulated antigen and the magnitude of the immune response following sub-cutaneous immunisation.

Animals↗
PubMed1999

Potential of polymeric lamellar substrate particles (PLSP) as adjuvants for vaccines.

In recent years microspheres or microparticles produced from biodegradable polymers such as poly(D,L-lactide) (PLA) and poly(D, L-lactide-co-glycolide) (PLGA) containing encapsulated vaccine antigens have been investigated for administration via parenteral, oral, and intranasal routes. These microparticles allow the controlled release of vaccines with an aim to reduce the number of doses for primary immunisation or to develop single dose vaccines. The polymer materials have been widely regarded as being of minimal toxicity. Evaluation of candidate systems in animal studies have shown antibody levels and cell responses similar to or greater than those observed with adjuvants such as alum. However, there are concerns regarding the integrity and immunogenicity of the antigen during the encapsulation process when the antigen is exposed to organic solvents, high shear stresses and the exposure of antigen to low pH which is caused by polymer degradation. An alternative approach would be to adsorb antigens to the surface of biodegradable polymer particles. Polymeric lamellar substrate particles (PLSP), produced by a simple precipitation of PLA, are suitable for this purpose. The adsorption of antigens onto these particles is a simple procedure. It avoids pH changes due to bulk polymer degradation and the use of solvents and therefore will be less damaging to the vaccine. Moreover, such systems will be much easier to scale up for a clinical study and eventual manufacture. The aim of this article is to discuss the preparation and physical characteristics of PLSP, antigen adsorption, in vivo efficacy of PLSP antigen systems and to consider the potential of PLSP as controlled release adjuvants for protein, peptide or viral vaccines.

Adjuvants, Immunologic↗
PubMed1998

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.

Drug Carriers↗
PubMed1998

The distribution of protein associated with poly(DL-lactide co-glycolide) microparticles and its degradation in simulated body fluids.

Poly(lactide co-glycolide) (PLG) microparticles with a mean size of less than 2 microns, prepared by the water-in oil-in water method, exhibited a maximum surface protein (ovalbumin) content in excess of 50% of the total loading. The surface-core distribution was found to be sensitive to stabiliser concentration and the type of albumin used in the formulation. The degradation of OVA was monitored following incubation of microparticles for 14 days in PBS and for 2 h in simulated gastric and intestinal fluids, respectively. OVA removed from the surface of particles, following incubation in PBS, was found to be intact as measured by SDS-PAGE. After 7 days in PBS at 37 degrees C, protein extracted from the microparticles was found to be partly hydrolysed with the prevalence of an antigen fragment at 36.1 kDa. The relative amount of intact OVA in 50:50 PLG microparticles decreased more rapidly than in the slower degrading 75:25 PLG microparticles. Importantly, the degradation of extracted OVA over 14 days was similar for microparticles incubated either with regular changes of release medium or in a dialysis tube. Almost all the OVA encapsulated in PLG microparticles remained intact after incubation in simulated gastric and intestinal media for 2 h. In contrast, the surface protein was rapidly degraded by trypsin and pepsin and was not detected by SDS-PAGE.

Body Fluids↗
PubMed1998

Resorbable lamellar particles of polylactide as adjuvants for influenza virus vaccines.

Lamellar particles and microspheres were produced by precipitation from solutions of resorbable, biocompatible, semi-crystalline poly(L-lactide)[PLA] and amorphous poly(DL lactide co-glycolide)[PLG] copolymer, respectively, to investigate their adjuvanticity towards adsorbed influenza virus. Both types of substrate were capable of adsorbing large quantities of virus (> 15% w/w) and retaining virus (> 60% of the initial load) over an 8 week time scale in-vitro. Potent immune responses were obtained in mice after the intra-muscular injection of adsorbed vaccine systems. The response to virus adsorbed on PLA lamellar particles was almost five times that obtained using PLG microspheres and fourteen times that using aqueous vaccine. The lamellar forms of PLA may function as an immunomodulator enhancing phagocytic activity due to their irregular shape and may be useful in improving the immune response to a variety of protein and viral antigens.

Adjuvants, Immunologic↗
PubMed1998

Current practice of local anaesthesia for routine ocular surgery.

We surveyed the current practice of local anaesthesia for routine ocular surgery by consultant anaesthetists in the Wessex region using a postal questionnaire. Most consultants were in agreement concerning the type and technique of block, and monitoring to be used during local anaesthesia for ocular surgery. The issue of teaching for trainee anaesthetists produced a varied response. We found that a different level of perioperative preparation and monitoring was used from that recommended by the Joint Working Party on the subject in 1993.

Anesthesia, Local↗
PubMed1997

Biodegradable polymeric microparticles for drug delivery and vaccine formulation: the surface attachment of hydrophilic species using the concept of poly(ethylene glycol) anchoring segments.

Poly(ethylene glycol)-dextran (PEG-DEX) conjugates have been used as a combined stabilizer and surface modifier to produce resorbable poly(DL-lactide-co-glycolide) (PLG) microparticles by an emulsification/solvent evaporation technique. The use of PEG or dextran polymers alone was incapable of producing microparticles. Particle size measurements revealed smaller mean particle sizes (480 nm) and improved polydispersity when using a 1.2% PEG substituted conjugate relative to a 9% substituted material (680 nm). PLG microparticles modified by post-adsorbed PEG-DEX conjugates flocculated in 0.01 M salt solutions, whereas PLG microparticles prepared using PEG-DEX as a surfactant were stable in at least 0.5 M NaCl solutions. Surface modification of PLG microparticles was confirmed by zeta potential measurements and surface analysis using X-ray photoelectron spectroscopy. The presence of surface exposed dextran was confirmed by an immunological detection method using a dextran-specific antiserum in an enzyme-linked immunosorbent assay. The findings support a model in which the PEG component of the PEG-DEX conjugate provides an anchor to the microparticle surface while the dextran component extends from the particle surface to contribute a steric stabilization function. This approach offers opportunities for attaching hydrophilic species such as targeting moieties to biodegradable microparticles to improve the interaction of drug carriers and vaccines with specific tissue sites.

Biocompatible Materials↗
PubMed1997

The immune response to a model antigen associated with PLG microparticles prepared using different surfactants.

The effect of different surfactants on the surface characteristics of poly(D,L-lactideco-glycolide) microparticles prepared by the emulsification/solvent evaporation technique was investigated and the immune response to a protein antigen (OVA) associated with these microparticles was measured. Three surfactants--polyvinyl alcohol (PVA, a conventional stabilizer of PLG microparticles), the non-ionic surfactant, poly(oxyethylene glycerol mono-oleate) [Tagat] and Bile salts (a natural emulsifer)--were used to produce OVA-loaded PLG microparticles. Antigen was detected at the surface of all three types of OVA-loaded microparticles, in amounts in excess of 40% of the total protein load. The levels of specific serum IgG antibody elicited to OVA were significantly higher (P < 0.05) after a single subcutaneous administration of antigen associated with the Bile salts and Tagat formulations compared to the PVA formulation. A strong correlation was revealed between the levels of antibody measured and the magnitude of negative surface charge of the particulate carrier. The pattern of the IgG antibody response to OVA was similar in all three cases, indicating that the degradation rate of the PLG polymer determined the duration of the response. The results demonstrate the potential of using different surfactants to produce PLG microparticles with increased adjuvant activity.

Animals↗
PubMed1996

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.

Alum Compounds↗
PubMed1996

Improving protein delivery from microparticles using blends of poly(DL lactide co-glycolide) and poly(ethylene oxide)-poly(propylene oxide) copolymers.

PURPOSE: Microparticles containing ovalbumin as a model for protein drugs were formulated from blends of poly(DL lactide-co-glycolide) and poly(ethylene oxide)-poly(propylene oxide) copolymers (Pluronic). The objectives were to achieve uniform release characteristics and improved protein delivery capacity. METHODS: The water- in oil -in oil emulsion/solvent extraction technique was used for microparticle production. RESULTS: A protein loading level of over 40% (w/w) was attained in microparticles having a mean diameter of approximately 5 microns. Linear protein release profiles over 25 days in vitro were exhibited by certain blend formulations incorporating hydrophilic Pluronic F127. The release profile tended to plateau after 10 days when the more hydrophobic Pluronic L121 copolymer was used to prepare microparticles. A delivery capacity of 3 micrograms OVA/mg particles/ day was achieved by formulation of microparticles using a 1:2 blend of PLG:Pluronic F127. CONCLUSIONS: The w/o/o formulation approach in combination with PLG:Pluronic blends shows potential for improving the delivery of therapeutic proteins and peptides from microparticulate systems. Novel vaccine formulations are also feasible by incorporation of Pluronic L121 in the microparticles as a co-adjuvant.

Chemistry, Pharmaceutical↗
PubMed1995

Synthetic delivery system for tuberculosis vaccines: immunological evaluation of the M. tuberculosis 38 kDa protein entrapped in biodegradable PLG microparticles.

Tuberculosis remains a major public health burden which could be ameliorated by effective and well-defined subunit vaccines, particularly because the protective efficacy of current M. bovis BCG vaccines is both unpredictable and variable. The immunodominant 38 kDa antigen from Mycobacterium tuberculosis was entrapped in biodegradable poly (DL-lactide co-glycolide) (PLG) microparticles which served as a delivery system. Both cellular and humoral immune responses were assessed and compared with those obtained after immunization with the 38 kDa protein emulsified in incomplete Freund's adjuvant (IFA). Vaccination of mice with a single dose of antigen-loaded microparticles resulted in specific IgG titres peaking after five weeks comparable to those achieved after vaccination with protein emulsified in incomplete Freund's adjuvant (IFA). T-cell responses were found to be superior to those induced with antigen/IFA. The T- and B-cell epitope specificities ad judged with synthetic peptides were identical following immunization with antigen in microparticles or IFA. Differences in adjuvanticity were revealed by measuring antigen-specific IgG1, IgG2a and antigen-induced IFN-gamma secretion in vitro: substantially higher titres of IgG2a were observed following immunization with antigen/microparticles than with 38 kDa protein/IFA. This was paralleled by a tenfold higher secretion of IFN-gamma in mice injected with antigen/microparticles. Reduction in colony-forming units was not consistent in mice immunized with 38 kDa protein entrapped in microparticles which were subsequently infected with live tubercle bacilli. Taken together these results indicate that biodegradable PLG microparticles constitute a favorable candidate vaccine delivery system worthy of further assessment in the quest to develop better and defined agents protecting against tuberculosis.

Amino Acid Sequence↗
PubMed1995

Aspects of the design and delivery of microparticles for vaccine applications.

Mortality and morbidity data continue to indicate there is a compelling need for the derivation of a new generation of vaccine delivery systems that can be usefully applied via injection and also mucosally. One technology that has potential for design as an effective vaccine delivery system is the formulation of biodegradable microparticles from the polymers and poly lactide co-glycolide (PLGA) in particular. The potential advantages of the delivery of vaccines within such microparticles is discussed. The potential for eliciting and optimising immunity after the mucosal delivery of biodegradable microparticles is also discussed.

Administration, Inhalation↗