Biomedical subjects

Steven P Schwendeman

Publications and source records attributed to Steven P Schwendeman.

11 recordsLinked to original sources

PubMed2006

Injectable polymer microspheres enhance immunogenicity of a contraceptive peptide vaccine.

Advanced contraceptive peptide vaccines suffer from the unavailability of adjuvants capable of enhancing the antibody response with acceptable safety. We sought to overcome this limitation by employing two novel poly(lactic-co-glycolic acid) (PLGA) microsphere formulations to deliver a synthetic human chorionic gonadotropin (hCG) peptide antigen co-synthesized with a T-cell epitope from tetanus toxoid (TT), C-TT2-CTP35: surface-conjugated immunogen to induce phagocytosis; and encapsulated peptide to provide a depot effect, with MgCO(3) co-encapsulated in the polymer to neutralize acidity from the biodegrading PLGA polyester. A single immunization of encapsulated peptide in rabbits elicited a stronger antibody response with equivalent duration relative to a positive control--three injections of the peptide administered in a squalene-based water-in-oil emulsion. Surface-conjugated peptide was less effective but enhanced antibody levels at 1/5 the dose, relative to soluble antigen. Most remarkable and unexpected was the finding that co-encapsulation of base was essential to attain the powerful adjuvant effect of the PLGA-MgCO(3) system, as the MgCO(3)-free microspheres were completely ineffective. A promising contraceptive hCG peptide vaccine with acceptable side effects (i.e., local tissue reactions) was achieved by minimizing PLGA and MgCO(3) doses, without significantly affecting antibody response.

Adjuvants, Immunologic↗

PubMed2006

Reduced nonprotein thiols inhibit activation and function of MMP-9: implications for chemoprevention.

Clinical studies demonstrate a positive correlation between the extent of matrix metalloproteinase (MMP) activation and malignant progression of precancerous lesions. Therefore, identification of effective, well-tolerated MMP inhibitors represents a rational chemopreventive strategy. A variety of agents, including proteinases and thiol-oxidizing compounds, activate MMPs by initiating release of the propeptide's cysteine sulfur "blockage" of the MMP active site. Despite the importance of the propeptide's cysteine thiol in preserving MMP latency, limited studies have evaluated the effects of reduced thiols on MMP function. This study investigated the effects of two naturally occurring nonprotein thiols, i.e., glutathione (GSH) and N-acetylcysteine (NAC), on activation, function, and cellular-extracellular matrix interactions of the basement-membrane-degrading gelatinase, MMP-9. Our results reveal that NAC and GSH employ protein S-thiolation to inhibit organomercurial activation of pro-MMP-9. Gelatinase activity assays showed that GSH and NAC significantly inhibited MMP-9 but not MMP-2 function, implying isoform structural specificity. Immunoblot analyses, which suggested GSH interacts with MMP-9's active-site Zn, were corroborated by computational molecular modeling. Cell invasion assays revealed that NAC enhanced endostatin's ability to inhibit human cancer cell invasion. Collectively, these data demonstrate that nonprotein thiols suppress MMP-9 activation and function and introduce the prospect for their use in chemopreventive applications.

Acetylcysteine↗

PubMed2006

Prediction of microclimate pH in poly(lactic-co-glycolic acid) films.

An equilibrium mathematical model that accurately predicts microclimate pH (mupH) in thin biodegradable polymer films of poly(lactic-co-glycolic acid) (PLGA) is described. mupH kinetics was shown to be primarily a function of: (i) kinetics of water-soluble acid content and composition in the polymer matrix and (ii) polymer/water partition coefficient of water-soluble degradation products (P(i)). Polymers were coated on standard pH glass electrodes, and mupH was measured potentiometrically. Water-soluble acid distribution and content in PLGA films were determined by pre-derivatization HPLC. Polymer degradation products partitioned favorably in the polymer phase relative to water (P(i) range: approximately 6-100), and P(i) increased with increasing hydrophobicity of the acidic species according to a linear free energy law related to reversed phase HPLC retention time for the corresponding derivatized bromophenacyl esters. The mupH predicted by the model was in excellent agreement with experimental mupH for several PLGAs as a function of time and PLGA lactic/glycolic acid ratio. These data may be useful to slowly release pH-sensitive PLGA-encapsulated bioactive substances and provide a general framework for predicting partitioning behavior of degradation products in biodegradable polymers.

Chromatography, High Pressure Liquid↗

PubMed2006

BSA degradation under acidic conditions: a model for protein instability during release from PLGA delivery systems.

Acidification of the internal poly(lactide-co-glycolide) (PLGA) microenvironment is considered one of the major protein stresses during controlled release from such delivery systems. A model protein, bovine serum albumin (BSA), was incubated at 37 degrees C for 28 days to simulate the environment within the aqueous pores of PLGA during the release phase and to determine how acidic microclimate conditions affect BSA stability. Size-exclusion high performance liquid chromatography (SE-HPLC), SDS-PAGE, and infrared spectroscopy were used to monitor BSA degradation. BSA was most stable at pH 7, but rapidly degraded via aggregation and hydrolysis at pH 2. These simulated degradation products were nearly identical to that of unreleased BSA found entrapped within PLGA 50/50 millicylinders. At pH 2, changes in BSA conformation detected by various spectroscopic techniques were consistent with acid denaturation of the protein. By contrast, at pH 5 and above, damage to BSA was insufficient to explain the instability of the protein in the polymer. Thus, these data confirm the hypothesis that acid-induced unfolding is the basis of BSA aggregation in PLGA and the acidic microclimate within PLGA is indeed a dominant stress for encapsulated BSA. To increase the stability of proteins within PLGA systems, formulations must protect against potentially extreme acidification such that native structure is maintained.

Drug Delivery Systems↗

PubMed2005

Biodegradable poly(lactic-co-glycolic acid) microparticles for injectable delivery of vaccine antigens.

Injectable biodegradable polymeric particles (usually microspheres) represent an exciting approach to control the release of vaccine antigens to reduce the number of doses in the immunization schedule and optimize the desired immune response via selective targeting of antigen to antigen presenting cells. After the first couple of decades of their study, much progress has been made towards the clinical use of antigen-loaded microspheres. Poly(lactide-co-glycolic acids) (PLGAs) have been studied most commonly for this purpose because of their proven safety record and established use in marketed products for controlled delivery of several peptide drugs. PLGA microspheres have many desirable features relative to standard aluminum-based adjuvants, including the microspheres' ability to induce cell-mediated immunity, a necessary requirement for emergent vaccines against HIV and cancer. This review examines several impediments to PLGA microparticle development, such as PLGA-encapsulated antigen instability and deficiency of animal models in predicting human response, and describes new trends in overcoming these important issues. PLGA microparticles have displayed unprecedented versatility and safety to accomplish release of one or multiple antigens of varying physical-chemical characteristics and immunologic requirements, and have now met numerous critical benchmarks in development of long-lasting immunity after a single injected dose.

Animals↗

PubMed2005

Mapping neutral microclimate pH in PLGA microspheres.

A quantitative ratiometric method based on laser scanning confocal microscopic imaging was developed to create a pixel-by-pixel neutral microclimate pH (5.8-8.0) map inside poly(lactic-co-glycolic acid) (PLGA) microspheres. The method was accurate to within +/-0.2 pH units. PLGA microspheres with encapsulated bovine serum albumin and the pH-sensitive dye, SNARF-1(R) dextran (MW=10 kDa), were prepared by a water-in-oil-in-water solvent evaporation method. The imaging method was used to monitor the pH changes in PLGA microspheres with or without co-encapsulation of acid-neutralizing MgCO3, and for three different microsphere sizes (2.0+/-0.3, 40+/-10 and 137+/-32 microm) during incubation under physiological conditions. The pH of microspheres was significantly higher with co-encapsulated base compared to base-free microspheres, whose microclimate pH was predominantly below the dye detection limit of pH=5.8. The small microspheres displayed a more neutral pH during incubation probably due to their much shorter diffusion path for polymer-dissolved acids compared with medium- and large-size microspheres. The pH mapping technique discussed here may be useful in routine formulation development and for elucidation of mechanisms of microclimate pH development and polymer erosion.

Drug Delivery Systems↗

PubMed2004

Determination of water-soluble acid distribution in poly(lactide-co-glycolide).

Determination of the kinetics of water-soluble degradation products inside poly(lactide-co-glycolide) (PLGA) delivery systems during polymer degradation is important to evaluate the polymer microclimate conditions, particularly microclimate pH changes for optimization of encapsulated drug stability. A pre-derivatization high-performance liquid chromatography (HPLC) method was developed for separation and quantification of water-soluble acid impurities and degradation products in PLGA. Thin PLGA films (approximately 200 microm) were incubated in PBS/0.02% Tween 80, pH 7.4, for 6 weeks. Water-soluble monomers and oligomers were obtained from polymer films after repeated CHCl(3)/H(2)O extraction and then derivatized into bromophenacyl esters. With the common chromophore, the esters were separated and quantified by HPLC with increased ultraviolet (UV) sensitivity at 254 nm. The total amount of water-soluble acids in the extract was validated by potentiometric titration with tetrabutyl ammonium hydroxide. During the first 3 weeks of incubation of PLGA 50:50 (inherent viscosity = 0.63 dL/g), the principal water-soluble acids in the polymer were glycolic, lactic, and lactoyllactic acids, and an unknown oligomer. After 4 weeks of incubation, a large fraction of higher molecular weight oligomers was observed. Pre-derivatization HPLC can be used to accurately measure water-soluble acid distribution, and may be invaluable to examine the degradation behavior of PLGAs, including the underlying mechanism of polymer microclimate pH development.

Acids↗

PubMed2004

Mechanistic evaluation of the glucose-induced reduction in initial burst release of octreotide acetate from poly(D,L-lactide-co-glycolide) microspheres.

One major obstacle for development of injectable biodegradable microspheres for controlled peptide and protein delivery is the high initial burst of drug release occurring over the first day of incubation. We describe here the significant reduction in initial burst release of a highly water-soluble model peptide, octreotide acetate, from poly(D,L-lactide-co-glycolide) microspheres by the co-encapsulation of a small amount of glucose (e.g., 0.2%w/w), i.e., from 30+/-20% burst - glucose to 8+/-3% + glucose (mean+/-SD, n=4). This reduction is unexpected since hydrophilic additives are known to increase porosity of microspheres, causing an increase in permeability to mass transport and a higher burst. Using the double emulsion-solvent evaporation method of encapsulation, the effect of glucose on initial burst in an acetate buffer pH 4 was found to depend on polymer concentration, discontinuous phase/continuous phase ratio, and glucose content. Extensive characterization studies were performed on two microsphere batches, +/-0.2% glucose, to elucidate the mechanism of this effect. However, no significant difference was observed with respect to specific surface area, porosity, internal and external morphology and drug distribution. Continuous monitoring of the first 24-h release of octreotide acetate from these two batches disclosed that even though their starting release rates were close, the microspheres + glucose exhibited a much lower release rate between 0.2 and 24h compared to those - glucose. The microspheres + glucose showed a denser periphery and a reduced water uptake at the end of 24-h release, indicating decreased permeability. However, this effect at times was offset as glucose content was further increased to 1%, causing an increase in surface area and porosity. In summary, we conclude that the effect of glucose on initial burst are determined by two factors: (1) increased initial burst due to increased osmotic pressure during encapsulation and drug release, and (2) decreased initial burst due to decreased permeability of microspheres.

Coated Materials, Biocompatible↗

PubMed2002

Characterization of the initial burst release of a model peptide from poly(D,L-lactide-co-glycolide) microspheres.

In order to study the mechanism of initial burst release from drug-loaded poly(D,L-lactide-co-glycolide) (PLGA) microspheres, a model peptide, octreotide acetate, was encapsulated in PLGA 50/50 (M(w) approximately 50,000) microspheres using a double emulsion-solvent evaporation method. A simple and accurate continuous monitoring system was developed to obtain a detailed release profile. After different incubation times in the release medium, the morphology and permeability of the microspheres were examined using scanning electron and confocal microscopy (after immersing the microspheres in a fluorescent dye solution for 30 min), respectively. Both the external and internal morphology of the microspheres changed substantially during release of >50% of the peptide over the first 24 h into an acetate buffer, pH 4 at 37 degrees C. After 5 h, a 1-3 microm "skin" layer with decreased porosity was observed forming around the microsphere surface. The density of the "skin" appeared to increase after 24 h with negligible surface pores present, suggesting the formation of a diffusion barrier. Similar morphological changes also occurred at pH 7.4, but more slowly. Correlated with these results, the confocal microscopy studies (at pH 4) showed that the amount of dye penetrated inside the microspheres sharply decreased with time. In summary, over the first 24 h of drug release, a non-porous film forms spontaneously at the surface of octreotide acetate-loaded PLGA microspheres in place of an initially porous surface. These rapid alterations in polymer morphology are correlated with a sharp decline in permeability and the cessation of the initial burst.

Biocompatible Materials↗

PubMed2002

Comparison of the effects of Mg(OH)2 and sucrose on the stability of bovine serum albumin encapsulated in injectable poly(D,L-lactide-co-glycolide) implants.

Incomplete release and poor stability of encapsulated proteins are common hurdles to overcome when developing poly(lactide-co-glycolide) (PLGA) controlled-release systems. Antacid excipients such as Mg(OH)2, which increase both microclimate pH and polymer water uptake, have been shown to prevent acid-induced instability of proteins encapsulated in PLGA. The purpose of this study was to delineate the effects of microclimate pH and polymer water content on the stability of encapsulated bovine serum albumin (BSA) by comparing the effects of Mg(OH)2 with those of another excipient, sucrose, which increases polymer water content without significantly affecting acid-base chemistry of the polymer. These two excipients, when encapsulated in PLGA at appropriate levels (3% Mg(OH)2 vs. 10% sucrose), were found to cause identical water sorption kinetics, thus allowing the effect of the two microclimate parameters to be determined. In contrast to their similar effects on polymer water sorption, Mg(OH)2 afforded a much greater stabilization effect on encapsulated BSA than did sucrose, with less than 7% aggregates for 3% Mg(OH)2 compared to 51% for 10% sucrose and 81% without either excipient after 4 weeks of incubation at 37 degrees C. When the protein stabilization rationale of neutralizing the acidic microenvironment by adding Mg(OH)2 was applied to the delivery of an important therapeutic protein, tissue plasminogen activator (t-PA), t-PA stability was also improved and the active protein was completely recovered during a one month period of in vitro release. These data demonstrated that although increased water uptake induced by antacid excipients may improve the stability of the encapsulated proteins, the homogeneous acid neutralization effect is unique to antacid excipients such as Mg(OH)2, which is necessary to maintain the stability of proteins in acidic PLGA specimens.

Drug Implants↗

PubMed2002

Recent advances in the stabilization of proteins encapsulated in injectable PLGA delivery systems.

Injectable controlled-release systems based on biodegradable copolymers of lactic and glycolic acids (PLGAs) have become widely used for delivery of protein therapeutics and vaccine antigens. Over the last five years, great strides have been made toward overcoming the difficulty of stabilizing PLGA-encapsulated proteins. In addition to stabilizing proteins during encapsulation with anhydrous methods, two approaches have proven highly effective to stabilize proteins during 1-month release incubation under physiological conditions: protein complexation with zinc and control of PLGA microclimate pH with antacid excipients. Described here are recent advances in the stabilization of proteins encapsulated in PLGA delivery systems.

Biocompatible Materials↗