The effect of poly(ethylene glycol)-poly(D,L-lactic acid) diblock copolymers on peptide acylation.

The combination of poly(ethylene glycol) (PEG) with a biodegradable poly(ester), such as poly(D,L-lactic acid) (PLA), is an approach that has been successfully used for the stabilization of proteins and peptides in several biodegradable delivery devices. The acylation of peptides inside degrading PLA microspheres has been described only recently as another instability mechanism related to the accumulation of polymer degradation products inside eroding PLA. We investigated whether the block copolymerization of PLA with PEG reduces peptide acylation inside degrading microspheres. Diblock copolymers consisting of poly(D,L-lactic acid) covalently bound to poly(ethylene glycol)-monomethyl ether (Me.PEG-PLA) were used for these investigations. Human atrial natriuretic peptide (ANP) was incorporated into microspheres manufactured from Me.PEG5-PLA45, a diblock copolymer with an overall PEG content of 10%. Peptide integrity inside the microspheres was monitored by HPLC-MS analysis during 4 weeks of microsphere degradation in isotonic phosphate buffer (pH 7.4) at 37 degrees C. Inside the degrading Me.PEG5-PLA45 microspheres, acylation products as well as an oxidation product of ANP were formed. The results demonstrate that the combination of PEG with PLA does not necessarily display a favorable effect concerning peptide acylation inside degrading polymer microspheres. However, they also suggested that the acylation reaction is mainly driven by the formation and accumulation of polymer degradation products inside the degrading microspheres.     

Degradation of and drug release from a novel 2,2-bis(2-oxazoline) linked poly(lactic acid) polymer.

The degradation rate of poly(lactic acid) (PLA) is typically modified by copolymerization of the glycolide with lactide. In the present study, the degradation rate of PDLLA was modified by a novel linking of PLA with 2,2'-bis(2-oxazoline). This modification resulted in formation of a more rapidly degrading poly(ester amide) (PEA) for controlled drug release. The hydrolytic degradation of PDLLA and PEA films was studied in PBS (pH 7.4, USP XXIV, 37 degrees C); the resulting decrease in molecular weight was determined by size exclusion chromatography and the weight loss of films was measured. Drug releases of guaifenesin (mw 198.2), timolol (mw 332.4), sodium salicylate (mw 160.1) and FITC-dextran (mw 4400) from PDLLA and PEA films and microspheres were examined in PBS (pH 7.4, 37 degrees C). The degradation rate of PEA was substantially greater than that of PDLLA. The release profiles of all small model drugs (mw <332.4) from PDLLA films were biphasic or triphasic, while the release profiles of small model drugs from PEA films varied extensively. Due to the faster weight loss of PEA, FITC-dextran (mw 4400) was released substantially more rapidly from PEA microspheres than from PDLLA microspheres. In conclusion, all model drugs, except guaifenesin, were released faster from PEA preparations than from PDLLA preparations.     

Comparative study on sustained release of human growth hormone from semi-crystalline poly(L-lactic acid) and amorphous poly(D,L-lactic-co-glycolic acid) microspheres: morphological effect on protein release.

Recombinant human growth hormone (rhGH) was encapsulated by a double emulsion solvent evaporation method within two biodegradable microspheres having different polymer compositions. Semi-crystalline poly(L-lactic acid) (PLA) and amorphous poly(D,L-lactic-co-glycolic acid) (PLGA) were used for the encapsulation of hGH. Protein release profiles from the two microspheres were comparatively evaluated with respect to their morphological difference. Both of the microspheres similarly exhibited rugged surface and porous internal structures, but their inner pore wall morphologies were quite different. The slowly degrading PLA microspheres had many nano-scale reticulated pores on the wall, while the relatively fast degrading PLGA microspheres had a non-porous and smooth wall structure. From the PLA microspheres, hGH was released out in a sustained manner with an initial approximately 20% burst, followed by constant release, and almost 100% complete release after a 1-month period. In contrast, the PLGA microspheres showed a similar burst level of approximately 20%, followed by much slower release, but incomplete release of approximately 50% after the same period. The different hGH release profiles between PLA and PLGA microspheres were attributed to different morphological characters of the pore wall structure. The inter-connected nano-porous structure of PLA microspheres was likely to be formed due to the preferable crystallization of PLA during the solvent evaporation process.     

Preparation and characterization of melittin-loaded poly (DL-lactic acid) or poly (DL-lactic-co-glycolic acid) microspheres made by the double emulsion method.

The water soluble peptide, melittin, isolated from bee venom and composed of twenty-six amino acids, was encapsulated in poly (DL-lactic acid, PLA) and poly (DL-lactic-co-glycolic acid, PLGA) microspheres prepared by a multiple emulsion [(W1/O)W2] solvent evaporation method. The aim of this work was to develop a controlled release injection that would deliver the melittin over a period of about one month. The influence of various preparation parameters, such as the type of polymer, its concentration, stabilizer PVA concentration, volume of internal water phase and level of drug loading on the characteristics of the microspheres and drug release was investigated. It was found that the microspheres of about 5 microm in size can be produced in high encapsulation (up to 90%), and the melittin content in the microspheres was up to 10% (w/w). The drug release profiles in vitro exhibited a significant burst release, followed by a lag phase of little or no release and then a phase of constant melittin release. The type of polymer used was a critical factor in controlling the release of melittin from the microspheres. In this study, the rate of peptide release from the microspheres correlated well with the rate of polymer degradation. Moreover, melittin was released completely during the study period of 30 days, which agreed well with the polymer degradation rate.     

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.     

Drug release characteristics of multi-reservoir type microspheres with poly(dl-lactide-co-glycolide) and poly(dl-lactide).

For the multi-reservoir type microspheres composed of poly(dl-lactide-co-glycolide) (PLGA) and poly(dl-lactide) (PLA), the influence of the drug-holding layer and the non-drug-holding layer on drug release profiles was studied. The microspheres with the blend of PLGA and PLA were prepared by the W/O type emulsion-solvent evaporation technique, and cisplatin was used as a model drug. The degree of water uptake and the erosion of each polymer were evaluated to clarify the mechanism of drug release for multi-reservoir type microspheres. The blending of PLA and PLGA provided two types of microspheres in terms of the drug distribution in a microsphere, depending on the ratio of the blend: the microspheres with the drug-holding layer covered by the non-drug layer and the microspheres with the drug on the outer region. The drug release in the early period was governed by the pattern of drug distribution. The drug release rate at a steady state was governed by the erosion of the drug-holding layer. The results of present study indicate that drug release from multi-reservoir type microspheres involves the following process: (a) rapid release of the drug near the surface of microspheres, (b) formation of micropores in the non-drug-holding layer by hydration and erosion, (c) degradation of the drug-holding layer, and (d) diffusion of the drug through micropores.     

Modification of release rates of cyclosporin A from polyl(L-lactic acid) microspheres by fatty acid esters and in-vivo evaluation of the microspheres.

Biodegradable microspheres containing cyclosporin A (CyA, cyclosporine) were prepared using poly(L-lactic acid) (PLA) and poly(lactide-co-glycolide)(PLGA) by a solvent evaporation method. CyA was efficiently entrapped in PLA, PLGA(50/50) and PLGA(75/25) microspheres in a range of 81-85%. CyA released constantly from PLA microspheres without any lag time, whereas the drug from PLGA(50/50) and PLGA(75/25) microspheres was released after lag time of about 1 and 3 weeks, respectively. Addition of fatty acid esters enhanced the release rates of CyA from PLA microspheres. In-vivo study was performed using rats with adjuvant-induced arthritis. PLA microspheres with ethyl myristate sustained high blood levels of CyA compared with the microspheres with no additives over 4 weeks. In addition, the PLA microspheres improved the symptoms such as the decrease in body weight and the increase in paw swelling occurred by adjuvant-induced arthritis in rats. Consequently, the release rate of CyA from PLA microspheres can be improved by adding fatty acid esters and PLA microspheres with fatty acid esters seem to be a useful dosage form for autoimmune disease therapy.     

A synthetic polymer matrix for the delayed or pulsatile release of water-soluble peptides.

The design of a polymeric peptide release system with a controlled delay time and a burst-free pre-release phase is described. In general, the system consists of a blend of a tyrosine-derived polyarylate and a fast-degrading copolymer of lactic and glycolic acid (PLGA). Due to the peptide-like structure of the polyarylate backbone, peptide-polymer interactions prevented the release of peptide from neat polyarylate films. The addition of PLGA acts as a 'delayed' excipient: as PLGA degrades, it generates acidic degradation products that cause a drop in the internal pH of the polyarylate matrix. This drop in pH weakens the peptide-polymer interactions and causes the release of peptide to commence. The initial molecular weight of PLGA can be used to control the length of time before degradation occurs. Consequently, this parameter can also be used to control the duration of the delay period prior to peptide release. As a specific model system, blends of poly(DTH adipate) with three different copolymers of lactic and glycolic acid were prepared and used for the delayed release of Integrilin, a synthetic water-soluble heptapeptide (clinically used in antithrombic injections) that acts as a highly potent glycoprotein IIb/IIIa antagonist. Blends composed of a 1:1 weight ratio of poly(DTH adipate) and PLGA and containing Integrilin (15%, w/w) were prepared. In vitro release studies were conducted in phosphate buffered solution at 37 degrees C and the release of Integrilin was followed by HPLC. As the initial molecular weight of PLGA varied from 12000 to 62000, the duration of the delay period prior to release increased from 5 to 28 days.     

Characterization and in vitro degradation of poly(2,3-(1,4-diethyl tartrate)-co-2,3-isopropyliden tartrate).

In the present study, a less known polyester based on tartaric acid was characterized with respect to its degradation mechanism. Poly(2,3-(1,4-diethyl tartrate)-co-2,3-isopropyliden tartrate) (PTA) differs from commonly used biodegradable polyesters, such as poly(lactides-co-glycolides) (PLGA) by the presence of additional cleavable bonds in the polymer side chains. This modification results in different polymer properties and influences polymer degradation. The hydrolytic degradation of PTA was studied in parallel to PLGA using disc-shape matrices, which were obtained by compression-molding. The discs were incubated in pH 7.4 phosphate buffer solution at 37 degrees C. The degraded samples were characterized for percentage mass loss, water absorption, decay of molecular weight and change in glass transition temperature. The results demonstrate that the degradation of PTA proceeds via bulk erosion similar to PLGA. However, the degradation of PTA implants is characterized by a rapid mass loss within a short period of time appearing after a definite lag phase without remarkable mass loss. This makes the polymer promising for pulsatile drug release systems.     

Influence of formulation parameters on the characteristics of poly(D, L-lactide-co-glycolide) microspheres containing poly(L-lysine) complexed plasmid DNA.

This study describes the influence of polymer type, surfactant type/concentration, and target drug loading on the particle size, plasmid DNA (pDNA) structure, drug loading efficiency, in vitro release, and protection from DNase I degradation of poly(D, L-lactide-co-glycolide) (PLGA) microspheres containing poly(L-lysine) (PLL) complexed pDNA. PLGA microspheres containing pDNA-PLL were prepared using the water-in-oil-in-water (w-o-w) technique with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) as surfactants in the external aqueous phase. A complex ratio of 1:0.33 (pDNA-PLL, w/w) enhanced the stability of pDNA during microsphere preparation. Higher pDNA-PLL loading efficiency (46.2%) and supercoiled structure (64.9%) of pDNA were obtained from hydrophobic PLGA (M(w) 31000) microspheres compared with hydrophilic PLGA or low-molecular-weight PLGA microspheres. The particle size decreased from 6.6 to 2.2 microm when the concentration of PVA was increased from 1 to 7%. At the same concentration of surfactant, PVA stabilized microspheres showed higher pDNA-PLL loading efficiency (46.2%) than PVP stabilized microspheres (24.1%). Encapsulated pDNA in PLGA microspheres was protected from enzymatic degradation and maintained in the supercoiled form. The pDNA-PLL microspheres showed in vitro release of 95.9 and 84.9% within 38 days from the low-molecular-weight PLGA and hydrophilic PLGA microspheres, respectively, compared to 54.2% release from the hydrophobic, higher-molecular-weight PLGA microspheres. The results suggest loading and release of pDNA-PLL complex can be influenced by surfactant concentration and polymer type.     

骨形态发生蛋白2/聚乳酸缓释微球的制备及表征

背景：聚乳酸具有良好的生物相容性,是优良的药物缓释载体。 目的：制备重组人骨形态发生蛋白2/聚乳酸缓释微球,考察其理化特性。 方法：采用复乳溶剂挥发法制备重组人骨形态发生蛋白2/聚乳酸缓释微球,进行扫描电镜、激光粒度、Zeta电位、溶胀性能检测及采用ELISA试剂盒检测包封率、载药率及体外释药率。 结果与结论：扫描电镜见重组人骨形态发生蛋白2/聚乳酸缓释微球微球近似圆形,形态较规则,分散性较好,表面光滑。激光粒度分析重组人骨形态发生蛋白2/聚乳酸缓释微球微平均粒径839.6 nm,Zeta电位（-32.93±3.74）mV,微球溶胀系数1.157±0.059,包封率及载药率分别为（88.943±2.878）%,（0.026±0.001）%;微球在第1天释药约10.199%,随后释药较恒定,至第19天累计释药率为54.643%。说明制备出的重组人骨形态发生蛋白2/聚乳酸缓释微球的粒径达到中华人民共和国药典第10版二部关于亚微球的定义标准及包封率不低于80%的要求,并且在体外具有很好的缓释功能。     

Influence of formulation parameters on the characteristics of poly( -lactide-co-glycolide) microspheres containing poly( -lysine) complexed plasmid DNA

This study describes the influence of polymer type, surfactant type/concentration, and target drug loading on the particle size, plasmid DNA (pDNA) structure, drug loading efficiency, in vitro release, and protection from DNase I degradation of poly( -lactide-co-glycolide) (PLGA) microspheres containing poly( -lysine) (PLL) complexed pDNA. PLGA microspheres containing pDNA–PLL were prepared using the water-in-oil-in-water (w–o–w) technique with poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVP) as surfactants in the external aqueous phase. A complex ratio of 1:0.33 (pDNA–PLL, w/w) enhanced the stability of pDNA during microsphere preparation. Higher pDNA–PLL loading efficiency (46.2%) and supercoiled structure (64.9%) of pDNA were obtained from hydrophobic PLGA (M_w 31 000) microspheres compared with hydrophilic PLGA or low-molecular-weight PLGA microspheres. The particle size decreased from 6.6 to 2.2 μm when the concentration of PVA was increased from 1 to 7%. At the same concentration of surfactant, PVA stabilized microspheres showed higher pDNA–PLL loading efficiency (46.2%) than PVP stabilized microspheres (24.1%). Encapsulated pDNA in PLGA microspheres was protected from enzymatic degradation and maintained in the supercoiled form. The pDNA–PLL microspheres showed in vitro release of 95.9 and 84.9% within 38 days from the low-molecular-weight PLGA and hydrophilic PLGA microspheres, respectively, compared to 54.2% release from the hydrophobic, higher-molecular-weight PLGA microspheres. The results suggest loading and release of pDNA–PLL complex can be influenced by surfactant concentration and polymer type.     

Brush-like branched biodegradable polyesters, part III. Protein release from microspheres of poly(vinyl alcohol)-graft-poly(D,L-lactic-co-glycolic acid).

Brush-like branched polyesters, obtained by grafting poly(lactic-co-glycolic acid), PLGA, onto water-soluble poly(vinyl alcohol) (PVAL) backbones, were investigated regarding their utility for the microencapsulation of proteins. Poly(vinyl alcohol)-graft-poly(lactic-co-glycolic acid), PVAL-g-PLGA, offers additional degrees of freedom to manipulate properties such as e.g. molecular weight, glass transition temperature and hydrophilicity. PLGA chain length was varied at a constant molecular weight (M(w)) of the PVAL backbone and secondly M(w) of the PVAL backbone was varied keeping the PLGA chain lengths constant. The most striking feature of these polymers is their high M(w). Microencapsulation of hydrophilic macromolecules, such as bovine serum albumin, ovalbumin, cytochrome c and FITC-dextran using a w/o/w double emulsion technique was investigated. Surface morphology, particle size, encapsulation efficiencies and protein release profiles were characterized as well. Microencapsulation of model compounds was feasible at temperatures of 0-4 degrees C with yields typically in the range of 60-85% and encapsulation efficiencies of 70-90%. Both, encapsulation efficiency and initial protein release (drug burst) were strongly affected by the glass transition temperature, T(g), of the polymer in contact with water, whereas the in vitro protein release profile depended on the PVAL-g-PLGA structure and composition. In contrast to PLGA, protein release patterns were mostly continuous with lower initial drug bursts. Shorter PLGA chains increased drug release in the erosion phase, whereas initial pore diffusion was affected by the M(w) of PVAL backbone. Release profiles from 2 to 12 weeks could be attained by modification of composition and molecular weight of PVAL-g-PLGA and merit further investigations under in vivo conditions. The in vitro cytotoxicity of PVAL-g-PLGA is comparable to PLGA and therefore, this new class of biodegradable polyesters has considerable potential for parenteral drug delivery systems.     

High glycolic poly (DL lactic co glycolic acid) nanoparticles for controlled release of meropenem

Nanospheres of low molecular weight poly lactic co glycolic acid (PLGA) with high glycolic acid content (10:90) and polylactic acid (PLA) are synthesized and loaded with meropenem, a broad spectrum antibiotic. The loading efficiency of the drug is 82 and 70% in PLGA 10:90 and PLA respectively. The rate of drug release is higher with PLGA 10:90 (3.2 μg/s) than with PLA (2.4 μg/s). Eighty and 60% of the encapsulated drug is released from the two polymers in 30 days respectively. Initial burst followed by sustained drug release is observed which is mathematically explained using a biphasic model. The drug release from the former polymer leads to two times lower E. coli growth than the release from the latter. The nanoparticles are biocompatible with no significant effect on the viability of 3T3 cells. This study indicates that PLGA 10:90 can be used for the delivery of antibiotics for interim period, especially for post orthopaedic surgeries.     

In vitro degradation of nanoparticles prepared from polymers based on DL-lactide, glycolide and poly(ethylene oxide)

Nanoparticles of poly(DL-lactic acid) (PDLLA), poly(DL-lactic-co-glycolic acid) (PLGA) and poly(ethylene oxide)-PLGA diblock copolymer (PEO-PLGA) were prepared by the salting-out method. The in vitro degradation of PDLLA, PLGA and PEO-PLGA nanoparticles in PBS (pH 7.4) at 37 degrees C was studied. The particle size, molecular weight of the polymers and the amount of lactic and glycolic acids formed were followed in time. PDLLA nanoparticles gradually degraded over a period of 2 years and retain their size during that period. A faster degradation was observed for PLGA nanoparticles, which was nearly complete after 10 weeks. PLGA nanoparticles retained their size during that period. In PEO-PLGA nanoparticles, the ester bond connecting the PEO and the PLGA segments was preferentially cleaved, which led to a relatively fast decrease in molecular weight and to (partial) aggregation, as multimodal size distributions were observed. PEO-PLGA nanoparticles were almost completely degraded within 8 weeks.     

Feeding liquid, non-ionic surfactant and cyclodextrin affect the properties of insulin-loaded poly(lactide-co-glycolide) microspheres prepared by spray-drying.

The potential of spray-drying technique for the encapsulation in poly(lactide-co-glycolide) (PLGA) microspheres of bovine insulin, a poorly stable peptide, has been investigated. Insulin-loaded microspheres were prepared by spray-drying different feeding liquids containing insulin and PLGA, that is a S/O dispersion, a W/O emulsion or an acetic acid solution. In the case of the emulsion, insulin was also co-encapsulated with either non-ionic surfactants such as polysorbate 20 and poloxamer 188, or complexing agents such as HPbetaCD. In the microspheres prepared from the acetic acid solution of insulin and PLGA, HPbetaCD was tested. Microspheres containing surfactants were aggregated, whereas good quality particles displaying a mean diameter in the range 12.1-27.9 microm were produced in the other cases. Insulin was efficiently loaded inside microspheres except for S/O formulation (only 22% of total insulin content was entrapped). The impact of the microencapsulation process on insulin chemical and conformational stability was assessed by HPLC, circular dichroism and turbidimetry studies. Under the adopted manufacture conditions, insulin was encapsulated in the native state and its chemical and conformational stability was preserved along the fabrication process. The formulations containing only insulin displayed low burst effects (6-11%), whereas the addition of surfactants resulted in much higher burst effects (49-54%) and faster release rate. The co-encapsulation of HPbetaCD slowed down the overall release rate and, in the case of microspheres prepared from the emulsion, allowed a constant insulin release up to 45 days. The study of insulin stability along the release phase showed that insulin was released in the intact form and un-released insulin was stable inside all the microsphere formulations. We conclude that insulin can be effectively encapsulated in PLGA microspheres by the spray-drying technique. Additives with complexing properties such as HPbetaCD have demonstrated a potential in optimizing the release rate of insulin when used in microspheres prepared from W/O emulsions.     

乳酸-羟基乙酸共聚物多肽蛋白类药物微球控释系统的突释与控制

背景:突释问题是限制多肽蛋白类微球广泛应用的一个关键技术问题,已经成为PLGA微球控释系统面临的一个亟待解决的问题.目的:分析近年来国内外对乳酸-羟基乙酸共聚物多肽蛋白类药物微球的突释与控制的研究,对突释的原因、影响突释的因素以及减少突释的方法与措施进行了详细的介绍.方法:应用计算机检索CNKI和PubMed数据库中1999-01/2010-12关于乳酸-羟基乙酸共聚物多肽蛋白类药物微球控释系统研究的文章,在标题和摘要中以"聚乳酸-羟基乙酸;多肽;蛋白;微球;突释;控制"或"PLGA; peptide; protein ; microspheres; burst release; control"为检索词进行检索.通过阅读标题和摘要进行初选,排出较陈旧和重复研究文献,保留符合纳入标准的文献24篇.结果与结论:对乳酸-羟基乙酸共聚物多肽蛋白类药物微球突释机制的理解,可以更好地实现对微球突释的控制,以扩大多肽蛋白类药物在临床上的应用.PLGA的性质、微球的制备方法、微球的制备参数都在不同程度上影响微球的突释,并且可能是多因素协同作用.通过对上述各种因素加以适当控制,可在一定程度上减少微球的突释率.通过该方面的机制研究对指导新药开发具有重要意义.     

Release of anti-restenosis drugs from poly(ethylene oxide)-poly(DL-lactic-co-glycolic acid) nanoparticles.

Dexamethasone- or rapamycin-loaded nanoparticles based on poly(ethylene oxide) and poly(dl-lactic-co-glycolic acid) block copolymers (PEO-PLGA) were prepared without additional stabilizer using the salting-out method. A fast release of drug in PBS (pH 7.4) at 37 degrees C resulting in 100% release within 5 h was observed for both drugs. The rate of drug release was substantially reduced by treating the particles with gelatin or albumin after drug loading, resulting in a linear drug release in time. It was shown that the rate of drug release is related to the amount of protein associated with the nanoparticles. After gelatin treatment of drug-loaded nanoparticles, sustained release of dexamethasone for 17 days and of rapamycin for 50 days could be achieved.     

Stability of bovine serum albumin complexed with PEG-poly(L-histidine) diblock copolymer in PLGA microspheres.

The aim of this study was to examine the stability of bovine serum albumin (BSA) in poly(DL-lactic acid-co-glycolic acid) (PLGA) microspheres upon addition of a new excipient, poly(ethylene glycol)-poly(L-histidine) diblock copolymer (PEG-PH). Poly(L-histidine) component can form an ionic complex with BSA under acidic conditions within a narrow pH range. To optimize the ionic complexation conditions for BSA with PEG-PH, the resulting complex sizes were monitored using the Zetasizer. PLGA microspheres containing BSA as a model protein were prepared by w/o/w double emulsion method. BSA stability in aqueous solutions and after release from PLGA microspheres was determined using circular dichroism (CD) spectroscopy for secondary structure analyses and fluorescence measurements for tertiary structure analyses. The release profile of BSA from the microspheres was monitored using UV spectrophotometry. The rate of PLGA degradation was monitored by gel permeation chromatography. The pH profile within microspheres was further evaluated by confocal microscopy using a pH-sensitive dye. Approximately 19 PEG-PH molecules and one BSA molecule coalesced to form an ionic complex around a pH range of 5.0-6.0. Plain BSA/PLGA and BSA/PEG-PH/PLGA microspheres had a mean size of 27-35 microm. PLGA microspheres with a BSA loading efficiency >80% were prepared using the double emulsion method. PEG-PH significantly improved the stability of BSA both in aqueous solutions and in PLGA microspheres. The release profiles of BSA from different formulations of PLGA microspheres were significantly different. PEG-PH effectively buffered the local acidity inside the microspheres and improved BSA release kinetics by reducing initial burst release and extending continuous release over a period of time, when encapsulated as an ionic complex. PLGA degradation rate was found to be delayed by PEG-PH. There was clear evidence that PEG-PH played multiple roles when complexed with BSA and incorporated into PLGA microspheres. PEG-PH is an effective excipient for preserving the structural stability of BSA in aqueous solution and BSA/PLGA microspheres formulation.     

One-month sustained release microspheres of 125I-bovine calcitonin. In vitro-in vivo studies.

To obtain a 1-month release formulation of 125I-bovine calcitonin, microspheres were prepared with three different PLA copolymers, PLGA I (mol. wt. [MW]=30000), polyethyleneglycol (PEG)-PLGA (MW=34000) and PLGA II (MW=12000) using the double emulsion method. The release of 125I-bovine calcitonin was assayed in vitro using dialysis bags at 37 degrees C in isotonic phosphate buffer (pH 7.4). The in vitro release results indicated a very slow release rate for an optimal 1-month sustained release formulation. 125I-bovine calcitonin microspheres were administered under the skin on the back of Wistar rats and the radioactivity at the injection site was subsequently measured over a 4-week period. The in vitro and in vivo profiles were affected by the weight average molecular weight of the copolymers. The 125I-bovine calcitonin release rate was faster from microspheres prepared with PLGA II (MW=12000) than from microspheres prepared with higher molecular weight copolymers (PLGA I and PEG-PLGA). Microspheres prepared with PLGA II (MW=12000) release 100% of the dose in 1 month, in vivo release profiles presented two phases, during the first 2 weeks approximately 70% of the 125I-bovine calcitonin injected was released, followed by a second slower phase.