Stabilization of Vinca Alkaloids Encapsulated in Poly(lactide-co-glycolide) Microspheres

Purpose. The purpose of this study was to stabilize the vinca alkaloids,vincristine sulfate (VCR) and vinblastine sulfate (VBL), inpoly(lactide-co-glycolide) (PLGA) microspheres and to release the drugs in asustained manner for more than a month. Methods. An oil-in-oil emulsion-solvent extraction method was usedto encapsulate VCR and VBL in PLGA50/50 microspheres. Stabilityand release kinetics of the drugs during the incubation at 37°C inPBS/Tween 80 were assessed by HPLC. Degradation products wereidentified with HPLC-MS. Results. VCR and VBL were encapsulated in PLGA microspheresunchanged. During the microsphere incubation, however, VCRdegraded inside the particles with a t_1/2 7.5 days. The degradationproduct was identified by LC-MS as the deformyl derivative, commonlyformed at acidic pH. VBL, which differs only by a stable methyl groupin place of the N-formyl group in VCR, was completely stable in thePLGA microclimate. The neutralization of acidic PLGA microclimateby addition of 3–10% Mg(OH)₂ completely inhibited deformylationof VCR during release, but introduced a new degradation productformed under the more alkaline conditions used during the preparation.The substitution of Mg(OH)₂ with a weaker base, ZnCO₃, inhibitedthe formation of both degradation products resulting in VCRstabilization of >92% for 4 weeks. The optimal formulations of VCR(containing ZnCO₃) and VBL (no additives) slowly and continuouslyreleased stable drugs for over a month. Conclusions. VCR and VBL were successfully stabilized and releasedin a sustained manner from PLGA microspheres. Co-encapsulation ofZnCO₃ stabilizes VCR against acid-catalyzed degradation duringrelease from the polymer and minimizes VCR decomposition duringencapsulation.     

Visual Evidence of Acidic Environment Within Degrading Poly(lactic-co-glycolic acid) (PLGA) Microspheres

Purpose. In the past decade, biodegradable polymers have becomethe materials of choice for a variety of biomaterials applications. Inparticular, poly(lactic-co-glycolic acid) (PLGA) microspheres havebeen extensively studied for controlled-release drug delivery. However,degradation of the polymer generates acidic monomers, andacidification of the inner polymer environment is a central issue in thedevelopment of these devices for drug delivery. Methods. To quantitatively determine the intrapolymer acidity, weentrapped pH-sensitive fluorescent dyes (conjugated to 10,000 Dadextrans) within the microspheres and imaged them with confocalfluorescence microscopy. The technique allows visualization of thespatial and temporal distribution of pH within the degradingmicrospheres (1). Results. Our experiments show the formation of a very acidicenvironment within the particles with the minimum pH as low as 1.5. Conclusions. The images show a pH gradient, with the most acidicenvironment at the center of the spheres and higher pH near the edges,which is characteristic of diffusion-controlled release of the acidicdegradation products.     

Stabilization of Proteins Encapsulated in Cylindrical Poly(lactide-co-glycolide) Implants: Mechanism of Stabilization by Basic Additives

Purpose. A previous study from our group has shown that in theacidic microclimate of poly(lactide-co-glycolide) (PLGA) implants,encapsulated BSA forms insoluble noncovalent aggregates and ishydrolyzed during in vitro release. Incorporation of Mg(OH)₂ stronglyinhibits these mechanisms of instability and facilitates continuousprotein release. The purpose of this study was to determine the proteinstabilization mechanism in the presence of basic additives. Methods. BSA, as a model protein, was encapsulated in PLGAmillicylinders by a solvent extrusion method. The release of BSA fromthe PLGA millicylinders with and without basic additives (Mg(OH)₂,Ca(OH)₂, ZnCO₃ and Ca₃(PO₄)₂) in a physiological buffer was carriedout at 37°C and quantified by a modified Bradford assay. The insolubleaggregates extracted from the polymer with acetone were reconstitutedin a denaturing (6 M urea) or denaturing/reducing solvent (6 M urea/10 mM DTT) to determine the type of aggregation. Results. Aggregation of encapsulated BSA was inhibited withincreasing amount of base co-encapsulated in the polymer, irrespective of thetype of base used. The pH drop in the release medium and extent ofacid-catalyzed PLGA degradation were both inhibited in the presenceof base. The resultant effect was also reflected in an increase in wateruptake and porosity of the devices. The inhibition and mechanism ofBSA aggregation was correlated with the basicity of the additive.For Ca(OH)₂, at 3% loading, covalent BSA aggregation due tothiol-disulfide interchange was observed (indicative of ionization ofalbumin's free thiol at high pH), whereas at 3% ZnCO₃ or Ca₃(PO₄)₂, ahigher percentage of non-covalent aggregates was observed comparedto Mg(OH)₂. Decreasing the loading of BSA at constant Mg(OH)₂content caused an increase in BSA aggregation. Conclusions. The mechanism by which Mg(OH)₂ stabilizesencapsulated BSA in PLGA implants is through neutralizing the acidicmicroclimate pH in the polymer. The successful neutralization afforded by thebasic additives requires a percolating network of pores connecting bothbase and protein. The microclimate pH inside PLGA implants can becontrolled by selecting the type of basic salt, which suggests a potentialapproach to optimize the stability of encapsulated pharmaceuticals inPLGA including therapeutic proteins.     

Stabilization of 10-Hydroxycamptothecin in Poly(lactide-co-glycolide) Microsphere Delivery Vehicles

Purpose. The purpose of this study was to investigate the potential of poly(lactide-co-glycolide) (PLGA) microspheres to stabilize and deliver the analogue of camptothecin, 10-hydroxycamptothecin (10-HCPT). Methods. 10-HCPT was encapsulated in PLGA 50:50 microspheres by using an oil-in-water emulsion-solvent evaporation method. The influence of encapsulation conditions (i.e., polymer molecular weight (M_w), polymer concentration, and carrier solvent composition) on the release of 10-HCPT from microspheres at 37°C under perfect sink conditions was examined. Analysis of the drug stability in the microspheres was performed by two methods:i) by extraction of 10-HCPT from microspheres and ii). by sampling release media before lactone— carboxylate conversion could take place. Results. Microspheres made, of low M_w polymer (inherent viscosity 0.15 dl/g) exhibited more continuous drug release than those prepared from polymers of higher M_w (i.v. = 0.58 and 1.07 dl/g). In addition, a high polymer concentration and the presence of cosolvent in the carrier solution to dissolve 10-HCPT were both necessary in the microsphere preparation in order to eliminate a large initial burst of the released 10-HCPT. An optimal microsphere formulation released 10-HCPT slowly and continuously for over two months with a relatively small initial burst of the released drug. Both analytical methods used to assess the stability of 10-HCPT revealed that the unreleased camptothecin analogue in the microspheres remained in its active lactone form (>95%) over the entire 2-month duration of study. Conclusions. PLGA carriers such as those described here may be clinically useful to stabilize and deliver camptothecins for the treatment of cancer.     

Acidic Microclimate pH Distribution in PLGA Microspheres Monitored by Confocal Laser Scanning Microscopy

PURPOSE: The acidic microclimate pH (micropH) distribution inside poly(lactic-co-glycolic acid) (PLGA) microspheres was monitored quantitatively as a function of several formulation variables. METHODS: A ratiometric method by confocal laser scanning microscopy with Lysosensor yellow/blue dextran was adapted from those previously reported, and micropH distribution kinetics inside microspheres was examined during incubation under physiologic conditions for 4 weeks. Effects of PLGA molecular weight (MW) and lactic/glycolic acid ratio, microspheres size and preparation method, and polymer blending with poly(ethylene glycol) (PEG) were evaluated. RESULTS: micropH kinetics was accurately sensed over a broadly acidic range (2.8 < micropH < 5.8) and was more acidic and variable inside PLGA with lower MW and lactic/glycolic acid ratio. Lower micropH was found in larger microspheres of lower MW polymers, but size effects for lactic-rich polymers were insignificant during 4 weeks. Microspheres prepared by the oil-in-oil emulsion method were less acidic than those prepared by double emulsion, and blending PLGA 50/50 with 20% PEG increased micropH significantly (micropH > 5 throughout incubation). CONCLUSIONS: Coupling this method with that previously developed (SNARF-1 dextran for micropH 5.8-8.0) should provide microclimate pH mapping over the entire useful pH range (2.8-8.0) for optimization of PLGA delivery of pH-sensitive bioactive substances.     

Release of Human Serum Albumin from Poly(lactide-co-glycolide) Microspheres

Human serum albumin (HSA) was encapsulated in a 50:50 copolymer of DL-lactide/glycolide in the form of microspheres. These microspheres were used as a model formulation to study the feasibility of controlling the release of large proteins over a 20- to 30-day period. We show that HSA can be successfully incorporated into microspheres and released intact from these microspheres into various buffer systems at 37°C. A continuous release of the protein could be achieved in physiological buffers at 37°C over a 20- to 30-day period from microspheres with high protein loadings (11.6%). These results demonstrate the potential of poly(DL-lactide-co-glycolide) microspheres for continuous delivery of large proteins.     

影响蛋白质药物在聚乳酸-羟基乙酸微球制备过程中稳定性与增加累积释放的方法

目的 介绍增加乳酸-羟基乙酸聚合物(PLGA)蛋白微球中药物稳定性与蛋白累积释放量的方法.方法根据国内外文献,较全面地总结了PLGA微球中稳定蛋白,解决不完全释放的策略.结果与结论 虽然PLGA微球制备与药物释放过程中存在不利于蛋白稳定的因素,但通过选用不同添加剂、优化体外释放介质及装置、改进制备工艺、开发复合材料及复合微球等方法可以有效保存其活性,增加累积释放量.     

多肽、蛋白质药物的聚乳酸及其共聚物微球研究进展

综述了以可生物降解的合成高分子材料-聚乳酸及其共聚物为载体的多肽，蛋白质药物微球的制备方法和影响因素。讨论了蛋白质在制备和释放过程中的稳定性。     

复乳法制备胰岛素PLGA纳米粒影响包封率因素考察

以Poloxamer188为乳化剂，乙酸乙酯为有机溶剂，采用复乳法制备了胰岛素乳酸／羟基乙酸共聚物（PLGA）纳米粒，考察了乳化剂和PLGA的浓度。内水相中胰岛素的浓度为pH，溶剂挥发方法和内水相中加入聚乙烯醇（PVA）等实验各因素对胰岛素PLGA纳米粒包封率的影响。结果表明，乳化剂的浓度较高，PLGA的浓度较小，内水相的pH接近胰岛素的pI(5．3），胰岛素的浓度较低，缩短有机溶剂挥发时间及内水相中加入PVA有利于提高胰岛素的包封率，经实验条件优化后制备的胰岛素PLGA纳米纳平均粒径为149．6nm，多分散性系数小于0．1，包封率提高到42．8％。     

Protein Instability in Poly(Lactic-co-Glycolic Acid) Microparticles

In this review the current knowledge of protein degradation during preparation, storage and release from poly(lactic-co-glycolic acid) (PLGA) microparticles is described, as well as stabilization approaches. Although we have focussed on PLGA microparticles, the degradation processes and mechanisms described here are valid for many other polymeric release systems. Optimized process conditions as well as stabilizing excipients need to be used to counteract several stress factors that compromise the integrity of protein structure during preparation, storage, and release. The use of various stabilization approaches has rendered some success in increasing protein stability, but, still, full preservation of the native protein structure remains a major challenge in the formulation of protein-loaded PLGA microparticles.     

Adsorption of Brain Proteins on the Surface of Poly (D,L-lactide-co-glycolide) (PLGA) Microspheres

Poly(D,L-lactide-co-glycolide) (PLGA) microspheres have been studied for intracerebral delivery of anticancer agents. To explore the biocompatibility nature of the polymer in brain, we have investigated the adsorption of brain proteins on the surfaces of PLGA microspheres. Microspheres were made by the solvent evaporation method using an oil/water (o/w) system. The brain protein adsorption experiment was performed by using a sonication eluting method. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to examine the brain proteins adsorbed. Ethyl cellulose microspheres were used in the study as a reference. The amount of brain proteins adsorbed on PLGA microspheres was also determined using a radiolabeling technique. The extent of brain proteins adsorbed on the PLGA microspheres was found to be lower than that adsorbed on the ethyl cellulose microspheres. The adsorption of brain proteins on PLGA microspheres, however, was significant, as indicated quantitatively by the ¹²⁵I labeling studies. The adsorption of brain proteins on the surface of the PLGA microspheres may be important when considering the use of this polymer as a brain implant delivery system.     

盐酸恩丹西酮乳酸羟基乙酸共聚物微球中药物的含量测定

目的:建立测定盐酸恩丹西酮乳酸羟基乙酸共聚物微球含量的方法.方法:采用二氯甲烷溶解微球后,再以水提取主药,用紫外分光光度法测定药物的含量.结果:盐酸恩丹西酮在2～20μg·ml-1的范围内,浓度与吸收度的线性关系良好,平均回收率为98.76%±0.64%,RSD为1.82%.结论:该方法操作简单、可靠,可用于快速测定微球中药物的含量.     

Potential Oral Delivery of 7-Ethyl-10-Hydroxy-Camptothecin (SN-38) using Poly(amidoamine) Dendrimers

Purpose  To investigate potential application of poly(amidoamine) (PAMAM) dendrimers for improving the delivery of SN-38. Methods  Complexes of SN-38 with generation 4 amine terminated PAMAM dendrimers were synthesized with varying amounts of drug. Stability of the complexes as well as influence of complexation on permeability across and cellular uptake by Caco-2 cells was evaluated. Results  The complexes were stable at pH 7.4 and drug was released at pH 5. A tenfold increase in permeability and more than hundredfold increase in cellular uptake of the complexes with respect to free SN-38 was observed. Conclusions  Studies suggest that complexation with PAMAM dendrimers has the potential to improve the oral bioavailability of SN-38.     

Drug delivery into the brain using poly(lactide-co-glycolide) microspheres

Among the strategies developed for drug delivery into the CNS, locally controlled drug release by the way of an implantable polymeric device has been developed in recent years. The first polymeric devices developed were macroscopic implants needing open surgery for implantation. Over the last few years, poly(lactide-co-glycolide) microspheres have been shown to be safe and promising for drug delivery into the brain. Poly(lactide-co-glycolide) is biodegradable and biocompatible with brain tissue. Due to their size, these microspheres can be easily implanted by stereotaxy in discrete, precise and functional areas of the brain without causing damage to the surrounding -tissue. Brain tumour treatments have been developed using this approach and clinical trials have been performed. Potential applications in neurodegenerative diseases have also been explored, particularly neurotrophic factor delivery and cell therapy.     

Preparation and in Vitro/in Vivo Evaluation of Insulin-Loaded Poly(Acryloyl-Hydroxyethyl Starch)-PLGA Composite Microspheres

Purpose. The purpose of this study was to develop and evaluate a novel composite microsphere delivery system composed of poly(D,L-lactide-co-glycolide) (PLGA) and poly(acryloyl hydroxyethyl starch) (acryloyl derivatized HES; AcHES) hydrogel using bovine insulin as a model therapeutic protein. Methods. Insulin was incorporated into the AcHES hydrogel microparticles by a swelling technique, and then the insulin-containing AcHES microparticles were encapsulated in a PLGA matrix using a solvent extraction/evaporation method. The composite microspheres were characterized for loading efficiency, particle size, and in vitro protein release. Protein stability was examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, high-performance liquid chromatography, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The hydrogel dispersion process was optimized to reduce the burst effect of microspheres and avoid hypoglycemic shock in the animal studies in which the serum glucose and insulin levels as well as animal body weight were monitored using a diabetic animal model. Results. Both the drug incorporation efficiency and the in vitro release profiles were found to depend upon the preparation conditions. Sonication effectively dispersed the hydrogel particles in the PLGA polymer solution, and the higher energy resulted in microspheres with a lower burst and sustained in vitro release. Average size of the microspheres was around 22 m and the size distribution was not influenced by sonication level. High-performance liquid chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis, along with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry showed the retention of insulin stability in the microspheres. Subcutaneous administration of microspheres provided glucose suppression <200 mg/dL for 810 days with hyperglycemia recurring by day 16. During the treatment, the time points with higher serum insulin level were consistent with a more significant glucose suppression. The microsphere-treated rats also grew virtually at the same rate as normal control until the insulin level declined and hyperglycemia returned. Multiple dosing given every 10 days demonstrated that the pharmacological effect and serum insulin levels from second or third doses were similar and comparable to that of the first dose. Conclusion. The AcHES-PLGA composite microsphere system provides satisfactory in vitro and in vivo sustained release performance for a model protein, insulin, to achieve 10-day glucose suppression.     

Effect of water on exenatide acylation in poly(lactide-co-glycolide) microspheres

Peptide or protein degradation often occurs when water flows into the dosage form. The aim of this study was to investigate the effect of water on exenatide acylation in poly(lactide-co-glycolide) (PLGA) microspheres. Exenatide-loaded PLGA microspheres were incubated at different relative humidities (RH) as well as in solutions of different pH for 20 days. The stability of exenatide was monitored using HPLC and HPLC–MS analysis. The alteration of exenatide conformation caused by water was investigated by FT-IR spectroscopy. Exenatide and glycolide were incubated in DMSO–water solutions to verify the effect of exenatide conformation state on the peptide acylation. Exenatide was relatively stable in microspheres at lower RH, and the absorbed water could act as a plasticizer and thus promote the peptide acylation by PLGA. However, when the microspheres were incubated at 100% RH, the excessively absorbed water could cause conformation recovery of exenatide and play an inhibitory effect on acylation. The formation of acylated exenatide incubated in acetate buffer saline of pH 6.0 was more than that of pH 4.5 and 3.0. Stability studies of exenatide in glycolide solutions showed that exenatide in nonnative monomer state was easier to be acylated by eletrophiles than that in aggregation state.     

PLGA的结构和分子量对纳曲酮微球体外释药的影响

制备了分子量、比旋度、摩尔比及分子链末端修饰不同的丙交酯-乙交酯共聚物,并测定理化参数.以其为载体制备纳曲酮微球,比较了体外释药速率.结果表明,用分子量较小、有光学活性、单体摩尔比较小、分子链末端未酯化的共聚物制备的微球体外释药速率较快.     

Dose and Load Studies for Subcutaneous and Oral Delivery of Poly(lactide-co-glycolide) Microspheres Containing Ovalbumin

Poly(lactide-co-glycolide) microspheres containing different loads of OVA (0.05, 0.1, 0.5 and 1.0% w/w) were manufactured by a w/o/w emulsion/solvent evaporation method. Low load efficiencies of less than 20% were observed. Normal size distributions with mean volume diameters ranging from 3.7 to 4.7 µm were obtained for different batches. The in vitro release of OVA from different loaded microspheres showed an expected burst release with all batches. The in vivo dose study (1, 10, 25, 50 µg of OVA) was performed by subcutaneous and oral inoculation in mice by single (0 week) or double (0 and 3 weeks) administration of PLGA 50/50 microspheres containing 0.1% OVA. Subcutaneous administration showed an immune response (serum Ig levels by ELISA) statistically (Fishers paired t-test; P < 0.05) above OVA saline negative controls at 3, 6 and 12 weeks after administration. Oral administration of microspheres produced statistically higher systemic immune responses at the higher doses. Single and double inoculation orally and subcutaneously produced similar serum antibody levels. The in vivo load study was performed by subcutaneous and oral administration to mice of 25 µg OVA contained in various loaded (0.05, 0.1, 0.5 and 1.0% w/w) microspheres. Serum immune responses at 3, 6, and 12 weeks after inoculation were statistically above OVA saline controls and were inversely proportional to the OVA load using either route. This observation suggested a relationship between the number of microspheres delivered and the in vivo serum response. Single subcutaneous administration of 0.05 or 0.1% OVA loaded PLGA 50/50 microspheres induced larger immune responses compared with complete Freunds adjuvant.     

The Stability of Recombinant Human Growth Hormone in Poly(lactic-co-glycolic acid) (PLGA) Microspheres

Purpose. The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. Methods. rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37°C. Results. rhGH PLGA formulations were produced with a low initial burst (<20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37°C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occured at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. Conclusions. rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37°C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.     

超氧化物歧化酶乳酸-羟乙酸共聚物微球的制备及其性质

利用复乳溶剂挥发法制备了超氧化物歧化酶（SOD）的乳酸－羟乙酸共聚物（PLGA）微球，考察了各工艺因素对微球粒径、包封率等的影响，通过扫描电子显微镜（SEM）、差示扫描量热分析（DSC）初步研究了其性质，结果表明，通过调整内水相的体积及浓度，分散相体积及PH值，可得到较高包封率，粒径在20－30μm，形态圆整，表面多孔的SOD微球，DSC表明SOD被有效地包入了PLGA微球中。