蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球研究进展

缓释微粒给药系统是蛋白质/多肽药物传输系统的一个重要研究方向，聚乳酸和乳酸-羟基乙酸共聚物是制备缓释微球最常用的载体材料。蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球常用的制备方法包括溶剂萃取/挥发法(复乳法)、相分离法和喷雾干燥法。本文总结了微球制备中面临的难点如蛋白质/多肽药物稳定性、包封率、药物突释和药物吸附等问题，并综述了保持药物结构稳定性和生物活性、提高包封率、改善药物释放曲线等微球制备方法和进展。     

多肽、蛋白质药物缓释微球研究进展

目的介绍多肽、蛋白质缓释微球的研究情况。方法通过查阅国内外文献，综述多肽、蛋白质微球的制备技术，影响微球质量和体外释药的因素以及药物的稳定性问题。结果与结论多肽、蛋白质药物缓释微球在药学领域有着广阔的发展前景。     

多肽、蛋白质药物缓释微球研究进展

目的 介绍多肽、蛋白质缓释微球的研究情况.方法 通过查阅国内外文献,综述多肽、蛋白质微球的制备技术,影响微球质量和体外释药的因素以及药物的稳定性问题.结果与结论 多肽、蛋白质药物缓释微球在药学领域有着广阔的发展前景.     

天然药物聚乳酸或聚乳酸-羟基乙酸共聚物微球的研究进展

通过查阅相关文献,综述天然药物聚乳酸、聚乳酸-羟基乙酸共聚物微球的特点、制备方法及给药途径,展望天然药物微球的发展前景.     

多肽蛋白质类药物微球制备方法研究进展

多肽蛋白质药物在人类疾病治疗中的作用日趋重要,开发多肽蛋白质类药物的微球给药系统,防止药物在体内很快降解,将药物有效递送至人体相应部位,这对多肽蛋白质药物的的开发和利用特别有意义。目前,微球制备中常用的方法有液中干燥法、喷雾干燥法、相分离法和乳化交联法等。     

多肽,蛋白质微球制剂的研究进展

多肽、蛋白质微球制剂的研究进展上海医药工业研究院（２００４３７）陈庆华，瞿文随着生物技术的飞速发展，近年来运用基因工程开发的肽类、蛋白质等生物大分子药物也不断涌现。临床应用证实这类药物对癌症、自身免疫、记忆缺陷、精神疾病、心血管疾病及一些代谢类疾病疗...     

多肽,蛋白质药物的微球给药系统研究进展

本文综述了近年来生物大分子药物微球给药系统的现状，包括长效注射剂，鼻腔、口服给药微球、毫微球的研究进展，探讨了各剂型的释药机理，特点和存在的问题。     

聚乳酸、聚乳酸乙醇酸共聚物微球的制备及体外释放影响因素研究进展

目的:对近年来以PLA、PLGA为载体的微球剂的研究进展进行综述。方法:查阅近10年来有关PLA、PLGA微球研究的国内外文献,介绍此类微球的制备方法和影响其体外释放等性质的主要因素。结果:PLA、PLGA的性质、药物的性质及微球的制备工艺等对微球的体外释放等性质均有重要的影响。结论:对以PLA、PLGA为载体制备的药物微球,有待于更进一步的研究和开发     

聚乳酸、聚乳酸乙醇酸共聚物微球的制备及影响其质量因素的研究进展

本文综述了近年来以聚乳酸、聚乳酸乙醇酸共聚物为载体的微示的研究进展,并介绍此类微球的制备方法及影响其质量的主要因素。研究表明,载体和药物的性质及微球的制备工艺等对微球的质量和体外释放等均有重要的影响。     

聚乳酸、聚乳酸乙醇酸共聚物微球的制备及体外释放影响因素研究进展

目的：对近年来以PLA，PLGA为载体的微球剂的研究进展进行综述。方法：查阅近10年来有关PLA，PLGA微球研究的国内外文献，介绍此类微球的制备方法和影响其体外释放等性质的主要因素。结果：PLA，PLGA的性质，药和折性质及微球的制备工艺等对微球的体外释放等性质均有重要的影响。结论：对以PLA，PLGA为载体制备的药物微球，有待于更进一步的研究和开发。     

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.     

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.     

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.     

Microencapsulation of PEGylated Adenovirus within PLGA Microspheres for Enhanced Stability and Gene Transfection Efficiency

Purpose Green fluorescent protein (GFP) encoding adenovirus (ADV) was surface modified with polyethylene glycol (PEG) for microencapsulation within poly(lactic-co-glycolic acid) (PLGA) microspheres with the aim of improving stability and gene transfection activity. Methods A series of PEGylated ADV (PEG-ADV) with different PEG seeding densities on the viral surface was prepared and the GFP expression efficiency of each PEG-ADV in the series determined. The physical stabilities of naked ADV and PEG-ADV were comparatively evaluated by exerting a high shear homogenization process or by exposure to low pH. Naked ADV or PEG-ADV was microencapsulated within PLGA microspheres using a water-in-oil-in-water (W/O/W) double emulsion and solvent evaporation method. In vitro cumulative ADV and PEG-ADV release profiles from PLGA microspheres were determined over a 10-day period. GFP transfection efficiencies into HeLa cells were quantified, and the relative extent of the immune response for ADV and PEG-ADV encapsulated within PLGA microspheres was analyzed using macrophage cells. Results The physical stability of PEGylated ADV was greatly enhanced relative to that of naked ADV under the simulated W/O/W formulation conditions, such as exposure to an aqueous/organic interface during high shear-stressed homogenization. PEG-ADV was also more stable than ADV at low pH. ADV and PEG-AD were both released from PLGA microspheres similarly in a sustained fashion. However, when the ADV and PEG-ADV encapsulated microspheres transfected into HeLa cells, PEG-ADV microspheres demonstrated a higher GFP gene transfection efficiency than ADV microspheres. The PEG-ADV microspheres also exhibited a reduced extent of innate immune response for macrophage cells. Conclusions PEGylated ADV could be more safely microencapsulated within PLGA microspheres than naked ADV due to their enhanced physical stability under the harsh formulation conditions and acidic microenvironmental conditions of the microsphere, thereby increasing gene transfection efficiency.     

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.     

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.     

The Acidic Microclimate in Poly(lactide-co-glycolide) Microspheres Stabilizes Camptothecins

Purpose. The camptothecin (CPT) analogue, 10-hydroxycamptothecin (10-HCPT) has been shown previously to remain in its acid-stable (and active) lactone form when encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres (1). The purpose of this study was to determine the principal mechanism(s) of 10-HCPT stabilization. Methods. CPTs were encapsulated in PLGA 50:50 microspheres by standard solvent evaporation techniques. Microspheres were eroded in pH 7.4 buffer at 37°C. The ratio of encapsulated lactone to carboxylate was determined by HPLC as a function of time, initial form of drug encapsulated, fraction of co-encapsulated Mg(OH)₂, CPT lipophilicity, and drug loading. Two techniques were developed to assess the microclimate pH, including: i) measurement of H⁺ content of the dissolved microspheres in an 80:20 acetonitrile/H₂O mixture and ii) confocal microscopy of an encapsulated pH-sensitive dye, fluorescein. Results. The encapsulated carboxylate converted rapidly to the lactone after exposure to the release media, indicating the lactone is favored at equilibrium in the microspheres. Upon co-encapsulation of Mg(OH)₂, the trend was reversed, i.e., the lactone rapidly converted to the carboxylate form. Measurement of -log(hydronium ion activity) (pa*_H) of dissolved microspheres with pH-electrode and pH mapping with fluorescein revealed the presence of an acidic microclimate. From the measurements of H+ and water contents of particles hydrated for 3 days, a microclimate pH was estimated to be in the neighborhood of 1.8. The co-encapsulation of Mg(OH)₂ could both increase the pa*_H reading and neutralize pH in various regions of the microsphere interior. Varying the drug lipophilicity and loading revealed that the precipitation of the lactone could also stabilize CPT. Conclusions. PLGA microspheres prepared by the standard solvent evaporation techniques develop an acidic microclimate that stabilizes the lactone form of CPTs. This microclimate may be neutralized by co-encapsulating a base such as Mg(OH)₂, as suggested by previous work with poly(ortho esters) (2).     

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.     

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。     

A Heterogeneously Structured Composite Based on Poly(lactic-co-glycolic acid) Microspheres and Poly(vinyl alcohol) Hydrogel Nanoparticles for Long-Term Protein Drug Delivery

Purpose. To prepare a heterogeneously structured composite based on poly (lactic-co-glycolic acid) (PLGA) microspheres and poly(vinyl alcohol) (PVA) hydrogel nanoparticles for long-term protein drug delivery. Methods. A heterogeneously structured composite in the form of PLGA microspheres containing PVA nanoparticles was prepared and named as PLGA-PVA composite microspheres. A model protein drug, bovine serum albumin (BSA), was encapsulated in the PVA nanoparticles first. The BSA-containing PVA nanoparticles was then loaded in the PLGA microspheres by using a phase separation method. The protein-containing PLGA-PVA composite microspheres were characterized with regard to morphology, size and size distribution, BSA loading efficiency, in vitroBSA release, and BSA stability. Results. The protein-containing PLGA-PVA composite microspheres possessed spherical shape and nonporous surface. The PLGA-PVA composite microspheres had normal or Gaussian size distribution. The particle size ranged from 71.5 m to 282.7 m. The average diameter of the composite microspheres was 180 m. The PLGA-PVA composite microspheres could release the protein (BSA) for two months. The protein stability study showed that BSA was protected during the composite microsphere preparation and stabilized inside the PLGA-PVA composite microspheres. Conclusions. The protein-containing PLGA-PVA composite may be suitable for long-term protein drug delivery.