利福平/聚乳酸-聚羟基乙酸缓释微球的制备及特性

背景：虽然国内外有很多制备利福平/聚乳酸-聚羟基乙酸共聚物(poly lactic acid-glycolic acid copolymer,PLGA)微球的报道,但这些微球粒径多在10 μm左右,不适合与磷酸钙骨水泥复合制备成具有良好降解性的抗结核修复材料。 目的：制备大粒径利福平/PLGA缓释微球,观察其理化特性和体外缓释特性。 方法：以PLGA为载体,将利福平分散于PLGA的有机溶剂中,采用复乳溶剂挥发法制备利福平/ PLGA缓释微球。光镜和扫描电镜下观察微球的形态特征,测定微球平均直径和跨距,高效液相色谱法测定载药量和包封率,以溶出法和高效液相色谱法观察其体外释药特性,并拟合药物体外释放曲线建立曲线方程。 结果与结论：利福平/PLGA微球电镜观察呈圆球形,分散性好,粘连少,粒径分布集中,平均粒径(80.0±9.4) μm。载药量、包封率分别为(33.18±1.36)%,(54.79±1.13)%。体外缓释试验显示突释期内微球释放度为(14.66±0.18)%,前3 d累计释放度(18.09±0.45)%,到42 d体外累积释放度达到(92.17±1.23)%。提示利福平/PLGA微球具有良好的缓释效果,是一种较为理想的抗结核药物的载体材料和释放系统;PLGA是良好的药物缓释载体,可以用来制备载药缓释微球。     

The release profiles and bioactivity of parathyroid hormone from poly(lactic-co-glycolic acid) microspheres

Poly(lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) or human parathyroid hormone (PTH)(1-34) were prepared using a double emulsion method with high encapsulation efficiency and controlled particle sizes. The microspheres were characterized with regard to their surface morphology, size, protein loading, degradation and release kinetics, and in vitro and in vivo assessments of biological activity of released PTH. PLGA5050 microspheres degraded rapidly after a 3-week lag time and were degraded completely within 4 months. In vitro BSA release kinetics from PLGA5050 microspheres were characterized by a burst effect followed by a slow release phase within 1-7 weeks and a second burst release at 8 weeks, which was consistent with the degradation study. The PTH incorporated PLGA5050 microspheres released detectable PTH in the initial 24h, and the released PTH was biologically active as evidenced by the stimulated release of cAMP from ROS 17/2.8 osteosarcoma cells as well as increased serum calcium levels when injected subcutaneously into mice. Both in vitro and in vivo assays demonstrated that the bioactivity of PTH was maintained largely during the fabrication of PLGA microspheres and upon release. These studies illustrate the feasibility of achieving local delivery of PTH to induce a biologically active response in bone by a microsphere encapsulation technique.     

PLGA microsphere/calcium phosphate cement composites for tissue engineering: in vitro release and degradation characteristics

Bone cements with biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres have already been proven to provide a macroporous calcium phosphate cement (CPC) during in situ microsphere degradation. Furthermore, in vitro/in vivo release studies with these PLGA microsphere/CPC composites (PLGA/CPCs) showed a sustained release of osteo-inductive growth factor when drug was distributed inside/onto the microspheres. The goal of this study was to elucidate the mechanism behind drug release from PLGA/CPC. For this, in vitro release and degradation characteristics of a low-molecular-weight PLGA/CPC (M(w) = 5 kg/mol) were determined using bovine serum albumin (BSA) as a model protein. Two loading mechanisms were applied; BSA was either adsorbed onto the microspheres or incorporated inside the microspheres during double-emulsion. BSA release from PLGA microspheres and CPC was also measured and used as reference. Results show fast degrading polymer microspheres which produced a macroporous scaffold within 4 weeks, but also showed a concomitant release of acidic degradation products. BSA release from the PLGA/CPC was similar to the CPC samples and showed a pattern consisting of a small initial release, followed by a period of almost no sustained release. Separate PLGA microspheres exhibited a high burst release and release efficiency that was higher with the adsorbed samples. Combining degradation and release data we can conclude that for the PLGA/CPC samples BSA re-adsorbed to the cement surface after being released from the microspheres, which was mediated by the pH decrease during microsphere degradation.     

Pegylated recombinant human epidermal growth factor (rhEGF) for sustained release from biodegradable PLGA microspheres

Recombinant human epidermal growth factor (rhEGF) was conjugated with polyethylene glycol (PEG) to improve its physical stability during microencapsulation in biodegradable poly(lactic-co-glycolic acid) microspheres. rhEGF was conjugated with N-hydroxysuccimide (NHS)-derivatized methoxy-PEG (mPEG) of MW 2000 and 5000 under various reaction conditions to optimize the extent of pegylation. Pegylated rhEGF showed much enhanced physical stability against homogenization. Pegylated rhEGF was encapsulated in PLGA microspheres by a double emulsion solvent evaporation method to achieve a sustained release. Pegylated rhEGF exhibited a tri-phasic release profile with a reduced initial burst, compared with unpegylated rhEGF. This study demonstrated that protein pegylation enhanced physical stability of protein and could be a good approach to achieve a sustained protein release profile from biodegradable microspheres.     

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。     

BSA-PLGA缓释微球的制备及优化条件的探索

目的以牛血清白蛋白(BSA)为模型蛋白、聚乳酸-聚乙二醇酸(PLGA)为包裹材料,探索微球的制备方法并优化制备工艺。方法采用复乳-溶剂挥发法制备BSA-PLGA微球,显微测量微球粒径,以微量BCA法测定微球的蛋白含量并计算包封率,进行体外释放,测定微球的累积释放量。探索BSA投药量、PLGA用量、PVA浓度、超声功率等因素对微球包封率、突释量的影响。结果通过正交实验设计,优化了微球的制备工艺,其优化条件是BSA投药量为10mg、PLGA用量为250mg、PVA浓度为1.5%、超声乳化功率为60周。结论通过控制不同的因素,可以得到较高包封率、较小突释、适当载药量和粒径的BSA-PLGA微球。     

Development of chondroitin sulfate encapsulated PLGA microsphere delivery systems with controllable multiple burst releases for treating osteoarthritis

The purpose of the study was to design and develop unique drug delivery systems with controllable multiple burst releases of drugs for treating osteoarthritis. Chondroitin sulfate (CS) was encapsulated into four types of PLGA materials, that is, PLGA 50:50, PLGA 65:35, PLGA 75:25, and PLGA 85:15. The effects of microsphere size and various combinations of blend PLGA microspheres on CS release were investigated. The cytotoxicity of the CS-encapsulated microspheres was investigated according to the ISO 10993 guideline. Our study showed that the encapsulation efficiency of CS into PLGA 50:50 microspheres varied with the size of microspheres; however, the encapsulation efficiencies of CS into PLGA microspheres were independent of the types of PLGA materials. The size of PLGA microspheres was shown to affect the rate of CS release. With the increase of microsphere size from 75-150 μm to 300-355 μm, the initial CS release decreased. Further increase in microsphere size led to an increase in the initial CS release. In addition, combination of different types of PLGA microspheres was shown to be capable of achieving multiple burst CS releases. Moreover, the CS encapsulated PLGA microspheres were shown to be non-cytotoxic. This study proved the concept of multiple burst drug releases that were achieved by encapsulating CS into different types of PLGA microspheres and delivering CS from systems consisting of mixed types of PLGA microspheres, which may be applied to treat osteoarthritis by mimicking multiple intra-joint injection of therapeutic agents.     

影响微球药物释放因素的研究

目的 观察影响微球药物释放的因素，为其应用提供理论基础。方法 以可生物降解的聚乳酸—聚乙醇酸共聚物(PLGA)和聚L—乳酸(PLIA)为载体，采用乳化—溶剂挥发法制备含细胞松弛素B(cytoB)微球，以HPLC测定cy-toB含量。结果 制备了不同球径的微球，其球径分别为150nm、500nm、1μm、5μm、10μm和20μm。体外释放实验证明，球径越小，药物释放速度越快；球径相同时，以PLIA为基材的微球比PLGA的释放慢。结论 可通过选择适当的微球大小和基质材料达到所期望的药物释放过程。     

In vitro and in vivo release of nerve growth factor from biodegradable poly-lactic-co-glycolic-acid microspheres

Regeneration of peripheral nerves after injury is suboptimal. We now report the long term delivery of nerve growth factor (NGF) by biodegradable poly-lactic-co-glycolic acid (PLGA) microspheres in vitro and in vivo. Lactic to glycolic acid ratios of 50:50 and 85:15 were fabricated using the double emulsion solvent, evaporation technique. Three different inherent viscosities (0.1 dL g(-1) : 1A, 0.4 dL g(-1) : 4A, 0.7 dL g(-1) : 7A) were analyzed. In vitro, release of NGF for 23 days was measured. Electron microscopy demonstrated intact spheres for at least 7 days (50:50 1A), 14 days (50:50 4A), or 35 days (50:50 7A and 85:15 7A). In vitro release kinetics was characterized by burst release, followed by release of NGF at a rate of 0.6-1.6% a day. Release curves for 50:50 1A and 85:15 7A differed significantly from other compositions (p < 0.01). In vivo, release was characterized by a novel radionuclide tracking assay. Release rates varied from 0.9 to 2.2% per day with linear kinetics. All but the 85:15 type of spheres showed different release profiles in vivo compared to in vitro conditions. On the basis of the surface morphology and release profiles, we found microspheres fabricated from 50:50 4A PLGA to be best suited for the use in a rat sciatic nerve injury model.     

Controlled release of gentamicin from calcium phosphate-poly(lactic acid-co-glycolic acid) composite bone cement

Modification of a self setting bone cement with biodegradable microspheres to achieve controlled local release of antibiotics without compromising mechanical properties was investigated. Different biodegradable microsphere batches were prepared from poly(lactic-co-glycolic acid) (PLGA) using a spray-drying technique to encapsulate gentamicin crobefate varying PLGA composition and drug loading. Microsphere properties such as surface morphology, particle size and antibiotic drug release profiles were characterized. Microspheres were mixed with an apatitic calcium phosphate bone cement to generate an antibiotic drug delivery system for treatment of bone defects. All batches of cement/microsphere composites showed an unchanged compressive strength of 60 MPa and no increase in setting time. Antibiotic release increased with increasing drug loading of the microspheres up to 30% (w/w). Drug burst of gentamicin crobefate in the microspheres was abolished in cement/microsphere composites yielding nearly zero order release profiles. Modification of calcium phosphate cements using biodegradable microspheres proved to be an efficient drug delivery system allowing a broad range of 10-30% drug loading with uncompromised mechanical properties.     

乳酸-羟基乙酸共聚物缓控释微球的制备及突释成因与影响因素

背景：乳酸-羟基乙酸共聚物是一种生物可降解高分子材料,以乳酸-羟基乙酸共聚物为原料制备的载药微球和纳米粒既可提高药物的稳定性,又能实现缓释、控释和靶向释放。 目的：分析乳酸-羟基乙酸共聚物缓控释微球的制备方法以及突释的成因、影响因素和改进方法。 方法：应用计算机检索1990/2010中国期刊全文数据库和PubMed数据库与乳酸-羟基乙酸共聚物缓控释微球的制备及突释联系紧密的文章。 结果与结论：目前乳酸-羟基乙酸共聚物缓释微球制备方法主要有单凝聚法、乳化-固化法、喷雾干燥法。造成其突释的原因首先是药物分子和聚合物分子之间的相互作用太弱,导致药物很容易从微球进入释放递质中,其次是在微球释放初期,药物从微球中的孔洞和缝隙中释放出来导致突释。影响突释程度的具体因素有乳酸-羟基乙酸共聚物的相对分子质量、浓度、微球载药量、主药理化性质、微球制备方法及制备参数等。虽然国内外对突释机制以及控制突释措施的研究都还处于初步阶段,通过对各影响因素加以适当优化与控制,可在一定程度上减少微球的突释率,突释问题应该能够得到解决和控制。     

Development of a sustained-release system for perivascular delivery of dipyridamole

Vascular access grafts implanted in dialysis patients are prone to failure in the long-term because of stenosis and occlusion caused by neointimal hyperplasia. Local delivery of antiproliferative drugs may be effective to prevent this consequence while minimizing the systemic side effects they cause. We developed a combination of poly(lactide-co-glycolide) (PLGA) microspheres with ReGel, an injectable copolymer, as a sustained-release system for perivascular delivery of an antiproliferative drug, dipyridamole. Dipyridamole-incorporated PLGA microspheres with various molecular weights (MWs) of PLGA were prepared by oil-in-water emulsion method. Encapsulation efficiency and surface morphology of microspheres were characterized. In vitro release kinetics of dipyridamole from ReGel or from microspheres/ReGel was experimentally determined. Without microspheres, 40% of the dipyridamole was released from ReGel as an initial burst in the first 3 days followed by continuous release in the subsequent 2 weeks. The use of PLGA microspheres decreased the initial burst and extended dipyridamole release from 23 to 35 days with increasing MW of PLGA. The highest MW PLGA showed a lag time of 17 days before consistent drug release occurred. Mixing microspheres and ReGel with two different MW PLGA achieved a continuous release for 35 days with little initial burst. In vivo release of dipyridamole from microspheres/ReGel exhibited a comparable release pattern to that seen in vitro. This injectable platform is a promising technique for sustained perivascular delivery of antiproliferative drugs.     

Preparation, characterization, and in vitro evaluation of physostigmine-loaded poly(ortho ester) and poly(ortho ester)/poly(D,L-lactide-co-glycolide) blend microspheres fabricated by spray drying

The physostigmine-loaded poly(ortho ester) (POE), poly(dl-lactide-co-glycolide) (PLGA) and POE/PLGA blend microspheres were fabricated by a spray drying technique. The in vitro degradation of, and physostigmine release from, the microspheres were investigated. SEM analysis showed that the POE and POE/PLGA blend particles were spherical. They were better dispersed when compared to the pure PLGA microspheres. Two glass transition temperature ( Tg ) values of the POE/PLGA blend microspheres were observed due to the phase separation of POE and PLGA in the blend system. XPS analysis proved that POE dominated the surfaces of POE/PLGA blend microspheres, indicating that the blend microspheres were coated with POE. The encapsulation efficiencies of all the microspheres were more than 95%. The incorporation of physostigmine reduced the Tg value of microspheres. The Tg value of the degrading microspheres increased with the release of physostigmine. For instance, POE blank microspheres and physostigmine-loaded POE microspheres had a Tg value of 67 degrees C and 48 degrees C, respectively. After 19 days in vitro incubation, Tg of the degrading POE microspheres increased to 55 degrees C. Weight loss studies showed that the degradation of the blend microspheres was accelerated with the presence of PLGA because its degradation products catalyzed the degradation of both POE and PLGA. The release rate of physostigmine increased with increase of PLGA content in the blend microspheres. The initial burst release of physostigmine was effectively suppressed by introducing POE to the blend microspheres. However, there was an optimized weight ratio of POE to PLGA (85:15 in weight), below which a high initial burst was induced. The POE/PLGA blend microspheres may make a good drug delivery system.     

Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: investigation for cartilage tissue engineering

Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage.     

Biodegradable polymer-silica xerogel composite microspheres for controlled release of gentamicin

Single and double layered composite microspheres were prepared by encapsulating gentamicin-loaded silica xerogels with biodegradable PLGA polymers (poly(DL-lactide-co-glycolide)). The in vitro drug release properties of both the composite microspheres were investigated. The single layered composite microspheres showed a high initial burst, followed by two sustained release stages lasting for approximately 6 weeks. The two sustained release stages of the single layered composite microspheres could be attributed to the swelling and bulk erosion of the polymer encapsulations, respectively. In comparison with the single layered composite microspheres, the double layered composite microspheres realized a much reduced initial burst together with three sustained release stages. The whole release period of the double layered composite microspheres could last more than 9 weeks. These distinct behaviors make the double layered composite microspheres promising as a new drug release material for localized drug delivery applications.     

Novel scaffolds fabricated from protein-loaded microspheres for tissue engineering

Biodegradable scaffolds play an important role in tissue engineering by providing physical and biochemical support for both differentiated and progenitor cells. Here, we describe a novel method for incorporating proteins in 3D biodegradable scaffolds by utilizing protein-loaded microspheres as the building blocks for scaffold formation. Poly(l,d-lactic-co-glycolic acid) (PLGA) microspheres containing bovine serum albumin (BSA) were fused into scaffolds using dichloromethane vapor for various time intervals. Microspheres containing 0, 0.4, 1.5, 4.3% BSA showed that increased protein loading required increased fusion time for scaffold fabrication. Protein release from the scaffolds was quantified in vitro over 20 days and compared to that of loose microspheres. Scaffolds had a slightly lower (up to 20%) release over the first 10 days, however, the cumulative release from both microspheres and scaffolds at the end of the study was not statistically different and the rate of release was the same, indicating that microsphere release can be predictive of scaffold kinetics. Scaffolds fused from larger (113.3 +/- 58.0 microm) rather than smaller (11.15 +/- 11.08 microm) microspheres, generated pores on the order of 200 microm as compared to 20 microm, respectively, showing control over pore size. In addition, four dyes (carbon black, acid green, red 27, and fast green FCF) were encapsulated in PLGA microspheres and fused into homogeneous and partitioned scaffolds, indicating control over spatial distribution within the scaffold. Finally, the scaffolds were seeded with fibroblast cells, which attached and were well spread over the polymer surface after 4h of incubation. These results highlight the versatility of this simple scaffold fusion method for incorporating essentially any combination of loaded microspheres into a 3D structure, making this a powerful tool for tissue engineering and drug delivery applications.     

Intraseptal implantation of NGF-releasing microspheres promote the survival of axotomized cholinergic neurons

Neurotrophic factors therapy requires their precise delivery to the targeted neuronal population. For this purpose, a wide range of strategies have been developed, and among them the stereotaxic implantation of biodegradable microparticles. To assess the in vivo activity of NGF-releasing PLGA microspheres, unloaded and NGF-loaded microparticles were implanted in the rat brain, near the septal cholinergic neurons, axotomized by an unilateral transection of the fornix-fimbria. Histological analysis at two and six weeks after implantation revealed a non-specific astro- and micro-glial reaction around the microspheres, identical for both unloaded and NGF-loaded microspheres. No neuronal toxicity was noticed, and healthy looking neurons were observed in contact with the microspheres. In the non-treated animals, the percentage of axotomized surviving neurons, when compared to the contralateral intact side, was 31 +/- 2 and 27 +/- 1% at two and six weeks, respectively. Unloaded microspheres caused no protective nor neurotoxic effects (40 +/- 9 and 39 +/- 6% of surviving cholinergic neurons at two and six weeks, respectively). In contrast, NGF-loaded microspheres showed a significant effect on the survival of axotomized cholinergic neurons at two and six weeks after implantation (66 +/- 9 and 61 +/- 5% when compared to the contralateral intact side, respectively). These results show that PLGA microparticles present no neurotoxicity and release sufficient amounts of bioactive NGF to significantly limit the lesion-induced disappearance of cholinergic neurons in the septum during at least six weeks. PLGA microparticles can be used in the future to administer neurotrophic factors in central nervous system disorders.     

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.     

Development and in vitro characterization of vascular endothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique

Poly(DL-lactide-co-glycolide) (PLGA)/polyethylene glycol (PEG) microspheres are one modality of controlled delivery of biologically active molecules that would further the development of engineered tissues. As a possible mechanism to stimulate angiogenesis within an engineered tissue, vascular endothelial growth factor (VEGF) and bovine serum albumin (BSA) were coencapsulated into microspheres fabricated from PEG and 50/50 PLGA using a solid-encapsulation/single-emulsion/solvent extraction technique. Two VEGF/BSA ratios were studied: 1:2000 and 1:10,000. Analysis consisted of the loading efficiency, particle size distribution, bright-field microscopy, scanning electron microscopy, release kinetics, and an in vitro human umbilical vein endothelial cell proliferation assay to assess biological activity of the released VEGF. Results show the microspheres could be manufactured, stored, and degraded over 28 days. The burst release rates for 1:2000 and 1:10,000 VEGF/BSA microspheres were 71.87 +/- 8.11 and 27.91 +/- 1.71 ng/mL (mean +/- standard error of the mean), respectively; steady-state release rates were 6.56 +/- 1.10 and 2.21 +/- 0.47 ng/mL, respectively. The microspheres released biologically active VEGF, and the VEGF increased the proliferation of HUVECs in culture (p <.05). The successful development of a novel, cost-effective, scalable technique for producing microspheres loaded with biologically active proteins is presented. Using the data obtained from these studies, a defined concentration of microspheres will deliver a quantifiable level of VEGF at a known release rate.     

In vitro characterization of vascular endothelial growth factor and dexamethasone releasing hydrogels for implantable probe coatings

Anti-fouling hydrogel coatings, copolymers of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol, were investigated for the purpose of improving biosensor biocompatibility. These coatings were modified to incorporate poly(lactide-co-glycolide) (PLGA) microspheres in order to release dexamethasone (DX) and/or vascular endothelial growth factor (VEGF). DX and VEGF release kinetics from microspheres, hydrogels, and microspheres embedded in hydrogels were determined in 2-week and 1-month studies. Overall, monolithic, non-degradable hydrogel drug release had an initial burst followed by release at a significantly lower amount. Microsphere drug release kinetics exhibited an initial burst followed by sustained release for 1 month. Embedding microspheres in hydrogels resulted in attenuated drug delivery. VEGF release from embedded microspheres, 1.1+/-0.3 ng, was negligible compared to release from hydrogels, 197+/-33 ng. After the initial burst from DX-loaded hydrogels, DX release from embedded microspheres was similar to that of hydrogels. The total DX release from hydrogels, 155+/-35 microg, was greater than that of embedded microspheres, 60+/-6 microg. From this study, hydrogel sensor coatings should be prepared incorporating VEGF in the hydrogel and DX either in the hydrogel or in DX microspheres embedded in the hydrogel.