牛血清白蛋白阳离子微球的制备及体外评价

目的 制备牛血清白蛋白(BSA)口服阳离子微球,考察天然阳离子物质壳聚糖(CHS)的加入对蛋白微球的粒径、电动电势、包封率、载药量及体外释放情况的影响。方法 以乳酸/羟基乙酸共聚物(PLGA)和壳聚糖(CHS)为载体材料,采用W/O/W复乳-溶剂挥发法制备牛血清白蛋白乳酸/羟基乙酸共聚物-壳聚糖(PLGA/CHS)阳离子微球。通过正交设计优化制备工艺,确定最佳处方。建立准确而简便的蛋白含量测定方法,并对微球进行体外评价。结果 最佳处方为：BSA浓度为150 g·L^-1、PLGA浓度为8%、外水相体积为80 mL、壳聚糖浓度为0.2%。制得的微球形态圆整,平均粒径为(6.9±5.5)μm,为表面荷正电的阳离子微球[ζ电势=(10.0±0.6)mV],包封率为(75.4±4.6)%,载药量为(9.3±0.2)%。体外释放结果表明,在模拟胃液和模拟肠液中,壳聚糖的加入均能减少突释,延缓药物的释放。结论 与PLGA微球相比,制得的PLGA/CHS阳离子微球表面带正电,具有较高的包封率和载药量,可以延缓药物释放,同时减少突释现象。     

扑尔敏PLGA缓释微球的制备及体外质量评价

目的以扑尔敏为模型药物,开发一种PLGA缓释微球给药系统。方法采用复乳-溶剂挥发法制备扑尔敏PLGA缓释微球,紫外分光光度法测定其载药量、药物释放。结果制得的扑尔敏PLGA缓释微球平均粒径为(112±57)μm,含药量为(798.33±145.00)μg,载药量为(0.32±0.12)%;药物9 d累积药物释放量可达87%。结论本研究扑尔敏PLGA缓释微球制备方法简单,药物能达到长期缓慢释放。     

海藻酸-壳聚糖-聚乳酸羟乙醇酸复合微球的制备及其对蛋白释放的调节

目的制备蛋白的海藻酸-壳聚糖-聚乳酸羟乙醇酸(PLGA)复合微球，以增加蛋白药物的包封率、减少突释和不完全释放。方法以牛血清白蛋白为模型药物采用修饰的乳化、醇洗法制备小粒径海藻酸微囊，再以壳聚糖孵育制得海藻酸-壳聚糖双层微囊，并进一步用PLGA包裹制得复合微球。采用微量BCA试剂盒测定蛋白浓度，考察其包封率及释放行为，改变各种制备因素调节微球的释放特性。结果复合微球粒径约30 μm，形态圆整。与单纯PLGA微球相比，包封率由60%-70%上升至80%以上。复合微球在磷酸盐缓冲液的1 h突释量由40%-50%下降至25%以下，在生理盐水中则进一步下降至5%以下。结论海藻酸-壳聚糖-PLGA复合微球提高了蛋白药物的包封率，减少了药物的突释，并可通过调节PLGA比例调节药物的释放。     

牛血清白蛋白阳离子微球的制备及体外评价

目的制备牛血清白蛋白（BSA）口服阳离子微球,考察天然阳离子物质壳聚糖（CHS）的加入对蛋白微球的粒径、电动电势、包封率、载药量及体外释放情况的影响。方法以乳酸／羟基乙酸共聚物（PLGA）和壳聚糖（CHS）为载体材料,采用W／O／W复乳-溶剂挥发法制备牛血清白蛋白乳酸／羟基乙酸共聚物-壳聚糖（PLGA／CHS）阳离子微球。通过正交设计优化制备工艺,确定最佳处方。建立准确而简便的蛋白含量测定方法,并对微球进行体外评价。结果最佳处方为：BSA浓度为150g·L^-1、PLGA浓度为8％、外水相体积为80mL、壳聚糖浓度为0．2％。制得的微球形态圆整,平均粒径为（6．9±5．5）μm,为表面荷正电的阳离子微球[ζ电势=00．0±0．6）mV],包封率为（75．4±4．6）％,载药量为（9．3±0．2）％。体外释放结果表明,在模拟胃液和模拟肠液中,壳聚糖的加入均能减少突释,延缓药物的释放。结论与PLGA微球相比,制得的PLGA／CHS阳离子微球表面带正电,具有较高的包封率和载药量,可以延缓药物释放,同时减少突释现象。     

低分子肝素聚乳酸-羟基乙酸缓释微球的研制

目的制备低分子肝素聚乳酸-羟基乙酸(LWMH-PLGA)缓释微球,观察微球表面形态,检测微球物理性能和体外释药行为。方法采用W1/O/W2复乳溶剂挥发法制作微球;通过扫描电镜观察微球的表面形态结构;利用天青A比色法测试微球中药物的载药量和包封率,并对微球中药物的体外释放行为进行研究。结果微球表面显现较多的孔隙,平均粒径为(2.55±0.94)μm,载药量为(14.79±1.03)%,包封率为(55.7±2.21)%;48 h的体外释放试验表明,LWMH累积释放率达到40%。结论 LWMH-PLGA微球能够稳定地释放药物LWMH,验证了PLGA微球作为LWMH控制释放载体的可行性。     

PLGA微球的制备及其用于脉冲给药的初步研究

目的：制备聚乳酸-羟基乙酸共聚物（PLGA）微球,并考察其用于脉冲式释药系统的可行性。方法：以牛血清白蛋白（BSA）为模型药物,用S/O/W（Solid-in-oil-in-water）法和S/O/O（Solid-in-oil-in-oil）法制备PLGA（75：25）和PLGA（50：50）微球,比较2种方法制备的微球的表面形态、包封率及载药量等,并考察2种微球的体外释放行为。结果：S/O/W法和S/O/O法制备的微球均圆整、无粘连、形态良好,但S/O/W法制备的微球表面较为平整,而S/O/O法表面均匀分布有较大的凹陷。S/O/W法制备的PLGA（75：25）和PLGA（50：50）微球包封率分别为（60.15±5.95）%、（49.50±3.69）%,载药量分别为（2.56±0.25）%、（2.10±0.16）%,10h内药物释放均为10%左右,而后随着聚合物的降解药物的释放量突然增加;S/O/O法所制微球包封率分别为（84.36±1.11）%、（77.94±1.42）%,载药量分别为（3.58±0.05）%、（3.31±0.06）%,24h内药物释放均可达50%左右,而后呈现较为平稳的释放行为。S/O/O法制备的微球包封率及载药量均较S/O/W法高;S/O/W法制备的PLGA微球药物释放呈现一定的脉冲行为,其中PLGA（75：25）微球体外释放行为受微球粒径的影响较大。结论：S/O/W法制备的PLGA微球具有一定的脉冲式释药效果,微球的粒径最好控制在120μm以下。     

微球中聚乳酸羟基乙酸共聚物浓度与微球结构、释药、降解的关系研究

目的:研究微球中聚乳酸羟基乙酸共聚物(PLGA)浓度与微球结构、释药、降解的关系。方法:以牛血清白蛋白(BSA)为药物,采用复乳法制备PLGA浓度分别为10%、15%、20%的BSA-PLGA微球,以包封率、载药量、粒径为指标考察PLGA浓度对3种微球性质的影响;采用扫描电子显微镜观察3种微球和降解40 d内的外观和内部形态;使用荧光蛋白-异硫氰酸荧光素牛血清白蛋白代替BSA作为模型药物制备PLGA微球,并采用激光共聚焦显微镜观察荧光蛋白在微球骨架内的分布情况;采用BCA法考察3种微球的体外释药情况;采用压汞仪考察降解28 d内20%PLGA所制微球的孔径、孔隙率、截面孔隙率的变化;采用凝胶渗透色谱法检测10%、20%PLGA所制微球降解28 d内分子质量及其降解模型拟合。结果:与10%、15%PLGA所制微球比较,20%PLGA所制微球的包封率[(81.96±1.84)%]和粒径[(139.50±0.21)μm]最大,载药量[(7.28±0.45)%]最低,截面孔隙率[(32.35±1.98)%]和孔径[(12.43±0.14)μm]最小,释药突释率最低,40 d内的释放速率相对较慢,降解后截面孔隙率最大,降解均遵循假一级模式(r2=0.065 3)。结论:在考察范围内,随着PLGA浓度的增加,微球的结构更致密,释药更平稳,降解更易形成中空结构。     

盐酸多西环素微球的制备及质量控制

目的:制备盐酸多西环素(DXY)微球并建立其质量控制方法。方法:以乳酸-羟基乙酸共聚物(PLGA)为载体材料,采用O/O型乳化溶剂挥发法制备微球,用光学显微镜观察微球的外观形态和粒径,采用紫外分光光度法测定微球的载药量和体外释放度等。结果:所制微球外观光滑圆整,平均粒径为(49±6.9)μm,跨距为0.9,平均载药量为(3.3±0.2)%,平均包封率为(52.4±3.2)%(n=3),0.5h的累积释放度为28%,并可持续释药30d以上。结论:DXY微球的制备工艺可行,质量可控。     

bFGF-PLGA缓释微球及其体外释药性能研究

目的以bFGF为缓释药物、PLGA为药物载体制备bFGF-PLGA缓释微球,观察微球表面形态,检测微球物理性能和体外释药行为。方法采用W1/O/W2复乳溶剂挥发法制作微球;通过扫描电镜观察微球的表面形态结构;利用ELISA法测试微球中药物的载药量和包封率,并对微球中药物的体外释放行为进行研究。结果微球表面圆滑均匀,平均粒径(0.75±0.08)μm,载药量[(59.9±1.9)×10-3]%,包封率为(79.9±2.8)%;在为期45 d的体外释放试验中,bFGF累积释放率达到80%。结论bFGF-PLGA微球能够稳定地在较长时间释放药物bFGF,验证了PL-GA微球作为bFGF控制释放载体的可行性。     

喷雾干燥法制备鼻用甲氨蝶呤壳聚糖微球及其特性的考察

目的以壳聚糖为载体材料,甲氨蝶呤为模型药物,制备鼻腔给药甲氨蝶呤壳聚糖微球。方法采用喷雾干燥法制备甲氨蝶呤壳聚糖微球;采用正交设计优化制备工艺,并对药物的包封率、收率、粒径以及释放行为进行考察。结果优化条件下所得微球粒径分布较为均匀,平均粒径为4.0~5.0μm,制成微球后药物可缓慢释放,符合鼻腔给药的要求。结论该方法适用于甲氨蝶呤壳聚糖微球的制备,壳聚糖是良好的鼻用制剂的载体材料,可用于制备脑部靶向鼻黏膜给药制剂。     

胸腺肽α1缓释注射微球的研究

制备胸腺肽α₁(Tα₁)的长效注射微球，并对微球的体外释放特性、体外活性及药效学进行考察。采用复乳法(W/O/W)制备了载Tα₁聚乳酸-羟基乙酸嵌段共聚物(PLGA)的微球；考察微球的粒径大小、外观及包封率等理化特性；以HPLC法测定微球的体外释放速率；采用CCK-8法评价微球制备工艺和体外释放过程中Tα₁的生物学活性；体内药效学研究中采用流式细胞仪检测免疫抑制模型大鼠给予Tα₁微球后所产生的CD₄⁺，CD₈⁺因子的量，根据CD₄⁺/CD₈⁺的比值变化评价体内药效。微球球形圆整，分散性好，两个优选处方(外水相中加入5%氯化钠和10%葡萄糖)的微球包封率分别为87.8%和90.2%；Tα₁微球1个月的体外累积释放可达90%以上。使用含10%葡萄糖的PVA溶液作为外水相，较好地保持了制备工艺过程中的Tα₁生物学活性，在体外释放过程中Tα₁的生物学活性略有下降；Tα₁微球可显著提高免疫抑制模型小鼠的免疫力。用可生物降解的聚合物PLGA作为载体材料，可以将Tα₁制备成缓释1个月的注射微球。     

Fucosphere--new microsphere carriers for peptide and protein delivery: preparation and in vitro characterization

PURPOSE: Fucoidan is a complex polysaccharide containing sugars and high amounts of sulphate derived from marine brown algaes. In this study, a new microsphere-delivery system based on cross-linking of fucoidan with chitosan, named Fucosphere, was evaluated as a drug carrier. Bovine serum albumin (BSA) was used as a model protein. The effect of fucoidan (1.5, 1.75, 2.0 and 2.5%), chitosan (0.25, 0.50 and 0.75%) and protein (0.25, 0.50 and 0.75%) concentrations, the origin of chitosan and the preparation methods of the particles on the microsphere characteristics were evaluated. METHODS: The microspheres were prepared by a simple method based on the cross-linking of the opposite charged biopolymers. The shape and surface morphologies of the particles were evaluated by scanning electron microscopy (SEM) and the size, charge and encapsulation capacity of the microspheres were determined. The released amount of BSA from the microspheres into phosphate buffered saline (PBS pH 7.4) was determined spectrophotometrically by the Bradford method. SDS-PAGE was performed to check the structural integrity of BSA after the preparation. RESULTS: Smooth and spherical microspheres between the size ranges of 0.61-1.28 microm were obtained. BSA was efficiently encapsulated into the microspheres (51.8-89.5%). All formulation parameters affected the encapsulation capacity of Fucosphere (p < 0.05). The highest encapsulation was obtained with microspheres containing 2.5% of fucoidan (89.5%). CONCLUSIONS: The extent of drug release from the microspheres was dependent on the concentrations of polymers and BSA, chitosan origin and type of preparation method. When the addition methods of protein compared, BSA encapsulated into Fucosphere released slower than the adsorbed protein (E) (p < 0.05). The electrophoretic mobility values of Fucospheres changed between +6.9 and +32.3 mV. In general, BSA release from Fucosphere showed a three-phasic release curve. In conclusion, this new fucoidan microsphere system may be a potential delivery of macromolecular drug such as peptide and protein.     

Effect of osmotic pressure in the solvent extraction phase on BSA release profile from PLGA microspheres

This study investigated the influence of osmotic pressure in the organic solvent extraction phase on release profile of bovine serum albumin (BSA) from poly(lactide-co-glycolide) (PLGA) microspheres. BSA-loaded PLGA microspheres with a target load of 10% were prepared by a double emulsion phase separation method. All the microsphere batches were fabricated in the same conditions except that in the organic solvent (CH2Cl2) evaporation step. Different concentrations of NaCl (0, 1.8, and 3.6%) or sucrose (20%) were used to generate a range of osmotic pressures in the extraction aqueous phase. These microspheres were characterized for incorporation efficiency, surface and internal morphology, particle size, protein stability, and in vitro release. The microspheres were spherical with particle size ranging from 16.8 to 27.8 microns. Higher osmotic pressure resulted in a denser internal structure although similar nonporous surface morphology was observed with all batches. No significant difference in encapsulation efficiency existed from batch to batch (87-94%). Sodium dodecyl sulfate-polyamide gel electrophoresis showed that BSA integrity was well retained. The release profile of the batch prepared with only water as the continuous (solvent extraction) phase exhibited a 79% burst release in the first 24 hr followed by a plateau and then a little release after 21 days. In the presence of NaCl or sucrose, the burst effect significantly decreased with increase in osmotic pressure in the extraction aqueous phase, which was then followed by sustained release for 35 days. A mass balance was made when the release terminated. Therefore, in the organic solvent extraction and evaporation step, increasing the osmotic pressure in the aqueous phase both reduced the burst release from the microspheres and improved the subsequent sustained release profile.     

Sustained release of low molecular weight heparin from PLGA microspheres prepared by a solid-in-oil-in-water emulsion method

Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres for the sustained release of low molecular weight heparin (LMWH) were prepared by a soild-in-oil-in-water (s/o/w) emulsion method. Prior to encapsulation, the LMWH micro-particles were fabricated by a modified freezing-induced phase separation method. The micro-particles were subsequently encapsulated into PLGA microspheres. Process optimization revealed that the NaCl concentration in the outer phase of s/o/w emulsion played a critical role in determining the properties of the microspheres. When the NaCl concentration increased from 0% to 5%, the encapsulation efficiency significantly increased from 51.5% to 76.8%. The initial burst release also decreased from 37.3% to 12.4%. In vitro release tests showed that LMWH released from PLGA microspheres in a sustained manner for about 14 days. Single injection of LMWH-loaded PLGA microspheres into rabbits resulted in an elevation of an anti-factor Xa activity for about 6 days. Furthermore, the integrity of the encapsulated LMWH was preserved during encapsulation process.     

Sustained release of low molecular weight heparin from PLGA microspheres prepared by a solid-in-oil-in-water emulsion method

Biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres for the sustained release of low molecular weight heparin (LMWH) were prepared by a soild-in-oil-in-water (s/o/w) emulsion method. Prior to encapsulation, the LMWH micro-particles were fabricated by a modified freezing-induced phase separation method. The micro-particles were subsequently encapsulated into PLGA microspheres. Process optimization revealed that the NaCl concentration in the outer phase of s/o/w emulsion played a critical role in determining the properties of the microspheres. When the NaCl concentration increased from 0% to 5%, the encapsulation efficiency significantly increased from 51.5% to 76.8%. The initial burst release also decreased from 37.3% to 12.4%. In?vitro release tests showed that LMWH released from PLGA microspheres in a sustained manner for about 14 days. Single injection of LMWH-loaded PLGA microspheres into rabbits resulted in an elevation of an anti-factor Xa activity for about 6 days. Furthermore, the integrity of the encapsulated LMWH was preserved during encapsulation process.     

Development of a single dose tetanus toxoid formulation based on polymeric microspheres: a comparative study of poly(D,L-lactic-co-glycolic acid) versus chitosan microspheres

Stable polymeric microspheres capable of controlled release of tetanus toxoid (TT) for periods ranging from days to over months were developed. TT was stabilized, encapsulated in microspheres prepared from poly(D,L)-lactide-co-glycolide (PLGA) and chitosan by using protein stabilizer (trehalose) and its immune response was compared. The influence of co-encapsulated protein stabilizer on tetanus toxoid's stability and release from the microspheres was studied. The protein stabilizer (trehalose) prevented structural losses and aggregation of microencapsulated TT. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polymer, we also co-incorporated into the polymer an antacid, (Mg(OH)2), which neutralized the acidity during degradation of the polymer and also prevented TT structural losses and aggregation. The in vitro release experiments with PLGA and chitosan microspheres were performed and the release of TT was increased up to 80-90%. The antigen integrity was investigated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by coomassie brilliant blue staining. The SDS-PAGE analysis confirmed that antigen integrity was not affected by the encapsulation procedure. In addition, the immunogenicity of PLGA and chitosan microspheres based single dose vaccine was evaluated in guinea pigs and compared with multiple doses of alum adsorbed TT. Results indicated that a single injection of PLGA and chitosan microspheres containing TT could maintain the antibody response at a level comparable to the booster injections of conventional alum adsorbed vaccines. The both PLGA and chitosan based stable vaccine formulations produced an equal immune response. Hence chitosan can be used to replace the expensive polymer PLGA. This approach should have potential application in the field of vaccine delivery.     

Preparation of superoxide dismutase loaded chitosan microspheres: characterization and release studies.

Superoxide dismutase (SOD) is the most potent antioxidant enzyme. In this study, SOD was encapsulated in chitosan microspheres to obtain suitable sustained protein delivery. Protein-loaded chitosan microspheres with various formulations were prepared based on complex coacervation process. Due to the inherent characteristic of SOD, high encapsulation efficiency could not be obtained with simple preparation method. The pH of chitosan solution is 3.0; when the chitosan microspheres were prepared with this solution, encapsulation was low. Therefore, several strategies have been tested to increase the encapsulation efficiency and good results have been obtained. 70-80% protein encapsulation efficiency was obtained. The addition of PEG to the protein solution enhanced the encapsulation efficiency also. Mean sizes of microspheres were between 1.38 and 1.94 microm. Factors affecting the release behaviour of SOD from microspheres have been studied. They included pH values of chitosan solution (the pH of chitosan solution is 3.0), addition of PEG to the protein solution and the use of adsorption technique. In general, biphasic release profiles were obtained with these formulations. The protein activity changed between 70 and 100% during the release. In general, the protein activity remained in acceptable limits. The SOD encapsulated chitosan microspheres can be prepared by changing the pH or addition of PEG, allowing the safe incorporation of protein for controlled release.     

Preparation,characterization and in vitro release study of BSA-loaded double-walled glucose-poly(lactide-co-glycolide) microspheres

The aim of this study was to prepare a model protein, bovine serum albumin (BSA) loaded double-walled microspheres using a fast degrading glucose core, hydroxyl-terminated poly(lactide-co-glycolide) (Glu-PLGA) and a moderate-degrading carboxyl-terminated PLGA polymers to reduce the initial burst release and to eliminate the lag phase from the release profile of PLGA microspheres. The double-walled microspheres were prepared using a modified water-in-oil-in-oil-in-water (w/o/o/w) method and single-polymer microspheres were prepared using a conventional water-in-oil-in-water (w/o/w) emulsion solvent evaporation method. The particle size, morphology, encapsulation efficiency, thermal properties, in vitro drug release and structural integrity of BSA were evaluated in this study. Double-walled microspheres prepared with Glu-PLGA and PLGA polymers with a mass ratio of 1:1 were non-porous, smooth-surfaced, and spherical in shape. A significant reduction of initial burst release was achieved for the double-walled microspheres compared to single-polymer microspheres. In addition, microspheres prepared using Glu-PLGA and PLGA polymers in a mass ratio of 1:1 exhibited continuous BSA release after the small initial burst without any lag phase. It can be concluded that the double-walled microspheres made of Glu-PLGA and PLGA polymers in a mass ratio of 1:1 can be a potential delivery system for pharmaceutical proteins.