【作者】 叶邦阜；

【导师】 胡富强；

【作者基本信息】 浙江大学 ， 药剂学， 2011， 硕士

【摘要】 固体脂质纳米粒(Solid Lipid Nanoparticles, SLN)是20世纪90年代发展起来的微粒给药系统,超临界流体辅助雾化法(Supercritical assisted atomization, SAA)是最近10年左右兴起的一种超临界微粒制备技术,本文将新兴的超临界辅助雾化技术应用于以脂质材料为载体的载药固体脂质纳米粒的制备,可提高SLN的制备效率、减少有机溶剂的残留,获得载药量高、粒径大小可控的载药SLN,有望成为脂质纳米粒制备工艺研究的新方向。根据超临界辅助雾化法(SAA)原理,我们设计制备了自制超临界辅助雾化制粒设备,选择乙醇为辅助溶剂,硬脂酸为载体材料,制备固体硬脂酸脂质纳米粒,考察脂质浓度、超临界流体CO2与载体溶液流量比、喷嘴孔径等工艺因素对SLN粒径的影响,筛选合适的处方工艺参数。结果显示,气液混合室中负载的脂质浓度越高,制备所得的粒径越大,当载体浓度在1%w/v以下时所得的粒径小于1μm,载体浓度在0.5%w/v以下时可制备得到500 nm以下的纳米粒。气液混合室中泵入超临界流体CO2的速率较快时(超临界CO2与载体溶液流量比加大),制得SLN的粒径较小；当泵入速率为10 mL/min以上时,制得微粒粒径小于1μm；当泵入速率提高至30 mL/min以上时(超临界CO2与载体溶液流量比为3：1以上),可制得平均粒径500 nm以下的固体脂质纳米粒。雾化膨胀的喷嘴孔径与所得的脂质微粒粒径呈正性相关,即喷嘴孔径越大,所得粒径越大并且大小分布不均一,反之相反；当喷嘴孔径为0.2mm或更小时,制得的纳米粒粒径小于400nm。以单硬脂酸甘油酯替换硬脂酸作为脂质载体材料时,制得SLN的理化性质相近,显示此SAA制粒系统也适用于单苷酯纳米粒的制备。在前述筛选优化的超临界技术工艺条件(脂质浓度0.5% w/v、CO2泵入速率30 mL/min、喷嘴孔径0.2 mm)的基础上,以大分子亲水药物胰岛素为模型药物,采用自制的超临界流体辅助雾化制粒设备,制备胰岛素脂质纳米粒。试验分别以硬脂酸和单硬脂酸甘油酯为载体材料,辅助以亲水性成分进行修饰,考察不同处方所制的SLN的粒径、电位、包封率、载药率等理化性质,并考察了样品的体外释放行为。结果显示,不同处方胰岛素SLN的平均粒径从180nm～300nm不等,包封率约为65%～80%,载药量约为2.8%～3.8%,经泊洛沙姆或PEG修饰的处方粒径更细小均一但药物包封率相对降低；原子力显微镜照片显示胰岛素SLN表面呈球形,外观较平整。体外释放研究结果表明,硬脂酸或单硬脂酸甘油酯为载体材料的SLN均在体外呈现约12小时缓慢释放药物的特性,同时未经修饰的处方释放较缓慢但容易释药不完全,经亲水性表面活性剂泊洛沙姆修饰后,纳米粒累积释药程度提高但伴随着较高的突释现象；在单硬脂酸甘油酯为载体的处方中以无表面活性的PEG 2000替代泊洛沙姆时,胰岛素SLN在获得较高释药率的同时突释率较低。综合胰岛素SLN的粒径、电位、包封率、载药量、体外释放特性以及预期的体内生理腔道吸收水平,以PEG修饰的单硬脂酸甘油酯为载体材料的胰岛素SLN是优选的处方：本研究进一步以链脲霉素、四氧嘧啶建立了糖尿病模型大鼠和糖尿病模型犬,考察了以SAA法优选处方制备的胰岛素-SLN的动物体内降血糖药效。实验以不给药为空白对照、皮下注射胰岛素为阳性对照,分别以20 IU/kg、15 IU/kg高低两种剂量口服给予大鼠或犬胰岛素脂质纳米粒,通过葡萄糖酶标试剂盒测定给药后24小时内的体内血糖经时变化情况。结果显示,胰岛素脂质纳米粒经大鼠口服后,在0.5h即可产生降血糖作用,并于8h后达到降血糖作用峰值,最大血糖下降幅度可达80%。根据AAC (0-24h)法计算大鼠不同剂量口服胰岛素脂质纳米粒的相对生物利用度分别为36.4%和33.3%。胰岛素脂质纳米粒给予糖尿病模型犬后同样有显著的降血糖作用,较好的控制了模型动物的血糖水平。按照血糖水平-时间曲线的曲线上面积AAC法评价的两种剂量(9IU、30 IU)的相对生物利用度分别为27.0%和21.9%。受限于吸收速率和程度的影响,口服纳米粒的起效时间和强度均不如注射给药方式,但前者的降血糖持续作用时间较皮下注射持久(前者药效持续达8小时以上),这可以归因于脂质纳米粒在体内缓慢释放胰岛素作用产生的治疗效果,体现预期的口服缓释制剂特征药动学,具备相当的临床开发应用价值。更多还原

【Abstract】 Solid Lipid Nanoparticles(SLN) is one of micronized drug carriers, which developed since 1990s, and Supercritical assisted atomization(SAA) is a novel supercritical technology for microparticles preparation appeared within recent 10 years. In this study, SAA technology was applied to prepare the SLN encapsulating drug. SAA was found to improve the productivity, reduce solvent residue, and to gain high drug-loading, size-controllable SLN, and seems to be a promising method for lipid nanoparticles preparation.A novel SAA particle-producing equipment was designed based on SAA principle. Using ethanol as an assisted solvent, stearic acid SLN was prepared by this equipment. The effect of lipid concentration, flow ratio of supercritical CO2 and lipid solution and pore size of nozzle, on the particle size of resulted SLN were investigated to explore the appropriate process conditions. The results showed that SLNs with bigger size were obtained when lipid concentration in saturator was higher. When lipid concentration was 1.0% w/v or lower, particle size was smaller than 1μm. When the lipid concentration was lower than 0.5% w/v, diameter of SLN was below 500 nm. Flow ratio of SCF CO2 to lipid solution was negatively correlated with particle size, SLN with below 1μm size was obtained when the flow ratio was 1:1. SLN with below 500 nm size was obtained when flow ratio was more than 3:1. However the particle size was positively correlated with pore size of nozzle. When nozzle size is 0.2 mm or lower, the size of SLN was lower than 400 nm, the size distribution was narrow. The mnostearin SLN prepared by SAA equipment had similar physio-chemical properties with stearic acid SLN.Based above optimal process conditions, insulin, a hydrophilic macromolecule drug, was used as model drug to produce SLN loading insulin (INS-SLN). During SLNs preparation, both stearic acid, monostearin, were choosed as matirx, with or without modified by hydrophilic polymers such as poloxamer and PEG 2000. Physico-chemical properties, such as particle size, zeta-potential, drug entrapment efficiency (EE), loading ratio (DL) and in vitro release profile of SLN were investigated. Mean particle size, EE and DL of INS-SLN was variable from 180 nm to 300 nm, EE from 65% to 80%, and DL 2.8% to 3.8% depending on the fomula, respectively. AFM image showed the INS-SLN was spherical with smooth surface. Unmodified formulation by hydrophilic polymers of SLN was found to be larger size and slower drug releasing profile, and nearly 15% of encapsulated INS couldn’t be release within 12 hrs in vitro. Poloxamer used as hydrophilic polymers was introduced to modify the lipid carriers. The modified INS-SLN was smaller (180 nm) and uniform size, but the EE was reduced, and burst release was increased significantly. When poloxamer was replaced by PEG 2000 in monostearin formulation, the particle size and releasing behavior were kept, meanwhile the EE and burst release were improved. Thus the PEG modified formulation was preferred formulation for further in vivo study.Diabetic rats and dogs were established through administration of streptozocin amd/or alloxan, to evaluate the in vivo efficacy of INS-SLN. S.c. injection of INS solution was used as positive control. After oral administration of INS-SLN for 0.5 hr, the blood glucose of rats was decreased, and the maximum hypoglycemic effect of 80% was reached in 8 hr. Relative bioavailability (F) of oral INS-SLN calculated by AAC(0-24h) were 36.4% and 33.3% for dosage of 15 IU/kg and 20 IU/kg. Similar results were observed in diabetic dogs’model, blood glucose was inhibited since 0.5h after administration and maintained for 8 hr or more. Relative bioavailability (F) based on AAC(0-24h) were 27.0% and 21.9% for dosage of 9 IU and 30 IU. Due to the absorption of SLN, the onset time and strength of oral INS-SLN were inferior to s.c. INS injection. However, INS-SLN showed Longer-time hypoglycemic effect (more than 8 hr), which should attribute to extended INS release of SLN. The results indicated clinical development possibility of INS-SLN.更多还原