羟基喜树碱长循环纳米粒及其两亲性载体的研究
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摘要
研究背景和目的
随着上世纪70年代,Birrenbach等首次提出纳米粒的概念和制备方法,药物制剂的研究进入了一个全新的微观领域----纳米制剂。与常规药物相比,纳米药物具有比表面积大、表面反应活性高、活性中心多、吸附能力强等特性,还可以改变药物在体内的分布,增加药物在靶器官的分布量,从而提高疗效。但普通纳米制剂静注后,血浆中的多种成分(如载脂蛋白、补体C蛋白等)会吸附于纳米粒的表面,使其易于被吞噬细胞识别,即机体的调理过程(opsonization),被单核巨噬细胞吞噬系统(mononuclear phagocyte system,MPS)吞噬并迅速从血液中清除。因而如何避免MPS对纳米粒的识别和吞噬是其能否被传输至其他器官或组织的关键。研究发现纳米粒的表面特性如亲脂性强弱、表面电位值等与调理素蛋白在其表面的吸附息息相关。一般来说纳米粒的表面亲脂性越强、表面电位值越低,调理素蛋白越容易吸附。由此可见纳米粒的表面特性对制备长循环纳米粒极为重要。目前针对纳米粒表面改性的研究主要集中在两个方面：(1)在纳米粒的表面包被亲水性的聚合物或表面活性剂；(2)制备含亲水性片断的两亲性高分子载体材料,这些材料含有PEG、PEO、poloxamer、poloxamine、Polysorbate-80及Briji-35等。其共同点是能在纳米粒亲脂性内核的表面形成水化保护层(protective clouds)并具备一定的立体位阻效应,能抑制调理素蛋白在纳米粒表面的吸附,即所制备的纳米粒具有“隐形”(stealths)特性,从而使得纳米粒不易被MPS识别,达到长循环目的。
包被有poloxamer等表面活性剂的纳米粒虽具备长循环的特质,但在体内微环境中,有学者认为血液中物质和包衣层物质间范德华力的存在容易引起包被层在体内循环中脱落,脱落的外源性物质作为一种外来性抗原对人体可能产生的潜在毒副作用值得关注并仍存在争议；在两亲性高分子载体合成方面,目前文献报道的共聚物几乎均为AB型或者ABA型,其中包含有亲水性的片段如PEG、PEO等,具体的载体材料有PEG-PLGA、PEG-PLA及PEG-PHDCA等。其制备多是将PEG或单甲基取代的PEG分子、PEO等与乳酸分子单体或氰基烷酸酯等单体聚合反应而成,其反应条件要求苛刻如要求惰性气体保护、反应管需融封、添加引发剂等。
本课题直接选用适宜分子量的PEG和PCL为原料,琥珀酸酐为桥联剂,制备含不同重复单元数量的PEG和PCL的两亲性共聚物载体材料。其中PCL为半结晶状的高分子材料,选用它作为两亲性共聚物载体的亲脂性部分,既可以作为亲脂性药物的储库,其所含有的晶状结构又有利于保持纳米粒形态的稳定。PCL应用历史悠久,如将其作为长效避孕药体内埋植剂的载体材料、组织工程材料等；PEG安全无毒,已被FDA批准应用于临床。本课题所选用的制备方法具有反应条件温和、简便易行和反应收率高等优点。
羟基喜树碱(10-hydroxycamptothecin,HCPT)是从珙桐科植物喜树中提取的一种微量生物碱,是拓扑异构酶Ⅰ的特异性抑制剂,广泛应用于肝癌、胃癌、白血病等多种恶性肿瘤的治疗。但其特殊的理化性质：水不溶脂难溶、内酯环结构不稳定等因素,限制了临床应用,普遍存在疗效降低、半衰期短的缺点。本研究以HCPT为模型药物,以两亲性的PEG-PCL多嵌段共聚物为载体材料,制备长循环纳米粒(long-circulating nanoparticles),同时以脂质材料包裹的羟基喜树碱半固体脂质纳米粒制剂为对照组,通过体外释放和大鼠体内药代动力学试验比较二者的长循环能力之间的差异,旨在寻求一种新型的长循环制剂载体材料和制备HCPT长循环纳米制剂。
材料与方法
1 HCPT-SSLN的制备与表征
选择固态和液态脂质为膜材,大豆磷脂为表面活性剂,采用乳化蒸发-低温固化法制备了羟基喜树碱半固体脂质纳米粒(HCPT-SSLN).以纳米粒的外观、粒径、包封率为指标,分别考察了表面活性剂种类、固态脂质／液态脂质摩尔比、药脂比、表面活性剂浓度、乳化蒸发搅拌时间、搅拌速度等因素对制备的影响；在此基础上,选定药脂比、固态脂质与液态脂质的摩尔比、表面活性剂浓度为3个因素,进行3因素3水平正交设计优选处方。用Malvern激光粒度仪测定HCPT-SSLN的表面电位、粒径及粒径分布；透射电镜观察HCPT-SSLN的形态；建立了414nm处紫外分光光度法测定开环形式HCPT含量的方法,用葡聚糖凝胶柱层析-UV法测定HCPT-SSLN的包封率和载药量。探讨了不同冷冻保护剂对冻干HCPT-SSLN的影响,并对冻干制剂进行了稳定性考察。以HCPT钠盐注射液作对比,选用昆明种小白鼠考察所制备的HCPT-SSLN的急性毒性。
2多嵌段PEG-PCL共聚物的合成研究
选用适宜分子量的PEG和PCL为起始原料,琥珀酸酐为桥联剂,合成多嵌段的PEG-PCL两亲性共聚物载体,并用FT-IR、1H-NMR、GPC、DSC、XRD分析对其进行表征；选用L969细胞株,以MTT和乳酸脱氢酶(LDH)活性指标考察所合成共聚物的生物相容性。
3 HCPT-PEG-PCL-NPs的制备与表征
以共聚物为载体材料,Span60和Poloxamer 188为乳化剂和稳定剂,用共溶剂挥发法制备了羟基喜树碱两亲性嵌段共聚物纳米粒(HCPT-PEG-PCL-NPs)。以纳米粒的外观形态、粒径、包封率、载药量为指标,考察了Span/PEG-PCL的摩尔比、投药量、Span/Poloxamer的摩尔比、搅拌速率、搅拌温度、水相用量、水化时间等因素对制备的影响；在此基础上,选定PEG-PCL种类、投药量、Span/Poloxamer的摩尔比、Span/PEG-PCL的摩尔比为4个因素,进行4因素3水平正交设计优化工艺。用Malvern激光粒度仪测定HCPT-SSLN的表面电位、粒径及粒径分布；透射电镜观察HCPT-SSLN的形态；建立了384nm处内酯形式存在的HCPT体外HPLC分析方法,用超滤离心-HPLC法测定HCPT-PEG-PCL-NPs的包封率和载药量。探讨了不同冷冻保护剂对冻干HCPT-PEG-PCL-NPs的影响,并对冻干制剂进行了XRD分析和初步的稳定性考察。
4 HCPT-NPs的体外释放研究
采用动态透析法,以pH7.4的磷酸盐缓冲溶液为释药介质,HCPT原料药的水溶液为对照组,37℃恒温避光恒速(100r／min)搅拌下进行HCPT-SSLN和HCPT-PEG-PCL-NPs的体外释药实验。在设定的时间点上取样测定其中的HCPT含量,计算累计释药量；采用不同的数学模型对不同纳米制剂的体外释药曲线进行拟合,探讨其可能的释药机制。
5 HCPT-NPs大鼠体内药代动力学研究
设定HCPT普通水针剂、HCPT-SSLN及4种两亲性共聚物载体材料制备的HCPT-PEG-PCL-NPs共6组；以54只Wistar大鼠为实验动物(n=9),给药剂量均为5mg／kg,经尾静脉单剂量注射给药后在设定的时间点上取血并测定HCPT的血药浓度；建立了测定血浆中HCPT含量的HPLC方法,并绘制血药浓度-时间曲线,计算药代动力学参数,对不同的HCPT-NPs的长循环作用进行评价和比较,优选最佳HCPT的长循环制剂及载体。
结果
1 HCPT-SSLN的制备与表征
结果表明,单因素实验中考察的各因素对HCPT-SSLN的粒径、包封率均有影响,其中表面活性剂浓度对SSLN的包封率影响最大且具显著性意义。优选的处方及工艺条件为：药脂比为1：20,固态／液态脂质摩尔比为2：1,表面活性剂浓度为5%,油相注入水相的滴速为2.0mL／min,乳化蒸发搅拌温度选取75℃,搅拌时间为2h,搅拌速度为800r／min。所制备的HCPT-SSLN的平均粒径为130.5nm,多分散度为0.19；平均毛电位为—33.1mV,平均pH为6.29,平均包封率为79.19%,平均载药量为2.51%；透射电镜结果表明,纳米粒为圆整的球状体,无明显团聚现象。UV法测定开环HCPT含量的线性范围为0.2～10.0μg/mL,回归方程为A=0.0681C—0.004,r=1.000。冻干实验结果表明：添加2.5%的乳糖为冻干保护剂,所制备的冻干纳米制剂外观为白色,冻干前后体积不变、无裂缝、脱壁等现象；有较好的复水性,复水后的纳米粒径为138.9nm,多分散系数为0.25,平均ξ电位为—36.2mV,平均pH为6.29,平均包封率为75.47%；稳定性考察结果表明HCPT-SSLN冻干品宜密封避光于4℃下保存。急性毒性实验结果表明,在最大耐受剂量214.6mg/kg(相当于人临床应用剂量17.89mg/kg)注射情况下,小鼠仍能保持良好状态,未出现急性毒性反应。
2多嵌段PEG-PCL共聚物的合成研究
所制备的PEG-PCL两亲性共聚物载体经FT-IR、1H-NMR、GPC、DSC、XRD等分析证实含有PEG和PCL片段,符合目标物质,且GPC的多分散指数均小于1.1,分子量分布较窄。细胞毒性实验表明所合成的载体基本无毒,有着较佳的生物相容性,毒性反应为1级,符合生物学评价标准。
3 HCPT-PEG-PCL-NPs的制备与表征
结果表明,单因素实验中考察的各因素对HCPT-PEG-PCL-NPs的粒径、包封率、载药量均有影响,其中投药量和Span/PEG-PCL的摩尔比为主要影响因素,具统计学意义。优选的处方及工艺条件为：投药量为5mg, Span/PEG-PCL的摩尔比为20：1,Span/Poloxamer的摩尔比为3：1,水相用量40mL,搅拌速度800r／min,有机相滴加至水相速度为2.0mL/min,水化时间2 h,最佳PEG-PCL载体为PEG4000-PCL2000。以共聚物PEG4000-PCL2000、PEG4000-PCL1250、PEG2000-PCL2000、PEG2000-PCL1250为载体制备的4种HCPT-PEG-PCL-NPs的平均粒径依次为116.1、110.0、119.9、99.1nm,平均毛电位依次为—22.4、—16.9、—33.5、—28.8 mV,平均包封率依次为88.29%、83.10%、80.67%、77.46%,平均载药量依次为2.96%、2.56%、2.31%、2.14%；透射电镜结果表明,4种纳米粒为圆形,粒径大小比较均一。384nm处内酯形式存在的HCPT体外HPLC分析方法,线性范围为0.05～5.0μg/mL,回归方程为A=78.215C—0.498,r=1.000。共聚物的表面电位值说明随着共聚物中PEG链段的增长而升高,随着亲脂性组分PCL链段的增长而降低。冻干实验结果表明：添加4%的海藻糖和4%的蔗糖为冻干保护剂,所制备的冻干纳米制剂外观为白色,冻干前后体积不变、无裂缝、脱壁等现象；有较好的复水性,以PEG4000-PCL2000为载体材料制备NPs冻干品,复水后的纳米粒径为124.5nm,多分散系数为0.15,平均毛电位为—25.2mV,平均包封率为85.24%；稳定性考察结果表明HCPT-PEG-PCL-NPs冻干品宜密封避光于常温下保存。XRD分析结果说明HCPT在所制备的纳米制剂中是以无定型的状态存在的。4 HCPT-NPs的体外释放研究
实验结果表明,HCPT原料药水溶液组24h即达最大释放量约80%,与它相比,HCPT-NPs均具有一定的缓释作用,其中以HCPT-SSLN最明显,但96h释放量趋于饱和,累积释药百分数不超过60%。以共聚物为载体制备的HCPT-NPs中,随着分子中PEG片段的增长,对应的NPs释药速率与累积释药百分数增高；随着分子中PCL片段的增长,其相应NPs的释药速率与累积释药百分数降低。所制备的HCPT-PEG-PCL-NPs体外释药均符合Weibull分布模型,其释药机理应是药物的被动扩散与载体基质的溶蚀协同作用。
5 HCPT-NPs大鼠体内药代动力学研究
结果表明,不同制剂在大鼠体内的药代动力学特征有显著不同(P<0.01),HCPT水针剂注射后体内代谢极快,其体内半衰期tl／2仅0.1418h,以共聚物载体PEG4000-PCL1250、PEG4000-PCL2000、PEG2000-PCL1250、PEG2000-PCL2000制备的4种HCPT-PEG-PCL-NPs与HCPT-SSLN的t1／2依次为它的18.07、9.08、5.25、5.14、4.28倍；表明HCPT-NPs均具备一定的长循环效果,尤以PEG4000-PCL1250为最佳(P<0.01),其释药速度快,释药量大且作用持久；其次为PEG4000-PCL2000 (P< 0.01),而PEG2000-PCL1250、PEG2000-PCL2000的长循环效应较差,半衰期与MRT值均无显著性差异(P>0.05)。
结论
本研究所制备的HCPT-NPs均能有效增进HCPT在制剂中的溶解度,粒径、形态、包封率与载药量均达到制剂要求,冻干后复水性较好,动物急性毒性实验安全。合成的多嵌段PEG-PCL共聚物经分析符合目标物质,有着良好的生物相容性。体外释药实验中HCPT-NPs均具有一定的缓释作用且符合Weibull分布模型,其释药机理应是药物的被动扩散与载体基质的溶蚀协同作用。大鼠药代动力学实验表明HCPT-NPs均具备一定的长循环作用,但因载体材料不同效果不同,尤以HCPT-PEG4000-PCL1250-NPs为最佳,采用两亲性嵌段共聚物材料为载体制备HCPT长循环制剂的思路是可行的。
Background & objective
Since 1970s, Birrenbach introduced the concept and preparative method of nanoparticle firstly, the study of the pharmaceutical preparation has entered into a new micro realm——nanometric praeparatum. Compared to routine drugs, nanometric praeparatums have many good characters such as large specific surface area, high surface reaction activity, plenty of active centers, strong adsorbability and so on. They can also change the intracorporal distribution of the medicine to increase the dose distribution in target organs and raise therapeutic effects. However, after the intravenous injection of general nano-medicament, a variety of components (such as apolipoprotein, complement protein C, etc) in plasma will adsorb on the surface of nanoparticles to make it be easily identified by the phagocytic cells. This is just the process of body opsonization, the nanoparticles can be phagocytosed by mononuclear phagocyte system and rapidly cleared from the blood. Therefore, how to avoid MPS identifying and phagocytosing the nanoparticles is the key for transmiting them to other organs or tissues. It is found that nanoparticles'surface properties (such as lipophilic strength, surface potential value, etc.) are closely related to the adsorption of opsonin proteins on their surface. Generally speaking, the stronger the surface lipophilicity is, the lower the value of surface potential is, the easier that opsonin proteins absorb nanoparticles. This shows that the surface characteristics of nanoparticles are extremely important for preparing long-circulating nanoparticles. Currently, the study of nanoparticles for surface modification mainly focuses on two aspects:(1)To coat the surface of nanoparticles with hydrophilic polymers or surfactants; (2)To prepare amphiphilic polymer carrier materials containing hydrophilic fragments which contain PEG, PEO, Poloxamer, Poloxamine, Polysorbate-80 and Briji-35,etc. They can all form hydration protective clouds on the surface of nanoparticles' lipophilic cores and have certain three-dimensional steric effects, which can inhibit opsonin protein absorbing on the nanoparticles surface. Therefore, the nanoparticles have "stealths" feature for being not easily identified by MPS to achieve the purpose of long-circulating.
Although the nanoparticles coated with surfactants (such as poloxamer, etc.) have long-circulating feature, in vivo micro-environment, some scholars believe that the vander wale force between blood materials and coating materials may easily lead the coated layer to fall off in the vivo circulation. As a foreign antigen, the deciduous exogenous substances may have the potential toxic effects on the human body and it is still in disputes. In the synthesis of amphiphilic polymer carrier, the copolymers reported are almost only the AB-type or ABA-type currently, which contain hydrophilic segments (such as PEG, PEO, etc.) and carrier materials (such as PEG-PLGA, PEG-PLA, PEG-PHDCA, etc.). The copolymers are mostly made through the polymerization reaction between PEG (or Monomethyl-substituted PEG molecule, PEO) and lactic acid monomers (or cyano-alkyl ester monomer, etc.). The reaction conditions are strict, such as requiring inert gas protection, sealing reaction tube, adding initiators, etc.
This study chose poly (ethylene glycerol) (PEG) and poly(s-caprolactone) (PCL) of appropriate molecular weight as raw materials, succinic anhydride for the bridging agent, synthesized the amphiphilic copolymer carrier materials with different numbers of repeating units of PEG and PCL. PCL is semi-crystalline polymer material, as the lipophilic part of amphiphilic copolymer carriers, it can not only store the lipophilic drug, but also be beneficial to the stability of nanoparticles' morphology by its crystalline structure. The applications of PCL has a long history, such as the carrier material of long-acting contraceptive implant in vivo, tissue engineering materials, etc.; PEG is safe and non-toxic, and has been approved by FDA for clinical application. The synthetic reaction in this study is mild, simple and the reaction yield is high.
10-hydroxycamptothecin (HCPT) is a kind of micro amount alkaloids extracted from Camptotheca acuminate Decaisne, and is one of the specific DNA topoisomerase I inhibitors. It is widely used to cure liver cancer, stomach cancer, leukemia and many malignant tumors. However, its clinical applications are confined by its special physico-chemical properties (for example, it is insolubilize in water and difficultly dissolves in lipids, the lactone band is also instable, etc.), so its curative effects decrease and the intracorporal half life is too short. This study choose it as model drug, and the amphiphilic PEG-PCL multiblock copolymers as the carrier material, to prepare long-circulating nanoparticles. Moreover, HCPT-SSLN (HCPT semisolid lipid nanoparticles) coated with solid and liquid lipids acted as the control group, their long-circulating abilities were compared by in vitro drug release experiment and in vivo pharmacokinetics tests. The goal of this study is to find a novel carrier for long-circulation and prepare long-circulating HCPT-NPs formulation.
Materials and methods
1 Preparation and characterization of HCPT-SSLN
HCPT-SSLN was prepared by the method of "emulsion evaporation at a high temperature and solidification at a low temperature", using solid & liquid lipids as carrier materials, soya lecithin as surfactant. According to the morphology, particle size and entrapment efficiency, the affected factors in the preparing technology, including "types of surfactants", "molar ratio of solid/liquid lipids", "ratio of HCPT/lipids", "concentration of surfactants", "stirring time", "stirring velocity", etc. were investigated. On this basis, "ratio of HCPT/lipids", "molar ratio of solid/ liquid lipids" and "concentration of surfactants" were selected as major factors to make L9 (34) orthogonal design for optimization of formulation and technology. The zeta potential, particle size and size distribution (polydispersion) of HCPT-SSLN were determined on a Malvern Zetasizer HSA 3000; the morphology was observed by transmission electron microscopy. In order to control the quality of HCPT-SSLN, the methods for determination of open-loop form of HCPT quantity by UV spectrophotometry at 414nm and for determination of the entrapment efficiency and drug loading of HCPT-SSLN by sephadex gel filtration chromatography-UV were established. Different cryoprotectants were investigated to prepare the lyophilized HCPT-SSLN powder, and then the stability of the NPs powder was studied. By the comparison of HCPT sodium injection, the animal acute toxicity of HCPT-SSLN was conducted using kunming mice by tail intravenous administration.
2 Synthesis of multiblock PEG-PCL copolymer
The multiblock polyethylene-polycaprolactone copolymer was synthesized, which PEG and PCL were used as starting meterials, succinic anhydride acted as cross-linking agent, DCC and DMAP as condensation agent and catalyst, respectively. Then FT-IR、1H-NMR、GPC、DSC、XRD analyses were used to characterize the copolymers. The cytotoxicity of the copolymers was evaluated with L929 cell line through MTT and LDH methods.
3 Preparation and characterization of HCPT-PEG-PCL-NPs
HCPT-PEG-PCL-NPs were prepared by co-solvent evaporation method, which copolymer was the carrier material, Span60 and Poloxamer 188 was the emulsifier and stabilizer. According to the morphology, particle size, entrapment efficiency and drug loading, the affected factors in the preparing technology, including "molar ratio of Span/PEG-PCL", "dosage of HCPT", "molar ratio of Span/Poloxamer", "stirring velocity", "stirring temperature", "volume of water phase", "hydration time", etc. were investigated. On this basis, "types of PEG-PCL", "dosage of HCPT", "molar ratio of Span/Poloxamer" and "molar ratio of Span/PEG-PCL" were selected as major factors to make L18(34) orthogonal design for optimization of formulation and technology. The zeta potential, particle size and size distribution (polydispersion) of HCPT-PEG-PCL-NPs were determined on a Malvern Zetasizer HSA 3000; the morphology was observed by transmission electron microscopy. In order to control the quality of HCPT-PEG-PCL-NPs, the methods for determination of lactone form of HCPT quantity by HPLC analysis in vitro at 384nm and for determination of the entrapment efficiency and drug loading of HCPT-PEG-PCL-NPs by centrifugal ultrafiltration—HPLC were established. Different cryoprotectants were investigated to prepare the lyophilized HCPT-PEG-PCL-NPs powder, and then the stability and XRD analysis of the NPs powder were studied.
4 In vitro drug release study of HCPT-NPs
In vitro drug release studies were carried out as follows. Drug-loaded nanoparticles suspensions and aqueous solution of HCPT (the control) were placed in a dialysis membrane bag with molecular weight cut-off 3500g/mol, tied, and dropped into 250mL of a phosphate buffer solution media (pH=7.4). The entire system was kept dark and at 37℃with magnetic stirring at 100r/min. At predetermined time intervals,3 mL of aqueous solution was withdrawn from the release media. Meanwhile, an equal volume release medium was added. Then the drug content of the sample was assayed by HPLC. Furthermore, the drug release mechanisms of HCPT-NPs were studied with methematics modles including Weibull distribution model, Ritger-Peppas modle, etc.
5 In vivo pharmacokinetic study of HCPT-NPs in rats
Six experiment groups including HCPT sodium injection, HCPT-SSLN and 4 kinds of HCPT-PEG-PCL-NPs formulations with the dosage of 5mg/kg were administrated to 54 Wistar rats (n=9) via the tail vein injection of a single dose. At predetermined time intervals, blood was taken and HCPT in plasma was extracted with ethyl acetate, and then measured by HPLC. The method for determination of lactone form of HCPT quantity by HPLC analysis in plasma was established, the plasma-drug concentration-time curve was drawn and the pharmacokinetic parameters were calculated by PKSolver 2.0 software. The long-circulating effects of different HCPT-NPs were evaluated and compared on order to choose the best long-circulating formulation and carrier material for HCPT.
Results
1 Preparation and characterization of HCPT-SSLN
It was showed that all the factors in single-factor experiment could influence the particle size and entrapment efficiency of HCPT-SSLN. Among them, only "concentration of surfactants" had significant effect on the entrapment efficiency of HCPT-SSLN. The optimal preparation parameters were as follows:ratio of HCPT/ lipids was 1:20, molar ratio of solid/liquid lipids was 2:1, concentration of surfactants was 5%, the velocity of oil phase into aqueous phase was 2.0mL/min, stirring temperature, time and velocity of emulsion evaporation was 75℃,2 h, 800 r/min, respectively. The average particle size, polydispersity, zeta potential, pH, entrapment efficiency and drug loading of the NPs was 130.5nm,0.19, 33.1mV,6.29,79.19%,2.51%, respectively. From the observation of transmission electron microscopy, the NPs exhibited a regular spherical shape. The regression equation of UV assay was A=0.0681C—0.004 (r=1.000) and the linear range was 0.2～10.0μg/mL. Furthermore, the lyophilized powder of HCPT-SSLN embodied good appearance and redissolving ability, in which 2.5% lactose added as cryoprotectant. After redissolving, the average particle size, polydispersity, zeta potential, pH and entrapment efficiency of the NPs was 138.9nm,0.25,—36.2mV, 6.29,75.47%, respectively. The stability study results showed that HCPT-SSLN lyophilized powder should be sealed and kept under dark at 4℃. In animal acute toxicity test, at the maximum tolerated dose of 214.6mg/kg, no poisonous effect and side effects were found..
2 Synthesis of multiblock PEG-PCL copolymer
The results of FT-IR、1H-NMR、GPC、DSC and XRD analyses showed that the multiblock PEG-PCL copolymer synthesized contained fragment PEG and PCL, in accordance with the target substance. The GPC polydispersities of the copolymers was less than 1.1, as there was a narrow molecular weight distribution. The result of cytotoxicity showed that the multiblock PEG-PCL copolymers had good biocompatibility and was in line with biological evaluation criteria.
3 Preparation and characterization of HCPT-PEG-PCL-NPs
It was showed that all the factors in single-factor experiment could influence the particle size, entrapment efficiency and drug loading of HCPT-PEG-PCL-NPs. Among them, "molar ratio of Span/PEG-PCL" and "dosage of HCPT" were major factors and had significant effect. The optimal preparation parameters were as follows:dosage of HCPT was 5mg, molar ratio of Span/PEG-PCL was 20:1, molar ratio of Span/Poloxamer was 3:1,40mL water, stirring velocity was 800r/min, the velocity of organic phase into aqueous phase was 2.0mL/min, hydration time was 2 h, the best carrier material was PEG4000-PCL2000. When using PEG4000-PCL2000, PEG4000-PCL1250, PEG2000-PCL2000, PEG2000-PCL1250 as the carrier material to prepare NPs, the average particle size of NPs in turn were 116.1,110.0,119.9, 99.1nm; the zeta potential were—22.4,—16.9,—33.5,—28.8 mV; the entrapment efficiency were 88.29%,.83.10%,80.67%,77.46%; and the drug loading were 2.96%,2.56%,2.31%,2.14%, respectively. From the observation of transmission electron microscopy,4 kinds of NPs exhibited a regular spherical shape. The regression equation of HPLC assay was A=78.215C—0.498 (r=1.000) and the linear range was 0.05～5.0μg/mL. With different molecular weight of hydrophilic fragment PEG and hydrophobic fragment PCL, the HCPT-PEG-PCL-NPs showed different zeta potential, the higher Mw of PEG, the higher zeta potential was; but the effect of PCL on the zeta potential was on the contrary. Furthermore, the lyophilized powder of HCPT-PEG-PCL-NPs embodied good appearance and redissolving ability, in which 4% trehalose and 4% sucrose used as cryoprotectants. After redissolving, the average particle size, polydispersity, zeta potential and entrapment efficiency of HCPT-PEG4000-PCL2000-NPs was 124.5nm,0.15,—25.2mV,85.24%, respectively. The stability study results showed that HCPT-PEG-PCL-NPs lyophilized powder should be sealed and kept under dark at room temperature. The XRD analysis results indicated that the existence of HCPT in HCPT-PEG-PCL-NPs was nano-amorphous state.
4 In vitro drug release study of HCPT-NPs
It was showed that the aqueous solution of HCPT (the control) reached the maxi-mum releaseas the cumulative release percentage was about 80% only in 24h. Compared with it, HCPT-NPs all showed a certain degree of sustained-release characteristics. Moreover, the most significant sustained-release characteristics was from HCPT-SSLN, but its cumulative release percentage tended to be saturation and less than 60% in 96h. With different molecular weight of hydrophilic fragment PEG and hydrophobic fragment PCL, the HCPT-PEG-PCL-NPs showed different release rate and cumulative release percentage, the higher Mw of PEG, the higher the release rate and cumulative release percentage were; but the effect of PCL on the release rate and cumulative release percentage was on the contrary. The release mechanisms of HCPT-NPs were fitted to Weibull modle, and it showed that the drug release process included passive diffusion and matrix-eroded procedure.
5 In vivo pharmacokinetic study of HCPT-NPs in rats
It was showed that there were significant difference among in vivo pharmacokinetic charac-teristics in rats of different HCPT-NPs (P<0.01). The in vivo metabolism of HCPT sodium injection was so fast that its in vivo half-life (t1/2) was only 0.1418h. However, the t,/2 of HCPT-PEG4000-PCL1250-NPs, HCPT-PEG2000-PCL1250-NPs, HCPT-PEG4000-PCL2000-NPs, HCPT-PEG2000-PCL2000-NPs and HCPT-SSLN were 18.07,9.08,5.25,5.14,4.28 times in turn as great as that of the control, which showed all HCPT-NPs prepared had a certain degree of long-circulating effect. The turn of all HCPT-NPs prepared which had stronger long-circulating effect was HCPT-SSLN≌HCPT-PEG2000-PCL2000-NPs≌HCPT-PEG2000-PCL1250-NPs (P>0.05) Conclusions
All HCPT-NPs exhibited a regular spherical shape and narrow distribution, which also had high entrapment efficiency and drug loading. Compared with the original HCPT, HCPT-NPs can enhance HCPT solubility in aqueous solution. The lyophilized powder of HCPT-NPs embodied good appearance and redissolving ability. On animal acute toxicity test, no poisonous result or other side-effects were found after Kunming mice administrated at the maximum tolerated dose of 214.6mg/kg via the tail vein; the same the cytotoxicity study showed that PEG-PCL copolymers had good biocompatibility. In vitro drug release studies showed that all HCPT-NPs embodied sustained release characteristics, the drug-release behavior was conformed to Weibull equation; its release mechanism may be cooperated by passive diffusion and matrix-eroded procedure. In vivo pharmacokinetic studies in rats showed all HCPT-NPs had a certain degree of long-circulating effect, but the effect were different depending on different carrier materials as the best long-circulating formulation of HCPT was HCPT-PEG4000-PCL2000-NPs, therefore, it is feasible to use amphiphilic block copolymer as carrier materials to prepare HCPT long-circulating formulations.
随着上世纪70年代,Birrenbach等首次提出纳米粒的概念和制备方法,药物制剂的研究进入了一个全新的微观领域----纳米制剂。与常规药物相比,纳米药物具有比表面积大、表面反应活性高、活性中心多、吸附能力强等特性,还可以改变药物在体内的分布,增加药物在靶器官的分布量,从而提高疗效。但普通纳米制剂静注后,血浆中的多种成分(如载脂蛋白、补体C蛋白等)会吸附于纳米粒的表面,使其易于被吞噬细胞识别,即机体的调理过程(opsonization),被单核巨噬细胞吞噬系统(mononuclear phagocyte system,MPS)吞噬并迅速从血液中清除。因而如何避免MPS对纳米粒的识别和吞噬是其能否被传输至其他器官或组织的关键。研究发现纳米粒的表面特性如亲脂性强弱、表面电位值等与调理素蛋白在其表面的吸附息息相关。一般来说纳米粒的表面亲脂性越强、表面电位值越低,调理素蛋白越容易吸附。由此可见纳米粒的表面特性对制备长循环纳米粒极为重要。目前针对纳米粒表面改性的研究主要集中在两个方面：(1)在纳米粒的表面包被亲水性的聚合物或表面活性剂；(2)制备含亲水性片断的两亲性高分子载体材料,这些材料含有PEG、PEO、poloxamer、poloxamine、Polysorbate-80及Briji-35等。其共同点是能在纳米粒亲脂性内核的表面形成水化保护层(protective clouds)并具备一定的立体位阻效应,能抑制调理素蛋白在纳米粒表面的吸附,即所制备的纳米粒具有“隐形”(stealths)特性,从而使得纳米粒不易被MPS识别,达到长循环目的。
包被有poloxamer等表面活性剂的纳米粒虽具备长循环的特质,但在体内微环境中,有学者认为血液中物质和包衣层物质间范德华力的存在容易引起包被层在体内循环中脱落,脱落的外源性物质作为一种外来性抗原对人体可能产生的潜在毒副作用值得关注并仍存在争议；在两亲性高分子载体合成方面,目前文献报道的共聚物几乎均为AB型或者ABA型,其中包含有亲水性的片段如PEG、PEO等,具体的载体材料有PEG-PLGA、PEG-PLA及PEG-PHDCA等。其制备多是将PEG或单甲基取代的PEG分子、PEO等与乳酸分子单体或氰基烷酸酯等单体聚合反应而成,其反应条件要求苛刻如要求惰性气体保护、反应管需融封、添加引发剂等。
本课题直接选用适宜分子量的PEG和PCL为原料,琥珀酸酐为桥联剂,制备含不同重复单元数量的PEG和PCL的两亲性共聚物载体材料。其中PCL为半结晶状的高分子材料,选用它作为两亲性共聚物载体的亲脂性部分,既可以作为亲脂性药物的储库,其所含有的晶状结构又有利于保持纳米粒形态的稳定。PCL应用历史悠久,如将其作为长效避孕药体内埋植剂的载体材料、组织工程材料等；PEG安全无毒,已被FDA批准应用于临床。本课题所选用的制备方法具有反应条件温和、简便易行和反应收率高等优点。
羟基喜树碱(10-hydroxycamptothecin,HCPT)是从珙桐科植物喜树中提取的一种微量生物碱,是拓扑异构酶Ⅰ的特异性抑制剂,广泛应用于肝癌、胃癌、白血病等多种恶性肿瘤的治疗。但其特殊的理化性质：水不溶脂难溶、内酯环结构不稳定等因素,限制了临床应用,普遍存在疗效降低、半衰期短的缺点。本研究以HCPT为模型药物,以两亲性的PEG-PCL多嵌段共聚物为载体材料,制备长循环纳米粒(long-circulating nanoparticles),同时以脂质材料包裹的羟基喜树碱半固体脂质纳米粒制剂为对照组,通过体外释放和大鼠体内药代动力学试验比较二者的长循环能力之间的差异,旨在寻求一种新型的长循环制剂载体材料和制备HCPT长循环纳米制剂。
材料与方法
1 HCPT-SSLN的制备与表征
选择固态和液态脂质为膜材,大豆磷脂为表面活性剂,采用乳化蒸发-低温固化法制备了羟基喜树碱半固体脂质纳米粒(HCPT-SSLN).以纳米粒的外观、粒径、包封率为指标,分别考察了表面活性剂种类、固态脂质／液态脂质摩尔比、药脂比、表面活性剂浓度、乳化蒸发搅拌时间、搅拌速度等因素对制备的影响；在此基础上,选定药脂比、固态脂质与液态脂质的摩尔比、表面活性剂浓度为3个因素,进行3因素3水平正交设计优选处方。用Malvern激光粒度仪测定HCPT-SSLN的表面电位、粒径及粒径分布；透射电镜观察HCPT-SSLN的形态；建立了414nm处紫外分光光度法测定开环形式HCPT含量的方法,用葡聚糖凝胶柱层析-UV法测定HCPT-SSLN的包封率和载药量。探讨了不同冷冻保护剂对冻干HCPT-SSLN的影响,并对冻干制剂进行了稳定性考察。以HCPT钠盐注射液作对比,选用昆明种小白鼠考察所制备的HCPT-SSLN的急性毒性。
2多嵌段PEG-PCL共聚物的合成研究
选用适宜分子量的PEG和PCL为起始原料,琥珀酸酐为桥联剂,合成多嵌段的PEG-PCL两亲性共聚物载体,并用FT-IR、1H-NMR、GPC、DSC、XRD分析对其进行表征；选用L969细胞株,以MTT和乳酸脱氢酶(LDH)活性指标考察所合成共聚物的生物相容性。
3 HCPT-PEG-PCL-NPs的制备与表征
以共聚物为载体材料,Span60和Poloxamer 188为乳化剂和稳定剂,用共溶剂挥发法制备了羟基喜树碱两亲性嵌段共聚物纳米粒(HCPT-PEG-PCL-NPs)。以纳米粒的外观形态、粒径、包封率、载药量为指标,考察了Span/PEG-PCL的摩尔比、投药量、Span/Poloxamer的摩尔比、搅拌速率、搅拌温度、水相用量、水化时间等因素对制备的影响；在此基础上,选定PEG-PCL种类、投药量、Span/Poloxamer的摩尔比、Span/PEG-PCL的摩尔比为4个因素,进行4因素3水平正交设计优化工艺。用Malvern激光粒度仪测定HCPT-SSLN的表面电位、粒径及粒径分布；透射电镜观察HCPT-SSLN的形态；建立了384nm处内酯形式存在的HCPT体外HPLC分析方法,用超滤离心-HPLC法测定HCPT-PEG-PCL-NPs的包封率和载药量。探讨了不同冷冻保护剂对冻干HCPT-PEG-PCL-NPs的影响,并对冻干制剂进行了XRD分析和初步的稳定性考察。
4 HCPT-NPs的体外释放研究
采用动态透析法,以pH7.4的磷酸盐缓冲溶液为释药介质,HCPT原料药的水溶液为对照组,37℃恒温避光恒速(100r／min)搅拌下进行HCPT-SSLN和HCPT-PEG-PCL-NPs的体外释药实验。在设定的时间点上取样测定其中的HCPT含量,计算累计释药量；采用不同的数学模型对不同纳米制剂的体外释药曲线进行拟合,探讨其可能的释药机制。
5 HCPT-NPs大鼠体内药代动力学研究
设定HCPT普通水针剂、HCPT-SSLN及4种两亲性共聚物载体材料制备的HCPT-PEG-PCL-NPs共6组；以54只Wistar大鼠为实验动物(n=9),给药剂量均为5mg／kg,经尾静脉单剂量注射给药后在设定的时间点上取血并测定HCPT的血药浓度；建立了测定血浆中HCPT含量的HPLC方法,并绘制血药浓度-时间曲线,计算药代动力学参数,对不同的HCPT-NPs的长循环作用进行评价和比较,优选最佳HCPT的长循环制剂及载体。
结果
1 HCPT-SSLN的制备与表征
结果表明,单因素实验中考察的各因素对HCPT-SSLN的粒径、包封率均有影响,其中表面活性剂浓度对SSLN的包封率影响最大且具显著性意义。优选的处方及工艺条件为：药脂比为1：20,固态／液态脂质摩尔比为2：1,表面活性剂浓度为5%,油相注入水相的滴速为2.0mL／min,乳化蒸发搅拌温度选取75℃,搅拌时间为2h,搅拌速度为800r／min。所制备的HCPT-SSLN的平均粒径为130.5nm,多分散度为0.19；平均毛电位为—33.1mV,平均pH为6.29,平均包封率为79.19%,平均载药量为2.51%；透射电镜结果表明,纳米粒为圆整的球状体,无明显团聚现象。UV法测定开环HCPT含量的线性范围为0.2～10.0μg/mL,回归方程为A=0.0681C—0.004,r=1.000。冻干实验结果表明：添加2.5%的乳糖为冻干保护剂,所制备的冻干纳米制剂外观为白色,冻干前后体积不变、无裂缝、脱壁等现象；有较好的复水性,复水后的纳米粒径为138.9nm,多分散系数为0.25,平均ξ电位为—36.2mV,平均pH为6.29,平均包封率为75.47%；稳定性考察结果表明HCPT-SSLN冻干品宜密封避光于4℃下保存。急性毒性实验结果表明,在最大耐受剂量214.6mg/kg(相当于人临床应用剂量17.89mg/kg)注射情况下,小鼠仍能保持良好状态,未出现急性毒性反应。
2多嵌段PEG-PCL共聚物的合成研究
所制备的PEG-PCL两亲性共聚物载体经FT-IR、1H-NMR、GPC、DSC、XRD等分析证实含有PEG和PCL片段,符合目标物质,且GPC的多分散指数均小于1.1,分子量分布较窄。细胞毒性实验表明所合成的载体基本无毒,有着较佳的生物相容性,毒性反应为1级,符合生物学评价标准。
3 HCPT-PEG-PCL-NPs的制备与表征
结果表明,单因素实验中考察的各因素对HCPT-PEG-PCL-NPs的粒径、包封率、载药量均有影响,其中投药量和Span/PEG-PCL的摩尔比为主要影响因素,具统计学意义。优选的处方及工艺条件为：投药量为5mg, Span/PEG-PCL的摩尔比为20：1,Span/Poloxamer的摩尔比为3：1,水相用量40mL,搅拌速度800r／min,有机相滴加至水相速度为2.0mL/min,水化时间2 h,最佳PEG-PCL载体为PEG4000-PCL2000。以共聚物PEG4000-PCL2000、PEG4000-PCL1250、PEG2000-PCL2000、PEG2000-PCL1250为载体制备的4种HCPT-PEG-PCL-NPs的平均粒径依次为116.1、110.0、119.9、99.1nm,平均毛电位依次为—22.4、—16.9、—33.5、—28.8 mV,平均包封率依次为88.29%、83.10%、80.67%、77.46%,平均载药量依次为2.96%、2.56%、2.31%、2.14%；透射电镜结果表明,4种纳米粒为圆形,粒径大小比较均一。384nm处内酯形式存在的HCPT体外HPLC分析方法,线性范围为0.05～5.0μg/mL,回归方程为A=78.215C—0.498,r=1.000。共聚物的表面电位值说明随着共聚物中PEG链段的增长而升高,随着亲脂性组分PCL链段的增长而降低。冻干实验结果表明：添加4%的海藻糖和4%的蔗糖为冻干保护剂,所制备的冻干纳米制剂外观为白色,冻干前后体积不变、无裂缝、脱壁等现象；有较好的复水性,以PEG4000-PCL2000为载体材料制备NPs冻干品,复水后的纳米粒径为124.5nm,多分散系数为0.15,平均毛电位为—25.2mV,平均包封率为85.24%；稳定性考察结果表明HCPT-PEG-PCL-NPs冻干品宜密封避光于常温下保存。XRD分析结果说明HCPT在所制备的纳米制剂中是以无定型的状态存在的。4 HCPT-NPs的体外释放研究
实验结果表明,HCPT原料药水溶液组24h即达最大释放量约80%,与它相比,HCPT-NPs均具有一定的缓释作用,其中以HCPT-SSLN最明显,但96h释放量趋于饱和,累积释药百分数不超过60%。以共聚物为载体制备的HCPT-NPs中,随着分子中PEG片段的增长,对应的NPs释药速率与累积释药百分数增高；随着分子中PCL片段的增长,其相应NPs的释药速率与累积释药百分数降低。所制备的HCPT-PEG-PCL-NPs体外释药均符合Weibull分布模型,其释药机理应是药物的被动扩散与载体基质的溶蚀协同作用。
5 HCPT-NPs大鼠体内药代动力学研究
结果表明,不同制剂在大鼠体内的药代动力学特征有显著不同(P<0.01),HCPT水针剂注射后体内代谢极快,其体内半衰期tl／2仅0.1418h,以共聚物载体PEG4000-PCL1250、PEG4000-PCL2000、PEG2000-PCL1250、PEG2000-PCL2000制备的4种HCPT-PEG-PCL-NPs与HCPT-SSLN的t1／2依次为它的18.07、9.08、5.25、5.14、4.28倍；表明HCPT-NPs均具备一定的长循环效果,尤以PEG4000-PCL1250为最佳(P<0.01),其释药速度快,释药量大且作用持久；其次为PEG4000-PCL2000 (P< 0.01),而PEG2000-PCL1250、PEG2000-PCL2000的长循环效应较差,半衰期与MRT值均无显著性差异(P>0.05)。
结论
本研究所制备的HCPT-NPs均能有效增进HCPT在制剂中的溶解度,粒径、形态、包封率与载药量均达到制剂要求,冻干后复水性较好,动物急性毒性实验安全。合成的多嵌段PEG-PCL共聚物经分析符合目标物质,有着良好的生物相容性。体外释药实验中HCPT-NPs均具有一定的缓释作用且符合Weibull分布模型,其释药机理应是药物的被动扩散与载体基质的溶蚀协同作用。大鼠药代动力学实验表明HCPT-NPs均具备一定的长循环作用,但因载体材料不同效果不同,尤以HCPT-PEG4000-PCL1250-NPs为最佳,采用两亲性嵌段共聚物材料为载体制备HCPT长循环制剂的思路是可行的。
Background & objective
Since 1970s, Birrenbach introduced the concept and preparative method of nanoparticle firstly, the study of the pharmaceutical preparation has entered into a new micro realm——nanometric praeparatum. Compared to routine drugs, nanometric praeparatums have many good characters such as large specific surface area, high surface reaction activity, plenty of active centers, strong adsorbability and so on. They can also change the intracorporal distribution of the medicine to increase the dose distribution in target organs and raise therapeutic effects. However, after the intravenous injection of general nano-medicament, a variety of components (such as apolipoprotein, complement protein C, etc) in plasma will adsorb on the surface of nanoparticles to make it be easily identified by the phagocytic cells. This is just the process of body opsonization, the nanoparticles can be phagocytosed by mononuclear phagocyte system and rapidly cleared from the blood. Therefore, how to avoid MPS identifying and phagocytosing the nanoparticles is the key for transmiting them to other organs or tissues. It is found that nanoparticles'surface properties (such as lipophilic strength, surface potential value, etc.) are closely related to the adsorption of opsonin proteins on their surface. Generally speaking, the stronger the surface lipophilicity is, the lower the value of surface potential is, the easier that opsonin proteins absorb nanoparticles. This shows that the surface characteristics of nanoparticles are extremely important for preparing long-circulating nanoparticles. Currently, the study of nanoparticles for surface modification mainly focuses on two aspects:(1)To coat the surface of nanoparticles with hydrophilic polymers or surfactants; (2)To prepare amphiphilic polymer carrier materials containing hydrophilic fragments which contain PEG, PEO, Poloxamer, Poloxamine, Polysorbate-80 and Briji-35,etc. They can all form hydration protective clouds on the surface of nanoparticles' lipophilic cores and have certain three-dimensional steric effects, which can inhibit opsonin protein absorbing on the nanoparticles surface. Therefore, the nanoparticles have "stealths" feature for being not easily identified by MPS to achieve the purpose of long-circulating.
Although the nanoparticles coated with surfactants (such as poloxamer, etc.) have long-circulating feature, in vivo micro-environment, some scholars believe that the vander wale force between blood materials and coating materials may easily lead the coated layer to fall off in the vivo circulation. As a foreign antigen, the deciduous exogenous substances may have the potential toxic effects on the human body and it is still in disputes. In the synthesis of amphiphilic polymer carrier, the copolymers reported are almost only the AB-type or ABA-type currently, which contain hydrophilic segments (such as PEG, PEO, etc.) and carrier materials (such as PEG-PLGA, PEG-PLA, PEG-PHDCA, etc.). The copolymers are mostly made through the polymerization reaction between PEG (or Monomethyl-substituted PEG molecule, PEO) and lactic acid monomers (or cyano-alkyl ester monomer, etc.). The reaction conditions are strict, such as requiring inert gas protection, sealing reaction tube, adding initiators, etc.
This study chose poly (ethylene glycerol) (PEG) and poly(s-caprolactone) (PCL) of appropriate molecular weight as raw materials, succinic anhydride for the bridging agent, synthesized the amphiphilic copolymer carrier materials with different numbers of repeating units of PEG and PCL. PCL is semi-crystalline polymer material, as the lipophilic part of amphiphilic copolymer carriers, it can not only store the lipophilic drug, but also be beneficial to the stability of nanoparticles' morphology by its crystalline structure. The applications of PCL has a long history, such as the carrier material of long-acting contraceptive implant in vivo, tissue engineering materials, etc.; PEG is safe and non-toxic, and has been approved by FDA for clinical application. The synthetic reaction in this study is mild, simple and the reaction yield is high.
10-hydroxycamptothecin (HCPT) is a kind of micro amount alkaloids extracted from Camptotheca acuminate Decaisne, and is one of the specific DNA topoisomerase I inhibitors. It is widely used to cure liver cancer, stomach cancer, leukemia and many malignant tumors. However, its clinical applications are confined by its special physico-chemical properties (for example, it is insolubilize in water and difficultly dissolves in lipids, the lactone band is also instable, etc.), so its curative effects decrease and the intracorporal half life is too short. This study choose it as model drug, and the amphiphilic PEG-PCL multiblock copolymers as the carrier material, to prepare long-circulating nanoparticles. Moreover, HCPT-SSLN (HCPT semisolid lipid nanoparticles) coated with solid and liquid lipids acted as the control group, their long-circulating abilities were compared by in vitro drug release experiment and in vivo pharmacokinetics tests. The goal of this study is to find a novel carrier for long-circulation and prepare long-circulating HCPT-NPs formulation.
Materials and methods
1 Preparation and characterization of HCPT-SSLN
HCPT-SSLN was prepared by the method of "emulsion evaporation at a high temperature and solidification at a low temperature", using solid & liquid lipids as carrier materials, soya lecithin as surfactant. According to the morphology, particle size and entrapment efficiency, the affected factors in the preparing technology, including "types of surfactants", "molar ratio of solid/liquid lipids", "ratio of HCPT/lipids", "concentration of surfactants", "stirring time", "stirring velocity", etc. were investigated. On this basis, "ratio of HCPT/lipids", "molar ratio of solid/ liquid lipids" and "concentration of surfactants" were selected as major factors to make L9 (34) orthogonal design for optimization of formulation and technology. The zeta potential, particle size and size distribution (polydispersion) of HCPT-SSLN were determined on a Malvern Zetasizer HSA 3000; the morphology was observed by transmission electron microscopy. In order to control the quality of HCPT-SSLN, the methods for determination of open-loop form of HCPT quantity by UV spectrophotometry at 414nm and for determination of the entrapment efficiency and drug loading of HCPT-SSLN by sephadex gel filtration chromatography-UV were established. Different cryoprotectants were investigated to prepare the lyophilized HCPT-SSLN powder, and then the stability of the NPs powder was studied. By the comparison of HCPT sodium injection, the animal acute toxicity of HCPT-SSLN was conducted using kunming mice by tail intravenous administration.
2 Synthesis of multiblock PEG-PCL copolymer
The multiblock polyethylene-polycaprolactone copolymer was synthesized, which PEG and PCL were used as starting meterials, succinic anhydride acted as cross-linking agent, DCC and DMAP as condensation agent and catalyst, respectively. Then FT-IR、1H-NMR、GPC、DSC、XRD analyses were used to characterize the copolymers. The cytotoxicity of the copolymers was evaluated with L929 cell line through MTT and LDH methods.
3 Preparation and characterization of HCPT-PEG-PCL-NPs
HCPT-PEG-PCL-NPs were prepared by co-solvent evaporation method, which copolymer was the carrier material, Span60 and Poloxamer 188 was the emulsifier and stabilizer. According to the morphology, particle size, entrapment efficiency and drug loading, the affected factors in the preparing technology, including "molar ratio of Span/PEG-PCL", "dosage of HCPT", "molar ratio of Span/Poloxamer", "stirring velocity", "stirring temperature", "volume of water phase", "hydration time", etc. were investigated. On this basis, "types of PEG-PCL", "dosage of HCPT", "molar ratio of Span/Poloxamer" and "molar ratio of Span/PEG-PCL" were selected as major factors to make L18(34) orthogonal design for optimization of formulation and technology. The zeta potential, particle size and size distribution (polydispersion) of HCPT-PEG-PCL-NPs were determined on a Malvern Zetasizer HSA 3000; the morphology was observed by transmission electron microscopy. In order to control the quality of HCPT-PEG-PCL-NPs, the methods for determination of lactone form of HCPT quantity by HPLC analysis in vitro at 384nm and for determination of the entrapment efficiency and drug loading of HCPT-PEG-PCL-NPs by centrifugal ultrafiltration—HPLC were established. Different cryoprotectants were investigated to prepare the lyophilized HCPT-PEG-PCL-NPs powder, and then the stability and XRD analysis of the NPs powder were studied.
4 In vitro drug release study of HCPT-NPs
In vitro drug release studies were carried out as follows. Drug-loaded nanoparticles suspensions and aqueous solution of HCPT (the control) were placed in a dialysis membrane bag with molecular weight cut-off 3500g/mol, tied, and dropped into 250mL of a phosphate buffer solution media (pH=7.4). The entire system was kept dark and at 37℃with magnetic stirring at 100r/min. At predetermined time intervals,3 mL of aqueous solution was withdrawn from the release media. Meanwhile, an equal volume release medium was added. Then the drug content of the sample was assayed by HPLC. Furthermore, the drug release mechanisms of HCPT-NPs were studied with methematics modles including Weibull distribution model, Ritger-Peppas modle, etc.
5 In vivo pharmacokinetic study of HCPT-NPs in rats
Six experiment groups including HCPT sodium injection, HCPT-SSLN and 4 kinds of HCPT-PEG-PCL-NPs formulations with the dosage of 5mg/kg were administrated to 54 Wistar rats (n=9) via the tail vein injection of a single dose. At predetermined time intervals, blood was taken and HCPT in plasma was extracted with ethyl acetate, and then measured by HPLC. The method for determination of lactone form of HCPT quantity by HPLC analysis in plasma was established, the plasma-drug concentration-time curve was drawn and the pharmacokinetic parameters were calculated by PKSolver 2.0 software. The long-circulating effects of different HCPT-NPs were evaluated and compared on order to choose the best long-circulating formulation and carrier material for HCPT.
Results
1 Preparation and characterization of HCPT-SSLN
It was showed that all the factors in single-factor experiment could influence the particle size and entrapment efficiency of HCPT-SSLN. Among them, only "concentration of surfactants" had significant effect on the entrapment efficiency of HCPT-SSLN. The optimal preparation parameters were as follows:ratio of HCPT/ lipids was 1:20, molar ratio of solid/liquid lipids was 2:1, concentration of surfactants was 5%, the velocity of oil phase into aqueous phase was 2.0mL/min, stirring temperature, time and velocity of emulsion evaporation was 75℃,2 h, 800 r/min, respectively. The average particle size, polydispersity, zeta potential, pH, entrapment efficiency and drug loading of the NPs was 130.5nm,0.19, 33.1mV,6.29,79.19%,2.51%, respectively. From the observation of transmission electron microscopy, the NPs exhibited a regular spherical shape. The regression equation of UV assay was A=0.0681C—0.004 (r=1.000) and the linear range was 0.2～10.0μg/mL. Furthermore, the lyophilized powder of HCPT-SSLN embodied good appearance and redissolving ability, in which 2.5% lactose added as cryoprotectant. After redissolving, the average particle size, polydispersity, zeta potential, pH and entrapment efficiency of the NPs was 138.9nm,0.25,—36.2mV, 6.29,75.47%, respectively. The stability study results showed that HCPT-SSLN lyophilized powder should be sealed and kept under dark at 4℃. In animal acute toxicity test, at the maximum tolerated dose of 214.6mg/kg, no poisonous effect and side effects were found..
2 Synthesis of multiblock PEG-PCL copolymer
The results of FT-IR、1H-NMR、GPC、DSC and XRD analyses showed that the multiblock PEG-PCL copolymer synthesized contained fragment PEG and PCL, in accordance with the target substance. The GPC polydispersities of the copolymers was less than 1.1, as there was a narrow molecular weight distribution. The result of cytotoxicity showed that the multiblock PEG-PCL copolymers had good biocompatibility and was in line with biological evaluation criteria.
3 Preparation and characterization of HCPT-PEG-PCL-NPs
It was showed that all the factors in single-factor experiment could influence the particle size, entrapment efficiency and drug loading of HCPT-PEG-PCL-NPs. Among them, "molar ratio of Span/PEG-PCL" and "dosage of HCPT" were major factors and had significant effect. The optimal preparation parameters were as follows:dosage of HCPT was 5mg, molar ratio of Span/PEG-PCL was 20:1, molar ratio of Span/Poloxamer was 3:1,40mL water, stirring velocity was 800r/min, the velocity of organic phase into aqueous phase was 2.0mL/min, hydration time was 2 h, the best carrier material was PEG4000-PCL2000. When using PEG4000-PCL2000, PEG4000-PCL1250, PEG2000-PCL2000, PEG2000-PCL1250 as the carrier material to prepare NPs, the average particle size of NPs in turn were 116.1,110.0,119.9, 99.1nm; the zeta potential were—22.4,—16.9,—33.5,—28.8 mV; the entrapment efficiency were 88.29%,.83.10%,80.67%,77.46%; and the drug loading were 2.96%,2.56%,2.31%,2.14%, respectively. From the observation of transmission electron microscopy,4 kinds of NPs exhibited a regular spherical shape. The regression equation of HPLC assay was A=78.215C—0.498 (r=1.000) and the linear range was 0.05～5.0μg/mL. With different molecular weight of hydrophilic fragment PEG and hydrophobic fragment PCL, the HCPT-PEG-PCL-NPs showed different zeta potential, the higher Mw of PEG, the higher zeta potential was; but the effect of PCL on the zeta potential was on the contrary. Furthermore, the lyophilized powder of HCPT-PEG-PCL-NPs embodied good appearance and redissolving ability, in which 4% trehalose and 4% sucrose used as cryoprotectants. After redissolving, the average particle size, polydispersity, zeta potential and entrapment efficiency of HCPT-PEG4000-PCL2000-NPs was 124.5nm,0.15,—25.2mV,85.24%, respectively. The stability study results showed that HCPT-PEG-PCL-NPs lyophilized powder should be sealed and kept under dark at room temperature. The XRD analysis results indicated that the existence of HCPT in HCPT-PEG-PCL-NPs was nano-amorphous state.
4 In vitro drug release study of HCPT-NPs
It was showed that the aqueous solution of HCPT (the control) reached the maxi-mum releaseas the cumulative release percentage was about 80% only in 24h. Compared with it, HCPT-NPs all showed a certain degree of sustained-release characteristics. Moreover, the most significant sustained-release characteristics was from HCPT-SSLN, but its cumulative release percentage tended to be saturation and less than 60% in 96h. With different molecular weight of hydrophilic fragment PEG and hydrophobic fragment PCL, the HCPT-PEG-PCL-NPs showed different release rate and cumulative release percentage, the higher Mw of PEG, the higher the release rate and cumulative release percentage were; but the effect of PCL on the release rate and cumulative release percentage was on the contrary. The release mechanisms of HCPT-NPs were fitted to Weibull modle, and it showed that the drug release process included passive diffusion and matrix-eroded procedure.
5 In vivo pharmacokinetic study of HCPT-NPs in rats
It was showed that there were significant difference among in vivo pharmacokinetic charac-teristics in rats of different HCPT-NPs (P<0.01). The in vivo metabolism of HCPT sodium injection was so fast that its in vivo half-life (t1/2) was only 0.1418h. However, the t,/2 of HCPT-PEG4000-PCL1250-NPs, HCPT-PEG2000-PCL1250-NPs, HCPT-PEG4000-PCL2000-NPs, HCPT-PEG2000-PCL2000-NPs and HCPT-SSLN were 18.07,9.08,5.25,5.14,4.28 times in turn as great as that of the control, which showed all HCPT-NPs prepared had a certain degree of long-circulating effect. The turn of all HCPT-NPs prepared which had stronger long-circulating effect was HCPT-SSLN≌HCPT-PEG2000-PCL2000-NPs≌HCPT-PEG2000-PCL1250-NPs (P>0.05)
All HCPT-NPs exhibited a regular spherical shape and narrow distribution, which also had high entrapment efficiency and drug loading. Compared with the original HCPT, HCPT-NPs can enhance HCPT solubility in aqueous solution. The lyophilized powder of HCPT-NPs embodied good appearance and redissolving ability. On animal acute toxicity test, no poisonous result or other side-effects were found after Kunming mice administrated at the maximum tolerated dose of 214.6mg/kg via the tail vein; the same the cytotoxicity study showed that PEG-PCL copolymers had good biocompatibility. In vitro drug release studies showed that all HCPT-NPs embodied sustained release characteristics, the drug-release behavior was conformed to Weibull equation; its release mechanism may be cooperated by passive diffusion and matrix-eroded procedure. In vivo pharmacokinetic studies in rats showed all HCPT-NPs had a certain degree of long-circulating effect, but the effect were different depending on different carrier materials as the best long-circulating formulation of HCPT was HCPT-PEG4000-PCL2000-NPs, therefore, it is feasible to use amphiphilic block copolymer as carrier materials to prepare HCPT long-circulating formulations.
引文
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[47]易以木,陈秀珍.靶向性羟基喜树碱豆磷脂纳米冻干粉针制剂及制备方法、用途[P].CN：200410013143.2,2004-05-13.
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[55]GBT16886.5-2003,医疗器械生物学评价[S]-第5部分：细胞毒性试验：体外法.
[56]张忠涛,王宇.白细胞介素21β对原代培养大鼠肝细胞的细胞毒性作用[J].中华普通外科杂志,2002,17(2)：90.
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[58]Harada A, Togawa H, Kataoka K. Physicochemical properties and nuclease resistance of antisense-oligodeoxynucleotides entrapped in the core of polyion complexmicelles composed of poly (ethylene glycol)-poly (L-lysine) block copolymers [J]. Eur. J. Pharm a. Sci.,2001,13 (1):35-42
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[41]董生,肖湘生,郝楠馨.羟基喜树碱明胶微球肝动脉化疗栓塞治疗原发性肝癌[J].中国医学计算机成像杂志,1999,5(1)：55-58.
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[43]龚明涛,张钧寿,沈益.羟基喜树碱纳米乳的制备及其抗癌作用初步研究[J].中国天然药物,2005,3(1)：41-43.
[44]袁建华,熊俊.羟基喜树碱乳剂及其制备方法[P].CN：03152834.1,2004-05-05.
[45]易以木,陈秀珍.羟基喜树碱葡聚糖纳米粒的制备方法[P].CN：01138252.X,2002-08-07.
[46]易以木,杨唐玉,吴汉强,等.羟基喜树碱聚乳酸纳米粒的制备方法[P].CN：01133700.1,2002-08-14.
[47]易以木,陈秀珍.靶向性羟基喜树碱豆磷脂纳米冻干粉针制剂及制备方法、用途[P].CN：200410013143.2,2004-05-13.
[48]Muller RH, Mehnert W, Lucks JS, et al. Solid lipid nanoparticles(SLN)-analternative colloidal carrier system for controlled drug delivery[J]. Eur J Pharm Biopharm,1995,41(1):62-69.
[49]Muller RH, Radtke M, Wissing SA. Nanostructured lipid matrices for improved microencapsulation of drugs[J].Int J Pharm,2002,242(1/2):121-128.
[50]Siekmann B,Westesen K. Melt-homogenized solid lipid nanoparticles stabled by the nonionic surfactant tyloxapol preparation and particle size determination[J]. Pharm Pharmaco Lett,1994,3(3):194-197.
[51]IL Gyun Shin, So Yeon Kim, Young Moo Lee, et al. Methoxypoly (ethylene glycol)/ε-cap rolactone amphiphilic block copolymeric micelle containing indomethacin. I.Preparation and characterization [J]. J.Contr. Release,1998,51: 1-11.
[52]Seo Yeon Kim, IL Gyun Shin, Young Moo Lee, et al. Methoxypoly (ethylene glycol) and ε-cap rolactone amphiphilic block copolymeric micelle containing indomethacin. Ⅱ. Micelle formation and drug release behaviours [J]. J. Contr. Release,1998,51:13-22.
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[54]Ts.Petrova, N.Manolova, I.Rashkov, et al. Synthesis and characterization of poly(oxythylene)-Poly(caprolactone) multiblock copolymers[J].Polymer International,1998,45:419-426.
[55]GBT16886.5-2003,医疗器械生物学评价[S]-第5部分：细胞毒性试验：体外法.
[56]张忠涛,王宇.白细胞介素21β对原代培养大鼠肝细胞的细胞毒性作用[J].中华普通外科杂志,2002,17(2)：90.
[57]Adams ML, Lavasanifar A, Kwon GS. Amphiphilic block copolymer for drug delivery [J]. J. Pharm a. Sci.,2003,92 (7):1343-1355.
[58]Harada A, Togawa H, Kataoka K. Physicochemical properties and nuclease resistance of antisense-oligodeoxynucleotides entrapped in the core of polyion complexmicelles composed of poly (ethylene glycol)-poly (L-lysine) block copolymers [J]. Eur. J. Pharm a. Sci.,2001,13 (1):35-42
[59]Kataoka K, Arada A, Nagasaki Y. Block copolymer micelles for drug delivery: design, characterization and biological significance[J]. Adv.Drug Deliv. Rev,2001,47:113.
[60]Burke TG, Staubus AE, Mishra AK. Liposomal stabilization of camptothecin's lactone ring[J]. J Am Chem Soc,1992,114:8318-8319.
[61]Thomas G, Avadesh K, Mansukh C, et al. Lipid bilayer partitioning and stability of Camptothecin drugs [J]. Biochemistry,1993,32(20):5352-5364.
[62]奕立标,朱家壁.喜树碱非离子表面活性剂载体组成对其形态和粒度分布的影响[J].中国药学杂志,2002,37(5)：357-359.
[63]Hong MS, Lim SJ, Lee MK, et al. Prolonged blood circulation of methotrexate by modulation of liposomal composition[J]. Drug Deliv,2001,8(4):231-237.
[64]Woodle MC. Surface-modified liposomes-assessment and characterization of increased stability and prolonged blood-circulation[J]. Chem Phys Lipids, 1993,64(1-3):249-262.
[65]Woodle MC, Matthay KK, Newman M. Versatility in lipid compositions showing prolonged circulation with sterically stabilized liposomes[J]. Biochem Biophys Acta,1992,1105(2):193-200.
[66]Muller H., Willis KM. Surface modification of i.v. injectable biodegradable nanoparticles with polozamer polymers and polozamine 908[J]. Int. J. Pharm.,1993,89:25.
[67]Kumaresh S.Soppimath, Tejraj M. Aminabhavi, Anandrao R.Kulkarni, et al. Bviodegradable polymeric nanoparticles as drug delivery devices[J]. Journal of controlled release,2001,70:1.
[68]程宇慧,廖工铁,侯世祥,等.去甲斑蝥酸钠白蛋白微球的研究[J].药学学报,1993,28(5)：384.
[69]Ammoury N, Fessi H, Devissaguet JP, et al. In vitro drug release kinetic pattern of indomethacin from poly(d,l-lactic) nanocapsules[J]. J Pharm Sci, 1990,79:7631.
[70]Muller RH, Ruhl D, Runge S.Biodegradation of solid lipid nanoparticles as a function of lipase incubation time[J]. Int J Pharm,1990,144:1151.
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