【作者】 张波；

【导师】 张娜； Sanjay Garg；

【作者基本信息】 山东大学 ， 药剂学， 2017， 博士

【摘要】 癌症是威胁人类健康的重要疾病之一,结直肠癌在全球发病率及死亡率均位列各种癌症疾病的第三位,并呈逐年上升趋势。由于结直肠癌起病隐匿,早期不易发现,超过一半的患者在确诊时即已发生转移,而转移性结直肠癌患者的5年生存率仅为10%。化学药物治疗是目前临床上治疗结直肠癌不可或缺的重要手段,尤其适用于无法接受手术切除或放射治疗等局部治疗手段的转移性结直肠癌患者。由于化疗药物单独使用时作用靶点单一,治疗效果有限,常引起多药耐药、肿瘤耐受及复发,不能满足临床治疗的要求。因此,目前临床上结直肠癌的一线化学治疗方案大部分均采用不同药物联合治疗方式,其中,奥沙利铂(OXA)与伊立替康(IRI)联用方案在美国国家癌症综合网络《结肠癌临床治疗指南》1中,推荐用于结直肠癌的一线治疗。OXA与IRI两种药物联用时,能够以不冋作用机制同时作用于细胞DNA,从而产生协同治疗效果。但由于奥沙利铂和盐酸伊立替康均存在一系列的缺点,如:低的药物耐受,非特异性,多药耐药,非期望的药代动力学和生物分布,在杀死肿瘤细胞时对正常组织也会造成严重损害,而二者的联合使用则会进一步加剧对机体的毒副作用,限制了其临床应用。另外,临床上的传统方法只是简单的将两种药物联合使用,无法控制药物在体内的释放,由于每种药物的药代动力学不同,而不同药物比例对疾病的治疗可能起到协同、相加甚至是拮抗作用,从而导致抗肿瘤效果的不确定性,影响治疗效果。因此,建立一种新型给药体系,提高结直肠癌治疗效果,减少两种药物联用的毒副作用,具有十分重要意义。药物递释系统为克服化疗药物所存在的弊端提供了一个很好的机遇,利用纳米载体实现药物递释及共递送的研究已成为抗肿瘤研究的热点。利用药物递释系统实现药物联合治疗一般有三种策略:单载纳米制剂加游离药物的"鸡尾酒策略"(free drug+nano);两种单载纳米制剂联用的"混合策略"(nano+nano);两种药物装载于同一纳米制剂的"共载策略"(co-encapsulation)。在体内递送过程中,由于鸡尾酒策略及混合策略中两种药物在某一部位的比例是任意的,而共载策略中两种药物在体内能够保持固定的比例,因此可保证两药发挥最大的治疗效果。基于脂质纳米载体(Lipid-based nanocarriers)是构建药物递送系统的重要载体材料,以脂质体和脂肪乳为典型代表,其安全性高,具有良好的生物相容性及生物可降解性,目前脂质体、脂肪乳已有多种药物批准上市,表现出巨大的应用前景。为提高结直肠癌治疗效果,并减轻化疗药物联用产生的毒副作用,本课题拟选择脂质体和脂肪乳两种具有较高临床转化前景的纳米载体,分别构建两种奥沙利铂和伊立替康共载的药物递释系统,以控制两种药物在体内释放,实现两种化疗药在肿瘤细胞内有效共递送,从而提高结直肠癌治疗效果。此外,进一步探讨在药物递释系统联合治疗中,混合策略及共载策略在抗肿瘤效果上的差异,分析其疗效差异原因,为药物联合治疗的递送给药设计提供理论依据。本课题主要研究内容及结果如下:第一部分奥沙利铂及盐酸伊立替康共载脂质体的制备、表征及体内外评价第一章分别采用HPLC及UV-Vis建立奥沙利铂和盐酸伊立替康的含量测定方法,两种药物在测定浓度下均呈现良好线性关系,且精密度、回收率均符合要求。采用透析法及葡聚糖凝胶G25两种手段分别建立奥沙利铂包封率测定方法,并采用葡聚糖凝胶G25建立盐酸伊立替康包封率测定方法,两种药物均可与脂质体实现分离,回收率符合要求。第二章通过Gaddum模型及CI指数法确定了奥沙利铂及盐酸伊立替康存在协同效应,且在OXA/IRI摩尔比在1:1～1:1.5时,两药具有最佳协同效应。选择乙醇注入法结合硫酸铵梯度法制备得到OXA及IRI共载脂质体,粒径小于200 nm,PDI小于0.2,呈圆整球形。该方法采用次序载药策略,能够精确控制两药比例。体外释放实验表明两种药物的释放行为包括初始的突释释放以及后续的持续释放两部分,且共载脂质体能够更好的控制两药比例在最佳协同范围。第三章体外细胞实验表明共载脂质体具有更强的细胞杀伤作用;以CT-26荷瘤小鼠为模型进行体内药效学实验,结果表明共载脂质体与混合溶液及混合脂质体相比,能够显著增强抗肿瘤效果(p<0.05)。对小鼠给予不同DiR处方进行近红外荧光成像扫描,表明脂质体能够增加药物在肿瘤部位的蓄积。以罗丹明B及FITC为荧光染料对脂质体进行标记,通过体外细胞摄取及体内荷瘤小鼠冰冻切片观察,对共载脂质体的共递送不同药物至同一细胞的能力进行评价,结果表明共载脂质体较混合制剂具有更高的共递送效率。荷瘤小鼠给予共载脂质体后体重变化明显高于混合溶液组,此外组织HE染色中混合溶液组可见心、肺、脾组织异常,均表明脂质体具有良好的安全性,能够降低药物的毒副作用。第二部分奥沙利铂及伊立替康共载脂肪乳的制备、表征及体内外评价第四章成功制备了制备奥沙利铂脂质复合物(OPPC)及伊立替康脂质复合物(IPPC),并分别确定了二者的优化工艺。所制备的OPPC及IPPC在7天内均能够保持较好的稳定性。通过DSC曲线对所制备的OPPC及IPPC进行表征,其吸热放热峰均与药物发生明显改变,证明两种复合物的成功制备。第五章采用药物脂质复合物技术成功制备了奥沙利铂及伊立替康共载的脂肪乳制剂,并确定其优化处方及工艺为:选择MCT为油相、处方比例5%;普郎尼克F68处方比例2%;甘油及油酸处方比例分别为2%、0.125%;乳化温度60℃;匀质压力1000 bar、循环次数15次。所制备的共载脂肪乳其粒径在100～120 nm之间,PDI小于0.2,zeta电位在-20 mV左右,且具有良好的稳定性。长期稳定性实验表明其于4℃条件下贮存,在三个月内粒径无明显变化,可保持稳定。体外释放实验表明脂肪乳能够控制药物的持续释放,至48 h时,两种药物在单载与共载脂肪乳的累积释放分别可达80%及70%以上,表现出整体相当的释放行为,表明脂肪乳可控制两种药物的同步释放并达到最佳协同比例。第六章体外细胞毒性实验表明空白脂肪乳表现出较好的安全性,共载脂肪乳具有较混合溶液及混合脂肪乳略强的细胞毒性,但无统计学差异。小鼠体内药效学实验表明,共载脂肪乳与较混合溶液及混合脂肪乳相比,表现出显著增强的抗肿瘤效果。此外,脂肪乳制剂能够降低OXA及IRI游离药物联用时的毒副作用。近红外荧光成像结果表明,脂肪乳可增加药物在肿瘤部位的蓄积,且能够延缓药物在肿瘤部位的滞留时间。以DiI及DiO对脂肪乳进行荧光标记进行体外细胞摄取及体内小鼠肿瘤冰冻切片评价,均表明共载脂肪乳能够以固定的比例同时共递送两种药物至同一细胞,表现出较高的共递送效率,从而保持二者的最佳协同治疗比例。更多还原

【Abstract】 Cancer is one of the most serious diseases that threaten the human’s health,for which the incidence and mortality of colorectal cancer(CRC)rank third among all of the cancer,and show an upward trend nowadays.Due to the symptoms on CRC is ambiguous and hard to be distinguished at the early stage,nearly half of the patients have suffered the tumor metastasis once diagnosis,and the 5-year survival rate of patients on metastatic CRC is only around 10%.Chemotherapy is one of the most important methods for the treatment of CRC.especially for those patients who are bearing with metastatic CRC and cannot accept a surgical resection or radiotherapy.However,the therapeutic efficacy of single chemotherapeutic drug is often limited and may cause multidrug resistance,tumor tolerance and recurrence,therefore,combinational stragegy is widely applied for the first-line treatment of CRC.Among that,oxaliplatin(OXA)and irinotecan(IRI)combinational scheme is recommended inthe "Guidelines for clinical treatment of CRC’’by Comprehensive National Cancer Network(NCCN).Although the combination on OXA and IRI can both act with DNA of the cells with different mechanisms and hence generate the synergistic effect,there are series of defects in both drugs.such as low drug tolerance,non-specific biodistribution,multidrug resistance and unexpected pharmacokinetics,which may cause serious damage to normal tissue when killing tumor cells.Additionally,the combination of both drugs will further aggravate the toxic side effects of the body,and hence limit its clinical applications.On the other hand,traditional approach in clinic is just simply a cocktail,and thus the release of drugs in viv ocan not be kept in control.Due to the different pharmacokinetics of each drug,different drug ratios may play a synergistic,additive,or even antagonistic effect,and thus their combination may lead to the uncertainty of therapeutic effect.Consequetly,it would becruical to build a noval strategy to improve the therapeutic effect on CRC and reduce the side effects of the two drugs.Drug delivery system(DDS)provides a good opportunity to overcome the disadvantages of the chemotherapeutics,and it has become a hot topic in pharmaceutical field to delivery or co-delivery the drugs by the proper nanocarriers.There are three strategies to achieve the combination therapy by DDS,first is "cocktail strategy",for which one free drug is combined with another drug loaded in nanocarriers;second is"mixture strategy",for which both drugs are delivered by separate nanocarriers;third is"co-encapsulation strategy",for which both drugs are co-encapsulated in the same nanocarrier.During the delivery process of the drugs in vivo,the ratios of both drugs in the specified site would be random in both cocktail and mixture strategies,while the ratio can be fixed in co-encapsulation strategy and thus an optimal synergistic effect can be achieved.Lipid-based nanocarriers are one kind of the fundamental carriers for building the DDS with the liposomes and lipid emulsions(LEs)as typical representatives.Lipid-based nanocarriers have desired safety,satisfied biocompatibility and biodegradability,and many liposomal and lipid emulsions’ products have been approved in clinics,showing great application prospects.In this study,in order to improve the therapeutic efficay of CRC and reduce the side effects of chemotherapeutics,liposomes and LEs are selected as the nanocarriers and two types DDS co-encapulated with OXA and IRI were built.The enhanced therapeutic effect of CRC was realized by controlling the release of two kinds of drugs in vivo and effectively delivery of the two drugs into the tumor cells synchronously.Furthermore,the differences in therapeutic efficacy betwen mixture strategy and co-encapulation strategy in DDS were further explored and analyzed,which would provide theoretical basis on the design of drug co-delivery systems.The main contents and results in this study are listed as follows:PART ONE:preparation,characterization and evaluation of OXA and IRI co-loaded liposomesChapter 1.Determination method of OXA and IRI were established by HPLC and UV-Vis,respectively.Both drugs showed good linear relationships under determinated concentrations,and the precisions and recoveries could meet the requirements.The encapsulation efficacy of OXA in liposomes was established by both dialysis and Sephadex G25 methods,aslo the encapsulation efficacy of IRI was established by Sephadex G25 method.Both drugs could be well seprated from the liposomes and the recoveries could meet the requirements.Chapter 2.The synergistic effect of OXA and IRI was verified by both Gaddum model and CI index methods,and both drugs have maximum synergistic effect at OXA/IRI molar-ratios from 1:1 to 1:1.5.OXA/IRI co-loaded liposomes were prepared by ethanol injection followed with ammonium sulfate gradient method,with particle size less than 200 nm,PDI less than 0.2,and well-formed spherical shape.In vitro release study showed that the release behavior of the two drugs include the initial burst release and subsequent sustained release of the release of the two parts,and co-loaded liposomes can better control the molar-ratios of two drugs within the optimal synergistic ranges.Chapter 3.In vitro cytotoxicity study showed that co-loaded liposomes had stronger cell killing ability.In vivo antu-tumor activity experiment was investigated in CT-26 tumor bearing mice,and co-loaded liposomes can significantly enhance the anti-tumor activity compared with mixture solution and mixture liposomes(p<0.05).Near-infrared fluorescence imaging was carried out after the mice were treated with different DiR formulaitons,and the results showed that liposomes could increase the accumulation of drugs in the tumor site.By labeling liposomes with Rhodamine B and FITC as fluorescent dyes,the co-delivery ability of the liposomes were evaluated through in vitro cell uptake and in vivo cryo-sections observation,the results showed that co-loaded liposomes had higher co-delivery efficiency than mixture formulations.Additionally,body weight of tumor bearing mice after administrated with co-loaded liposomes were obviously higher that of mixture solutions,as well as abnormal morphology of the mice could be seen in heart,lung and spleen tissue by HE staining for mixture solution group,indicated that liposomes have superior safety and can decrease the side effects of the drugs.PART TWO:preparation,characterization and evaluation of OXA and IRI co-loaded LEsChapter 4.Oxaliplatin phophoslipid complex(OPPC)and irinotecan phophoslipid complex(IPPC)was successfully formulated,and the optimal procedure was investigated.The prepared OPPC and IPPC exhibited good stability within measured 7 days.OPPC and IPPC were then characterized by DSC curve,in which the endothermic and exothermic peaks were significantly changed compared with the free drug,proving the successful formulation of the complexes.Chapter 5.OXA and IRI co-loaded LEs was successfully prepared by drug-phophoslipid complex technique,and the the optimal formulation is:MCT selected as the oil phase and the proportion of MCT 5%;Pluronic F68 2%;glycerol 2%and oleic acid 0.125%,respectively;emulsification temperature at 60℃;homogenization pressure at 1000 bar for15 cycles.The prepared co-loaded LEs have particle size around 100-120 nm,PDI less than 0.2,zeta potential around-20 mV,also with good stability.Long-term stability study showed that the co-loaded LEs could remain stable without obvious particle size changes after stored under 4 degrees for three months.In vitro release study showed that LEs could control the sustained release of drugs,and the cumulative release of both drugs in both single and co-loaded LEs was more than 80%and 70%within 48 h,respectively.Comparative release behaviors of both drugs indicate that LEs can control the simultaneous release of two drugs and achieve the optimal synergy.Chapter 6.In vitro cytotoxicity study showed that blank LEs had desired safety,although co-loaded LEs had lower cell viability than mixture solution and mixture LEs,no statistical difference existed(p>0.05).In vivo anti-tumor activity experiment showed that co-loaded liposomes can significantly enhance the anti-tumor activity compared with mixture solution and mixture LEs(p<0.05),additionally,LEs could reduce the side effects of the mixtue solution.Near-infrared fluorescence imaging also verified that LEs could increase the accumulation of drugs in the tumor site and sustain the metabolism of drugs.By labeling liposomes with Dil and DiO as fluorescent dyes,the co-delivery ability of LEs were evaluated through in vitro cell uptake and in vivo cryo-sections observation,the results also showed that co-loaded LEs had superior co-delivery efficiency than mixture formulations,herein generate the optimal synergistic effect.更多还原