亚油酸和聚苹果酸双接枝壳聚糖新型纳米载体材料及其增强抗肿瘤药物功效研究

【作者】 赵子明；

【导师】 印春华；

【作者基本信息】 复旦大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 恶性肿瘤是严重威胁人类健康的重大疾病,化疗药物对肿瘤细胞的选择性差,毒副作用大,治疗效果不理想。开发新型给药系统（Drug Delivery System,DDS）提高现有抗肿瘤药物疗效,减少毒副作用,可降低新药开发的风险,药物的安全性也更加可靠,已成为癌症治疗研究的热点。纳米给药系统由于可缓、控释药物及靶向给药而受到广泛关注,纳米给药系统的载体材料必须具有良好的安全性才能进一步用于临床或生产上市。壳聚糖因良好的生物相容性和生物可降解性而获得广泛应用,是美国FDA批准的载体材料之一。本研究目的在于开发一种两亲性壳聚糖衍生物纳米粒,并对其进行PEG、叶酸（Folic acid,FA）和生物素（Biotin,BT）功能化修饰,使其成为一种安全、高效、可主动靶向肿瘤组织的抗癌药物给药载体。以酰氯化反应制备亚油酸（LA）和聚苹果酸（PMLA）双接枝壳聚糖（LMC）,希夫碱反应与EDC·HCl脱水缩合反应制备功能化修饰的LMC衍生物:PEG修饰的LMC（PEG-LMC）、叶酸修饰的PEG-LMC（FA-PEG-LMC）及生物素修饰的PEG-LMC（BT-PEG-LMC）;LMC及其衍生物在水中自组装形成纳米粒;以紫杉醇（PTX）为难溶性抗肿瘤模型药物研究LMC及其衍生物纳米粒的理化性质、载药特点及体外释药规律;以H22荷瘤小鼠为模型,考察载PTX LMC及其衍生物纳米粒的抑瘤率;以SMMC-7721为肿瘤细胞模型、HEK-293为正常细胞模型研究LMC及其衍生物纳米粒的体外细胞摄取;以正常小鼠和H22荷瘤小鼠为模型,研究LMC及其衍生物纳米粒的体内组织分布及靶向性。1 LMC及其功能化修饰衍生物的制备与表征以D,L-天冬氨酸为起始单体,乳酸为引发剂,采用内酯开环聚合法合成一种含乳酸末端的聚苹果酸苄酯（PMLABz）;以甲基磺酸为溶剂,酰氯法活化亚油酸和聚苹果酸苄酯,使其与壳聚糖的羟基以酯键连接或与氨基以酰胺键连接,制备LMC。考察亚油酸和聚苹果酸的投料比、投料顺序、反应温度及反应时间对LMC接枝反应的影响,确定LMC的制备纯化工艺:亚油酸与壳聚糖单元摩尔比为0.2～1.0,苹果酸单体与壳聚糖单元摩尔比为1.0,室温反应6 h,以甲基磺酸脱保护基,利用LMC的pH敏感性纯化产物。DMSO-醋酸酐法制备mPEG醛,考察醋酸酐与mPEG的摩尔比、反应温度与反应时间对mPEG醛活化率的影响,制得活化率80%以上的mPEG醛。mPEG醛与LMC在DMSO和pH8.0硼酸缓冲液混合溶剂中反应,硼氢化钠还原,离心纯化制得PEG-LMC。经EDC·HC1介导的缩合反应将叶酸和生物素接枝至PEG-LMC上,制得FA-PEG-LMC和BT-PEG-LMC。确定其制备工艺:以EDC-HC1、NHS、DMAP催化反应,透析法纯化产物。采用FTIR、¹H NMR、x射线衍射、DSC和元素分析表征了LMC及其衍生物的结构。元素分析结果表明亚油酸的取代度（DS）介于36.1～60.5之间,PMLA的DS介于0.7-1.1之间,PEG、FA和BT的DS分别为12.4,6.0和6.1。2 LMC及其衍生物纳米粒的制备、表征及载药、释药特性以PTX为模型药物,研究LMC及功能化修饰的LMC衍生物自组装纳米粒作为难溶性抗癌药物载体的特点。超声法制备LMC及其衍生物自组装纳米粒;透射电子显微镜（TEM）和扫描电子显微镜（SEM）考察纳米粒的形态,动态激光光散射法（DLLS）测定纳米粒的粒径与Zeta电势,稳态荧光探针法测定纳米粒的临界聚集浓度（CAC）;制备载PTX LMC及其衍生物纳米粒,考察亲疏水组分比例与PEG、FA和BT修饰对包封率、载药量和体外释放行为的影响。LMC及其衍生物纳米粒在水中的平均粒径为190～350 ilm,生理条件下Zeta电势为-2～-20mV。LMC的粒径随LA取代度的增加而减小,随PMLA链长的增加而增大,且碱性条件下的粒径大于酸性条件。LMC纳米粒的Zeta电势受介质pH值影响,pH<6.0时带正电,6.0<pH<7.0时电荷接近0,pH>7.0时带负电。PEG修饰增大LMC纳米粒的粒径,且可屏蔽纳米粒表面电荷,使其Zeta电势绝对值降低。FA和BT修饰对PEG-LMC的粒径和Zeta电势无明显影响。LMC的CAC随LA取代度的增加而减小,PEG、FA和BT修饰不明显改变CAC。LMC及其衍生物纳米粒对PTX的包封率均超过70%,最高可达90%;载药量随LA取代度的增加而增加,最高可达9.9%,PEG修饰使载药量略有降低。载PTX LMC纳米粒4 h的累积释放率约为50～60%,8 h的累积释放率约为70-80%,可持续释放药物达24 h。LA取代度较大、PMLA链较长的LMC纳米粒释药速度较慢,PEG修饰可增强LMC纳米粒的缓释能力。3载紫杉醇纳米粒的抗肿瘤作用、体内外靶向性及安全性考察载PTX LMC及其衍生物纳米粒对H22荷瘤小鼠的抑瘤效果,结果抑瘤率强弱顺序为:FA-PEG-LMC>BT-PEG-LMC>PEG-LMC>LMC>PTX溶液,其中FA-PEG-LMC与BT-PEG-LMC的抑瘤率分别为82.5%和80.6%,可有效抑制H22肿瘤的生长。细胞摄取试验考察LMC及其衍生物纳米粒的体外肿瘤细胞靶向性。制备罗丹明B标记的LMC纳米粒（RB-LMC NP）、PEG-LMC纳米粒（RB-PEG-LMCNP）、叶酸修饰的LMC纳米粒（RB-FA-LMC NP）、叶酸修饰的PEG-LMC纳米粒（RB-FA-PEG-LMC NP）、生物素修饰的LMC纳米粒（RB-BT-LMC NP）和生物素修饰的PEG-LMC纳米粒（RB-BT-PEG-LMC NP）,考察其在SMMC-7721细胞和HEK-293细胞中的摄取。结果表明SMMC-7721肿瘤细胞对LMC及其衍生物纳米粒的摄取能力强于HEK-293正常细胞;纳米粒浓度低于1000μg·mL^-1时,SMMC-7721细胞对纳米粒的摄取量随纳米粒浓度的增加而增加;纳米粒浓度低于500μg·mL^-1时,HEK-293细胞对纳米粒的摄取量随纳米粒浓度的增加而增加;4 h内,SMMC-7721和HEK-293细胞对纳米粒的摄取量随共培育时间的延长而增加。FA和BT修饰的LMC纳米粒在SMMC-7721细胞中的摄取量显著高于LMC纳米粒（FA-LMC和B^LMC的摄取量分别为LMC的3～5倍和2～3倍）,在HEK-293正常细胞中的摄取量显著低于SMMC-7721肿瘤细胞,表明FA和BT修饰的LMC纳米粒可选择性地主动靶向肿瘤细胞。PEG修饰对LMC纳米粒的摄取无明显影响,但同时修饰配体和PEG的FA-PEG-LMC和BT-PEG-LMC的细胞摄取量低于FA-LMC和BT-LMC。游离叶酸及生物素可抑制相应FA和BT修饰的纳米粒的摄取,对FA-PEG-LMC和BT-PEG-LMC纳米粒的抑制率高于FA-LMC和B%LMC,表明FA-LMC和BT-LMC对细胞表面叶酸和生物素受体亲和力更强。以正常小鼠和荷H22肿瘤小鼠为模型,考察载PTX的LMC纳米粒、PEG-LMC纳米粒、FA-PEG-LMC纳米粒与BT-PEG-LMC纳米粒的体内组织分布和肿瘤靶向性。HPLC测定体内药物浓度,方法专属性、精密度、回收率均较好。PEG-LMC组血液中药物浓度时间曲线下面积（AUC）为LMC组的3.05倍,肝中AUC为LMC组的61%,脾中AUC与LMC组相近,表明PEG修饰可显著延长纳米粒在血液中的循环时间,减少肝巨噬细胞的吞噬,但无法避免脾巨噬细胞的吞噬;LMC、PEG-LMC、FA-PEG-LMC与BT-PEG-LMC纳米粒均具有一定的肿瘤靶向性,肿瘤相对摄取率（R_e）分别为1.36、2.51、3.80、2.95,表明FA-PEG-LMC纳米粒的肿瘤靶向性最佳;LMC、PEG-LMC、FA-PEG-LMC与BT-PEG-LMC在心、肾中的药物分布显著低于PTX溶液组,可降低PTX对心、肾的毒性。溶血试验和急性毒性试验考察LMC纳米粒的安全性,结果LMC的溶血率低于5%;小鼠对LMC的最大耐受剂量为1250 mg·kg^-1,耐受倍数为125倍,表明LMC作为注射给药载体安全性较高。更多还原

【Abstract】 In the case of cancer therapy, chemotherapy is often impotent in effective treatment due to unfavorable specificity towards tumors and great side effects. Therefore, development of novel drug delivery systems （DDS） which are characterized by improved anti-tumor effect, reduced side effects, decreased cost during R&D of new drugs, and favorable safety has around great attention. Among them nanoparticles delivery systems show distinct beneficial attributes, including controlled and sustained drug release and targeted drug delivery. It is a prerequisite that the polymeric nanoparticles subjected to clinical investigations and marketing should possess desired biocompatibility and safety. Chitosan shows wide applications due to its favorable biocompatibility and biodegradability, and is an approved excipient by FDA. The current investigation aims at developing a novel kind of amphiphilic chitosan derivative nanoparticles, which are further modified with PEG, folic acid （FA） and biotin （BT）, thus achieving an effective, safe, and actively targeted delivery carrier for anti-tumor drugs.Linoleic acid （LA） and poly（malic acid） double-grafted chitosan （LMC） was prepared through acrylation reaction. Functionally modified LMC derivatives including PEG modified LMC （PEG-LMC）, FA modified PEG-LMC （FA-PEG-LMC）, and BT modified PEG-LMC （BT-PEG-LMC） were synthesized through Schiff base reaction and EDC·HCl mediated covalent bonding. The amphiphilic LMC and LMC derivatives self-assembled into nanoparticles in water. PTX as a model insoluble anticancer drug was loaded in LMC and LMC derivatives nanoparticles, which were characterized by drug loading efficiency, loading capacity, and in vitro release. In vitro cellular uptake of LMC and LMC derivatives nanoparticles were evaluated in SMMC-7721 cells and HEK-293 cells, and the tumor inhibition effect, biodistribution, and targeted effect were monitored in H22 bearing mice.1 Preparation and characterization of LMC and LMC derivativesPMLABz was synthesized through ring-open polymerization of lactone with D,L-aspartic acid as monomer and lactic acid as initiator. LA and PMLABz were activated using the acrylation method in MeSO₃H, and were conjugated with hydroxyl and amino groups on chitosan through ester and amide bonding, respectively, thus achieving the LMC. Effect of feed ratio and feed sequence of LA/poly（malic acid）, reaction temperature, and reaction time on the graft reaction was assessed. The optimal molar ratio of LA/saccharide ring was set to be 0.2-1.0, molar ratio of malic acid/saccharide ring to be 1.0, reaction time to be 6 h at room temperature, and MeSO₃H was used for hydrolysis of benzyl groups.mPEG aldehyde with an activation degree of above 80% was synthesized using the DMSO-acetic anhydride method, and the effect of acetic anhydride/mPEG molar ratio, reaction temperature and reaction time on the activation degree was evaluated. Optimally, mPEG aldehyde was allowed to conjugate with LMC in a mixed solution of DMSO and pH 8.0 borate suffer, which was deoxidized with NaBH₄ and purified through centrifugation.Folic acid and biotin were separately conjugated to PEG-LMC via amide bond mediated by EDC·HCl, achieving the FA-PEG-LMC and BT-PEG-LMC.FTIR, ¹H NMR, XRD, DSC, and elemental analysis confirmed synthesis of LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC. Elemental analysis demonstrated that the degree of substitution （DS） of LA was 36.1-60.5, DS of PMLA was 0.7-1.1, DS of PEG was 12.4, DS of FA was 6.0, and DS of BT was 6.1.2 Preparation, characterization of blank and PTX-loaded LMC and LMC derivatives nanoparticlesLMC and LMC derivatives self-assembled nanoparticles with different LA and PMLA substitution degrees were prepared by sonication. Morphology of the nanoparticles was observed using TEM and SEM, and particle size and Zeta potential were monitored using DLLS. CAC was determined using fluorescence spectroscopy with pyrene as a hydrophobic probe. PTX as a model anticancer drug was loaded in the nanoparticles, and the effect of nanoparticle composition and modification of PEG, FA, and BT on encapsulation efficiency, loading capacity and in vitro release behavior was investigated.Average particle size of LMC and its derivatives nanoparticles at pH 7.4 was 190-350 nm, and Zeta potential was -2～-20 mV. An increase in DS of LA or decrease in PMLA chain length led to decreased particle size of LMC, and particle size in alkaline conditions was larger than in acidic conditions. Zeta potential of the LMC nanoparticles was sensitive towards pH values in that they were positively charged at pH lower than 6.0, nearly uncharged within the pH range of 6.0-7.0, and negatively charged at pH higher than 7.0. The modification of PEG led to an increase in particle size and a decrease in the absolute value of Zeta potential. The modification of FA or BT had no significant effect on the particle size and Zeta potential of PEG-LMC NP. The CAC of LMC was deceased as the DS of LA enhanced and the modification of PEG, FA, and BT didn’t increase CAC. Loading efficiency of LMC and its derivatives nanoparticles for PTX was above 70%, and the loading capacity increased with LA substitution degree which reached maximal value of 9.9%. The modification of PEG slightly decreased the loading capacity of LMC. A sustained release of PTX was achieved within 24 h, and an increase in LA substitution degree and PMLA chain length could slow down the release rate of PTX. The modification of PEG could enhance the slow-release effect of LMC.3 Anti-tumor effect, tumor-targeting and safety assessment of LMC and LMC derivatives nanoparticlesAnti-tumor effect of PTX loaded LMC and LMC derivatives nanoparticles were investigated in H22 bearing mice. Both unmodified and modified PTX loaded LMC nanoparticles showed significant anti-tumor effect, while PEG modification and ligand modification resulted in improved anti-tumor effect. The tumor inhibition rate （TIR） was: FA-PEG-LMC>BT-PEG-LMC>PEG-LMC>LMC>PTX solution, with TIR of 82.5% and 80.6% for FA-PEG-LMC and BT-PEG-LMC, respectively.Cellular uptake study of LMC and LMC derivatives nanoparticles were performed in SMMC-7721 cells and HEK-293 cells to evaluate the in vitro tumor targeting effect. Rhodamine B labeled LMC （RB-LMC）, PEG-LMC （RB-PEG-LMC）, FA-LMC （RB-FA-LMC）, FA-PEG-LMC （RB-FA-PEG-LMC）, BT-LMC （RB-BT-LMC）, and BT-PEG-LMC （RB-BT-PEG-LMC） nanoparticles were used for assessment. The uptake of LMC and its derivatives nanoparticles in SMMC-7721 cells was much higher than that in HEK-293 cells. Cellular uptake of SMMC-7721 increased with nanoparticle concentration within 1000μg·mL^-1, and cellular uptake of HEK-293 increased with nanoparticle concentration within 500μg·mL^-1. Cellular uptake of SMMC-7721 and HEK-293 increased with incubation time in within 4 h. The uptake amount of FA-LMC and BT-LMC nanoparticles was 3-5 fold and 2-3 fold of that of LMC nanoparticls and the uptake amount of FA and BT modified LMC nanoparticles in HEK-293 was significantly lower than that of SMMC-7721. It meant that FA and BT modified LMC nanoparticles could actively target to tumor cells. The modification of PEG had no obviously effect on the uptake of LMC nanoparticles, but the uptake amount of FA-PEG-LMC and BT-PEG-LMC was lower than that of FA-LMC and BT-LMC. Free folic acid and biotin inhibited uptake of FA and BT modified LMC nanoparticles, respectively. The inhibition on FA-PEG-LMC and BT-PEG-LMC was stronger than that on FA-LMC and BT-LMC, which meant that FA-LMC and BT-LMC had higher affinity with FA and BT receptors on cell surface.Biodistribution of PTX loaded LMC, PEG-LMC, FA-PEG-LMC, and BT-PEG-LMC nanoparticles were investigated in normal and H22 bearing mice. HPLC was used for PTX quantification, and the method was precise and reliable. PEG modification prolonged retention of LMC nanoparticles in the blood circulation and decreased phagocytosis by macrophages in the liver. However, phagocytotis by the spleen was not prevented. Both unmodified and modified LMC nanoparticles showed tumor-targeting effect, while FA and BT modification yielded an additional active targeting towards tumor with FA-PEG-LMC showing the maximal tumor-target effect of R_e of 3.8. Besides, distribution of LMC and LMC derivatives nanoparticles in the heart and spleen was remarkably lower than PTX injection, indicating that toxicity of PTX towards heart and spleen was successfully reduced following encapsulation in LMC and LMC derivatives nanoparticles.Finally, safety assessment was carried out on LMC nanoparticles in terms of hemolysis and acute toxicity. Hemolysis ratio of LMC was below 5%, and the maximum tolerance amount of LMC in mice was 1250 mg·kg^-1, which suggested it a safety drug carrier for intravenous injection.更多还原