【作者】 王亮；

【导师】 张仕勇；

【作者基本信息】 四川大学 ， 有机化学， 2021， 硕士

【摘要】 纳米载药系统在肿瘤治疗领域显示出了强劲的发展势头。但载体和负载药物的毒副作用严重阻碍了纳米药物的进一步发展和临床应用。目前研究多停留在单一的改善载体或者负载药物的毒性层面,关于同时降低载体和负载药物的毒性的研究报道非常少。此外在降低毒性的同时保持药效不降低、治疗效果不打折也是非常具有挑战性的。针对以上纳米载药系统中存在的问题,本论文设计了基于生物正交策略的交联硫辛酸纳米载药系统用于肿瘤的协同治疗。主要研究内容包括:(1)交联硫辛酸纳米胶囊(cross-linked lipoic acid nanocapsule,cLANCs)的制备。通过改良的乳液法制备了水包油型(O/W)纳米乳液,经二硫苏糖醇(DTT)交联并除去分散相,最终得到了仅由硫辛酸构筑的具有空腔结构的cLANCs。通过紫外分光光度计(UV-vis)中相关峰的变化证明了交联的成功发生。动态光散射(DLS)和透射电镜(TEM)结果显示cLANCs具有规则的球形结构,水动力直径为79 nm,符合高渗透长滞留效应(EPR)的粒径需求。通过稀释和血清稳定性实验证明了cLANCs具有强健的稳定性。通过高浓度下对正常细胞的毒性和对血细胞的溶血和凝血能力评估证实了cLANCs优异的生物相容性。(2)双组分生物正交纳米体系的制备。通过将抗肿瘤药物康普瑞汀A4(CA4)衍生物的两个前体预先溶解于低沸点易挥发的分散相中,制备得到了负载前药的交联硫辛酸纳米胶囊(1+2)@cLANCs,通过UV-vis谱图的变化证明前体的成功包载。同时以cLANCs为模板,通过加入铜盐和还原剂制备得到金属纳米催化剂Cu@cLANCs。DLS、TEM和Zeta电势的结果表明双组分生物正交纳米体系具有相似的表面性质。前体的包载量和催化剂的负载比分别通过高效液相色谱(HPLC)和电感耦合原子发射光谱(ICP-AES)测定得到。通过两组分的泄露实验验证明了cLANCs赋予了纳米前药和纳米催化剂强健的稳定性。特别地,当两者共存时,泄露量(用单位时间激活的原药占理论值的百分比表示)几乎可忽略不计,支持双组分生物正交纳米体系同时给药。在溶液实验中相当于肿瘤组织浓度下GSH的作用下,双组分生物正交纳米体系可以快速且高效地进行释药;而在相当于正常组织浓度GSH作用下,药物释放量相当少。(3)体外/体内协同抗肿瘤研究。在细胞实验中,通过对作用后的细胞裂解液进行HR-MS测试,证实了双组分生物正交纳米体系可以响应肿瘤细胞内高GSH含量进行前体和催化剂的释放进而继发点击反应生成抗肿瘤药物。相同含量下,双组分生物正交纳米体系具有比游离原药更高的细胞毒性,原因在于cLANCs的降解产物和原位生成药物的协同作用,并通过协同指数CI进行了证实。细胞周期和细胞凋亡实验证实了协同作用的机制在于硫辛酸加强了CA4类似物的周期阻滞效果并最终导致细胞凋亡。组织分布实验表明,相同含量下双组分生物正交纳米体系相比游离原药组,在肿瘤组织处具有更高的原药积累量,同时在其它脏器最低的原药积累量。体内抗肿瘤实验表明双组分生物正交纳米体系具有最好的肿瘤抑制率,同时治疗结束后的脏器没有明显的组织损伤,证实了其生物安全性。更多还原

【Abstract】 Nanoscale drug delivery systems(nano-DDSs)have shown strong development momentum in the field of cancer treatment.However,the toxic and side effects of the loaded drugs as well as the carriers seriously hinder the further development and clinical application of nanomedicines.Current studies have largely focused on improving the toxicity of carriers or loaded drugs,and there are very few reports on reducing the toxicity of carriers and loaded drugs at the same time.In addition,it is also very challenging to keep the efficacy of drugs not reduced and the therapeutic effect is not discounted while reducing the toxicity.In view of the above problems in nano-drug delivery system,a cross-linked lipoic acid nano-drug delivery system based on bio-orthogonal strategy was designed for the synergistic treatment of colorectal cancer.The main research contents include:(1)Preparation of cross-linked lipoic acid nanocapsules(cLANCs).O/W nanoemulsions were prepared by a modified emulsion method followed crosslinked by DTT and the dispersed phase removal,and finally,cLANCs with cavity structure constructed only from lipoic acid were obtained.The successful occurrence of crosslinking was demonstrated by the change of UV-vis and ~1H NMR correlation peaks.DLS and TEM results showed that cLANCs had regular spherical structure with a hydrodynamic diameter of 79 nm,which met the particle size requirement of EPR effect.The robust stability of cLANCs was demonstrated by dilution and serum stability experiments.The excellent biocompatibility of cLANCs was confirmed by toxicity to normal cells at high concentrations and evaluation of hemolysis and coagulation ability of blood cells.(2)Preparation of two-component bioorthogonal nanosystems.Prodrug-loaded crosslinked lipoic acid nanocapsules(1+2)@cLANCs were prepared by pre-dissolving the two inert precursors of the antitumor drug CA4 derivative in a low boiling point volatile dispersed phase.The successful entrapment was demonstrated by the change in UV-vis spectra.At the same time,taking cLANCs as a template,metal nanocatalysts Cu@cLANCs can be prepared by adding copper salts and reducing agents.XPS analysis demonstrated the presence of nano-copper.The results of DLS,TEM and Zeta potential indicate that the two-component bioorthogonal nanosystems have similar surface properties.The drug loading content of precursors and catalysts were determined by HPLC and ICP-AES,respectively.The leak test of the two components proved that cLANCs endowed the nanoprodrug and nanocatalyst with robust stability.In particular,when coexist,the amount of leakage(expressed as the percentage of the theoretical value of the drug activated per unit time)is almost negligible,supporting the simultaneous administration of two-component bioorthogonal nanosystems.In solution experiments,the two-component bioorthogonal nanosystem can release drug rapidly and efficiently under the glutathione(GSH)level of tumor cells,while the drug release amount is quite small under the glutathione(GSH)level of normal cells.(3)In vitro/in vivo synergistic anti-tumor study.In in-vitro experiments,the high-resolution mass spectrometry(HR-MS)analysis indicated that the final product was inside the cell lysate,hence confirming that the two-component bioorthogonal nanosystem could release precursors and catalysts and then generate antitumor drugs by secondary click reaction in tumor cells.At the same content,the two-component bioorthogonal nanosystem had higher cytotoxicity than the free drug due to the synergistic effect of the degradation products of cLANCs with the drugs generated in situ,which was confirmed by the combination index(CI).Cell cycle and apoptosis experiments confirmed that the synergistic mechanism was that lipoic acid could reinforce the cycle arrest effect of CA4 analogues and eventually led to apoptosis.Tissue distribution experiments showed that the two-component bio-orthogonal nanosystem with the same content had a higher accumulation of crude drugs in tumor tissues and the lowest accumulation of crude drugs in other organs compared with the free drug group.In vivo anti-tumor experiments showed that the two-component bioorthogonal nanosystem had the best tumor inhibition rate,and no obvious tissue damage were observed in the organs after the end of treatment,which confirmed the biological safety.更多还原