紫杉醇Pluronic P105聚合物胶束的制备、表征与逆转肿瘤多药耐药性的体外研究

药物递送系统是克服肿瘤多药耐药性(MDR)的一种新策略。本文以聚合物胶束系统和难溶性药物紫杉醇(PTX)为研究对象，旨在制备一种新型的PTX给药系统，既能增溶难溶性药物，又具有克服肿瘤MDR的能力。以Pluronic P105为载体，采用固体分散-水化法制备PTX聚合物胶束，并以星点设计-效应面优化法进行处方优化。对其粒径、体外释放等性质进行表征后，以人耐药卵巢癌细胞SKOV-3/PTX为细胞模型，体外评价PTX聚合物胶束的细胞摄取及其逆转肿瘤细胞耐药性的作用。结果显示，聚合物胶束制剂的载药量约为1.1%、药物浓度约为700 μg·mL^-1、平均粒径约为24 nm。胶束制剂与普通制剂(Taxol)在6 h内的累积释放分别为45.4%和95.2%，前者具有较强的缓释作用；胶束制剂与Taxol对SKOV-3/PTX的IC₅₀值分别为1.14和5.11 μg·mL^-1，二者的耐药逆转指数(RRI)分别为9.65和2.15。胶束制剂可促进耐药细胞对P-糖蛋白(P-gp)底物(PTX或Rhodamine-123)的摄取。结果表明，Pluronic P105可有效增溶难溶性药物PTX，并形成具有较强缓释作用的纳米级聚合物胶束制剂，该制剂可显著提高PTX对人卵巢癌耐药细胞的细胞毒性，能逆转其耐药性。     

溶酶体酸性微环境敏感型粒径可变纳米粒的构建及评价

目的 构建一种以功能性支化聚乙烯亚胺(bPEI)为基本骨架材料的粒径可变纳米粒,用于递送药物至黑色素瘤的深部区域.方法 通过酰胺键修饰特定量的琥珀酸酐至bPEI骨架上,得到具有电荷翻转功能的材料NSP,再引入聚N-异丙基丙烯酰胺(PNIPAM)制备粒径可变纳米粒(PSP),分别考察其粒径、Zeta电位和释药行为;考察B16F10细胞对PSP的摄取及胞内内涵-溶酶体对PSP的影响;通过构建体外三维肿瘤球模型来考察纳米粒的穿透能力.结果 载阿霉素粒径可变纳米粒(D/PSP)在pH7.4缓冲液中的粒径、PDI和Zeta电位分别为31.2±4.8 nm、0.106 ±0.015、-2.26-±0.06 mV.在内涵-溶酶体所对应的pH5.0条件下,D/PSP的粒径可膨胀至891.6±26.3 nm,PDI为0.256±0.123,Zeta电位为15.61±0.19 mV.B16F10细胞对D/PSP的摄取在一定范围内呈孵育浓度依赖性及时间依赖性,其主要的入胞机制为网格蛋白介导和能量依赖型的内吞途径.结论 成功制备了具有较好的溶酶体酸性微环境响应性的PSP,D/PSP具有较强的肿瘤球穿透能力.     

多西他赛pH敏感嵌段共聚物胶束的制备

本文在合成pH敏感两亲性嵌段共聚物聚(2-乙基-2-噁唑啉)-聚乳酸(PEOz-PDLLA)的基础上，采用薄膜分散法制备多西他赛pH敏感嵌段共聚物胶束，利用芘荧光探针技术测定胶束的临界胶束浓度(CMC)；通过高效液相色谱测定胶束的载药量及包封率；分别利用透射电镜、动态光散射法和zeta电位分析仪对胶束的形态、粒径和表面电位进行了表征；采用透析法考察了载药聚合物胶束的体外释放行为。结果表明，胶束的临界胶束浓度值为1.0×10^-3 g·L^-1；载药量可达15.0%，包封率为91.1%；胶束的粒度分布很窄，平均粒径为28.7nm；胶束粒子为圆球形且分散良好，其表面zeta电位值为(1.19±0.12)mV；在pH 7.4释放介质中，多西他赛胶束具有缓释作用；而在pH 5.0条件下，胶束释药明显加快，体现出PEOz-PDLLA胶束释药行为的pH敏感性。综合上述研究可见，PEOz-PDLLA嵌段共聚物胶束作为疏水性抗肿瘤药物的给药系统具有很好的应用前景。     

正电化修饰的HPMA聚合物-阿霉素接合物的制备表征及对肿瘤细胞摄取的影响

目的:制备2种正电化修饰的N-(2-羟丙基)甲基丙烯酰胺(HPMA)聚合物-阿霉素接合物并表征,分别考察2种接合物的正电基团含量对肿瘤细胞摄取的影响。方法:制备侧链带伯胺基的HPMA聚合物-阿霉素接合物(pHPMA-DOX-APMA)和侧链带胍基的HPMA聚合物-阿霉素接合物(pHPMA-DOX-GPMA),对其药剂学性质如正电基团含量,载药量,Zeta电位和分子量进行表征,进一步考察不同正电基团含量的接合物对MCF-7细胞摄取和毒性的影响。结果:通过自由基聚合反应,2种接合物成功合成。其中pHPMA-DOX-APMA伯胺基含量为0.44~1.57 mmol·g^-1,载药量为7.15%~9.25%;pHPMA-DOX-GPMA胍基含量为0.11~0.54 mmol·g^-1,载药量为7.55%~9.07%;相对分子质量分别为33~38 kDa和32~37 kDa。通过BCA法和MTT法研究分别发现在pHPMA-DOX-APMA中的伯胺基团含量为1.570 mmol·g^-1及pHPMA-DOX-GPMA中的胍基含量为0.260 mmol·g^-1时,肿瘤细胞对阿霉素的摄取量显著增加,二者的IC₅₀与pHPMA-DOX相比显著降低(P<0.05)。结论:成功制备了2种正电化修饰的HPMA聚合物-阿霉素接合物;适当的正电化修饰对阿霉素的肿瘤细胞摄取有促进作用。     

替诺福韦阳离子脂质体的制备及其对人体肝细胞SMMC-7721摄取和细胞毒性研究

目的:研究替诺福韦阳离子脂质体的制备及其促进肝实质细胞摄取的情况和细胞毒性作用。方法:采用叔丁醇冻干法制备替诺福韦阳离子脂质体,测定其包封率及理化性质;以SMMC-7721细胞为模型,研究脂质体对肝实质细胞摄取替诺福韦的促进作用,MTT法检测不同条件下载药脂质体对细胞的毒性情况。结果:制备的脂质体包封率为(88.3±1.6)%,粒径为(278.4±67.6)nm,Zeta电势为(31±5)mV;经半乳糖基及PEG修饰的脂质体较游离药物进入肝实质细胞的浓度明显升高,且时间延长;当替诺福韦脂质体、脂质浓度分别为7.5、30μg·mL-1时,细胞存活率在80%以上,毒性较小。结论:所制备的阳离子脂质体具有显著增加细胞摄取替诺福韦和保护替诺福韦的作用,有望成为抗病毒药物如替诺福韦等的高效传递系统。     

聚古罗糖醛酸硫酸酯-铜纳米胶束制备及抗肿瘤活性研究

目的 制备一种基于化学动力学疗法的聚古罗糖醛酸硫酸酯(PGS)-铜纳米胶束,进行抗肿瘤活性研究。方法 采用聚离子复合法,将PGS与铜离子及过氧化氢反应制备聚电解质纳米胶束PGS-Cu,分光光度法测定铜离子含量和羟基自由基,CCK-8法测定细胞活力。结果 制备的PGS-Cu纳米粒径为109.54±14.29 nm,分布系数为0.23±0.02,Zeta电位为-47.58±1.71 mV。酸性环境下,PGS-Cu纳米胶束铜离子累积释放量显著高于中性环境,并产生.OH。100 μg.mL_^-1的PGS-Cu纳米胶束作用下,人源三阴性乳腺癌细胞(MDA-MB-231)、小鼠乳腺癌细胞(4T1)和小鼠正常成纤维细胞(NIH/3T3)的活力分别为14.87%,62.24%和90.77%,表明PGS-Cu纳米胶束对乳腺癌细胞有显著的细胞毒性,且对MDA-MB-231细胞抑制效果最佳。此外,4T1细胞在pH 7.4和6.0时的活力分别为62.24%和18.33%,说明该纳米胶束具有pH响应性。结论成功构建了一种基于化学动力学疗法、以多糖为载体的聚电解纳米胶束,该纳米胶束具有pH响应性和肿瘤靶向性,能有效抑制乳腺癌细胞生长,发挥抗肿瘤活性。     

姜黄素纳米胶束的制备及体外抗肿瘤作用研究

目的 制备姜黄素(Cur)/甲氧基聚乙二醇-聚己内酯(MP)载药纳米胶束,并研究其体外抗肿瘤作用。方法 采用薄膜分散法制备Cur/MP纳米胶束,筛选不同药材质量比优化处方,在扫描电镜下观察载药纳米胶束的形态;采用CCK-8法考察Cur、Cur/MP胶束对人非小细胞肺癌A549细胞的抑制率;以香豆素6为荧光探针考察细胞对胶束的摄取行为。结果 按最优处方制备3批胶束的平均粒径为35.71±0.2 nm,平均电位为-16.8±0.2 mV,平均包封率为97.52%±1.00%,平均载药量5.25%±0.80%;Cur/MP胶束在72 h内释放度最大可达74.16%,释放缓慢,无突释现象,遵循一级动力学方程;与Cur溶液比较,Cur/MP纳米胶束对A549细胞的抑制作用增强;荧光显微镜下可观察到,随着时间的延长,细胞对药物的摄取增加。结论 Cur/MP胶束制备工艺简单,可增强药物对A549细胞的抑制作用;聚合物纳米胶束的pH敏感性有利于其在体内肿瘤组织中的蓄积及肿瘤细胞内的摄取。     

喜树碱/聚天冬氨酸衍生物-接枝-聚乙二醇共聚物胶束的制备及性质研究

目的制备喜树碱/聚天冬氨酸衍生物-接枝-聚乙二醇的聚合物胶束,并对其化学结构进行表征。方法采用透析法制备喜树碱/聚合物胶束,采用动态激光散射法、透射电镜和核磁共振法测定胶束的粒径、形态及药物分布,采用高效液相色谱法测定喜树碱含量。结果喜树碱/聚合物胶束近似球形,粒径小于50nm,分布较窄,喜树碱被包裹在胶束内,在pH7.4的缓冲液中孵育12h,溶液组喜树碱70%被破坏,而被胶束组喜树碱仅有20%被破坏;在小鼠血浆中孵育12h,溶液组喜树碱90%多被破坏,而胶束组喜树碱约23%被破坏,喜树碱/聚合物胶束在pH7.4的介质中释药接近Higuchi模型。结论聚天冬氨酸衍生物-接枝-聚乙二醇聚合物胶束对喜树碱具有保护作用。     

包载香豆素-6的PEO-(hb-PG)-g-PCL杂臂聚合物胶束的体外评价,细胞摄取以及跨膜转运(英文)

当今聚合技术的发展为合成各种非线形两亲性聚合物提供了手段,对于包载模型药物的杂臂聚合物胶束的摄取和跨膜转运却鲜有报道。本文对两种不同分子量的PEO-(hb-PG)-g-PCL杂臂聚合物胶束的物理性质和Caco-2细胞对其包载的香豆素-6(C6)的摄取量进行了比较,随后选择了PEO113-(hb-PG)15-g-PCL22聚合物胶束来研究PMs-C6在Caco-2单层的摄取和转运。细胞摄取和跨膜转运试验发现,对于Caco-2细胞单层,PMs是很好的摄取和跨膜转运促进剂。对内吞通路的研究发现,PMs-C6的摄取是通过小窝蛋白介导的内吞途径而非网格蛋白介导的内吞途径进行的。本文中制备的PEO-(hb-PG)-g-PCL杂臂聚合物胶束有较小的临界胶束浓度,对Caco-2细胞无显著细胞毒性,以及强大的增加Caco-2细胞单层对疏水性荧光探针C6摄取的作用提示很有潜力用于口服药物递送。     

Cellular uptake and cytotoxicity of shell crosslinked stearic acid-grafted chitosan oligosaccharide micelles encapsulating doxorubicin

Stearic acid-grafted chitosan oligosaccharide (CSO-SA) with 3.48% amino-substituted degree (SD%) was synthesized by coupling reaction. The CSO-SA could self-aggregate to form micelle with a critical micelle concentration (CMC) at 0.035 mg/mL in the aqueous phase. The CSO-SA self-aggregate micelles indicated spatial structure with multi-hydrophobic core. One CSO-SA chain could form 2.8 hydrophobic cores. Cellular uptakes of CSO-SA micelles by using A549, LLC, and SKOV3 cells as model tumor cell lines showed the faster cellular internalization of CSO-SA micelles, and the cellular uptakes on the LLC and SKOV3 cells were higher than that on the A549 cells. Doxorubicin (DOX) was then used as a model drug to incorporate into CSO-SA micelles. To reduce the initial burst drug release from CSO-SA micelles loading DOX (CSO-SA/DOX), the shell of CSO-SA micelles was crosslinked by glutaraldehyde. The shell crosslinking of CSO-SA micelles reduced the micelle size and surface potential, but it did not significantly affect the cellular uptake and drug encapsulation efficiency of CSO-SA micelles. The cellular inhibition experiments demonstrated that the cytotoxicity of DOX was increased by the encapsulation of CSO-SA micelles. CSO-SA/DOX displayed the best antitumor efficacy in SKOV3 cell line due to the higher cellular uptake percentage of CSO-SA micelles and the lower sensitivity of free drug to the cells. The cytotoxicities of shell crosslinked CSO-SA/DOX were highly enhanced in all cell lines than those of unmodified CSO-SA/DOX.     

Linear-like polypeptide-based micelle with pH-sensitive detachable PEG to deliver dimeric camptothecin for cancer therapy

Nano drug delivery systems have made significant progress in delivering anticancer drugs camptothecin (CPT). However, many challenges for CPT delivery remain, including low drug loading efficiency, premature drug leakage, and poor cellular internalization. Herein, we report a novel dual-sensitive polypeptide-based micelle with remarkably high drug loading of CPT for cancer therapy. This self-assembled micelle possesses the following essential components for CPT: (1) pH-sensitive PEG (OHC-PEG-CHO) for prolonging blood circulation and allowing biocompatibility by shielding the cationic micelles, which can be detached under the tumor acidic microenvironment and facilitates the cellular uptake; (2) polypeptide polylysine-polyphenylalanine (PKF) synthesized via ring-opening polymerization for micelle formation and CPT analogue loading; (3) dimeric CPT (DCPT) with redox-sensitive linker for increasing CPT loading and ensuring drug release at tumor sites. Interestingly, the linear-like morphology of PEG-PKF/DCPT micelles was able to enhance their cellular internalization when compared with the spherical blank PKF micelles. Also, the anticancer efficacy of DCPT against lung cancer cells was significantly improved by the micelle formation. In conclusion, this work provides a promising strategy facilitating the safety and effective application of CPT in cancer therapy.     

PEG-I-dC16酸敏释药胶束的制备及体外释放研究

目的:构建酸敏释药胶束并考查其酸敏释药特性。方法:用亚胺键连接PEG和苯棕榈酸脂肪链,用透析法制备载阿霉素胶束,对其粒径,载药量和包封率进行考察,用紫外分光光度法测定载药胶束在不同pH值条件下的释放。结果:载药胶束粒径为60~70 nm,PEG相对分子质量为2000 Da的胶束载药量和包封率分别为(12.7±1.1)%和(49.8±2.2)%,PEG相对分子质量为5000 Da的胶束载药量和包封率分别为(10.7±0.3)%和(39.9±2.1)%。体外释放研究表明酸敏释药胶束在pH 6.5时的累积释放率比pH 7.4时大,但在pH 5.0条件下其累积释放较pH 7.4时还要小,可能原因是胶束解聚太快致药物与材料形成复合物沉淀所致。结论:以酸敏感亚胺键连接的两亲材料载药胶束具有一定的酸敏释药特性。     

多西他赛pH敏感型聚(2-乙基-2-噁唑啉)-聚(D,L-丙交酯)自组装胶束的制备及其性质

目的利用两亲性嵌段共聚物聚(2-乙基-2-噁唑啉)-聚(D,L-丙交酯)[poly(2-ethyl-2-oxazo-line)-poly(D,L-lactide),PEOz-PDLLA]的自组装性能制备pH敏感型多西他赛胶束,并对其相关性质进行考察。方法运用阳离子开环聚合反应得到PEOz-PDLLA,通过FITR、1H-NMR和凝胶色谱法对其结构进行表征,采用电位滴定法测定共聚物pKa,应用荧光探针技术确定临界胶束浓度(criticalm icelle concentration,CMC)。动态光散射法和Zeta电位测试仪测定胶束的粒径和Zeta电位。以薄膜分散法包载多西他赛,并用透析法研究载药胶束的体外释放度。结果PEOz-PDLLA的亲水/疏水段分子质量比值为0.76,pKa为6.41,CMC为0.8×10-3g.L-1。载药胶束包封率为94.9%、载药量质量分数为8.7%、平均粒径为(35.3±4.9)nm、Zeta电位为(25.51±2.14)mV,在pH5.0的释放介质中释药速度加快。结论PEOz-PDLLA嵌段共聚物可自组装形成胶束,高效包载多西他赛,体外释放具有pH敏感性。     

pH Triggered Doxorubicin Delivery of PEGylated Glycolipid Conjugate Micelles for Tumor Targeting Therapy

The main objective of this study was aimed at tumor microenvironment-responsive vesicle for targeting delivery of the anticancer drug, doxorubicin (DOX). A glucolipid-like conjugate (CS) was synthesized by the chemical reaction between chitosan and stearic acid, and polyethylene glycol (PEG) was then conjugated with CS via a pH-responsive cis-aconityl linkage to produce acid-sensitive PEGylated CS conjugates (PCCS). The conjugates with a critical micelle concentration (CMC) of 181.8 μg/mL could form micelles in aqueous phase, and presented excellent DOX loading capacity with a drug encapsulation efficiency up to 87.6%. Moreover, the PCCS micelles showed a weakly acid-triggered PEG cleavage manner. In vitro drug release from DOX-loaded PCCS micelles indicated a relatively faster DOX release in weakly acidic environments (pH 5.0 and 6.5). The CS micelles had excellent cellular uptake ability, which could be significantly reduced by the PEGylation. However, the cellular uptake ability of PCCS was enhanced comparing with insensitive PEGylated CS (PCS) micelles in weakly acidic condition imitating tumor tissue. Taking PCS micelles as a comparative group, the PCCS drug delivery system was demonstrated to show much more accumulation in tumor tissue, followed by a relatively better performance in antitumor activity together with a security benefit on xenograft tumor model.     

A new peptide ligand for colon cancer targeted delivery of micelles

Abstract

Ligands are an imperative part of targeted drug delivery systems, and choosing a ligand with high affinity is a subject of considerable interest. In this study, we first synthesized a 12-residue peptide (TK) that interacts with integrin α₆β₁ overexpressed on colonic cancer cells. The molecular binding affinity assay indicated that TK had a high binding affinity for integrin α₆β₁. The results of cellular and tumor spheroid uptake suggested that TK peptide not only increases Caco-2 cells uptake, but also effectively increases penetration of the tumor spheroids. TK-conjugated PEG-PLA was synthesized to prepare a novel PEG-PLA micelles loading DOX or coumarin-6 (TK-MS/DOX or TK-MS/C6). The obtained TK-MS/DOX exhibited uniform, spherical shape with a size of 23.80?±?0.32?nm and zeta potential of 12.21?±?0.31 mV. The release behavior of DOX from micelles were observed no significant changes after TK modification, however, the release profile exhibited pH-sensitive properties. Compared with MS/DOX, TK-MS/DOX exhibited significantly stronger cytotoxicity for Caco-2. Confocal laser microscopy and flow cytometry data further indicated that the targeting micelles not only had higher uptake by Caco-2 cells, but also more effectively penetrated the tumor spheroids. Therefore, TK peptide appears to be suitable as a targeting ligand with potential applications in colonic targeted therapy.     

Targeted Delivery of Doxorubicin via CD147-Mediated ROS/pH Dual-Sensitive Nanomicelles for the Efficient Therapy of Hepatocellular Carcinoma

Low accumulation in tumor sites and slow intracellular drug release remain as the obstacles for nanoparticles to achieve effective delivery of chemotherapeutic drugs. In this study, multifunctional micelles were designed to deliver doxorubicin (Dox) to tumor sites to provide more efficient therapy against hepatic carcinoma. The micelles were based on pH-responsive carboxymethyl chitosan (CMCh) modified with a reactive oxygen species (ROS)-responsive segment phenylboronic acid pinacol ester (BAPE) and an active targeted ligand CD147 monoclonal antibody. The Dox-loaded micelles provided rapid and complete drug release in pH 5.3 incubation conditions with 1 mM H₂O₂. In addition, an in vitro cell uptake study revealed that CD147 modification significantly enhanced cellular internalization due to the high affinity to CD147 receptors, which are overexpressed on tumor cells. An in vivo study revealed that CD147-modified micellar formulations exhibited high accumulation in tumor sites and markedly enhanced antiproliferation effects with fewer side effects than other formulations. In conclusion, this CD147 receptor targeted delivery system with ROS/pH dual sensitivity provides a promising strategy for the treatment of hepatic carcinoma.     

pH敏感释药两亲性壳聚糖共聚物胶束的制备及其性质考察

目的在合成了两亲性接枝共聚物丁酰基-羧甲基-壳聚糖(butyryl-carboxymethyl-chitosan,BR-CM-CS)的基础上,采用化学键合载药方式结合透析法制备了阿霉素pH敏感两亲性共聚物胶束并对其相关性质进行考察。方法利用芘荧光探针技术测定胶束的临界胶束浓度(CMC);通过透析法结合紫外分光光度法测定胶束的载药量及包封率;分别利用透射电镜(TEM)、扫描电镜(SEM)、动态光散射法(DLS)和zeta电位分析仪对胶束及其冷冻干燥产品的形态、粒径和表面电位进行了表征;采用透析法考察了载药聚合物胶束的体外释放行为。结果胶束的CMC值为1.0 mg.L-1,载药量可达12.5%,包封率为89.1%;胶束的粒度分布很窄,平均粒径为205.2 nm;胶束粒子为类球形且分散良好,其表面zeta电位值为25.94 mV;胶束释药行为体现pH敏感性。结论以壳聚糖为载体的化学腙键释药胶束作为抗肿瘤药物的传递系统具有可行性及良好的应用前景。