NGR配体修饰的紫杉醇PEG-PLGA胶束抗肿瘤作用的体外体内研究(英文)

本研究制备了NGR(asparagine-glycine-arginine)配体修饰的紫杉醇PEG-PLGA胶束(NGR-PM-PTX),并对其靶向肿瘤新生血管内皮细胞及肿瘤细胞所表达的氨肽酶N进行了研究。采用薄膜法制备NGR-PM-PTX胶束。通过针对人脐静脉内皮细胞(HUVEC),人纤维肉瘤细胞(HT1080)和人乳腺癌细胞(MCF-7)的流式细胞试验和激光共聚焦实验,在体外细胞水平上研究并证实了NGR配体修饰的聚合物胶束对于上述细胞的靶向效果。HT1080荷瘤裸鼠的体内药效学研究结果进一步证实NGR-PM-PTX的抗肿瘤药效。     

9-硝基喜树碱包合物脂质体的制备与评价

目的:制备9-硝基喜树碱包合物脂质体以提高其抗肿瘤效果。方法:饱和水溶液法制备9-硝基喜树碱包合物,考查制备包合物脂质体的最佳方法和工艺,制备9-硝基喜树碱包合物隐形脂质体并评价其药剂学性质及抗肿瘤效果。结果:9-NC包合物脂质体最佳工艺为乙醇注入法以10 mg胆固醇,720 mg磷脂,1.0 g包合物处方制备。所制备的9-NC包合物脂质体平均粒径为(112.10±0.81) nm,包封率为(60.11±1.08)%,且体外抗肿瘤效果显著提高。结论:包合物脂质体是喜树碱类药物的抗肿瘤应用的有效载体。     

主动靶向脂质体的相变温度与靶细胞摄取的相关性研究

分别采用二硬脂酰磷脂酰胆碱(DSPC)、氢化大豆磷脂(HSPC)、二棕榈酰磷脂酰胆碱(DPPC)和二肉豆蔻酰磷脂酰胆碱(DMPC)为载体,制备了NGR肽(Asn-Gly-Arg)修饰的主动靶向脂质体,并用其负载香豆素-6.结果表明,4种脂质体的粒径为120～160 nm,粒度分布均匀,ζ电位接近中性,包封率均在95％以上.差示扫描量热分析(DSC)表明,4种脂质体的相变温度(Tm)值由高至低分别为DSPC脂质体＞HSPC脂质体＞DPPC脂质体＞DMPC脂质体.以CD13高表达的HT1080细胞为模型,采用流式细胞仪、激光共聚焦显微镜观察评价脂质体的入胞效果.结果显示,脂质体的入胞效率与Tm值呈正相关.     

RGD类似物修饰的阿霉素隐形脂质体的制备及体外细胞结合试验

目的研究用精氨酸-甘氨酸-天冬氨酸(RGD)类似物(RGDm)修饰隐形脂质体(SL)，以增加抗癌药物在肿瘤部位积蓄的同时，增加抗癌药物向肿瘤细胞内的传递。方法合成RGDm，将其通过PEG链与二硬脂酰磷脂酰乙醇胺(DSPE)连接形成导向化合物DSPE-PEG-RGDm，在此基础上制备RGDm修饰的隐形脂质体(RGDm-SL)，阿霉素(DOX)作为模型药物通过硫酸铵梯度法装载。体外实验中，用pH探针(BCECF-AM)标记黑色素瘤细胞，通过细胞黏附试验考察导向化合物与肿瘤细胞的黏附情况；通过流式细胞实验和激光共聚焦显微实验考察肿瘤细胞对SL包封的阿霉素(SL-DOX)及RGDm-SL包封的阿霉素(RGDm-SL-DOX)的结合或摄取情况。结果与DSPE-PEG相比，黑色素瘤细胞与导向化合物DSPE-PEG-RGDm的黏附显著增加，过量游离RGDm的加入能抑制其黏附；与SL-DOX相比，RGDm-SL-DOX与黑色素瘤细胞共同孵育后，细胞对阿霉素的结合及摄取均显著增加。结论RGDm修饰的隐形脂质体可作为肿瘤靶向的载体通过受体介导的方式促进抗肿瘤药物向肿瘤细胞内的传递。     

吐温80对9-硝基喜树碱脂质体药物动力学性质的影响

目的:考察吐温80对9-硝基喜树碱(9-NC)脂质体体内药物动力学以及内酯型/羧酸盐型平衡的影响。方法:采用薄膜法制备9-NC脂质体以及吐温80修饰的9-NC脂质体;12只大鼠随机分为两组,按1.5 mg.kg-1剂量分别给予9-NC普通脂质体和吐温80修饰的脂质体,于不同时间点取血,处理后测定9-NC内酯型浓度和总浓度(内酯/羧酸盐)。采用统计矩模型利用3P97程序计算药物动力学参数。结果:采用表面活性剂吐温80进行修饰后,9-NC内酯型和总浓度的AUC分别提高了1.47倍和1.65倍,内酯型和总浓度的清除率CL和表观分布容积Vss显著下降(P<0.01)。此外,总浓度的MRT以及t1/2延长(P<0.05)。结论:吐温80修饰使得9-NC内酯型比例有所下降,但没有显著性差异,9-NC吐温修饰对9-NC脂质体具有一定的长循环效果。     

穿膜肽介导紫杉醇脂质体对肿瘤的治疗作用

目的制备和表征F3修饰紫杉醇脂质体,并进行细胞学和体内抗肿瘤评价。方法采用薄膜分散法制备脂质体,人肺腺癌细胞A549进行摄取评价;小鼠肝癌细胞H22荷瘤小鼠进行体内抗肿瘤活性评价。结果 F3修饰脂质体粒径为90 nm;该修饰后脂质体被细胞摄取能力明显增强,进而提高了药物的抗肿瘤活性,给药16天后,F3-Lipo组瘤体增长仅为11倍,相比于生理盐水组26倍,Taxol 18倍,未修饰脂质体14倍,有更好的抗肿瘤效果。结论 F3穿膜肽可以明显提高载体入胞能力,进而改善药物治疗作用。     

柔红霉素隐形脂质体的药剂学性质

本文研究了柔红霉素隐形脂质体的药剂学性质。考察了柔红霉素隐形脂质体的形态和粒径分布;考察了柔红霉素隐形脂质体的包封率和脂质体在Hepes缓冲液(pH 7.5)和大鼠血清中的体外释放行为。结果表明所制备的柔红霉素隐形脂质体包封率高(>85%),平均粒径为56.3 nm,体外释放慢。结论认为制备的隐形脂质体包封率较高,药剂学性质较好,适于临床使用。     

RGD修饰载紫杉醇脂质体用于抗脑胶质瘤的体外研究

目的制备RGD修饰载紫杉醇脂质体并考察其体外抗脑胶质瘤治疗效果。方法采用薄膜分散法制备RGD修饰载紫杉醇脂质体（RGD—LP-PTX）,考察其理化性质。用鼠源性脑胶质瘤c6细胞考察肿瘤细胞对脂质体的摄取效率。MTT实验考察脂质体对肿瘤细胞的增殖抑制率。结果制备得到的RGD—LP粒径在120nm左右,PD,〈0.3。C6细胞对RGD．LP的摄取能力显著〉LP;RGD-LP-PTX对C6细胞的生长的抑制作用显著强于LP。结论经过RGD修饰后,脂质体具有良好的脑胶质瘤细胞靶向性,且能够显著增强栽药脂质体对脑胶质瘤细胞的增殖抑制作用。     

结肠癌靶向米托蒽醌脂质体的制备及其抗肿瘤活性研究

目的:比较透明质酸(HA)修饰的脂质体(HA-LP)与普通脂质体(LP)的抗转移及抗肿瘤活性。方法:采用薄膜水化法制备空白脂质体,通过透明质酸上的羧基与膜材中DSPE的氨基反应将HA连接在脂质体上,运用硫酸铵梯度法包载模型药物米托蒽醌;通过透射电镜表征脂质体形态;免疫荧光法检测结肠癌细胞(CT26 cells)中透明质酸受体CD44表达情况;高内涵扫描仪检测结肠癌细胞对脂质体的摄取情况;划痕实验考察2组脂质体抑制细胞转移的能力;体内抗肿瘤实验考察2组脂质体的抑瘤效果。结果:所制备的脂质体形态均一,包封率均大于95%。免疫荧光结果显示,结肠癌细胞中CD44受体高度表达。相同的药物浓度下,HA-LP相较于未修饰的脂质体具有明显增加的细胞摄取。划痕实验结果表明,HA可能与受体CD44存在某种相互作用,抑制了结肠癌细胞的转移,表现出HA-LP具有更强的抗转移活性。体内抗肿瘤结果显示,HA-LP对小鼠荷结肠癌实体瘤的抑瘤率为62.1%,显著高于普通脂质体组(46.6%,P<0.05)。结论:HA-LP与细胞膜上的受体CD44作用,显著增加了其细胞摄取,并抑制了结肠癌细胞的转移,体内抑瘤结果同样显示,具有肿瘤靶向功能的HA-LP具有更强的抑制肿瘤生长的能力。     

甘草次酸修饰的阿霉素靶向脂质体的制备及体外抑瘤活性考察

目的：制备甘草次酸（GA）靶向阿霉素脂质体（GA-DOX-Lip）,并初步考察其体外抑瘤活性。方法：采用硫酸铵梯度载药法制备GA-DOX-Lip,单因素考察其处方工艺;采用微柱离心法和粒径仪考察脂质体包封率及其理化性质;通过荧光显微镜和流式细胞仪考察BEL-7402肝癌细胞对脂质体的摄取情况并用MTT法评价其体外杀伤效果。结果：GA-DOX-Lip形态圆整,粒径约为120 nm,包封率达到95%以上,72 h释放约20%;体外细胞摄取量和细胞杀伤效果均显著高于阿霉素普通脂质体。结论：甘草次酸修饰的脂质体有望成为肝肿瘤靶向的新型载体,促进抗肿瘤药物向肿瘤细胞内的递送。     

9-硝基喜树碱纳米脂质载体系统的释放、细胞摄取及组织分布特性

目的研究9-硝基喜树碱纳米脂质载体系统的体外释放、巨噬细胞摄取及体内组织分布特性。方法以BLB/C小鼠腹腔巨噬细胞为细胞模型进行9-硝基喜树碱纳米脂质载体系统的体外细胞吞噬实验；以一级昆明种小鼠为动物模型进行体内组织分布实验；并进行多晶X-射线衍射及体外释放实验的测定。结果9-硝基喜树碱可能以无定形物分散在纳米脂质载体中，液态脂质的加入并没有改变纳米粒固态内核的性质；体外释放实验表明纳米脂质载体系统具有一定的缓释特性；PEG的修饰使巨噬细胞对纳米粒的摄取减少，且能够增强纳米粒抵抗蛋白吸附的能力；纳米脂质载体系统能够延长9-硝基喜树碱在血中的滞留时间，并改变其在小鼠体内的分布特征。结论由Myrj59制备的纳米脂质载体系统具有缓释特性，且具有良好的长循环性及肝、肺靶向性。     

Synthesis and evaluation of novel Tc‐99 m labeled NGR‐containing hexapeptides as tumor imaging agents

Asparagine‐glycine‐arginine (NGR)‐containing peptides targeting aminopeptidase N (APN)/CD13 can be an excellent candidate for targeting ligands in molecular tumor imaging. In this study, we developed two NGR‐containing hexapeptides, and evaluated the diagnostic performance of Tc‐99 m labeled hexapeptides as molecular imaging agents in an HT‐1080 fibrosarcoma‐bearing murine model. Peptides were synthesized using Fmoc solid‐phase peptide synthesis. Radiochemical purity of Tc‐99 m was evaluated using instant thin‐layer chromatography. The uptake of two NGR‐containing hexapeptides within HT‐1080 cells was evaluated in vitro. In HT‐1080 fibrosarcoma tumor‐bearing mice, gamma images were acquired. A biodistribution study was performed to calculate percentage of the injected dose per gram of tissue (%ID/g). Two hexapeptides, glutamic acid‐cysteine‐glycine (ECG)‐NGR and NGR‐ECG were successfully synthesized. After radiolabeling procedures with Tc‐99 m, the complexes Tc‐99 m hexapeptides were prepared in high yield. The uptake of Tc‐99 m ECG‐NGR within the tumor cells had been assured by in vitro studies. The gamma camera imaging in the murine model showed that Tc‐99 m ECG‐NGR was accumulated substantially in the subcutaneously engrafted tumor. However, Tc‐99 m NGR‐ECG was accumulated minimally in the tumor. Two NGR‐containing hexapeptides, ECG‐NGR and NGR‐ECG were developed as molecular imaging agents to target APN/CD13 in HT‐1080 fibrosarcoma. Tc‐99 m ECG‐NGR showed a significant uptake in the tumor, and it is a good candidate for tumor imaging.     

藏药余甘子鞣质部位对HT1080细胞迁移与侵袭能力的影响研究

目的：探讨藏药余甘子鞣质部位对人纤维肉瘤细胞（HT1080）迁移和侵袭能力的影响。方法采用四甲基偶氮唑盐（MTT）法检测余甘子鞣质部位对HT1080细胞活力的影响。分别采用划痕实验和Transwell侵袭实验法检测余甘子鞣质部位对HT1080细胞迁移和侵袭能力的影响。结果余甘子鞣质部位作用48h能够显著抑制HT1080细胞的增殖,其半数抑制浓度（IC50）为0.174g·L^-1。与对照组比较,余甘子鞣质部位质量浓度为0.1g·L^-1,作用3h后即可明显降低HT1080细胞迁移面积;在质量浓度为0.025g·L^-1时可明显降低HT1080细胞透过胶膜的数量,降低其侵袭能力。结论余甘子鞣质部位可有效抑制人纤维肉瘤细胞迁移和侵袭能力。     

Uptake characteristics of NGR-coupled stealth PEI/pDNA nanoparticles loaded with PLGA-PEG-PLGA tri-block copolymer for targeted delivery to human monocyte-derived dendritic cells

We have investigated the in vitro uptake, toxicity, phenotypic consequences and transfection efficiency of a stealth NGR/PEG/PDBA-coupled-SHA-PEI/pDNA targeting polyplex loaded with PLGA-PEG-PLGA tri-block copolymer in human monocyte-derived dendritic cells (DCs). Modification with PEG effectively shielded and reduced non-specific phagocytosis by immature DCs to approximately 20%. Coupling the NGR cell-specific peptide to the PEGylated polyplex (NGR/PEG/PDBA-SHA-PEI/pDNA) however resulted in specific and enhanced phagocytosis in DCs without any observable toxicity at the optimum concentration of 0.25% of the copolymer. DNase treatment had no effect on DNA integrity in the encapsulated polyplex. Confocal microscopy confirmed intracellular localization of the targeting NGR/PEG/PDBA-SHA-PEI/pDNA microparticles, resulting in more enhanced uptake of the radiolabeled plasmid DNA and approximately 5- and 10-fold increase over the control tri-block Pluronic F68 copolymer and the non-targeting polyplex, respectively. More importantly, phagocytosis of the targeting microparticles neither altered the functionality of immature DCs nor the phenotypic expression of DC-specific cell surface molecules, CD80, CD86, CD40 and CD54 (ICAM-1), suggesting that uptake of the targeting microparticles by themselves did not induce DC maturation. Taken together, these results suggest that PLGA-PEG-PLGA encapsulation of this stealth targeting polyplex has no negative effects on key properties of immature DCs and should pave the way for targeting DCs for vaccination purposes.     

9-nitrocamptothecin polymeric nanoparticles: cytotoxicity and pharmacokinetic studies of lactone and total forms of drug in rats

The objective of this study was to evaluate the cytotoxicity and pharmacokinetics of total and lactone forms of 9-nitrocamptothecin (9-NC), an effective antineoplastic drug, after intravenous injection of drug incorporated into poly (DL-lactic-glycolic acid) nanoparticles (NPs). Drug-loaded NPs (9-NC.NP) were prepared by the nanoprecipitation method and examined for particle characteristics and in-vitro release in phosphate buffered saline. The best formulation showed a narrow size with an average diameter of 207+/-26 nm and a drug loading of more than 33.5%. The drug release profile showed a sustained 9-NC release up to 160 h. For a pharmacokinetic study, the concentration of 9-NC as the lactone form (9-NC.lac) and as the total of the lactone and carboxylate forms (9-NC.tot) in plasma was determined by using reverse-phase high performance liquid chromatography after intravenous administration of 9-NC.NP and a control solution to cannulated Wistar rats. In-vitro cytotoxic activity of 9-NC.NP and control solution was evaluated on the human ovarian cancer cell line (A2780sn) by MTT cell cytotoxicity assay. Results of in-vivo studies showed that NP encapsulation markedly increased the plasma concentration of both lactone and total forms of 9-NC compared with free drug. In comparison with free drug, NPs resulted in 3.63-fold and 5.40-fold increases in area under the plasma concentration-versus-time curve (AUC(0-infinity)) for lactone and total forms of 9-NC, respectively. The values of mean residence time and elimination half-life (T(1/2)) were also significantly higher for NPs than for free drug. The in-vitro cytotoxicity study revealed that the IC50 value of NPs decreased 10-fold compared with the drug solution. Prepared NPs described here were considered potentially useful in both stabilizing and delivering 9-NC and enhancing the efficacy of this drug for cancer treatment for which high drug retention in the body, protection from the drug-active lactone form, and gradual drug release appeared to be related.     

Preparation and comparative evaluation of 99mTc‐HYNIC‐cNGR and 99mTc‐HYNIC‐PEG2‐cNGR as tumor‐targeting molecular imaging probes

The tripeptide sequence asparagine‐glycine‐arginine (NGR) specifically recognizes aminopeptidase N (APN or CD13) receptors highly expressed on tumor cells and vasculature. Thus, NGR peptides can precisely deliver therapeutic and diagnostic compounds to CD13 expressing cancer sites. In this regard, 2 NGR peptide ligands, HYNIC‐c(NGR) and HYNIC‐PEG₂‐c(NGR), were synthesized, radiolabeled with ^99mTc, and evaluated in CD13‐positive human fibrosarcoma HT‐1080 tumor xenografts. The radiotracers, ^99mTc‐HYNIC‐c(NGR) and ^99mTc‐HYNIC‐PEG₂‐c(NGR), could be prepared in approximately 95% radiochemical purity and exhibited excellent in vitro and in vivo stability. The radiotracers were hydrophilic in nature with log P values being ?2.33 ± 0.05 and ?2.61 ± 0.08. The uptake of 2 radiotracers ^99mTc‐HYNIC‐c(NGR) and ^99mTc‐HYNIC‐PEG₂‐c(NGR) was similar in nude mice bearing human fibrosarcoma HT‐1080 tumor xenografts, which was significantly reduced (P < .05) during blocking studies. The 2 radiotracers being hydrophilic cleared rapidly from blood, liver, and intestine and were excreted through renal pathway. The pharmacokinetics of ^99mTc‐labeled HYNIC peptide could not be modulated through introduction of PEG₂ unit, thus posing a challenge for studies with other linkers towards enhanced tumor uptake and retention.     

Comparative evaluation of 68Ga‐labeled NODAGA,DOTAGA, and HBED‐CC‐conjugated cNGR peptide chelates as tumor‐targeted molecular imaging probes

The biological behavior of ⁶⁸Ga‐based radiopharmaceuticals can be significantly affected by the chelators’ attributes (size, charge, lipophilicity). Thus, this study aimed at examining the influence of three different chelators, DOTAGA, NODAGA, and HBED‐CC on the distribution pattern of ⁶⁸Ga‐labeled NGR peptides targeting CD13 receptors. ⁶⁸Ga‐DOTAGA‐c(NGR), ⁶⁸Ga‐NODAGA‐c(NGR), and ⁶⁸Ga‐HBED‐CC‐c(NGR) were observed to be hydrophilic with respective log p values being −3.5 ± 0.2, −3.3 ± 0.08, and −2.8 ± 0.14. The three radiotracers exhibited nearly similar uptake in human fibrosarcoma HT‐1080 tumor cells with 86%, 63%, and 33% reduction during blocking studies with unlabeled cNGR peptide for ⁶⁸Ga‐DOTAGA‐c(NGR), ⁶⁸Ga‐NODAGA‐c(NGR), and ⁶⁸Ga‐HBED‐CC‐c(NGR), respectively, indicating higher receptor specificity of the first two radiotracers. The neutral radiotracer ⁶⁸Ga‐NODAGA‐c(NGR) demonstrated better target‐to‐non‐target ratios during in vivo studies compared to its negatively charged counterparts, ⁶⁸Ga‐DOTAGA‐c(NGR) and ⁶⁸Ga‐HBED‐CC‐c(NGR). The three radiotracers had similar HT‐1080 tumor uptake and being hydrophilic exhibited renal excretion with minimal uptake in non‐target organs. Significant reduction (p < .005) in HT‐1080 tumor uptake of the radiotracers was observed during blocking studies. It may be inferred from these studies that the three radiotracers are promising probes for in vivo imaging of CD13 receptor expressing cancer sites; however, ⁶⁸Ga‐NODAGA‐c(NGR) is a better candidate.     

9-硝基喜树碱在大鼠组织中的分布及内酯稳定性

考察9-硝基喜树碱(9-NC)静脉注射后在人鼠组织中的分布及内酯稳定性.建立了HPLC法间时测定组织和血浆中9-NC内酯浓度和总浓度.大鼠静脉注射9-NC溶液后测定各时间点组织中内酯浓度、总浓度和内酯比例.大多数组织中的9-NC内酯比例明显高于血浆;肝中的内酯比例最低,甚至低于血浆;血浆、肾和小肠中的内酯比例随时问延长而下降.9-NC在肝以外的组织中内酯稳定性显著优于血浆.     

Pharmacokinetics of lactone, carboxylate and total 9-nitrocamptothecin with different doses and administration routes in rats

9-Nitrocamptothecin (9-NC) is a newly developed poorly soluble derivative of camptothecin and has a wide spectrum of anticancer activity in preclinical evaluation. The effects of the dose and administration route on pharmacokinetics and lactone/carboxylate equilibrium of 9-NC were studied in rats. A single intravenous dose of 1.5, 3 or 6 mg/kg of 9-NC solution was given to male rats (n = 6 per dose level). In another study, a single dose of 6 mg/kg 9-NC solution was given orally to rats (n = 6). Plasma samples were drawn at predetermined intervals and the concentrations of lactone, carboxylate and total 9-NC were determined by a validated HPLC method. Pharmacokinetic analysis was performed using non-compartmental analysis. Analysis of variance showed that the pharmacokinetic characteristics of lactone, carboxylate and total 9-NC were all independent of dose (p > 0.05). Based on the AUC measurements, the lactone 9-NC constituted 52% +/- 4%, 49% +/- 6% and 55% +/- 6% of the circulating total 9-NC in rats after intravenous administration of 1.5, 3, 6 mg/kg 9-NC solution, respectively. After oral administration of 6 mg/kg, the pharmacokinetics parameters were significantly different from those of intravenous administration at the same dose (p < 0.05). The lactone ratio was 60% +/- 14%. The absolute bioavailability of lactone and total 9-NC were calculated to be 23.4% and 22.7%, respectively. In conclusion, the pharmacokinetics of lactone, carboxylate and total 9-NC are not dose-dependent. Lactone, carboxylate and total 9-NC are poorly absorbed following oral administration. Both the dose and the route of administration have little effect on the lactone/carboxylate equilibrium of 9-NC in rats in vivo. But the route of administration plays an important part on the pharmacokinetics of 9-NC.