APP/PS1小鼠中PEG-PLA纳米粒的表面蛋白冠组成及其脑内递送的实验研究

摘要 目的：研究阿尔茨海默病（Alzhemer''s disease,AD）模型鼠中聚乙二醇聚乳酸（poly(ethylene glycol)-poly(l-lactide),PEG-PLA）纳米粒表面蛋白冠组成及其对脑内递送特性的影响。方法：制备PEG-PLA纳米粒,测定纳米粒的zeta电位及粒径,采用透射电子显微镜观察纳米粒形态。通过双光子显微镜观察APP/PS1小鼠与野生型（Wild Type,WT）小鼠脑内PEG-PLA纳米粒分布特性。采用液相色谱-质谱联用（LC-MS）技术对PEG-PLA纳米粒分别与APP/PS1小鼠和WT小鼠血浆孵育形成的两种不同蛋白冠进行蛋白组学分析。结果：制备的PEG-PLA纳米粒粒径均一,分散性较好。静脉注射PEG-PLA后,APP/PS1小鼠脑内纳米粒量明显高于WT小鼠。蛋白质组学结果显示,APP/PS1小鼠血浆孵育组PEG-PLA纳米粒表面蛋白冠中凝聚素（Clusterin）明显高于WT小鼠血浆孵育组,该蛋白与纳米粒逃避机体清除有关。此外,纳米粒蛋白冠中血管性血友病因子（Von Willebrand factor）、玻连蛋白（Vitronectin）、肌球蛋白重链-9（Myosin-9）等参与细胞粘附作用相关蛋白在APP/PS1小鼠血浆孵育组也明显多于WT小鼠血浆孵育组。结论：PEG-PLA纳米粒在AD模型小鼠中表现出的高入脑量,可能与AD疾病影响纳米粒蛋白冠组成有关。     

抗肿瘤多药耐药纳米粒的制备及其对MCF-7/ADR 细胞的体外评价

目的:肿瘤的多药耐药现象会显著降低肿瘤细胞内药物浓度,本研究通过制备抗肿瘤多药耐药的靶向给药系统来逆转肿瘤的耐药性以提升细胞对药物的敏感性,从而降低该现象对癌症治疗的阻碍。方法:本文使用乳化溶剂挥发法制备以含姜黄素两亲性嵌段共聚物载体、以紫杉醇和磁性粒为核心的抗肿瘤多药耐药纳米粒,使用透射电镜和动态粒径散射仪等对纳米粒进行表征和磁响应性测试后,使用MTT法测定纳米粒对肿瘤耐药细胞MCF-7/ADR的抑制率以探究给药系统的耐药逆转性能。结果:制备的抗肿瘤多耐药纳米粒粒径为105 nm左右,磁响应性良好。所制得载紫杉醇纳米粒包封率为74.74%,载药率为12.40%。纳米粒可以通过磁场和生物素受体介导作用促进肿瘤细胞对粒子的内化,以增加抗癌药物的蓄积。与游离紫杉醇相比,逆转细胞耐药指数达8.5。结论:纳米系统在维持自身稳定性同时,能够凭借协同作用和靶向作用较大程度提升药物对耐药肿瘤细胞的杀伤效果。     

酸敏阿霉素脂质纳米粒的制备及其体外抗肿瘤活性研究

目的:本研究旨在制备具有被动靶向和酸敏特性的脂质混合纳米粒,以期提高阿霉素(doxorubicin,DOX)的靶向递药效率,降低DOX的毒副作用,提高抗肿瘤活性。方法:采用微乳法制备磷酸钙纳米粒核,薄膜分散法制备脂质混合纳米粒,硫酸铵梯度法包封DOX。采用透射电镜观察外观形态,用zeta电位及纳米粒度分析仪测定纳米粒的粒径及zeta电位,透析法评价阿霉素脂质纳米粒体外释药特征。用MTT方法研究阿霉素脂质混合纳米粒对A549细胞的细胞毒性。采用流式细胞仪和激光共聚焦显微镜观察A549细胞对阿霉素脂质纳米粒的摄取。结果:体外释药结果显示阿霉素脂质纳米粒具有酸敏特性。流式结果说明A549细胞对阿霉素脂质纳米粒的摄取具有明显的时间依赖性,激光共聚焦显示阿霉素脂质纳米粒能将阿霉素递送至细胞核中。结论:阿霉素脂质体对A549细胞有明显的细胞毒性,为进一步进行体内实验提供了基础。     

聚乙二醇-聚乳酸嵌段共聚物在药物递送系统中的应用

聚乙二醇-聚乳酸嵌段共聚物具备良好的生物相容性和生物可降解性,是良好的纳米级药物载体。嵌段共聚物具有载药能力强、粒径小、体内循环时间长、主动靶向性和被动靶向性等特点,因此在药物递送系统中得到广泛应用。简要介绍了聚乙二醇-聚乳酸嵌段共聚物的合成和性质,及其作为脂质体、胶束、微球等载体在药物递送系统中的最新进展。     

乳铁蛋白修饰纳米粒包载的α-倒捻子素对AD模型动物脑内特征性病理改变的影响

目的:采用乳铁蛋白修饰的PEG-PLA纳米粒为递药工具包载α-M,探讨其对快速老化SAMP8小鼠AD相关脑内病理特征的改善作用。方法:用乳化/溶剂蒸发法制备载α-M的PEG-PLA纳米粒NP(α-M),将巯基化的乳铁蛋白连接于纳米粒表面,得到Lf-NP(α-M)。7月龄SAMP8系小鼠尾静脉给予注射生理盐水、Lf-NP(α-M)或空白纳米粒溶液,每日一次,连续两周。正常老化小鼠SAMR为模型对照组,通过对脑组织进行免疫组化分析,观察Lf-NP(α-M)对SAMP8小鼠脑内炎症、Aβ沉积等AD特征性病理变化的影响。结果:0.5、2 mg/kgα-M对SAMP8小鼠脑内小胶质细胞激活、星形胶质细胞增生以及Aβ沉积均无显著影响;0.5mg/kg Lf-NP(α-M)可抑制小胶质细胞的激活(P0.001),2 mg/m L Lf-NP(α-M)显著抑制星形胶质细胞增生以及Aβ沉积(P0.05)。结论:乳铁蛋白修饰的包载α-M的可降解纳米粒脑靶向递药系统成功有效,显著提高α-M的成药性并改善AD模型小鼠脑内特征性病理改变。     

叶酸-壳聚糖Prdx6 shRNA纳米粒的构建及其对胃癌细胞增殖的影响

目的:探讨叶酸-壳聚糖Prdx6 shRNA纳米粒对胃癌细胞生长的影响。方法:制备靶向性叶酸-壳聚糖Prdx6 shRNA纳米粒,原子力显微镜观察其形态,激光粒度分析仪测定纳米粒的粒径;倒置荧光显微镜观察叶酸-壳聚糖Prdx6 shRNA纳米粒的转染效率;采用蛋白质印迹法检测胃癌细胞Prdx6蛋白的表达变化;CCK8细胞增殖实验检测胃癌细胞的存活率。结果:1制备成功叶酸-壳聚糖Prdx6 shRNA向纳米粒。2荧光显微镜下观察靶向性叶酸-壳聚糖Prdx6 shRNA纳米粒转染胃癌细胞的效率明显高于非靶向纳米粒;胃癌细胞转染靶向组纳米粒后Prdx6蛋白的表达显著低于非靶向组。3与对照组相比,叶酸-壳聚糖Prdx6 shRNA纳米粒能够明显抑制胃癌细胞的增殖(P0.01)。结论:1叶酸-壳聚糖Prdx6 shRNA纳米粒可高效转染胃癌细胞。2转染叶酸-壳聚糖Prdx6 shRNA纳米粒后胃癌细胞的生长明显受抑制。     

转铁蛋白修饰磁性纳米粒的制备及其体外抗肿瘤活性研究

目的:本研究旨在构建一种转铁蛋白修饰负载阿霉素(DOX)的磁纳米粒靶向递药系统,以提高阿霉素作用的靶向性。方法:采用化学共沉淀法制备转铁蛋白修饰负载阿霉素的磁性纳米粒(DOX@MNP),采用zeta电位及纳米粒度分析仪测定DOX@MNP的粒径及其zeta电位,透析法评价DOX@MNP的体外释药特征。通过MTT实验,研究DOX@MNP与游离DOX对A549细胞的细胞毒性,通过激光共聚焦显微镜和流式细胞仪观察A549细胞对DOX@MNP与游离DOX的摄取情况。结果:DOX@MNP的释药具有p H依赖性。MTT实验结果显示,DOX@MNP与游离DOX具有相当的细胞毒性;激光共聚焦显微镜和流式细胞仪检测结果显示A549细胞对DOX和DOX@MNP的摄取没有明显差异。结论:本文构建了一种转铁蛋白修饰包载阿霉素的磁纳米粒,体外结果显示其具有与游离DOX相当的细胞毒性,为进一步进行体内实验奠定了基础。     

载药纳米粒作为脑靶向给药系统的研究

血脑屏障使大部分的活性药物很难由血液进入脑内发挥作用。载药纳米粒具有脑靶向性,可显著提高药物在脑内浓度,成为药物突破血脑屏障的有效途径。本文综述了近年来载药纳米粒透过血脑屏障的研究进展,并对纳米粒载中药入脑提出展望。     

载药纳米粒作为脑靶向给药系统的研究

血脑屏障使大部分的活性药物很难由血液进入脑内发挥作用。载药纳米粒具有脑靶向性,可显著提高药物在脑内浓度,成为药物突破血脑屏障的有效途径。本文综述了近年来载药纳米粒透过血脑屏障的研究进展,并对纳米粒载中药入脑提出展望。     

叶酸白蛋白靶向纳米粒的肿瘤细胞吸收及叶酸受体相关基因表达研究

实验选用叶酸受体表达阳性FR(+)卵巢癌细胞株SKOV3与表达阴性的FR(-)肺癌细胞株A549,通过肿瘤细胞对叶酸白蛋白靶向纳米粒的吸收及叶酸受体相关基因的表达,研究分析叶酸白蛋白靶向纳米粒叶酸受体吸收特征及叶酸受体胞吞途径的参与机制。结果表明:随着叶酸白蛋白靶向纳米粒给药时间的增加,SKOV3细胞FR1基因的表达显著增强,而A549细胞未见FR1基因表达。同时SKOV3细胞FR1基因的表达程度与细胞摄取叶酸白蛋白靶向纳米粒的量成正相关。叶酸代谢通路相关的基因(RFC、FPGS、GGH)表达,研究显示叶酸白蛋白靶向纳米粒能够启动细胞膜叶酸通道相关蛋白的表达。     

Temporal control over cellular targeting through hybridization of folate-targeted dendrimers and PEG-PLA nanoparticles

Polymeric nanoparticles (NPs) and dendrimers are two major classes of nanomaterials that have demonstrated great potential for targeted drug delivery. However, their targeting efficacy has not yet met clinical needs, largely because of a lack of control over their targeting kinetics, which often results in rapid clearance and off-target drug delivery. To address this issue, we have designed a novel hybrid NP (nanohybrid) platform that allows targeting kinetics to be effectively controlled through hybridization of targeted dendrimers with polymeric NPs. Folate (FA)-targeted generation 4 poly(amidoamine) dendrimers were encapsulated into poly(ethylene glycol)-b-poly(D,L-lactide) (PEG-PLA) NPs using a double emulsion method, forming nanohybrids with a uniform size (~100 nm in diameter) at high encapsulation efficiencies (69-85%). Targeted dendrimers encapsulated within the NPs selectively interacted with FA receptor (FR)-overexpressing KB cells upon release in a temporally controlled manner. The targeting kinetics of the nanohybrids were modulated using three different molecular weights (MW) of the PLA block (23, 30, and 45 kDa). The release rates of the dendrimers from the nanohybrids were inversely proportional to the MW of the PLA block, which dictated their binding and internalization kinetics with KB cells. Our results provide evidence that selective cellular interactions can be kinetically controlled by the nanohybrid design, which can potentially enhance targeting efficacy of nanocarriers.     

Paclitaxel-loaded poly(gamma-glutamic acid)-poly(lactide) nanoparticles as a targeted drug delivery system against cultured HepG2 cells

The study was to develop paclitaxel-loaded formulations using a novel type of self-assembled nanoparticles that was composed of block copolymers synthesized from poly(gamma-glutamic acid) and poly(lactide) via a simple coupling reaction. The nanoparticles (the NPs) were prepared with various feed weight ratios of paclitaxel to block copolymer (the P/BC ratio). The morphology of all prepared nanoparticles was spherical and the surfaces were smooth. Increasing the P/BC ratio significantly increased the drug loading content of the prepared nanoparticles, but remarkably reduced the drug loading efficiency. The release rate of paclitaxel from the NPs decreased significantly as the P/BC ratio increased. For the potential of targeting liver cancer cells, galactosamine was further conjugated on the prepared nanoparticles (the Gal-NPs) as a targeting moiety. It was found that the activity in inhibiting the growth of HepG2 cells (a liver cancer cell line) by the Gal-NPs was comparable to that of a clinically available paclitaxel formulation, while the NPs displayed a significantly less activity. This may be attributed to the fact that the Gal-NPs had a specific interaction with HepG2 cells via ligand-receptor recognition. Cells treated with distinct paclitaxel formulations resulted in arrest in the G2/M phase. The arrest of cells in the G2/M phase was highly suggestive of interference by paclitaxel with spindle formation and was consistent with the morphological findings presented herein. In conclusion, the active targeting nature of the Gal-NPs prepared in the study may be used as a potential drug delivery system for the targeted delivery to liver cancers.     

PEG-PLA nanoparticles decorated with small-molecule PSMA ligand for targeted delivery of galbanic acid and docetaxel to prostate cancer cells

Prostate cancer (PCa) is one of the most prevalent non-drug delivery system cutaneous malignancies. Undoubtedly, introducing novel treatment options to achieve higher therapeutic index will be worthwhile. In this study, we report for the first time, a novel targeted self-assembled based on PEG-PLA nanoparticles (PEG-PLA NPs) containing galbanic acid (GBA) and docetaxel, which was targeted using ((S)-2-(3-((S)-5-amino-1-carboxypentyl) ureido) pentanedioic acid (ACUPA), a small molecule inhibitor targeting prostate-specific membrane antigen (PSMA), in prostate cancer cell line. The prepared NPs were characterized by different analytical methods. The MTT assay was used to compare the anti-proliferation of drugs-loaded PEG-PLA NPs and ACUPA-PEG-PLA against LNCaP (PSMA⁺) and PC3 (PSMA⁻) cells. PEG-PLA NPs with an average size of 130–140 nm had an enhanced release of GBA and docetaxel at pH 5.5 compared with pH 7.5. Spectrofluorometric analysis suggested that ACUPA-modified PEG-PLA could effectively enhance the drug uptake in PSMA⁺ prostate cancer cells. Cytotoxicity studies showed that the targeted NPs loaded with different concentrations of GBA and fixed concentration of docetaxel (4 nM) have shown higher toxicity (IC₅₀ 30 ± 3 µM) than both free GBA (80 ± 4.5 µM) and nontargeted NPs (IC₅₀ 40 ± 4.6 µM) in LNCaP cells. Collectively, these findings suggest that ACUPA-conjugated PEG-PLA nanosystem containing GBA and docetaxel is a viable delivery carrier for various cancer-targeting PSMA that suffer from short circulation half-life and limited therapeutic efficacy.     

Diblock copolymers based on dihydroxyacetone and ethylene glycol: synthesis, characterization, and nanoparticle formulation

Polymeric biomaterials have played an integral role in tissue engineering, biomedical devices, and targeted drug delivery. Block copolymers are especially important because their physical and chemical properties can be controlled by adjusting the ratio, size, and type of constituting blocks. Herein, the synthesis and characterization of diblock copolymers composed of poly(ethylene glycol) and a polycarbonate based on the metabolic intermediate, dihydroxyacetone, are reported. The length of the dihydroxyacetone-based block was controlled by adjusting the reactant feed ratios and initiator injection conditions. Intermediates and final products were characterized via (1)H NMR, GPC, DSC, TGA, and diffusion-ordered NMR spectroscopy. The dihydroxyacetone-based hompolymer is insoluble in water and most organic solvents, but is hydrophilic in nature. This, coupled with poly(ethylene glycol)'s solubility characteristics, allows the block copolymer to form nanoparticles in aqueous and organic anti-solvents. Dynamic light scattering and TEM results indicated the formation of spherical nanoparticles.     

Preparation of micelle-forming polymer-drug conjugates.

Adriamycin, a hydrophobic anticancer drug, was conjugated with poly(ethylene oxide)-poly(aspartic acid) block copolymers composed of various lengths of each block copolymer segment ranging from 1000 to 12,000 in molecular weight and from 10 to 80 units, respectively. Conjugation was achieved without precipitation by adjusting the ratio of adriamycin to aspartic acid residues of the block copolymer and the quantity of DMF used for the reaction. Thus obtained conjugates showed high water solubility irrespective of a large amount of the conjugated adriamycin. Furthermore, these conjugates were found to form micellar structures with a hydrophobic inner core and a hydrophilic outer shell. This micellar architecture may be utilized for effective drug targeting.     

生物可降解嵌段共聚物在给药载体中的应用

生物可降解嵌段聚合物因具有双亲性 ,靶向药物到特定部位等优点大大推动了作为给药载体系统的发展。本文综述了生物可降解嵌段聚合物在表面修饰、水凝胶、胶束、生物大分子载体系统中的应用     

靶向肿瘤新生血管纳米粒的构建和质量控制

目的：制备F56多肽修饰的长春新碱纳米粒（F56-VCR-NP）,并建立其质量控制方法。方法：乳化－溶剂挥发法优化制备F56．VCR-NP：HPLC法测定其载药量、包封率,透射电镜下观察其形态,激光粒度分析仪测定其粒径和Zeta电位,CBQCA试剂盒测定纳米粒表面多肽密度,XPS进行表面元素分析。结果：优化制备的F56－VCR-NP粒径约为153nm,Zeta电位为－20．8mv,包封率为21．4％,载药量为1．9％,多肽连接效率为26．3％。结论：以聚乙二醇－聚乳酸（PEG-PLA）为原料,长春新碱为模型药物,成功制备出纳米粒子,并建立起有效的质量控制方法,对该实验样品进行了表征。结果表明此类纳米粒子尺寸均匀,表面多价连接F56多肽,载药量和包封率稳定可控,工艺成熟。     

Preparation of Intravenous Stealthy Acyclovir Nanoparticles with Increased Mean Residence Time

A major cause of thromboplebitis, during acyclovir (ACV) parenteral administration is the high pH of its reconstituted solution (pH 11). Its plasma half life is 2.5 h, requiring repeated administration which may result in excess of drug solubility leading to possible renal damage and acute renal failure. The present study reports the efficiency of stealthy ACV nanoparticles (NPs) to increase the mean residence time of the drug 29 times. It caused a marked decrease in thrombophlebitis when injected into rabbit’s ear vein. The polymers used were (Poly lactic acid, polylactic-co-glycolic (PLGA) 85/15, PLGA 75/25, PLGA 50/50). Particles were evaluated for their encapsulation efficiency, morphology, particle size and size distribution, zeta potential, and in vitro drug release. Small NPs (280–300 nm) with 60% drug release after 48 h were obtained. Among the block copolymer used, poloxamer 407 was of superior coating properties with a coat thickness in the range of 1.5–8.3 nm and a decreased surface charge.