壳聚糖季铵盐纳米粒的制备及其应用研究进展

壳聚糖季铵盐是一种常见的壳聚糖修饰物,属水溶性壳聚糖衍生物.它是聚阳离子多聚物,具有低毒性、良好的生物降解性和生物相容性、稳定性强等优势,因而其应用范围相当广泛.此文主要就近几年壳聚糖季铵盐纳米粒的制备方法及该纳米粒在基因递送载体、疫苗佐剂、给药系统等应用领域的最新研究进展作了综述.     

Resistance training changes LDL metabolism in normolipidemic subjects: a study with a nanoemulsion mimetic of LDL

Objective

To evaluate the effects of resistance training (RT) on the metabolism of an LDL-like nanoemulsion and on lipid transfer to HDL, an important step of HDL metabolism.

Methods

LDL-like nanoemulsion plasma kinetics was studied in 15 healthy men under regular RT for 1–4 years (age = 25 ± 5 years, VO₂peak = 50 ± 6 mL/kg/min) and in 15 healthy sedentary men (28 ± 7 years, VO₂peak = 35 ± 9 mL/kg/min). LDL-like nanoemulsion labeled with ¹⁴C-cholesteryl-ester and ³H-free-cholesterol was injected intravenously, plasma samples were collected over 24-h to determine decay curves and fractional clearance rates (FCR). Lipid transfer to HDL was determined in vitro by incubating of plasma samples with nanoemulsions (lipid donors) labeled with radioactive free-cholesterol, cholesteryl-ester, triacylglycerols and phospholipids. HDL size, paraoxonase-1 activity and oxidized LDL levels were also determined.

Results

The two groups showed similar LDL and HDL-cholesterol and triacylglycerols, but oxidized LDL was lower in RT (30 ± 9 vs. 61 ± 19 U/L, p = 0.0005). In RT, the nanoemulsion ¹⁴C-cholesteryl-ester was removed twice as fast than in sedentary individuals (FCR: 0.068 ± 0.023 vs. 0.037 ± 0.028, p = 0.002), as well as ³H-free-cholesterol (0.041 ± 0.025 vs. 0.022 ± 0.023, p = 0.04). While both nanoemulsion labels were removed at the same rate in sedentary individuals, RT ³H-free-cholesterol was removed slower than ¹⁴C-cholesteryl-ester (p = 0.005). HDL size, paraoxonase 1 and the transfer rates to HDL of the four lipids were the same in both groups.

Conclusions

RT accelerated the clearance of LDL-like nanoemulsion, which probably accounts for the oxidized LDL levels reduction in RT. RT also changed the balance of free and esterified cholesterol FCR's. However, RT had no effect on HDL metabolism related parameters.     

超临界反溶剂法制备叶酸介导吡柔比星葡聚糖纳米粒的工艺研究

目的:优化可主动靶向癌细胞的叶酸(FA)介导吡柔比星(PRB)葡聚糖(DEX)纳米粒(FA-PRB-DEX-NPs)的制备工艺。方法:采用超临界反溶剂法制备FA-PRB-DEX-NPs,采用二水平设计法,以PRB与DEX质量比(X1)、DEX浓度(X2)、混合液流速(X3)、萃取釜温度(X4)、萃取釜压力(X5)为因素,以粒径、载药量、包封率等为指标筛选出最佳工艺条件,计算叶酸偶联度,通过扫描电镜、红外光谱、X射线衍射分析和差示扫描量热分析法表征纳米粒性质。结果:最佳工艺条件为X1=0.5、X2=9.98mg·mL-1、X3=3.3mL·min-1、X4=50℃、X5=15MPa。以此所制纳米粒的平均粒径、载药量和包封率分别为(178±15.8)nm、7.73%、33.2%,FA偶联度为2.97%。各表征方法结果显示纳米粒与原药比较粒径更小,呈无定形非晶态。结论:筛选的制备工艺成功地制备出了可主动靶向癌细胞表面的FA-PRB-DEX-NPs,且所采用的超临界反溶剂法方法简单,更适合工艺化生产。     

Apoptosis of A549 cells by small interfering RNA targeting survivin delivery using poly-β-amino ester/guanidinylated O-carboxymethyl chitosan nanoparticles

Gene-based therapeutics has emerged as a promising approach for human cancer therapy. Among a variety of non-viral vectors, polymer vectors are particularly attractive due to their safety and multivalent groups on their surface. This study focuses on guanidinylated O-carboxymethyl chitosan(GOCMCS) along with poly-β-amino ester(PBAE) for si RNA delivery. Binding efficiency of PBAE/si RNA/GOCMCS nanoparticles were characterized by gel electrophoresis. The si RNA-loaded nanoparticles were found to be stable in the presence of RNase A, serum and BALF respectively. Fine particle fraction(FPF) which was determined by a two-stage impinger(TSI) was 57.8% ± 2.6%. The particle size and zeta potential of the nanoparticles were 153.8 ± 12.54 nm and + 12.2 ± 4.94 m V. In vitro cell transfection studies were carried out with A549 cells. The cellular uptake was significantly increased. When the cells were incubated with si Survivin-loaded nanoparticles, it could induce 26.83% ± 0.59% apoptosis of A549 cells and the gene silencing level of survivin expression in A549 cells were 30.93% ± 2.27%. The results suggested that PBAE/GOCMCS nanoparticle was a very promising gene delivery carrier.     

Cdk5 knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity

Blocking the programmed death-ligand 1 (PD-L1) on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy. However, only a minority of patients presented immune responses in clinical trials. To develop an alternative treatment method based on immune checkpoint blockade, we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells via specifically knocking out Cyclin-dependent kinase 5 (Cdk5) gene in vivo. The expression of PD-L1 on tumor cells was significantly attenuated by knocking out Cdk5, leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer. Importantly, we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8⁺ T cells was significantly increased while regulatory T cells (Tregs) was decreased. It may be the first case to exhibit direct in vivo PD-L1 downregulation via CRISPR-Cas9 genome editing technology for cancer therapy. It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.     

纳米炭在乳腺癌前哨淋巴结示踪的实验研究

目的比较和评价纳米炭混悬注射液(卡纳琳)及亚甲蓝注射液进行乳癌淋巴结示踪的作用差别,为临床乳腺癌患者的治疗提供相关手段。方法取40只雌性成年大耳兔,将VX2细胞株制成的瘤组织块悬液注射于兔乳腺下方制成乳腺癌模型,并将成癌模型分成卡纳琳组及亚甲蓝组,分别行淋巴结活检。记录第一枚淋巴结成功染色枚数、染色所需时间、褪色所需时间、染色淋巴结总枚数,并观察染色效果。结果两组第1枚淋巴结染色成功率相同,所需时间无统计学差异;卡纳琳组淋巴结染色总枚数明显多于亚甲蓝组,差异有统计学意义(P0.05);卡纳琳组褪色时间明显慢于亚甲蓝组,且染色效果优于亚甲蓝。结论用VX2细胞株制成的瘤组织块悬液注射于兔乳腺下方可成功制成理想的兔转移性乳腺癌模型。纳米炭混悬液行淋巴结示踪染色效果优于亚甲蓝,为更理想的乳腺癌手术淋巴示踪剂。     

工程化蛋白纳米笼探针在肿瘤分子影像中的应用

开发具有高特异性、高灵敏度、低毒性的影像探针是推动肿瘤分子影像技术发展的核心。天然表达纯化的蛋白纳米笼作为一种能够在生物体内外自组装形成特定笼状结构的纳米粒子,具有粒度高度分散均一、优良生物相容性和生物可降解性以及工程化改造方法简单多样等优势,已被广泛应用于开发各类肿瘤分子影像探针。该文从天然蛋白纳米笼的种类和特性、工程化改造方法以及其在肿瘤分子影像的应用几个方面来综述其研究进展。     

可注射性纳米粒/凝胶复合物缓释SDF-1α体系促进颅骨再生的研究

目的采用壳聚糖(CS)为主要原料制备可缓释基质细胞衍生因子-1α (SDF-1α)的纳米粒,将其与壳聚糖/β-甘油磷酸钠(CS/β-GP)水凝胶复合,通过持续释放SDF-1α诱导大鼠颅骨再生修复情况。方法分别制备SDF-1α/壳聚糖/羧甲基壳聚糖纳米粒(SDF-1α/CS/CMCS NPs)和CS/β-GP水凝胶,将SDF-1α和SDF-1α/CS/CMCS NPs分别混入CS/β-GP水凝胶中并观察其对SDF-1α的缓释效果。将CS/β-GP水凝胶负载SDF-1α (Gel/SDF-1α组)和SDF-1α/CS/CMCSNPs (Gel/SDF-1α-NPs组)植入大鼠颅骨缺损,未植入材料组作为空白对照组。采用Micro-CT、HE染色及马松三色(MT)染色等方法观察和分析8周后大鼠颅骨再生修复的效果。结果纳米粒/凝胶复合物缓释SDF-1α体系对SDF-1α具有明显的持续释放作用。Micro-CT结果分析显示:Ge1/SDF-1α-NPs组骨再生效果显著优于空白对照组和Ge1/SDF-1α组。HE和MT组织学分析结果也显示:Ge1/SDF-1α-NPs组较空白对照组和Ge1/SDF-1α组可以获得更多的新骨形成。结论纳米粒/水凝胶复合物缓释SDF-1α体系可以通过持续释放SDF-1α促进大鼠颅骨再生修复。     

RGD肽表面修饰壳聚糖载基因纳米粒子的体外实验研究

目的将Arg—Gly—Asp（RGD）肽偶联到壳聚糖（CH）材料表面,并制备成包载质粒DNA的纳米粒子,以未偶连RGD的壳聚糖载质粒DNA作为对照,进行体外内皮细胞转染,观察其是否能提高对内皮细胞的转染效率。方法以1-乙基-3-（3-二甲基氨基丙基）碳化二亚胺盐酸盐（EDC）和N-羟基丁二酰亚胺（NHS）为偶联剂,通过酰胺键将RGD肽偶联到壳聚糖表面,对其进行表征,并以未偶连RGD的壳聚糖作为对照,制备载pEGFP-C1质粒DNA纳米粒子,比较2者对Hy926细胞的转染效率。结果壳聚糖-RGD（CH-RGD）载基因纳米粒子转染Hy926细胞的效率明显高于未偶连RGD的壳聚糖载基因纳米粒子（35．7％VS14．3％．P〈0．001）。结论RGD肽表面修饰壳聚糖载基因纳米粒子可用于体外细胞转染,其对细胞的转染效率明显优于未偶连RGD的壳聚糖。     

负载两性霉素B壳聚糖-聚乳酸纳米粒的制备及其释药性能*☆

背景：两性霉素B为治疗深部真菌感染的首选药物,但该药无法通过血脑屏障而对隐球菌性脑膜炎的治疗效果甚微。利用纳米粒子作为药物载体的优势,通过相分离透析技术制备负载两性霉素B的壳聚糖-聚乳酸纳米粒子,有望克服两性霉素B的不足。 目的：对负载两性霉素B的壳聚糖-聚乳酸纳米粒进行表征,分析其体外药物释放能力。 设计、时间及地点：重复测量设计,于 2008-11/2009-04 在国家纳米科学中心纳米医学与生物实验室完成。 材料：壳聚糖,平均相对分子质量为3.4×105,脱乙酰度为93%,为上海卡伯工贸有限公司产品。两性霉素B为Sigma公司产品。 方法：在二甲基亚砜溶液中,在三乙胺存在下,通过壳聚糖和D,L-丙交酯的开环聚合反应能够生成壳聚糖-聚乳酸共聚物。该共聚物由亲水壳聚糖段和疏水聚乳酸段组成,在水中能够组装形成纳米粒子。两性霉素B通过相分离透析技术包载于纳米粒子中。 主要观察指标：激光粒度分析仪测定纳米颗粒的粒径大小、粒径分布,环境扫描电镜观察纳米颗粒的外观形态,紫外光谱分析负载两性霉素B的壳聚糖-聚乳酸纳米粒的包封率、载药量和释药性能。 结果：壳聚糖-聚乳酸纳米粒和负载两性霉素B的壳聚糖-聚乳酸纳米粒,其粒径分别为114 nm和153 nm(当丙交酯与壳聚糖摩尔比为11∶1时)。纳米粒子粒径分布较窄,呈球形。共聚物中丙交酯与壳聚糖摩尔比影响药物的包封率和载药量,随着丙交酯与壳聚糖摩尔比从11∶1到20∶1,包封率从(62.3±3.5)%增加到(90.7±2.8)%,载药量从(7.8±1.2)%增加到(12.3±1.4)%。随着聚乳酸段质量比增加,纳米粒子尺寸、包封率和载药量增加,而药物释放降低。 结论：开环聚合制备壳聚糖-聚乳酸共聚物及用相分离透析方法制备负载两性霉素B纳米粒简便可靠,负载两性霉素B后纳米粒径明显变大,且纳米粒对两性霉素B有很高的包封率,体外释药具有明显的缓释作用。 关键词：两性霉素B;壳聚糖;聚乳酸;纳米粒子;包封率;体外释放