脂质体辅助递送CTLA-4 siRNA通过激活系统性抗肿瘤免疫反应抑制肾细胞癌生长

目的:探索携带CTLA-4 siRNA的适配子偶联脂质体颗粒是否可以激活肿瘤部位的抗肿瘤免疫反应，抑制肾细胞癌的生长。方法:采用薄膜水化法制备脂质体；使用透射电子显微镜观察脂质体的形态和结构；用Zetasizer测量Zeta电位；共孵育实验观察靶细胞对Lipo-siRNA的摄取；qPCR检测Lipo-siRNA对CTLA-4基因的沉默；小鼠移植瘤模型检测Lipo-siRNA的体内抑瘤能力；流式细胞术检测肿瘤浸润T细胞的激活状态；免疫荧光法检测肿瘤浸润T细胞的数目。结果:成功制备Lipo-siRNA，电镜结果显示其具有双层球状结构；Zetasizer测得其Zeta电位为(+20.53±2.66)mV；荧光显微镜观察结果表明Lipo-siRNA可以被靶细胞有效摄取，qPCR检测Lipo-siRNA可以显著降低CTLA-4基因的表达（P＜0.001）；小鼠移植瘤模型显示Lipo-siRNA较对照组而言可以显著抑制肿瘤生长（P＜0.001），降低肿瘤细胞中CTLA-4的表达（P＜0.001），提升肿瘤浸润T细胞的数量（P＜0.000 1），并且提高了肿瘤浸润T细胞中IL-2（P＜0.000 1）和IFN-γ（P＜0.000 1）的表达水平。结论:适配子偶联脂质体可以携带CTLA-4 siRNA靶向肿瘤细胞，激活肿瘤部位的抗肿瘤免疫反应，抑制肿瘤的生长，对肾细胞癌的治疗具有潜在的临床应用价值。     

RPV‐modified epirubicin and dioscin co‐delivery liposomes suppress non‐small cell lung cancer growth by limiting nutrition supply

Chemotherapy for non‐small cell lung cancer (NSCLC) is far from satisfactory, mainly due to poor targeting of antitumor drugs and self‐adaptations of the tumors. Angiogenesis, vasculogenic mimicry (VM) channels, migration, and invasion are the main ways for tumors to obtain nutrition. Herein, RPV‐modified epirubicin and dioscin co‐delivery liposomes were successfully prepared. These liposomes showed ideal physicochemical properties, enhanced tumor targeting and accumulation in tumor sites, and inhibited VM channel formation, tumor angiogenesis, migration and invasion. The liposomes also downregulated VM‐related and angiogenesis‐related proteins in vitro. Furthermore, when tested in vivo, the targeted co‐delivery liposomes increased selective accumulation of drugs in tumor sites and showed extended stability in blood circulation. In conclusion, RPV‐modified epirubicin and dioscin co‐delivery liposomes showed strong antitumor efficacy in vivo and could thus be considered a promising strategy for NSCLC treatment.     

注射用青藤碱脂质体温敏凝胶的制备及其药动学研究

目的通过脂质体与温敏凝胶相结合,制备新型注射用关节腔给药系统,并研究其药物代谢动力学(简称药动学)。方法采用逆向蒸发法制备青藤碱脂质体,通过单因素考察、星点设计-效应面法优化处方。选取壳聚糖/β-甘油磷酸(CS/β-GP)为温敏凝胶制备体系,并采用透析袋法对青藤碱脂质体温敏凝胶的体外释放度进行测定。建立家兔关节腔给药模型,应用DAS 3.1.0软件进行数据处理进而研究其药动学。结果制备青藤碱脂质体最优处方为药脂比1∶6,大豆磷脂与胆固醇比6∶1,油水比1∶6。体外释放结果显示,青藤碱脂质体温敏凝胶缓释效果明显,至114 h还未释放完全。家兔体内药动学研究表明,青藤碱脂质体温敏凝胶的生物利用度约为青藤碱注射液的2.0倍(P0.01),达峰时间显著延长,约为青藤碱注射液的3.0倍(P0.01),血药峰浓度降低(P0.05),缓释效果优于青藤碱注射剂。结论青藤碱脂质体温敏凝胶性状稳定,缓释效果明显,注射于家兔关节腔后,经体液侵蚀后自行消解,可用于关节腔注射给药。     

载As2O3 pH敏感钙砷复合物脂质体的制备及体外评价

目的 制备pH敏感释药的As₂O₃脂质体，并进行体外评价。方法 采用薄膜分散法制备含钙离子脂质体，然后用离子沉淀法孵育制备钙砷复合物脂质体（CaAs-LP）。测定CaAs-LP的粒径、Zeta电位及多分散系数（PDI）；透射电子显微镜观察脂质体的形态；电感耦合等离子体发射光谱仪测定纳米药物的载药量与包封率；透析袋法考察其体外释药特性。噻唑蓝（MTT）法考察未载药脂质体及CaAs-LP对人源性乳腺癌MCF-7细胞、人源性脑胶质瘤U87细胞和人源性肝癌HepG2细胞的毒性；共聚焦显微镜考察U87细胞对CaAs-LP的摄取效率。结果 制备的CaAs-LP呈规整类球型，粒径约为（117.16±1.94）nm，包封率和载药量分别为（74.31±2.11）%、（8.31±0.13）%。体外释放研究表明，CaAs-LP具有明显的缓释以及pH响应释药特征。未载药的脂质体在MCF-7、U87、HepG2和L02细胞中的生物相容性良好；CaAs-LP抑制肿瘤细胞生长的作用较原药有所上升，半数抑制浓度（IC₅₀）值分别为11.91、4.90、19.41、27.59 μmol/L。细胞摄取研究表明肝癌细胞对脂质体具有良好的摄取。结论 CaAs-LP具备显著的缓释以及pH响应释药的特性，在肿瘤治疗方面具有较好的应用前景。     

星点设计-效应面法优化pH值响应及线粒体靶向双功能金丝桃苷脂质体的处方及其体外评价

目的 优化线粒体靶向金丝桃苷脂质体（DLD/Hyp-Lip）制备的最佳处方，并研究研究其在胎牛血清中的稳定性及体外释放行为，考察其线粒体靶向性。方法 采用薄膜分散法制备DLD/Hyp-Lip，以包封率和载药量为考察指标进行单因素实验，考察磷脂总量与金丝桃苷（hyperoside，Hyp）用量比、二硬脂酰磷脂酰乙醇胺-聚乙二醇（DSPE-PEG）与DLD用量比等条件对DLD/Hyp-Lip的影响，结合星点设计-效应面法优化DLD/Hyp-Lip处方。使用透射电子显微镜和粒径仪观察测定脂质体粒子外观、平均粒径和Zeta电位，采用血清稳定性实验和体外释药、线粒体靶向性对该载药系统进行评价。结果 DLD/Hyp-Lip最佳处方为磷脂总量和金丝桃苷用量比为12.50：1，磷脂总量与胆固醇用量比为6.00：1，DSPE-PEG与DLD用量比为3：5；测得金丝桃苷包封率为（95.57±0.56）%，载药量为（8.55±0.57）%。所制备的DLD/Hyp-Lip外观良好，平均粒径为（124.9±3.4）nm，Zeta电位为（-6.2±1.9）mV；在胎牛血清中性状稳定，在体外释放介质中24 h累积释放量达到40%。线粒体靶向实验表明DLD/Hyp-Lip可以促进药物聚集在线粒体部位。结论 采用此方法能够精准有效的优化DLD/Hyp-Lip的制备工艺，该方法操作简单方便，可以用于DLD/Hyp-Lip制备与处方的优化，制备的DLD/Hyp-Lip包封率高，粒径小，分布均匀，且具有良好的缓释作用，为DLD/Hyp-Lip的进一步体内研究奠定了基础。载金丝桃苷的DLD/Hyp-Lip具有良好的肝癌细胞线粒体靶向性，是一种潜在高效的肝癌细胞线粒体靶向给药系统。     

Multicompartment systems: A putative carrier for combined drug delivery and targeting

In this review, we discuss recent developments in multicompartment systems commonly referred to as vesosomes, as well as their method of preparation, surface modifications, and clinical potential. Vesosomal systems are able to entrap more than one drug moiety and can be customized for site-specific delivery. We focus in particular on the possible reticuloendothelial system (RES) – mediated accumulation of vesosomes, and their application in tumor targeting, as areas for further investigation.     

Variable cooperativity in SNARE-mediated membrane fusion

The soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23–30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.Membrane fusion is an essential reaction common to intracellular trafficking and exocytosis in eukaryotic cells. Although the process involves an intricate interplay of several proteins, the fusion of membranes is dependent on the conserved family of proteins known as soluble N-ethylmaleimide–sensitive factor attachment protein receptors, or SNAREs (1, 2). In the important case of the fusion of synaptic vesicles (SVs), the SNAREs responsible are vesicular synaptobrevin 2 (syb) and plasma membrane proteins SNAP-25A (SN25) and syntaxin-1A (syx). A critical intermediate seems to be an acceptor complex consisting of a three-helix bundle formed by a 1:1 syx:SN25 complex, which serves as a binding site for syb (3, 4). According to the zipper hypothesis, the N termini of syb and the 1:1 syx:SN25 complex nucleate to form a parallel four-helix bundle called the SNARE complex. The directional assembly then proceeds toward the C termini, resulting in a pulling force between the membranes that leads to their fusion (4, 5). There is some consensus that the highly exergonic nature of the assembly of the SNARE complex provides the energy for overcoming the barrier for fusion (6, 7), although identification of putative fusion intermediates at molecular resolution as well as force measurement experiments suggest multiple energy barriers are present (7–10).The question of whether and how SNAREs cooperate to mediate fusion has received substantial attention. Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11, 12), much attention has been given to the notion of a preferred number of SNARE complexes, with estimates varying from a single SNARE complex (13) to 15 (14), although more recent estimates vary between two and eight (12, 15–18). Unfortunately, this large disparity in results has not been appropriately explained, and it remains unclear whether the differences are a result of inherent properties of the particular set of SNAREs involved or rather originate from the biophysical characteristics of the fusing vesicles.A commonly used approach to investigate how SNAREs work is by reconstituting complementary SNAREs into liposomes (19). We have previously demonstrated that SNAREs can mediate both lipid and contents mixing with similar kinetics (13), an important functional criterion for establishing membrane fusion (20). Using the SNAREs responsible for exocytosis of SVs as a model, we investigate here how the density of SNAREs affects fusion of liposomes to obtain mechanistic information on cooperativity. A rigorous way to address this is to vary the SNARE density in one or both membranes. Whereas it is experimentally straightforward for syb, this approach is problematic for SN25 and syx because they tend to associate into “off-pathway” complexes, compromising kinetic analysis (6, 21). To circumvent this, Pobbati et al. (22) introduced a stabilized acceptor complex that contains a C-terminal peptide of synaptobrevin (syb 49–96) (22). This 1:1 syx:SN25 complex (herein referred to as the ∆N complex) is stable and contains a free binding site for syb, allowing one to precisely define its concentration. Therefore, we used the ∆N complex in our experiments to rule out the effect of any side reactions related to the assembly/disassembly of the acceptor complex and thus simplify the kinetic analysis on fusion.     

Direct in vivo gene transfer to healing rat patellar ligament by intra-arterial delivery of haemagglutinating virus of Japan liposomes

BACKGROUND: Manipulation of ligament healing has been a major focus of orthopaedic research. In recent years, gene transfer to healing ligament appears to be a feasible method for manipulating the healing process. In this study, we investigated the feasibility of gene transfer to healing rat patellar ligament by intra-arterial delivery. METHODS: An attempt was made to transfer a reporter gene (Escherichia coli, beta-galactosidase gene) to healing rat patellar ligament using the haemagglutinating virus of Japan (HVJ) liposome-mediated gene transfer method. Three days after cutting the patellar tendons of 25 14-week-old male Wistar rats, HVJ-liposome complexes containing beta-galactosidase (beta-gal) cDNA were injected into the femoral artery of 15 Wistar rats as the experimental group. HVJ liposomes without DNA were injected into the femoral artery of 10 Wistar rats as the control group. Three rats from the experimental group and two control rats were killed 3, 7, 14, 28 and 56 days after the injection. RESULTS: After X-gal staining, the rate of transfection in the experimental group (mean +/- SEM) was found to be 12.1% +/- 0.590%, 8.7% +/- 0.217%, 10.2% +/- 0.227%, 3.2% +/- 0.247% and 0.7% +/- 0.060% at post-injection days 3, 7, 14, 28 and 56 respectively. In control sections the number of blue-stained cells were very few at any point. CONCLUSION: We succeeded in introducing a reporter gene into healing rat patellar ligament by infra-arterial delivery of HVJ-liposome complexes. This method appears to have the potential to be applicable for soft-tissue healing studies and also healing studies of other tissues and organs.