A novel liposome adjuvant DPC mediates Mycobacterium tuberculosis subunit vaccine well to induce cell-mediated immunity and high protective efficacy in mice

Tuberculosis (TB) is a serious disease around the world, and protein based subunit vaccine is supposed to be a kind of promising novel vaccine against it. However, there is no effective adjuvant available in clinic to activate cell-mediated immune responses which is required for TB subunit vaccine. Therefore, it is imperative to develop new adjuvant. Here we reported an adjuvant composed of dimethyl dioctadecylammonium (DDA), Poly I:C and cholesterol (DPC for short). DDA can form a kind of cationic liposome with the ability to deliver and present antigen and can induce Th1 type cell-mediated immune response. Poly I:C, a ligand of TLR3 receptor, could attenuate the pathologic reaction induced by following Mycobacterium tuberculosis challenge. Cholesterol, which could enhance rigidity of lipid bilayer, is added to DDA and Poly I:C to improve the stability of the adjuvant. The particle size and Zeta-potential of DPC were analyzed in vitro. Furthermore, DPC was mixed with a TB fusion protein ESAT6-Ag85B-MPT64(190-198)-Mtb8.4-Rv2626c (LT70) to construct a subunit vaccine. The subunit vaccine-induced immune responses and protective efficacy against M. tuberculosis H37Rv infection in C57BL/6 mice were investigated. The results showed that the DPC adjuvant with particle size of 400 nm and zeta potential of 40 mV was in good stability. LT70 in the adjuvant of DPC generated strong antigen-specific humoral and cell-mediated immunity, and induced long-term higher protective efficacy against M. tuberculosis infection (5.41 ± 0.38 log₁₀ CFU) than traditional vaccine Bacillus Calmette–Guerin (BCG) (6.01 ± 0.33 log₁₀ CFU) and PBS control (6.53 ± 0.26 log₁₀ CFU) at 30 weeks post-vaccination. In conclusion, DPC would be a promising vaccine adjuvant with the ability to stimulate Th1 type cell-mediated immunity, and could be used in TB subunit vaccine.     

Dibucaine in Ionic-Gradient Liposomes: Biophysical,Toxicological, and Activity Characterization

Administration of local anesthetics is one of the most effective pain control techniques for postoperative analgesia. However, anesthetic agents easily diffuse into the injection site, limiting the time of anesthesia. One approach to prolong analgesia is to entrap local anesthetic agents in nanostructured carriers (e.g., liposomes). Here, we report that using an ammonium sulphate gradient was the best strategy to improve the encapsulation (62.6%) of dibucaine (DBC) into liposomes. Light scattering and nanotracking analyses were used to characterize vesicle properties, such as, size, polydispersity, zeta potentials, and number. In vitro kinetic experiments revealed the sustained release of DBC (50% in 7 h) from the liposomes. In addition, in vitro (3T3 cells in culture) and in vivo (zebrafish) toxicity assays revealed that ionic-gradient liposomes were able to reduce DBC cyto/cardiotoxicity and morphological changes in zebrafish larvae. Moreover, the anesthesia time attained after infiltrative administration in mice was longer with encapsulated DBC (27 h) than that with free DBC (11 h), at 320 μM (0.012%), confirming it as a promising long-acting liposome formulation for parenteral drug administration of DBC.     

基于pH梯度载药技术的咪喹莫特脂质体的制备工艺研究

目的 根据咪喹莫特的理化性质,利用pH梯度主动载药技术制备脂质体,考察其性状、粒径、表面电荷及体外释药特征。方法 葡聚糖凝胶滤过法测定脂质体的包封率,以包封率与成型性为主要指标筛选制备方法,考察水化液的种类、pH值、离子强度及pH梯度载药、磷脂-胆固醇比例、脂药比、维生素E用量对包封率的影响;正交试验优化咪喹莫特脂质体的处方,考察脂质体样品在0~4℃下的稳定性。结果 按处方咪喹莫特50 mg、大豆卵磷脂400 mg、胆固醇130 mg、油酸10 mg、维生素E 5 mg、柠檬酸pH 2.5缓冲液5 mL,采用薄膜分散法工艺制备脂质体样品,并进行pH梯度主动载药,pH值调至7.0。制得的咪喹莫特脂质体呈白色均匀的混悬液,脂质体微粒圆整,分散性好,粒径（347±21）nm,包封率（81.2±1.9）%,Zeta电位（-12.19±1.7）mV。结论 pH梯度主动载药技术适于咪喹莫特脂质体的制备。     

紫杉醇脂质体与紫杉醇分别联合卡铂治疗卵巢癌疗效的Meta分析

摘 要 目的：系统评价紫杉醇脂质体与普通紫杉醇制剂分别联合卡铂治疗卵巢癌的有效性和安全性。方法: 计算机检索中国知识资源总库（CNKI）、中国生物医学文献数据库（CBM）、中文科技期刊数据库（VIP）、中国学术期刊数据库（万方数据）、PubMed、Cochrane Library、Embase 中紫杉醇脂质体联合卡铂与紫杉醇联合卡铂化疗治疗卵巢癌的临床随机对照试验,检索范围均为建库至2017年7月12日。2名研究者根据Cochrane系统评价手册5.1.0,按纳入与排除标准独立进行文献筛选、资料提取、质量评价,并使用RevMan5.3软件Meta分析。 结果:共纳入8篇随机对照试验,共计793例受试者。结果显示,在卵巢癌的治疗中,紫杉醇脂质体联合卡铂与紫杉醇联合卡铂比较,客观缓解率有统计学意义（P=0.02）。在不良反应方面,紫杉醇脂质体联合卡铂与紫杉醇联合卡铂比较：血小板减少（P=0.02）、恶心呕吐（P＜0.000 01）、肌肉关节痛（P＜0.000 01）、皮疹（P＜0.000 01）、呼吸困难（P=0.000 8）和面部潮红（P=0.001 0）,差异有统计学意义;而白细胞减少（P=0.13）、血红蛋白减少（P=0.28）、腹泻便秘（P=0.15）、脱发（P=0.62）,差异无统计学意义。结论:紫杉醇脂质体联合卡铂化疗治疗卵巢癌的疗效优于紫杉醇联合卡铂化疗,且能减轻患者不良反应,提高用药有效性和安全性。     

Selective induction of apoptosis in MCF7 cancer-cell by targeted liposomes functionalised with mannose-6-phosphate

Liposomes are versatile platforms to carry anticancer drugs in targeted drug delivery; they can be surface modified by different strategies and, when coupled with targeting ligands, are able to increase cellular internalisation and organelle-specific drug delivery. An interesting strategy of antitumoral therapy could involve the use of lysosomotropic ligand-targeted liposomes loaded with molecules, which can induce lysosomal membrane permeabilization (LMP), leakage of cathepsins into the cytoplasm and subsequent apoptosis. We have previously demonstrated the ability of liposomes functionalised with a mannose-6-phosphate to reach lysosomes; in this research we compare the behaviour of M6P-modified and non-functionalised liposomes in MCF7 tumour cell and in HDF normal cells. With this aim, we first demonstrated by Western blotting the overexpression of mannose-6-phosphate/insulin-like growth factor (M6P/IGF-II) receptor in MCF7. Then, we prepared calcein-loaded liposomes and we revealed the increased uptake of M6P-functionalised liposomes in MCF7 cells respect to HDF cells by flow cytometry analysis. Finally, we loaded functionalised and not functionalised liposomes with N-hexanoyl-d-erythro-sphingosine (C6Cer), able to initiate LMP-induced apoptosis; after having studied the stability of both vesicles in the presence of serum by Dynamic Light Scattering and Spectrophotometric turbidity measurements, we showed that ceramide-loaded M6P-liposomes significantly increased apoptosis in MCF7 with respect to HDF cells.     

Improvement of chemosensitivity and inhibition of migration via targeting tumor epithelial-to-mesenchymal transition cells by ADH-1-modified liposomes

How to overcome drug resistance and prevent tumor metastasis is key to the success of malignant tumor therapy. In this paper, ADH-1 peptide-modified liposomes (A-LP) have been successfully constructed for restoring chemosensitivity and suppressing cancer cell migration. With a particle size of about 90?nm, this functionalized nanocarrier was loaded with fluorescent probe or paclitaxel (PTX). Cellular uptake studies showed that A-LP facilitated the delivery of anticancer drug to tumor cells undergoing EMT. Interestingly, this nanocarrier enhanced chemosensitivity by assessing the cell activity using CCK-8 assay. Further, the results of Wound scratch assay and Transwell migration assay showed the inhibition effect of this nanocarrier on tumor cell migration. Moreover, this nanocarrier exhibited significant tumor-targeting ability and anti-tumor efficacy in vivo. Collectively, A-LP might be a novel targeted drug delivery system to enhance the efficacy of chemotherapy and prevent tumor metastasis.     

Enhanced selective cellular uptake and cytotoxicity of epidermal growth factor-conjugated liposomes containing curcumin on EGFR-overexpressed pancreatic cancer cells

Pancreatic cancer is one of the most malignant cancers with a high mortality rate. Some types of pancreatic cancer cells overexpress epidermal growth factor receptor (EGFR), which is a potential target for anticancer agents. In this study, we examined the effect of epidermal growth factor (EGF)-conjugated liposomes containing curcumin (EGF-LP-Cur) on three different EGFR-expressed human pancreatic cancer cell lines, BxPC-3, Panc-1 and Mia Paca-2. We have demonstrated that it is feasible to prepare liposomal vesicles of EGF-LP-Cur and that it is stable in the liquid vehicle at ambient conditions for three weeks. In addition, the formulation of curcumin had higher cytotoxicity on BxPC-3 than on any other cells. It is also shown that the cellular uptake of curcumin on BxPC-3, which is essential for the cytotoxicity, is associated with EGFR-mediated mechanism of action. In summary, our results have showed that targeting EGFR with EGF-conjugated curcumin liposomes enhanced the antitumor activity of curcumin against human pancreatic cancer cells.     

Multiseed liposomal drug delivery system using micelle gradient as driving force to improve amphiphilic drug retention and its anti-tumor efficacy

To improve drug retention in carriers for amphiphilic asulacrine (ASL), a novel active loading method using micelle gradient was developed to fabricate the ASL-loaded multiseed liposomes (ASL-ML). The empty ML were prepared by hydrating a thin film with empty micelles. Then the micelles in liposomal compartment acting as ‘micelle pool’ drove the drug to be loaded after the outer micelles were removed. Some reasoning studies including critical micelle concentration (CMC) determination, influencing factors tests on entrapment efficiency (EE), structure visualization, and drug release were carried out to explore the mechanism of active loading, ASL location, and the structure of ASL-ML. Comparisons were made between pre-loading and active loading method. Finally, the extended drug retention capacity of ML was evaluated through pharmacokinetic, drug tissue irritancy, and in vivo anti-tumor activity studies. Comprehensive results from fluorescent and transmission electron microscope (TEM) observation, encapsulation efficiency (EE) comparison, and release studies demonstrated the formation of ML-shell structure for ASL-ML without inter-carrier fusion. The location of drug mainly in inner micelles as well as the superiority of post-loading to the pre-loading method , in which drug in micelles shifted onto the bilayer membrane was an additional positive of this delivery system. It was observed that the drug amphiphilicity and interaction of micelles with drug were the two prerequisites for this active loading method. The extended retention capacity of ML has been verified through the prolonged half-life, reduced paw-lick responses in rats, and enhanced tumor inhibition in model mice. In conclusion, ASL-ML prepared by active loading method can effectively load drug into micelles with expected structure and improve drug retention.     

Stabilized tetraether lipids based particles guided prophyrins photodynamic therapy

Photodynamic therapy (PDT) that involves ergonomically delivered light in the presence of archetypical photosensitizer such as Protoporphyrin IX (PpIX) is a time-honored missile strategy in cancer therapeutics. Yet, the premature release of PpIX is one of the most abundant dilemma encounters the therapeutic outcomes of PDT due to associated toxicity and redistribution to serum proteins. In this study, ultrastable tetraether lipids (TELs) based liposomes were developed. PpIX molecules were identified to reside physically in the monolayer; thereby the inherent π-π stacking that leads to aggregation of PpIX in aqueous milieu was dramatically improved. TEL_29.9?mol% and TEL_62mol% based liposomes revealed PpIX sustained release diffusion pattern from spherical particles as confirmed by converged fitting to Baker &; Lonsdale model. Stability in presence of human serum albumins, a key element for PDT accomplishment was emphasized. The epitome candidates were selected for vascular photodynamic (vPDT) in in-Ovo chick chorioallantoic membrane. Profoundly, TEL_62mol% based liposomes proved to be the most effective liposomes that demonstrated localized effect within the irradiated area without eliciting quiescent vasculatures damages. Cellular photodynamic therapy (cPDT) revealed that various radiant exposure doses of 134, 202, 403 or 672?mJ.cm^?2 could deliberately modulate the photo-responses of PpIX in TEL-liposomes.     

Co-delivery of a CD4 T cell helper epitope via covalent liposome attachment with a surface-arrayed B cell target antigen fosters higher affinity antibody responses

Liposomal vaccines incorporating adjuvant and CD4 T cell helper peptides enhance antibody responses against weakly immunogenic B cell epitopes such as found in the membrane proximal external region (MPER) of the HIV-1 gp41 subunit. While the inclusion of exogenous helper peptides in vaccine formulations facilitates stronger and more durable antibody responses, the helper peptide incorporation strategy per se may influence the overall magnitude and quality of B cell target antigen immunogenicity. Both variability in individual peptide encapsulation as well as the potential for liposome surface-associated helper peptides to misdirect the humoral response are potential parameters impacting outcome. In this study, we used MPER/liposome vaccines as a model system to examine how the mode of the potent LACK T helper peptide formulation modulates antibody responses against the MPER antigen. We directly compared liposome surface-arrayed palmitoyl LACK (pLACK) versus soluble LACK (sLACK) encapsulated in the liposomes and free in solution. Independent of LACK formulation methods, dendritic cell activation and LACK presentation were equivalent in vivo. The frequency of MPER-specific GC B cells promoted by sLACK was higher than that stimulated by pLACK formulation, a finding associated with a significantly greater frequency of LACK-specific GC B cells induced by pLACK. While there were no significant differences in the quantity of MPER-specific serological responses, the MPER-specific antibody titer trended higher with sLACK formulated vaccines at the lower dose of LACK. However, pLACK generated relatively greater MPER-specific antibody affinities than those induced by sLACK-formulated vaccines. Overall, the results suggest that liposomal surface-associated LACK enhances immunogenicity of LACK through better engagement of LACK-specific B cells. Of note, this is not detrimental to the induction of MPER-specific immune responses; rather, the elicitation of higher affinity anti-MPER antibodies benefits from augmented help delivered via covalent linkage of the pLACK CD4 T cell epitope in conjunction with MPER/liposome presentation.