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.     

鳖甲肽脂质体药代动力学及肝靶向分析

目的:制备含DiR碘化物荧光染料的长循环脂质体(DiR-SSL),采用SD大鼠和BALB/c裸鼠进行荧光成像的体内靶向性研究,利用空白SSL包封含5-羧基荧光素(5-FAM)的鳖甲肽(HGRFG-5-FAM,HF)制备HGRFG-5-FAM-SSL(HFL),研究HF及HFL分别给药于大鼠的药代动力学。方法:采用薄膜分散法制备DiR-SSL及HFL,取2组SD大鼠分别尾静脉注射HF及HFL,其HF质量浓度及剂量一致,于不同时间点采血,利用多功能微孔板检测仪分析HF血药浓度,计算药代动力学参数;将DiR-SSL尾静脉注射于BALB/c裸鼠,分别于1,2,5,7,24 h活体成像,读取荧光强度值;取正常SD大鼠尾静脉注射DiR-SSL,24 h后解剖心、肝、脾、肺、肾于荧光成像系统成像并读取荧光强度值,将肝组织冷冻切片进行激光共聚焦扫描。结果:HF注射到SD大鼠体内,其半衰期(T_(1/2))仅0.826 h,而HFL在相同剂量下,T1/2和药时曲线下面积(AUC_(0-∞))分别为HF的16.253,11.899倍;在BALB/c裸鼠活体成像中,荧光主要集中于肝脏位置,随着时间增加荧光强度逐渐减弱;SD大鼠各脏器中的DiR-SSL荧光在肝脏中最强,注射24 h后DiR-SSL仍能在肝脏储留。结论:SSL递药系统能显著延长HF在SD大鼠体内的T_(1/2)。SSL递药系统具有肝靶向作用,提示该系统可作为药物载体进行肝靶向递药。     

新型抗结核药物制剂研究进展

结核病(tuberculosis,TB)是一种由结核分枝杆菌(Mycobacterium tuberculosis,MTB)引起的慢性传染病,它积聚在各个器官,特别是肺部。目前临床上常用的抗结核药物主要以胶囊剂、片剂或注射剂为主,但这些常规剂型往往难以在结核病核心病灶区域有效分布,药物通过全身循环向其他组织和器官非特异性分布,导致局部药物浓度低,易产生不良反应,进而影响抗结核药的治疗效果。微球、脂质体、纳米粒等新剂型能够选择性地将药物浓集于结核病变部位,从而达到降低药物不良反应,提高患者依从性的目的。因此,本文旨在对抗结核药物新剂型的相关研究进行综述。     

三氧化二砷新剂型的研究进展

三氧化二砷是急性早幼粒细胞白血病的一线用药,对多种实体瘤也具有抑制作用。然而,三氧化二砷肾脏清除速率快,无组织特异性分布,靶向性较差,易产生心、肝和肾等毒性作用,限制了其在实体瘤的临床应用。近年来,科学家开发大量三氧化二砷新剂型以提高其疗效,降低其不良反应,包括脂质体、微球、纳米粒、乳剂和悬浮剂等。本文对三氧化二砷的新剂型研究进展进行梳理,以期为三氧化二砷新剂型的合理开发和应用提供新思路。     

盐酸倍他洛尔-蒙脱石脂质体的制备与性能考察

目的:优化盐酸倍他洛尔-蒙脱石脂质体(Mt-BH-LP)的处方及制备工艺,考察其体外释放性能和渗透性能。方法:采用乙醇注入法-硫酸铵梯度法制备Mt-BH-LP;以包封率和载药量为评价指标,采用正交试验优化Mt-BH-LP的处方;通过透析法考察Mt-BH-LP的体外释放性能;以家兔离体角膜为模型,采用体外改良的Franz扩散池法研究Mt-BH-LP的渗透性,以人永生化角膜上皮细胞(i HCEC)为模型,研究Mt-BH-LP的渗透性。结果:Mt-BH-LP处方工艺条件为卵磷脂-BH(5∶1),卵磷脂-胆固醇(9∶1),硫酸铵浓度0.15 mol·L~(-1),卵磷脂质量225 mg。Mt-BH-LP包封率(75.85±2.15)%,载药量(11.41±0.29)%,平均粒径(218±22.32)nm,Zeta电位(17.03±0.25)m V;体外释放试验表明Mt-BH-LP在10 h累计释放度达到60.2%,离体角膜透过试验结果表明Mt-BH-LP的角膜水化值[(76.72±2.68)%]在正常范围内。结论:采用乙醇注入法-硫酸铵梯度法制备的Mt-BH-LP包封率和载药量较高,缓释性能良好,具有良好的开发与应用前景。     

Cationic liposomes formulated with a novel whole Leishmania lysate (WLL) as a vaccine for leishmaniasis in murine model

Although there have been numerous attempts to develop a successful vaccine against leishmaniasis, based on the clinical trial in this field, no vaccine against Leishmania in routine way can be found for globally effective vaccination in human. Amongst, first generation vaccines consisting of parasite fractions or whole killed Leishmania showed more successful results in clinical trials. It seems that the main reason for the low efficacy of these vaccines is lack of a suitable adjuvant. In this study, a crude extract of detergent-solubilized L. major promastigotes as a novel developed antigen (whole Leishmania lysate (WLL)) was formulated in liposomal form. The cationic liposomes consisting of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) were used to deliver WLL. Liposomes formulations containing different WLL concentrations (prepared from 10³, 10⁴, 10⁵, 10⁶ and 10⁷ parasites) were prepared and characterized for particle size, surface charge, proteins, DNA and phospholipids contents. Moreover, to explore the type of immune response generated and extend of immunization, in vivo and in vitro tests including evaluation of lesion development, parasite burden in the foot and spleen, Th1 and Th2 cytokine analysis, and titration of IgG isotypes before and after the challenge were used. The maximum immunization was provided by WLL06 as depicted by the reduction of footpad swelling andparasite load, increase in anti-Leishmania IgG2a production, though no significant difference was observed between mice which received WLL05 vs WLL06. While maximum immunization was seen in WLL06 group, most of the liposomal WLL formulations induced a mixed Th1/Th2 response. Hence, a more protective immune response is expected to be induced when an immune potentiator adjuvant such as CpG ODNs would be co-deliverd in WLL liposomal formulations.     

A Novel Approach to Increase the Stability of Liposomal Containers via In Prep Coating by Poly[N‐(2‐Hydroxypropyl)Methacrylamide] with Covalently Attached Cholesterol Groups

Highly stable liposomes are developed by coating phosphatidylcholine liposomes with amphiphilic N‐(2‐hydroxypropyl)methacrylamide copolymer. Two approaches in the preparation of coated liposomes are employed: the copolymer is added during (“in prep”) or after (“ex post”) the liposome formation. The influence of polymer concentration and coating method is evaluated using the cryogenic transmission electron microscopy, dynamic light scattering, and small‐angle X‐ray scattering techniques. The in prep modification significantly increases, up to four weeks, the stability of liposomes against aggregation and makes the liposomal membrane nonpermeable toward an inorganic salt. Such enhanced longevity is attributed to the different structure of in prep coated liposomal membranes.     

星点设计-效应面法优化溴新斯的明多囊脂质体的制备工艺

目的采用星点设计-效应面法优化溴新斯的明多囊脂质体的制备工艺。方法采用复乳法制备溴新斯的明多囊脂质体,以包封率为评价指标,用星点设计优化处方,并进行多元线性回归与二项式方程拟合,采用效应面法选取较佳工艺条件,并进行预测。结果以磷脂与胆固醇为1.41∶1投料、主药浓度为22.98 g·L-1、三油酸甘油酯为24.93 mmol·L-1的优化条件制备溴新斯的明多囊脂质体,包封率为80.34%±1.53%,与二项式拟合方程预测结果相差小于2.5%。结论应用星点设计-效应面法优化溴新斯的明多囊脂质体的制备工艺,能快速方便地得到最佳制备工艺,预测性良好。     

薄膜分散法制备白藜芦醇长循环热敏前体脂质体及其表征研究

目的:对白藜芦醇长循环热敏前体脂质体制备的研究以及对其性质进行分析。方法:先用薄膜分散法制备白藜芦醇长循环热敏脂质体后,再用冷冻干燥法来制备白藜芦醇长循环热敏脂质体的前体。采用电势测定仪、HPLC等方法对该脂质体的包封率、粒径、稳定性、载药量、电位、释放度等来展开系统的检查。结果:白藜芦醇长循环热敏前体脂质体水合后形成白藜芦醇长循环热敏脂质体,粒径均值为(107.9±3.6)nm,Zeta电位的均值为(-12.2±1.6)m V,包封率可达89.4%;该脂质体在相变温度42℃下药物释放达到94%以上。结论:采用长循环热敏来制备的白藜芦醇前体脂质体含量与包封率检查方法准确、快速、简单且方法简便易行。载药量大,包封率好,工艺比较稳定。本实验可为新型白藜芦醇静脉注射用热敏脂质体的研究提供基础。