Brucine-loaded liposomes composed of HSPC and DPPC at different ratios: in vitro and in vivo evaluation

Objective: The objective of this study is to test the hypothesis that the phase transition temperature (T_m), the main property of liposomes, can be easily controlled by changing the molar ratio of hydrogenated soy phosphatidylcholine (HSPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphacholine (DPPC) after drug encapsulation.

Materials and methods: Brucine, an antitumor alkaloid, was encapsulated into the liposomes with different HSPC/DPPC compositions. The T_ms of the brucine-loaded liposomes (BLs) were determined by differential scanning calorimetry (DSC). Then the physicochemical properties and pharmacokinetics of the BLs with different HSPC/DPPC compositions were investigated and compared.

Results: The results of DSC revealed that HSPC and DPPC can combine into one phase. The findings of molecular modeling study suggested that HSPC interacts with DPPC via electrostatic interaction. The molar ratio of HSPC/DPPC influenced the sizes of BLs but had little effect on the entrapment efficiency (EE). The stability of BLs was improved with the increase of the HSPC ratios, especially with the presence of plasma. Following i.v. administration, it was found that AUC values of BLs in vivo were directly related to the HSPC/DPPC ratios of BLs, namely the T_ms of BLs.

Discussion: The behavior of liposomes, especially in vivo pharmacokinetic behavior, can be controlled by the modification of T_m.

Conclusion: The characterization of BLs in vitro and in vivo had demonstrated that the T_m could be flexibly modified for liposomes composed of both HSPC and DPPC. Using HSPC/DPPC composition may be an efficient strategy to control the T_m, thus control the in vivo pharmacokinetic behavio, of BLs.     

高效液相色谱法测定环维黄杨星D脂质体的含量

目的:建立高效液相色谱法测定环维黄杨星D(CVB-D)脂质体中药物的含量。方法:采用高效液相色谱法在205 nm波长处测定峰面积,计算CVB-D脂质体中CVB-D的含量。结果:CVB-D在0.0202~0.2020mg·mL-1的浓度范围内与峰面积呈良好的线性关系,回归方程:A=461.6C+7.5885,r=0.9994(n=5),平均回收率为99.13%,RSD为0.77%(n=9)。结论:本方法灵敏、准确度高、专属性好,且稳定性、精密度均符合要求,可用于CVB-D脂质体的含量测定。     

Electrochemically Stimulated Release from Liposomes Embedded in a Polyelectrolyte Multilayer

Triggered release of an entrapped dye from vesicles embedded in a polyelectrolyte multilayer (PEM), as a consequence of the electrochemically induced local pH change in the vicinity of the electrode, is reported. The PEM was deposited on an indium tin oxide (ITO) electrode wherein lipid vesicles filled with a fluorescent dye were embedded. The use of vesicles with a strong negative charge and the polyelectrolyte species of the PEM matrix with a polycation as topmost layer enabled the generation of a stable layer of liposomes in the PEM.     

TG-DHA高含量脱腥鱼油对脂代谢的调节作用

目的：探究TG-DHA高含量脱腥鱼油的制备及对高脂饮食小鼠脂代谢的调节作用。方法：脂质体包埋技术制备TG-DHA高含量脱腥鱼油。将雄性C57BL/6J小鼠（6周龄）随机分为对照组（C）、模型组（M）、TG-DHA高含量鱼油组（O-DHA）、TG-DHA高含量脱腥鱼油组（L-DHA）。连续灌胃8周后,检测小鼠血清、肝脏脂质水平;测定肝脏脂代谢相关基因脂肪酸合成酶（Fatty acid synthase,FAS）、固醇调节元件结合蛋白1c （Sterol regulatory element binding protein-1c,SREBP-1c）、肉碱棕榈酰转移酶1（Carnitine palmitoyl transferase,CPT1）、脂蛋白脂酶（Lipoprotein lipase,LPL） mRNA表达量。结果：脂质体包埋掩盖了己醛、2,4-庚二烯醛等鱼油中的腥味物质。TG-DHA高含量脱腥鱼油中的DHA含量为73.74%,粒径为159.50 nm,Zeta电位为-43.10 mV,稳定性良好,且在动物体内易于被消化吸收。L-DHA可显著改善高脂饮食小鼠血清和肝脏脂质水平（P<0.05）,O-DHA可显著改善高脂饮食小鼠血清水平和肝脏中的TG含量（P<0.05）,二者均能极显著降低FAS mRNA表达（P<0.01）,显著上调CPT1 mRNA表达（P<0.05）,且L-DHA效果更好。结论：TG-DHA高含量脱腥鱼油可改善高脂饮食导致的脂代谢紊乱,其机制与下调肝脏脂肪酸合成,促进脂肪酸分解代谢有关。     

Pharmaceutical Development of Nanostructured Vesicular Hydrogel Formulations of Rifampicin for Wound Healing

Chronic wounds exhibit elevated levels of inflammatory cytokines, resulting in the release of proteolytic enzymes which delay wound-healing processes. In recent years, rifampicin has gained significant attention in the treatment of chronic wounds due to an interesting combination of antibacterial and anti-inflammatory effects. Unfortunately, rifampicin is sensitive to hydrolysis and oxidation. As a result, no topical drug product for wound-healing applications has been approved. To address this medical need two nanostructured hydrogel formulations of rifampicin were developed. The liposomal vesicles were embedded into hydroxypropyl methylcellulose (HPMC) gel or a combination of hyaluronic acid and marine collagen. To protect rifampicin from degradation in aqueous environments, a freeze-drying method was developed. Before freeze-drying, two well-defined hydrogel preparations were obtained. After freeze-drying, the visual appearance, chemical stability, residual moisture content, and redispersion time of both preparations were within acceptable limits. However, the morphological characterization revealed an increase in the vesicle size for collagen–hyaluronic acid hydrogel. This was confirmed by subsequent release studies. Interactions of marine collagen with phosphatidylcholine were held responsible for this effect. The HPMC hydrogel formulation remained stable over 6 months of storage. Moving forward, this product fulfills all criteria to be evaluated in preclinical and clinical studies.     

蛋白修饰植物甾醇脂质体的制备及体外消化研究

本研究以植物甾醇(PS)替代胆固醇与卵磷脂(PC)为原料,以粒径、多分散系数PdI、电位和包封率为指标,采用乙醇注入法制备蛋白修饰的植物甾醇脂质体(SPI-LS),通过单因素优化脂质体制备工艺,研究了SPI-LS的复溶性及其在水中的溶解度,并考察了植物甾醇包埋前后体外消化生物活性。结果表明,最佳制备工艺为:PS:PC为1:4,乙醇体积比为25%,蛋白含量为1%;脂质体复溶后外观与冻干前无显著差异,溶解度可达1.971 mg/mL;经3 h体外胃肠消化后,SPI-LS中植物甾醇的生物活性仍可保持在50%以上,是未经包埋植物甾醇的三倍之多。故优化制备条件得到的SPI-LS复溶性良好,并能有效保持植物甾醇的生物活性。     

Insights into the Action Mechanism of the Antimicrobial Peptide Lasioglossin III

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.     

多囊泡型二氢杨梅素脂质体的制备、表征及其抑菌性能

耐药金色葡萄球菌的出现和高效抗生素的缺乏已经对食品质量与公共安全造成威胁,因此,亟需寻找一种新的治疗策略来应对日益严峻的细菌挑战。本研究采用水热提取法从藤茶提取二氢杨梅素（DMY）,并以脂质体为药物载体、聚乙二醇4000为修饰剂成功制备出多囊泡型二氢杨梅素脂质体（DMY-lips）。使用紫外分光光度计、傅里叶红外光谱仪、X射线粉末衍射仪和同步热分析仪分析确定了DMY的成功包覆,并通过透射电子显微镜和纳米粒度测试仪证实了脂质体的多囊泡结构。该脂质体粒径均一,平均粒径为155 nm,载药率为42.93%。此外,抑菌实验证实脂质体的包覆提高了DMY的抑菌活性和抑菌时间,这主要是由于脂质体提高了DMY的水解度和膜渗透度。DMY-lips对金色葡萄球菌的最小抑菌浓度为0.05 mg/ml。生物扫描电镜和电导率测试实验表明DMY-lips可以破坏金色葡萄球菌的细胞壁和细胞膜,导致菌体膜内内容物流出而死亡。因此,该多囊泡型二氢杨梅素脂质体在制药行业具有巨大潜力,并有望缓解医疗系统对化学抗生素的依赖。     

Comparison of different separative techniques in the quantitative determination of active compound enclosed in liposomal systems

The suitability of three different separative techniques, dialysis, gel filtration and centrifugation, for determining the percentage of active compound included (PAI) in liposomal systems was assessed. Two model compounds, glucose and vitamin E acetate were encapsulated in dipalmitoylphosphatidylcholine (DPPC), soybean lecithin (SL) and hydrogenated soybean lecithin (HSL) multilamellar vesicles (MLV). Vitamin E acetate PAI values from DPPC MLV liposomes obtained by dialysis, gel filtration and centrifugation, were compared with those determined by differential scanning calorimetry. Glucose PAI values from DPPC MLV liposomes, obtained using the same separative techniques, were compared with that calculated by taking into account the glucose content of the liposome internal aqueous phase on the basis of liposome mean size determined by light scattering.
Vitamin E acetate and glucose PAI values from SL and HSL liposomes were compared with those obtained for DPPC liposomes. Dialysis proved suitable for PAI determination for both lipophilic and hydrophilic compounds, centrifugation was found to be suitable only for the determination of lipophilic compound PAI values while gel filtration using Sephadex G-25M proved inadequate for the determination of PAI values for both lipophilic and hydrophilic compounds in the experimental conditions used in this study.