不同表面活性剂对两性霉素B脂质体体内外性质的影响

目的寻找与DSPE-PEG功能相似的表面活性剂修饰两性霉素B脂质体，以增加其在体内的稳定性，改善其体内分布，降低毒副作用。方法用薄膜超声法制备两性霉素B脂质体；比较用DSPE-PEG，Tween 80和Brij 35修饰后两性霉素B脂质体包封率、粒径分布、稳定性及组织分布的变化。结果两性霉素B脂质体的包封率最高为（91.2±1.6）％。修饰后的两性霉素B脂质体包封率提高，粒径减小，稳定性增加，肝、脾和肾中两性霉素B的浓度降低，脑中AmB的浓度提高。结论DSPE-PEG和Brij 35能提高脂质体逃避网状内皮系统吞噬的能力；Tween 80能增加两性霉素B在大鼠脑组织中的分布。     

培美曲塞-磷脂偶联物修饰的靶向性舒尼替尼与长春瑞滨脂质体的研制及其在体外对耐药性乳腺癌的抑制效应

乳腺癌是女性最易罹患的疾病,而肿瘤多药耐药性通常是化疗失败的主要原因。本研究以培美曲塞（PMT）和DSPE-PEG2000-NH2为原料合成了新的靶向性偶联物DSPE—PEG2000．PMT,并将其修饰到脂质体表面,制备了同时包封有舒尼替尼与长春瑞滨的靶向性脂质体,以增强化疗药物对多药耐药性乳腺癌的治疗效果。经过质谱分析证实,合成的靶向性载体材料DSPE．PEG2000-PMT与目标产物相符。建立了可同时检测舒尼替尼和长春瑞滨含量的高效液相色谱分析方法,检测波长为215nm,柱温30℃,流动相为乙腈-0．05MKH2P04（pH3．5）-三乙胺（35：65：0．3,v／v／v）。舒尼替尼和长春瑞滨的最低检测浓度分别为25ng／mL和5ng／mL,最低定量浓度均为0．25μg／mL。两药在0．5-25．0μg／mL范围内线性良好。各脂质体包封率均大于90％,粒径均-（～90nm）,Zeta电位略显负电性。在体外耐药乳腺癌MCF-7／Adr细胞中评价了靶向性舒尼替尼与长春瑞滨脂质体的抗增殖效应。结果显示,同对照组相比,靶向性舒尼替尼与长春瑞滨脂质体对MCF-7／Adr细胞具有最强的抑制增殖效应。以靶向性香豆素脂质体为荧光探针,考察了靶向性脂质体在耐药乳腺癌MCF-7／Adr细胞中的靶向性,同非靶向性制剂相比,靶向脂质体在耐药性癌细胞中摄取最多。因此,制备的靶向性舒尼替尼与长春瑞滨脂质体是-种新的靶向制剂,能够被耐药乳腺癌细胞靶向性摄取,可在体外显著抑制耐药性乳腺癌生长,从而为耐药乳腺癌的化学治疗提供了-种新的策略。     

Hydration of surfactant-modified and PEGylated cationic cholesterol-based liposomes and corresponding lipoplexes by monitoring a fluorescent probe and the dielectric relaxation time

For the optimization of plasmid DNA (pDNA)-cationic lipid complexes and lipoplex delivery, proper indexes of the physicochemical properties of lipoplexes are required. In general, the characteristics of lipoplexes are defined by particle size and zeta-potential at various mixing ratios of cationic liposomes and pDNA. In this study, we characterized the hydration level of surfactant-modified and PEGylated cationic cholesterol-based (OH-Chol) liposomes and their lipoplexes by monitoring both the fluorescent probe laurdan and the dielectric relaxation time. Fluorescence measurement using laurdan detected hydration of the headgroup of lipids in surfactant-modified liposomes and PEGylated DOTAP-liposomes, but hardly any fluorescence was detected in PEGylated OH-Chol-liposomes because the PEG layers may extend and cover the fluorescent maker. On the other hand, the measurement of dielectric relaxation time of water molecules revealed total hydration, including hydration of the PEG layer and the headgroup of cationic lipids. Furthermore, we found an inverse correlation between hydration level and cellular uptake of PEGylated lipoplexes (R=0.946). This finding indicated that the dielectric relaxation time of water molecules provides an important indicator of hydration of liposome and lipoplexes along with the fluorescence intensity of laurdan.     

色胺酮纳米胶束的制备及其性质研究

目的制备色胺酮纳米胶束,改善色胺酮的水溶性,并进行体外性质考察。方法以二硬脂酰基磷脂酰乙醇胺-聚乙二醇2000(DSPE-PEG2000)为载体,用溶剂挥发法制备色胺酮纳米胶束,通过正交实验筛选制备胶束的最佳条件,核磁共振氢谱(1 HNMR)验证色胺酮包载于纳米胶束,用芘荧光探针法测定其临界胶束浓度(CMC),用紫外分光光度计测定其包封率和载药率,动态光散射法测定胶束的粒径,以粒径、外观形态和包封率为指标考察胶束的稳定性。结果色胺酮纳米胶束的CMC为8.93×10-6mol·L~(-1),色胺酮与聚合物投药比为0.442 9∶1(mol∶mol),真空干燥1h,水化5min时,胶束的包封率为32.24%±1.37%,载药率为5.468%±0.39%。色胺酮纳米胶束平均粒径为112.5nm,平均分散系数为0.208,4℃条件下胶束可稳定15d以上。结论制备色胺酮纳米胶束,将色胺酮的溶解度提高至1.625mmol·L~(-1),为改善色胺酮生物利用度的研究奠定了基础。     

Preparation and anti-MDR tumors study of folate and TPGS dual-modified DSPE-PEG micelles loaded with docetaxel

Multidrug resistance (MDR) operated by P-glycoprotein (P-gp) is one of the major causes in the treatment failure of cancers. In this work,docetaxel-loaded mixed micelles comprised of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene-glycol)₂₀₀₀ (DSPE-PEG₂₀₀₀),D-α-Tocopherylpolyethylene glycol 1000 succinate (TPGS₁₀₀₀) and DSPE-PEG₂₀₀₀-folate were developed to overcome MDR and reduce the side effect of docetaxel in cancer therapy. The diameters of micelles ranged from 13 to 26 nm and the encapsulation efficiencies were all above 85%. The influences of DSPE-PEG₂₀₀₀ and TPGS₁₀₀₀ ratios on the micellar characteristics and anti-resistant tumors effects were evaluated. Micelles with high TPGS₁₀₀₀ amount showed an increased cellular uptake and stronger cytotoxicity against MDR KBv cells. Moreover, the micelles modified by targeting ligand of folic acid exhibited better antitumor effect on folate receptor over-expressing KBv cells.The study provides a method for overcoming MDR in cancer therapy.     

N-oleoylethanolamine − phosphatidylcholine complex loaded,DSPE-PEG integrated liposomes for efficient stroke

Causing more and more deaths, stroke has been a leading cause of death worldwide. However, success in clinical stroke trials has remained elusive. N-oleoylethanolamine (OEA) was an endogenous highly hydrophobic molecule with outstanding neuroprotective effect. In this article, hydrogen bonds were successfully formed between OEA and soybean phosphatidylcholine (SPC). The synthetic OEA-SPC complex and DSPE-PEG were self-assembled into liposomes (OEA NPs), with OEA-SPC loaded in the core and PEG formed a hydrophilic shell. Hence, highly hydrophobic OEA was loaded into liposomes as amorphous state with a drug loading of 8.21 ± 0.18 wt%. With fairly uniform size and well-distributed character, the OEA NPs were systemically assessed as an intravenous formulation for stroke therapy. The results indicated that the administration of OEA NPs could significantly improve the survival rate and the Garcia score of the MCAO rats compared with free OEA. The TTC-stained brain slices declared that the cerebral infarct volume and the edema degree induced by MCAO could be decreased to an extremely low level via the administration of OEA NPs. The Morris water maze (MWM) test suggested that the spatial learning and memory of the MCAO rats could also be ameliorated by OEA NPs. The immunofluorescence assay stated that the apoptosis of the neurons and the inflammation within the brain were greatly inhibited. The results suggest that the OEA NPs have a great chance to develop OEA as a potential anti-stroke formulation for clinic application.     

Core-shell nanoparticles for targeted and combination antiretroviral activity in gut-homing T cells

A major sanctuary site for HIV infection is the gut-associated lymphoid tissue (GALT). The α4β7 integrin gut homing receptor is a promising therapeutic target for the virus reservoir because it leads to migration of infected cells to the GALT and facilitates HIV infection. Here, we developed a core-shell nanoparticle incorporating the α4β7 monoclonal antibody (mAb) as a dual-functional ligand for selectively targeting a protease inhibitor (PI) to gut-homing T cells in the GALT while simultaneously blocking HIV infection. Our nanoparticles significantly reduced cytotoxicity of the PI and enhanced its in vitro antiviral activity in combination with α4β7 mAb. We demonstrate targeting function of our nanocarriers in a human T cell line and primary cells isolated from macaque ileum, and observed higher in vivo biodistribution to the murine small intestines where they accumulate in α4β7+ cells. Our LCNP shows the potential to co-deliver ARVs and mAbs for eradicating HIV reservoirs.     

Selective death of human breast cancer cells by lytic immunoliposomes: Correlation with their HER2 expression level

Trastuzumab (Herceptin™) targets the human epidermal growth factor receptor 2 (HER2), which is overexpressed in 20–30% of breast and ovarian cancers carrying a bad prognosis. Our purpose was to target HER2-overexpressing human breast cancer cells with pegylated immunoliposomes bearing trastuzumab and containing melittin, which has recently shown anticancer properties. Using a panel of human breast cancer cells with different HER2 expression levels, these immunoliposomes decreased cancer cells viability in a dose–response manner and in correlation to their level of HER2 expression. Specific binding of the immunoliposomes to SKBr3 breast cancer cells was shown by ImageStream-based analysis. The morphological changes observed in the treated cells suggested a cytolytic process. This preclinical approach may suppose an effective strategy for the treatment of HER2-overexpressing tumors, and can support the development of an early phases I–II clinical trial. Trastuzumab resistant breast cancer cells (JIMT-1), can also be targeted using this approach.     

The antiangiogenic efficacy of NGR-modified PEG–DSPE micelles containing paclitaxel (NGR-M-PTX) for the treatment of glioma in rats

Aminopeptidase N (APN), recognized by Asn-Gly-Arg (NGR) peptides, is expressed in the pericytes associated with the BBB, and the main objective of this study is to confirm the hypothesis that NGR-modified DSPE–PEG micelles containing paclitaxel (NGR-M-PTX) can bind to and kill brain tumor angiogenic blood vessels and penetrate into the brain tumor interstitial space, resulting in direct cell death. NGR-M-PTX is prepared by a thin-film hydration method. The in vitro targeting characteristics of NGR-modified micelles on BMEC (murine brain microvascular endothelial cells) were investigated. The effect of NGR-M-PTX on BMEC proliferation and the cytotoxicity of NGR-M-PTX in C6 glioma cells were also tested. The antitumor activity NGR-M-PTX was evaluated in C6 glioma tumor–bearing rats in vivo. The particle size of NGR-M-PTX was approximately 54.2?nm. The drug encapsulation efficiency of NGR-M-PTX was 82.11?±?2.82%. The cellular coumarin-6 level of NGR-M-coumarin-6 in the BMEC was about 2.2-fold higher than that of M-coumarin-6. BMEC proliferation was significantly inhibited by NGR-M-PTX. NGR-M-PTX had a much lower IC₅₀ value than M-PTX and free drug. The growth of C6 glioma tumor was markedly inhibited by NGR-M-PTX compared with Taxol. In conclusion, our results show that antiangiogenic therapy using NGR-M-PTX exhibits potent in vivo antitumor activity in a C6 glioma–bearing animal model.     

Specific targeting to B cells by lipid-based nanoparticles conjugated with a novel CD22-ScFv

The CD22 antigen is a viable target for therapeutic intervention for B-cell lymphomas. Several therapeutic anti-CD22 antibodies as well as an anti-CD22-based immunotoxin (HA22) are currently under investigation in clinical settings. Coupling of anti-CD22 reagents with a nano-drug delivery vehicle is projected to significantly improve treatment efficacies. Therefore, we generated a mutant of the targeting segment of HA22 (a CD22 scFv) to increase its soluble expression (mut-HA22), and conjugated it to the surface of sonicated liposomes to generate immunoliposomes (mut-HA22-liposomes). We examined liposome binding and uptake by CD22⁺ B-lymphocytes (BJAB) by using calcein and/or rhodamine PE-labeled liposomes. We also tested the effect of targeting on cellular toxicity with doxorubicin-loaded liposomes. We report that: (i) Binding of mut-HA22-liposomes to BJAB cells was significantly greater than liposomes not conjugated with mut-HA22 (control liposomes), and mut-HA22-liposomes bind to and are taken in by BJAB cells in a dose and temperature-dependent manner, respectively; (ii) This binding occurred via the interaction with the cellular CD22 as pre-incubation of the cells with mut-HA22 blocked subsequent liposome binding; (iii) Intracellular localization of mut-HA22-liposomes at 37 °C but not at 4 °C indicated that our targeted liposomes were taken up through an energy dependent process via receptor-mediated endocytosis; and (iv) Mut-HA22-liposomes loaded with doxorubicin exhibited at least 2-3 fold more accumulation of doxorubicin in BJAB cells as compared to control liposomes. Moreover, these liposomes showed at least a 2-4 fold enhanced killing of BJAB or Raji cells (CD22⁺), but not SUP-T1 cells (CD22⁻). Taken together these data suggest that these 2nd-generation liposomes may serve as promising carriers for targeted drug delivery to treat patients suffering from B-cell lymphoma.