Lipid membrane composition influences drug release from dioleoylphosphatidylethanolamine-based liposomes on exposure to ultrasound

The effect of membrane composition on calcein release from dioleoylphosphatidylethanolamine (DOPE)-based liposomes on exposure to low doses of 1.13 MHz focused ultrasound (US) was investigated by multivariate analysis, with the goal of designing liposomes for US-mediated drug delivery. Regression analysis revealed a strong correlation between sonosensitivity and the non-bilayer forming lipids DOPE and pegylated distearoylphosphatidylethanolamine (DSPE-PEG 2000), with DOPE having the strongest impact. Unlike most of the previously studied distearoylphosphatidylethanolamine (DSPE)-based liposomes, all the current DOPE-based liposome formulations were found stable in 20% serum in terms of drug retention.     

具有活性羧基末端的长循环脂质体的制备和分布

目的　研究长循环免疫脂质体(immunoliposomes,IML)的制备方法, 体外靶细胞杀伤活性和在小鼠体内的组织分布。方法　合成和纯化了1个带末端羧基的磷脂酰乙醇胺(PE)的聚乙二醇衍生物(DPPE-PEG3000-COOH),掺入脂质体中制成长循环脂质体;通过羧基活泼酯化,将膀胱癌单克隆抗体BDI-1或小鼠IgG共价连接到该脂质体表面制成免疫脂质体,体外肿瘤细胞杀伤实验检测载阿霉素免疫脂质体(ADM-BDI-1-IML)特异杀伤靶细胞的能力。用同位素氚示踪法测量免疫脂质体在小鼠的组织分布。结果　抗体在脂质体上的结合率可达30％。体外肿瘤细胞杀伤实验证明载阿霉素免疫脂质体有选择性杀伤靶细胞人膀胱癌细胞EJ的能力。和普通脂质体相比,免疫脂质体在血中的滞留时间明显延长,并减少了在肝、脾的聚集。结论　长循环免疫脂质体在血中有较长的滞留时间,在体外有特异寻靶活性,载阿霉素免疫脂质体有选择性杀伤靶细胞的活性,这些性质为其在体内主动寻靶和选择性杀伤靶肿瘤细胞提供了必要条件。     

Long-circulating, pH-sensitive liposomes sterically stabilized by copolymers bearing short blocks of lipid-mimetic units

A major hurdle towards in vivo utilization of pH-sensitive liposomes is their prompt sequestration by reticuloendothelial system and hence short circulation time. Prolonged circulation of liposomes is usually achieved by incorporation of pegylated lipids, which have been frequently reported to deteriorate the acid-triggered release. In this study we evaluate the ability of four novel nonionic copolymers, bearing short blocks of lipid-mimetic units to provide steric stabilization of DOPE:CHEMs liposomes. The vesicles were prepared using the lipid film hydration method and extrusion, yielding liposomes of 120–160 nm in size. Their pH-sensitivity was monitored via the release of encapsulated calcein. The incorporation of the block copolymers at concentration up to 10 mol% did not deteriorate the pH-sensitivity of the liposomes. A selected formulation was tested for stability in presence of 25% human plasma and proved to significantly outclass the plain DOPE:CHEMs liposomes. The ability of calcein-loaded liposomes to deliver their cargo inside EJ cells was investigated using fluorescent microscopy and the results show that the surface-modified vesicles are as effective to ensure intracellular delivery as plain liposomes. The pharmacokinetics and organ distribution of a selected formulation, containing a copolymer bearing four lipid anchors was investigated in comparison to plain liposomes and PEG (2000)–DSPE stabilized liposomes. The juxtaposition of the blood clearance curves and the calculated pharmacokinetic parameters show that the block copolymer confers superior longevity in vivo. The block copolymers utilized in this study can be consider as promising sterically stabilizing agents for pH-sensitive liposomes.     

Cytofectin amine head group modification and degree of liposome pegylation: factors influencing gene transfer

The effectiveness of liposome-mediated gene transfer methods hinges, in part, on the nature of the interaction between the DNA cargo and the liposomes. Here we have examined the effect of quaternization of the cytofectin cationic head group on this interaction and the effect of concentration of the biocompatible, protective polymer polyethylene glycol(2000) (PEG(2000)) on transfection activity. Thus 3β[N-(N',N'-dimethylaminopropane)-carbamoyl] cholesterol (Chol-T) and 3β[N-(N',N',N'-trimethylammonium propane)-carbamoyl] cholesterol iodide (Chol-Q), differing only in the degree of head group methylation, have been formulated into liposomes with polyethylene glycol(2000)-distearoylphosphatidyl ethanolamine (DSPE PEG(2000)) and the neutral co-lipid dioleoylphosphatidylethanolamine (DOPE). Their DNA-binding characteristics have been determined and the gene transfer capabilities of resulting lipoplexes have been examined in HEK 293 human embryonic kidney cells. Quaternary ammonium Chol-Q liposomes were found to bind DNA more avidly than their tertiary amine Chol-T counterparts. The inclusion of PEG(2000) in liposome formulations resulted in an increase in the optimal liposome-DNA binding ratio. Chol-T liposomes promoted transgene activity levels 5 times greater than those obtained with Chol-Q lipoplexes. Furthermore, a drop in transfection activity of only 17% was noted on increase of liposome pegylation from 2 to 5 mole percent. The study's findings suggest that strong association between cationic liposomes and DNA may lead to reduced levels of transfection activity as a result of poor release of nucleic acid after cellular uptake.     

聚乙二醇对盐酸伊立替康脂质体体外释放及在不同稀释介质中稳定性的影响

目的:考察不同聚乙二醇含量对盐酸伊立替康脂质体体外释放特性及在不同稀释介质中稳定性的影响。方法:采用乙二胺四乙酸铵梯度法,以聚乙二醇2000含量分别为0、8、14、20、26 mg/ml的聚乙二醇2000-二硬脂酰磷脂酰乙醇胺(m PEG2000-DSPE)制备盐酸伊立替康脂质体,测定其包封率和粒径,评价其体外24 h内的累积释放度和在生理氯化钠溶液与5%葡萄糖注射液中的稳定性。结果:随着m PEG2000-DSPE含量的增加,盐酸伊立替康脂质体体外24 h累积释放度逐渐减小;以生理氯化钠溶液及5%葡萄糖注射液稀释后,随着m PEG2000-DSPE含量的增加,盐酸伊立替康脂质体的包封率和粒径变化均减小;当聚乙二醇2000质量浓度增加至20、26 mg/ml时,脂质体包封率和粒径基本不再变化。结论:m PEG2000-DSPE的加入可减慢盐酸伊立替康脂质体的体外释放,提高其在不同稀释介质中的稳定性。     

pH-sensitive, serum-stable and long-circulating liposomes as a new drug delivery system

The lack of stability in blood and the short blood circulation time of pH-sensitive liposomes are major drawbacks for their application in-vivo. To develop pH-sensitive, serum-stable and long-circulating liposomes as drug delivery systems, the impact of polyethylene glycol-derived phosphatidylethanolamine (DSPE-PEG) on the properties of pH-sensitive liposomes was investigated. pH-sensitive liposomes were prepared with dioleoylphosphatidylethanolamine (DOPE) and oleic acid (DOPE/oleic acid liposome) or DOPE and 1,2-dipalmitoylsuccinylglycerol (DOPE/DPSG liposome). The inclusion of DSPE-PEG enhanced the serum stability of both DOPE/oleic acid and DOPE/DPSG liposomes, but also shifted the pH-response curve of pH-sensitive liposomes to more acidic regions and reduced the maximum leakage percentage. The impact of DSPE-PEG, however, was much lower in the DOPE/DPSG liposomes than in the DOPE/oleic acid liposomes. In tumour tissue homogenates, where the pH is lower than normal healthy tissues, the pH-sensitive DOPE/DPSG liposomes released the entrapped markers rapidly, in comparison with pH-insensitive dipalmitoylphosphatidylcholine/cholesterol/DSPE-PEG liposomes. Moreover, the release rate was not affected by the content of DSPE-PEG. The blood circulation time of methotrexate incorporated in DOPE/UDPSG liposomes was significantly prolonged with increasing content of DSPE-PEG. Taken together, the liposomes composed of DOPE, DPSG and DSPE-PEG (up to 5%) were pH sensitive, plasma stable and had a long circulation time in the blood. The complete destabilization of the liposomes at tumour tissues suggests that the liposomes might be useful for the targeted delivery of drugs such as anticancer agents.     

pH-Sensitive PEGylated liposomes functionalized with a fibronectin-mimetic peptide show enhanced intracellular delivery to colon cancer cell

pH-sensitive liposomes undergo rapid destabilization under mildly acidic conditions such as those found in endocytotic vesicles. Though this makes them promising drug carriers, their application is limited due to their rapid clearance from circulation by the reticulo-endothelial system. Researchers have therefore used pH-sensitive liposomes that are sterically stabilized by polyethylene glycol (PEG) molecules (stealth liposomes) on the liposome surface. The goal of this study is to bring bio-functionality to pH-sensitive PEGylated liposomes in order to facilitate their potential use as a targeted drug delivery agent. To improve the selectivity of these nanoparticles, we included a targeting moiety, PR_b which specifically recognizes and binds to integrin α(5)β(1) expressing cells. PR_b (KSSPHSRN(SG)(5)RGDSP) is a novel fibronectin-mimetic peptide sequence that mimics the cell adhesion domain of fibronectin. Integrin α(5)β(1) is expressed on several types of cancer cells, including colon cancer, and plays an important role in tumor growth and metastasis. We have thoroughly studied the release of calcein from pH-sensitive PEGylated liposomes by varying the lipid composition of the liposomes in the absence and presence of the targeting peptide, PR_b, and accounting for the first time for the effect of both pH and time (photo-bleaching effect) on the fluorescence signal of calcein. We have demonstrated that we can design PR_b-targeted pH-sensitive PEGylated liposomes, which can undergo destabilization under mildly acidic conditions and have shown that incorporating the PR_b peptide does not significantly affect the pH-sensitivity of the liposomes. PR_b-targeted pH-sensitive PEGylated liposomes bind to CT26.WT colon carcinoma cells that express integrin α(5)β(1), undergo cellular internalization, and release their load intracellularly in a short period of time as compared to other formulations. Our studies demonstrate that PR_b-functionalized pH-sensitive targeted delivery systems have the potential to deliver a payload directly to cancer cells in an efficient and specific manner.     

Characterization of Cationic Liposome Formulations Designed to Exhibit Extended Plasma Residence Times and Tumor Vasculature Targeting Properties

Cationic liposomes exhibit a propensity to selectively target tumor-associated blood vessels demonstrating potential value as anti-cancer drug delivery vehicles. Their utility however, is hampered by their biological instability and rapid elimination following i.v. administration. Efforts to circumvent rapid plasma elimination have, to date, focused on decreasing cationic lipid content and incorporating polyethylene glycol (PEG)-modified lipids. In this study we wanted to determine whether highly charged cationic liposomes with surface-associated PEG could be designed to exhibit extended circulation lifetimes, while retaining tumor vascular targeting properties in an HT29 colorectal cancer xenograft model. Cationic liposomes prepared of DSPC, cationic lipids (DODAC, DOTAP, or DC-CHOL), and DSPE-PEG₂₀₀₀ were studied. Our results demonstrate that formulations prepared with 50 mol% DODAC or DC-CHOL, and 20 mol% DSPE-PEG₂₀₀₀ exhibited circulation half-lives ranging from 6.5 to 12.5 h. Biodistribution studies demonstrated that DC-CHOL formulations prepared with DSPE-PEG₂₀₀₀ accumulated threefold higher in s.c. HT29 tumors than its PEG-free counterpart. Fluorescence microscopy studies suggested that the presence of DSPE-PEG₂₀₀₀ did not adversely affect liposomal tumor vasculature targeting. We show for the first time that it is achievable to design highly charged, highly pegylated (20 mol% DSPE-PEG₂₀₀₀) cationic liposomes which exhibit both extended circulation lifetimes and tumor vascular targeting properties     

Reconsideration of drug release from temperature-sensitive liposomes

The liposomal phase transition temperature was monitored in unstirred suspensions using a differential scanning calorimeter. The main and pre-transition temperatures under conditions of stirring were measured by the change in 90 degrees light scattering using a fluorescence spectrophotometer. Both methods show the same main transition temperature either with or without stirring. Temperature sensitive liposomes were made of DPPC (dipalmitoylphosphatidylcholine), DMPC (dimylisitoylphosphatidylcholine) or DSPC (distearoylphosphatidylcholine). The calcein release profile from the liposomes depends on the stirring time of the liposome suspension at the main transition temperature. For 1 h incubation, the leakage profile with and without stirring is similar. It had been hypothesized that temperature sensitive liposomes released drug at the main-transition temperature. However, calcein leakage from liposomes is observed also at the pre-transition temperature. Thus, a liposomal encapsulated drug will likely leak from DPPC liposomes at body temperature (37 degrees C), even if the liposomes were designed to have a higher main transition temperature.     

Pharmaco attributes of dioleoylphosphatidylethanolamine/cholesterylhemisuccinate liposomes containing different types of cleavable lipopolymers.

Various amounts of one of three different types of cleavable methoxy polyethylene glycol (mPEG)-phospholipids or of a non-cleavable counterpart (mPEG-DSPE) were included into pH-sensitive liposome formulations containing dioleoylphosphatidylethanolamine (DOPE) and cholesterylhemisuccinate (CHEMS) at a 6:4 molar ratio, and the effect on plasma clearance and contents release rates was determined. The cleavable lipopolymers were all based on a distearoylphosphatidyl lipid anchor, which was linked to mPEG via dithiodipropionateaminoethanol (mPEG-DTP-DSPE), dithio-3-hexanol (mPEG-DTH-DSPA), or Gly-Phe-Leu-Gly-aminoethanol (mPEG-GFLG-DSPE) linkers. In contrast to the first-generation thiolytically cleavable lipopolymer, mPEG-DTP-DSPE, the second generation conjugates contained a hindered disulfide or enzymatically cleavable tetrapeptide, respectively, as the points of scission. In the absence of mPEG-lipid, DOPE/CHEMS liposomes had rapid clearance half-lives. As the mol% of mPEG-lipid in the liposomes increased, the rate of clearance of DOPE/CHEMS liposomes in mice decreased. Zeta-potential measurements showed that decreased clearance was correlated with a decrease in the apparent surface charge of the liposomes, which approached neutrality as the content of mPEG-lipids increased to above 15 mol%. At these levels, liposomes containing mPEG-DTP-DSPE were cleared from blood circulation faster than liposomes containing other, less vulnerable lipopolymers. Liposomes with the peptide-linked lipopolymer exhibited the slowest clearance. The presence of either cleavable or non-cleavable mPEG-lipids at concentrations of 5 mol% or higher in the DOPE/CHEMS liposomes inhibited the release of doxorubicin from these liposomes in response to acid pH.     

胰岛素与二棕榈酰磷脂酰胆碱脂质体的相互作用

目的　研究胰岛素与二棕榈酰磷脂酰胆碱脂质体的相互作用。方法　用反相蒸发法制备脂质体,研究胰岛素对脂质体的包封率、粒径大小及分布的影响,脂质体对胰岛素荧光发射光谱的影响、胰岛素诱发包埋钙黄绿素脂质体的泄漏。结果　胰岛素对粒径为170～190 nm的脂质体的粒径大小及分布影响不大,胰岛素的酪氨酸基团对中性磷脂膜插入不深,胰岛素与磷脂发生作用,在进入脂质体双层过程中扰动脂质体膜,导致钙黄绿素泄漏,随着多肽浓度的增加,脂质体所包封的钙黄绿素的泄漏加快。结论　多肽或蛋白类药物,例如胰岛素,可以通过亲水或疏水的相互作用扰动脂质体的结构完整性。对多肽与脂质体包封的过程要给予足够的重视。     

Stability of poly(Acrylic Acid)‐grafted phospholipid liposomes in gastrointestinal conditions

Major limitations in the use of liposomes for oral formulation relate to their physical and chemical instability in the GI tract. In this study, the conjugate (PAA‐DSPE) of poly(acrylic acid) and distearoylphosphatidylethanolamine (DSPE) was synthesized, and liposomes were prepared with the mixture of PAA‐DSPE and DSPE to improve the stability of liposomes in the GI tract. The prepared PAA‐DSPE was characterized by FT‐IR and ¹³C‐NMR to confirm the coupling between PAA and DSPE. In the elemental analysis, the coupling ratio of PAA and DSPE in PAA‐DSPE was calculated as 1:1.73. The average size of a PAA‐DSPE/DSPE liposome, measured by dynamic light scattering, was in the range of 300–500 nm, and the minimum average size was 320 nm at 6 mol% of PAA‐DSPE. The stability of the prepared liposomes was evaluated in different pH (2, 5, 7.4) solutions, and in different concentrations of bile acid (0, 0.1, 1, 10%) and pancreatin solutions (0, 0.03, 0.3, 3%). The stability of liposomes in different conditions was determined by measuring the fluorescence intensity of 5(6)‐CF leaked from liposomes. The amount of the 5(6)‐CF leakage from the PAA‐DSPE/DSPE liposomes of 6 mol% PAA‐DSPE was the lowest in all the cases of acidic, bile, and pancreatin solutions. In conclusion, the optimum amount of PAA‐DSPE in liposome was 6 mol%, and PAA‐DSPE/DSPE liposomes could improve the stability in the GI tract. Drug Dev. Res. 61:13–18, 2004. © 2004 Wiley‐Liss, Inc.     

聚乙二醇POPA磷脂衍生物构建的酶敏脂质体

以磷酰胺键将聚乙二醇高分子MePEG2000-NH₂与磷脂POPA连接在一起, 合成聚乙二醇磷脂衍生物, 以聚乙二醇磷脂衍生物为主要膜材构建酸敏脂质体。采用荧光分析法系统研究了聚乙二醇磷脂衍生物脂质体在酸性条件下对荧光染料的释药特性。以聚乙二醇磷脂衍生物构建的酸敏脂质体,在pH 6.5～7.5时稳定,其稳定性与制备脂质体的磷脂种类及胆固醇含量密切相关,在pH 5.0时发生显著的荧光泄漏,泄漏率与环境酸性的强度及处于酸性的时间呈正相关。聚乙二醇磷脂衍生物构建的脂质体具有开发成酸敏释药脂质体的前景。
     

Effect of surface ligand density on cytotoxicity and pharmacokinetic profile of docetaxel loaded liposomes

Various biotin-modified liposomes incorporated with docetaxel (DTX) were prepared to study the effect of surface biotin density on the pharmacokinetic profile of the liposome. Four types of liposomes such as PEG modified liposome (PDL), 0.5% (mol) biotin modified liposome (0.5BDL), 1% (mol) biotin modified liposome (1BDL) and 2% (mol) biotin modified liposome (2BDL) were prepared using thin film dispersion method. The prepared liposomes were characterized by measuring encapsulation efficiency (EE), particle size, Zeta-potential, physical stability and drug release profiles in vitro. MTT assay was performed to elevate the cytotoxicity of liposomes on MCF-7 cells. In vivo evaluation was further performed to investigate the effect of biotin surface density on the pharmacokinetic profiles. All the prepared liposomes exhibited high encapsulation efficiency, small particle size, narrow particle distribution and sustained release profiles in vitro. In MTT assay, 0.5BDL showed largest tumor cell toxicity, compared with DTX solution. All liposomes containing DTX showed prolonged blood circulation in vivo, and 0.5BDL showed the longest circulation time among the biotin modified liposome. Surface modification of liposome had a negative impact on the circulation of liposomes in the blood, which needs to be considered when designing the ligand mediated targeting delivery systems. A proper amount of biotin liposome with 0.5% molar ratio is expected to produce the best anti-tumor effect.     

Enhanced Delivery and Antitumor Activity of Doxorubicin Using Long-Circulating Thermosensitive Liposomes Containing Amphipathic Polyethylene Glycol in Combination with Local Hyperthermia

Enhanced delivery of doxorubicin (DXR) to a solid tumor subjected to local hyperthermia was achieved by using long-circulating, thermosensitive liposomes (TSL) composed of dipalmitoyl phosphatidylcholine (DPPC)/distearoyl phosphatidylcholine (DSPC) (9:1, m/m) and 3 mol% amphipathic polyethylene glycol (PEG) in colon 26-bearing mice. Inclusion of 3 mol% of distearoyl phosphatidylethanolamine derivatives of PEG (DSPE-PEG, amphipathic PEG) with a mean molecular weight of 1000 or 5000 in DPPC/DSPC liposomes resulted in decreased reticuloendothelial system (RES) uptake and a concomitant prolongation of circulation time, affording sustained increased blood levels of the liposomes. Concomitantly, DXR levels in blood were also kept high over a long period. The presence of amphipathic PEG did not interfere with the encapsulation of DXR by the pH gradient method (>90% trapping efficiency) or with the temperature-dependent drug release from the liposomes. The optimal size of these liposomes was 180 – 200 nm in mean diameter for thermosensitive drug release and prolonged circulation time. The DXR levels in the tumor after injection of long-circulating TSL (DXR-PEG1000TSL or DXR-PEG5000TSL, at a dose of 5 mg DXR/ kg) with local hyperthermia were much higher than after treatment with DXR-TSL lacking PEG or with free DXR, reaching 7.0 – 8.5 DXR µg/g tumor (approximately 2 times or 6 times higher than that of DXR-TSL or free DXR, respectively). Furthermore, the combination of DXR-PEGTSL and hyperthermia effectively retarded tumor growth and increased survival time. Our results indicate that the combination of drug-loaded, long-circulating, thermosensitive liposomes with local hyperthermia at the tumor site could be clinically useful for delivering a wide range of chemotherapeutic agents in the treatment of solid tumors.     

A targeted liposome delivery system for combretastatin A4: formulation optimization through drug loading and in vitro release studies

Efficient liposomal therapeutics require high drug loading and low leakage. The objective of this study is to develop a targeted liposome delivery system for combretastatin A4 (CA4), a novel antivascular agent, with high loading and stable drug encapsulation. Liposomes composed of hydrogenated soybean phosphatidylcholine (HSPC), cholesterol, and distearoyl phosphoethanolamine-PEG-2000 conjugate (DSPE-PEG) were prepared by the lipid film hydration and extrusion process. Cyclic arginine-glycine-aspartic acid (RGD) peptides with affinity for alphav beta3-integrins overexpressed on tumor vascular endothelial cells were coupled to the distal end of polyethylene glycol (PEG) on the liposomes sterically stabilized with PEG (non-targeted liposomes; LCLs). Effect of lipid concentration, drug-to-lipid ratio, cholesterol, and DSPE-PEG content in the formulation on CA4 loading and its release from the liposomes was studied. Total liposomal CA4 levels obtained increased with increasing lipid concentration in the formulation. As the drug-to-lipid ratio increased from 10:100 to 20:100, total drug in the liposome formulation increased from 1.05+/-0.11 mg/mL to 1.55+/-0.13 mg/mL, respectively. When the drug-to-lipid ratio was further raised to 40:100, the total drug in liposome formulation did not increase, but the amount of free drug increased significantly, thereby decreasing the percent of entrapped drug. Increasing cholesterol content in the formulation decreased drug loading. In vitro drug leakage from the liposomes increased with increase in drug-to-lipid ratio or DSPE-PEG content in the formulation; whereas increasing cholesterol content of the formulation up to 30 mol-percent, decreased CA4 leakage from the liposomes. Ligand coupling to the liposome surface increased drug leakage as a function of ligand density. Optimized liposome formulation with 100 mM lipid concentration, 20:100 drug-to-lipid ratio, 30 mol-percent cholesterol, 4 mol-percent DSPE-PEG, and 1 mol-percent DSPE-PEG-maleimide content yielded 1.77+/-0.14 mg/mL liposomal CA4 with 85.70+/-1.71% of this being entrapped in the liposomes. These liposomes, with measured size of 123.84+/-41.23 nm, released no significant amount of the encapsulated drug over 48 h at 37 degrees C.     

掺入不同形式聚乙二醇的大黄素脂质体的制备与评价

目的：制备掺入甲氧基聚乙二醇2000（mPEG2000）、二硬脂酰乙醇胺-聚乙二醇2000（DSPE-PEG2000）、胆固醇-聚乙二醇2000（Chol-PEG2000）3种形式聚乙二醇（Polyethylene glycol,PEG）的大黄素脂质体,并对其制剂学性质与体外释放性能进行评价。方法：采用薄膜分散-超声法制备掺入不同形式PEG的大黄素脂质体,分别为甲氧基聚乙二醇化大黄素脂质体（mPEG-Emo-Lip）、二硬脂酰乙醇胺-聚乙二醇化大黄素脂质体（DSPE-Emo-Lip）、胆固醇-聚乙二醇化大黄素脂质体（Chol-Emo-Lip）;各脂质体采用激光散射法测定粒径,透射电镜扫描法观察形态,微柱离心法考察包封率（entrapped efficiency,EE%）、载药量（eoading capacity,LC）与泄漏率（leakage,LK%）等制剂学性质;采用动态透析法考察各脂质体在pH 7.4、pH 5.8以及含5% FBS的PBS释放介质中的释放性能。结果：3种脂质体的粒径在120~140 nm之间,分布较均匀,其中mPEG-Emo-Lip粒径稍大;各脂质体包封率大于88%,载药量为32.15~32.53 mg·g^-1,泄漏率低;在pH 7.4、pH 5.8以及含5% FBS的PBS释放介质中的释放度均为DSPE-Emo-Lip > mPEG-Emo-Lip > Chol-Emo-Lip。结论：采用薄膜分散-超声法制备含有不同形式PEG的3种大黄素脂质体,均具有较好的制剂学性质,其中Chol-Emo-Lip具有较强的缓释性能。     

A two-component drug delivery system using Her-2-targeting thermosensitive liposomes

We report on a new method for enhancing the specificity of drug delivery for tumor cells, using thermosensitive immunoliposomes. The liposomes are conjugated to the antibody trastuzumab (Herceptin^®), which targets the human epidermal growth factor receptor 2 (Her-2), a cell membrane receptor overexpressed in many human cancers. Being thermosensitive, the liposomes only release their contents when heated slightly above body temperature, allowing for the possibility of tissue targeting through localized hyperthermia. Using self-quenching calcein, we demonstrate the release of liposome contents into cell endosomes after brief heating to 42°C. To further increase targeting specificity, we incorporate the concept of a two-component delivery system that requires the interaction of two different liposomes within the same endosome for cytoplasmic delivery. Experimental evaluation of the technique using fluorescently labeled liposomes shows that a two-component delivery system, combined with intracellular disruption of liposomes by hyperthermia, significantly increases specificity for Her-2-overexpressing tumor cells.     

Encapsulation of ATP into liposomes by different methods: optimization of the procedure

Different methods and conditions for ATP incorporation into PEGylated liposomes were compared in order to obtain a preparation with a maximized ATP content. Such a preparation may find the application for the in vivo treatment of ischemic tissues suffering from an insufficient ATP supply. Several different methods of liposome preparation and purification were used and HPLC was employed to determine the concentration of ATP in the liposomes. Thin lipid film hydration produced vesicles with the lowest ATP encapsulation (ca. 5 mol%). A pH gradient method yielded liposomes with ca. 10 mol% of ATP. Reverse phase evaporation and freezing-thawing methods resulted in a maximum entrapment of ATP on the level of 36-38 mol%. The freezing-thawing method was chosen for further investigation because of its simplicity and absence of a need to use organic solvents. The separation of the non-entrapped ATP by gel-filtration, centrifugation or dialysis yielded virtually identical liposomal preparations. The incorporation of PEG (as PEG-distearoyl phosphatidylethanolamine, PEG-DSPE) into the liposomal membrane decreases the quantity of the entrapped ATP (from 38 mol% for liposomes with 0.5 mol% of PEG-DSPE to only 17 mol% for liposomes with 5 mol% of PEG-DSPE).     

PEGylated estradiol benzoate liposomes as a potential local vascular delivery system for treatment of restenosis

This study was directed towards the preparation and optimization of PEGylated (PEG, poly(ethylene glycol)) estradiol benzoate (ESB)-loaded liposomes to be used for the treatment of restenosis by local vascular delivery. Various liposomal formulations were prepared by thin film hydration method followed by sonication. Response surface methodology was applied to study the influence of three different independent variables, on the response of entrapment efficiency (%EE). Liposomes were characterized in terms of size, zeta potential, %EE and release profile. Incorporation of ESB was higher in egg phosphatidylcholine (EPC) liposomes, whereas the drug was displaced from liposomes, as the cholesterol (Chol) content of liposome increased. The optimum formulation composed of EPC/dioleyloxy trimethylammonium propane/distearoylphosphatidylethanolamine-PEG2000 with a molar proportion of 8.5:1:0.5 had the highest EE. In?vivo studies in the balloon-injured rat carotid arteries revealed the potential of ESB-loaded liposomes as efficient local and controlled drug delivery systems to reduce restenosis.