Liposomal topotecan formulation with a low polyethylene glycol grafting density: pharmacokinetics and antitumour activity

Objectives PEGylated liposomes could evade recognition by the reticulo‐endothelial system and prolong the circulation time of vesicles, resulting in enhanced targeting efficiency and antitumour effect. Typically, vesicles are modified with distearoylphosphatidylethanolamine (DSPE)‐polyethylene glycol (PEG) at a high PEG grafting density. However, long circulation time and slow drug release rate might induce severe hand‐foot syndrome in clinical practice. In this study, a liposomal topotecan formulation with a low PEG grafting density was prepared and its pharmacokinetics, acute toxicity and antitumour effect were investigated. Methods Topotecan was loaded into liposomes using an ammonium sulfate gradient. The resulting formulation was injected to healthy Wistar rats at different dose levels to investigate whether its clearance followed linear kinetics. Biodistribution was performed in Lewis lung cancer‐bearing mice. The acute toxicity was evaluated in healthy mice and beagle dogs. To compare the antitumour effects of different formulations and dose schedule, RM‐1 prostate, Lewis lung, H446 and L1210 cancer models were used. Key findings Topotecan could be encapsulated into low DSPE‐PEG liposomes with ～100% loading efficiency. The clearance of the liposomal formulation followed linear kinetics at a dose level ranging from 0.5 to 4 mg/kg despite the fact that the vesicles were coated at a low PEG density. Compared with free topotecan the liposomal formulation preferentially accumulated into tumour zones instead of normal tissues. Both formulations could rapidly accumulate into liver and tumour, but the liposomal formulation was cleared from tissues at a slow rate relative to the conventional formulation. In rats and beagle dogs, liposomal formulations could not induce skin toxicity. In all the tumour models, smaller split doses were more therapeutically active than larger doses when the overall dose intensity was equivalent. Conclusions This has been the first report that plasma kinetics of a liposomal formulation with a low PEG density followed linear kinetics. Moreover, due to its short circulation half‐life, the formulation did not induce skin toxicity. Our data revealed that the dose schedule of liposomal drugs should be adjusted in accordance with the biophysical and biological properties of the formulations to achieve the optimal therapeutic efficacy.     

Accelerated blood clearance of pegylated liposomal topotecan: Influence of polyethylene glycol grafting density and animal species

Upon repeated administration, empty pegylated liposomes lose long‐circulating characteristics, referred to as accelerated blood clearance (ABC) phenomenon. However, pegylated liposomal cytotoxic drug formulations could not elicit the phenomenon. In the study, it was found that repeated injection of pegylated liposomal topotecan could induce ABC phenomenon in Wistar rats, beagle dogs, and mice, which might be associated with the formation of empty liposomes in circulation because of the rapid drug release rate. In rats, the 9% polyethylene glycol (PEG) formulation induced more severe ABC phenomenon than 3% PEG formulation despite the similar anti‐PEG immunoglobulin M (IgM) levels following the first dose. Antibody neutralization experiments revealed that high PEG formulation was easily neutralized by IgM. Repeated administration of 3% PEG formulation in dogs could result in more severe ABC phenomenon. It seems that slow infusion was liable to cause ABC phenomenon. In all animal species, considerable intraindividual variability of IgM levels could be observed. Our observations may have important implications for the development, evaluation, and therapeutic use of pegylated liposomal cytotoxic drug formulations because using the current drug loading technology, most of the cytotoxic drugs could not be stably loaded in liposomes and rapid drug leakage from liposomes might occur in circulation. © 2012 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 101:3864–3876, 2012     

The functional roles of poly(ethylene glycol)‐lipid and lysolipid in the drug retention and release from lysolipid‐containing thermosensitive liposomes in vitro and in vivo

Triggered release of liposomal contents following tumor accumulation and mild local heating is pursued as a means of improving the therapeutic index of chemotherapeutic drugs. Lysolipid‐containing thermosensitive liposomes (LTSLs) are composed of dipalmitoylphosphatidylcholine (DPPC), the lysolipid monostearoylphosphatidylcholine (MSPC), and poly(ethylene glycol)‐conjugated distearoylphosphatidylethanolamine (DSPE‐PEG₂₀₀₀). We investigated the roles of DSPE‐PEG₂₀₀₀ and lysolipid in the functional performance of the LTSL–doxorubicin formulation. Varying PEG‐lipid concentration (0–5 mol%) or bilayer orientation did not affect the release; however, lysolipid (0–10 mol%) had a concentration‐dependent effect on drug release at 42°C in vitro. Pharmacokinetics of various LTSL formulations were compared in mice with body temperature controlled at 37°C. As expected, incorporation of the PEG‐lipid increased doxorubicin plasma half‐life; however, PEG‐lipid orientation (bilayer vs. external leaflet) did not significantly improve circulation lifetime or drug retention in LTSL. Approximately 70% of lysolipid was lost within 1 h postinjection of LTSL, which could be due to interactions with the large membrane pool of the biological milieu. Considering that the present LTSL–doxorubicin formulation exhibits significant therapeutic activity when used in conjunction with mild heating, our current study provided critical insights into how the physicochemical properties of LTSL can be tailored to achieve better therapeutic activity. © 2009 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 99: 2295–2308, 2010     

Lipid composition and grafted PEG affect in vivo activity of liposomal mitoxantrone

Mitoxantrone was encapsulated into pegylated SUVs using ammonium sulfate gradient method. Four formulations (LM-s, LM-p, LM-m and LM-m-L) were prepared, which were made from different PCs and exhibited different PEG grafting density. In vitro release studies revealed that drug release rate increased with decreased T(m) of PCs, and reduced PEG polymer coverage. In circulation, the trend towards increased circulation time as T(m) of PCs and PEG lipid content are elevated is observed. However, it was found that the order of toxicity in balb/c mice was Lm-s相似文献   

离子载体A23187介导pH梯度法制备重酒石酸长春瑞滨长循环脂质体

目的:采用A23187制备重酒石酸长春瑞滨长循环脂质体,优化了处方工艺,并考察了含量、包封率、药脂比和体外释放等检测指标。方法:采用A23187介导的pH梯度法制备了重酒石酸长春瑞滨脂质体;用HPLC法检测了脂质体中重酒石酸长春瑞滨的含量和脂质(HSPC)的含量,考察了药脂比;采用阳离子交换树脂分离脂质体和游离药物,HPLC法检测包封率;以4 mmol.L-1NH4Cl-PBS(pH 7.4)为体外释放介质考察了脂质体的体外释放行为。结果:重酒石酸长春瑞滨脂质体包封率为96.1%,药脂比为1∶5(w/w);高药脂比有利于延长药物体外释放的时间。结论:采用A23187介导的pH梯度法制备重酒石酸长春瑞滨脂质体工艺可行、载药量大、包封率高;所建立体外释放的检测方法快速、准确。     

Inhalable Liposomes of Low Molecular Weight Heparin for the Treatment of Venous Thromboembolism

This study tests the feasibility of inhalable pegylated liposomal formulations of low molecular weight heparin (LMWH) for treatment of two clinical manifestations of vascular thromboembolism: deep vein thrombosis (DVT) and pulmonary embolism (PE). Conventional distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and long-circulating pegylated (DSPE–PEG-2000 and DSPE-PEG-5000) liposomes were prepared by hydration method. Formulations were evaluated for particle size, entrapment efficiency, stability, pulmonary absorption, anticoagulant, and thrombolytic effects in rats. Pulmonary absorption was monitored by measuring plasma antifactor Xa activity; anticoagulant and thrombolytic effects were studied by measuring reduction in thrombus weight and amount of dissolved radioactive clot in the blood, respectively. Pegylated liposomal were smaller and showed greater drug entrapment efficiency than conventional liposomes. All formulations produced an increase in pulmonary absorption and circulation time of LMWH upon first dosing. Three repeated dosings of conventional liposomes resulted in decreased half-life and bioavailability; no changes in these parameters were observed with pegylated liposomes. PEG-2000 liposomes were effective in reducing thrombus weight when administered every 48 h over 8 days. In terms of thrombolytic effects and dosing frequency, PEG-2000 liposomes administered via the pulmonary route at a dose of 100U/kg were as effective as 50 U/kg LMWH administered subcutaneously. This paper suggests that inhalable pegylated liposomes of LMWH could be a potential noninvasive approach for DVT and PE treatment. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci99:4554–4564,     

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     

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.     

Novel sulfobutyl ether cyclodextrin gradient leads to highly active liposomal irinotecan formulation

Objectives Liposomal delivery of irinotecan could provide protection against drug hydrolysis, deliver more active lactone form to tumours and prolong irinotecan exposure time. Nevertheless, conventional drug‐loading technologies have typically resulted in undesired drug retention properties. To resolve the problem, a modified gradient loading method was developed and the resulting formulations were evaluated in a systemic manner. Methods Irinotecan was loaded into liposomes using a novel sulfobutyl ether beta‐cyclodextrin (sbe‐CD) gradient. The effect of drug‐to‐lipid ratio (D/L) and polyethylene glycol (PEG) grafting density were investigated. Drug release experiments were performed in ammonium‐containing medium based on the fluorescence dequenching phenomenon of irinotecan. Pharmacokinetic studies were performed in normal balb/c mice treated with different formulations. To compare the anti‐tumour effect of different formulations, an RM‐1 prostate cancer model was used. Acute toxicity studies were performed in healthy female c57 mice. Key findings Irinotecan could be encapsulated into liposomes with > 90% loading efficiency at a high drug‐to‐lipid mass ratio (> 0.5). In‐vitro release experiments revealed that sbe‐CD anion was more able to retain irinotecan than sulfate. Moreover, the elevated D/L ratio elicited decreased drug release kinetics. Both trends had also been observed when the effects of anions and D/L ratio on half‐life of irinotecan were assessed. Pegylated liposomal irinotecan loaded with sbe‐CD/triethylammonium gradient had irinotecan half‐life values ranging from 9.4 to 13.1 h, surpassing vesicles prepared by the triethylammonium sulfate method (～4.5 h). In the RM‐1 tumour model, all the liposomal irinotecan formulations were more therapeutically active than free irinotecan and the formulation with a high D/L ratio was the most efficacious. Moreover, the high D/L formulation might be less toxic than free irinotecan based on acute toxicity studies. Conclusions The novel sbe‐CD gradient could mediate effective irinotecan loading and improve irinotecan retention, thus resulting in highly active liposomal irinotecan formulations. The improvement in drug retention might be associated with the formation of complicated aggregates inside vesicles.     

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.     

Ni2+-mediated mitoxantrone encapsulation: improved efficacy of fast release formulation

Despite that in solution we cannot detect the interaction between Ni(2+) and mitoxantrone (MIT), Ni(2+) could mediate effective and stable MIT loading into large unilamellar vesicles (LUVs). The presence of nigericin had almost no influences on MIT encapsulation. However, in the presence of NH(3), the drug loading kinetics significantly altered. UV-vis spectrum analysis revealed that the absorption profile of liposomal MIT prepared with NiSO(4) gradient method was markedly different from that of liposomal MIT prepared with pH gradient method and that of free MIT. Three liposomal formulations were prepared, which were made from DMPC/chol, DPPC/chol and HSPC/chol and named LM-m, LM-p and LM-s. The in vitro release T(1/2) values for the formulations were 15.0, 28.2 and 38.5h, respectively. Following an intravenous injection into BDF1 mice at a dose of 4 mg/kg, the MIT plasma levels at 24h time point were 3.3, 11.3 and 12.7 microg/mL, considerably compared to that of free MIT group. In L1210 ascitic model, LM-m therapy resulted in approximately 60% long-term survivor (>60 days), and increased survival times in comparison with other treatments. However, both LM-p and LM-s formulations were less therapeutically active than free MIT. In conclusion, transmembrane NiSO(4) gradient could mediate effective MIT loading, and the formulation prepared with fluid lipid had fast release rate and improved efficacy in L1210 ascitic tumor model.     

IN VIVO TRAFFICKING OF LONG-CIRCULATING LIPOSOMES IN TUMOUR-BEARING MICE DETERMINED BY POSITRON EMISSION TOMOGRAPHY

Various kinds of long-circulating liposome, such as ganglioside GM1-, polyethyleneglycol- (PEG-), and glucuronide-modified liposomes, have been developed for passive targeting of liposomal drugs to tumours. To evaluate the in vivo behaviour of such long-circulating liposomes, we investigated the liposomal trafficking, especially early trafficking just after injection of liposomes, by a non-invasive method using positron emission tomography (PET). Liposomes composed of dipalmitoylphosphatidylcholine, cholesterol, and modifier, namely, GM1, distearoylphosphatidylethanolamine (DSPE)–PEG or palmityl-D -glucuronide (PGlcUA), were labelled with [2-¹⁸F]-2-fluoro-2-deoxy-D -glucose ([2-¹⁸F]FDG), and administered to mice bearing Meth A sarcoma after having been sized to 100 nm. A PET scan was started immediately after injection of liposomes and continued for 120 min. PET images and time–activity curves indicated that PEG liposomes and PGlcUA liposomes were efficiently accumulated in tumour tissues time dependently from immediately after injection. In contrast, GM1 liposomes accumulated less in the tumour as was also the case for control liposomes that contained dipalmitoylphosphatidylglycerol (DPPG) instead of a modifier. Long-circulating liposomes including GM1 liposomes, however, remained in the blood circulation and avoided liver trapping compared with control DPPG liposomes. These data suggest that PGlcUA and PEG liposomes start to accumulate in the tumour just after injection, whereas GM1 liposomes may accumulate in the tumour after a longer period of circulation.     

Cholesterol as a bilayer anchor for PEGylation and targeting ligand in folate-receptor-targeted liposomes

Phospholipids have been extensively evaluated as an anchor for both PEGylation and receptor-targeting in liposomal formulations. However, cholesterol, another important component in biomembranes, has not been fully investigated as an alternative anchor. In this study, the potential role of cholesterol for anchoring PEG and folate was investigated. Cholesterol derivatives were synthesized for PEGylation (mPEG-cholesterol) and folate receptor (FR) targeting (folate-PEG-cholesterol) and incorporated into the bilayer of FR-targeted liposomal doxorubicin. The colloidal stability of these cholesterol derivative-containing liposomes was superior to non-PEGylated liposomes, indicating that steric barrier provided by mPEG-cholesterol can efficiently inhibit aggregation of liposomes. FR-targeting activity of these liposomes was demonstrated by in vitro cell-binding studies on FR-overexpressing KB cells. In addition, in vivo circulation of cholesterol-anchored liposomes was prolonged compared to non-PEGylated liposomes. These studies suggest that cholesterol is a viable bilayer anchor for synthesis of PEGylated and FR-targeted liposomes.     

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.     

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.     

Design and development of folate appended liposomes for enhanced delivery of 5-FU to tumor cells

Folate appended sterically-stabilized liposomes (FA-SL) were investigated for tumor targeting. Liposomes were prepared using HSPC, cholesterol and FA-polyethylene glycol (PEG)-SA. The liposomes with polyethylene glycol (PEG) without folic acid which has similar lipid composition were used for comparison. Liposomal preparations were characterized for shape, size and percent entrapment. The average size of liposomes was found to be in range 124-163 nm and maximum drug entrapment was found to be 34.2-40.3%. In vitro drug release from the formulations is obeying fickian release kinetics. Cellular uptake and IC(50) values of the FR-targeted formulation were determined in vitro in FR (+) B16F10 melanoma cells. In vitro cell binding of FA-SL exhibits 11-folds higher binding to B16F10 melanoma cells in comparison to SL. In vivo cytotoxicy assay on FR targeted liposomes gave IC(50) of 1.87 microM and non-targeted liposomes gave IC(50) of 4.02 microM. In therapeutic experiments 5-fluorouracil (5-FU), SL and FA-SL were administered at the dose of 10 mg 5-FU/kg body weight to B16F10 tumor bearing Balb/c mice. Administration of FA-SL formulation results in effective reduction in tumor growth as compared with free 5-FU and SL. Results indicate that folic acid appended SL bearing 5-FU are significantly (P < 0.01) active against primary tumor and metastasis than non-targeted sterically-SL. Thus, it could be concluded that folate coupled liposomal formulations enhanced drug uptake by tumor cells.     

Prolongation of the Circulation Time of Doxorubicin Encapsulated in Liposomes Containing a Polyethylene Glycol-Derivatized Phospholipid: Pharmacokinetic Studies in Rodents and Dogs

The pharmacokinetics of doxorubicin (DOX) encapsulated in liposomes containing polyethylene glycol-derivatized distearoylphosphatidylethanolamine (PEG/DSPE) were investigated in rodents and dogs. The plasma levels of DOX obtained with PEG/DSPE-containing liposomes were consistently higher than those without PEG/DSPE or when PEG/DSPE was replaced with hydrogenated phosphatidylinositol (HPI). Despite the inclusion of PEG/DSPE in liposomes, there was a significant drop in the plasma levels of DOX when the main phospholipid component, hydrogenated phosphatidylcholine, was replaced with lipids of lower phase transition temperature (dipalmitoylphosphatidylcholine, egg phosphatidylcholine), indicating that phase transition temperature affects the pharmacokinetics of liposome-encapsulated DOX. In beagle dogs, clearance was significantly slower for DOX encapsulated in PEG/ DSPE-containing liposomes than in HPI-containing liposomes, with distribution half-lives of 29 and 13 hr, respectively. In both instances, almost 100% of the drug measured in plasma was liposome-associated. The apparent volume of distribution was only slightly above the estimated plasma volume of the dogs, indicating that drug leakage from circulating liposomes is insignificant and that the distribution of liposomal drug is limited mostly to the intravascular compartment in healthy animals.     

The effects of polyethyleneglycol (PEG)-derived lipid on the activity of target-sensitive immunoliposome

In this study, serum stability and target-sensitivity of phosphatidylethanolamine (PE) immunoliposomes prepared with dioleoylphosphatidylethanolamine (DOPE), HYB-241 monoclonal antibody that targets p-glycoproteins, and various levels of polyethyleneglycol 2000 dioleoylphosphatidylethanolamine (PEG(2000)-DOPE) were determined. Incubation of calcein-laden pegylated immunoliposomes prepared with different levels of PEG(2000)-DOPE (0.3, 0.5 and 1.0 mol%) with p-glycoprotein rich bovine brain microvessel endothelial cells in 10% serum cell culture medium, all resulted in time-dependent release of calcein from the liposomes. The release of calcein was greatest for immunoliposomes prepared with 0.3 mol% PEG(2000)-DOPE (66% in 1 h). Contrarily, the release of calcein from the other two immunoliposomes reached only approximately 10-3% after same period of incubation. When serum-induced leakage of calcein was investigated for the above liposome preparations, liposomes prepared with 0.3 and 0.5 mol% PEG(2000)-DOPE had the highest leakage level (10% in 1 h). Contrarily, the release of calcein from liposomes prepared with 1.0 mol% PEG(2000)-DOPE reached only 3% after same period of incubation. Together, it would appear that release of calcein from the immunoliposomes prepared with 0.3 mol% PEG(2000)-DOPE is a result of both serum-induced and target-induced destabilization of liposomes. The net release of calcein due to target-induced destabilization of liposomes is calculated to be at approximately 56%. In contrast, there is no target-induced leakage of calcein from immunoliposomes prepared with either 0.5 or 1.0 mol% PEG(2000)-DOPE.     

Ultrasound-mediated destabilization and drug release from liposomes comprising dioleoylphosphatidylethanolamine

Novel sonosensitive doxorubicin-containing liposomes comprising dioleoylphosphatidylethanolamine (DOPE) as the main lipid constituent were developed and characterized in terms of ultrasound-mediated drug release in vitro. The liposome formulation showed high sonosensitivity; where approximately 95% doxorubicin was released from liposomes after 6 min of 40 kHz US exposure in buffered sucrose solution. This represented a 30% increase in release extent in absolute terms compared to liposomes comprising the saturated lipid analogue distearoylphosphatidylethanolamine (DSPE), and a 9-fold improvement in release extent when compared to standard pegylated liposomal doxorubicin, respectively. Ultrasound release experiments in the presence of serum showed a significantly reduction in sonosensitivity of DSPE-based liposomes, whilst the release properties of DOPE-based liposomes were essentially maintained. Dynamic light scattering measurements and cryo-transmission electron microscopy of DOPE-based liposomes after ultrasound treatment indicated liposome disruption and formation of various lipid structures, corroborating the high release extent. The results point to the potential of DOPE-based liposomes as a new class of drug carriers for ultrasound-mediated drug delivery.     

Characterization of Long-Circulating Cationic Nanoparticle Formulations Consisting of a Two-Stage PEGylation Step for the Delivery of siRNA in a Breast Cancer Tumor Model

Polyethylene glycol (PEG) has been used widely in liposomal formulations as a strategy to inhibit opsonization by plasma proteins and to prolong liposome plasma circulation time. PEG can be incorporated onto the surface of liposomes either during the spontaneous self‐assembling process or inserted after vesicle formation. The advantages of employing the PEG postinsertion method include improved drug encapsulation efficiency and the ability to incorporate PEG conjugates for enhanced cell binding and uptake. In this study, we propose to evaluate a cationic lipid nanoparticle formulation containing two PEGylation steps: pre‐ and post‐siRNA insertion. Our results indicate that formulations consisting of the extra PEG post‐insertion step significantly increased siRNA circulation in the plasma by two‐folds in comparison with the formulations consisting of only the single PEGylation step. Moreover, this formulation was able to efficiently carry siRNA to the tumor site, increase siRNA stability and significantly downregulate luciferase mRNA expression by >50% when compared with the controls in an intraperitoneal and subcutaneous breast cancer tumor model. Overall, our cationic lipid nanoparticle formulation displayed enhanced plasma circulation, reduced liver accumulation, enhanced tumor targeting, and effective gene knockdown‐–demonstrating excellent utility for the delivery of siRNA.