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.     

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.     

In vivo monitoring of liposomal release in tumours following ultrasound stimulation

Dioeleoylphosphatidylethanolamine (DOPE)-based liposomes were recently reported as a new class of liposomes for ultrasound (US)-mediated drug delivery. The liposomes showed both high stability and in vitro US-mediated drug release (sonosensitivity). In the current study, in vivo proof-of-principle of US triggered release in tumoured mice was demonstrated using optical imaging. Confocal non-thermal US was used to deliver cavitation to tumours in a well-controlled manner. To detect in vivo release, the near infrared fluorochrome Al (III) Phthalocyanine Chloride Tetrasulphonic acid (AlPcS₄) was encapsulated into both DOPE-based liposomes and control liposomes based on hydrogenated soy phosphatidylcholine (HSPC). Encapsulation causes concentration dependent quenching of fluorescence that is recovered upon AlPcS₄ release from the liposomes. Exposure of tumours to US resulted in a significant increase in fluorescence in mice administered with DOPE-based liposomes, but no change in the mice treated with HSPC-based liposomes. Thus, DOPE-based liposomes showed superior sonosensitivity compared to HSPC-based liposomes in vivo.     

DC-Chol/DOPE cationic liposomes: A comparative study of the influence factors on plasmid pDNA and siRNA gene delivery

Cationic liposomes (CLs) composed of 3β-[N-(N′,N′-dimethylaminoethane) carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposomes) have been classified as one of the most efficient gene delivery systems. Our study aims to examine the effect of the molar ratio of DC-Chol/DOPE, PEGylation and serum on the pDNA (plasmid pDNA) and siRNA (small interfering RNA) transfection of DC-Chol/DOPE liposomes. The results showed that the most efficient DC-Chol/DOPE liposomes for pDNA or siRNA delivery were at a 1:2 or 1:1 molar ratio of DC-Chol/DOPE, respectively. The transfection efficiency of DC-Chol/DOPE liposomes increased along with increased weight ratio of DC-Chol/siRNA. However, the pDNA transfection efficiency decreased along with increased weight ratio of DC-Chol/pDNA from 3/1. As expected, PEGylation decreased siRNA and pDNA transfection efficiency of DC-Chol/DOPE liposomes. In PEGylated DC-Chol/DOPE liposomes, increased weight ratio of DC-Chol/pDNA from 3/1 did not lead to higher pDNA transfection efficiency, whereas increased weight ratio of DC-Chol/siRNA resulted in increased siRNA transfection efficiency. Furthermore, the serum did not significantly inhibit the pDNA and siRNA transfection efficiency of DC-Chol/DOPE liposomes. In conclusion, our results elucidated the influence factors of DC-Chol/DOPE liposome transfection and would reveal that siRNA and pDNA transfection mechanisms were different in DC-Chol/DOPE liposomes.     

Encapsulation enhancement and stabilization of insulin in cationic liposomes

The purpose of this study was to enhance encapsulation efficiency and sustained-release delivery for parenteral administration of a protein drug. To reduce the administration frequency of protein drugs, it is necessary to develop sustained delivery systems. In this study, protein drug-loaded cationic liposomes were formulated with dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), dioleoyl-3-trimethylammonium-propane (DOTAP), and cholesterol (CH) at a molar ratio of DOPE/DOTAP/CH of 2/1.5/2. Five mol% of distearoylphosphatidyl ethanolamine polyethylene glycol (DSPE-PEG) was added prior to encapsulation of the drug into liposomes. Insulin was chosen as a model protein drug and encapsulation efficiency was evaluated in various liposomes with and without DSPE-PEG. Scanning electron microscopy was used to examine the insulin-loaded cationic liposomes. Structural analysis was performed using spectropolarimetry. Additionally, the stability and cytotoxicity of insulin-loaded cationic liposomes were evaluated. Liposomes coated with DSPE-PEG showed higher insulin encapsulation efficiency than did those without DSPE-PEG, but not significantly. Moreover, among the liposomes coated with DSPE-PEG, those hydrated with 10% sucrose showed higher encapsulation efficiency than did liposomes hydrated in either phosphate-buffered saline or 5% dextrose. In vitro release of insulin was prolonged by cationic liposomes. Our findings suggest that cationic liposomes may be a potential sustained-release delivery system for parenteral administration of protein and peptide drugs to prolong efficacy and improve bioavailability.     

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.     

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.     

Cationic liposome (DC-Chol/DOPE=1:2) and a modified ethanol injection method to prepare liposomes, increased gene expression

Cationic liposomes composed of 3beta-[N-(N',N'-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) (DC-Chol/DOPE liposome, molar ratio, 1:1 or 3:2) prepared by the dry-film method have been often used as non-viral gene delivery vectors. The formulation and preparation of DC-Chol/DOPE liposomes, as well as the formation of their lipoplexes were investigated in an attempt to improve transfection efficiency in vitro. A more efficient transfection in medium with serum was achieved using DC-Chol/DOPE liposomes (molar ratio, 1:2) than those (3:2), and preparation method by a modified ethanol injection than the dry-film. The most efficient DC-Chol/DOPE liposome for gene transfer was molar ratio (1:2) and prepared by a modified ethanol injection method. The enhanced transfection might be related to an increase in the release of DNA in the cytoplasm by the large lipoplex during incubation in optiMEM, not to an increased cellular association with the lipoplex. The use of a modified ethanol injection method might enhance the role of DOPE that is aid in destabilization of the plasma membrane and/or endosome. These findings suggested that cationic liposomes rich in DOPE prepared by a modified ethanol injection method will help to improve the efficacy of liposome vector systems for gene delivery.     

Direct measurement of the extravasation of polyethyleneglycol-coated liposomes into solid tumor tissue by in vivo fluorescence microscopy

The extravasation of liposomes of different sizes into solid tumors after i.v. injection was visualized by in vivo fluorescence microscopy in mouse neuroblastoma C-1300-bearing mice. Liposomes composed of distearoylphosphatidylcholine/cholesterol (1/1 molar ratio) and 6 mol% distearoylphosphatidylethanolamine derivative of polyethyleneglycol (PEG) were prepared. The PEG-coated liposomes were fluorescently labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) as a liposome marker or with doxorubicin (DXR) as an aqueous-phase marker. Liposomes with an average diameter of 100–200 nm showed the greatest tumor accumulation. With time after injection of DiI-labeled liposomes, the tumor interstitial fluorescence intensity increased. Most fluorescent spots were located outside and around the vessel wall, indicating extravasation of intact liposomes. The perivascular distribution was heterogeneous. We also obtained the same fluorescence localization pattern with DXR released from extravasated liposomes after injection of DXR-encapsulated liposomes. No fluorescence from extravasated liposomes was detected in normal s.c. tissue; the fluorescent spots were observed only in the vessel wall. Our results indicate that small-size long-circulating liposomes are able to traverse the endothelium of blood vessels in tumors and extravasate into interstitial spaces. Moreover, encapsulated drug was released from extravasated liposomes in the tumor.     

Synthesis of transferrin (Tf) conjugated liposomes via Staudinger ligation

Staudinger ligation was evaluated as a strategy for synthesizing receptor targeted liposomes. First, an activated lipid derivative was synthesized by reacting dioleoyl phosphatidylethanolamine (DOPE) and 2-(diphenylphosphino) terephthalic acid 1-methyl 4-penta-fluorophenyldiester. Second, transferrin (Tf) was activated with p-azidophenyl isothiocyanate. Third, liposomes containing the activated lipid were prepared and then coupled to the activated Tf via the Staudinger reaction. These liposomes were evaluated in KB cells for cellular uptake and cytotoxicity, and in mice for pharmacokinetic properties. Tf-derivatized liposomes encapsulating calcein prepared by this conjugation method effectively targeted Tf receptor expressing KB cells. In addition, the Tf-targeted liposomes entrapping doxorubicin showed greatly enhanced in vitro cytotoxicity relative to non-targeted control liposomes. Pharmacokinetic parameters indicated that these liposomes retained long circulating properties relative to the free drug. In summary, Staudinger ligation is an effective method for the synthesis of receptor targeted liposomes.     

High Sensitivity Differential Scanning Calorimetry Study of DNA-Cationic Liposome Complexes

PURPOSE: To investigate plasmid DNA interactions with liposomes prepared from dimyristoylglyceroethylphosphocholine (EDMPC) and DOPE using high sensitivity differential scanning calorimetry (HSDSC). MATERIALS AND METHODS: Large unilamellar liposomes of EDMPC with DOPE (mol ratio 0-50%) were prepared. Plasmid DNA was added to give a final DNA/lipid (-/+) charge ratio of 0.5. Samples were placed into an HSDSC and cooled to 3 degrees C, held isothermally for 30 min and then the temperature was ramped to 120 degrees C at a rate of 1 degree C/min. RESULTS: On heating EDMPC liposomes, the main phase transition occurred at 21.2 degrees C, with a low temperature shoulder on the endothermic peak. At low DOPE concentrations the main phase transition temperatures and enthalpies of transition were lower than for pure EDMPC, with a peak corresponding to a pure EDMPC phase occurring at DOPE concentrations of 12-17 mol%. At 50 mol%, no main transition endotherm was observed. DNA solution produced two endothermic peaks with numerous 'satellite' peaks indicating thermal denaturation. DNA binding to EDMPC changed the shape of the thermogram, indicating alteration in lipid packing within the bilayer. DNA induced demixing in the bilayers of DOPE-containing liposomes. CONCLUSION: HSDSC provided information for characterizing liposome formulations and DNA interactions with such vesicles.     

pH-sensitive liposomes--principle and application in cancer therapy

The purpose of this review is to provide an insight into the different aspects of pH-sensitive liposomes. The review consists of 6 parts: the first introduces different types of medications made in liposomal drug delivery to overcome several drawbacks; the second elaborates the development of pH-sensitive liposomes; the third explains diverse mechanisms associated with the endocytosis and the cytosolic delivery of the drugs through pH-sensitive liposomes; the fourth describes the role and importance of pH-sensitive lipid dioleoylphosphatidylethanolamine (DOPE) and research carried on it; the fifth explains successful strategies used so far using the mechanism of pH sensitivity for fusogenic activity; the final part is a compilation of research that has played a significant role in emphasizing the success of pH-sensitive liposomes as an efficient drug delivery system in the treatment of malignant tumours. pH-Sensitive liposomes have been extensively studied in recent years as an amicable alternative to conventional liposomes in effectively targeting and accumulating anti-cancer drugs in tumours. This research suggests that pH-sensitive liposomes are more efficient in delivering anti-cancer drugs than conventional and long-circulating liposomes due to their fusogenic property. Research focused on the clinical and therapeutic side of pH-sensitive liposomes would enable their commercial utility in cancer treatment.     

Development of pH-sensitive liposomes that efficiently retain encapsulated doxorubicin (DXR) in blood

We have reported that targeted, pH-sensitive sterically stabilized liposomes are able to increase the cytotoxicity of DXR in vitro against B lymphoma cells, but the rate of release of DXR in plasma was too rapid to permit the results to be extended to in vivo applications. The purpose of the study reported here is two-fold. First, to understand the mechanism of the rapid release of DXR from pH-sensitive sterically stabilized liposomes (PSL) in human plasma. Second, to reformulate the above liposomes to improve their drug retention, while retaining their pH sensitivity. The stability of the PSL formulations in human plasma was evaluated by comparing the rate of release of encapsulated DXR with that of HPTS, a water-soluble fluorescent marker. Since DXR, but not HPTS, a water soluble-less membrane permeable fluorescence marker, was rapidly released from liposomes in the presence of plasma, the rapid release of DXR is likely caused by the diffusion of DXR molecules through the lipid bilayer, not by the disruption of the membrane. In order to develop more stable PSL formulations, various molar ratios of the membrane rigidifying lipid, hydrogenated soy HSPC and/or CHOL, were added to the lipid composition and the rate of release of encapsulated solutes and pH-sensitivity were evaluated. The compositions that showed the best drug retention and pH-sensitivity were a mixture of DOPE/HSPC/CHEMS/CHOL/mPEG(2000)-DSPE at a molar ratio of 4:2:2:2:0.3 and DOPE/HSPC/CHEMS/CHOL at a molar ratio of 4:2:2:2. Our formulations, if targeted to internalizing antigens on cancer cells, may increase intracellular drug release rates within acidic compartment, resulting in a further increase in the therapeutic efficacy of targeted anticancer drug-containing liposomes.     

A nano-liposome vaccine carrying E75, a HER-2/neu-derived peptide,exhibits significant antitumour activity in mice

E75 (HER-2/neu-369–377), is an immunogenic peptide which is highly expressed in breast cancer patients. The purpose of this study was to develop an effective vaccine delivery/adjuvant system by attachment of this peptide to the surface of liposomes consisting of phospholipids including distearoylphosphocholine (DSPC) and distearoyl phosphoglycerol (DSPG) with high transition temperature (Tm) and dioleoylphosphatidylethanolamine (DOPE) (a pH-sensitive lipid for cytosolic antigen delivery) to improve antitumour immune activity against the E75 peptide. For this purpose, the E75 peptide was incorporated into liposomes consisting of DSPC/DSPG/cholesterol (Chol)/DOPE (15/2/3/5 molar ratio) through conjugation with distearoylphosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (maleimide-PEG₂₀₀₀-DSPE). Immunization of BALB/c mice was performed three times with different forms of liposomal formulations at 2-week intervals and antitumour immunity responses were evaluated. Results of ELISpot and flow cytometry analysis showed that mice vaccinated with DSPC/DSPG/Chol/DOPE/E75 have significantly enhanced the antigen-specific IFN-γ response of CD8⁺ T cells and generated cytotoxic T lymphocytes (CTL) antitumour responses. CTL responses induced by this formulation resulted in inhibition of tumour progression and longer survival time in the mice TUBO tumour model. The results revealed that the liposomes consist of DSPC/DSPG/Chol/DOPE could be suitable candidates for vaccine delivery of E75 peptide for the prevention and therapy of HER2-positive breast cancer and merit further investigation.     

Development and characterization of site specific target sensitive liposomes for the delivery of thrombolytic agents

In recent times, search for potent and highly selective thrombolytic agents with minimal side effects has become a major area of research. The aim of the present study was to develop and characterize target sensitive (TS) liposomes encapsulating streptokinase, a thrombolytic agent. The developed TS liposomes were composed of dioleylphophatidyl ethanolamine (DOPE) and dipalmityl-c(RGDfK) (10:1mol/mol). Dipalmityl-c(RGDfK) was synthesized using typical carbodiimide chemistry using palmitic acid and c(RGDfK), while lysine was used as a spacer. Liposomes were of 100-120nm size. In vitro drug release study showed that nearly 40% drug of the entrapped drug was released in 12h in the PBS (pH 7.4), however on incubation with activated platelet about 90% of drug was released within 45min. The results suggested target sensitivity of the liposomes. Further, targeting potential was confirmed using fluorescent microscopy and flow cytometry. Clot lysis study revealed that TS liposomes could not only reduce the clot lysis time but also increase the extent of clot lysis as compared to non-liposomal streptokinase solution. In conclusion, the present liposomal formulation will target the thrombolytic agent to the activated platelets in the thrombus and hence will improve the therapeutic efficacy of the drug.     

In vitro and in vivo transfection of melanoma cells B16-F10 mediated by cholesterol-based cationic liposomes

In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.     

In Vitro and In Vivo Transfection of Melanoma Cells B16-F10 Mediated by Cholesterol-based Cationic Liposomes

In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.     

Inhibition of VEGF expression in A431 and MDA-MB-231 tumour cells by cationic lipid-mediated siRNA delivery

In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid ( Percot et al., 2004 ), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.     

A novel cationic cardiolipin analogue for gene delivery

The optically active R and S isomers of cationic cardiolipin analogues (CCA) were synthesized and evaluated as a liposome based transfection reagent. Both isomers form stable liposomes with mean diameters of about 120 nm without any additional lipid ingredients. No significant change in particle size distribution profile was observed over one-month storage at room temperature (20-25 degrees C). The gel to liquid crystalline phase transition temperature (Tm) of cationic liposomes comprised of both R and S isomers was approximately 2 degrees C, as measured by differential scanning calorimetry (DSC). Both isomers also formed stable liposomes when combined with DOPE. In vitro transfection efficiency of the CCA/DOPE liposomes complexed to plasmid DNA was evaluated using a luciferase reporter gene. Both liposomes composed of R and S isomers of the cationic cardiolipin displayed higher transfection efficiency than commercially available Lipofectin. Further in vivo studies are warranted.     

Preparation of pH-sensitive,long-circulating and EGFR-targeted immunoliposomes

A long-circulating formulation of pH-sensitive liposomes (PSLs) with antibodies against epidermal growth factor receptor (EGFR) attached was designed, expecting an increase in binding and delivery of liposomes to the target cells including non-small cell lung cancer (NSCLC) cells. Physicochemical properties of the PSLs were measured by SEM and DLS. Leakage of a self-quenching fluorescent probe, calcein, from the liposome was studied for the evaluation of pH-sensitivity. Encapsulation efficiency of gemcitabine (an anti-cancer drug) in PSLs was about 67%. Average size of liposomes was 88 nm in diameter. The PSL of DOPE/CHEMS (6:4 molar ratio) formulation showed a dramatic pH-sensitivity at/around pH 5.5, whereas non-PSL of DPPC/Chol or PC/CHEMS formulation did not. Anti-proliferation effect of gemcitabine-encapsulating PSLs & Ab-PSLs in A549 cells was 2-fold higher than the free drug, which was further elucidated by the apoptosis of the cells by gemcitabine (∼10% apoptosis for PSL or Ab-PSL formulation vs. ∼1% for free drug or non-PSL formulation) using FACS analysis. These data demonstrate delivery of gemcitabine to tumor cells can be improved by long-circulating PSLs or Ab-PSLs formulations in vitro.