Non-cationic Liposomes Research Articles

Cytoplasmic delivery of small interfering RNA by photoresponsive non-cationic liposomes

Small interfering RNA (siRNA) can specifically suppress gene expression by cleaving mRNA in the cytoplasm, termed RNA interference (RNAi). Although a nanoparticle-based siRNA delivery system has been approved for clinical use, the efficiency of cytoplasmic delivery of siRNA is still low. Recently, our group has established a highly efficient siRNA encapsulation method using non-cationic liposomes (LPs), which are more biocompatible than conventional cationic LPs. While non-cationic LPs containing siRNA were taken up by cells in vitro, the cytoplasmic release of siRNA and subsequent phenomena involving RNAi were not observed. It was considered that cytoplasmic delivery of siRNA, and siRNA release from LPs, should occur sequentially. We utilized amphiphilic photosensitizer Al (III) phthalocyanine chloride disulfonic acid (AlPcS2a), which can generate singlet oxygen under exposure to red light. When SK-HEP-1 cells expressing luciferase were treated with non-cationic LPs containing both siRNA and AlPcS2a, RNAi could be observed upon red light exposure, suggesting that singlet oxygen efficiently disrupts the membranous structures of endo/lysosomes and LPs. Thus, the combined use of AlPcS2a and red light is effective for utilizing non-cationic LPs for cytoplasmic delivery of siRNA.

Cationized liposomal keto-mycolic acids isolated from Mycobacterium bovis bacillus Calmette-Guérin induce antitumor immunity in a syngeneic murine bladder cancer model.

Intravesical therapy using Mycobacterium bovis bacillus Calmette-Guérin (BCG) is the most established cancer immunotherapy for bladder cancer. However, its underlying mechanisms are unknown. Mycolic acid (MA), the most abundant lipid of the BCG cell wall, is suspected to be one of the essential active components of this immunogenicity. Here, we developed cationic liposomes incorporating three subclasses (α, keto, and methoxy) of MA purified separately from BCG, using the dendron-bearing lipid D22. The cationic liposomes using D22 were efficiently taken up by the murine bladder cancer cell line MB49 in vitro, but the non-cationic liposomes were not. Lip-kMA, a cationic liposome containing keto-MA, presented strong antitumor activity in two murine syngeneic graft models using the murine bladder cancer cell lines MB49 and MBT-2 in comparison to both Lip-aMA and Lip-mMA, which contained α-MA and methoxy-MA, respectively. Interestingly, Lip-kMA(D12), which was made of D12 instead of D22, did not exhibit antitumor activity in the murine syngeneic graft model using MB49 cells, although it was successfully taken up by MB49 cells in vitro. Histologically, compared to the number of infiltrating CD4 lymphocytes, the number of CD8 lymphocytes was higher in the tumors treated with Lip-kMA. Antitumor effects of Lip-kMA were not observed in nude mice, whereas weak but significant effects were observed in beige mice with natural killer activity deficiency. Thus, a cationized liposome containing keto-MA derived from BCG induced in vivo antitumor immunity. These findings will provide new insights into lipid immunogenicity and the underlying mechanisms of BCG immunotherapy.

DNA Oligonucleotide-Functionalized Liposomes: Bioconjugate Chemistry, Biointerfaces, and Applications.

Interfacing DNA with liposomes has produced a diverse range of programmable soft materials, devices, and drug delivery vehicles. By simply controlling liposomal composition, bilayer fluidity, lipid domain formation, and surface charge can be systematically varied. Recent development in DNA research has produced not only sophisticated nanostructures but also new functions including ligand binding and catalysis. For noncationic liposomes, a DNA is typically covalently linked to a hydrophobic or lipid moiety that can be inserted into lipid membranes. In this article, we discuss fundamental biointerfaces formed between DNA and noncationic liposomes. The methods to prepare such conjugates and the interactions at the membrane interfaces are also discussed. The effect of DNA lateral diffusion on fluid bilayer membranes and the effect of membrane on DNA assembly are emphasized. DNA hybridization can be programmed to promote fusion of lipid membranes. Representative applications of this conjugate for drug delivery, biosensor development, and directed assembly of materials are briefly described toward the end. Some future research directions are also proposed to further understand this biointerface.

Solubilization of α-galactosylceramide in aqueous medium: Impact on Natural Killer T cell activation and antitumor responses

The potent antitumor effect of α-galactosylceramide (α-GalCer) is based on its recognition by invariant Natural Killer T cells (iNKT) after its capture and presentation by antigen presenting cells including dendritic cells (DCs). Synthetic α-GalCer has already been tested in advanced cancer patients but no or only moderate clinical responses were obtained.To optimize α-GalCer efficacy, we have postulated that alternative formulations impacting its molecular organization in aqueous medium could modify DC uptake and iNKT-based immune responses. To this end, we have developed two strategies: (1) the formulation of α-GalCer in non-cationic liposomes and (2) the synthesis of a water-soluble α-GalCer analogue by anchoring a polyethyleneglycol moiety on its sugar head. The biological activities of these new preparations were compared to that induced by the classically used Polysorbate 20 α-GalCer micelles. Both formulations retained their uptake by DCs and activated iNKT cells both in vitro and in vivo. Despite a lower cytokine production, the formulations induced a potent immune response able to control lung murine carcinoma. In conclusion, it is possible to increase α-GalCer solubility in aqueous solution without limiting its antitumor properties.

One-step scalable preparation method for non-cationic liposomes with high siRNA content

Cationic liposomes (LPs) have been utilized for short interfering RNA (siRNA) delivery in vitro and in vivo owing to their high affinity for siRNA via electrostatic binding. However, both cytotoxicity and non-specific adsorption of cationic LPs in the body have prevented clinical siRNA applications. These situations have led to siRNA encapsulation in non-cationic LPs. We found that the instillation of neutral phospholipids dissolved in ethanol into aqueous solutions containing siRNA and CaCl2 resulted in high siRNA encapsulation (siRNA encapsulation efficiency: ∼80%; siRNA weight ratio: ∼10wt% of LPs). The products were monodispersed, ∼200nm, and negatively charged. Furthermore, when phospholipids with a high-phase transition temperature or cholesterol were used, the encapsulation efficiency and siRNA content remained high. Although anionic LPs could not encapsulate siRNAs using this method, the use of cholesterol-conjugated siRNA helped achieve substantial siRNA encapsulation in anionic LPs. These non-cationic siRNA-containing LPs did not show cytotoxicity in vitro, and could be formed with polyethylene glycol-conjugated phospholipids. When conjugated with targeting ligand, the non-cationic siRNA-containing LPs could suppress the expression of target gene in vitro. These data demonstrate that our preparation method would be suitable for large-scale LP production for systemic siRNA delivery.

Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes

The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques.

Dendrosome-dendriplex inside liposomes: as a gene delivery system

Dendrosomes are lipid vesicular entities containing entrapped dendrimer-DNA complexes and possessing low toxicity, acceptable transfection efficiency, and good in vivo tolerance. Herein, an attempt was made to explore the potential of dendrosomes as a gene delivery system combining the advantages of both polyamidoamine (PAMAM) dendrimer (nucleic acid condensation, facilitated endosomal release) and of non-cationic liposomes (increased cellular uptake, low cytotoxicity), and at the same time overcoming the drawbacks of these system (low encapsulation efficiency of non-cationic liposome and toxicity of dendrimers). Dendrosomes were assembled by loading optimized DNA-dendrimer complexes into liposomes prepared by solvating of dried lipid films made of DOPE/EggPC/Cholesterol (4.74:4.75:1.5 mole ratio). Dendrosomes were characterized in terms of size, zeta, encapsulation efficiency and the ability to protect the system from DNA degradation. The transfection efficiency and toxicity of the preparations were evaluated in HeLa cells using flow cytometry and CellTiter-Blue® methods. The efficient transfection and low toxicity makes them an appealing alternative to be further explored for gene delivery in vivo.

Ki-67 as a Molecular Target for Therapy in an In vitro Three-Dimensional Model for Ovarian Cancer

Targeting molecular markers and pathways implicated in cancer cell growth is a promising avenue for developing effective therapies. Although the Ki-67 protein (pKi-67) is a key marker associated with aggressively proliferating cancer cells and poor prognosis, its full potential as a therapeutic target has never before been successfully shown. In this regard, its nuclear localization presents a major hurdle because of the need for intracellular and intranuclear delivery of targeting and therapeutic moieties. Using a liposomally encapsulated construct, we show for the first time the specific delivery of a Ki-67-directed antibody and subsequent light-triggered death in the human ovarian cancer cell line OVCAR-5. Photoimmunoconjugate-encapsulating liposomes (PICEL) were constructed from anti-pKi-67 antibodies conjugated to fluorescein 5(6)-isothiocyanate, as a photoactivatable agent, followed by encapsulation in noncationic liposomes. Nucleolar localization of the PICELs was confirmed by confocal imaging. Photodynamic activation with PICELs specifically killed pKi-67-positive cancer cells both in monolayer and in three-dimensional (3D) cultures of OVCAR-5 cells, with the antibody TuBB-9 targeting a physiologically active form of pKi-67 but not with MIB-1, directed to a different epitope. This is the first demonstration of (a) the exploitation of Ki-67 as a molecular target for therapy and (b) specific delivery of an antibody to the nucleolus in monolayer cancer cells and in an in vitro 3D model system. In view of the ubiquity of pKi-67 in proliferating cells in cancer and the specificity of targeting in 3D multicellular acini, these findings are promising and the approach merits further investigation.

Remote-loading labeling of liposomes with 99mTc-BMEDA and its stability evaluation: effects of lipid formulation and pH/chemical gradient

Efficient, convenient, and stable radiolabeling plays a critical role for the monitoring of liposome behavior via either blood sampling, organ distribution, or noninvasive nuclear imaging. The direct labeling of liposome-carrying drugs without any prior modification undoubtedly is convenient and optimal for liposomal drug testing. In this article, we investigated the effect of various lipid formulations and pH/chemical gradients on the radiolabeling efficiency and entrapment stability of technetium-99m (99mTc) remotely loaded into liposomes, using 99mTc-N,N-bis(2-mercaptoethyl)-N’,N’-diethyl-ethylenediamine (99mTc-BMEDA) complex. The tested liposomes either contained unsaturated lipid or possessed various surface charges. 99mTc could be efficiently loaded into various premanufactured liposomes containing either an ammonium sulfate pH, citrate pH, or glutathione (GSH) chemical gradient. 99mTc-entrapment stabilities of these liposomes in phosphate-buffered saline (PBS; pH 7.4) buffer at 25°C were mainly dependent on the pH/chemical gradient, but not lipid formulation. Stability sequence was ammonium sulfate pH-gradient>citrate pH-gradient>GSH-gradient. Stabilities of 99mTc-liposomes in 50% fetal bovine serum (FBS)/PBS (pH 7.4) buffer at 37°C are dependent on both lipid formulation and pH/chemical gradient. Specifically, 99mTc labeling of the ammonium sulfate pH-gradient liposomes were less stable in 50% FBS/PBS than in PBS, whereas noncationic liposomes with citrate pH- or GSH-gradient displayed higher stability, except that anionic citrate pH-gradient liposomes showed no stability difference in these two media. Cationic liposomes aggregated in 50% FBS/PBS, forming a new discrete fraction with larger particle sizes. These in vitro characterization results have indicated the optimism of using 99mTc-BMEDA for labeling pH/GSH gradient liposomes without the requirement of modifying lipid formulation for liposomal therapeutic-agent development.

Efficiency of cytoplasmic delivery by non-cationic liposomes to cells in vitro: A confocal laser scanning microscopy study

It is necessary to understand liposomal uptake mechanisms and intracellular distribution in order to design more efficient gene (drug) carrier systems. Until now, a few studies have been carried out using confocal laser scanning microscopy (CLSM) to investigate the cellular uptake and transfection mediated with liposomes. So, by CLSM, we demonstrated that artificial virus-like envelope (AVE) vesicles labeled with rhodamine-PE (Rh-PE), carbocyanine (DiI) and carboxyfluorescein (CF) were investigated into the cytoplasm of two human cell lines, Mewo (human melanoma cell line) and HepG2 (human hepatoma cell line) cells grown in DMEM medium supplemented with different percentages (0%, 30%, and 100%) fetal calf serum (FCS). The liposome uptake was dependent on the cell line, in view that the whole process of liposomes associated with cells (uptake) is a two-step process involving binding and endocytosis. Based upon the various assays used to measure cellular uptake of liposomes, we conclude the efficacy of cytoplasmic delivery by AVE-liposomes to cells in culture.

Advances in Targeting Drug Delivery to Glomerular Mesangial Cells by Long Circulating Cationic Liposomes for the Treatment of Glomerulonephritis

Newly designed polyethylene glycol (PEG)-modified cationic liposomes, containing a novel cationic lipid TRX-20 (3,5-dipentadecyloxybenzamidine hydrochloride), bind specifically to cultured human mesangial cells, and not to endothelial cells. In this study, we investigated targeting the delivery of PEG-modified liposomes containing TRX-20 (TRX-liposomes) to mesangial cells and evaluated their pharmacokinetic behavior in a rat experimental glomerulonephritis model, using prednisolone phosphate (PSLP) as a model drug. TRX-liposomes were injected intravenously into experimental glomerulonephritic rats and normal rats to compare its pharmacokinetic behavior with that of non-cationic liposomes (PEG-liposomes). Rhodamine-labeled liposomes were used to evaluate the accumulation in inflamed kidneys. Pharmacological effects of three formulations of PSLP (i.e., a single injection of two liposomal formulations and daily injections of PSLP in saline solution) were estimated in terms of suppressing glomerular cell proliferation in the rat nephritis model. TRX-liposomes markedly accumulated in the glomeruli of inflamed kidneys, but did not accumulate in the glomeruli of normal kidneys. Although the PEG-liposomes also accumulated in the glomeruli of the inflamed kidneys, their pharmacological behavior was quite different from that of the TRX-liposomes, which were internalized by the target cells. In a comparison among the three formulations of PSLP, the dose of TRX-liposomes required for significant suppression of glomerular cell proliferation was much less (dose of 0.032 mg/kg and above) than that required for the same effect by the PSLP saline solution (3.2 mg/kg daily; 12.8 mg/kg total) and PEG-liposomes (0.32 mg/kg). Interestingly, significant suppression of mesangial cell activation, as assessed by the expression of alpha-smooth muscle actin, was observed in nephritic rats treated with TRX-liposomes, but not in the other two treatment groups. The pharmaceutical properties of TRX-liposomes due to their preferential binding to mesangial cells and long circulation time make this a likely candidate system for targeted drug delivery to the inflamed glomeruli of glomerulonephritis.

Complexes of non-cationic liposomes and histone H1 mediate efficient transfection of DNA without encapsulation

Transfection competent complexes were assembled using a three component system. The constituents of the basic system were plasmid DNA, cationic DNA binding protein (NLS-H1) and anionic liposomes (dioleoyl phosphatidylethanolamine (DOPE) or phosphatidylserine (PS)). In contrast to cationic liposome/DNA binary complexes, all of the DNA in these ternary complexes was sensitive to DNase I degradation and ethidium bromide intercalation. Transmission electron microscopy revealed that these ternary complexes formed unique structures in which the DNA was located either on the outside of individual liposomes or bridging two or more liposomes. This provides evidence that plasmid DNA encapsulation is not essential for transfection competency.