pH-sensitive Fusogenic Liposomes Research Articles

Plasma stable, pH-sensitive fusogenic polymer-modified liposomes: A promising carrier for mitoxantrone

pH-sensitive liposomes are designed to undergo acid-triggered destabilization. In the present study, we prepared polymer-modified, plasma stable, pH-sensitive fusogenic mitoxantrone liposomes to increase efficacy and selectivity on cancer cell lines. Conventional liposomes were prepared using cholesterol and dipalmitoyl-sn-glycero-3-phosphatidylethanolamine. Dioleoylphosphatidylethanolamine and a cholesteryl derivative, poly(monomethylitaconate)-co-poly(N,N-dimethylaminoethyl methacrylate) (PMMI-co-PDMAEMA), were used for the preparation of pH-sensitive fusogenic liposomes. Using polyethylene glycol (PEG)-poly(monomethylitaconate)-CholC6 (PEG-PMMI-CholC6) copolymers instead of cholesterol introduced pH-sensitive and plasma stability properties simultaneously in prepared liposomes. All formulations were prepared by thin film hydration method and subsequently, pH-sensitivity and stability in human serum were evaluated. The ability of pH-sensitive fusogenic liposomes to enhance the mitoxantrone cytotoxicity and selectivity in cancerous cell lines was assessed invitro compared to normal cell line using human breast cancer cell line (MCF-7), human prostate cancer cell line (PC-3), and human umbilical vein endothelial cells line. Results revealed that both PMMI-co-PDMAEMA and PEG-PMMI-CholC6-based formulations showed pH-sensitive property and were found to rapidly release mitoxantrone under mildly acidic conditions. Nevertheless, only the PEG-PMMI-CholC6-based liposomes preserved pH-sensitivity after incubation in plasma. Mitoxantrone loaded-pH-sensitive fusogenic liposomes exhibited a higher cytotoxicity than the control conventional liposomes on MCF-7 and PC-3 cell lines. On the contrary, both pH-sensitive fusogenic liposomes showed lower cytotoxic effect on human umbilical vein endothelial cell line. Plasma stable, pH-sensitive fusogenic liposomes are promising carriers for enhancing the efficiency and selectivity, besides reduction of the side effects of anticancer agents.

PH-Sensitive fusogenic polymer-modified liposomes as a carrier of antigenic proteins for activation of cellular immunity

By modification of liposomes with poly(glycidol) derivatives such as succinylated poly(glycidol) and 3-methylglutarylated poly(glycidol), we have developed functional liposomes that generate fusion ability at mildly acidic pH. We investigated the feasibility of these polymer-modified liposomes as a carrier of antigenic proteins for induction of cellular immunity. These pH-sensitive fusogenic liposomes encapsulating ovalbumin (OVA) were applied to DC2.4 cells, a murine dendritic cell line. Observation with confocal laser scanning microscopy showed that these polymer-modified liposomes were taken up efficiently by the cells, thereafter delivering their contents into the cytosol, probably through fusion with endosomal membranes. Murine bone marrow-derived dendritic cells treated with polymer-modified liposomes encapsulating OVA stimulated CD8-OVA1.3 cells more strongly than OT4H.1D5 cells, indicating that the liposomes induced MHC class I-restricted presentation. Furthermore, administration of the polymer-modified, OVA-loaded liposomes from nasal cavities of mice induced stronger cellular immune responses than the OVA-loaded plain liposomes. Because the ability of the polymer-modified liposomes to activate cellular immunity was comparable to that of Freund's complete adjuvant, which is a widely used adjuvant, they potentially have use in production of efficient vaccines for immunotherapy.

Effect of Transferrin As a Ligand of pH-Sensitive Fusogenic Liposome−Lipoplex Hybrid Complexes

We previously developed potent nonviral vectors based on complexation of lipoplexes and pH-sensitive fusogenic liposomes, which achieve efficient transfection through membrane fusion with intracellular acidic compartments such as endosomes. Because transferrin receptor is known to be overexpressed in cancer cells, in this study, we investigated the effect of transferrin as a ligand for transfection of various cancer-derived cell lines mediated by the liposome-lipoplex hybrid complexes. Results showed that these hybrid complexes with transferrin exhibited higher transfection efficiency toward these cells than complexes without transferrin, but the extent of the transferrin-induced enhancement was dependent on the cell line. Conjugation of transferrin increased their transfection activity for HeLa and KB cells, although it only slightly enhanced transfection for HT1080, HepG2, and K562. Transferrin receptors in HT1080, HepG2, and K562 cells were internalized slowly, whereas those in HeLa and KB cells were internalized quickly and actively. These results indicate that transfection mediated by the ligand-attached hybrid complex does not correlate with the amount of transferrin receptor in the cell surface but correlate with the activity of internalization of transferrin receptor into the cells.

Gene delivery to dendritic cells mediated by complexes of lipoplexes and pH-sensitive fusogenic polymer-modified liposomes

Dendritic cells (DCs) are potent professional antigen presenting cells that are useful for cancer immunotherapy. We previously reported the preparation and characterization of complexes of lipoplexes with pH-sensitive fusogenic liposomes, which comprise polymers based on poly(glycidol) with carboxyl groups, to transfect various malignant cell lines. The present study applied this kind of vectors to transfection of a murine DC line DC2.4. We first optimized the ratios of their components for efficient transfection. We subsequently investigated the effects of ligands and pH-sensitive polymers to improve transfection activities. Our results indicate that the anionic surface derived from pH-sensitive polymers might be recognized by scavenger receptors on DC2.4 cells. In addition, no effects on transfection or cell association were observed by attaching ligands such as transferrin and mannan. We found that more sensitive pH-responding polymers led to higher transfection activities into DC2.4 cells, which suggest that endosomal escape is important for transfection into DC2.4 cells. These complexes with pH-sensitive fusogenic polymers exhibited higher transfection activity toward DC2.4 cells than some commercial reagents and hence may be useful as a gene vector for DCs.

Targetability and intracellular delivery of anti-BCG antibody-modified, pH-sensitive fusogenic immunoliposomes to tumor cells

We prepared tumor-specific immunoliposomes by coupling anti-BCG monoclonal antibodies to pH-sensitive fusogenic liposomes modified with succinylated polyglycidol (sucPG), in order to obtain efficient binding to, and endocytotic internalization into, the tumor cells. Mouse colon carcinoma 26 cells, which are known to share a common antigen with BCG, were used in in vitro experiments. BCG-sucPG immunoliposomes showed fusion ability under acidic conditions. Fluorescence microscopic observation indicated that BCG-sucPG immunoliposomes bound to colon 26 tumor cells and induced receptor-mediated endocytosis at 37 °C. Fusion assay by resonance energy transfer using N-(7-nitro-2-1,3-benzoxadiazol-4-yl) diacyl phosphatidylethanolamine and N-(lissamine rhodamine B sulfonyl) diacyl phosphatidylethanolamine suggested that fusion between BCG-sucPG immunoliposomes and endosomal and/or lysozomal membrane did occur. These results imply that the BCG-sucPG immunoliposomes transfer their content into the cytoplasm by fusing with the endosomal and/or lysozomal membrane after recognition of target cells and subsequent internalization into the cells by endocytosis.

Monogalactosyldiacylglycerol Confers Fusogenicity to Liposomal Delivery Systems and Facilitates Targeting to Hepatocytes

Fusogenic liposomes typically rely on unsaturated phosphatidyleth-anolamines, usually dioleoylphosphatidylethanolamine (DOPE), to confer the ability to fuse with cell membranes and deliver drug intracellularly. Targeted fusogenic liposomes require an additional component to facilitate binding to, and internalization within, specific cell populations. This report examines the ability of the unsaturated galactolipid monogalactosyldiacylglycerol (MGDG) to replace DOPE in pH-sensitive fusogenic liposomes and to simultaneously confer enhanced binding to hepatocytes. Liposomes prepared using the pH-sensitive lipid N-succinylDOPE and either DOPE, MGDG or a non-fusogenic control galactolipid were compared for (i) propensity to undergo either pH-or calcium-induced membrane fusion; (ii) ability to deliver encapsulated gen-tamicin to infections of Salmonella typhimurium in murine macrophages; and (iii) binding and uptake into human hepatoma cells. Liposomes composed of MGDG/N-succinylDOPE were very similar to those of DOPE/N-succinyl-DOPE for both the fusion rate and final extent of fusion induced by lowered pH. In contrast, MGDG/N-succinylDOPE liposomes were substantially less susceptible than DOPE/N-succinylDOPE liposomes to fusion at physiological pH induced by addition of calcium. The ability of the MGDG/N-succinylDOPE formulation to deliver the membrane-impermeable antibiotic gentami-cin to infections of S. typhimurium in macrophages was equal or superior to that achieved by the DOPE/N-succinylDOPE preparation. The binding and uptake of MGDG/N-succinylDOPE liposomes into HepG2 hepatoma cells was 3.5-fold greater than that of the DOPE/N-succinylDOPE formulation and 7.6-fold greater than that of a galactolipid control preparation. Taken together, these data demonstrate the potential to achieve both fusogenicity and hepato-cyte targeting by the addition of a single component, MGDG, to liposomal drug delivery systems.