Fusogenic Liposomes Research Articles

Resveratrol Encapsulated in Novel Fusogenic Liposomes Activates Nrf2 and Attenuates Oxidative Stress in Cerebromicrovascular Endothelial Cells From Aged Rats

Resveratrol (3,4',5-trihydroxystilbene) is a plant-derived polyphenolic trans-stilbenoid, which exerts multifaceted antiaging effects. Here, we propose a novel delivery system for resveratrol, which significantly increases its cellular uptake into aged cells. Combination of resveratrol with a positively charged lipid component to "conventional" liposomes converts these lipid vesicles to a robust fusogenic system. To study their cellular uptake and cellular effects, we treated primary cerebromicrovascular endothelial cells isolated from aged F344xBN rats with resveratrol encapsulated in fusogenic liposomes (FL-RSV). To demonstrate effective cellular uptake of FL-RSV, accumulation of the lipophilic tracer dye, DiR, and resveratrol in cerebromicrovascular endothelial cells was confirmed using flow cytometry and confocal microscopy and high-performance liquid chromatography electrochemical detection. Treatment of aged cerebromicrovascular endothelial cells with FL-RSV activated Nrf2 (assessed with a reporter gene assay), significantly decreased cellular production of reactive oxygen species (assessed by a flow cytometry-based H2DCFDA fluorescence method), and inhibited apoptosis. Taken together, encapsulation of resveratrol into novel fusogenic liposomes significantly enhances the delivery of resveratrol into aged cells, which subsequently results in rapid activation of cellular Nrf2-driven antioxidant defense mechanisms. Our studies provide proof-of-concept for the development of a novel, translationally relevant interventional strategy for prevention and/or control of oxidative stress-related pathophysiological conditions in aging.

Fusogenic pH sensitive liposomal formulation for rapamycin: Improvement of antiproliferative effect

Context: Liposomes are increasingly employed to deliver chemotherapeutic agents, antisense oligonucleotides, and genes to various therapeutic targets.Objective: The present investigation evaluates the ability of fusogenic pH-sensitive liposomes of rapamycin in increasing its antiproliferative effect on human breast adenocarcinoma (MCF-7) cell line.Materials and methods: Cholesterol (Chol) and dipalmitoylphosphatidylcholine (DPPC) (DPPC:Chol, 7:3) were used to prepare conventional rapamycin liposomes by a modified ethanol injection method. Dioleoylphosphatidylethanolamine (DOPE) was used to produce fusogenic and pH-sensitive properties in liposomes simultaneously (DPPC:Chol:DOPE, 7:3:4.2). The prepared liposomes were characterized by their size, zeta potential, encapsulation efficiency percent (EE%), and chemical stability during 6 months. The antiproliferative effects of both types of rapamycin liposomes (10, 25, and 50 nmol/L) with optimized formulations were assessed on MCF-7 cells, as cancerous cells, and human umbilical vein endothelial cells (HUVEC), as healthy cells, employing the diphenyltetrazolium bromide (MTT) assay for 72 h.Results and discussion: The particle size, zeta potential, and EE% of the liposomes were 165 ± 12.3 and 178 ± 15.4 nm, −39.6 ± 1.3, and −41.2 ± 2.1 mV as well as 76.9 ± 2.6 and 76.9 ± 2.6% in conventional and fusogenic pH-sensitive liposomes, respectively. Physicochemical stability results indicated that both liposome types were relatively stable at 4 °C than 25 °C. In vitro antiproliferative evaluation showed that fusogenic pH-sensitive liposomes had better antiproliferative effects on MCF-7 cells compared to the conventional liposomes. Conversely, fusogenic pH-sensitive liposomes had less cytotoxicity on HUVEC cell line.

Plasma membrane functionalization using highly fusogenic immune activator liposomes

Cell surface functionalization and target molecule incorporation into living cell membranes without functional damage represent major biotechnological challenges. One possible way to achieve these goals is to induce cell membrane fusion with an artificial membrane containing molecules equipped with reactive groups or ligands. In this work we developed a carrier system to incorporate lipopolysaccharide (LPS), an immune cell activating molecule from Gram-negative bacteria, into mammalian membranes. LPS is not present in untreated mammalian cells which hence are not detectable by the immune system. Here, we demonstrate the successful incorporation of LPS into fusogenic liposomes (FLs) and subsequent incorporation into mammalian plasma membranes using these FLs. Additionally, the presence of LPS in cell membranes was probed by the addition of non-activated macrophages. A high concentration of LPS in the plasma membrane of immortalized fibroblasts activated the immune cells, which in turn started to eliminate LPS-exhibiting cells. Our method for cellular membrane functionalization is a promising tool for biomedical applications and could provide the basis for specific cell targeting approaches.

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.

In Situ Modification of Plain Liposomes with Lipidated Coiled Coil Forming Peptides Induces Membrane Fusion

Complementary coiled coil forming lipidated peptides embedded in liposomal membranes are able to induce rapid, controlled, and targeted membrane fusion. Traditionally, such fusogenic liposomes are prepared by mixing lipids and lipidated peptides in organic solvent (e.g., chloroform). Here we prepared fusogenic liposomes in situ, i.e., by addition of a lipidated peptide solution to plain liposomes. As the lipid anchor is vital for the correct insertion of lipidated peptides into liposomal membranes, a small library of lipidated coiled coil forming peptides was designed in which the lipid structure was varied. The fusogenicity was screened using lipid and content mixing assays showing that cholesterol modified coiled coil peptides induced the most efficient fusion of membranes. Importantly, both lipid and content mixing experiments demonstrated that the in situ modification of plain liposomes with the cholesterol modified peptides yielded highly fusogenic liposomes. This work shows that existing membranes can be activated with lipidated coiled coil forming peptides, which might lead to highly potent applications such as the fusion of liposomes with cells.

Application of pH-sensitive fusogenic polymer-modified liposomes for development of mucosal vaccines

To evaluate the usefulness of pH-sensitive fusogenic polymer (succinylated poly(glycidol) (SucPG) and 3-methylglutarylated poly(glycidol) (MGluPG))-modified liposomes as mucosal vaccine in the induction of a protective immune responses was evaluated. Mice were nasally immunized with OVA-containing SucPG-modified liposomes. After immunization, significant Ag-specific Abs were detected in the serum and intestine. When sera were analyzed for isotype distribution, antigen-specific IgG1 Ab responses were noted in mice immunized with OVA-containing polymer-unmodified liposomes, whereas immunization with OVA-containing SucPG-modified liposomes resulted in the induction of OVA-specific IgG1, IgG2a and IgG3 Ab responses. In spleen lymphocytes from mice immunized with OVA-containing SucPG-modified liposomes, both IFN-γ and IL-4 mRNA were detected. The same result was obtained also in the mouse immunized with OVA-containing MGluPG-modified liposomes. Furthermore, we examined the induction of immune responses in chickens following intraocular immunization with Salmonella Enteritidis Ag-containing MGluPG-modified liposomes, and the protective effect against the challenge with S. Enteritidis. Immunization with S. Enteritidis Ag-containing MGluPG-modified liposomes induced significant Ab responses against S. Enteritidis in the serum and intestine. Less fecal excretion of bacteria was observed in chickens immunized with S. Enteritidis Ag-containing MGluPG-modified liposomes after challenge. The numbers of bacteria in the caecum were also lower in immunized chickens than in unimmunized controls.

Studies on intracellular delivery of carboxyl-coated CdTe quantum dots mediated by fusogenic liposomes.

The use of Quantum Dots (QDs) as fluorescent probes for understanding biological functions has emerged as an advantageous alternative over application of conventional fluorescent dyes. Intracellular delivery of QDs is currently a specific field of research. When QDs are tracking a specific target in live cells, they are mostly applied for extracellular membrane labeling. In order to study intracellular molecules and structures it is necessary to deliver free QDs into the cell cytosol. In this work, we adapted the freeze and thaw method to encapsulate water dispersed carboxyl-coated CdTe QDs into liposomes of different compositions, including cationic liposomes with fusogenic properties. We showed that labeled liposomes were able to fuse with live human stem cells and red blood cells in an endocytic-independent way. We followed the interactions of liposomes containing QDs with the cells. The results were minutely discussed and showed that QDs were delivered, but they were not freely diffused in the cytosol of those cells. We believe that this approach has the potential to be applied as a general route for encapsulation and delivery of any membrane-impermeant material into living cells.

Stabilizing endothelium of donor hearts with fusogenic liposomes reduces myocardial injury and dysfunction

BackgroundMyocardial injury after heart transplantation is a consequence of pathophysiologic events initiated by local ischemia/reperfusion injury that is further aggravated by the inflammatory response due to blood exposure to the pump’s artificial surfaces during cardiopulmonary bypass. The purpose of the present study was to determine the effectiveness of fusogenic lipid vesicles (FLVs) in enhancing the cardioprotective effect of St. Thomas organ preservation solution (ST). We hypothesized that donor hearts preserved with ST+FLVs will stabilize the endothelium during reperfusion, which, in turn, will reduce both endothelial barrier dysfunction and myocardial damage. MethodsTo examine the effect of ST+FLVs therapy in vitro, C3b deposition and adhesion molecule expression studies were performed on human umbilical vein endothelial cells challenged with plastic contact-activated plasma. To assess the therapy in vivo, a cervical heterotopic working heart transplantation model in rats was used. Donor hearts were preserved for 1 h at 27°C (15 min) and 4°C (45 min) and, after transplantation, were followed up for 2 h. Left ventricular function and the blood cardiac troponin I levels were quantified. ResultsHuman umbilical vein endothelial cells treated with ST+FLVs had reduced C3b deposition and expression of adhesion molecules compared with ST alone (P < 0.05). Donor hearts receiving ST+FLVs therapy had reduced left ventricular dysfunction and cardiac troponin I compared with ST alone. ConclusionsWe concluded that FLVs enhanced the cardioprotective effect of ST and reduced postischemic left ventricular dysfunction and myocardial damage. The mechanism of protection appears to be associated with the stabilization of endothelial cell membranes owing to incorporation of FLV-derived lipids.

Acid-Triggered Release via dePEGylation of Fusogenic Liposomes Mediated by Heterobifunctional Phenyl-Substituted Vinyl Ethers with Tunable pH-Sensitivity

A new family of heterobifunctional phenyl-substituted vinyl ether (PIVE) coupling agents with tunable acid-sensitivity has been developed. The PIVE compounds are designed to hydrolyze under acidic conditions with hydrolysis rates that can be varied by rational selection of the phenyl ring substituent. These reagents were incorporated within 2-methoxypoly(ethylene glycol) PEG-conjugated 1,3-dioctadecyl-rac-glycerol lipids to produce the acid-cleavable lipopolymers mPEG-[H-PIVE]-DOG, mPEG-[F-PIVE]-DOG, mPEG-[Me-PIVE]-DOG, and mPEG-[MeO-PIVE]-DOG. These lipopolymers were hydrolyzed under acidic conditions (pH 3.5 or 4.5) at rates that were dependent on the electron donating or withdrawing character of the α-phenyl vinyl ether substituent, while remaining stable at pH 7.4. Blending of these compounds with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in a 10:90 mPEG-PIVE-Lipid:DOPE ratio produced stable liposomes at neutral pH; however, acidification of the solution led to dePEGylation and release of the liposomal cargo in a manner that correlated with the PIVE proton affinity. Specifically, we observed 70% calcein release within 12 h from mPEG-[MeO-PIVE]-DOG-containing liposomes at pH 4.5, whereas only 22% calcein release was observed from mPEG-[F-PIVE]-DOG:DOPE liposomes over this same time scale and pH. These results indicate that dePEGylation following acidification is a triggering mechanism that can be rationally designed and controlled through the appropriate selection of PIVE moieties.

Phosphatase‐Triggered Fusogenic Liposomes for Cytoplasmic Delivery of Cell‐Impermeable Compounds

Phosphatase‐Triggered Fusogenic Liposomes for Cytoplasmic Delivery of Cell‐Impermeable Compounds

Phosphatase-triggered fusogenic liposomes for cytoplasmic delivery of cell-impermeable compounds.

License to fuse! A phosphorylated fusion peptide can mediate membrane fusion when the phosphates (green triangles, see scheme) are removed by phosphatases (blue spheres), delivering the contents of the liposome into the cytosol. This phosphatase-triggered approach may be useful to create target-specific lipid nanocarriers.

Bleomycin in octaarginine-modified fusogenic liposomes results in improved tumor growth inhibition

Bleomycin (BLM) is an example of an anticancer drug that should be delivered into cytosol for its efficient therapeutic action. With this in mind, we developed octaarginine (R8)-modified fusogenic DOPE-liposomes (R8-DOPE-BLM). R8-modification dramatically increased (up to 50-fold) the cell-liposome interaction. R8-DOPE-liposomes were internalized via macropinocytosis and did not end up in the lysosomes. R8-DOPE-BLM led to a significantly stronger cell death and DNA damage in vitro relative to all controls. R8-DOPE-BLM demonstrated a prominent anticancer effect in the BALB/c mice bearing 4T1 tumors. Thus, R8-DOPE-BLM provided efficient intracellular delivery of BLM leading to strong tumor growth inhibition in vivo.

Safety Evaluation of Nanoliposomes Containing Cyclosporine A After Ocular Administration

Aim: Cyclosporine A (CyA) is an anti inflammatory drug which modulates immune system. We prepared two liposomal formulations of CyA for ophthalmic administration. In the present study, the safety of these formulations was tested in rabbits.Materials and methods: Fusogenic and positively charged liposomes were prepared by solvent evaporation method. CyA concentration in ophthalmic preparations was 0.2% w/v. Twenty New Zealand albino rabbits (40 eyes) were divided into two groups. Right eyes were treated and left eyes served as control. Eyes were examined clinically in 1, 3, 7, 14 and 28th days of the study. Eleven eyes were enucleated for pathological evaluation at the end of the study.Results: Mean size and charge of positively charged and fusogenic liposomes were 110.7 ± 1.5 and 114.9 ± 15.2 nm; 19.0 ± 2.2 and 2.5 ± 0.5 mV, respectively. Clinically, there were transient conjunctival injections and corneal epithelial defects in a few cases which were not limited to the treated eyes. Positively charged liposomes caused conjunctival injection in two cases, while this complication occurred in only one eye after administration of fusogenic liposomes. Histopathological examination revealed the presence of follicular conjunctivitis in two eyes with conjunctival injection. Inflammation in cases received the fusogenic form of the drug was mild and non-specific.Conclusions: The results of this study suggest that fusogenic liposomal preparation of CyA is safe and can be useful for future studies.

Fluorescent Lipids: Functional Parts of Fusogenic Liposomes and Tools for Cell Membrane Labeling and Visualization

In this paper a rapid and highly efficient method for controlled incorporation of fluorescent lipids into living mammalian cells is introduced. Here, the fluorescent molecules have two consecutive functions: First, they trigger rapid membrane fusion between cellular plasma membranes and the lipid bilayers of their carrier particles, so called fusogenic liposomes, and second, after insertion into cellular membranes these molecules enable fluorescence imaging of cell membranes and membrane traffic processes. We tested the fluorescent derivatives of the following essential membrane lipids for membrane fusion: Ceramide, sphingomyelin, phosphocholine, phosphatidylinositol-bisphosphate, ganglioside, cholesterol, and cholesteryl ester. Our results show that all probed lipids could more efficiently be incorporated into the plasma membrane of living cells than by using other methods. Moreover, labeling occurred in a gentle manner under classical cell culture conditions reducing cellular stress responses. Staining procedures were monitored by fluorescence microscopy and it was observed that sphingolipids and cholesterol containing free hydroxyl groups exhibit a decreased distribution velocity as well as a longer persistence in the plasma membrane compared to lipids without hydroxyl groups like phospholipids or other artificial lipid analogs. After membrane staining, the fluorescent molecules were sorted into membranes of cell organelles according to their chemical properties and biological functions without any influence of the delivery system.

Co-immunization with interlukin-18 enhances the protective efficacy of liposomes encapsulated recombinant Cu–Zn superoxide dismutase protein against Brucella abortus

Brucellosis is a worldwide zoonotic disease caused by Brucella abortus and a number of closely related species. Brucellosis has severe impact on the health and economic prosperity of the developing countries due to the persistent nature of infection and unavailability of effective control measures. The Cu–Zn superoxide dismuatse (SOD) protein of Brucella have been extensively studied as a major antigen involved in bacterial evading mechanism of host defence. Being a critical pro-inflammatory cytokine interleukin-18 (IL-18) plays key role in induction of immune mediated protection against intracellular pathogens. In the present study, we aimed to investigate the immunogenic potential of fusogenic liposomes (escheriosomes) encapsulated recombinant Cu–Zn SOD (rSOD) protein alone or in combination with recombinant IL-18 (rIL-18). Escheriosomes encapsulated rSOD mediated immune responses were further increased upon co-immunization with rIL-18. Furthermore, immunization with escheriosomes encapsulated rSOD alone or in combination with rIL-18, increased resistance in mice against challenge with B. abortus 544.

Abstract 3224: Targeted non-invasive therapy of prostate cancer using fusogenic liposomes

Abstract Prostate cancer is the most common cancer in Canadian men. Most chemotherapeutic drugs do not preferentially target the tumour and therefore subject the patient's healthy cells to significant toxicity. There is therefore an urgent need for a novel strategy to deliver chemotherapeutic drugs specifically to the tumour. P14 is a fusion associated small transmembrane (FAST) protein which mediates efficient cell-cell interaction. When we incorporate this p14 FAST protein into liposomes we increase the fusion of the liposome to cell membranes. I hypothesize that targeted ablation of prostate cancer may be achieved by delivering chemotherapeutics encapsulated inside fusogenic liposomes targeted to the gastrin releasing peptide receptors. The objectives of the study are to i) validate the fusogenic properties of p14-containg liposomes ii) express and purify p14-bomesin fusion protein, iii) assemble targeted fusogenic lipsoomes iv) and assess the targeted ablation of human prostate cancer in a xenograft model. To validate the fusogenic properties of p14-containing liposomes in our cell lines, we encapsulated fluorescent dextran inside liposomes containing either no p14, mutant p14 (fusogenic incompetent) or an active p14. We incubated the liposomes on human fibrosarcoma cells (HT1080) and measured the uptake of fluorescent dextran using fluorescent microscopy. To construct a fusion protein, we used PCR amplification to fuse bombesin to the C-terminus of the p14 protein. Our analysis demonstrates that the incorporation of the p14 protein significantly increased the fluorescent dextran uptake in the cytoplasm of the HT1080 cells compared to the liposomes that contained no p14 and the mutant p14. Protein purification of the p14-bombesin fusion protein is still in progress. I propose to use these targeted fusogenic liposomes for future studies to evaluate the ability of bombesin-coated fusogenic liposomes to target prostate cells in vitro and deliver a toxic therapeutic payload to prostate tumours in vivo and assess tumour reduction. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3224. doi:10.1158/1538-7445.AM2011-3224

Membrane fusion of pH-sensitive liposomes – a quantitative study using giant unilamellar vesicles

Drug delivery systems based on liposome carriers that are designed for induction of membrane fusion in response to the microenvironment have been investigated for more than two decades. However, most studies have focused on self-fusion among large unilamellar vesicles (LUVs) in solution, a system with limited biological relevance which suffers from averaging effects and non-trivial interpretation of results. Here we present a fusion assay capable of visualizing and quantifying heterogeneous fusion between fusogenic liposomes and stable giant unilamellar vesicles (GUVs), suitable for studying both lipid and content mixing. Fusion was visualized and quantified using fluorescence microscopy and a FRET-based lipid mixing assay and the number of fusion events with single GUVs was estimated. Fusogenic pH-sensitive oleic acid (OA):DOPE liposomes were used as a model system and fusion was quantified with giant vesicles containing 5 or 10 mol% positively charged lipids. It was shown that the number of fusion events with single GUVs after 30 min at room temperature is approximately 100 or 200 per 100 μm2 of GUV surface, for 5 or 10 mol%, respectively. Furthermore, the mixing of the aqueous content during fusion was visualized and quantified, which showed that the content of the fusogenic liposomes is transferred essentially without leakage. Fusion was confirmed to be pH-dependent and is enhanced by electrostatic attraction between the fusing vesicles, which was demonstrated by the absence of fusion in experiments performed with negatively charged GUVs.

Encapsulation in fusogenic liposomes broadens the spectrum of action of vancomycin against Gram-negative bacteria

Many antibacterial agents, including the glycopeptides, are inactive against Gram-negative bacteria because of their inability to cross the outer membrane of these cells. Different chemical and technological approaches have been described to circumvent such limitation. In this study, we aimed to apply the strategy of fusogenic liposomes, up to now used to carry biological compounds and materials inside cells, to localise a glycopeptide antibiotic, vancomycin (VAN), to the periplasmic space, thus allowing it to exert its bactericidal activity. Small unilamellar liposome vesicles were prepared by an extrusion procedure (SUVETs) from a phospholipid–cholesterol hemisuccinate mixture known for its fusogenic properties with the eukaryotic cell membrane. VAN was loaded with high efficiency into these vesicles and in microbiological experiments in vitro was shown to be able to inhibit to a different extent the growth of wild and standard Gram-negative bacterial strains. Minimum inhibitory concentrations as low as 6mg/L were observed, for instance against clinical isolates of Escherichia coli and Acinetobacter baumannii. In comparison, neither the free antibiotic nor VAN-loaded ‘classical’ (non-fusogenic) liposomes showed any activity against the same bacteria. Scanning and transmission electron microscopy studies allowed confirmation that the produced SUVETs were able to adhere to and fuse with the external membrane of E. coli. According to preliminary experiments, this technological strategy can be proposed as a potentially successful way to enlarge the spectrum of activity of VAN.

Novel Fusogenic Liposomes for Fluorescent Cell Labeling and Membrane Modification

Efficient delivery of biomolecules into membranes of living cells as well as cell surface modifications are major biotechnological challenges. Here, novel liposome systems based on neutral and cationic lipids in combination with lipids modified by aromatic groups are introduced for such applications. The fusion efficiency of these liposome systems was tested on single cells in culture like HEK293, myofibroblasts, cortical neurons, human macrophages, smooth muscle cells, and even on tissue. Fusogenic liposomes enabled highly efficient incorporation of molecules into mammalian cell membranes within 1 to 30 min at fully unchanged cell growth conditions and did not affect cell behavior. We hypothesize that membrane fusions were induced in all cases by the interaction of the positively charged lipids and the delocalized electron system of the aromatic group generating local dipoles and membrane instabilities. Selected applications ranging from basic research to biotechnology are envisaged here.

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.