Recent development in transdermal drug delivery systems has led to an improvement of systemic and local efficacies. In the cosmetic field, liposomes have long been used as a container of cosmetic agents for their protection. Considering that these agents should be released from the liposomes at appropriate sites during their penetration into the skin, the use of skin environment-sensitive liposomes for transdermal penetration is beneficial for improving cosmetic efficacy. For this study, we prepared novel functional liposomes modified with methacrylate-based copolymers poly(MD-co-MAA-co-LT)s, which have sensitivity to both temperature and pH. Poly(MD-co-MAA-co-LT)s changed their water-solubility in response to both pH and temperature. Poly(MD-co-MAA-co-LT)-modified liposomes showed content release under conditions with acidic pH and temperatures higher than 35 °C, which correspond to endosome/lysosome environments of the melanocytes at the stratum basale of the skin. Polymer-modified liposomes were taken up efficiently by a murine melanoma cell line, B16–F10 cells, which delivered their contents into endosomes and cytosol. Polymer-modified liposomes could penetrate into the deep layers of skin models and reached the stratum basale. Results demonstrate that poly(MD-co-MAA-co-LT)-modified liposomes are promising as a system for delivering cosmetic agents to melanocytes.

Efficient vaccine carriers for cancer immunotherapy require two functions: antigen delivery to dendritic cells (DCs) and the activation of DCs, a so-called adjuvant effect. We previously reported antigen delivery system using liposomes modified with pH-sensitive polymers, such as 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG), for the induction of antigen-specific immune responses. We reported that inclusion of cationic lipids to MGlu-HPG-modified liposomes activates DCs and enhances antitumor effects. In this study, CpG-DNA, a ligand to Toll-like receptor 9 (TLR9) expressing in endosomes of DCs, was introduced to MGlu-HPG-modified liposomes containing cationic lipids using two complexation methods (Pre-mix and Post-mix) for additional activation of antigen-specific immunity. For Pre-mix, thin membrane of lipids and polymers were dispersed by a mixture of antigen/CpG-DNA. For Post-mix, CpG-DNA was added to pre-formed liposomes. Both Pre-mix and Post-mix delivered CpG-DNA to DC endosomes, where TLR9 is expressing, more efficiently than free CpG-DNA solution did. These liposomes promoted cytokine production from DCs and the expression of co-stimulatory molecules in vitro and induced antigen-specific immune responses in vivo. Both Pre-mix and Post-mix exhibited strong antitumor effects compared with conventional pH-sensitive polymer-modified liposomes. Results show that inclusion of multiple adjuvant molecules into pH-sensitive polymer-modified liposomes and suitable CpG-DNA complexation methods are important to design potent vaccine carriers.

•Dual-stimuli-sensitive methacrylate-based polymers were synthesized.•Polymer-modified liposome showed content release at acidic pH with high temperature.•Polymer-modified liposomes achieved content release at inside of cells.•No such temperature-dependent enhancement was observed for unmodified liposomes.Dual-signal-sensitive copolymers were synthesized by copolymerization of methoxy diethylene glycol methacrylate, methacrylic acid, and lauroxy tetraethylene glycol methacrylate, which respectively provide temperature sensitivity, pH sensitivity, and anchoring to liposome surfaces. These novel copolymers, with water solubility that differs depending on temperature and pH, are soluble in water under neutral pH and low-temperature conditions, but they become water-insoluble and form aggregates under acidic pH and high-temperature conditions. Liposomes modified with these copolymers exhibited enhanced content release at weakly acidic pH with increasing temperature, although no temperature-dependent content release was observed in neutral conditions. Interaction between the copolymers and the lipid monolayer at the air–water interface revealed that the copolymer chains penetrate more deeply into the monolayer with increasing temperature at acidic pH than at neutral pH, where the penetration of copolymer chains was moderate and temperature-independent at neutral pH. Interaction of the copolymer-modified liposomes with HeLa cells demonstrated that the copolymer-modified liposomes were adsorbed quickly and efficiently onto the cell surface and that they were internalized more gradually than the unmodified liposomes through endocytosis. Furthermore, the copolymer-modified liposomes enhanced the content release in endosomes with increasing temperature, but no such temperature-dependent enhancement of content release was observed for unmodified liposomes.Download high-res image (197KB)Download full-size image

The accurate delivery of antigens into the cytosol of antigen-presenting cells, such as dendritic cells, is crucially important for the induction of cellular immunity in efficient cancer immunotherapy. To date, the cytoplasmic delivery of antigens has been achieved with various delivery systems. Among them, pH-sensitive liposomes are a promising option because of their pH-responsive membrane disruption or fusion abilities, which cause the encapsulated antigen to be transferred into the cytosol. Recently, liposomes modified with pH-sensitive polymers have been used as highly effective antigen delivery systems. The control of the pH-responsive fusion ability and the intracellular distribution of antigens, the induction of humoral or cellular immunity in vivo, the induction of protective immunity against pathogens, and the treatment of tumor-bearing mice have been achieved using these liposomes. The design and function of these pH-sensitive polymer-modified liposomes are outlined in this review.

For establishment of cancer immunotherapy, antigen carriers are needed which have functions not only to deliver antigen into cytosol of dendritic cells (DCs), which induces antigen-specific cellular immune responses, but also to activate DCs. We previously reported cytoplasmic delivery of antigen using liposomes modified with pH-sensitive polymers such as carboxylated poly(glycidol)s or dextran. Modification using these polymers provides stable liposomes with pH-sensitive fusogenic/membrane-disruptive ability. For this study, bioactive polysaccharide-based pH-sensitive polymers were constructed to achieve not only cytoplasmic delivery of antigen but also activation of DCs. Curdlan and mannan were used as bioactive polysaccharides because they are known to activate DCs via their respective interactions with Dectin-1 and Dectin-2. Carboxylated curdlan and mannan promoted Th1 cytokine production from DCs, indicating the activation of DCs by these polysaccharide derivatives. These polymer-modified liposomes released their contents at weakly acidic pH and delivered model antigenic proteins into cytosol of DCs. Subcutaneous administration of curdlan derivative-modified or mannan derivative-modified liposomes induced strong antigen-specific immune responses and stronger antitumor effects than those of liposomes modified with dextran derivative. Therefore, bioactive polysaccharide-modified liposomes that achieve both cytoplasmic delivery of antigen and activation of DCs are promising for cancer immunotherapy.