Preparation of photothermal-chemotherapy nanohybrids by complexation of gold nanorods with polyamidoamine dendrimers having poly(ethylene glycol) and hydrophobic chains.

The combination of anticancer drugs and laser hyperthermia could lead to efficient cancer treatment with less-adverse effects. This study combined anticancer drug-loaded functional dendrimers and light-responsive gold nanorods to fabricate nanohybrids that can provide anticancer-drug delivery and subsequent heat generation under near-infrared laser irradiation. A condensation reaction was used to conjugate poly(ethylene glycol)-modified polyamidoamine dendrimers to carboxylated gold nanorod surfaces. Oleoyl groups were incorporated into dendrimers to improve the drug loading capacity. Doxorubicin loading capacity was improved by incorporation of oleoyl chains into dendrimers in the nanohybrid, indicating increased hydrophobic interaction between anticancer drugs and nanohybrids. The nanohybrids exhibited heat generation properties under near infrared laser irradiation. They released anticancer drugs over time. The combination of doxorubicin-loaded nanohybrids and laser irradiation showed markedly better cytotoxicity than that of the nanohybrids used with lasers and drug-loaded nanohybrids without the use of lasers. After intravenous or intratumoral injection of nanohybrids to tumor-bearing mice, a sharp temperature increase was observed at the tumor site under laser irradiation. Especially, intratumorally injected doxorubicin-loaded nanohybrids showed almost complete tumor growth suppression under laser irradiation. The results demonstrate that functional dendrimer-gold nanorod nanohybrids are promising as multi-functional nanomaterials to achieve synergistic effects of anticancer drugs and heat ablation to support effective cancer treatments.

Chondroitin Sulfate-Based pH-Sensitive Polymer-Modified Liposomes for Intracellular Antigen Delivery and Induction of Cancer Immunity.

Induction of cancer-specific cytotoxic T lymphocytes is crucially important to complement therapeutic effects of immune checkpoint inhibitors and to achieve efficient cancer immunotherapy. To induce cancer-specific cytotoxic T lymphocytes, cancer antigen carriers must have multiple functions to deliver cancer antigens to antigen presenting cells, release antigens into cytosol, and promote the maturation of these cells. We earlier achieved cytosolic delivery of antigens and induction of antigen-specific cytotoxic T lymphocytes using carboxylated polyglycidol or polysaccharide derivative-modified liposomes that can induce membrane fusion with endosomes in response to weakly acidic pH. Furthermore, pH-sensitivity and adjuvant properties of these polymers were enhanced strongly by introduction of hydrophobic carboxylated units to dextran. Against our expectations, these polymer-modified liposomes only slightly induce cancer immunity, probably because of the high hydrophobicity of spacer units. This study used a polysaccharide with charged groups (chondroitin sulfate) instead of dextran as a backbone to reduce hydrophobicity. Chondroitin sulfate derivative-modified liposomes showed almost equal pH-sensitivity to that of dextran derivative-modified liposomes and achieved selective delivery to dendritic cells, whereas dextran derivative-modified liposomes were highly taken up by both dendritic cells and fibroblasts. Chondroitin sulfate derivative-modified liposomes delivered model antigenic proteins into cytosol of dendritic cells and promoted cytokine production from the cells, leading to tumor regression on tumor-bearing mice after subcutaneous administration. Results demonstrate that charged groups having polysaccharide as a backbone can be used in an effective strategy to balance strong hydrophobicity of spacer units with their utilization for immunity-inducing systems.

Designing immunostimulatory double stranded messenger RNA with maintained translational activity through hybridization with poly A sequences for effective vaccination.

Messenger (m)RNA vaccines require a safe and potent immunostimulatory adjuvant. In this study, we introduced immunostimulatory properties directly into mRNA molecules by hybridizing them with complementary RNA to create highly immunogenic double stranded (ds)RNAs. These dsRNA formulations, comprised entirely of RNA, are expected to be safe and highly efficient due to antigen expression and immunostimulation occurring simultaneously in the same antigen presenting cells. In this strategy, design of dsRNA is important. Indeed, hybridization using full-length antisense (as)RNA drastically reduced translational efficiency. In contrast, by limiting the hybridized portion to the mRNA poly A region, efficient translation and intense immunostimulation was simultaneously obtained. The immune response to the poly U-hybridized mRNAs (mRNA:pU) was mediated through Toll-like receptor (TLR)-3 and retinoic acid-inducible gene (RIG)-I. We also demonstrated that mRNA:pU activation of mouse and human dendritic cells was significantly more effective than activation using single stranded mRNA. In vivo mouse immunization experiments using ovalbumin showed that mRNA:pU significantly enhanced the intensity of specific cellular and humoral immune responses, compared to single stranded mRNA. Our novel mRNA:pU formulation can be delivered using a variety of mRNA carriers depending on the purpose and delivery route, providing a versatile platform for improving mRNA vaccine efficiency.

Potentiation of pH-sensitive polymer-modified liposomes with cationic lipid inclusion as antigen delivery carriers for cancer immunotherapy.

Cationic lipid-incorporated liposomes modified with pH-sensitive polymers were prepared by introducing 3, 5-didodecyloxybenzamidine as a cationic lipid to egg yolk phosphatidylcholine liposomes modified with 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG) as a pH-sensitive polymer. These liposomes were stable at neutral pH, but were destabilized below pH 6.0 because MGlu-HPG changed its characteristics from hydrophilic to hydrophobic in response to the pH decrease. Cationic lipid inclusion improved their pH sensitivity at weakly acidic pH and association of liposomes with murine dendritic cell (DC) lines. Cationic lipid-incorporated liposomes delivered entrapped ovalbumin (OVA) molecules not only to cytosol but also to endosome/lysosome. Treatment with cationic lipid-incorporated liposomes induced up-regulation of antigen presentation-involved molecules on DCs, the promotion of cytokine production, and antigen presentation via both major histocompatibility complex (MHC) class I and II molecules. Especially, antigen presentation via MHC class II was promoted by cationic lipid inclusion, which might correspond to efficient endosome/lysosome delivery of OVA. Subcutaneous administration of OVA-loaded cationic lipid-incorporated liposomes induced antigen-specific antibody production in serum and Th1-dominant immune responses in the spleen. Furthermore, administration of the cationic lipid-incorporated liposomes to mice bearing E.G7-OVA tumor more significantly reduced the tumor volume than liposomes without cationic lipids. Therefore, cationic lipid inclusion into pH-sensitive polymer-modified liposomes, which can achieve both efficient antigen intracellular delivery and activation of antigen presenting cell, is an effective approach to develop antigen carriers for efficient cancer immunotherapy.

PEGylated PAMAM dendrimer-doxorubicin conjugate-hybridized gold nanorod for combined photothermal-chemotherapy.

We prepared pH-sensitive drug-dendrimer conjugate-hybridized gold nanorod as a promising platform for combined cancer photothermal-chemotherapy under in vitro and in vivo conditions. Poly(ethylene glycol)-attached PAMAM G4 dendrimers (PEG-PAMAM) were first covalently linked on the surface of mercaptohexadecanoic acid-functionalized gold nanorod (MHA-AuNR), with subsequent conjugation of anti-cancer drug doxorubicin (DOX) to dendrimer layer using an acid-labile-hydrazone linkage to afford PEG-DOX-PAMAM-AuNR particles. The particles with a high PEG-PAMAM dendrimer coverage density (0.28 per nm(2) AuNR) showed uniform sizes and excellent colloidal stability. In vitro drug release studies demonstrated that DOX released from PEG-DOX-PAMAM-AuNR was negligible under normal physiological pH, but it was enhanced significantly at a weak acidic pH value. The efficient intracellular acid-triggered DOX release inside of lysosomes was confirmed using confocal laser scanning microscopy analysis. Furthermore, the combined photothermal-chemo treatment of cancer cells using PEG-DOX-PAMAM-AuNR for synergistic hyperthermia ablation and chemotherapy was demonstrated both in vitro and in vivo to exhibit higher therapeutic efficacy than either single treatment alone, underscoring the great potential of PEG-DOX-PAMAM-AuNR particles for cancer therapy.