Antifibrotic effects of pentoxifylline improve the efficacy of gemcitabine in human pancreatic tumor xenografts.

We investigated the combinatorial effects of pentoxifylline (PTX) on the efficacy of gemcitabine (GEM) in a human pancreatic tumor xenograft model. PTX significantly improved the efficacy of GEM, as shown by a 50% reduction in tumor growth rate at 4 weeks of treatment compared with that in animals given GEM alone. The fluorescent drug doxorubicin (DOX) was used to test whether drug delivery was improved by PTX, contributing to the improved efficacy of GEM. PTX given for 2 weeks prior to giving DOX improved drug distribution by 1.8- to 2.2-fold with no changes in vessel density, suggesting that improvement in drug delivery was not related to the vascular mechanism. Instead, collagen I content in tumor stroma was significantly reduced, as was the expression of alpha-smooth muscle actin of cancer-associated fibroblasts and connective tissue growth factor (CTGF) by PTX pretreatment. Overall, our data demonstrated that increased efficacy of GEM by PTX was associated with improved drug delivery to tumor tissue, which may be attributed to decreased expression of CTGF and subsequent reduction in the stromal collagen matrix in the pancreatic ductal adenocarcinoma tumor. These results support the usefulness of PTX in combination with chemotherapy for targeting drug delivery barriers associated with the stromal matrix, which should be further evaluated for clinical development.

GM-CSF-loaded chitosan hydrogel as an immunoadjuvant enhances antigen-specific immune responses with reduced toxicity.

BACKGROUND: The application of vaccine adjuvants has been vigorously studied for a diverse range of diseases in order to improve immune responses and reduce toxicity. However, most adjuvants have limited uses in clinical practice due to their toxicity. METHODS: Therefore, to reduce health risks associated with the use of such adjuvants, we developed an advanced non-toxic adjuvant utilizing biodegradable chitosan hydrogel (CH-HG) containing ovalbumin (OVA) and granulocyte-macrophage colony-stimulating factor (GM-CSF) as a local antigen delivery system. RESULTS: After subcutaneous injection into mice, OVA/GM-CSF-loaded CH-HG demonstrated improved safety and enhanced OVA-specific antibody production compared to oil-based adjuvants such as Complete Freund's adjuvant (CFA) or Incomplete Freund's adjuvant (IFA). Moreover, CH-HG system-mediated immune responses was characterized by increased number of OVA-specific CD4(+) and CD8(+) INF-γ(+) T cells, leading to enhanced humoral and cellular immunity. CONCLUSIONS: In this study, the improved safety and enhanced immune response characteristics of our novel adjuvant system suggest the possibility of the extended use of adjuvants in clinical practice with reduced apprehension about toxic side effects.

Topical application of liposomal cobalamin hydrogel for atopic dermatitis therapy.

Topical vitamin B12 was shown to be effective for atopic dermatitis. However, vitamin B12 itself is light sensitive and has low skin permeability, thus reducing its therapeutic effectiveness. In the present study, we prepared a liposomal hydrogel of adenosylcobalamin (AdCbl), a vitamin B12 derivative, and investigated possible beneficial effects of AdCbl on atopic dermatitis using an NC/Nga murine atopic dermatitis model. AdCbl was loaded into liposomes prepared by a thin film hydration method using a pH gradient method that employed citric acid buffer solution. This resulted in AdCbl-loaded liposomes that were 106.4 +/- 2.2 nm in size. The loading efficiency was 40% (of the initial AdCbl amount). Lipo-AdCbl had enhanced skin permeability, being about 17-fold compared with AdCbl-gel. Topical administration of Lipo-AdCbl-gel to 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis-like skin lesions in NC/Nga mice ameliorated lesion intensity scores, dorsal skin thickness, and total serum IgE in a concentration-dependent manner. Other preparations, including AdCbl solution, AdCbl cream, liposomes alone, and a mixture of AdCbl solution and liposomes had little effect. Taken together, our findings indicate that Lipo-AdCbl-gel has protective effects against atopic dermatitis symptoms, and suggest that it may be of benefit in the treatment of human inflammatory skin diseases.

NIR irradiation-controlled drug release utilizing injectable hydrogels containing gold-labeled liposomes for the treatment of melanoma cancer.

Drug-based chemotherapy is associated with serious side effects. We developed a chemotherapeutic system comprising a chitosan hydrogel (CH-HG) containing gold cluster-labeled liposomal doxorubicin (DOX) (CH-HG-GLDOX) as an injectable drug depot system. CH-HG-GLDOX can be directly injected into tumor tissue without a surgical procedure, allowing this system to act as a reservoir for liposomal DOX. CH-HG-GLDOX enhanced the retention time of DOX in tumor tissue and controlled its release in response to near-infrared (NIR) irradiation, resulting in significant inhibition of tumor growth and reduced DOX-related toxicity. The combined effect of CH-HG-GLDOX and poly (D,L-lactide-co-glycolic acid) nanoparticle-based vaccines increased cytotoxic CD8+ T cell immunity, leading to enhanced synergistic therapeutic efficacy. CH-HG-GLDOX provides an advanced therapeutic approach for local drug delivery and controlled release of DOX, resulting in reduced toxicity. Here, we suggest a combination strategy for chemo- and immunotherapies, as well as in nanomedicine applications. STATEMENT OF SIGNIFICANCE: We developed an injectable hydrogel containing gold cluster-labeled liposomes for sustained drug release at the tumor site. Moreover, we demonstrated the combined therapeutic efficacy of a hydrogel system and a nanoparticle-based immunotherapeutic vaccine for melanoma cancer. Thus, we show a potential combination approach for chemo- and immunotherapies for cancer treatment.

Efficacy of Combination Therapy with Linalool and Doxorubicin Encapsulated by Liposomes as a Two-in-One Hybrid Carrier System for Epithelial Ovarian Carcinoma.

BACKGROUND: Epithelial ovarian cancer (EOC) is a fatal gynecologic malignancy that is usually treated with chemotherapy after surgery. However, patients who receive chemotherapy experience severe side effects because of the inherent toxicity and high dose of chemotherapeutics. To overcome these issues, we suggest a combination therapeutic strategy using liposomes encapsulating linalool nanoemulsions (LN-NEs) and doxorubicin (DOX), a chemotherapeutic drug, to increase their synergistic antitumor efficacy and reduce the incidence of side effects from chemotherapeutics for EOC. METHODS: The physical properties of LN-NE-DOX-liposomes were characterized by light scattering with a particle size analyzer. Cell viability was determined by MTT assay. Therapeutic efficacy was evaluated in a mouse HeyA8 EOC tumor model of ovarian carcinoma. Additionally, biochemical toxicity was analyzed for levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and blood urea nitrogen (BUN) using BALB/c nude mice. RESULTS: The size of the liposomes encapsulating LN-NEs and DOX (LN-NE-DOX-liposomes) was 267.0 ± 4.6 nm, with a loading efficiency of 55.1 ± 3.1% and 27.2 ± 0.9% for linalool and DOX, respectively. Cell viability after treatment with LN-NE-DOX-liposomes was significantly decreased compared to that of cells treated with DOX liposomes, and apoptosis was significantly increased. Additionally, LN-NE-DOX-liposomes significantly inhibited HeyA8 EOC tumor growth compared to that of the control (p < 0.01) and DOX-liposome-treated groups (p < 0.05), while decreasing cell proliferation (Ki67) and microvessel density (CD31), and promoting apoptosis (caspase-3) compared to the control (p < 0.05). Moreover, the liposomal formulations induced no significant differences in biochemical toxicity (AST, ALT, and BUN) compared to healthy control mice, indicating that the liposomal formulations showed no overt toxicity in mice. CONCLUSION: This study demonstrates that the production of LN-NE-DOX-liposomes is a pivotal approach for EOC treatment, suggesting a novel combination therapeutic strategy.

Epigallocatechin-3-gallate enhances CD8+ T cell-mediated antitumor immunity induced by DNA vaccination.

Immunotherapy and chemotherapy are generally effective against small tumors in animal models of cancer. However, these treatment regimens are generally ineffective against large, bulky tumors. We have found that a multimodality treatment regimen using DNA vaccination in combination with chemotherapeutic agent epigallocatechin-3-gallate (EGCG), a compound found in green tea, is effective in inhibiting large tumor growth. EGCG was found to induce tumor cellular apoptosis in a dose-dependent manner. The combination of EGCG and DNA vaccination led to an enhanced tumor-specific T-cell immune response and enhanced antitumor effects, resulting in a higher cure rate than either immunotherapy or EGCG alone. In addition, combined DNA vaccination and oral EGCG treatment provided long-term antitumor protection in cured mice. Cured animals rejected a challenge of E7-expressing tumors, such as TC-1 and B16E7, but not a challenge of B16 7 weeks after the combined treatment, showing antigen-specific immune responses. These results suggest that multimodality treatment strategies, such as combining immunotherapy with a tumor-killing cancer drug, may be a more effective anticancer strategy than single-modality treatments.

Enhanced Antitumor Immunity Using a Tumor Cell Lysate-Encapsulated CO2-Generating Liposomal Carrier System and Photothermal Irradiation.

Dendritic cell (DC)-based cancer immunotherapies have been studied extensively. In cancer immunotherapy, the initial key step is the delivery of tumor-specific antigens, leading to the maturation and activation of DCs. To promote effective antigen delivery, liposome-based delivery systems for tumor-specific antigens have been investigated, and although promising, a triggered release of the antigen from the liposome is required to attain an optimum immune response. In this study, we developed CO2-bubble-generating thermosensitive liposomes (BG-TSLs) that encapsulate whole tumor cell lysates (TCLs). The release of the lysate from BG-TSLs can be triggered using near-infrared (NIR) irradiation. We also developed BG-TSLs able to encapsulate doxorubicin (DOX) for combination therapy. The DOX-BG-TSLs and TCL-BG-TSLs have a mean particle size of 114.17 ± 8.28 nm and 123.8 ± 10.2 nm and a surface charge of -22.56 ± 1.3 mV and -28.9 ± 0.8 mV, respectively. CO2 bubble generation within TCL-BG-TSLs and DOX-BG-TSLs by NIR irradiation led to the burst release of TCL or DOX. TCL release from TCL-BG-TSLs promoted dendritic cell maturation and activation, leading to the emergence of antigen-specific cytotoxic CD8+ T cells. The combination of TCL-BG-TSLs with DOX-BG-TSLs showed a significantly greater antitumor efficacy in B16F10 tumor-bearing mice compared to that seen in the control mice (P < 0.001). Taken together, our liposomal delivery system, combined with NIR irradiation, could enhance the therapeutic efficacy of cancer immunotherapies.

A chitosan hydrogel-based cancer drug delivery system exhibits synergistic antitumor effects by combining with a vaccinia viral vaccine.

Cancer treatment combining chemotherapy and immunotherapy has been vigorously exploited to further improve cancer therapeutic efficacy. This study investigated a new chemoimmunotherapy approach utilizing hydrogel as a local anti-cancer drug delivery system. Chitosan hydrogel containing doxorubicin (CH-DOX) and vaccinia virus vaccine expressing Sig/E7/LAMP-1 (Vac-Sig/E7/LAMP-1) were used as chemoimmunotherapeutic agents. It was found that intratumoral injection of CH-DOX effectively inhibited tumor growth itself and, in addition, exhibited a synergistic antitumor effect in combination with a vaccinia virus-based vaccine. This combination did not decrease but rather increased the number of tumor-specific CD8(+) T cells primed by vaccinia virus-mediated vaccination; the resulting antitumor effects were further improved up to 60 days as compared with monotherapy after tumor challenge, and the survival of tumor-bearing mice was dramatically prolonged. This study is a pioneer report that demonstrates the use of a biodegradable hydrogel system as an anti-cancer drug delivery system for successful chemoimmunotherapy. It is hoped that, this study can provide a foundation for a rational approach to improve antitumor efficacy of chemoimmunotherapy.

Pitch-tunable pillar arrays for high-throughput culture and immunohistological analysis of tumor spheroids.

Tumor spheroids are multicellular, three-dimensional (3D) cell culture models closely mimicking the microenvironments of human tumors in vivo, thereby providing enhanced predictability, clinical relevancy of drug efficacy and the mechanism of action. Conventional confocal microscopic imaging remains inappropriate for immunohistological analysis due to current technical limits in immunostaining using antibodies and imaging cells grown in 3D multicellular contexts. Preparation of microsections of these spheroids represents a best alternative, yet their sub-millimeter size and fragility make it less practical for high-throughput screening. To address these problems, we developed a pitch-tunable 5 × 5 mini-pillar array chip for culturing and sectioning tumor spheroids in a high throughput manner. Tumor spheroids were 3D cultured in an alginate matrix using a twenty-five mini-pillar array which aligns to a 96-well. At least a few tens of spheroids per pillar were cultured and as many as 25 different treatment conditions per chip were evaluated, which indicated the high throughput manner of the 5 × 5 pillar array chip. The twenty-five mini-pillars were then rearranged to a transferring pitch so that spheroid-containing gel caps from all pillars can be embedded into a specimen block. Tissue array sections were then prepared and stained for immunohistological examination. The utility of this pitch-tunable pillar array was demonstrated by evaluating drug distribution and expression levels of several proteins following drug treatment in 3D tumor spheroids. Overall, our mini-pillar array provides a novel platform that can be useful for culturing tumor spheroids as well as for immunohistological analysis in a multiplexed and high throughput manner.

CD44-Targeting PLGA Nanoparticles Incorporating Paclitaxel and FAK siRNA Overcome Chemoresistance in Epithelial Ovarian Cancer.

Chemotherapy is commonly used in the treatment of ovarian cancer, yet most ovarian cancers harbor inherent resistance or develop acquired resistance. Therefore, novel therapeutic approaches to overcome chemoresistance are required. In this study, we developed a hyaluronic acid-labeled poly(d,l-lactide-co-glycolide) nanoparticle (HA-PLGA-NP) encapsulating both paclitaxel (PTX) and focal adhesion kinase (FAK) siRNA as a selective delivery system against chemoresistant ovarian cancer. The mean size and zeta potential of the HA-PLGA-NP were 220 nm and -7.3 mV, respectively. Incorporation efficiencies for PTX and FAK siRNA in the HA-PLGA-NPs were 77% and 85%, respectively. HA-PLGA-NP showed higher binding efficiency for CD44-positive tumor cells as compared with CD44-negative cells. HA-PLGA (PTX+FAK siRNA)-NP caused increased cytotoxicity and apoptosis in drug-resistant tumor cells. Treatment of human epithelial ovarian cancer tumor models HeyA8-MDR (P < 0.001) and SKOV3-TR (P < 0.001) with HA-PLGA (PTX+FAK siRNA)-NP resulted in significant inhibition of tumor growth. Moreover, in a drug-resistant, patient-derived xenograft (PDX) model, HA-PLGA (PTX+FAK siRNA)-NP significantly inhibited tumor growth compared with PTX alone (P < 0.002). Taken together, HA-PLGA-NP acts as an effective and selective delivery system for both the chemotherapeutic and the siRNA in order to overcome chemoresistance in ovarian carcinoma.Significance: These findings demonstrate the efficacy of a novel, selective, two-in-one delivery system to overcome chemoresistance in epithelial ovarian cancer. Cancer Res; 78(21); 6247-56. ©2018 AACR.

Selective delivery of PLXDC1 small interfering RNA to endothelial cells for anti-angiogenesis tumor therapy using CD44-targeted chitosan nanoparticles for epithelial ovarian cancer.

Angiogenesis plays an essential role in the growth and metastasis of tumor cells, and the modulation of angiogenesis can be an effective approach for cancer therapy. We focused on silencing the angiogenic gene PLXDC1 as an important factor for anti-angiogenesis tumor therapy. Herein, we developed PLXDC1 small interfering siRNA (siRNA)-incorporated chitosan nanoparticle (CH-NP/siRNA) coated with hyaluronic acid (HA) to target the CD44 receptor on tumor endothelial cells. This study aimed to improve targeted delivery and enhance therapeutic efficacy for tumor anti-angiogenesis. The HA-CH-NP/siRNA was 200 ± 10 nm in size with a zeta potential of 26.4 mV. The loading efficiency of siRNA to the HA-CH-NP/siRNA was up to 60%. The selective binding of HA-CH-NP/siRNA to CD44-positive tumor endothelial cells increased by 2.1-fold compared with that of the CD44 nontargeted CH-NP/siRNA. PLXDC1 silencing by the HA-CH-NP/siRNA significantly inhibited tumor growth in A2780 tumor-bearing mice compared with that in the control group (p < .01), and mRNA expression of PLXDC1 was significantly reduced in the HA-CH-NP/siRNA-treated group. Furthermore, treatment with HA-CH-NP/siRNA resulted in significant inhibition of cell proliferation (p < .001), reduced microvessel density (p < .001), and increased cell apoptosis (p < .001). This study demonstrates that HA-CH-NP/siRNA is a highly selective delivery platform for siRNA, and has broad potential to be used in anti-angiogenesis tumor therapy.

Doxorubicin-encapsulated thermosensitive liposomes modified with poly(N-isopropylacrylamide-co-acrylamide): drug release behavior and stability in the presence of serum.

In the field of the temperature sensitive drug delivery systems, we studied on the surface modification of liposomes by using poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and polyethyleneglycol (PEG) to increase the release of doxorubicin (DOX) from liposomes and prolong the stability of liposomes in the presence of serum. The release of DOX from the PNIPAM-AAM/PEG modified liposomes is enhanced around the transition temperature of the polymer. In addition, the stability of the PNIPAM-AAM/PEG modified liposomes in serum shows a high level comparing with polymer unmodified liposomes. These results suggest that the modification on the surface of liposomes with both PNIPAM-AAM and PEG enhances the drug release from liposomes and reduces the protein adsorption in serum.

Hyperthermia-induced antitumor activity of thermosensitive polymer modified temperature-sensitive liposomes.

Temperature-sensitive liposomes (TS-liposomes) have been studied for chemotherapeutic purposes to enhance the release of anticancer drugs at tumor sites. In this study, we prepared poly(N-isopropylacrylamide-co-acrylamide) (PNIPAM-AAM) and polyethylene glycol (PEG)-modified TS-liposomes (PETS-liposomes). PETS-liposomes significantly increased in vitro drug release in serum compared with PEG-fixed or PNIPAM-AAM-modified liposomes. Furthermore, incorporation of both PNIPAM-AAM and PEG into PETS-liposomes enhanced the stabilities of liposomes in serum by inhibiting protein adsorption. In addition, to investigate the therapeutic efficacy of doxorubicin (DOX)-loaded PETS-liposomes, the in vivo antitumor activity of liposomes in combination with hyperthermia was evaluated in a B16F10 melanoma tumor-bearing mouse model. PETS-liposomes showed much higher levels of tumor growth inhibition than PEG-fixed or PNIPAM-AAM-modified TS-liposomes. Moreover, the antitumor activity of PETS-liposomes was enhanced significantly when they were administered in combination with hyperthermia. PETS-liposomes were found to be highly efficacious carriers for the in vivo delivery of anticancer drugs, and to have potential anticancer applications in combination with hyperthermia.

In vivo distribution and antitumor activity of heparin-stabilized doxorubicin-loaded liposomes.

The purpose of this study was to investigate the effect of heparin conjugation to the surface of doxorubicin (DOX)-loaded liposomes on the circulation time, biodistribution and antitumor activity after intravenous injection in murine B16F10 melanoma tumor-bearing mice. The heparin-conjugated liposomes (heparin-liposomes) were prepared by fixation of the negatively charged heparin to the positively charged liposomes. The existence of heparin on the liposomal surface was confirmed by measuring the changes in the particle size, zeta potential and heparin amount of the liposomes. The stability of the heparin-liposomes in serum was higher than that of the control liposomes, due to the heparin-liposomes being better protected from the adsorption of serum proteins. The DOX-loaded heparin-liposomes showed high drug levels for up to 64 h after the intravenous injection and the half-life of DOX was approximately 8.4- or 1.5-fold higher than that of the control liposomes or polyethyleneglycol-fixed liposomes (PEG-liposomes), respectively. The heparin-liposomes accumulated to a greater extent in the tumor than the control or PEG-liposomes as a result of their lower uptake by the reticuloendothelial system cells in the liver and spleen. In addition, the DOX-loaded heparin-liposomes retarded the growth of the tumor effectively compared with the control or PEG-liposomes. These results indicate the promising potential of heparin-liposomes as a new sterically stabilized liposomal delivery system for the enhancement of the therapeutic efficacy of chemotherapeutic agents.

Toll-like receptor 3-induced immune response by poly(d,l-lactide-co-glycolide) nanoparticles for dendritic cell-based cancer immunotherapy.

Dendritic cells (DCs) are potent professional antigen-presenting cells that are capable of initiating a primary immune response and activating T cells, and they play a pivotal role in the immune responses of the host to cancer. Prior to antigen presentation, efficient antigen and adjuvant uptake by DCs is necessary to induce their maturation and cytokine generation. Nanoparticles (NPs) are capable of intracellular delivery of both antigen and adjuvant to DCs. Here, we developed an advanced poly(d,l-lactide-co-glycolide) (PLGA)-NP encapsulating both ovalbumin (OVA) as a model antigen and polyinosinic-polycytidylic acid sodium salt (Toll-like receptor 3 ligand) as an adjuvant to increase intracellular delivery and promote DC maturation. The PLGA-NPs were taken up by DCs, and their uptake greatly facilitated major histocompatibility class I antigen presentation in vitro. Moreover, vaccination with PLGA-NP-treated DCs led to the generation of ovalbumin-specific CD8+ T cells, and the resulting antitumor efficacy was significantly increased in EG.7 and TC-1 tumor-bearing mice compared to control mice (P<0.01). Taken together, these findings demonstrated that the PLGA-NP platform may be an effective method for delivering tumor-specific antigens or adjuvants to DCs.

Marbofloxacin-encapsulated microparticles provide sustained drug release for treatment of veterinary diseases.

Fluoroquinolone antibiotics with concentration-dependent killing effects and a well-established broad spectrum of activity are used commonly to treat infectious diseases caused by bacteria. However, frequent and excessive administration of these antibiotics is a serious problem, and leads to increased number of drug-resistant bacteria. Thus, there is an urgent need for novel fluoroquinolone antibiotic formulations that minimize the risk of resistance while maximizing their efficacy. In this study, we developed intramuscularly injectable polymeric microparticles (MPs) that encapsulated with marbofloxacin (MAR) and were composed of poly(D,L-lactide-co-glycolic acid) (PLGA) and poloxamer (POL). MAR-encapsulated MP (MAR-MP) had a spherical shape with particle size ranging from 80 µm to 120 µm. Drug loading efficiency varied from 55 to 85% (w/w) at increasing amount of hydrophilic agent, POL. Drug release from MAR-MP demonstrated a significant and sustained increase at increased ratios of POL to PLGA. These results indicate that MAR-MP is an improved drug delivery carrier for fluoroquinolone antibiotics, which can reduce the number of doses needed and sustain a high release rate of MAR for 2-3 days. As a novel and highly effective drug delivery platform, MAR-MP has great potential for use in a broad range of applications for the treatment of various veterinary diseases.

Linalool-Incorporated Nanoparticles as a Novel Anticancer Agent for Epithelial Ovarian Carcinoma.

Although cytotoxic chemotherapy is widely used against epithelial ovarian cancer (EOC), adverse side effects and emergence of resistance can limit its utility. Therefore, new drugs with systemic delivery platforms are urgently needed for this disease. In this study, we developed linalool-incorporated nanoparticles (LIN-NP) as a novel anticancer agent. We prepared LIN-NPs by the self-assembly water-in-oil-in-water (w/o/w) emulsion method. LIN-NP-mediated cytotoxicity and apoptosis was assessed in EOC cells, and the role of reactive oxygen species (ROS) generation as the mechanism of action was evaluated. In addition, therapeutic efficacy of LIN-NP was assessed in cell lines and patient-derived xenograft (PDX) models for EOC. LIN-NPs had significant cytotoxicity and apoptotic activity against EOC cells, including A2780, HeyA8, and SKOV3ip1. LIN-NP treatment increased apoptosis in EOC cells through ROS generation and a subsequent decrease in mitochondrial membrane potential and increase in caspase-3 levels. In addition, 100 mg/kg LIN-NPs significantly decreased tumor weight in the HeyA8 (P < 0.001) and SKOV3ip1 (P = 0.006) in vivo models. Although treatment with 50 mg/kg LIN-NP did not decrease tumor weight compared with the control group, combination treatment with paclitaxel significantly decreased tumor weight compared with paclitaxel alone in SKOV3ip1 xenografts (P = 0.004) and the patient-derived xenograft model (P = 0.020). We have developed LIN-NPs that induce ROS generation as a novel anticancer agent for EOC. These findings have broad applications for cancer therapy. Mol Cancer Ther; 15(4); 618-27. ©2016 AACR.

In vivo stepwise immunomodulation using chitosan nanoparticles as a platform nanotechnology for cancer immunotherapy.

Dentritic cell (DC)-based cancer immunotherapy faces challenges in both efficacy and practicality. However, DC-based vaccination requires multiple injections and elaborates ex vivo manipulation, which substantially limits their use. Therefore, we sought to develop a chitosan nanoparticle (CH-NP)-based platform for the next generation of vaccines to bypass the ex vivo manipulation and induce immune responses via active delivery of polyinosinic-polycytidylic acid sodium salt (poly I:C) to target Toll-like receptor 3 (TLR3) in endosomes. We developed CH-NPs encapsulating ovalbumin (OVA) as a model antigen and poly I:C as the adjuvant in an ionic complex. These CH-NPs showed increased in vivo intracellular delivery to the DCs in comparison with controls after injection into tumor-bearing mice, and promoted DC maturation, leading to emergence of antigen-specific cytotoxic CD8+ T cells. Finally, the CH-NPs showed significantly greater antitumor efficacy in EG.7 and TC-1 tumor-bearing mice compared to the control (p < 0.01). Taken together, these data show that the CH-NP platform can be used as an immune response modulatory vaccine for active cancer immunotherapy without ex vivo manipulation, thus resulting in increased anticancer efficacy.

Polycations enhance emulsion-mediated in vitro and in vivo transfection.

To enhance the in vitro and in vivo transfection activity of the cationic lipid emulsion (LE), three natural polycations, protamine sulfate (PS), poly-L-lysine and spermine, were selected as DNA condensing active agents. Formation of the LE/polycation/DNA ternary complexes was identified by using agarose gel retardation study. The structure of these complexes was characterized by measuring the complex size and the decrease of the DNA fluorescence in the presence of ethidium bromide (EtBr). By adding a polycation, the particle size of the complex decreased, and DNA in the complex became highly condensed and resistant to intercalation of EtBr. Among the polycations, PS yielded the most highly compacted ternary complex. In vitro and in vivo transfection activities of the complexes were determined using various cell lines and Balb/c mouse intravenously and intranasally, respectively. The transfection activity of the ternary complex increases by at least 2.5-5-fold in vitro cell culture system in the presence of 80% serum as well as in vivo mouse system, as compared with LE/DNA binary complexes. More importantly, after intravenous and intranasal administrations, the in vivo transfection efficiency of the LE/PS/DNA complex was ca. 30 and 50 times higher than that of the liposome (LP)/DNA complex in spleen and lung, respectively. On the other hand, cell toxicity of the ternary complex is lower than that of binary complex. Thus, we conclude that the pre-condensation of DNA with polycations can be a promising approach to further increase in vitro and in vivo transfection efficiency of cationic lipid emulsion.

Gold cluster-labeled thermosensitive liposmes enhance triggered drug release in the tumor microenvironment by a photothermal effect.

Stimulus-triggered drug release based on the liposomal drug delivery platform has been studied vigorously to increase drug release at the target site. Although the delivery system has been developed, an effective carrier system is needed to achieve effective therapeutic efficacy. Therefore, we focused on the development of gold cluster bound thermosensitive liposomes (G-TSL), which are capable of triggered drug release when stimulated by external near-infrared (NIR) irradiation in the tumor microenvironment. The size of doxorubicin (DOX)-loaded G-TSL (DOX/G-TSL) was 171.5 ± 8.3 nm, and the efficiency of DOX encapsulation was up to 90%. The release of DOX from DOX/G-TSL was increased 70% by NIR irradiation (1.50 W/cm(2) for 0.5 min) compared to non-gold-coated TSL. Consequentially, the gold cluster on the TSL enabled the light-controlled DOX release through the photothermal conversion of the energy of NIR-absorbed light, leading to membrane destabilization. Cell cytotoxicity of DOX/G-TSL was also increased by their NIR irradiation-triggered DOX release compared to non-NIR-irradiated DOX/G-TSL. In addition, we demonstrated the therapeutic efficacy of DOX/G-TSL against the MDA-MB-231 tumor model. The NIR-irradiated DOX/G-TSL treatment showed greater therapeutic efficacy than that of the non-NIR-irradiated DOX/G-TSL and control (p<0.05). Taken together, DOX/G-TSL has the potential for remote-triggered drug release upon stimulation with NIR irradiation in the tumor microenvironment, and may be applied to a broad range of photothermal-based disease therapies.