Joung Woo Choi

Multifunctional Nanoparticles for Targeted Chemophotothermal Treatment of Cancer Cells

This work was financially supported by NRF through the National Core Research Center for Nanomedical Technology (Grant No. R15-2004-024-00000-0) and by Basic Science Research Program through the NRF funded by the Ministry of Education, Science and Technology (2010-0003946).

Active targeting and safety profile of PEG-modified adenovirus conjugated with herceptin

PEGylation of adenovirus (Ad) increases plasma retention and reduces immunogenicity, but decreases the accessibility of virus particles to target cells. We tested whether PEGylated Ad conjugated to Herceptin (Ad-PEG-HER) can be used to treat Her2/neu-positive cells in vitro and in vivo to demonstrate the therapeutic feasibility of this Ad formulation. Ad-PEG-HER transduced Her2/neu-overexpressing cancer cells through a specific interaction between Herceptin and Her2/neu. Ad-PEG-HER treatment resulted in higher plasma retention and lower neutralizing antibody and IL-6 production than naked Ad. This formulation was extended to generate a Her2/neu-targeted, PEGylated oncolytic Ad (DWP418-PEG-HER). DWP418-PEG-HER specifically killed Her2/neu-positive cells and performed better than non-targeted and naked Ad in vivo. DWP418-PEG-HER showed a 10(10)-fold increase in the liver to tumor biodistribution compared with naked Ad. Immunohistochemical staining confirmed accumulation of Ad E1A in tumors. These data suggest that targeted gene therapy with the PEGylated Ad conjugated with Herceptin might shed a light on its therapeutic application for metastatic cancer in the future.

Therapeutic targeting of chitosan-PEG-folate-complexed oncolytic adenovirus for active and systemic cancer gene therapy

Adenovirus (Ad)-based cancer therapies have shown much promise. However, until now, Ad has only been delivered directly to primary tumors because the therapeutic efficacy of systemic delivery is limited by the immune response of the host, short blood circulation times, and non-specific liver uptake of Ad. In order to circumvent the issues regarding systemic delivery and to increase the safety and efficacy of Ad therapies, the surface of oncolytic Ad was coated with cationic polymer chitosan via ionic crosslinking (Ad/chitosan), after which polyethylene glycol (PEG) and/or folic acid (FA) was chemically conjugated onto the surface of Ad/chitosan, generating Ad/chitosan-FA, Ad/chitosan-PEG, and Ad/chitosan-PEG-FA nanocomplex. The FA-coordinated Ad nanocomplexes (Ad/chitosan-FA & Ad/chitosan-PEG-FA) elicited folate receptor (FR)-selective cancer cell killing efficacy. In vivo administration of Ad/chitosan-PEG or Ad/chitosan-PEG-FA into mice demonstrated that PEGylation greatly increased blood circulation time, resulting in 9.0-fold and 48.9-fold increases at 24h after injection compared with naked Ad, respectively. In addition, generation of Ad-specific neutralizing antibodies in mice treated with Ad/chitosan-PEG-FA was markedly decreased by 75.3% compared with naked Ad. The quantitative polymerase chain reaction assay results showed a 285.0-fold increase in tumor tissues and a 378-fold reduction of Ad/chitosan-PEG-FA in liver tissues compared with naked Ad. Bioluminescence imaging study further supported the enhanced tumor-to-liver ratio of Ad/chitosan-PEG-FA. Consequently, systemic delivery of Ad/chitosan-PEG-FA significantly inhibited the growth of FR-positive tumor, decreasing 52.8% compared to the naked Ad-treated group. Importantly, PEGylated oncolytic Ad nanocomplexes showed no elevation of both alanine transaminase and aspartate transaminase levels, demonstrating that systemically delivered Ad-related hepatic damage can be completely eliminated with PEG conjugation. In sum, these results demonstrate that conjugation of chitosan-PEG-FA to oncolytic Ad significantly improves antitumor efficacy and safety profiles, suggesting that Ad/chitosan-PEG-FA has potential as a therapeutic agent to target FR-positive cancer via systemic administration.

pH-sensitive oncolytic adenovirus hybrid targeting acidic tumor microenvironment and angiogenesis

Although oncolytic adenoviruses (Ads) are an attractive option for cancer gene therapy, the intravenous administration of naked Ad still encounters unfavorable host responses, non-specific interactions, and heterogeneity in targeted cancer cells. To overcome these obstacles and achieve specific targeting of the tumor microenvironment, Ad was coated with the pH-sensitive block copolymer, methoxy poly(ethylene glycol)-b-poly(l-histidine-co-l-phenylalanine) (PEGbPHF). The physicochemical properties of the generated nanocomplex, Ad/PEGbPHF, were assessed. At pH6.4, GFP-expressing Ad/PEGbPHF induced significantly higher GFP expression than naked Ad in both coxsackie and adenovirus receptor (CAR)-positive and -negative cells. To assess the therapeutic efficacy of the Ad/PEGbPHF complex platform, an oncolytic Ad expressing VEGF promoter-targeting transcriptional repressor (KOX) was used to form complexes. At pH6.4, KOX/PEGbPHF significantly suppressed VEGF gene expression, cancer cell migration, vessel sprouting, and cancer cell killing effect compared to naked KOX or KOX/PEGbPHF at pH7.4, demonstrating that KOX/PEGbPHF can overcome the lack of CAR that is frequently observed in tumor tissues. The antitumor activity of KOX/PEGbPHF systemically administered to a tumor xenograft model was significantly higher than that of naked KOX. Furthermore, KOX/PEGbPHF showed lower hepatic toxicity and did not induce an innate immune response against Ad. Altogether, these results demonstrate that pH-sensitive polymer-coated Ad complex significantly increases net positive charge upon exposure to hypoxic tumor microenvironment, allowing passive targeting to the tumor tissue. It may offer superior potential for systemic therapy, due to its improved tumor selectivity, increased therapeutic efficacy, and lower toxicity compared to naked KOX.

Utilizing adenovirus vectors for gene delivery in cancer

Introduction: Adenovirus (Ad) is a promising candidate vector for cancer gene therapy because of its unique characteristics, which include efficient infection, high loading capacity and lack of insertional mutagenesis. However, systemic administration of Ad is hampered by the hosts immune response, hepatocytoxicity, short half-life of the vector and low accumulation at the target site. For these reasons, clinical applications of Ad are currently restricted. Areas covered: In this review, we focus on recent developments in Ad nanocomplex systems that improve the transduction and targeting efficacy of Ad vectors in cancer gene therapy. We discuss the development of different Ad delivery systems, including surface modification of Ad, smart Ad/nanohybrid systems and hydrogels for sustained release of Ad. Expert opinion: The fusion of bioengineering and biopharmaceutical technologies can provide solutions to the obstacles encountered during systemic delivery of Ads. The in vivo transgene expression efficiency of Ad nanocomplex systems is typically high, and animal tumor models demonstrate that systemic administration of these Ad complexes can arrest tumor growth. However, further optimization of these smart Ad nanocomplex systems is needed to increase their effectiveness and safety for clinical application in cancer gene therapy.

Capacitance-based real time monitoring of receptor-mediated endocytosis.

Receptor-mediated endocytosis is essential for targeted gene/drug delivery to a specific cell type. In this study, we developed a capacitance sensor to monitor receptor-mediated endocytosis in real time. The capacitance sensor was able to detect a capacitance peak in different cell lines during the internalization of adenoviruses or antibodies via receptor-mediated endocytosis. In contrast, the capacitance declined without a capacitance peak when nanoparticles were taken up via non-specific pinocytosis. Thus, our capacitance sensor represents a potential capacitance-based means of discrimination between receptor-mediated endocytosis and non-specific pinocytosis. Moreover, we developed a capacitance sensor array to demonstrate capacitance-based high-throughput screening. We showed that the capacitance sensor array could rapidly identify antibodies or ligands with high specificity for target molecules. We propose that the capacitance sensor array will provide a valuable tool for high-throughput screening.

Potent antitumor effect of neurotensin receptor-targeted oncolytic adenovirus co-expressing decorin and Wnt antagonist in an orthotopic pancreatic tumor model

Pancreatic cancer is highly aggressive, malignant, and notoriously difficult to cure using conventional cancer therapies. These conventional therapies have significant limitations due to excessive extracellular matrix (ECM) of pancreatic cancer and poor cancer specificity. The excess ECM prevents infiltration of drugs into the inner layer of the solid tumor. Therefore, novel treatment modalities that can specifically target the tumor and degrade the ECM are required for effective therapy. In the present study, we used ECM-degrading and Wnt signal-disrupting oncolytic adenovirus (oAd/DCN/LRP) to achieve a desirable therapeutic outcome against pancreatic cancer. In addition, to overcome the limitations in systemic delivery of oncolytic Ad (oAd) and to specifically target pancreatic cancer, neurotensin peptide (NT)-conjugated polyethylene glycol (PEG) was chemically crosslinked to the surface of Ad, generating a systemically injectable hybrid system, oAd/DCN/LRP-PEG-NT. We tested the targeting and therapeutic efficacy of oAd/DCN/LRP-PEG-NT toward neurotensin receptor 1 (NTR)-overexpressing pancreatic cancer cells, both in vitro and in vivo. The oAd/DCN/LRP-PEG-NT elicited increased NTR-selective cancer cell killing and transduction efficiency when compared with a cognate control lacking NT (oAd/DCN/LRP-PEG). Furthermore, systemic administration of oAd/DCN/LRP-PEG-NT significantly decreased induction of innate and adaptive immune responses against Ad, and blood retention time was markedly prolonged by PEGylation. Moreover, NTR-targeting oAd elicited greater in vivo tumor growth suppression when compared with naked oAd and 9.5 × 10(6)-fold increased tumor-to-liver ratio. This significantly enhanced antitumor effect of oAd/DCN/LRP-PEG-NT was mediated by active viral replication and viral spreading, which was facilitated by ECM degradation and inhibition of Wnt signaling-related factors (Wnt, β-catenin, and/or vimentin) in the tumor tissues. Taken together, these results demonstrate that oAd/DCN/LRP-PEG-NT has strong therapeutic potential for systemic treatment of NTR-overexpressing pancreatic cancer due to its NTR-targeting ability, enhanced therapeutic efficacy, and safety.

Safety profiles and antitumor efficacy of oncolytic adenovirus coated with bioreducible polymer in the treatment of a CAR negative tumor model

Adenovirus (Ad) vectors show promise as cancer gene therapy delivery vehicles, but immunogenic safety concerns and coxsackie and adenovirus receptor (CAR)-dependency have limited their use. Alternately, biocompatible and bioreducible nonviral vectors, including arginine-grafted cationic polymers, have been shown to deliver nucleic acids through a cell penetration peptide (CPP) and protein transduction domain (PTD) effect. We utilized the advantages of both viral and nonviral vectors to develop a hybrid gene delivery vehicle by coating Ad with mPEG-PEI-g-Arg-S-S-Arg-g-PEI-mPEG (Ad/PPSA). Characterization of Ad/PPSA particle size and zeta potential showed an overall size and cationic charge increase in a polymer concentration-dependent manner. Ad/PPSA also showed a marked transduction efficiency increase in both CAR-negative and -positive cells compared to naked Ad. Competition assays demonstrated that Ad/PPSA produced higher transgene expression levels than naked Ad and achieved CAR-independent transduction. Oncolytic Ad (DWP418)/PPSA was able to overcome the nonspecificity of polymer-only therapies by demonstrating cancer-specific killing effects. Furthermore, the DWP418/PPSA nanocomplex elicited a 2.24-fold greater antitumor efficacy than naked Ad in vivo. This was supported by immunohistochemical confirmation of Ad E1As accumulation in MCF7 xenografted tumors. Lastly, intravenous injection of DWP418/PPSA elicited less innate immune response compared to naked Ad, evaluated by interleukin-6 cytokine release into the serum. The increased antitumor effect and improved vector targeting to both CAR-negative and -positive cells make DWP418/PPSA a promising tool for cancer gene therapy.

Dual tumor targeting with pH-sensitive and bioreducible polymer-complexed oncolytic adenovirus

Oncolytic adenoviruses (Ads) have shown great promise in cancer gene therapy but their efficacy has been compromised by potent immunological, biochemical, and specific tumor-targeting limitations. To take full advantage of the innate cancer-specific killing potency of oncolytic Ads but also exploit the subtleties of the tumor microenvironment, we have generated a pH-sensitive and bio-reducible polymer (PPCBA)-coated oncolytic Ad. Ad-PPCBA complexes showed higher cellular uptake at pH 6.0 than pH 7.4 in both high and low coxsackie and adenovirus receptor-(CAR)-expressing cells, thereby demonstrating Ad-PPCBAs ability to target the low pH hypoxic tumor microenvironment and overcome CAR dependence for target cell uptake. Endocytic mechanism studies indicated that Ad-PPCBA internalization is mediated by macropinocytosis instead of the CAR-dependent endocytic pathway that internalizes naked Ad. VEGF-specific shRNA-expressing oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) elicited much more potent suppression of U87 human brain cancer cell VEGF gene expression in vitro, and human breast cancer MCF7 cell/Matrigel plug vascularization in a mouse model, when cancer cells had been previously infected at pH 6.0 versus pH 7.4. Moreover, intratumorally and intravenously injected RdB/shVEGF-PPCBA nanocomplexes elicited significantly higher therapeutic efficacy than naked virus in U87-tumor mouse xenograft models, reducing IL-6, ALT, and AST serum levels. These data demonstrated PPCBAs biocompatibility and capability to shield the Ad surface to prevent innate immune response against Ad after both intratumoral and systemic administration. Taken together, these results demonstrate that smart, tumor-specific, oncolytic Ad-PPCBA complexes can be exploited to treat both primary and metastatic tumors.

Polymeric oncolytic adenovirus for cancer gene therapy

Oncolytic adenovirus (Ad) vectors present a promising modality to treat cancer. Many clinical trials have been done with either naked oncolytic Ad or combination with chemotherapies. However, the systemic injection of oncolytic Ad in clinical applications is restricted due to significant liver toxicity and immunogenicity. To overcome these issues, Ad has been engineered physically or chemically with numerous polymers for shielding the Ad surface, accomplishing extended blood circulation time and reduced immunogenicity as well as hepatotoxicity. In this review, we describe and classify the characteristics of polymer modified oncolytic Ad following each strategy for cancer treatment. Furthermore, this review concludes with the highlights of various polymer-coated Ads and their prospects, and directions for future research.