-Wook Chung

Dual Stimuli-Responsive poly(N-isopropylacrylamide)-b- poly(L-histidine) Chimeric Materials for the Controlled Delivery of Doxorubicin into Liver Carcinoma

A series of dual stimuli responsive synthetic polymer bioconjugate chimeric materials, poly(N-isopropylacrylamide)55-block-poly(L-histidine)n [p(NIPAM)55-b-p(His)n] (n=50, 75, 100, 125), have been synthesized by employing reversible addition-fragmentation chain transfer polymerization of NIPAM, followed by ring-opening polymerization of α-amino acid N-carboxyanhydrides. The dual stimuli responsive properties of the resulting biocompatiable and membrenolytic p(NIPAM)55-b-p(His)n polymers are investigated for their use as a stimuli responsive drug carrier for tumor targeting. Highly uniform self-assembled micelles (∼55 nm) fabricated by p(NIPAM)55-b-p(His)n polymers display sharp thermal and pH responses in aqueous media. An anticancer drug, doxorubicin (Dox), is effectively encapsulated in the micelles and the controlled Dox release is investigated in different temperature and pH conditions. Antitumor effect of the released Dox is also assessed using the HepG2 human hepatocellular carcinoma cell lines. Dox molecules released from the [p(NIPAM)55-b-p(His)n] micelles remain biologically active and have stimuli responsive capability to kill cancer cells. The self-assembling ability of these hybrid materials into uniform micelles and their efficiency to encapsulate Dox makes them a promising drug carrier to cancer cells. The new chimeric materials thus display tunable properties that can make them useful for a molecular switching device and controlled drug delivery applications needing responses to temperature and pH for the improvement of cancer chemotherapy.

Self-assembled nanoparticles of hyaluronic acid/poly(dl-lactide-co-glycolide) block copolymer

We synthesized block copolymer composed of hyaluronic acid (HA) and poly(DL-lactide-co-glycolide) (PLGA) (HAbLG) for antitumor targeting. (1)H NMR was employed to confirm synthesis of block copolymer. At (1)H NMR study, HabLG nanoparticles showed HA intrinsic peaks only at D(2)O, indicating that they contained HA as a hydrophilic outer-shell and PLGA as a inner-core. Anti-tumor activity was studied using CD44-overexpressing HCT-116 human colon carcinoma cells. Addition of doxorubicin (DOX)-incorporated nanoparticles to tumor cells resulted in the expression of a strong red fluorescence color while they expressed very weak fluorescence when CD44 receptor was blocked with free HA. Flow cytometry data also showed similar results, indicating that the fluorescence intensity of tumor cells treated with nanoparticles was significantly decreased when CD44 receptor was blocked. These results indicate that HAbLG nanoparticles were able to target CD44-overexpressing tumor cells via receptor-mediated endocytosis.

Antitumor activity of sorafenib-incorporated nanoparticles of dextran/poly(dl-lactide-co-glycolide) block copolymer

Sorafenib-incoporated nanoparticles were prepared using a block copolymer that is composed of dextran and poly(DL-lactide-co-glycolide) [Dexb LG] for antitumor drug delivery. Sorafenib-incorporated nanoparticles were prepared by a nanoprecipitation-dialysis method. Sorafenib-incorporated Dexb LG nanoparticles were uniformly distributed in an aqueous solution regardless of the content of sorafenib. Transmission electron microscopy of the sorafenib-incorporated Dexb LG nanoparticles revealed a spherical shape with a diameter < 300 nm. Sorafenib-incorporated Dexb LG nanoparticles at a polymer/drug weight ratio of 40:5 showed a relatively uniform size and morphology. Higher initial drug feeding was associated with increased drug content in nanoparticles and in nanoparticle size. A drug release study revealed a decreased drug release rate with increasing drug content. In an in vitro anti-proliferation assay using human cholangiocarcinoma cells, sorafenib-incorporated Dexb LG nanoparticles showed a similar antitumor activity as sorafenib. Sorafenib-incorporated Dexb LG nanoparticles are promising candidates as vehicles for antitumor drug targeting.

Effect of 5-aminolevulinic acid-based photodynamic therapy via reactive oxygen species in human cholangiocarcinoma cells

Cancer cells have been reported to exhibit an enhanced capacity for protoporphyrin IX (PpIX) synthesis facilitated by the administration of 5-aminolevulinic acid (ALA). We investigated the effect of ALA-based photodynamic therapy (PDT) on human cholangiocarcinoma cells (HuCC-T1). Since protoporphyrin IX (PpIX), a metabolite of ALA, can produce reactive oxygen species (ROS) under irradiation and then induce phototoxicity, ALA-based PDT is a promising candidate for the treatment of cholangiocarcinoma. When various concentrations of ALA (0.05–2 mM) were used to treat HuCC-T1 cells for 6 or 24 hours, the intracellular PpIX level increased according to the ALA concentration and treatment time. Furthermore, an increased amount of PpIX in HuCC-T1 cells induced increased production of ROS by irradiation, resulting in increased phototoxicity.

5-aminolevulinic acid-incorporated nanoparticles of methoxy poly(ethylene glycol)-chitosan copolymer for photodynamic therapy

Purpose The aim of this study was to make 5-aminolevulinic acid (5-ALA)-incorporated nanoparticles using methoxy polyethylene glycol/chitosan (PEG-Chito) copolymer for application in photodynamic therapy for colon cancer cells. Methods 5-ALA-incorporated (PEG-Chito-5-ALA) nanoparticles were prepared by ion complex formation between 5-ALA and chitosan. Protoporphyrin IX accumulation in the tumor cells and phototoxicity induced by PEG-Chito-5-ALA nanoparticles were assessed using CT26 cells in vitro. Results PEG-Chito-5-ALA nanoparticles have spherical shapes with sizes diameters 200 nm. More specifically, microscopic observation revealed a core-shell structure of PEG-Chito-5-ALA nanoparticles. 1H NMR spectra showed that 5-ALA was incorporated in the core of the nanoparticles. In the absence of light irradiation, all components such as 5-ALA, empty nanoparticles, and PEG-Chito-5-ALA nanoparticles did not affect the viability of cells. However, 5-ALA or PEG-Chito-5-ALA nanoparticles induced tumor cell death under light irradiation, and the viability of tumor cells was dose-dependently decreased according to the increase in irradiation time. In particular, PEG-Chito-5-ALA nanoparticles induced increased phototoxicity and higher protoporphyrin IX accumulation into the tumor cells than did 5-ALA alone. Furthermore, PEG-Chito-5-ALA nanoparticles accelerated apoptosis/necrosis of tumor cells, compared to 5-ALA alone. Conclusion PEG-Chito-5-ALA nanoparticles showed superior delivery capacity of 5-ALA and phototoxicity against tumor cells. These results show that PEG-Chito-5-ALA nanoparticles are promising candidates for photodynamic therapy of colon cancer cells.

5-aminolevulinic acid-incorporated poly(vinyl alcohol) nanofiber-coated metal stent for application in photodynamic therapy

Background The study investigated the use of combined photodynamic therapy (PDT) and stent placement for the treatment of cholangiocarcinoma (CC). For this purpose, 5-aminolevulinic acid (ALA) was incorporated into poly(vinyl alcohol) (PVA) nanofiber, and coated onto metal stents. Their efficacy was assessed in PDT towards HuCC-T1 CC cells. Methods Fabrication of ALA-PVA nanofiber, and simultaneous coating onto metal stents, was performed through electrospinning. The dark-toxicity, generation of protoporphyrin IX (PpIX), and PDT effect of ALA and ALA-PVA nanofiber were studied in vitro, using HuCC-T1 CC cells. Results The ALA-PVA nanofibers were coated onto metal stents less than 1000 nm in diameter. ALA-only displayed marginal cytotoxicity; ALA-PVA nanofiber showed less cytotoxicity. PpIX generation was not sigficantly different between ALA and ALA-PVA nanofiber treatments. PVA itself did not generate PpIX in tumor cells. ALA and ALA-PVA nanofiber displayed a similar PDT effect on tumor cells. Cell viability was decreased, dose-dependently, until ALA concentration reached 100 μg/mL. Necrosis and apoptosis of tumor cells occurred similarly for ALA and ALA- PVA nanofiber treatments. Conclusion The ALA-PVA nanofiber-coated stent is a promising candidate for therapeutic use with cholangiocarcinoma.

Anti-tumor activity of all-trans retinoic acid-incorporated glycol chitosan nanoparticles against HuCC-T1 human cholangiocarcinoma cells.

The aim of this study is to investigate antitumor activity of all-trans retinoic acid (RA)-incorporated glycol chitosan (GC) nanoparticles. RA-incorporated GC nanoparticles were prepared by electrostatic interaction between RA and amine group of GC. RA-incorporated GC nanoparticles have spherical shape and their particle size was 317 ± 34.5 nm. They were simply reconstituted into aqueous solution without changes of intrinsic properties. RA-incorporated GC nanoparticles were evidently inhibited the proliferation of HuCC-T1 cholangiocarcinoma cells at higher than 20 μg/ml of RA concentration while empty GC vegicles did not affect to the viablity of tumor cells. Apoptosis and necrosis analysis of tumor cells with treatment of RA or RA-incorporated GC nanoparticles also supported these results. Invasion test using Matrigel also showed that invasion of tumor cells was significantly inhibited at higher than 20 μg/ml of RA concentration. Wound healing assay also showed that RA-incorporated GC nanoparticles were inhibited migration of tumor cells as similar to RA itself. Our results suggested that RA-incorporated GC nanoparticles is a promising vehicles for RA delivery to HuCC-T1 cholangiocarcinoma cells.

Dual Stimuli-Responsive Vesicular Nanospheres Fabricated by Lipopolymer Hybrids for Tumor-Targeted Photodynamic Therapy

Smart delivery system of photosensitizer chlorin e6 (Ce6) has been developed for targeted photodynamic therapy (PDT). Simple self-assemblies of the mixtures comprising soybean lecithin derived phosphatidylcholine (PC), phosphatidylethanolamine-poly(L-histidine)40 (PE-p(His)40), and folic acid (FA) conjugated phosphatidylethanolamine-poly(N-isopropylacrylamide)40 (PE-p(NIPAM)40-FA) in different ratios yield smart nanospheres characterized by (i) stable and uniform particle size (∼100 nm), (ii) positive surface charge, (iii) high hydrophobic drug (Ce6) loading efficiency up to 45%, (iv) covalently linked targeting moiety, (v) low cytotoxicity, and (vi) smartness showing p(His) block oriented pH and p(NIPAM) oriented temperature responsiveness. The Ce6-encapsulated vesicular nanospheres (Ce6@VNS) were used to confirm the efficiency of cellular uptake, intracellular distribution, and phototoxicity against KB tumor cells compared to free Ce6 at different temperature and pH conditions. The Ce6@VNS system showed significant photodynamic therapeutic efficiency on KB cells than free Ce6. A receptor-mediated inhibition study proved the site-specific delivery of Ce6 in targeted tumor cells.

Poly(L-histidine)-tagged 5-aminolevulinic acid prodrugs: new photosensitizing precursors of protoporphyrin IX for photodynamic colon cancer therapy

Background 5-Aminolevulinic acid (ALA) and its derivatives have been widely used in photodynamic therapy. The main drawback associated with ALA-based photodynamic therapy (ALA-PDT) and ALA fluorescence diagnosis results from the hydrophilic nature of ALA and lack of selectivity for tumor versus nontumor cells. The application of certain triggers, such as pH, into conventional sensitizers for controllable 1O2 release is a promising strategy for tumor-targeted treatment. Methods A series of pH-sensitive ALA-poly(L-histidine) [p(L-His)n] prodrugs were synthesized via ring opening polymerization of 1-benzyl-N-carboxy-L-histidine anhydride initiated by the amine hydrochloride group of ALA itself. As an alternative to ALA for PDT, the synthesized prodrugs were used to treat a cultured human colon cancer HCT116 cell line under different pH conditions. The effect of ALA-p(L-His)n derivatives was evaluated by monitoring the fluorescence intensity of protoporphyrin IX, and measuring the cell survival rate after suitable light irradiation. Results The cytotoxicity and dark toxicity of ALA and synthesized ALA-p(L-His) derivatives in HEK293T and HCT116 cells in the absence of light at pH 7.4 and 6.8 shows that the cell viability was relatively higher than 100%. ALA-p(L-His)n showed high phototoxicity and selectivity in different pH conditions compared with ALA alone. Because the length of the histidine chain increases in the ALA-p(L-His)n prodrugs, the PDT effect was found to be more powerful. In particular, high phototoxicity was observed when the cells were treated with ALA-p(L-His)15, compared with treatment using ALA alone. Conclusion The newly synthesized ALA-p(L-His)n derivatives are an effective alternative to ALA for enhancing protoporphyrin IX production and the selectivity of the phototoxic effect in tumor cells.

Lipo-Poly(L-histidine) Hybrid Materials with pH-Sensitivity, Intracellular Delivery Efficiency, and Intrinsic Targetability to Cancer Cells

Biocompatible lipo-histidine hybrid materials conjugated with IR820 dye show pH-sensitivity, efficient intracellular delivery of doxorubicin (Dox), and intrinsic targetability to cancer cells. These new materials form highly uniform Dox-loaded nanosized vesicles via a self-assembly process showing good stability under physiological conditions. The Dox-loaded micelles are effective for suppressing MCF-7 tumors, as demonstrated in vitro and in vivo. The combined mechanisms of the EPR effect, active internalization, endosomal-triggered release, and drug escape from endosomes, and a long blood circulation time, clearly prove that the IR820 lipopeptide DDS is a safe theranostic agent for imaging-guided cancer therapy.