Won Ho Kong

Nanographene oxide-hyaluronic acid conjugate for photothermal ablation therapy of skin cancer.

Melanoma skin cancer is one of the most dangerous skin cancers and the main cause of skin-cancer-related mortality. Hyaluronic acid (HA) has been used as an effective transdermal delivery carrier of chemical drugs and biopharmaceuticals. In this work, a nanographene oxide-HA conjugate (NGO-HA) was synthesized for photothermal ablation therapy of melanoma skin cancer using a near-infrared (NIR) laser. Confocal microscopy and ex vivo bioimaging clearly visualized the remarkable transdermal delivery of NGO-HA to tumor tissues in the skin of mice, which might be ascribed to highly expressed HA receptors and relatively leaky structures around tumor tissues, enabling the enhanced permeation and retention of nanoparticles. The NIR irradiation resulted in complete ablation of tumor tissues with no recurrence of tumorigenesis. The antitumor effect was confirmed by ELISA for caspase-3 activity and histological and immunohistochemical analyses with TUNEL assay for tumor apoptosis. Taken together, we could confirm the feasibility of transdermal NGO-HA for photothermal ablation therapy of melanoma skin cancers.

Bioimaging of hyaluronic acid derivatives using nanosized carbon dots.

Fluorescent nanosized carbon dots (Cdots) are an emerging bioimaging agent with excellent chemical inertness and marginal cytotoxicity in comparison to widely used semiconductor quantum dots. In this work, we report the application of Cdots for real time bioimaging of target specific delivery of hyaluronic acid (HA) derivatives. Polyethylene glycol (PEG) diamine-capped Cdots were synthesized by the pyrolysis of citric acid in a hot solvent. The synthesized Cdots showed strong fluorescence under UV excitation with emission properties dependending on the excitation wavelength. HA-Cdot conjugates were synthesized by amide bond formation between amine groups of Cdot and carboxylic groups of HA. After confirmation of the negligible cytotoxicity of Cdots and HA-Cdot conjugates, in vitro bioimaging was carried out for target specific intracellular delivery of the HA-Cdot conjugates by HA receptor-mediated endocytosis. Furthermore, in vivo real-time bioimaging of Cdots and HA-Cdot conjugates exhibited the target specific delivery of HA-Cdot conjugates to the liver with abundant HA receptors. Taken together, we could confirm the feasibility of HA derivatives as a target-specific drug delivery carrier for the treatment of liver diseases and Cdots as a promising bioimaging agent.

Photodynamic therapy of melanoma skin cancer using carbon dot - chlorin e6 - hyaluronate conjugate.

UNLABELLED Despite wide application of photodynamic therapy (PDT) for the treatment of melanoma skin cancers, there are strong biomedical unmet needs for the effective generation of singlet oxygen after targeted delivery of photosensitizers. Here, we investigated a facile PDT of melanoma skin cancer using transdermal carbon dot - chlorine e6 - hyaluronate (Cdot-Ce6-HA) conjugates. The Cdot-Ce6-HA conjugate was synthesized by the coupling reaction of diaminohexane modified HA (DAH-HA) with the carboxylic group of Ce6. The singlet oxygen generation of Cdot-Ce6-HA conjugates in aqueous solution was more significant than that of free Ce6. The enhanced transdermal and intracellular delivery of Cdot-Ce6-HA conjugates to B16F10 melanoma cells in tumor model mice were corroborated by confocal microscopy and two-photon microscopy. The laser irradiation after topical treatment with Cdot-Ce6-HA conjugates resulted in complete suppression of melanoma skin cancers. The antitumor effect was confirmed by histological analysis with H&E staining and TUNEL assay for tumor apoptosis. Taken together, we could confirm the feasibility of Cdot-Ce6-HA conjugate for transdermal PDT of melanoma skin cancers. STATEMENT OF SIGNIFICANCE To our knowledge, this is the first report on a facile transdermal photodynamic therapy (PDT) of melanoma skin cancer using carbon dot - chlorine e6 - hyaluronate (Cdot-Ce6-HA) conjugates. We found that the singlet oxygen generation of Cdot-Ce6-HA conjugates in aqueous solution was more significant than that of free Ce6. Confocal microscopy and two-photon microscopy clearly confirmed the enhanced transdermal and intracellular delivery of Cdot-Ce6-HA conjugates to B16F10 melanoma cells in tumor model mice. Taken together, we could confirm the feasibility of Cdot-Ce6-HA conjugate for transdermal PDT of melanoma skin cancers.

Cationic solid lipid nanoparticles derived from apolipoprotein-free LDLs for target specific systemic treatment of liver fibrosis

Low density lipoprotein (LDL) plays an important role in transporting fat molecules including cholesterols in the body. In this work, cationic solid lipid nanoparticles (CSLNs), bioinspired and reconstituted from natural LDLs, were designed and applied to target specific systemic delivery of connective tissue growth factor siRNA (siCTGF) for the treatment of liver fibrosis. They could form a nuclease-resistant stable nano-complex with siRNA, which was efficiently internalized into cells achieving targeted gene silencing in the presence of serum with a remarkably low cytotoxicity. After intravenous injection, CSLN/siCTGF complex was target specifically delivered to the liver and resulted in a significant reduction in collagen content and pro-fibrogenic factors like tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukin-6 (IL-6), and CTGF with the dramatic improvement of patho-physiological symptoms in liver fibrosis model rats. The bio-distribution study by fluorescence bioimaging and single-photon emission computed tomography (SPECT) confirmed the target specific delivery and accumulation of CSLN/siCTGF complexes to the liver tissues.

Hyaluronic acid-tumor necrosis factor-related apoptosis-inducing ligand conjugate for targeted treatment of liver fibrosis.

Liver fibrosis is a chronic liver disease caused by viral infection and/or metabolic, genetic and cholestatic disorders. The inhibition of hepatic stellate cell (HSC) activation and the selective apoptosis of activated HSCs can be a good strategy to treat liver fibrosis. The activated HSCs are known to be more susceptible to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induced apoptosis than normal HSCs because death receptor 5 is overexpressed on the cell surface. In this work, a target-specific and long-acting hyaluronic acid (HA)-TRAIL conjugate was successfully developed for the treatment of liver fibrosis. The HA-TRAIL conjugate was synthesized by a coupling reaction between aldehyde-modified HA and the N-terminal amine group of TRAIL. The biological activity of the HA-TRAIL conjugate was confirmed by an in vitro anti-proliferation assay and caspase-3 expression in human colon cancer HCT116 cells. In vivo real-time bioimaging exhibited the target-specific delivery of near-infrared fluorescence dye-labeled HA-TRAIL conjugate to the liver in mice. According to pharmacokinetic analysis, the HA-TRAIL conjugate was detected for more than 4days after single intravenous injection into Sprague-Dawley (SD) rats. Finally, we could confirm the antifibrotic effect of HA-TRAIL conjugate in an N-nitrosodimethylamine-induced liver fibrosis model SD rats.

Noncovalenly PEGylated CTGF siRNA/PDMAEMA complex for pulmonary treatment of bleomycin-induced lung fibrosis

On the basis of wide biomedical applications of methacrylate polymers, we previously developed noncovalently post-PEGylated ternary complex of siRNA using poly(dimethylamino)ethylmethacrylate (PDMAEMA) and its copolymer with poly(α-methylether-ω-methacrylate-ethyleneglycol) [PMAPEG]. In this work, we investigated the antifibrotic effect of connective tissue growth factor siRNA (siCTGF)/PDMAEMA/PDMAEMA-b-PMAPEG complex for the treatment of bleomycin-induced pulmonary fibrosis. After orotracheal administration to fibrotic Sprague Dawley (SD) model rats, FAM-labeled siCTGF complex was effectively delivered to the cells in the lung. The siCTGF ternary complex resulted in a significant reduction in target gene expression, collagen deposition, inflammatory cytokines production, and drastic attenuation of pulmonary fibrosis in pathophysiological analysis. Furthermore, the survival rate was remarkably increased to the statistically significant level in comparison with the scrambled siCTGF treatment group.

Nano graphene oxide–hyaluronic acid conjugate for target specific cancer drug delivery

A nano graphene oxide–hyaluronic acid (NGO–HA) conjugate was successfully prepared for target specific delivery of an anti-cancer drug loaded by π–π stacking via HA receptor mediated endocytosis. In vitro tests confirmed the pH dependent drug release and target specific anti-cancer effect of the complex.

Bioimaging and pulmonary applications of self-assembled Flt1 peptide-hyaluronic acid conjugate nanoparticles.

Despite wide exploitation of corticosteroid drugs for the treatment of asthma, the poor therapeutic effect on a neutrophilic subtype of asthma prohibits the full recovery of asthma patients. In this work, dexamethasone (Dexa) was loaded in Flt1 peptide-hyaluronic acid (HA) conjugate nanoparticles to overcome the limitation of corticosteroid resistance for the treatment of neutrophilic pulmonary inflammation. Flt1 peptide-HA conjugates are self-assembled to nanoparticles because of hydrophobic Flt1 peptide conjugated to HA by benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexafluorophosphate (BOP) chemistry. In vitro bioimaging showed efficient internalization of Flt1 peptide-HA conjugate nanoparticles into lung epithelial cells by HA-receptor mediated endocytosis. Also, ex vivo imaging for the biodistribution in ICR mice revealed long-term retention of Flt1 peptide-HA conjugate nanoparticles in deep lung tissues possibly due to mucoadhesive property of HA. On the basis of bioimaging results for pulmonary drug delivery applications, we prepared Dexa-loaded Flt1 peptide-HA conjugate nanoparticles. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) confirmed the formation of nanoparticles, which reduced cytokine levels of lipopolysaccharide (LPS)-stimulated cells more efficiently than free Dexa. Furthermore, according to the bronchoalveolar lavage (BAL) cellularity and histological analysis, Dexa loaded Flt1 peptide-HA conjugate nanoparticles showed remarkable therapeutic effects in both eosinophilic and neutrophilic asthma model mice.

Hyaluronic acid–siRNA conjugates complexed with cationic solid lipid nanoparticles for target specific gene silencing

Despite extensive investigations on siRNA delivery systems for the past decade, there has been no clinically available product until now. In this work, reducible hyaluronic acid (HA)–siRNA conjugate was successfully synthesized and used to make a complex with cationic solid lipid nanoparticles (CSLNs) for the development of a liver specific siRNA delivery system. The reducible HA–siRNA conjugate was synthesized by the disulfide–thiol exchange reaction between pyridyldithiol modified HA and thiolated siRNA. The remaining pyridyldithiol was further blocked with cysteine. The biomimetic CSLNs were prepared by reconstituting the composition of natural apolipoprotein-free low density lipoproteins (LDLs). The formation of the HA–siRNA/CSLN complex was confirmed by gel electrophoresis (GE), dynamic light scattering (DLS), and atomic force microscopy (AFM). The HA–siRNA/CSLN complex showed remarkably low cytotoxicity and high transfection efficiency in the presence of serum. The therapeutic index (LC50/IC50) of the HA–siRNA/CSLN complex was statistically much higher than that of a HA–siRNA conjugate or siRNA complexed with commercially available siRNA transfection reagents like in vivo jetPEI and INTERFERin, as well as an siRNA/CSLN complex. The HA–siRNA/CSLN complex can be effectively applied as a model system for the treatment of liver diseases, such as liver fibrosis and liver cancer.