Joung-Pyo Nam

Encapsulation of paclitaxel into lauric acid-O-carboxymethyl chitosan-transferrin micelles for hydrophobic drug delivery and site-specific targeted delivery

Transferrin/PEG/O-carboxymethyl chitosan/fatty acid/paclitaxel (TPOCFP) micelles were tested for suitability as a drug carrier characterized by low cytotoxicity, sustained release, high cellular uptake, and site-specific targeted delivery of hydrophobic drugs. Characterization, drug content, encapsulation efficiency, and in vitro drug release were investigated. When the feeding amount of paclitaxel (PTX) was increased, the drug content increased, but loading efficiency decreased. TPOCFP micelles had a spherical shape, with a particle size of approximately 140-649 nm. In vitro cell cytotoxicity and hemolysis assays were conducted to confirm the safety of the micelles. Anticancer activity and confocal laser scanning microscopy (CLSM) were used to confirm the targeting efficiency of target ligand-modified TPOCFP micelles. Anticancer activity and CLSM results clearly demonstrated that transferrin-modified TPOCFP micelles were quickly taken up by the cell. The endocytic pathway of TPOCFP micelles was analyzed by flow cytometry, revealing transfection via receptor-mediated endocytosis. These results suggest that PTX-encapsulated TPOCFP micelles may be used as an effective cancer-targeting drug delivery system for chemotherapy.

Target gene delivery from targeting ligand conjugated chitosan–PEI copolymer for cancer therapy

In this study, we designed a novel carrier which was having low cytotoxicity, site-specific target function, and high transfection efficiency using low molecular weight water soluble O-carboxymethyl chitosan (OCMCh), branched low molecular weight poly(ethyleneimine) (bPEI), and targeting ligand (epitope type, HER-2/neu). OCMCh/bPEI/targeting ligand, HPOCP copolymer, and targeting ligand-modified polyamphoteric polymer, and were prepared by chemical reaction and characterized by (1)H NMR and FT-IR. The binding affinity, protecting efficiency, and releasing ability of gene/HPOCP polyplex were confirmed by gel retardation assay. The pDNA(pEGFP)/HPOCP polyplexes showed high gene transfection efficiency in HCT 119 cell. In addition, siRNA/HPOCP polyplexes formed spherical shape and have particle sizes from 100 to 300nm. The siRNA/HPOCP polyplexes have lower cytotoxicity than PEI in the all of siRNA concentrations ranging from 0 to 2μg/μL in HEK 293 cells. The cell viability of siRNA/HPOCP polyplexes was performed in SK-Br3 cells with VEGF siRNA or BCL2 siRNA. In addition, confocal laser-scanning microscopy and flow cytometry assay were performed for cellular localization and cellular uptake efficiency of siRNA/HPOCP polyplexes. The results of the present study demonstrate that HPOCP copolymer is a good candidate as gene delivery carriers for gene delivery system or gene therapy.

Branched polyethylenimine-grafted-carboxymethyl chitosan copolymer enhances the delivery of pDNA or siRNA in vitro and in vivo

To generate a good carrier for gene transfection, O-carboxymethyl chitosan-graft-branched polyethylenimine (OCMPEI) copolymers were synthesized by increasing the weight percentage of branched polyethylenimine conjugated to the carboxyl groups of O-carboxymethyl chitosan. These spherical polyplexes with plasmid deoxyribonucleic acid (pDNA) or small interfering ribonucleic acid (siRNA) had diameters of ∼200–300 nm or ∼10–25 nm, respectively, and displayed significant transfection efficiency in normal and tumor cells. In particular, expression of green fluorescent protein (GFP) following pDNA transfection was effectively suppressed by delivery of GFP-specific siRNA with the same copolymer. The optimized copolymer and polyplexes were nontoxic in vitro and in vivo. The use of endocytosis inhibitors to investigate the mechanisms of transfection of the polyplexes suggested the involvement of macropinocytosis. An in vivo study in mice showed excellent GFP expression in the lung, kidney, and liver. The results demonstrated that the OCMPEI copolymer prepared in this study is a promising carrier for in vitro and in vivo gene delivery applications.

Targeting delivery of tocopherol and doxorubicin grafted-chitosan polymeric micelles for cancer therapy: In vitro and in vivo evaluation

In this study, we report the development of a novel, redox-sensitive chitosan-based targeted drug delivery system, containing two drugs. We determined whether the synthesized polymeric micelles (HPTOC-DOX) were suitable as a drug carrier. The formation of HPTOC-DOX micelles was confirmed by (1)H NMR. HPTOC-DOX formed micelles of approximately 151.9-311.2nm in size in aqueous solution. Analysis of the drug release profile of HPTOC-DOX in different pH conditions (pH 5.2, 6.2, and 7.4) indicated that DOX was released from HPTOC-DOX micelles at acidic pH (5.2 or 6.2), while almost no DOX was released at pH 7.4. In vitro cell cytotoxicity and hemolysis assays indicated that HPTOC-DOX micelles safely deliver anti-cancer drugs and decrease the cytotoxicity of DOX. In vitro anti-cancer activity assays, confocal laser scanning microscopy analysis of SK-BR-3 cells, and in vivo anti-tumor activity in SK-BR-3-derived tumor-bearing mice were used to evaluate synergistic drug effects and the effect of the targeting peptide (anti-human epidermal growth factor receptor 2 [HER2] target peptide, epitope form; LTVSPWY) on receptor-mediated endocytosis.

Antimicrobial Action of Water-Soluble β-Chitosan against Clinical Multi-Drug Resistant Bacteria

Recently, the number of patients infected by drug-resistant pathogenic microbes has increased remarkably worldwide, and a number of studies have reported new antibiotics from natural sources. Among them, chitosan, with a high molecular weight and α-conformation, exhibits potent antimicrobial activity, but useful applications as an antibiotic are limited by its cytotoxicity and insolubility at physiological pH. In the present study, the antibacterial activity of low molecular weight water-soluble (LMWS) α-chitosan (α1k, α5k, and α10k with molecular masses of 1, 5, and 10 kDa, respectively) and β-chitosan (β1k, β5k, and β10k) was compared using a range of pathogenic bacteria containing drug-resistant bacteria isolated from patients at different pH. Interestingly, β5k and β10k exhibited potent antibacterial activity, even at pH 7.4, whereas only α10k was effective at pH 7.4. The active target of β-chitosan is the bacterial membrane, where the leakage of calcein is induced in artificial PE/PG vesicles, bacterial mimetic membrane. Moreover, scanning electron microscopy showed that they caused significant morphological changes on the bacterial surfaces. An in vivo study utilizing a bacteria-infected mouse model found that LMWS β-chitosan could be used as a candidate in anti-infective or wound healing therapeutic applications.

Preparation of pullulan-g-poly(L-lysine) and it’s evaluation as a gene carrier

In this study, polypeptide/polysaccharide graft copolymers pullulan-g-poly(L-lysine) (PULPLL) was prepared through the ring opening polymerization of ɛ-benzoxycarbonyl L-lysine N-carboxyanhydrides (Z-L-lysine NCA) in the presence of pullulan. The PULPLL copolymer nanoparticles, which are prepared by the diafiltration method, were investigated by using Fourier transform infrared spectroscopy (FTIR), proton nuclear magnetic resonance spectra (1H NMR), dynamic light scattering (DLS), transmission electron microscope (TEM), and fluorescence spectroscopy. The PULPLL copolymers formed self-aggregated nanoparticles in the aqueous milieu. The physicochemical characterization revealed that the nanoparticle size ranged between 60 and 500 nanometers, and the critical aggregation concentration (CAC) is 0.001∼0.1 g/L, depending on the degree of substitution. With negligible cytotoxicity and higher transfection efficiency, PULPLL nanoparticles may be safe gene carriers.

Evaluation of polyethylene glycol-conjugated novel polymeric anti-tumor drug for cancer therapy

A novel polymeric prodrug (PXPEG) was prepared to enhance the solubility of an anti-cancer drug, paclitaxel, in aqueous solutions and decrease the cytotoxicity by PEGylation, which means PEG attached to another molecule. In addition, the targeting ligand, transferrin (TF), was modified to PXPEG to enhance the therapeutic efficacy. The targeting ligand-modified PXPEG (TFPXPEG) was examined by (1)H-NMR to confirm the successful synthesis. The synthesized TFPXPEG had better solubility than the free drug against aqueous solution. The particle size of TFPXPEG was approximately 197.2nm and it had a spherical shape. The MTT assay showed that the anti-tumor efficiency of TFPXPEG was better than that of TF-unmodified PXPEG. In the KB tumor-bearing mouse model, the tumor volume of TFPXPEG treated groups was decreased dramatically by more than 2 fold or 3 fold compared to the PBS or PXPEG treated groups. The in vitro and in vivo evaluation showed that TFPXPEG had better efficacy than that of PXPEG due to the targeting effect of targeting ligands, such as TF.

Anticancer effect of gene/peptide co-delivery system using transferrin-grafted LMWSC.

A series of ternary complex was designed to deliver psiRNA-bcl2 and (KLA)4 peptide into cancer cells for cancer therapy. The delivered psiRNA-bcl2 induced gene-silencing in a nucleus of cancer cells, while (KLA)4 peptide inhibited cancer growth via mitochondrial apoptosis, indicating that the ternary complexes exerted very strong synergistic effects on cancer growth suppression by acting on psiRNA-bcl2 and (KLA)4 peptide simultaneously. The ternary complexes having a targeting-ligand, transferrin (TfP), were found to be especially effective at binding to the TfP receptor rich cancer cells, HCT119. The plasmid DNA (pDNA) in ternary complexes was completely condensed at various content of LMWSC-PEG-TfP (32-64 times more than pDNA) and released into cells. pDNA in the complexes was protected from DNase present on the exterior of cells. The size (165-248 nm) of ternary complexes with LMWSC-PEG-TfP was increased, but surface charges (3-4.5 mV) were decreased. These results likely occurred because the free amine-group of LMWSC decreased in response to conjugated transferrin. Moreover, transfected ternary complexes with LMWSC-PEG-TfP were not expressed in the normal cells (HEK293), but were over expressed in HCT119 cells. These findings indicate that the ternary complexes can be specifically targeted to HCT119 cancer cells. The useful complexes for gene and peptide delivery had high anticancer activities via a synergistic effect due to co-operative action of psiRNA and (KLA)4 peptide in HCT119 cells.

Stent linker effect in a porcine coronary restenosis model.

In this study, we aimed to evaluate the mechanical effects of different stent linker designs on in-stent restenosis in porcine coronary arteries. We fabricated stents with an open-cell structure composed of nine main cells and three linker structures in model 1 (I-type), model 2 (S-types) and model 3 (U-types)) as well as Model 4, which is similar to a commercial bare metal stent design. The stent cells were 70 mm thick and wide, with a common symmetrical wave pattern. As the radial force increased, the number of main cells increased and the length of linker decreased. Radial force was higher in model 1, with a linear I-linker, than in models with S- or U-linkers. The flexibility measured by three-point bending showed a force of 1.09 N in model 1, 0.35 N in model 2, 0.19 N in model 3, and 0.31 N in model 4. The recoil results were similar in all models except model 4 and were related to the shape of the main cells. The foreshortening results were related to linker shape, with the lowest foreshortening observed in model 3 (U-linker). Restenosis areas in the porcine restenosis model 4 weeks after implantation were 35.4 ± 8.39% (model 1), 30.4 ± 7.56% (model 2), 40.6 ± 9.87% (model 3) and 45.1 ± 12.33% (model 4). In-stent restenosis rates measured by intravascular ultrasound (IVUS) and micro-computed tomography (micro-CT) showed similar trends as percent area stenosis measured by micro-CT. Model 2, with optimized flexibility and radial force due to its S-linker, showed significantly reduced restenosis in the animal model compared to stents with different linker designs. These results suggest that the optimal stent structure has a minimum radial force for vascular support and maximum flexibility for vascular conformability. The importance of the effects of these differences in stent design and their potential relationship with restenosis remains to be determined.

Bile acid conjugated chitosan oligosaccharide nanoparticles for paclitaxel carrier

To develop a paclitaxel carrier based on chitosan, chitosan oligosaccharide (COS) was chemically modified with bile acid (deoxycholic acid and lithocholic acid) as a hydrophobic group. Paclitaxel was loaded in bile acid conjugated chitosan oligosaccharide (CBs) nanoparticles by a dialysis method. We confirmed that the paclitaxel-loaded COS (CBs-Tx) nanoparticles could be successfully prepared with a yield of 80%–90% and paclitaxel encapsulation of 54%–70%. The size of CB nanoparticles was in the range of 200–300 nm, and it increased to 300–400 nm after paclitaxel loading with the narrow size distribution maintained. Paclitaxel-loaded CBs (CBs-Tx) nanoparticles showed remarkably high anticancer activity compared with paclitaxel in cremophore EL (CrEL)-ethanol against B16F10 cells. The antitumor efficacy in vivo was shown with significant inhibition of the tumor growth in both paclitaxel-treated groups. The effect on tumor size by the paclitaxel in the CrEL-ethanol formulation appeared to be slightly larger than that in CBs-Tx. The decrease in cytotoxicity and the increase in antitumor activity may lead to the improvement in the therapeutic index in clinical use compared to commercial paclitaxel. The efficacy of CBs-Tx nanoparticles suggests that bile acid as a hydrophobic group may have a potential application of effectively loading hydrophobic drugs such as paclitaxel.