Kyoung-Je Jang

Enhanced chitosan–DNA interaction by 2-acrylamido-2-methylpropane coupling for an efficient transfection in cancer cells

Gene therapy is the treatment of human disorders by the introduction of genetic material to specific target cells of a patient. Chitosan and its derivatives show excellent biological properties including biocompatibility, biodegradability and nonallergenicity. Primary amines of chitosan are responsible for its cationic nature and hence binding and protection of DNA for intracellular delivery. But the transfection efficiency of chitosan based gene transporters is severely hampered by its poor physical properties such as low water solubility and high viscosity. In this study, primary amines of low molecular weight (LMW) chitosan were coupled with 2-acrylamido-2-methylpropane sulphonic acid (AMP) making it water soluble for its application in gene delivery. AMP modified chitosan (CSAMP) showed an enhanced interaction with DNA and a higher buffering capacity due to AMP amines leading to a higher transfection efficiency in cancer cells (A549, HeLa and HepG2) compared to native chitosan and Lipofectamine®. In vivo studies in Balb/c through intravenous injection demonstrated a higher luciferase expression compared to LMW chitosan.

Vibrio vulnificus VvpE inhibits mucin 2 expression by hypermethylation via lipid raft-mediated ROS signaling in intestinal epithelial cells

Mucin is an important physical barrier against enteric pathogens. VvpE is an elastase encoded by Gram-negative bacterium Vibrio vulnificus; however, the functional role of VvpE in intestinal mucin (Muc) production is yet to be elucidated. The recombinant protein (r) VvpE significantly reduced the level of Muc2 in human mucus-secreting HT29-MTX cells. The repression of Muc2 induced by rVvpE was highly susceptible to the knockdown of intelectin-1b (ITLN) and sequestration of cholesterol by methyl-β-cyclodextrin. We found that rVvpE induces the recruitment of NADPH oxidase 2 and neutrophil cytosolic factor 1 into the membrane lipid rafts coupled with ITLN to facilitate the production of reactive oxygen species (ROS). The bacterial signaling of rVvpE through ROS production is uniquely mediated by the phosphorylation of ERK, which was downregulated by the silencing of the PKCδ. Moreover, rVvpE induced region-specific methylation in the Muc2 promoter to promote the transcriptional repression of Muc2. In two mouse models of V. vulnificus infection, the mutation of the vvpE gene from V. vulnificus exhibited an increased survival rate and maintained the level of Muc2 expression in intestine. These results demonstrate that VvpE inhibits Muc2 expression by hypermethylation via lipid raft-mediated ROS signaling in the intestinal epithelial cells.

Development and Characterization of Horse Bone-derived Natural Calcium Phosphate Powders

Purpose: This study was to develop an effective process for fabricating biocompatible calcium phosphate powders (CPPs) using horse bones, and to investigate the characteristics of them. Methods: The characteristics of horse bone powders (HBPs) were investigated according to the different osseous tissue types (compact bone and cancellous bone), bone types (spine and tibia), pretreatment methods (cold water, H2O2 , and hot water), sintering time (4, 8 and 12h), and sintering temperature (600, 900, 1100 and1300°C). In addition, the grinding methods were compared based on the wet grinding (ball mill) and dry grinding (blade grinder) method to make it as powders. Finally, their cytotoxicity and cell viability were checked. Results: Regardless of the types of osseous tissues and bones, HBPs were well fabricated as biocompatible CPPs. It was also found that the pretreatment methods did not influence on the resultants, showing well-fabricated HBPs. Considering the processing time, the hot water method was the most suitable compared to other pretreatment methods. Further, 12h-sintering time was sufficient to remove residual organic compounds. The sintering temperatures greatly affected the properties of bone powders fabricated. The x-ray diffraction (XRD) peak of horse bone sintered at 600°C was most closed to that of hydroxyapatite (HA). Our bioactivity study demonstrated that the HBPs fabricated by sintering horse bones at 1300°C showed the best performance in terms of cell viability whereas the HBPs 1100°C showed the cytotoxicity. Conclusions: Using various types of horse bone tissues, biocompatible CPPs were successfully developed. We conclude that the HBPs may have a great potential as biomaterials for various biological applications including bone tissue engineering.

Effects of pulsing of light on the dentinogenesis of dental pulp stem cells in vitro

Low power light (LPL) treatment has been widely used in various clinical trials, which has been known to reduce pain and inflammation and to promote wound healing. LPL was also shown to enhance differentiation of stem cells into specific lineages. However, most studies have used high power light in mW order, and there was lack of studies about the effects of very low power light in μW. In this study, we applied 810 nm LPL of 128 μW/cm2 energy density in vitro. Upon this value, continuous wave (CW) irradiation did not induce any significant changes for differentiation of human dental pulp stem cells (hDPSCs). However, the membrane hyperpolarization, alkaline phosphatase activity, and intracellular oxidative stress were largely enhanced in the pulsed wave (PW) with 30% of duty cycle and 300–3000 Hz frequencies-LPL in which LED driver work in the form of square wave. After 21 days of daily LPL treatment, Western blot revealed the dentinogenesis in this condition in vitro. This study demonstrates that the very low power light at 810 nm enhanced significant differentiation of hDPSCs in the PW mode and there were duty cycle dependency as well as pulsing frequency dependency in the efficiency.

JNK2 silencing and caspase-9 activation by hyperosmotic polymer inhibits tumor progression

c-Jun N-terminal kinase 2 (JNK2) is primarily responsible for the oncogenic transformation of the transcription factor c-Jun. Expression of the proto-oncogene c-Jun progresses the cell cycle from G1 to S phase, but when its expression becomes awry it leads to uncontrolled proliferation and angiogenesis. Delivering a JNK2 siRNA (siJNK2) in tumor tissue was anticipated to reverse the condition with subsequent onset of apoptosis which predominantly requires an efficient delivering system capable of penetrating through the compact tumor mass. In the present study, it was demonstrated that polymannitol-based vector (PMGT) with inherent hyperosmotic properties was able to penetrate through and deliver the siJNK2 in the subcutaneous tumor of xenograft mice. Hyperosmotic activity of polymannitol was shown to account for the enhanced therapeutic delivery both in vitro and in vivo because of the induction of cyclooxygenase-2 (COX-2) which stimulates caveolin-1 for caveolae-mediated endocytosis of the polyplexes. Further suppression of JNK2 and hence c-Jun expression led to the activation of caspase-9 to induce apoptosis and inhibition of tumor growth in xenograft mice model. The study exemplifies PMGT as an efficient vector for delivering therapeutic molecules in compact tumor tissue and suppression of JNK2 introduces a strategy to inhibit tumor progression.

Directional Matrix Nanotopography with Varied Sizes for Engineering Wound Healing

Topographic features play a crucial role in the regulation of physiologically relevant cell and tissue functions. Here, an analysis of feature-size-dependent cell-nanoarchitecture interactions is reported using an array of scaffolds in the form of uniformly spaced ridge/groove structures for engineering wound healing. The ridge and groove widths of nanopatterns are varied from 300 to 800 nm and the nanotopography features are classified into three size ranges: dense (300-400 nm), intermediate (500-600 nm), and sparse (700-800 nm). On these matrices, fibroblasts demonstrate a biphasic trend of cell body and nucleus elongation showing the maximum at intermediate feature density, whereas maximum migration speed is observed at the dense case with monotonic decrease upon increasing feature size. The directional organization of cell-synthesized fibronectin fibers can be regulated differently via the nanotopographical features. In an in vitro wound healing model, the covering rate of cell-free regions is maximized on the dense nanotopography and decreased with increasing feature size, showing direct correlation with the trend of migration speed. It is demonstrated that the properties of repaired tissue matrices in the process of wound healing may be controlled via the feature-size-dependent cell-nanoarchitecture interactions, which can be an important consideration for designing tissue engineering scaffolds.

Synergistic effects of hyperosmotic polymannitol based non-viral vectors and nanotopographical cues for enhanced gene delivery

Here, we report the synergistic effects of hyperosmotic and nanotopographical cues designed using non-viral vectors and nanopatterned matrices for gene delivery. We show that efficiency of gene delivery can be further enhanced by two factors in combination, indicating the importance of synergistic cues in designing non-viral gene delivery platforms and strategies for gene therapy.