Chimera Protein Engineering
Stem cells, particularly, iPSCs have become the popular choice for regenerative medicine in recent years. However, several critical issues, for example defined culture condition, bulk culture system, homogeneous culture etc., are limiting the progress of the field. Suitable technology has been sought for years to overcome relevant issues. Our lab has been pioneering on solving some of these factors and advancing the field. Some of the remarkable contributions are as follows:
E-cadherin-Fc chimeric protein that provides defined matrix for stem cell culture.
E-cadherin-Fc matrix provides one-stop non-stressed cell purification method.
LIF-Fc provides long-lasting stemness for stem cell culture without regular replenishment of LIF.
Sugar Containing Polymer Engineering
Biomimetic glycopolymers recognized by cell surface receptors, transporter and other functional protein were synthesized and cell-polymers interactions were evaluated. Galactose- or glucose-carrying polystyrene derivatives were shown to be highly recognized by hepatocytes and/or erythrocytes via specific receptors or transporters and were applied to cell and tissue engineering.
N-p-Vinylbenzyl-O-β-D-galactopyranosyl-(1,4)-D-gluconamide (PVLA) has been used as an asialoglycoprotein model polymer. PVLA, water-soluble polystyrene derivatives bearing galactose residues preferentially adsorb to plastic plates made of polystyrene. Hepatocytes expressing asialoglycoprotein receptors are capable of binding to PVLA coated hydrophobic plastic dishes.
The hepatocyte recognizes the structure of oligosaccharides via asialoglycoprotein receptors and synthesized lactose-carrying styrene polymer (PVLA) as a asialoglycoprotein model. On PVLA-coated dish, the specific functions as well as attachment of hepatocyte were successfully maintained. Moreover, cultured hepatocytes on PVLA substratum started gradual movement, and then remarkably formed multilayer spheroids which had long-term survival of 4weeks. Hepatocytes in the aggregation exhibited better maintenance of specific hepatocyte-functions such as the synthesis of albumin and secretion of bile acid, and retained mitochondrial enzyme activity than those in the monolayer culture on collagen and fibronectin. Furthermore, DNA synthesis in cultured hepatocytes was correlated to the cell-shape which could be controlled by the concentration of PVLA substratum. These results also suggested that the PVLA could potentiate the regulation of the differentiation and proliferation of hepatocytes.
Directed toward pharmacological applications of PVLA, its body distribution, clearance from blood and specific binding to receptors were investigated using radiolabeled PVLA. 125I-labeled PVLA was prepared by the co-polymerization. When 125I-labeled PVLA was injected into rats through their tail veins, the radioactivity was distributed highly to liver, less to thyroid gland, cecum-large intestine, urine, feces and blood, and much less to lung, heart, kidney, spleen, pancreas, small intestine and urinary bladder. It was clarified that about 97% of PVLA was distributed to parenchymal liver cells and only 3% to nonparenchymal liver cells. Specific binding between 125I-labeled PVLA and asialoglycoprotein receptors on parenchymal liver cells was demonstrated by its inhibition with asialofetuin. The bond dissociation constant estimated by Scatchard analysis was Kd = 1.4 x 10-9M. The binding was as strong as those of several naturally occurring asialoglycoproteins. These properties of PVLA, as liver-specific targeting materials using galactose ligands as recognition signals to asialoglycoprotein receptors, are discussed with the conformational structures of PVLA which can carry drugs in their hydrophobic regions.
Drug and Gene Delivery by Carbonate-Apatite
Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to treat critical diseases such as cancer and liver cirrhosis. Elucidation of the genetic basis of human diseases with complete sequencing of human genome revealed many vital genes as possible targets in gene therapy programs. DNA and RNA interference (RNAi) can be harnessed to rapidly develop novel drugs against any disease target organs. Here pH-sensitive carbonate apatite has been developed to efficiently deliver for these genes into the mammalian cells by virtue of its high affinity interactions with the genes and the desirable size of the resulting gene/apatite complex for effective cellular endocytosis. Therefore, the carbonate apatite being highly stable in typical physiological pH could easily be dissolved in endosomal acidic pH following endocytosis, thus quickly releasing the associated drugs in cytoplasm for effective therapeutic action. We showed for the first time that the inorganic crystal had characteristics of nano-scale effective for passive targetting, enhanced cellular uptake and quick release of genes in response to endosomal low pH, resulting in enhanced colorectal tumor inhibitory effects both in vitro and in vivo. The extraordinary proliferation inhibitory effect in a number of cancer cell lines was achieved by virtue of its pH sensitivity resulting in quick release of the drugs from the carrier and probably overcoming the MDR and getting advantages over different slow release carrier systems. Thus, we propose a novel nano-based pH targeting therapeutic strategy against malignancies, which is highly promising for preclinical and clinical cancer therapy and liver cirrhosis and hepatitis.