허혈성 뇌졸중 모델에 덱사메타손이 접합된 폴리아미도아민 덴드리머를 이용한 HO-1 유전자 전달

Abstract

Polyamidoamine (PAMAM) dendrimers which have repeating unit from core have been studied as a gene carrier due to the property that it forms DNA/polymer complex through electrostatic interaction with a number of primary amines in surface of PAMAM dendrimers and phosphate groups of nucleic acid. However, although low generation PAMAM dendrimers have low cytotoxicity, they have relatively lower transfection efficiency than high molecular weight PAMAM dendrimers. Thus, to overcome drawback, we developed an effective PAMAM based carrier by conjugating moiety which is used to improve delivery efficiency. Dexamethasone, a glucocorticoid steroid, translocates into the nucleus when it is bound to its glucocorticoid receptor and has anti-inflammatory effect. In addition, the previous paper has reported that the glucocorticoid receptor dilated the nuclear pore complexes, which is beneficial for DNA delivery into the nucleus. These properties facilitate transport of plasmid DNA more effectively into cells. In this research, dexamethasone conjugated to PAMAM generation 2 (PAMAM G2-dexa) may play a role as a gene carrier which has not only high transfection efficiency but also anti-inflammatory effect. In addition, we expected that PAMAM G2-dexa would have property of PAMAM G2 used as carrier based low toxicity polymer and concurrently forms more stable complex due to amphipathic characteristic by conjugation of hydrophilic PAMAM and hydrophobic dexamethasone. The DNA/polymer complex formation was evaluated by various physical characterizations such as Gel retardation, Heparin competition assay. Also, anti-inflammatory effect of the dexamethasone conjugated PAMAM (PAMAM G2-dexa) was confirmed by pro-inflammatory cytokine ELISA such as IL-6 and TNF-alpha. To confirm therapeutic effect using PAMAM G2-dexa in clinical application, the carrier was applied for treatment with therapeutic gene. Heme oxygenase-1 (HO-1) is an antioxidant enzyme which catalyzes the degradation of heme and is induced in response to ischemic environment. Delivery of the HO-1 gene produces the downstream products of bilivedin, CO, and Fe mediated anti-inflammatory effect. In research result, when the recombinant HO-1 gene was delivered with PAMAM G2-dexa in stoke model, the results showed that the infarct size was significantly decreased, compared to MCAO model. In summary, PAMAM G2-dexa had high transfection efficiency and anti-inflammatory effect, which is a good carrier for gene and drug combined therapy. Delivery of therapeutic gene such as HO-1 had a higher therapeutic effect to treat ischemic stroke. Therefore, PAMAM G2-dexa/HO-1 complexes may be useful for the treatment of ischemic diseases.