Branched polyethylenimine (bPEI) was non-viral vector and well known as high transfection materials and it has many amine group. However, utilization of bPEI was limited due to high toxicity. To solve these problem, bPEI was introduced to chitosan back bone by coupling agent. The chemical structure of ix
bPEI-graft-LMWSC (LMPEI) was analyzed by 1H-NMR and synthesized successfully. In addition, hyaluronic acid (HA), natural anion polymer, was introduced to binary complexes (pDNA/LMPEI) to target the specific cancer cell. The ternary complexes of pDNA/LMPEI/HA was prepared by electrostatic charge interaction and their binding affinity and DNase protection assay were conducted by gel retardation assay. The pDNA from ternary complexes was completely bound and pDNA from DNase was protected by LMPEI and HA. The particle size and distribution of pDNA/LMPEI showed that it had a unimodal size distribution and particle size was decreased according to quantity of raised LMPEI (1:1-16, pDNA/LMPEI). The weight ratio of binary complexes fixed to 1:16 (pDNA/LMPEI) as most small particle size ratio. To optimize quantity of HA to binary complexes, particle size of ternary complexes with 1:16:0.1-16 (pDNA/LMPEI/HA) were measured and found that optimized weight ratio of ternary complexes were 1:16:1 (pDNA/LMPEI2%/HA) and 1:16:2 (pDNA/LMPEI4%/HA). Their zeta potential showed that surface charge in case of ternary complexes was declined because HA was coated to binary complexes. Moreover, morphology property of them displayed to spherical shape. Their transfection assay was evaluated in HEK293, HCT116, and CT26 cell lines. In case of HEK293 cell, transfection of binary complexes was increased than ternary complexes x
because surface charge of ternary complexes was decreased by coated HA. On the other hands, the transfection assay of ternary complexes in HCT116 showed to be increased more than binary complexes without HA because of overexpressed CD44 receptor in HCT116 cells. Besides, to demonstrate targeting effect of ternary complexes in HCT116, competition assay was accomplished with treated free HA. As shown to the results, transfection of ternary complexes was remarkably decreased when free HA was treated to cancer cell. The cytotoxicity of them was confirmed by MTT assay and cell viability of ternary complexes was improved than binary complexes due to reduced positive charge by coated HA. The intracellular uptake and targeting effect of ternary complexes were investigated in HCT116 (CD44-receptor positive cancer cell) and CT26 (CD44-receptor negative cancer cell) cell lines by fluorescence microscopy. The pDNA from ternary complexes with propidium iodide (PI)-labeled pDNA was located to cytoplasm and nucleus more than binary complexes in HCT116 cell line. However, pDNA from that in case of CT26 cell line showed poor cell internalization. It means that ternary complexes can target against CD44-receptor overexpressed cancer cell line. These results suggest that ternary complexes are a superb gene carrier with low toxicity and high gene transfection.