**2.2 Cells and animals**

188 Liver Tumors

an anionic state when pH > 6.9. His-alginate combined CPX1 at physiological pH (7.2) via its anion interact with the cation on the surface of CPX1. In tumor micro-environment, the pH varied from 6.2-6.7 according to different physical states. At such a pH, Hisalginate turned it anionic state to a cationic one and dissociated from CPX2. To enhance the ability of CPX2 to escape from the reticuloendothelial system, PEG 2000 was conjugated to His-alginate to form PEG-His-alginate (PHA). The construct scheme was

Poly-GC and Poly-AT were synthesized by Invitrogen (Shanghai, China) with a length of 20 bases. Cationic gelatin was synthesized by using Ethylenediamine-modification according to the reported methods (Matsumoto et al., 2006). The conversion rate of carboxyl groups to amino groups was measured by the TNBS method to characterize the product (Eklund, 1976). PEG-His-modified alginate was synthesized according the following methods: A total of 500 mg of alginate (Sigma) was dissolved in 20 ml of 10 mM N,N,N',N' tetramethylethylenediamine (TEMED) / HCl buffer solution (pH 4.7). A total of 1.0 g of EDC was added to this solution and stirred at 40 ◦C for 24 h. Then, different1.5 g histidine were added and stirred for another 24 h at 40 ◦C. The resulting His-alginate were dialyzed for 4 days against Milli Q water and lyophilized. The resulted His-alginate was examined for its pKa value. The following Pegylation was achieved by conjugating a 2000 Da mPEG-NH2 to the His-alginate using a Pegylation kit (YARE Chem, Shanghai, China). Rhodamine-labled

shown in Figure 1.

Fig. 1. Fabrication scheme of CXP1 and CPX2.

**2. Materials and methods** 

**2.1 Materials synthesis** 

Hepa 1-6 is a mouse hepatoma cell line derived from the BW7756 mouse hepatoma which arose in C57/L mouse (ATCC). Hepa 1-6 cells were cultured in DMEM supplemented with 10% FCS. Female ICR mice (18-20g) of the same background were purchased from the experimental animal center of Nanjing Medical University. All animals received human care according to Chinese legal requirements. To generate the allograft model of liver cancer, Hepa 1-6 cells (1×107 cells/ml, 10 μl) were injected into the hepatic lobe of anesthetized mice using a microinjector (Cheng et al., 2011).
