Supplementary Materials for

Exploring advantages/disadvantages and improvements in overcoming gene delivery barriers of amino acid modified trimethylated chitosan

Hao Zheng, Cui Tang*, Chunhua Yin

State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China

*Corresponding author: Tel: +86 21 6564 3556; fax: +86 21 5552 2771.

E-mail address: (C. Tang)

Methods

1.1 Characterization of NC at various N/P ratios

Cationic polymers and pDNA were dissolved in water (pH 6.0) at the concentration of 2 mg/mL and 0.2 mg/mL, respectively. To achieve NC with different N/P ratios, different volumes of polymer solutions were added to certain volume of pDNA solution under vortex. NC was incubated at 37 °C for 30 min before determination of particle sizes and Zeta potentials.

1.2 Cytotoxicity of endocytic inhibitors

HEK 293 cells were seeded onto 96-well plate and allowed to grow for 24 h. Chlorpromazine (10 μg/mL) and genistein (200 μg/mL) were added and incubated with cells for 24 h. After changed with fresh DMEM, MTT solution (5 mg/mL dissolved in PBS) was added and incubated for 3 h at 37 °C. DMSO was used to dissolve the formazan crystal following discarding medium. Absorbance at 570 nm was measured and the relative cell viabilities of test groups were set as comparison with control group that treated with PBS.

Results and Discussion

2.1 Characterization of NC at various N/P ratios

As indicated in the Fig. S1, increased N/P ratio correlated with smaller particle sizes and augmented Zeta potentials. The N/P ratio required for TMC, TR, TC, and TH to effectively compact pDNA and form nanosized-complexes were 5, 5, 3, and 5, respectively. The reason why less TC was required to encapsulate pDNA and form NC probably resided in the intra-crosslinking effect of sulphydryl. However, when the N/P ratio exceeded 5, the particle sizes and Zeta potentials of various NC remained unchanged. In this study, when the polymer/pDNA weight ratio was 10, the corresponding N/P ratio of TNC, TRNC, TCNC, and THNC were 15, 15, 11, and 11, respectively. Since these N/P ratios were more than 5, the polymer/pDNA weight ratio of 10 for all tested NC was considered to be in their optimum range of the polymer:pDNA ratios in terms of particle sizes and Zeta potentials. Moreover, the same weight ratio to form NC was supposed to eliminate unwanted interferences arising from the difference in the amount used of cationic polymers. Therefore, the polymer/pDNA weight ratio of 10 was suitable for the subsequent serial analysis.

Fig. S1 Particle sizes (A) and Zeta potentials (B) of distinct NC at various N/P ratios.

2.2 Cytotoxicity of endocytic inhibitors

As shown in Fig. S2, the cell viabilities with the treatment of chlorpromazine and genistein were comparable to that treated with PBS (P > 0.05), suggesting that these endocytic inhibitors would not adversely affect the uptake process, providing warranty for the analysis of endocytic pathways of NC.

Fig. S2 Relative cell viability after chlorpromazine and genistein treatment.