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: tangcui@fudan.edu.cn (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.