Deciphering the role of chondroitin sulphate in increasing the transfection efficiency of amphipathic peptide basednanocomplexes

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1 Deciphering the role of chondroitin sulphate in increasing the transfection efficiency of amphipathic peptide basednanocomplexes aniel Nisakar 1#, Manika Vij 1,2#, Tanuja Pandey 1, Poornemaa Natarajan 1, Rajpal Sharma 1, Sarita Mishra 1,2, Munia Ganguli 1,2*. CSIR- Institute of Genomics and Integrative Biology, South Campus, Mathura Road, Opp: Sukhdev Vihar Bus Depot, New Delhi , India. Academy of Scientific and Innovative Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi , India. Supplementary Information Pages : S1 - S9 Figures: Fig.S1 - Fig.S9

2 (A) S1 (B) Figure S1: (A) Polydispersity index (PDI) values of Mgpe9 / Mgpe10 nanocomplexes formed at charge ratio 5 in presence/absence of exogenously added Chondroitin Sulfate A (CS) (CS/peptide wt/wt ratio = 0.25 and 1.0) using Dynamic Light Scattering. (B) Nuclease degradation assay was performed to check the protection ability of (i) Mgpe9 / (ii) Mgpe10 nanocomplexes (NC) formed at charge ratio 5 in presence/absence of exogenously added Chondroitin Sulfate A (CS) (CS/peptide wt/wt ratio = 0.25).

3 Figure S2: Cell viability upon addition of nanocomplexes prepared using Mgpe9/Mgpe10 peptide in presence/ absence of Chondroitin Sulfate A (CS) (CS/peptide wt/wt ratio = 0.25) was assessed using CellTitre Glo Luminescent Cell Viability assay for (A) 4h; (B) 24h and (C) At different charge ratios (for Mgpe10 nanocomplexes only) in CHO-K1 cells. Lipofectamine2000 was used for comparison. Untreated cells were taken as 100% viable. Three independent experiments were performed (in duplicates) and p value significance was calculated between Lipofectamine2000 and coated nanocomplexes for both the peptides (Mgpe9 and Mgpe10). p-value <0.05= *(slightly significant); <0.01= ** (moderately significant); <0.001=*** (highly significant); ns= not significant. S2

4 (A) (B) S3 Nuclear co-localization Pearson s co-efficient Mgpe Mgpe9 + CS (0.25) Mgpe Mgpe10 + CS (0.25) Figure S3: (A) Confocal microscopy was performed to check for nuclear accumulation of nanocomplexes Mgpe9/Mgpe10 in presence/absence of Chondroitin Sulfate A (CS) (CS/peptide wt/wt ratio = 0.25). DAPI dye was used to stain the nuclei of cells. (B) Table to depict the Pearson's coefficient values for nuclear accumulation of nanocomplexes as estimated through ImageJ software.

5 Mgpe9 -CS S4 Figure S4: z-stack images for uncoated Mgpe9 nanocomplexes to depict nuclear accumulation.

6 Mgpe9 +CS S5 Figure S5: z-stack images for CS coated Mgpe9 nanocomplexes to depict nuclear accumulation.

7 Mgpe10 - CS S6 Figure S6: z-stack images for uncoated Mgpe10 nanocomplexes to depict nuclear accumulation.

8 Mgpe10 +CS S7 Figure S7: z-stack images for CS coated Mgpe10 nanocomplexes to depict nuclear accumulation.

9 Figure S8: (A) Effect of exogenous Chondroitin Sulfate A (CS) on the transfection efficiency of uncoated and coated (wt/wt CS/peptide 0.25, 1.0) Mgpe10 nanocomplexes in absence and presence of 10% Fetal bovine serum (FBS). (B) Effect of exogenous Chondroitin Sulfate A (CS) on the transfection efficiency of uncoated and coated (wt/wt CS/peptide 0.25) Mgpe10 nanocomplexes in presence of different serum concentrations. p- value <0.05= *(slightly significant); <0.01= ** (moderately significant); <0.001= *** (highly significant); ns= not significant. (A) S8 ** *** (B) *** **

10 Figure S9: Effect of exogenous Chondroitin Sulfate A (CS) on transfection efficiency of coated and uncoated Mgpe10 nanocomplexes prepared at charge ratio 5, 24h and 48h post-transfection. Lipofectamine2000 was used for comparison. p-value <0.05= *(slightly significant); <0.01= ** (moderately significant); <0.001= *** (highly significant); ns= not significant. S9