Supplementary data. Paromita Kundu, Manasi Das, Kalpalata Tripathy ǂ, Sanjeeb K Sahoo *,

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1 Supplementary data Delivery of dual drug loaded lipid based nanoparticles across blood brain barrier impart enhanced neuroprotection in a rotenone induced mouse model of Parkinson s disease Paromita Kundu, Manasi Das, Kalpalata Tripathy ǂ, Sanjeeb K Sahoo *, A Oligomer VNPs CN CNPs PN PNPs CPN CPNPs B Fibrils VNPs CN CNPs PN PNPs CPN CPNPs C Figure S1. The histogram analysis of (A) oligomers (B) Fibrils at different treatment depicting size distribution. (C) ThT assay showing the effect of different treatments on α-synuclein aggregation as oligomers or fibrils. ThT fluorescence intensity of the samples was measured at an Ex: 450 nm, Em: 482 nm using a fluorescence spectrophotometer. Data represented as mean ± S.E.M (n = 3). ***p < corresponds to different treatments vs oligomers or fibrils. ##p < 0.01, ###p < corresponds to CPNPs vs CPN. 1

2 Figure S2. Dose dependent cytotoxicity of rotenone in PC12 cells for 48 hrs as determined by MTT assay. Data are presented as mean ± SEM (n = 4). ***p < different treatments vs control. 2

3 Figure S3. Effect of different treatments on cellular GSH and lipid peroxidation. (A) Measurement of GSH content in PC12 cells following different treatments for 48 hrs. Total cell lysate (from 1 x 10 6 cells) were treated with 5 % TCA to precipitate down proteins and the supernatant was taken for GSH analysis by adding 0.6 mm DTNB to give a yellow colour which was measured at 412 nm by UV/Vis spectrophotometer. Cellular GSH is expressed as nmoles/mg protein and presented as mean ± S.E.M, (n = 3). p< 0.05 is considered significant. ###p corresponds to rotenone vs control and *p or **p corresponds to different treatment vs rotenone. (B) Study of lipid peroxidation in PC12 cells following different treatment for 48 hrs. Lipid peroxidation is expressed as TBARS formed in nmoles/mg protein and presented as mean ± S.E.M, (n = 3). p< 0.05 considered significant. ###p corresponds to rotenone vs control and **p or ***p corresponds to different treatment vs rotenone. 3

4 Methods Thioflavin T (ThT) Binding Assay Binding of αs either as oligomers or fibrils by ThT was evaluated by fluorescence spectrophotometer (LS 55, Perkin Elmer, MA, USA) following previous published protocol. 1 Briefly, 50 µl of 10 µm stock of αs samples (prepared for oligomer or fibrils study) was added to 150 µl of 50 µm ThT solution (dissolved in Glycine-NaOH buffer, ph 8.5) and incubated for 15 min in the dark at room temperature. Finally, ThT fluorescence intensity of samples were measured (Ex: 450 nm, Em: 482 nm) using the fluorescence spectrophotometer. Data represented as mean ± S.E.M (n = 3). Glutathione (GSH) Assay GSH content was estimated by measuring the absorbance of DTNB following the protocol of Moron et al. 2 Briefly, PC12 cells seeded in poly-l-lysine coated T-25 flask (Corning Inc., NY, USA) was treated with rotenone (2 µg/ml) or co-administered with rotenone (2 µg/ml) along with curcumin or piperine (2 µg/ml, in single or in combination) both in native as well as in nanoformulations for 48 hrs. Following incubation period, the yellow colored complex developed by the reaction of GSH and DTNB was measured at 412 nm using a UV-visible spectrophotometer (Specord-210, Analytikjena, Germany) and expressed as nmoles/mg protein. Data represented as mean ± S.E.M (n = 3). Lipid Peroxidation Assay Lipid peroxidation assay was performed by measuring the formation of thiobarbituric acid reactive substances (TBARS) according to the protocol of Ohkawa et al. 3 Briefly, 1 4

5 10 6 PC12 cells seeded in poly-l-lysine coated T-25 flask were treated with rotenone (2 µg/ml) or co-administered with rotenone (2 µg/ml) along with curcumin or piperine (2 µg/ml, in single or in combination) both in native as well as in nanoformulations for 48 hrs. Following incubation period, the cells were processed for measuring TBARS at 532 nm using UV-visible spectrophotometer. TBARS presence in the sample was calculated from its extinction coefficient ε = M -1 cm -1 and expressed as nmoles TBARS formed per mg protein. References [1] Ahsan, N., Mishra, S., Jain, M. K., Surolia, A., and Gupta, S. (2015) Curcumin Pyrazole and its derivative (N-(3-Nitrophenylpyrazole) Curcumin inhibit aggregation, disrupt fibrils and modulate toxicity of Wild type and Mutant alpha-synuclein, Sci. Rep. 5, [2] Moron, M. S., Depierre, J. W., and Mannervik, B. (1979) Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lung and liver, Biochim. Biophys. Acta. 582, [3] Ohkawa, H., Ohishi, N., and Yagi, K. (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95,