Supporting Information

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1 Supporting Information Gemcitabine and Antisense-microRNA Co-encapsulated PLGA-PEG Polymer Nanoparticles for Hepatocellular Carcinoma Therapy Rammohan Devulapally, Kira Foygel, Thillai V Sekar, Juergen K. Willmann, and Ramasamy Paulmurugan* Molecular Imaging Program at Stanford (MIPS), Canary Center at Stanford for Cancer Early Detection, Bio-X Program, School of Medicine, Stanford University Stanford, California, U.S.A. * paulmur8@stanford.edu S- 1

2 Contents 1. Supplementary figure S1 S-3 2. Materials S-4 3. Methods S-4 4. References S-7 S- 2

3 1. Supplementary Figure S1 Figure S1: PI-Staining based FACS analysis of (a) Hep3B and (b) HepG2 cell lines treated with Gemcitabine (3 µm) and antisense-mir-21 (15 nm) loaded PLGA-PEG-NPs. FACS data was analyzed in a Flowjo linear scale mode. S- 3

4 2. Materials GEM hydrochloride salt (>99% purity) was purchased from LC labs (Woburn, MA, USA). Poly(D,L-lactide-co-glycolide) (Resomer RG 502 H, PLGA-COOH, 50:50, MW 7,000-17,000), N-Hydroxysuccinimide (NHS, 98% pure), N-Ethyl-N -(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC, 99.0% pure) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Hetero-bi-functional PEG polymer NH 2 -PEG- COOH (MW 3400) was purchased from JenKem Technology (Allen, TX, USA). Anti-miR- 21 or antisense-mir-21 (>95% purity) was custom synthesized by PAN facility at Stanford University. Dulbecco's Modified Eagle Medium (DMEM) cell culture media, fetal bovine serum (FBS), streptomycin and penicillin antibiotics were purchased from Invitrogen (Carlsbad, CA, USA). HCC (Hep3B and HepG2) cell lines were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA). 3. Methods: 3.1 Dynamic Light Scattering (DLS) measurements NPs zeta potential and size measurements were acquired by DLS (Zetasizer Nano ZS90, Malvern Instruments, U.K.). The z-average was determined using cumulant analysis. Zeta potential measurements were obtained using an aqueous dip cell by Smoluchowski model. 3.2 Transmission Electron Microscopy (TEM) We have used FEI TITAN kV environmental transmission electron microscope (ETEM) at Stanford Nano Shared facilities for obtaining TEM images of NPs. Because of PLGA-PEG NPs are not showing contrast for TEM, we have negatively stained the NPs with 1% phosphotungstic acid (PTA, ph 7.5) by mixing a drop of NPs. After 3 min of S- 4

5 incubation, a drop of PTA-stained NPs was carefully added on a carbon film-coated copper grid. After 3 min of incubation, the excess solution was drained off, air-dried and images of the NPs were obtained by operating TEM at 80 kv. ImageJ software was used for size analysis. 3.3 Entrapment efficiency estimation for anti-mir-21 Entrapment efficiency was estimated using similar procedure described in our previous work 1. Briefly, antisense-mir-21 encapsulated PLGA-PEG NPs were lyophilized by freeze drier at 80 o C, and dried NPs were disintegrated by addition of CH 2 Cl 2. The antisense-mir-21 was isolated using DNAse/RNase free water. The isolated antisensemir-21 was quantified after resolving in agarose gel. The co-encapsulated Cy5- antisense-mir-21 fluorescence image was obtained using IVIS-Lumina imaging system by imaging at the 570 nm excitation and Cy5 emission filter. The agarose gel images were quantified using densitometry, and the Cy5-fluorescence images were calculated using IVIS-Lumina imaging software. The entrapment efficiency of antisense-mir-21 loaded in the NPs was calculated using the following formula published previously by us Entrapment efficiency estimation for GEM Similar extraction procedure as stated above for antisense-mir-21 was adopted for extracting GEM from PLGA-PEG NPs. Entrapment efficiency was calculated by using the UV absorbance (Agilent Cary 60 UV-Vis Spectrophotometer) at 268 nm, indicative of GEM concentrations. The Entrapment efficiency of GEM loaded in NPs was estimated using the following formula: Entrapment efficiency (%) = Mass of GEM dried NPs (W) / Mass of total GEM used (M) x 100. S- 5

6 3.5 Protein extraction and immunoblot analysis For immunoblot assay, we have used the protocol described in our previous work 1. Briefly, for assessing the level of Bcl2, BAX, PTEN, and GAPDH expression, cells (Hep3b and HepG2) after respective treatment conditions (with GEM and antisensemir-21 loaded and co-loaded NPs) for 48 h were collected lysed in RIPA buffer containing protease inhibitor cocktail. The lysates were incubated for 1 h in an ice bath and vortex mixed for every 15 min for 5 sec and centrifuged at rpm at 4 C for 15 min. The proteins collected from the supernatant were measured using Bradford assay kit (Bio-Rad). All proteins samples (50 µg each) were incubated in loading-dye at 95 o C for 5 min for cleavage of disulfides linkages. Freshly prepared proteins sample along with pre-stained protein marker (New England Biolabs, Ipswich, MA) were resolved by 4-12% gradient SDS/PAGE (Invitrogen) at 80V and transferred onto a nitrocellulose membrane (0.45 µm pore size, Schleicher & Schuell) by electro blotting at 45V. The membrane was carefully removed from electro blot and washed three times (5 min each) with Tris-buffered saline-tween 20 (TBS-T buffer) and blocked in 5% of non-fat dry milk in TBS-T buffer for 1 h and incubated with respective antibody on a rocking shaker for overnight at 4 C. Next day, the membrane was washed with TBS-T buffer as above and incubated with HRP-conjugated anti-rabbit antibody (1:4000) in TBS-T buffer for 1 h at RT and washed with TBS-T buffer as above. Chemiluminescent HRP substrate LuminoGlo (Cell Signaling, Beverly, MA) was added to the membrane and imaged with IVIS-Lumina imaging system. Images were analyzed by IVIS-Lumina Image software. The same membrane was stripped using stripping buffer and used for another antibody and finally with GAPDH to control for protein loading. S- 6

7 References: 1. Devulapally, R.; Sekar, N. M.; Sekar, T. V.; Foygel, K.; Massoud, T. F.; Willmann, J. K.; Paulmurugan, R., Polymer Nanoparticles Mediated Codelivery of AntimiR-10b and AntimiR-21 for Achieving Triple Negative Breast Cancer Therapy. ACS Nano 2015, 9 (3), Devulapally, R.; Sekar, T. V.; Paulmurugan, R., Formulation of Anti-miR-21 and 4-Hydroxytamoxifen Co-loaded Biodegradable Polymer Nanoparticles and Their Antiproliferative Effect on Breast Cancer Cells. Mol. Pharmaceutics 2015, 12 (6), S- 7