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Lipid-based Cell Penetrating Nano-assembly for RNAi-mediated Anti-angiogenic Cancer Therapy

Supplementary Information

Poulami Majumder, Sukanya Bhunia, Arabinda Chaudhuri

Materials and MethodsGeneral Procedures and ReagentsEDCI and Tetrakis-(triphenylphosphin)-palladium(0) were purchased from Sigma Aldrich (USA). Column chromatography was performed with silica gel from Acme Synthetic Chemicals, India, 60-120 mesh). MALDI TOF spectral data were acquired by using Shimadzu Biotech Axima Performance mass spectrometer. 1H NMR spectra were recorded on a Varian FT 200 MHz Spectrometer.Cholesterol, Protamine sulfate (fraction X from Salmon), Annexin V-FITC apoptosis detection kit, cell culture media and fetal bovine serum were purchased from Sigma, St. Louis, USA. Verso® one-step RT-PCR kit and On-target plus Smart-pool siRNAs were from Dharmacon (Lafayette, CO). Blood vessel staining kit was purchased from Millipore (USA). Rabbit polyclonal anti-VEGF antibody was obtained from Abcam (UK). Alkaline phosphatase conjugated goat anti-rabbit and BCIP/NBT substrate were supplied by Pierce Biotechnology (Rockford, USA) and Calbiochem (USA), respectively. B16F10 (murine melanoma) cells were procured from ATCC (USA) and were grown at 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) with 10% FBS in a humidified atmosphere with 5% CO2/95% air. HUVEC (Human Umbilical Vein Endothelial Cells) were purchased from Lonza (USA) and maintained in EBM-2 upto 5th passages. 6-8 weeks old C57BL/6 mice (each weighing 20-22 g) were purchased from Centre for Cellular and Molecular Biology, Hyderabad, India. All the in vivo experiments were performed in accordance with the Institutional Bio-Safety and Ethical Committee Guidelines using an approved animal protocol.

1. Synthesis of monoallyl ester of succinic acid

Synthetic scheme for preparing the monoallyl ester of succinic acid has been shown in FigureS1. Succinic anhydride (1g, 0.01 mole) was suspended in dry toluene (20 mL) and allyl alcohol (0.5 mL, 0.009 mole) and DMAP (1.1 g, 0.009 mole) were added. The suspension was refluxed for 4 h. Excess chloroform (100 mL) was added, the resulting solution was washed successively with 1 (N) HCl (2 x 50 mL) and saturated sodium chloride (1 x 50 mL). The chloroform layer was dried over sodium sulfate and concentrated on a rotary evaporator. Column chromatographic purification of the residue using silica gel (60-120 mesh size) and 2-2.5% methanol in chloroform (v/v) as the eluent afforded monoallyl ester of succinic acid as a gummy colorless liquid (1.73 g, 80% yield, Rf = 0.5, 95:05, chloroform: methanol, v/v).

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2018

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Figure S1. Synthetic scheme of monoallyl ester of succinic acid

ESIMS: 181 [M + Na] + for C7O4H10 (Figure S2)

1H NMR (200 MHz, CDCl3): δ/ppm = 2.6-2.7 (m, 4H, -CH2-CH2-COOH); 4.6 (d, 2H, -COO-CH2-CH-CH2); 5.2-5.4 (dd, 2H, -COO-CH2-CH-CH2); 5.8-6.0 (m, 1H, -COO-CH2-CH-CH2) (Figure S3).

2.Synthesis of TIPA

Fmoc Solid Phase Peptide Synthesis strategy (as shown schematically in Figure S4) was used to synthesize TIPA. H-Arg(pbf)-2-chlorotrityl resin (200 mg, 0.53 mmol/g loading) in the peptide synthesizer (CS Bio, USA) vessel was first swelled in dry DMF for 2 h. The α-COOH group of the second arginine residue was then coupled with the α-NH2 group of the resin-attached first Arg residue by mixing the pre-swelled arginine pre-loaded resin with a solution of Fmoc-Arg(pbf)-OH (3.6 eqv), HATU (4 eqv), HOBt (4 eqv) and DIPEA (8 eqv) in dry DMF (10 mL) followed by agitating the peptide synthesizer vessel under nitrogen for 30 min. This coupling method was repeated twice to ensure complete conjugation. The Fmoc- protecting group was removed by stirring with a solution of piperidine/DMF (1:4, v/v) under nitrogen for 15 mins (2X). This peptide coupling strategy was repeated using all the necessary & appropriately protected (as shown in Figure S4) additional amino acids. After addition of the last amino acid, Fmoc-group was removed from the last amino acid residue of the resulting resin-attached undecapeptide by stirring with a solution of piperidine/DMF (1:4, v/v) under nitrogen for 15 mins (2X). Succinic acid monoallyl ester (3.6 eqv) was then coupled to the α-NH2 group of the last amino acid residue using HATU (4 eqv), HOBt (4 eqv) and DIPEA (8 eqv) in dry DCM-dry DMF (10 mL) for 30 mins (2X). Allyl group was removed1 with Pd(PPh3)4 in CHCl3/AcOH/N-methylmorpholine (37:2:1, v/v) under N2, by occasional gentle agitation for 2 h. Washing was done with DIPEA-DMF (5:95, v/v, 15 mL) followed by di-ethyl-dithiocarbamic acid sodium salt (0.05% in dry DMF). After allyl ester deprotection, N-2-aminoethyl-N, N-di-n-octadecylamine (5 eqv), HATU, HOBt and DIPEA were added and agitated for 6 h. The peptide was cleaved off the resin with TFA/phenol/water/TIS (85:10:2.5:2.5, v/v) for 4 h on ice. The reaction was terminated by addition of diethyl ether until a white precipitate separated. Chloride ion exchange chromatography over Amberlyst IRA-400 resin and repeated precipitation from MeOH/acetone afforded pure TIPA (20 mg, 22% yield) as white solid. TIPA was characterized by the molecular ion peak in MALDI-TOF MS and the purity was ascertained by reversed phase analytical HPLC using methanol as mobile phase. MALDI-MS and HPLC profiles are shown in Figures S5 and S6, respectively.

MALDI-MS: 2207 for [M] + (Figure S5)

3. Preparation of TIPA Nano-assemblies (TIPA-NAs)Nano-assemblies of TIPA (TIPA-NAs) were prepared using (n-C16H33)2N+(CH3) CH2CH2N+(CH3)3 2Cl- (designated herein as DCL, a readily available di-cationic amphiphile in our laboratory), cholesterol and TIPA in 1:1:0.05 mole ratio. Control nano-assemblies (Control-NA) were formulated with DCL and cholesterol in 1:1 molar ratio. Appropriate amounts of the components were dissolved in a mixture of chloroform and methanol (3:1, v/v), the lipid film was dried with a thin flow of moisture free nitrogen gas and kept under high vacuum for 8 h.

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siRNA pre-condensed with protamine sulfate (14:1, protamine: siRNA, w/w) in RNase free water was used to hydrate the dried lipid film. Lipid: siRNA ratio was maintained at 25:1 (w/w). The resulting suspension was vortexed, sonicated and exposed to 7 freeze/thaw cycles by alternately placing it at -78 ˚C and 37 ˚C baths. Unencapsulated siRNA was removed from the suspension by centrifugation at 5000 rpm using a 30 KD Amicon Ultra (EMD Millipore, USA). Finally, the nano-assemblies were obtained through extrusion using polycarbonate membranes (Avanti Polar Lipids, USA) of pore size 100 nm. The same protocol was used for preparing VEGFsiRNA, scrambled siRNA and FAM-siRNA-loaded TIPA-NA and control-NA formulations. Empty TIPA-NAs and Control-NAs were similarly prepared without loading any siRNA. As a control for siRNA encapsulation (pre-loading), electrostatic complexes (post-loading) were prepared by mixing empty TIPA-NAs or control-NAs with equivalent amount of siRNA for 20 minutes at 37 ˚C. Unbound siRNAs were similarly removed by centrifugation.

4. Measurement of siRNA encapsulation efficiencysiRNA encapsulation efficiencies of all formulations were determined using a standard curve prepared with known concentrations of FAM-labeled (6-fluorescein amidite) non-silencing siRNA. Samples were diluted to the concentration range of the standard curve and fluorescence intensities were measured at 490 and 520 nm (excitation and emission maxima of FAM, respectively) in presence of 0.1% SDS using a FLx 800 microplate fluorescence reader (Bio-Tek instruments, UK). Encapsulation efficiency was calculated as: Entrapment efficiency (%) = siRNAf/siRNAt × 100; where siRNAt is the total amount of siRNA used for preparation of the initial mixture and siRNAf is the amount of siRNA recovered after the lipid assemblies were disrupted with SDS2. The results shown are the average of the triplicate experiments performed on the same day.

5. Dynamic Light ScatteringHydrodynamic diameters and zeta potentials of the nano-assemblies and electrostatic complexes were measured by photon correlation spectroscopy and electrophoretic mobility on a Zeta sizer 3000HSA (Malvern UK) with a sample refractive index of 1.59 and a viscosity of 0.89. While measuring the zeta potentials, the following parameters were considered: viscosity, 0.89 cP; dielectric constant, 79; temperature, 25 °C; F(Ka), 1.50 (Smoluchowski). The system was calibrated by using DTS0050 standard from Malvern, UK. Measurements were done 10 times with the zerofield correction.

6. Gel retardation assayTIPA-NAs containing 1 µg of encapsulated non-silencing siRNA, electrostatic complex of TIPA-NA and equivalent amount of siRNA as well as 1 µg naked siRNA were loaded on a 0.75% (w/v) agarose gel containing ethidium bromide. The samples were electrophoresed in 0.5X TAE buffer for 45 min at 80 V. UV illumination was used to visualize individual bands.

7. Cellular viabilityHUVEC and B16F10 cells were seeded in 96-well plates at a density of 1.0 × 104 per well and incubated for 18 h. Cells were treated with scrambled siRNA encapsulated in TIPA-NAs and Control-NA formulations with varying siRNA concentrations in serum free media. Media was discarded 4 h post incubation and was incubated for another 24 h with fresh media containing 10% FBS. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyldiphenyltetrazolium bromide (MTT)

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solution (5 mg/mL) was then added to the cells and incubated for 4 h. An enzyme-linked immunosorbent assay reader was used to read the absorbance of formazan at 550 nm in each well and to determine the quantity of mitochondrial reductases in the viable cells. Results were expressed as percent viability = [A550(treated cells) - background/A550(untreated cells) - background] ×100.

8. Cellular uptake HUVEC and B16F10 Cells (1.2×104 per well) were seeded in 96-well plates (Corning Inc., Corning, NY) 18 h before treatment. Cells were treated with nano-assemblies containing encapsulated FAM-labeled non-silencing siRNA in serum-free DMEM for B16F10 cells and serum-free EBM-2 for HUVEC. Final siRNA concentration was 80 nM. Cells were washed with PBS (3 x 100 μL) after 4 h of incubation and imaged under an inverted fluorescence microscope (Nikon, Japan).

9. RT PCRB16F10 and HUVEC cells were (~1 x 106 cells per flask) treated with VEGFsiRNA encapsulated TIPA-NAs, Control-NAs or scrambled siRNA-loaded TIPA-NAs. In each case, final siRNA concentration was 120 nM. Serum free medium was replaced with complete growth medium after 4 h of incubation. Total RNAs were extracted from the cells 24 h post-treatment using Trizol solution (Invitrogen). First-Strand cDNAs were synthesized from the corresponding mRNAs by Reverse Transcription reaction and cDNAs were amplified using forward and reverse primers for VEGF by Polymerase chain reaction using Verso® one-step RT-PCR kit (Dharmacon, Lafayette, CO) according to the manufacturer’s protocol. 18S was used as the internal control for PCR. Finally, the amplified DNAs were resolved in 2% agarose gel. Primer sequences used are shown in Table S2.

10. Western blotB16F10 cells and HUVEC cells were treated as described above in section 8. After 24 h, the cells were detached from the flasks using a cell scrapper. Whole cell lysates were prepared by lysing the cells. Total protein content in each sample was determined by BCA method. Cell lysates were loaded and separated using 12% polyacrylamide gel in SDS-PAGE. Proteins were transferred onto nitrocellulose membranes (Thermo Scientific) using wet blotting. Membrane was blocked for 1.5 h with 3% BSA solution in PBS-T (phosphate buffer saline containing 0.05% Tween-20). Blot was then incubated with a rabbit polyclonal antibody against VEGF (Abcam) at 1:1000 dilution for 18 h at 4 ˚C. Following PBS-T washes, membranes were incubated with goat anti-rabbit secondary antibody conjugated to alkaline phosphatase (Pierce) at 1:5000 dilutions for 2 hours. Protein bands were visualized using BCIP-NBT solution (Calbiochem) according to the manufacturer’s protocol.

11. Flow cytometric apoptosis detection assayHUVEC and B16F10 cells were treated as described in section 8. Cells were trypsinized 30 h post-treatment, washed with PBS and suspended in 500 µL 1X binding buffer containing 5 µL of annexin-V FITC and 10 µL of PI. The suspension was incubated for 15 min in dark and analyzed by flow cytometer (BD FACS Canto II).

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12. Scratch wound assayHUVEC cells were seeded at a density of ~5.0×104 cells per well in a 24-well plate 18 h before treatment. A scratch was made using a sterile pipette tip immediately before treatment. Cells were treated as described in section 8. Media was replaced with fresh complete EBM2 media 4 h post-incubation and cells were allowed to migrate toward the scratched portion for 24 h. Scratched area were imaged using an inverted microscope (5X magnification).

13. Biodistribution studies~1.5 x 105 B16F10 cells in 250 µL Hank’s buffer salt solution were injected subcutaneously with a syringe with 30-gauge needle in the right flank of 6-8 weeks old female C57BL/6J mice (each weighing 20-22 g) on day 0. On day 14, mice bearing aggressive melanoma tumor (volume ~100 mm3) were administered intraperitoneally with FAM-labeled non-silencing siRNA (0.57 mg/kgBW) encapsulated within TIPA-NAs and Control-NAs. Mice were sacrificed 24 h post injection, organs and tissues were harvested and washed in cold saline. Tissues and organs were homogenized with the addition of 800 μL of lysis buffer (0.1 M Tris-HCl, 2 mM EDTA and 0.2% Triton X-100, pH 7.8) using a mechanical homogenizer. The homogenates were centrifuged at 14,000 rpm for 10 min at 4 ˚C and fluorescence of 100 μL from the supernatant was measured at excitation and emission wavelength of 490 nm and 520 nm, respectively using FLx 800 microplate fluorescence reader (Bio-Tek instruments, INC, UK). To correct for tissue autofluorescence, untreated control tissues were similarly treated and fluorescence observed from such untreated tissue homogenates were subtracted from that of treated tissues. The remaining counts were converted to % of injected dose/g of tissue weight with the help of a known standard curve of FAM-siRNA.

14. Solid Tumor growth inhibition study~1.5 x 105 B16F10 cells in 250 µL Hank’s buffer salt solution were injected subcutaneously with a syringe with 30-gauge needle in the right flank of 6-8 weeks old female C57BL/6J mice (each weighing 20-22 g) on day 0. On day 14, mice were randomly sorted into groups and each group (n = 5) was administered intravenously with: VEGFsiRNA-loaded TIPA-NAs; scrambled siRNA-loaded TIPA-NAs, VEGFsiRNA-loaded Control-NAs and 5% glucose (vehicle) on day 14, 16, 18, 20 and 22 post tumor inoculation. Each animal received 0.25 mg/kgBW of siRNA per injection. Tumor volumes (V = 1/2.ab2 where, a = maximum length of the tumor and b = minimum length of the tumor measured perpendicular to each other) were measured with a slide calipers for up to 23 days. Results represent the means +/- SD (for n = 5).

15. Immunohistochemical studiesTumor-bearing mice were treated as described above. Mice were sacrificed on day 24 post-tumor cell inoculation and tumors were excised. 10-micron thick tumor cryosections were prepared on glass slide of in cryostat instrument (Leica). The slides were fixed in 4% methanol-free formaldehyde in PBS. Fixed slides were then stained for: blood vessel marker vWF (von Willebrand factor) with a blood vessel staining kit (Chemicon, USA), for VE-cadherin (endothelial cells, red) with anti-VE-cadherin antibody (1:100 dilution) and a TUNEL assay kit (for observing cells undergoing apoptosis). The stained slides were put under an inverted fluorescence microscope and imaged. Tumor microvessel densities were detected by immunostaining cryosections with anti-CD31 primary antibody (at 1:100 dilution) for 90 min

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and goat anti-rabbit HRP-conjugated secondary antibody for 60 min. Cryosections were observed in bright field using inverted microscope.

16. Statistical analysisError bars represent mean values ± SD. The statistical significance between two treatment groups was determined using two-tailed student’s t-test. *P<0.05 were considered statistically significant.

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Figure S2. ESIMS of monoallyl ester of succinic acid

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Figure S3. 1H NMR Spectrum of monoallyl ester of succinic acid

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Figure S4. Synthetic scheme for preparing TIPA.

Reagents and conditions:(i) Fmoc-Arg(pbf)-OH (4 equiv), HATU (3.6 equiv), HOBt (3.6 equiv), DIPEA (8 equiv), 35 min (×2); (ii) piperidine: DMF (1:4), 10 min (×2);(iii) Fmoc-Gln(Trt)-OH (4 equiv), HATU (3.6 equiv), HOBt (3.6 equiv), DIPEA, 35 min (×2);(iv) Fmoc-Lys(Boc)-OH (4 equiv), HATU (3.6 equiv), HOBt (3.6 equiv), DIPEA, 35 min (×2);(v) Fmoc-Gly-OH(4 equiv), HATU (3.6 equiv), HOBt (3.6 equiv), DIPEA, 35 min (×2);(vi) Fmoc-Tyr(2-Cl-Trt)-OH (4 equiv), HATU (3.6 equiv), HOBt (3.6 equiv), DIPEA, 35 min (×2);(vii) mono allyl ester of succinic acid (4 equiv), HATU (3.6 equiv), HOBt (3.6 equiv), DIPEA, 35 min (×2);(viii) Pd(PPh3)4(3 equiv), CHCl3/AcOH/N-Methyl Morpholine (7.4:0.4:0.2), 120 min (×1);(ix) TFA/DCM,0 °C, 2 h; (x) HATU (3.6 equiv), HOBt (3.6 equiv), DIPEA, 120 min (×2);(xi) TFA/phenol/water/TIS (85:10:2.5:2.5), 0 °C, 4 h; (xii) Amberlyst A-26 resin for Cl- ion exchange.

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Figure S5. MALDI-MS of TIPA.

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System: Varian Prostar 210Column: Metasil AQ 10U C18 120A, 250 x 10 mm

Mobile Phase: MethanolFlow Rate: 1.0 mL/min (0-20 min)

Typical Column Pressure: 60-65 BarsTemperature: 250C; Detection: UV at 260 nm

Figure S6. HPLC chromatogram of TIPA.

Figure S7. Relative electrophoretic mobilities of siRNAs electrostatically complexed with empty TIPA-NA (lane 1), siRNA encapsulated within TIPA-NA (lane 2) and naked siRNA (lane 3). In each case 1 µg of non-silencing siRNA was used.

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Figure S8. Cellular uptake of FAM-labeled non-silencing siRNA-loaded TIPA-NA is significantly higher than that for FAM siRNA-loaded Control-NAs. Epifluorescence microscopic images of HUVEC (a) and B16F10 (b) cells were taken 4 h post-incubation with the nano-assemblies. Scale bar 50 μm in each case.

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Figure S9. Scrambled siRNA-loaded TIPA-NAs and control-NAs are non-toxic towards endothelial (a) and tumor (b) cells. Cellular viabilities of HUVEC (a) and B16F10 (b) cells treated with scrambled siRNA-loaded TIPA-NA and control-NA, 24 h post-treatment.

Figure S10. Scrambled siRNA-loaded TIPA-NAs is essentially incompetent to induce significant apoptosis in both HUVEC (a) and B16F10 (b) cells as revealed in the FITC-labeled Annexin-V binding-based flow cytometric monitoring of cells in early and late apoptotic stage. (c) Bar graph representing the degree of apoptosis (early and late) induced by different nano-assembly formulations in B16F10 cells. * p<0.05 as determined by two-tailed student’s t-test.

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Figure S11. Scratch-wound assay performed in HUVEC cells revealed significantly less cellular migration in cells treated with VEGFsiRNA-loaded TIPA-NAs when compared to that for cells treated with the indicated control formulations.

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Figure S12. Microvessel densities (CD31-positive cells) are significantly reduced in tumor cryosections prepared from mice treated with VEGFsiRNA-loaded TIPA-NAs when compared to those prepared from mice treated with the indicated control formulations. Additional images (apart from Figure 4e). Scale bar 100 μm.

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Figure S13. Immunohistochemical staining (for the tumor endothelial cell markers vWF and VE-cadherin and for TUNEL-positive apoptotic cells) of tumor cryosections prepared from mice treated with VEGFsiRNA-loaded TIPA-NAs and other indicated control NAs. Significant co-localization of TUNEL-positive and VE-cadherin positive cells demonstrate significant apoptosis inducing properties of VEGFsiRNA-loaded TIPA-NAs. Additional images (apart from Figure 4f). Scale bar 100 μm.

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Table S1. Hydrodynamic diameters and zeta potentials of TIPA and Control nano-assemblies

with and without encapsulated siRNAs. The corresponding values for the electrostatic complexes

of siRNA &TIPA-NA and siRNA & Control NA are shown in the bottom two rows.

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Table S2. Primer sequences used in the RT-PCR experiments.

Supplementary Reference

1. N. Nasongkla, X. Shuai, H. Ai, B. D. Weinberg, J. Pink, D. A. Boothman and J. Gao, Angew Chem Int Ed Engl, 2004, 43, 6323-6327.

2. P. Majumder, S. Bhunia, J. Bhattacharyya and A. Chaudhuri, J Control Release, 2014, 180, 100-108.