Supporting Information for

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1 Supporting Information for Nanoscale Coordination Polymers Co-deliver Chemotherapeutics and sirnas to Eradicate Tumors of Cisplatin-Resistant Ovarian Cancer Chunbai He 1, Christopher Poon 1, Christina Chan 1, S. Diane Yamada 2, and Wenbin Lin 1, * 1 Department of Chemistry, The University of Chicago, Chicago, IL 6637, USA 2 Department of Obstetrics and Gynecology, Section of Gynecologic Oncology, The University of Chicago, Chicago, IL 6637, USA. wenbinlin@uchicago.edu Methods Mass spectroscopy analysis of DSPE-siRNA The matrix solution was prepared by dissolving 2,5 dihydroxybenzoic acid (1 mg,.65 mmol) in a mixture of 1 ml acetonitrile/.1% trifluoroacetic acid (3:7, v/v). DSPE-siRNA (1 g, 76.9 pmol) was dissolved in 1 L of the matrix solution. After vortexing, the sample/matrix solution (1 L) was spotted onto a Bruker MTP 384 ground steel plate and dried at room temperature. The mass spectrometry analysis was carried out using a Bruker Ultraflextreme matrix-assisted laser desorption/ionization mass spectrometer (MALDI-TOF) MS. The analysis was carried out using a positive reflective ion mode at a mass range of 5-16 Da. Flow cytometry for analyzing cell apoptosis induced by NCP-1/siRNAs SKOV-3, A278/CDDP, and A278 cells were seeded at cells per well in 6-well plates and cultured for 24 hours. The culture media were then replaced with 2 ml of fresh culture media containing 1% FBS, and PBS (control), free cisplatin plus sirna solution, Zn control/sirnas, NCP-1, or NCP-1/siRNAs was added to the cells at a cisplatin concentration of 5 µm or equivalent nanoparticle concentration of 2 µg/ml. Cells incubated with saline served as the control. Following incubation for 24 hours, the floating and adherent cells were collected by cell scraper and stained with Alexa Fluor 488 Annexin V/dead cell apoptosis kit with Alexa Fluor 488 annexin V and PI (Invitrogen, USA) according to the manufacturer s instructions. Apoptosis was examined on a flow cytometer (LSRII Blue, BD, USA). In vivo pharmacokinetics of NCP-1/siRNAs in red and white blood cell layer and plasma layer BALB/c mouse was intraperitoneally injected with Alexa Fluor 647 labeled NCP- 1/siRNAs (NCP-1/Alexa-siRNAs) at 1 mg/kg cisplatin dose (or.25 mg/kg sirnas dose). Blood was drawn from the mouse at 3 h, 5 h, and 8 h post-injection. Whole blood was centrifuged at 3 rpm for 1 min to separate the red and white blood cell layer and plasma layer. The two layers were digested with nitric acid and analyzed for Pt concentration by inductively coupled plasmamass spectrometer (ICP-MS). Gel retardation assay of NCP-1/siRNAs in blood serum S1

2 BALB/c mouse was intraperitoneally injected with NCP-1/Alexa-siRNAs) at 1 mg/kg cisplatin dose (or.25 mg/kg sirnas dose). Blood was drawn from the mouse at 3 hours postinjection. The blood was centrifuged at 12, rpm for 4 min to obtain the serum. The association of sirna with NCP-1 in blood serum was determined with gel retardation assay on 4% (w/v) agarose gel electrophoresis containing.25 g/ml of EB at 56 V for 1 h. Lane 1 was loaded with free Alexa Fluor sirnas, while lane 2 was loaded with NCP-1/Alexa-siRNAs. In vitro stability and drug release of NCP-2 Particle stability was evaluated in vitro in phosphate buffered saline (PBS) buffer with bovine serum albumin (BSA) binding and time dependent drug release. BSA binding analysis was done by dispersing.5 mg of NCP-2 in 1 ml PBS containing BSA (3 nm) at 37 º C. DLS measurements were taken every hour for 24 hours to determine the size of nanoparticles in the suspension over time. The particle sizes, PDI, and count rate remained constant over a 24-hour period. In vitro release profiles of cisplatin and GMP from NCP-2 were performed in 4 ml in PBS buffer with or without 5 mm cysteine at 37 º C in ph 7.4. DOPA-NCP-2 or NCP-2 (2 mg) were suspended in 4 ml of 1x PBS buffer solution with or without 5 mm cysteine in a 1, MWCO pleated dialysis bag. The dialysis bag containing the nanoparticle suspension was added into the beaker containing 4 ml of 1x PBS buffer, incubated at 37 º C at ph 7.4, while stirring. 1 ml aliquots of solution were taken from the solution at preset time intervals, and a fresh 1 ml of buffer solution with or without cysteine was added to the beaker. The removed aliquot was digested in nitric acid or HCl and analyzed by ICP-MS for Pt or UV-Vis for GMP. Only 6.3% Pt release was measured from the suspension containing NCP-2 after 96 hours, while the suspension with DOPA- NCP-2 showed rapid burst release, with 47% total Pt release before 12 hours. Only 11% GMP release was observed after 12 hours for the NCP-2 suspension as compared to 29% for the DOPA- NCP-2 suspension. Drug release of NCP-2 was also simulated in the presence of 5mM cysteine with 92% Pt release and 63% GMP release before 12 hours, indicating that the NCPs underwent reductive degradation to release drugs; NCP-2 exhibited a similar drug release pattern in both PBS and PBS with cysteine. PEGylation of particles made it difficult for cysteine to penetrate the lipid layer, improving the stability of the particles in the bloodstream. Once particles entered the cells, the lipid coating incorporated into cell and plasma membranes, allowing cysteine to penetrate the core particle, triggering release of the drugs. In vitro cytotoxicity of NCP-2/siRNAs In vitro cytotoxicity assays were carried out on A278/CDDP and SKOV-3 ovarian cancer cells. In 96-well plates, A278/CDDP or SKOV-3 cells were seeded at a density of 2 cells/well in a total of 1 µl RPMI-164 or McCoy containing 1% FBS. The cells were incubated for 24 hours at 37 º C prior to drug treatment. The culture medium was then replaced by fresh medium. Different concentrations of cisplatin, GMP, free cisplatin/gmp mixture [at the same NCP-1 and GMP-NCP (NCP carrying GMP) drug dose], Zn-NCP (NCP carrying no drugs), NCP-1, NCP-2, GMP-NCP, and NCP-2/siRNAs were added and incubated at 37 º C and 5% CO2 for 72 hours. Cell viability was measured by MTS assay (Promega, USA) according to the manufacturer s instructions. IC5 values were measured. The combination index (CI) was calculated using the following equation S2

3 where D1 and D2 are concentrations of drug 1 and drug 2, respectively, in combination at a specific drug effect level (e.g., at a 5% inhibition concentration), while Dm1 and Dm2 are the concentrations of single agents required to reach the same drug effect level. The CI values were plotted against the drug effect levels (ICx values). CI values less than 1 indicate synergism. Cell apoptosis induced by NCP-2/siRNAs Six-well plates with coverslips were seeded with A278/CDDP cells at a density of cells/well. The cells were incubated at 37 º C and 5% CO2 for 24 hours prior to drug treatment. Dispersion of free drugs and RhoB-doped particles were incubated with A278/CDDP cells at 37 º C and 5% CO2 for 24 hours. Then, the cells were washed with PBS, fixed with iced 4% paraformaldehyde, and stained with Alexa Fluor 488 conjugated Annexin V (Invitrogen, USA) according to the manufacturer s instructions. The cells were imaged using a confocal laser scanning microscope (CLSM, Olympus FV1, Japan) at excitation wavelengths of 45 nm, 488 nm, 546 nm, and 647 nm to visualize nuclei (blue fluorescence), cell apoptosis (green fluorescence), nanoparticle internalization from chlorin e6 (red florescence), and sirnas uptake (violet fluorescence). A278/CDDP or SKOV-3 cells were seeded at cells/well in 6-well plates containing 2 ml total volume of cell culture medium for 24 hours at 37 º C and 5% CO2. The culture medium was replaced with fresh medium containing different drug treatments at a cisplatin concentration of.1 µm for A278/CDDP cells and 1. µm for SKOV-3 cells and/or a GMP concentration of.2 µm for A278/CDDP cells and 2. µm for SKOV-3 cells. Following incubation of 24 hours, the floating and adherent cells were collected and stained with Annexin V/dead cell apoptosis kit with Alexa Fluor 488 annexin V and propidium iodide (PI, Invitrogen, USA) according to manufacturer s instructions. The apoptosis was analyzed on a flow cytometer (LSRII 3-8, BD, USA). In vitro gene silencing of NCP-2/siRNAs In 24-well plates, SKOV-3 cells were seeded at cells/well and incubated for 24 hours. The medium was replaced with 1 ml serum-free medium containing NCP-2/siRNAs at an sirnas dose of 1 nm per type of sirna. After a 4-hour incubation, the medium was replaced with McCoy with 1% FBS and incubated for an additional 2 hours. Then the cells were lysed, and the Bcl-2 amount was quantified by enzyme-linked immunosorbent assays (ELISA, R&D Systems, USA) according to manufacturer s instructions. Bcl-2 knockdown efficiency of NCP-2/siRNAs was compared with PBS and NCP-2 by ELISA in SKOV-3 cells. NCP-2/siRNAs effectively knockdown 42. ± 7.7% of Bcl-2 gene expression. Slightly decreased Bcl-2 expression levels (by 16.8 ± 4.9%) in NCP-2 might be attributed to the cytotoxicity induced by cisplatin and gemcitabine. In vivo pharmacokinetic and biodistribution of NCP-2 S3

4 BALB/c mice bearing CT26 tumors were intravenously injected with NCP-2 at 1 mg/kg cisplatin dose (or 2 mg/kg GMP dose). Mice were sacrificed at 5 minutes, 1 hour, 3 hours, 8 hours, 24 hours, and 48 hours post-injection. Their livers, lungs, spleens, kidneys, hearts, bladders, tumors, and blood were harvested and analyzed for Pt concentration using ICP-MS. GMP concentrations in plasma were further analyzed using LC-MS (Agilent 646 QQQ MS-MS) according to the following procedure. 2 µl ice-cold acetonitrile was added to 5 µl plasma, which was then vortexed, mixed, and centrifuged. The resulting supernatant was evaporated and reconstituted in 1 µl of water. 2 µl samples were used for injections. The autosampler and column temperatures were kept at 4 and 3 º C, respectively. The samples were separated via a PGC Hypercarb column (1 2.1 ID, 5 µm, Thermo Fisher Scientific) fitted with a guard column (Hypercarb 1 2.1, 5 µm, Thermo Fisher Scientific). A gradient mobile phase of (A) 1 mm ammonium acetate at ph 1 and (B) acetonitrile were used with an initial mobile phase of 95% solvent A and 5% solvent B at a flow rate of.3 ml/min. After 2 minutes, solvent A was gradually decreased to 8% over.2 minutes and held at this condition for 5.6 minutes. The gradient was then returned to 95% solvent A over.2 minutes and held at this condition for an additional 7 minutes, for a total run time of 15 minutes. The mass to charge transition was monitored from 342 to 231. Supporting Figures Intens. [a.u.] x m/z Figure S1. MALDI-TOF mass spectrum shows strong peaks at Da for single strand sirna and its DSPE conjugate and weak peaks at Da for the DSPE conjugate of double stranded sirna. S4

5 Figure S2. NCP-1/siRNAs( ), DSPE-siRNA( ), and free sirna( ) stability in FBS. The different treatment groups were incubated with FBS for.5, 2, 12, and 24 hours. The sirna stability after incubation was evaluated by electrophoresis and quantified by measuring the intensity of sirna band with Image Lab software. Figure S3. GC spectrum confirming CO2 generation from PtBp in PBS containing 5 mm cysteine (Cys). S5

6 survivin protein (ng/ml) Transfection time (h) Figure S4. Time-dependent gene transfection of NCP-1/sisurivivin in SKOV-3 cells. NCP- 1/sisurvivin was dosed at a sirna dose of 6 nm and different transfection time was allowed. The survivin protein production was determined by ELISA (n = 3). Cell viability % free cisplatin+sirnas NCP-1 NCP-1/Bcl-2 sirna NCP-1/survivin sirna NCP-1/siRNAs cisplatin M) Figure S5. In vitro cytotoxicity of free cisplatin+sirnas( ), NCP-1( ), NCP-1/siBcl-2( ), NCP- 1/sisurvivin( ), and NCP-1/siRNAs( ) in SKOV-3 cells after a 72-hour incubation. Data are mean ± S.D. (n=3). S6

7 Cell viability % free cisplatin+sirnas NCP-1 NCP-1/Bcl-2 sirna NCP-1/survivin sirna NCP-1/siRNAs cisplatin M) Figure S6. In vitro cytotoxicity of free cisplatin+sirnas( ), NCP-1( ), NCP-1/siBcl-2( ), NCP- 1/sisurvivin( ), and NCP-1/siRNAs( ) in A278 cells after a 72-hour incubation. Data are mean ± S.D. (n=3). Cell viability % free cisplatin+sirnas NCP-1 NCP-1/Bcl-2 sirna NCP-1/survivin sirna NCP-1/siRNAs cisplatin M) Figure S7. In vitro cytotoxicity of free cisplatin+sirnas( ), NCP-1( ), NCP-1/siBcl-2( ), NCP- 1/sisurvivin( ), and NCP-1/siRNAs( ) in A278/CDDP cells after a 72-hour incubation. Data are mean ± S.D. (n=3). S7

8 Figure S8. Annexin V/PI analysis of SKOV-3 cells after a 24-hour incubation with (a) saline (control), (b) ZnControl/siRNAs, (c) free cisplatin plus sirna, (d) NCP-1, and (e) NCP-1/siRNAs. Quadrants counterclockwise from lower left to upper left represent healthy, early apoptotic, late apoptotic, and necrotic cells, respectively. S8

9 Figure S9. Annexin V/PI analysis of A278/CDDP cells after 24-hour incubation with (a) saline (control), (b) ZnControl/siRNAs, (c) free cisplatin plus sirna, (d) NCP-1, and (e) NCP-1/siRNAs. Quadrants counterclockwise from lower left to upper left represent healthy, early apoptotic, late apoptotic, and necrotic cells, respectively. S9

10 Figure S1. Annexin V/PI analysis of A278 cells after 24-hour incubation with (a) saline (control), (b) ZnControl/siRNAs, (c) free cisplatin plus sirna, (d) NCP-1, and (e) NCP-1/siRNAs. Quadrants counterclockwise from lower left to upper left represent healthy, early apoptotic, late apoptotic, and necrotic cells, respectively. A B 1 C 8 5 min 1 h 3 h 5 h 5 min 12 8 h 24 h 36 h 48 h 1 h h 8 h h 4 48 h Pt (ID%) Pt (ID%) Pt (ID%) 5 min 1h 3h 8h 24h 48h liver lung spleen kidneybladdertumor blood liver spleen lung kidney bladder ovary tumor blood Liver Lung Kidney Spleen Bladder Tumor Blood Figure S11. Pt injected dose percentage (ID%) tissue distribution of NCP-1/siRNAs at a cisplatin dose of 3 mg/kg and an sirna dose of.75 mg/kg by i.v. (A) or i.p. (B) injection. (C) Pt injected dose percentage (ID%) tissue distribution of NCP-2/siRNAs at a cisplatin dose of 1. mg/kg and a GMP dose of 2. mg/kg by i.v. injection. Data are expressed as means±sd (N=3). S1

11 1 8 Red/White Blood Cells Layer Plasma Layer Pt (ID%) h 5 h 8 h Figure S12. Pt injected dose percentage (ID %) of NCP-1/siRNAs at a cisplatin dose of 1. mg/kg and a sirnas dose of.25 mg/kg by i.p injection in red/white blood cells layer and plasma layer after centrifugation of whole blood. Figure S13. Gel retardation of NCP-1/siRNAs in blood serum after 3 h post i.p injection (4% agarose gel, 56 V, 1 h). Lane 1-2 free sirna, NCP-1/siRNAs. S11

12 3 27 Body weight (g) PBS (i.v.) free cisplatin+sirnas (i.v.) ZnControl/siRNAs (i.v.) NCP-1 (i.v.) NCP-1/siRNAs (i.v.) NCP-1/siRNAs (i.p.) NCP-1/ctrl sirna (i.p.) Day post tumor inoculation Figure S14. Body weights of SKOV-3 tumor-bearing mice after treatment with PBS by i.v.( ), free cisplatin+sirnas by i.v.( ), ZnControl/siRNAs by i.v.( ), NCP-1 by i.v.( ), and NCP- 1/siRNAs by i.v.( ), NCP-1/siRNAs by i.p.( ), and NCP-1/ctrl sirna by i.p( ) at a cisplatin dose of.75 mg/kg equivalent to an sirna dose of.18 mg/kg. (n=5 for NCP-1/ctrl sirna and n=6 for all the other groups). 9 TUNEL-positive cells (%) 6 3 PBS i.v. free cis+sirnas i.v. NCP-1 i.v. NCP-1/siRNAs i.v. Figure S15. The percentage of TUNEL-positive cells in tumor tissues of SKOV-3 tumor-bearing mice treated with an i.v. injection of NCP-1/siRNAs (n=3). S12

13 Figure S16. Histological sections of excised organs from SKOV-3 tumor-bearing mice i.v. injected with PBS and NCP-1/siRNAs. Bar = 1 m. Figure S17. An A278/CDDP intraperitoneal xenograft mouse treated with PBS survived to Day 92 post tumor inoculation but had a large tumor growing directly on the ovary as indicated by the red circle. S13

14 Figure S18. Abdominal views of A278/CDDP intraperitoneal xenograft mice treated with NCP- 1/siRNAs Day 92 post tumor inoculation. Tumors were completely eradicated in all of the mice. a).8 b) Absorbance Wavelength (nm) Absorbance y =.54 x R 2 = Concentration (ug/ml) c) Weight Change (%) NCP-1 GMP GMP-NCP DOPA-NCP-2 NCP Temperature ( o C) Figure S19. (a) and (b) UV-Vis analysis and (c) TGA analysis of DOPA-NCP-2 to determine GMP wt% loading. S14

15 Figure S2. TEM micrographs of (A, B) DOPA-NCP-2, (C, D) NCP-2, and (E, F) NCP-2/siRNAs. Size (d. nm) Z-avg PDI Count Rate B Size (d. nm) A Number-Avg PDI Count Rate 2 Time (h) Time (h) Figure S21. Stability tests of NCP-2 after PEGylation in PBS buffer with BSA at 37 ºC. Z-avg (A) and Number-avg (B) were evaluated over a 24-hour period. S15

16 A Pt Released (%) DOPA-NCP-2 NCP-2 DOPA-NCP-2 w/ cysteine NCP-2 w/ cysteine GMP Released (%) B DOPA-NCP-2 NCP-2 DOPA-NCP-2 w/ cysteine NCP-2 w/ cysteine Time (h) Time (h) Figure S22. Pt (A) and GMP (B) release profiles of DOPA-NCP-2 and NCP-2 in PBS buffer with and without 5 mm cysteine at 37 º C. 1.2 Relative Bcl-2 expression PBS NCP-2 NCP-2/siRNAs Figure S23. Relative Bcl-2 expression levels in SKOV-3 ovarian cancer cells transfected with PBS, NCP-2, and NCP-2/siRNAs at an sirna concentration of 1 nm for 4 hours (n=3). S16

17 A 12 B 12 C 1.6 Viability (%) Cisplatin Zn-NCP NCP-1 NCP-2 NCP-2/siRNAs Cisplatin Concentration ( M) Viability (%) Cisplatin Zn-NCP NCP-1 NCP-2 NCP-2/siRNAs GMP Concentration ( M) Combination Index NCP-2 vs. NCP-1 NCP-2 vs. free drugs NCP-2/siRNAs vs. NPC-1 NCP-2/siRNAs vs. free drug Drug Effect Level (%) Figure S24. In vitro cytotoxicity plots (A, B) and combination index (CI) (C) of cisplatin/gmp combinations on SKOV-3 cells. The cell viabilities of SKOV-3 cells were measured after 72-hour exposures to Zn-NCP, NCP-1, NCP-2, NCP-2/siRNAs, or free drugs (cisplatin or GMP). Data are mean ± S.D. (n=6). A B C Viability (%) 14 Cisplatin Cisplatin/GMP Zn-NCP NCP-1 NCP-2 NCP-2/siRNAs Cisplatin Concentration ( M) Viability (%) 14 Cisplatin Cisplatin/GMP Zn-NCP NCP-1 NCP-2 NCP-2/siRNAs GMP Concentration ( M) Combination Index NCP-2 vs. NCP-1 NCP-2 vs. free drug NCP-2/siRNAs vs. NCP-2 NCP-2/siRNAs vs. free drug Cisplatin/GMP vs. NCP-1 Cisplatin/GMP vs. free drug Drug Effect Level (%) Figure S25. In vitro cytotoxicity plots (A, B) and combination index (CI) (C) of cisplatin/gmp combinations on A278/CDDP cells. The cell viabilities of A278/CDDP cells were measured after 72-hour exposures to Zn-NCP, NCP-1, NCP-2, NCP-2/siRNAs, or free drugs (cisplatin or GMP). Data are mean ± S.D. (n=6). S17

18 Figure S26. CLSM images showing the apoptosis induced by saline, cisplatin, GMP, RhoB-Zn Control, RhoB-NCP-1, RhoB-GMP-NCP, and RhoB-NCP-2 in A278/CDDP ovarian cancer cells. GMP-NCP nanoparticles carry mono-chemotherapeutic GMP. The first column shows DAPI-stained nuclei. The second column shows Annexin-5-stained cells. The third column shows RhoB-labeled particles (only present in NCP particles). The fourth column shows a merged image of all three stainings. The fifth column is the DIC. Bar = 2 m. S18

19 Figure S27. CLSM images showing the apoptosis induced by RhoB-NCP-2/siRNAs in A278/CDDP ovarian cancer cells. The first column shows DAPI-stained nuclei. The second column shows Annexin-5-stained cells. The third column shows RhoB-labeled particles (only present in NCP particles). The fourth column shows Alexa Fluor 647-labeled sirna. The fifth column is a merged image of all four stainings. The sixth column is the DIC. Bar = 2 m. Control PBS Cisplatin GMP Zn Zn-NCP Control %.9% % 1.26% % 23.48% %.15% PI % 32.2% %.1% % %.% 73.89% PEG-NCP-1 PEG-NCP-3 GMP-NCP PEG-NCP-4-3 NCP-2 NCP-2/siRNAs NCP-4/siRNAs % 1.52% % 42.54% % 44.19% 2.7% 4.31% %.23% % 12.48% % 41.27% % 87.14% Annexin V Figure S28. Flow cytometry analysis of saline, cisplatin, GMP, Zn-NCP, NCP-1, GMP-NCP, NCP-2, and NCP-2/siRNAs in SKOV-3 ovarian cancer cells. GMP-NCP nanoparticles carry mono-chemotherapeutic GMP. Quadrants counterclockwise from lower left to upper left represent healthy, early apoptotic, late apoptotic, and necrotic cells, respectively. Supporting Tables Table S1 Percentages of healthy, apoptotic, and necrotic SKOV-3, A278/CDDP, and A278 cells after a 24-hour treatment of saline (control), NCP-1, NCP-1/siRNAs, Zn control, Zn control/sirnas, and free cisplatin. Healthy (%) Apoptosis (%) Necrosis (%) SKOV-3 Control 1... S19

20 Free cisplatin+sirna NCP NCP-1/siRNAs Zn Control/siRNAs A278/CDDP Control Free cisplatin+sirna NCP NCP-1/siRNAs Zn Control/siRNAs A278 Control Free cisplatin+sirna NCP NCP-1/siRNAs Zn Control/siRNAs Table S2 Primer sequences of β-actin, Bcl-2, and survivin for Real-Time PCR. Primer F β-actin 5 - CCACCCATGGCAAATTCCATGGCA- 3 Bcl GTGGAGGAGCTCTTCAGGGA-3 survivin 5 - GGCATGGGTGCCCCGACGTT-3 Primer R 5 - TCTAGACGGCAGGTCAGGTCCACC-3 5 -AGGCACCCAGGGTGATGCAA-3 5 -AGAGGCCTCAATCCATGGCA-3 Table S3 Sizes, polydispersities, zeta potentials, and drug loadings of NCP-2/siRNAs nanoparticles. Z-Ave diameter Number-Ave PDI Zeta Potential (nm) diameter (nm) (mv) DOPA-NCP ±.1 # 28.±1. #.116 NA NCP-2 83.±1. $ 51.2±.1 $ ±.22 $ NCP-2/siRNAs 84.1±.9 $ 48.8±3.2 $ ±.6 $ # Measured in THF. $ Measured in PBS buffer. Data are expressed as means±sd. Table S4. Cisplatin IC5 values of cisplatin, GMP, NCP-1, NCP-2, GMP-NCP, and NCP- 2/siRNAs in A278/CDDP and SKOV-3 cells (numbers in parentheses refer to GMP concentrations). GMP-NCP nanoparticles carry mono-chemotherapeutic GMP. Cisplatin(μM) GMP (μm) Zn Control * (μm) A278/CDDP ±1.456 (.274±.49) >25 (>5) NCP-1 (μm) GMP-NCP (μm) NCP-2 ( M) 9.283±1.424 (.364±.96).82±.17 (.163±.34) NCP- 2/siRNAs ( M).24±.2 (.51±.4) S2

21 SKOV-3 2.6±.4 (3.44±.98) >5 (>5) 8.18±1.1 (2.48±.31).7±.18 (1.43±.36).22±.4 (.46±.8) *Zn Control does not contain cisplatin or GMP to serve as a control to study the toxicity of NCP formulations. The number of Zn Control particles used was the same as NCP-2 under the studied concentrations. Data are expressed as means±sd. Table S5. Percentage of apoptotic cells determined by flow cytometry of SKOV-3 ovarian cancer cells. GMP-NCP nanoparticles carry mono-chemotherapeutic GMP. Samples % Apoptotic Cell Saline.99 Cisplatin 1.82 GMP 55.5 Zn-NCP.16 NCP GMP-NCP 55.2 NCP NCP-2/siRNAs S21