Supporting Information for. Post-Synthetic Modifications of Iron-Carboxylate Nanoscale Metal-Organic Frameworks for Imaging and Drug Delivery

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1 Supporting Information for Post-Synthetic Modifications of Iron-Carboxylate Nanoscale Metal-Organic Frameworks for Imaging and Drug Delivery Kathryn M. L. Taylor-Pashow, Joseph Della Rocca, Zhigang Xie, Sylvie Tran, and Wenbin Lin* Department of Chemistry, CB#3290, University of North Carolina, Chapel Hill, NC Materials and Methods. All starting materials were purchased from Aldrich and used without further purification. All solvents were purchased from Fisher. Microwave reactions were carried out in a CEM MARS 5 microwave. Thermogravimetric analysis (TGA) was performed using a Shimadzu TGA-50 equipped with a platinum pan and heated at a rate of 3 C per minute under air. Powder X-ray diffraction (PXRD) analyses were carried out using a Rigaku Multiflex powder diffractometer using Cu radiation or on a Bruker SMART APEX II diffractometer using Cu radiation. In the latter, the PXRD patterns were processed with the APEX 2 package using phase ID plugin. A Hitachi 4700 field emission scanning electron microscope (SEM) and a JEM 100CX-II transmission electron microscope (TEM) were used to determine particle size and morphology. A Cressington 108 Auto Sputter Coater equipped with a Au/Pd (80/20) target and an MTM-10 thickness monitor was used to coat the samples with a 5 nm thick conductive layer before taking SEM images. Each SEM sample was prepared by suspending the nanoparticles in ethanol. A drop of the suspension was then placed on a glass slide and the solvent was allowed to evaporate. TEM samples were also prepared from ethanolic particle dispersions on amorphous carbon coated copper grids. A Varian 820-MS Inductively Coupled Plasma-Mass Spectrometer was used to measure Fe, and Pt concentrations. 2. Synthesis of MIL-101 (Fe) Nanoparticles. Synthesis of MIL-101 (Fe) Nanoparticles (1). Fe 3 O(H 2 O) 2 Cl(BDC) 3 nanoparticles (1) were prepared using microwave heating g (0.346 mmol) of terephthalic acid and S1

2 g (0.346 mmol) of FeCl 3 H 2 O were dissolved in 15 ml of DMF. The solution was placed in a HP500 microwave vessel, and sealed. The reaction was then rapidly heated to 150 C, and was held at this temperature for 10 minutes. After cooling to room temperature, the particles were isolated by centrifuging, and were washed with DMF and ethanol. Synthesis of amino-mil Nanoparticles (1a and 2). Amino-functionalized MIL nanoparticles (1a and 2) were prepared using a similar procedure as above except a certain percentage of BDC was replaced with amino-bdc. The synthetic procedure for the MIL nanoparticles containing 100% amino-bdc is illustrated here g ( mmol) of 2-aminoterephthalic acid and g ( mmol) of FeCl 3 H 2 O were dissolved in 30 ml of DMF. The solution was placed in a HP500 microwave vessel, and sealed. The reaction was then rapidly heated to 150 C, and was held at this temperature for 15 minutes. After cooling to room temperature, the particles were isolated by centrifuging, and were washed with DMF and ethanol. As shown in Table S1, nanoparticles of 1a and 2 with different percents of amino-bdc incorporation were prepared and their phases determined by PXRD. Particles 1a are further divided into 1a-1 through 1a-5 depending on the molar ratio of amino-bdc in the feed, whereas Particles 2 are further divided into 2-1 through 2-3 depending on the molar ratio of amino-bdc in the feed. Table S1. Composition and phase of Fe(III) NMOFs. Compound mol% amino-bdc in the feed mol% incorporated amino-bdc determined by NMR S2 Phase as determined by PXRD MIL-101 1a MIL-101 1a MIL-101 1a MIL-101 1a MIL-101 1a MIL MIL-88B MIL-88B MIL-88B Since these particle synthesis reactions always gave low yields (~20%), the actual amount of incorporated 2-aminoterephthalic acid was determined by NMR. The particles were first digested using silica-supported triaminetetraacetate sodium salt. After the

3 digestion was complete, the silica was removed by centrifuging and the supernatant was dried. A methylation reaction was then performed by re-dissolving the residue obtained from drying the supernatant in methanol and adding a catalytic amount of concentrated sulfuric acid. The reaction was then heated to reflux overnight. The product was then extracted with ethyl acetate. NMR was then used to determine the ratio between the methyl esters of terephthalic acid and 2-aminoterephthalic acid. In most cases the incorporation of 2-aminoterephthalic acid was slightly higher than the ratio added (Table S1). Figure S1. SEM images of Fe 3 O(H 2 O) 2 Cl(BDC) 3 nanoparticles (1). Figure S2. SEM images of Fe 3 O(H 2 O) 2 Cl(BDC) 3 nanoparticles synthesized with 5 mol% NH 2 -BDC (1a-1). S3

4 Figure S3. SEM images of Fe 3 O(H 2 O) 2 Cl(BDC) 3 nanoparticles synthesized with 10 mol% NH 2 -BDC (1a-2). Figure S4. SEM images of Fe 3 O(H 2 O) 2 Cl(BDC) 3 nanoparticles synthesized with 12.5 mol% NH 2 -BDC (1a-3). Figure S5. SEM images of Fe 3 O(H 2 O) 2 Cl(BDC) 3 nanoparticles synthesized with 15 mol% NH 2 -BDC (1a-4). S4

5 Figure S6. SEM images of Fe 3 O(H 2 O) 2 Cl(BDC) 3 nanoparticles synthesized with 17.5 mol% NH 2 -BDC (1a-5). Figure S7. SEM images of Fe 3 O(DMF) 3 Cl(BDC) 3 nanoparticles synthesized with 20 mol% NH 2 -BDC (2-1). Figure S8. SEM images of Fe 3 O(DMF) 3 Cl(BDC) 3 nanoparticles synthesized with 50 mol% NH 2 -BDC (2-2). S5

6 Figure S9. SEM images of Fe 3 O(DMF) 3 Cl(BDC) 3 nanoparticles synthesized with 100 mol% NH 2 -BDC (2-3). Intensity (arb.) Theta Figure S10. PXRD pattern of 1 (red) compared to the simulated pattern for MIL-101 (Cr) (blue). S6

7 Theta Figure S11. PXRD pattern of NMOFs 2-3 (red) compared to the simulated pattern for MIL-88B (blue). Intensity (arb.) Intensity (arb.) Theta Figure S12. PXRD patterns of NMOFs 1a-1 (red), 1a-2 (green), 1a-3 (pink), 1a-4 (black), and 1a-5 (light blue) compared to the simulated pattern for MIL-101 (Cr) (dark blue). S7

8 Intensity (arb.) Theta Figure S13. PXRD pattern of NMOFs 2-1 (red) and 2-2 (green) compared to the simulated pattern for MIL-88B (blue) Volume (cc/g) Relative Pressure, P/Po Figure S14. Nitrogen sorption isotherm of NMOFs 1 synthesized using oven (blueadsorption, pink-desorption) and microwave heating (green-adsorption, red-desorption). NMOF 1 has a Langmuir surface area ranging from 3700 m 2 /g to 4535 m 2 /g. S8

9 Volume (cc/g) Relative Pressure, P/Po Figure S15. Nitrogen sorption isotherm of NMOFs 2-3 (blue-adsorption, pinkdesorption). NMOF 2-3 has a Langmuir surface area of 2550 m 2 /g. 3. Post-Synthetic Modifications of MIL-101 (Fe) Nanoparticles. BODIPY Functionalized amino-mil-101 Nanoparticles (1b). 6.5 ml of a suspension of 1a-5 in ethanol was centrifuged to isolate 23.3 mg of particles. The particles were washed twice with THF before being re-dispersed in10 ml of THF mg (74.2 µmol) of Br-BODIPY was dissolved in 4 ml of THF and was added to the particle suspension. The reaction was then stirred at r.t. for 2 days. The particles were then isolated by centrifuging, and were washed with THF until a colorless supernatant was observed. The particles were washed once with ethanol and stored in ethanol. Figure S16. SEM image of NMOF 1a-5 as synthesized (left) and after Br-Bodipy loading (right) S9

10 Figure S17. TGA analysis of as synthesized 1a-5 nanoparticles (red line) and after Bodipy loading (blue line). Figure S18. Powder X-ray diffraction patterns of simulated MIL101 (Cr) (blue line), as synthesized 1a-5 (red line) and 1-b (orange line) ESCP-Grafted amino-mil-101 Nanoparticles (1c). This method involved first synthesizing the c,c,t-[pt(iv)cl 2 (NH 3 ) 2 (OEt)(O 2 CCH 2 CH 2 CO 2 H)] complex using a previously published procedure. 1 For loading into 1a-5, 22 mg ( mmol) of c,c,t- 1 Feazell, R. P.; Nakayama Ratchford, N.; Dai, H.; Lippard, S. J., Soluble single walled carbon nanotubes as longboat delivery systems for platinum(iv) anticancer drug design. J. Am. Chem. Soc. 2007, 129 (27), S10

11 [Pt(IV)Cl 2 (NH 3 ) 2 (OEt)(O 2 CCH 2 CH 2 CO 2 H)] was dissolved in 3 ml of anhydrous DMF along with 9.3 mg ( mmol) of 1,1-carbonyldiimidazole. The reaction was then heated at 60 C for 1 hour, under Ar. After cooling to room temperature, 85 mg of dried 1a-5 was then added and the reaction was stirred at room temperature for 2 days. The product was then isolated by centrifuging, and was washed with DMF and ethanol before being dialyzed against distilled water for 5 hours, changing water once an hour. Figure S19. SEM images of NMOFs 1a-5 (as synthesized (left) and after ESCP loading (right). Intensity (arb.) Figure S20. PXRD of NMOFs 1a (with 17.4 mol% incorporated amino-bdc) as synthesized (blue) and after Pt loading using CDI coupling method (red). 4. Silica Coating Procedures Theta Silica coated 1a-5 nanoparticles. An ethanol suspension of NMOF 1a-5 containing 10 mg of nanoparticles was isolated by centrifugation and redispersed into 4 ml of water. Sodium S11

12 silicate (Na 2 SiO 3 5H 2 O, 47.5 mg, mmol) was dissolved into 2 ml of water and the ph of the silicate solution was adjusted to approximately 7 using HCl. The silicate solution was added to the nanoparticle suspension and the reaction was diluted to a final volume of 20 ml in water. The suspension was stirred for 3 hours at 60 C. The particles were isolated by centrifugation, washed with water and ethanol, and redispersed in ethanol. The same procedure was used to coat NMOFs 1b and 1c with silica as well. Figure S21. SEM micrographs of 1a-5@silica (left), 1b@silica (center), 1c@silica (right). Figure S22. PXRD patterns for simulated MIL101 (Cr) (blue), 1a-5 (red) and 1a- 5@silica (green) S12

13 Figure S23. TGA analysis of 1b (blue) and (red). Figure S24. TGA analysis of 1c (red) and (blue) Figure S25. EDX spectra of (left) and (right). Table S2: EDX data for and S13

14 Sample Fe (atomic %) Si (atomic %) Ratio (Si:Fe) RGD peptide functionalization of nanoparticles. Tri(ethoxy)silylpropyl carbomoyl c(rgdfk) was synthesized according to an established procedure. Silica coated Pt-MIL101 nanoparticles were suspended in absolute ethanol at a concentration of 2 mg/ml. An aliquot of the c (RGDfK) solution was added to the suspension (10 mass %). The ph of the resulting suspension was diluted to approximately 3 using 5M HCl (aq). The mixture was magnetically stirred in the dark for 24 hours. The functionalized particles were isolated by centrifugation, washed twice with ethanol and redispersed in ethanol. Figure S26. TGA weight loss analysis for 1c (red), 1c@silica (blue) and 1c@silica-cRGD (green) Figure S27. SEM micrograph of 1c@silica-cRGD S14

15 5. Release Profiles Release Profile of 1b, 1c, and An aliquot of the NMOF suspension (2mg) was isolated by centrifugation and re-suspended into 2mL of PBS. The aliquot was added to a 3500MW cutoff dialysis bag (Pierce). The bag was then placed into 250mL of PBS incubated at 37 C. The system was incubated at 37 C for 2 days, with aliquots removed periodically. The concentration of the Bodipy-BDC conjugate was measured by fluorescence spectrometry. The platinum concentrations were measured by ICP-MS. Figure S28. Release profile of 1b@silica (red) as measured by fluorescence spectroscopy. 6. In vitro studies Cell Lines: HT-29 human colon adenocarcinoma cells (ATC # HTB 28) were purchased from the Tissue Culture Facility of the Lineberger Comprehensive Cancer Center. Cells were cultured in McCoy s 5A media (Cellgro) with 10% FBS (Sigma) and 2% penicillin/streptomycin (Cellgro). Cell viability assays: HT-29 cells were grown in 96 well plates at 2000 cells/well in 100μL total media. After 18 hours of incubation, the media was removed and replaced with 100μL fresh media containing drug solution. Drug concentrations were varied as indicated and all measurements were performed in quadruplicate. Cell viability was measured after 72 hours using Cell Titer 96 Aqueous One Solution Assay (Promega) according to the manufacturer s protocol. S15

16 Figure S29. Cell viability assay of HT-29 human colon adenocarcinoma cells incubated with cisplatin (red), (purple) and (green). The IC 50 values were 20 μm (cisplatin), 21 μm and 29 μm Figure S30. Cell viability assay of HT-29 human colon adenocarcinoma cells incubated with cisplatin (blue), and 1c (red). The IC 50 values were 14 μm (cisplatin), 23 μm (1c). Confocal Microscopy: Microscope coverslips were silanized according to a reported protocol. 2 HT-29 cells were plated in 6 well plates with microscope coverslips at a density of 300,000 cells/well in 4mL media. The plate was returned to the incubator for 18 hours to promote cell attachment. The media was removed, washed once with phosphate buffered saline, and replaced with fresh media containing nanoparticle-dye 2 Cold Spring Harb. Protoc.; 2008; doi: /pdb.prot4988 S16

17 conjugates as indicated. The plate was returned to the incubator for 90 minutes. The media was then removed and each well washed with 1mL of PBS, before adding 1 ml media. Cell nuclei were stained with DRAQ5 (Biostatus Limited). The coverslips were removed from the wells and imaged at the Microscopy Services Laboratory of the Department of Pathology and Laboratory Medicine at UNC-Chapel Hill. An Olympus FV500 Confocal Laser Scanning Microscope was used with DIC settings. The DRAQ5 nuclear stain was imaged with 633nm excitation and a 660 nm long pass emission filter. The Bodipy-MIL101 conjugate was imaged using 488nm excitation and a 560 nm long pass emission filter. The composite images were made using photoshop. The images of the Bodipy-BDC conjugate were taken using a Zeiss LSM5 Pascal inverted confocal laser scanning microscope. The dye was imaged using 488nm excitation and a 560 long pass emission filter. A). B). C). S17

18 Figure S31. Laser Scanning Confocal Fluorescence studies of HT-29 cells incubated with no nanoparticle (A), 0.19 mg/ml (equivalent to 17 μm Bodipy) (B), and 0.38 mg/ml (equivalent to 34 μm Bodipy) (C). The Bodipy fluorescent channel is left (green), the DRAQ 5 nuclear stain is center (blue) and the DIC image is right. All scale bars indicate 25 μm. Figure S32. Laser Scanning Confocal microscopy images of 17μM Bodipy-BDC (top) and 34 μm Bodipy-BDC (bottom). The Bodipy-BDC flourescent channel is left and the DIC image is on the right. All scale bars are 25 μm. S18