Hyaluronic Acid-Modified Polymeric Gatekeepers on Biodegradable Mesoporous Silica Nanoparticles for Targeted Cancer Therapy

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1 Supporting Information Hyaluronic Acid-Modified Polymeric Gatekeepers on Biodegradable Mesoporous Silica Nanoparticles for Targeted Cancer Therapy L.Palanikumar, 1 Jimin Kim, 1 Jun Yong Oh, 1 Huyeon Choi, 1 Myoung-Hwan Park, 2 * Chaekyu Kim, 1 * Ja-Hyoung Ryu 1, * 1 Department of Chemistry, School of Natural Science, Ulsan National Institute of Science and Technology, Ulsan 44919, Republic of Korea 2 Department of Chemistry, Sahmyook University, Seoul 01795, Republic of Korea mpark@syu.ac.kr (M-H. Park), chaekyu@unist.ac.kr (C. Kim), and jhryu@unist.ac.kr (J.-H. Ryu) Experimental Section General. All chemicals and reagents were obtained from TCI, Korea and Sigma Aldrich, Korea unless otherwise specified. 2-Aminoethyl methacrylate hydrochloride, polyethylene glycol methacrylate, and D,L-dithiothreitol (DTT) was obtained from Sigma-Aldrich and 2,2 -Dithiodipyridine was obtained from TCI, Japan. Si(OC 4 H 9 ) 4 (TBOS) was purchased from TCI, Japan. Doxorubicin hydrochloride (Dox) was obtained from Acorn Pharma, China. Dulbecco s modified Eagle s medium, RPMI-1640 medium, penicillin-streptomycin, 0.25% trypsin-edta, and fetal bovine serum were obtained from Life Technologies, Korea. PEG-PDS-NH 2 random copolymer synthesis. 2-Aminoethyl methacrylate hydrochloride (42.3 mg, mmol) was dissolved in methanol and pyridyldithioethyl methacrylate (200 mg, S1

2 0.784 mmol), poly(ethylene glycol) methacrylate (248 mg, mmol), and azobisisobutyroni -trile (2 mg, mmol) was added to mixture. Then degassed by Ar purging for 30 min and stirred 70 C for 12 h. To remove unreacted reactants and purify the polymer, the mixture was precipitated in cold diethyl ether, yielding a random copolymer as confirmed by 1 H NMR (400 MHz, CDCL 3 ): δ 8.45, 7.66, 7.10, , , 3.55, 3.02, , 1.25, To increase the positive surface charge of the copolymer and its water solubility, 1 equivalent of HCl corresponding to ratio of PDS groups was added to the distilled polymer solution and sonicated to yield a homogenous dispersion. Synthesis of biodegradable mesoporous silica nanoparticles. Biodegradable MSNs were prepared according to a previous report. 1 Approximately g and 2.0 g of triethanolamine and hexadecyl trimethyl ammonium bromide (C 16 TMABr), respectively, were added to 240 ml of water, the ph of the solution was adjusted to 9.5, and stirred continuously for 30 min at 80 C. Subsequently, 11 mmol of tetrabutoxysilane (TBOS) was added to the above solution and continuously stirred for 6 h at 80 C. The obtained colloidal solution was then filtered and washed several times to remove the surfactant. Installation of polymer gatekeepers in the Dox loaded MSN. Approximately 5 mg of MSNs were dispersed in 1 ml of aqueous solution containing 5 mg of Dox and allowed to load into the MSNs for 24 h at 25 C continuous stirring. For the one-pot synthesis, approximately 10 mg of polymer solution was added to the above mixture and stirred continuously for 24 h to afford polymer capping. To encapsulate the Dox loaded PMSNs, the shells were crosslinked by adding 10 or 50 mol% of DTT with respect to the PDS groups in the polymer and stirred overnight at room temperature. Subsequently, the samples were centrifuged, washed by re- S2

3 dispersion cycles to remove unloaded Dox, and collected for future use. Analysis of the UVvis spectra was used to determine the Dox loading using the following equations 2, Entrapment efficiency (%) = (Mass of drug in the MSNs) / (Initial mass of drug) Drug loading (%) = (Mass of drug in the MSNs) / (Mass of drug the loaded MSNPs) The DLS, zeta potential, and TEM were used to characterize the polymer capping on the surface of the MSN. HA grafting over the surface of PMSNs The unreacted amine groups onto PMSNs can be reacted with carboxylic units onto hyaluronic acid (HA) as our targeting ligand following the previous report. 3 Hyaluronic acid (1 mg) was dissolved in 1 ml of water and 1-ethyl-3-(3- dimethylaminopropyl)carbodiimide hydrochloride (EDC) (10.9 mg, mmol) was added and then N-Hydroxysuccinimide (NHS) (8.0 mg, mmol) to activate carboxylic acid of hyaluronic acid. Approximately 5 mg of PMSNs were dispersed well in 1 mg/ml of hyaluronic acid solution which was reactivated with EDC/NHS coupling agents, allowed to stir for 24 h to initiate the conjugation between PMSNs and HA. After the stipulated time point, the conjugated samples were collected by centrifugation, washed twice with water and phosphate buffer solution. The conjugation of HA over the surface of PMSNs were confirmed by carbazole assay. Drug release profile analysis. The in vitro release of Dox from the PMSNs was investigated with varying GSH concentrations. The release of Dox from the nanoparticles was measured using fluorescence with excitation at 480 nm and emission at 580 nm. Cell culture. HeK293T (human embryonic kidney), HeLa (human cervical cancer), and NIH 3T3 (fibroblast) cells were cultured in DMEM medium containing 10% FBS, penicillin (100 U ml -1 )-streptomycin (100 µg ml -1 ), and L-glutamine (2 mm) in a humidified atmosphere with 5% CO 2 at 37 C. S3

4 Cell viability analysis. HeLa and NIH3T3 cells were cultured in sterile 96-well Nunc (Thermo Fisher Scientific Inc.) microtiter plate at a seeding density of 5 x 10 3 cells/well and allowed to settle for 24 h at 37 C and 5% CO 2 in DMEM medium. To determine viability, the cells were treated with different concentrations of plain MSNs and PMSNs (1, 5, 10, 20, 30, 50, 75, 100, and 200 µg/ml), and Dox loaded PMSNs and HA-PMSNs (concentrations of 0.01, 0.25, 0.5, 1.00, and 2.00 µg/ml of drugs). Cell viability was measured at 24 h using the alamar blue assay, with each data point measured in triplicate. Fluorescence measurements were performed using the plate reader (Tecan Infinite Series, Germany) by setting the excitation wavelength to 565 nm and monitoring emission at 590 nm. Confocal microscope imaging studies. HeLa and NIH3T3 cells were seeded in well glass cover slips at a seeding density of 2 x 10 5 cells/well. After 24 h, cells were treated with Dox loaded HA-PMSNs (10 µg/ml of Dox). The cellular uptake was monitored in the cover glass (Lab Tek II glass chamber cover glass, Thermo Fisher Scientific Inc) periodically using a FV1000 Olympus microscope connected to CO 2 incubator. Flow-cytometry analysis. To analyze the cellular uptake of non-ligand and ligand decorated PMSNs, HeLa cells were seeded at a density of 5 x 10 3 cells in 24 well plates. After 24 h, the cells were treated with non-ligand and ligand decorated PMSNs and HA-PMSNs for 3 h. Subsequently, the cellular uptake of Dox was analyzed using fluorescence activated cell sorting(facs caliber, BD Bioscience). S4

5 Figures: a) Quantity Adsorbed (cm 3 /g STP) Adsorption Desorption Relative Pressure (P/P o ) Figure S1. BET and BJH analysis of the biodegradable MSNs b) Pore Area (m 2 /g.nm) BJH Pore volume Pore Diameter (nm) i h i h i h e e e f f CDCl3 j g ether a b b c d g ether a b c e, e, e DCM d f i, i, i THF Acetone j h, h, h Figure S2. 1 H NMR spectroscopic characterization of the PEG-PDS-NH 2 polymers S5

6 a) b) mol% crosslinked mol% crosslinked Absorbance Absorbance Pyridothione Wavelength (nm) c) HA-Carbazole assay Wavelength (nm) Absorbance Wavelength (nm) Figure S3. UV-visible spectroscopic analysis of the crosslinking density of PEG-PDS-NH 2 at different mol % of DTT addition (10 mol% and 50 mol%) resulting at (a) 26 mol%, and (b) 62 mol%. (c) The amount of HA conjugation in PMSNs was quantified by using carbazole assay of the supernatant after centrifuging PMSNs. The crosslinking density was calculated using the known molar excitation coefficient of 8.08 x 10 3 M -1 cm -1 at 343 nm. The percentage of crosslinking was calculated by assuming that formation of a single, crosslinking disulfide bond would require cleavage of two PDS units and produce two pyridothione molecules. S6

7 0.05 Dox loaded MSNs 0.04 Absorbance Wavelength (nm) Figure S4. Dox loading capacity analysis in MSNs. UV-Visible spectra for Dox.HCl of 1000 times diluted solution of the supernatant after loading and calculation of loading capacity using the absorbance peak of Dox.HCl by beer s law. To calculate the Dox loading capacity in detail, from Figure S8: Approximately 5 mg of MSNs were dispersed in 1 ml of aqueous solution containing 5 mg of Dox and allowed to load into the MSNs for 24 h at 25 C continuous stirring and centrifuged. Then the Dox loading capacity were calculated using the Beer s law using the following calculation from 1000 times dilute solution of the supernatant containing unloaded Dox, A= ɛ b [c], where ɛ is Molar absorption coefficient; c = Concentration; b = Path length (1 cm); ɛ at 480 nm for Dox = 11,500 M -1 cm 1 Therefore the amount of Dox in supernatant solution [c] = Intensity / ɛ b = 0.02 / M -1 = x 10-6 mol / L Amount of Dox in supernatant = [c] x Dox molecular weight x dilution factor = x 10-6 mol / L x g x 3.85 ml (final volume of the supernatant after washing ) x 1000 (dilution factor) = mg. To calculate the amount of Dox in MSNs, we used the following method, Initial weight of Dox - final weight of Dox in supernatant = 5.0 mg mg = 1.03 mg Drug loading capacity = 1.03 / 5 = 20.6 wt% (approx. 20 wt%) Encapsulation efficiency = 1.03 / 5 = 20.6 wt% S7

8 % of Max PMSN HA-PMSN FL3-H Figure S5. Flow cytometry analysis of a control (red line), Dox loaded PMSNs (blue line), and HA-PMSNs (green line) after 3 h of incubation in HeLa cancer cells Figure S6. Confocal microscope imaging to check the uptake of Dox-PMSNs without HA after 1h incubation in A) HeLa and B) NIH 3T3 cells. Scale bar represents 10 µm. S8

9 100 MSNs PMSNs Cell Viability (%) Ctrl Concentration (µg/ml) Figure S7. Cell viability of the biocompatible MSNs and PMSNs in HeLa cells. References [1] H. Yamada, C. Urata, Y. Aoyama, S. Osada, Y. Yamauchi, and K. Kuroda. Chem. Mater. 2012, 24, DOI: /cm [2] B. Chang, J. Guo, C. Liu, J. Qian, and W. Yang. J. Mater. Chem. 2010, 20, DOI: /C0JM01237H [3] P. V. Almeida, M.-A. Shahbazi, E. Mäkilä, M.Kaasalainen, J. Salonen, J. Hirvonen, H. A. Santos,. Amine-modified hyaluronic acid-functionalized porous silicon nanoparticles for targeting breast cancer tumors. Nanoscale. 2014, 6, DOI: /c4nr02187h S9