Supporting Information
Biodegradable Oxamide-Phenylene-Based Mesoporous Organosilica Nanoparticles with Unprecedented Drug Payloads for Delivery in Cells
Jonas G. Croissant,[a, b] Yevhen Fatieiev,[a] Khachatur Julfakyan,[a] Jie Lu,[c] Abdul-Hamid Emwas,[d] Dalaver H. Anjum,[d] Haneen Omar,[a] Fuyuhiko Tamanoi,[c] Jeffrey I. Zink,[b] and Niveen M. Khashab.*[a]
[a] Smart Hybrid Materials Laboratory, Advanced Membranes and Porous Materials Center, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia
E-mail: [email protected]
[b] Department of Chemistry and Biochemistry, California NanoSystems Institute, Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California, Unites States
E-mail: [email protected]
[c] Department of Microbiology, Immunology and Molecular Genetics, California NanoSystems Institute, Jonsson Comprehensive Cancer Center, University of California Los Angeles, Los Angeles, California, USA.
[d] Imaging and Characterization Laboratory, King Abdullah University of Science and Technology, Thuwal, Saudi Arabia.
I. GENERAL PROCEDURES
I.1- Materials. Absolute ethanol, ammonium nitrate, sodium hydroxide, 1,4- bis(trimethoxysilyl)-benzene, hydrochloric acid, dimethyl sulfoxide (DMSO), fluorescein isothiocyanate (FITC, 90%), and cetyltrimethylammonium bromide (CTAB) were purchased from Sigma-Aldrich. Oxalyl chloride was purchased from Alfa Aesar. Anhydrous dichloromethane (DCM) was purchased from Acros. 3-trihydroxysilylpropyl methylphosphonate was purchased from Gelest. All chemicals were used without further purification. Deionized water was used in all synthetic experiments. Phosphate buffered solutions (PBS, pH 7.4) and trypsin were purchased from Invitrogen (USA).
I.2- Methods. 1H NMR and 13C NMR spectra were performed at 500 MHz with NB/CDCl3
solutions at 5 mg mL-1 with an Avance III Bruker Corporation instrument. IR spectra were recorded on a Thermo Scientific spectrometer (Nicolet iS10). Absorption spectra were recorded on a Varian Cary 5000 spectrophotometer and fluorescence data were collected on a
were performed using Malvern Nano ZS and ZetaPALS Brookhaven and Zetasizer Nano ZS Malvern instruments. Transition electron microscopy (TEM) images were recorded with a Technai 12 T (FEI Co.) microscope operated at 120 kV. Scanning electron microscopy (SEM) images were recorded with a field emission Nova Nano 630 microscope (FEI Co.). Thermal gravimetric analyses were done on TG 209 F1 machine. The elemental mapping of particles was carried out with another TEM of model Titan G2 80-300CT from FEI Company (Hillsboro, OR) which was equipped with a post-column energy filter of model GIF tridium 863 from Gatan Inc. (Pleasanton, CA). The GIF was used in EELS mode in conjunction with HAADF-STEM. Moreover Si-L23 (99 eV), S-L23 (165 eV), C-K (283 eV), N-K (401 eV) and O-K (532 eV) energy-loss edges were utilized in making Si, S, C, N and O elemental maps, respectively. A typical EELS spectrum showing the energy loss edges of these elements extracted from a spectrum image dataset is shown in Figure S9. While the elemental maps of above listed elements from a typical size NP is shown in Figure 2E. Zeiss LSM 710 upright confocal microscope. N2 adsorption-desorption isotherm and corresponding pore size distribution were acquired to characterize the porous structure of MON on a Micromeritics ASAP 2420 instrument. NMR experiments were carried out in a Bruker Avance III 400 MHz spectrometer (BrukerBioSpin, Rheinstetten, Germany). All samples were prepared by packing 40-50 mg of the samples in standard 4 mm zirconia rotors from Bruker and sealed at the open end with a Vespel cap. The 13C CP MAS NMR spectra were recorded at a resonance frequency of 100.622 MHz using a Double Resonance Broadband BB/1H 4 mm Bruker CP/MAS probe. To achieve a sufficient signal-to-noise ratio, at least 12 k transients were collected with 10 s recycle delay. To distinguish between real peaks and spinning side bands we recorded 13C spectra at different spinning rate (10 KHz, and 12 KHz). The temperature for all experiments was kept at 298 °K. The cross-polarization CP contact time was set to 3 ms employing ramp100 for variable amplitude CP. In order to employ comparative analysis we run the NMR spectra under the same experimental and instrumental conditions. Bruker Topspin 3.0 software was used for data collection and for spectral analysis.
II. SYNTHESIS AND CHARACTERIZATION OF THE BRIDGED ALKOXYSILANE II.1- Synthesis of the precursor. First, a 50 mL round-bottom flask was dried under vacuum using a heat gun. Dry DCM (10 mL) was added to the flask via canula followed by the addition of TEA (420 L, 3 mmol). Then, APTES (834 L, 2.1 mmol) was added to this
added dropwise (500 L, 1 mmol, 2 M). After the complete addition, the reaction mixture was removed from the ice bath and stirred at room temperature for 1 h; the colorless solution gradually became yellowish. The solvent were evaporated under reduced pressure by a rotary evaporator, and the obtained product was crystalline (475 mg, 95 %).
Figure S1. Synthetic route for the design of the bridged organoalkoxysilane precursor.
II.2- Characterizations of the precursor. NMR, FTIR, and mass spectroscopies were performed on the as-prepared bridged organoalkoxysilane precursor.
1H-NMR: (CDCl3, 500 MHz) δ (ppm) 7.57 (s, 2H), 3.80 (q, J = 7 Hz, 12H), 3.29 (t, J = 6.7 Hz, 4H), 1.66 (m, J = 7.7 Hz, 4H), 1.20 (t, J = 7.1 Hz, 18H), 0.63 (t, J = 8.3 Hz, 4H). 13C- NMR: (CDCl3, 125 MHz) δ (ppm) 159.9, 58.6, 42.1, 22.9, 18.3, 7.8. MS (ESI+) m/z (%):
496.3 (100) [MH+], 451.2 (55) [MH+- one ethoxy fragment]. HRMS (ESI+):
m/z calcd for C20H44N2O8Si2: 496.2636, found 496.2636. FTIR (cm-1): 3317, 3017, 2973, 2936, 2895, 1668, 1522, 1449, 1371, 1276, 1203, 1110, 950, 783, 703, 572, 481.
Figure S2. FTIR spectrum of the bridged organoalkoxysilane precursor.
Figure S3. Mass spectrum of the bridged organoalkoxysilane precursor (A), and the isotopic distribution (B).
III- SYNTHESES AND CHARACTERIZATION OF MON
Figure S4. DLS analysis of oxamide-phenylene MON in aqueous solution.
Figure S5. N2-adsorption-desorption isotherms, BET calculations (A) and BJH pore size (B) distributions for MON.
Figure S6. UV-visible spectrum of oxamide-phenylene MON, depicting the expected absorption of phenylene moieties.
Figure S7. Wide angle XRD pattern of MON containing peaks arising from the molecular periodicity of phenylenes through 7.2 nm domains. *Indicates the siloxane amorphous network.
Figure S8. FTIR spectra of the oxamide precursor and oxamide-phenylene MON confirming the incorporation of preserved oxamide functions within the silsesquioxane network. The broad carbonyl (1655 cm-1) and amine (1521 cm-1) vibration modes with relatively low intensities strongly suggests the absence of H-bonds between oxamide moieties.
Figure S9. Typical background-subtracted electron energy-loss spectrum of a representative oxamide-phenylene MON.
Figure S10. TGA analysis of oxamide-phenylene MON, confirming the high organic content in the nanomaterial.
IV- DEGRADATION STUDIES OF MON
IV.1- MON control with denatured trypsin. MON (1 mg) were dispersed in PBS (0.75 mL) in an Eppendorf tube at pH 7.4, the denatured trypsin (heated at 95 °C for 7 min) was added (0.25%, 0.75 mL), and were stirred gently for 48 h. The final concentration of trypsin was 0.05 mM. The sample was then centrifuged, washed with deionized water, and dispersed in deionized water for DLS analysis.
IV.2- P PMO NPs control with trypsin. P PMO NPs (1 mg) were dispersed in PBS (0.75 mL) in an Eppendorf tube at pH 7.4, trypsin (heated at 95 °C for 7 min) was added (0.25%, 0.75 mL), and were stirred gently for 48 h. The final concentration of trypsin was 0.05 mM.
The sample was then centrifuged, washed with deionized water, and dispersed in deionized water for DLS analysis.
Figure S11. FTIR spectra of the trypsin protein and surface-adsorbed proteins on MON.
Figure S12. Statistical analyses of TEM images of NPs (140 NPs) sizes before and after degradation in the presence of trypsin proteins after 48 h and aqueous washing (A). The distribution of degraded MON nano-objects is enlarged for clarity (B).
Figure S13. DLS (Zetasizer Nano ZS, Malvern instrument) analyses of MON before, after 24 h of mixing with trypsin in PBS at 37 °C at pH 7.4, and after 48 h of mixing followed by aqueous washing.
Figure S14. SS NMR 13C CP-MAS spectrum of MON demonstrating the presence phenylene- propyloxamide bridges in the material framework. *Asterisks indicate the spinning side bands of the phenylene peak at 134.9 ppm. 1: Si-OCH2CH3, 2: CH3CH2OH. The oxamide carbon at 162.0 ppm signal is enlarged for clarity. The lower intensity of this peak arises from the absence of proton in the local environment, which in turns removed the CP effect.
Figure S15. SS NMR 13C CP-MAS spectrum of MON (red) and degraded MON (blue) (A).
The zoomed 185 to 155 ppm area demonstrates the cleavage of oxamide (162.0 ppm) into carboxylate groups (173.2 ppm) via trypsin proteins (B).
Figure S16. Zeta potential of MON before and after protein-mediated degradation. The charge reversal of MON is consistent with the complete cleavage of oxamide groups favoured on the particle surface, thus generating propyl-ammonium groups per cleaved oxamide.
Figure S17. FTIR spectra of oxamide-phenylene MON before and after degradation, validating the key role of oxamide moieties, as shown by the change in intensities of the C=O and N-H modes and the large carbonyl shift.
Figure S18. Control experiment based on the degradation of the oxamide precursor by trypsin in neutral water (A). The product is thus hydrolysed and condenses affording the so-called trypsin-degraded oxamide condensed precursor. FTIR spectra comparison of degraded oxamide-phenylene MON and the trypsin-degraded oxamide condensed precursor, validating the key role of trypsin in the degradation of oxamide moieties, as shown by the identical changes in intensities for the C=O and N-H modes and the identical carbonyl shift.
Figure S19. UV-visible spectra of the FITC alkoxysilane and MON-FITC NPs confirming the functionalization of the dye unto MON nanospheres.
Figure S20. FTIR spectra comparison between MON and MON-FITC NPs confirming the grafting of the fluorescein carbamate unto MON nanospheres.
Figure S21. TEM micrographs of MON after FITC functionalization, depicting the integrity of NPs. Aggregates (mostly physically, and partly chemically see DLS Figure S23) are formed on the TEM grid due to the high hydrophobicity of the hybrid NPs.
Figure S22. TEM micrographs of aliquots taken from the trypsin protein and MON-FITC NPs mixture after 48 h of mixing, depicting the degradation of NPs. Initial aggregates
remained less degraded. The major proportion of degraded nano-objects is shown in Figure S23.
Figure S23. DLS hydrodynamic diameter distribution before and after the degradation of MON-FITC NPs, demonstrating that proteins can still react on surface-functionalized MON.
Figure S24. Degradation percentages of MON and P PMO NPs. The results were estimated via weighing the samples before (10 mg) and after 3 days of degradation with trypsin in PBS at 37 °C.
V- DRUG LOADING AND RELEASE STUDIES
V.1- Medium DOX loading in MON. MON (7 mg) were dispersed in deionized water (1 mL) in an Eppendorf tube at pH 7.4 with DOX (3.5 mg), and were stirred gently overnight.
The sample was then centrifuged, washed with deionized water four times, and dispersed in deionized water. Supernatants were gathered for the drug loading capacity calculation via UV-visible analysis.
V.2- Medium CPT loading in MON. MON (2.7 mg) were dispersed in DMSO (1 mL) in an Eppendorf tube at pH 7.4 with CPT (2.6 mg), and were stirred gently overnight. The sample was then centrifuged, washed with deionized water four times, and dispersed in deionized water. Supernatants were gathered for the drug loading capacity calculation via UV-visible analysis.
V.3- High DOX loading in NPs. MON or P PMO NPs (1.7 mg) were dispersed in deionized water (1 mL) in an Eppendorf tube at pH 7.4 with DOX (3.0 mg), and were stirred gently overnight. The sample was then centrifuged, washed with deionized water four times, and dispersed in deionized water. Supernatants were gathered for the drug loading capacity calculation via UV-visible analysis.
V.4- High CPT loading in NPs. MON or P PMO NPs (4.7 mg) were dispersed in DMSO (1 mL) in an Eppendorf tube at pH 7.4 with CPT (7.6 mg), and were stirred gently overnight.
The sample was then centrifuged, washed with deionized water four times, and dispersed in deionized water. Supernatants were gathered for the drug loading capacity calculation via UV-visible analysis.
Figure S25. Photographs of an aqueous dispersion of DOX-loaded (11 wt%) MON before (left) and after (right) centrifugation, displaying the stable drug loading into nanocarriers (A).
UV-visible spectrum of DOX-loaded MON in water (B).
Figure S26. UV-visible spectrum of CPT-loaded (31 wt%) MON in water, confirming the successful drug loading of nanocarriers.
Figure S27. UV-visible spectra of DOX-loaded (40 wt%) (A) and CPT-loaded (46 wt%) MON (B) in water, confirming the successful drug loadings of nanocarriers.
Figure S28. Measuring of the drug leakage overtime from drug-loaded (medium and high loadings) MON via UV-visible spectroscopy. The absence of drug molecules in supernatants confirms the stability of drug-loaded nanocarriers.
Figure S29. Study of the stability of DOX-loaded MON remaining negatively-charged (A), started to aggregate but did not degrade after one month in aqueous solution, as shown by
DLS (B) and TEM data (C-D). The DOX leakage was not observed after one month in solution.
Figure 30. Release experiments of DOX-loaded phenylene (P PMO, left-hand side) and of oxamide-phenylene-bridged MON (right-hand side) before and after addition of HCl aliquots to change the pH from 7 to 5.5. The very low drug release from MON strongly suggest the role of oxamide-drug interactions to prevent passive drug leakage, and the pH responsiveness observed for P PMO NPs.
Figure S31. Stability study of DOX-loaded MON in the presence of trypsin after 48 h (A).
The NPs were slightly degraded as shown by TEM micrographs (B), which triggered the release of DOX drugs (Figure S31).
Figure S32. UV-visible spectra of the supernatants of DOX- or CPT-loaded MON in the presence of trypsin proteins for two days, confirming the NPs degradation-mediated release of drugs which is a plausible mechanism of the in-vitro drug deliveries.
Figure S33. UV-visible spectra of propidium iodide-loaded MON (red curve), and MON (blue curve) in aqueous solution, confirming the loading of cargos.
VI- IN-VITRO STUDIES
VI.1- Cytoxicity study. Human Lung cancer cell line, A549, obtained from the American Type Culture Collection were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % fetal calf serum (Sigma), 2 % L-glutamine, 1 % penicillin, and 1 % streptomycin stock solutions, incubated at 37 °C under a 5 % CO2 95 % air atmosphere. The media were routinely changed every 3 days, and the cells were passaged by trypsinization before confluence.
VI.2- Confocal imaging study. Cells were incubated with nanoparticles for overnight at 37
°C under a 5 % CO2 95 % air atmosphere in an 8-chamber Lab-Tek chamber slide system with covers (Nalge Nunc International), washed with PBS, fixed with -20C methanol for 1 hour, and examined with fluorescent microscope (Carl Zeiss).
VI.3- Drug delivery study. Cells were cultured in 96-well plates (3000 cells/well) with fresh DMEM at 37 °C in a 5 % CO2/95 % air atmosphere for 24 h, then washed with PBS and the medium was changed to a fresh medium along with nanoparticles, loaded with dye or drugs, at the indicated concentrations. 24 h later, the cells were washed with PBS to remove nanoparticles that were not taken up by the cells, and continued incubating in DMEM for an additional 48 h. And then 10 % WST-8 solution (cell counting kit-8, Dojindo Molecular Technologies, Inc.) was added to cells and incubated for another 2 h. The absorbance of each well was measured at 450 nm with a plate reader. Since the absorbance is proportional to the number of viable cells in the medium, the viable cell number was determined using a previously prepared calibration curve.
Figure S34. Confocal fluorescence cellular imaging of propidium iodide-loaded MON, demonstrating the endocytosis of NPs with the red nuclei staining in A549 cells.
Figure S35. MON-FITC-Phosphonate preparation and zeta potential results supporting the surface coating (A). Confocal fluorescence cellular imaging of MON-FITC-Phosphonate NPs, demonstrating the endocytosis of NPs in PANC-1 cells (B).
