Supporting Information Ultrahigh Nanoparticle Stability against Salt ...
Supporting Information
Ultrahigh Nanoparticle Stability against Salt, pH and Solvent with Retained Surface Accessibility via Depletion Stabilization Xu Zhang†,‡, Mark R. Servos‡ and Juewen Liu†* †
Department of Chemistry and Waterloo Institute for Nanotechnology, ‡Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
Email: [email protected]
1. Experimental section Chemicals and materials. Glycerol, ethylene glycol, ethanol, dimethylformamide, isopropanol, acetonitrile, formamide and PEG 200, 400, 2k, 4k, 8k, 20k, and 35k were purchased from VWR. AuNPs (13 nm) were synthesized based on the standard citrate reduction procedures,S1 and its concentration was estimated to be ~10 nM. AuNPs (50 nm and 100 nm) were purchased from Ted Pella Inc. (Ridding, CA). Thiolated DNA with a 5-FAM (6-carboxyfluorescein) label (5FAM-ATGCGGAGGAAGGTTTT-SH) was purchased from Integrated DNA Technologies Inc (Coralville, IA). FAM-labeled PEG 10000 was purchased from Nanocs Inc. (New York, NY). FITC-labeled 50 nm silica nanoparticles were purchased from Kisker Biotech GmbH & Co. Carboxyl graphene oxide was purchased from Advanced Chemical Supplies (Medford, MA). Quantum dots (green fluorescence with carboxyl surface, catalog number: FN-525-C-1MG and red fluorescence with hydroxyl surface, catalog number: FN-630-H-1MG) and magnetic nanoparticles (10 nm iron oxide nanoparticles, 5 mg/mL dispersed in 0.25 M tetramethylammonium hydroxide, catalog number: IO-A10-1) were purchased from Cytodiagnostics Inc. (Burlington, Ontario, Canada). 1,2-dioleoyl-sn-glycero-3-phospho-(1'-racglycerol) (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N(lissamine rhodamine B sulfonyl) (ammonium salt) were purchased from Ananti Polar Lipids (Alabaster, Al). Adenosine, guanosine, cytidine, uridine, MgCl2, NaCl, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonate (HEPES) and sodium citrate were purchased from Mandel Scientific (Guelph, Ontario, Canada). Doxorubicin, HAuCl4, AgNO3, FeCl3, TbCl3, SrCl2, BaCl2, CoCl2, NiSO4, CuSO4, ZnCl2, CdCl2, KCN, and tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich. Milli-Q water was used for all experiments. Evaluation of nanoparticle stability. Different concentrations of PEG with various MWs were mixed with 10 nM AuNPs in a total volume of 100 µL. Afterwards, MgCl2 (1 M) was added into the mixture to achieve a final concentration of 10 mM Mg2+. The color change of the mixtures was recorded using a digital camera (Canon Powershot SD1200 IS). The same procedure was applied to GO (final concentration = 0.6 mg/mL), 50 nm silica nanoparticles (final concentration = 0.42 mg/mL), and DOPG liposome (final concentration = 0.1 mg/mL)
S1
other nanomaterials to test their stability against salt in the presence of PEGs. The green emitting quantum dots were diluted 60 times from the original stock (~20 M), and the red emitting quantum dots were diluted 100 times from the original stock (~20 M). The magnetic nanoparticles were diluted 10 times from the original stock at a concentration of 5 mg/mL. In order to better demonstrate the aggregation of nanoparticles, a brief centrifugation (600 rpm, 10 sec) was performed before imaging using a blue light transilluminator (Invitrogen Safe Imager 2.0, excitation wavelength = 470 nm). To test the range of Mg2+ that AuNPs can tolerate in 10% PEG 20000, Mg2+ was added from a 4 M MgCl2 stock solution. Next, we examined if PEG could protect AuNPs from aggregation induced by heavy metal ions (Ag+, Fe3+, Tb3+, Sr2+, Ba2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+), ribonucleosides (A, C, G, U) and doxorubicin. Each tube containing 90 µL of AuNP solution (10 nM) and 10 µL Milli-Q water was spiked with a heavy metal ion (2-10 mM), nucleoside (Adenosine: 5 µM; Guanosine: 250 µM; Cytidine: 2 mM; Uridine: 20 mM), or doxorubicin (2 g/mL) to generate distinctive color change indicating AuNP aggregation. The use of such a high concentration of uridine reflects its weak binding affinity to AuNPs, while adenosine induced AuNP aggregation just with a concentration of 5 µM. For comparison, the same amount of metal salt or nucleoside solution was added into AuNP solution that contained 2% PEG 20000. In a similar way, the PEG protection of AuNPs at extreme pHs was studied, where the pH of the AuNP solutions were adjusted by adding 1 M HCl or NaOH. To test the stabilization of AuNPs in organic solvents, 13 nm AuNPs were added to a final of 67% ethanol, dimethylformamide, isopropanol, acetonitrile and formamide. Blue or purple colored aggregates were observed with isopropanol and acetonitrile, which were then tested in the presence of PEG. For this purpose, 200 µL of the solvent was mixed with 100 µL AuNPs with or without 2% PEG 20000. The tubes were imaged using the digital camera. The adsorption isotherm of FAM-labeled PEG 10000. Various concentrations of FAMlabeled PEG 10000 (in 5 mM HEPES buffer, pH 7.6) were mixed with AuNPs (5 nM) to measure the loading capacity, where adsorbed PEG showed fluorescence quenching. The standard curve was obtained by directly monitoring the fluorescence signal of FAM-PEG 10000 of different concentrations. UV-vis characterization of AuNP aggregation. AuNPs (5 nM) were dispersed in various concentrations of PEG 20000 and their UV-vis spectra were measured using an Agilent 8453A spectrometer after adding increasing concentrations of MgCl2. Experiments were run in triplicates. Adsorption kinetics of doxorubicin onto AuNPs in the presence of PEG 20000. In the 96well plate, 20 µL of doxorubicin (1.72 µM) was spiked in 80 µL of 5 mM HEPES buffer containing various concentrations of PEG 20000 (0%, 4%, or 10%). After reading background fluorescence for ~3 min, 2 µL of AuNP solution (final concentration: 0.2 nM AuNPs) was
S2
added and the change of fluorescence signal over time was recorded with a microplate reader (Tecan Infinite F200Pro). Adsorption of SH-DNA on AuNP in the presence of PEG 20000. Based on the observations that PEG 20000 can stabilize AuNPs against high salt concentrations, we hypothesized that PEG 20000 might be able to maximize the loading of thiolated DNA without performing the salt aging protocol that requires gradual addition of NaCl over 1-2 days.S2 We evaluated the loading capacity of DNA on 50 nm AuNPs. In a 50 µL AuNP aqueous solution (0.075 nM), 1 µL of 100 µM the thiolated and FAM-labeled DNA was added so that the final concentration of the DNA was 2 µM. Then 2% PEG 20000 was added and mixed before adjusting NaCl concentration. After 2 hr incubation, the loaded DNA was quantified. To quantify the loading capacity of SH-DNA on AuNPs, the prepared DNA-SH-AuNP conjugates were collected by centrifugation and rinsed with 5 mM HEPES buffer for five times before re-dispersing into 100 µL of 5 mM HEPES buffer containing 10 mM KCN to dissolve AuNPs and release SH-DNAFAM strands. Finally, 2 µL of treated sample was mixed with 98 µL of 5 mM HEPES buffer for fluorescence measurement with the plate reader. The same DNA was used to prepare a standard curve for calibration.
Figure S1. (A) Color of AuNPs in the presence of various solutes and 10 mM Mg2+. (B) Color of AuNPs in various concentrations of high MW PEGs (no Mg2+ added). 2. Additional AuNP stability data. Small molecule solvents and low MW PEGs cannot protect AuNPs. In Figure 2A of the paper, we showed that AuNPs were stable in up to 50% glycerol and EG when the Na+ concentration was below 10 mM. Addition of 10 mM Mg2+, however, induced immediate aggregation and AuNP color changing
S3
to blue (Figure S1A). The same is true for PEG 200 and 400. Therefore, PEG molecular weight is the determining factor for its stabilization effect. High MW PEGs do not induce AuNP aggregation at low salt. Figure S1B shows the picture of Figure 2B in the paper before adding 10 mM Mg2+. All the AuNPs remained dispersed and no PEG induced aggregation was observed.
Fluorescence (a.u.)
3. Kinetics of thiolated DNA adsorption in 2% PEG 20000. In the paper we demonstrated that a high loading of thiolated DNA can be achieved in the presence of 2% PEG 20000 without performing the salt aging protocol, since a high concentration of salt can be added all at once. One important question is whether the kinetics of DNA adsorption is affected by PEG or not. To test this, we added 1 nM 13 nm AuNPs to 4 nM DNA (FAM-ATGCGGAGGAAGGTTTT-SH) dissolved in 5 mM HEPES buffer (pH 7.6) with 120 mM NaCl (Figure S2, black curve). Attachment of this thiolated DNA onto AuNPs was accompanied with fluorescence quenching. Adsorption equilibrium was established in ~10 min. In the presence of 2% PEG 20000, the kinetics of adsorption was slower but a similar degree of adsorption was achieved in ~1 hr (red curve). With higher salt concentrations, the rate of adsorption became faster and the final fluorescence intensity was also lower, consistent with higher loading capacity. Therefore, although at the same salt concentration, the rate of adsorption is slower with PEG, much higher salt can be added to accelerate the adsorption reaction and drive the reaction to completion. If too much salt is added in the absence of PEG, AuNPs would aggregate irreversibly. With 2% PEG 20000, 13 nm AuNPs are stable in at least 1 M NaCl.
No PEG, 120 mM NaCl 2% PEG 2k, 120 mM NaCl 2% PEG 2k, 300 mM NaCl 2% PEG 2k, 500 mM NaCl
80 60 40 20 0
20
40
60
Time (min) Figure 2. Kinetics of thiolated and FAM labelled DNA adsorption by AuNPs in 2% PEG 20000 as a function of salt. 4. TEM characterization of AuNPs. The as-prepared 13 nm AuNPs were dropped onto a holey carbon coated copper grid and imaged using a transmission electron microscope (Philips CM10). As shown in Figure S3, the AuNPs are very homogeneous with an average diameter of ~13 nm.
S4
Figure S3. A TEM micrograph of 13 nm citrate-capped AuNPs used in this work.
Additional references S1 S2
J. J. Storhoff, R. Elghanian, R. C. Mucic, C. A. Mirkin and R. L. Letsinger, J. Am. Chem. Soc., 1998, 120, 1959. S. J. Hurst, A. K. R. Lytton-Jean and C. A. Mirkin, Anal. Chem., 2006, 78, 8313.
S5
Ultrahigh Nanoparticle Stability against Salt, pH and Solvent with Retained Surface Accessibility via Depletion Stabilization Xu Zhang†,‡, Mark R. Servos‡ and Juewen Liu†* †
Department of Chemistry and Waterloo Institute for Nanotechnology, ‡Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1.
Email: [email protected]
1. Experimental section Chemicals and materials. Glycerol, ethylene glycol, ethanol, dimethylformamide, isopropanol, acetonitrile, formamide and PEG 200, 400, 2k, 4k, 8k, 20k, and 35k were purchased from VWR. AuNPs (13 nm) were synthesized based on the standard citrate reduction procedures,S1 and its concentration was estimated to be ~10 nM. AuNPs (50 nm and 100 nm) were purchased from Ted Pella Inc. (Ridding, CA). Thiolated DNA with a 5-FAM (6-carboxyfluorescein) label (5FAM-ATGCGGAGGAAGGTTTT-SH) was purchased from Integrated DNA Technologies Inc (Coralville, IA). FAM-labeled PEG 10000 was purchased from Nanocs Inc. (New York, NY). FITC-labeled 50 nm silica nanoparticles were purchased from Kisker Biotech GmbH & Co. Carboxyl graphene oxide was purchased from Advanced Chemical Supplies (Medford, MA). Quantum dots (green fluorescence with carboxyl surface, catalog number: FN-525-C-1MG and red fluorescence with hydroxyl surface, catalog number: FN-630-H-1MG) and magnetic nanoparticles (10 nm iron oxide nanoparticles, 5 mg/mL dispersed in 0.25 M tetramethylammonium hydroxide, catalog number: IO-A10-1) were purchased from Cytodiagnostics Inc. (Burlington, Ontario, Canada). 1,2-dioleoyl-sn-glycero-3-phospho-(1'-racglycerol) (sodium salt) (DOPG) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N(lissamine rhodamine B sulfonyl) (ammonium salt) were purchased from Ananti Polar Lipids (Alabaster, Al). Adenosine, guanosine, cytidine, uridine, MgCl2, NaCl, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonate (HEPES) and sodium citrate were purchased from Mandel Scientific (Guelph, Ontario, Canada). Doxorubicin, HAuCl4, AgNO3, FeCl3, TbCl3, SrCl2, BaCl2, CoCl2, NiSO4, CuSO4, ZnCl2, CdCl2, KCN, and tris(2-carboxyethyl)phosphine (TCEP) were purchased from Sigma-Aldrich. Milli-Q water was used for all experiments. Evaluation of nanoparticle stability. Different concentrations of PEG with various MWs were mixed with 10 nM AuNPs in a total volume of 100 µL. Afterwards, MgCl2 (1 M) was added into the mixture to achieve a final concentration of 10 mM Mg2+. The color change of the mixtures was recorded using a digital camera (Canon Powershot SD1200 IS). The same procedure was applied to GO (final concentration = 0.6 mg/mL), 50 nm silica nanoparticles (final concentration = 0.42 mg/mL), and DOPG liposome (final concentration = 0.1 mg/mL)
S1
other nanomaterials to test their stability against salt in the presence of PEGs. The green emitting quantum dots were diluted 60 times from the original stock (~20 M), and the red emitting quantum dots were diluted 100 times from the original stock (~20 M). The magnetic nanoparticles were diluted 10 times from the original stock at a concentration of 5 mg/mL. In order to better demonstrate the aggregation of nanoparticles, a brief centrifugation (600 rpm, 10 sec) was performed before imaging using a blue light transilluminator (Invitrogen Safe Imager 2.0, excitation wavelength = 470 nm). To test the range of Mg2+ that AuNPs can tolerate in 10% PEG 20000, Mg2+ was added from a 4 M MgCl2 stock solution. Next, we examined if PEG could protect AuNPs from aggregation induced by heavy metal ions (Ag+, Fe3+, Tb3+, Sr2+, Ba2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+), ribonucleosides (A, C, G, U) and doxorubicin. Each tube containing 90 µL of AuNP solution (10 nM) and 10 µL Milli-Q water was spiked with a heavy metal ion (2-10 mM), nucleoside (Adenosine: 5 µM; Guanosine: 250 µM; Cytidine: 2 mM; Uridine: 20 mM), or doxorubicin (2 g/mL) to generate distinctive color change indicating AuNP aggregation. The use of such a high concentration of uridine reflects its weak binding affinity to AuNPs, while adenosine induced AuNP aggregation just with a concentration of 5 µM. For comparison, the same amount of metal salt or nucleoside solution was added into AuNP solution that contained 2% PEG 20000. In a similar way, the PEG protection of AuNPs at extreme pHs was studied, where the pH of the AuNP solutions were adjusted by adding 1 M HCl or NaOH. To test the stabilization of AuNPs in organic solvents, 13 nm AuNPs were added to a final of 67% ethanol, dimethylformamide, isopropanol, acetonitrile and formamide. Blue or purple colored aggregates were observed with isopropanol and acetonitrile, which were then tested in the presence of PEG. For this purpose, 200 µL of the solvent was mixed with 100 µL AuNPs with or without 2% PEG 20000. The tubes were imaged using the digital camera. The adsorption isotherm of FAM-labeled PEG 10000. Various concentrations of FAMlabeled PEG 10000 (in 5 mM HEPES buffer, pH 7.6) were mixed with AuNPs (5 nM) to measure the loading capacity, where adsorbed PEG showed fluorescence quenching. The standard curve was obtained by directly monitoring the fluorescence signal of FAM-PEG 10000 of different concentrations. UV-vis characterization of AuNP aggregation. AuNPs (5 nM) were dispersed in various concentrations of PEG 20000 and their UV-vis spectra were measured using an Agilent 8453A spectrometer after adding increasing concentrations of MgCl2. Experiments were run in triplicates. Adsorption kinetics of doxorubicin onto AuNPs in the presence of PEG 20000. In the 96well plate, 20 µL of doxorubicin (1.72 µM) was spiked in 80 µL of 5 mM HEPES buffer containing various concentrations of PEG 20000 (0%, 4%, or 10%). After reading background fluorescence for ~3 min, 2 µL of AuNP solution (final concentration: 0.2 nM AuNPs) was
S2
added and the change of fluorescence signal over time was recorded with a microplate reader (Tecan Infinite F200Pro). Adsorption of SH-DNA on AuNP in the presence of PEG 20000. Based on the observations that PEG 20000 can stabilize AuNPs against high salt concentrations, we hypothesized that PEG 20000 might be able to maximize the loading of thiolated DNA without performing the salt aging protocol that requires gradual addition of NaCl over 1-2 days.S2 We evaluated the loading capacity of DNA on 50 nm AuNPs. In a 50 µL AuNP aqueous solution (0.075 nM), 1 µL of 100 µM the thiolated and FAM-labeled DNA was added so that the final concentration of the DNA was 2 µM. Then 2% PEG 20000 was added and mixed before adjusting NaCl concentration. After 2 hr incubation, the loaded DNA was quantified. To quantify the loading capacity of SH-DNA on AuNPs, the prepared DNA-SH-AuNP conjugates were collected by centrifugation and rinsed with 5 mM HEPES buffer for five times before re-dispersing into 100 µL of 5 mM HEPES buffer containing 10 mM KCN to dissolve AuNPs and release SH-DNAFAM strands. Finally, 2 µL of treated sample was mixed with 98 µL of 5 mM HEPES buffer for fluorescence measurement with the plate reader. The same DNA was used to prepare a standard curve for calibration.
Figure S1. (A) Color of AuNPs in the presence of various solutes and 10 mM Mg2+. (B) Color of AuNPs in various concentrations of high MW PEGs (no Mg2+ added). 2. Additional AuNP stability data. Small molecule solvents and low MW PEGs cannot protect AuNPs. In Figure 2A of the paper, we showed that AuNPs were stable in up to 50% glycerol and EG when the Na+ concentration was below 10 mM. Addition of 10 mM Mg2+, however, induced immediate aggregation and AuNP color changing
S3
to blue (Figure S1A). The same is true for PEG 200 and 400. Therefore, PEG molecular weight is the determining factor for its stabilization effect. High MW PEGs do not induce AuNP aggregation at low salt. Figure S1B shows the picture of Figure 2B in the paper before adding 10 mM Mg2+. All the AuNPs remained dispersed and no PEG induced aggregation was observed.
Fluorescence (a.u.)
3. Kinetics of thiolated DNA adsorption in 2% PEG 20000. In the paper we demonstrated that a high loading of thiolated DNA can be achieved in the presence of 2% PEG 20000 without performing the salt aging protocol, since a high concentration of salt can be added all at once. One important question is whether the kinetics of DNA adsorption is affected by PEG or not. To test this, we added 1 nM 13 nm AuNPs to 4 nM DNA (FAM-ATGCGGAGGAAGGTTTT-SH) dissolved in 5 mM HEPES buffer (pH 7.6) with 120 mM NaCl (Figure S2, black curve). Attachment of this thiolated DNA onto AuNPs was accompanied with fluorescence quenching. Adsorption equilibrium was established in ~10 min. In the presence of 2% PEG 20000, the kinetics of adsorption was slower but a similar degree of adsorption was achieved in ~1 hr (red curve). With higher salt concentrations, the rate of adsorption became faster and the final fluorescence intensity was also lower, consistent with higher loading capacity. Therefore, although at the same salt concentration, the rate of adsorption is slower with PEG, much higher salt can be added to accelerate the adsorption reaction and drive the reaction to completion. If too much salt is added in the absence of PEG, AuNPs would aggregate irreversibly. With 2% PEG 20000, 13 nm AuNPs are stable in at least 1 M NaCl.
No PEG, 120 mM NaCl 2% PEG 2k, 120 mM NaCl 2% PEG 2k, 300 mM NaCl 2% PEG 2k, 500 mM NaCl
80 60 40 20 0
20
40
60
Time (min) Figure 2. Kinetics of thiolated and FAM labelled DNA adsorption by AuNPs in 2% PEG 20000 as a function of salt. 4. TEM characterization of AuNPs. The as-prepared 13 nm AuNPs were dropped onto a holey carbon coated copper grid and imaged using a transmission electron microscope (Philips CM10). As shown in Figure S3, the AuNPs are very homogeneous with an average diameter of ~13 nm.
S4
Figure S3. A TEM micrograph of 13 nm citrate-capped AuNPs used in this work.
Additional references S1 S2
J. J. Storhoff, R. Elghanian, R. C. Mucic, C. A. Mirkin and R. L. Letsinger, J. Am. Chem. Soc., 1998, 120, 1959. S. J. Hurst, A. K. R. Lytton-Jean and C. A. Mirkin, Anal. Chem., 2006, 78, 8313.
S5
