Supporting Information: Direct Aqueous-Phase Synthesis of Sub-10 ...
Supporting Information:
Direct Aqueous-Phase Synthesis of Sub-10 nm “Luminous Pearls” with Enhanced in Vivo Renewable Near-Infrared Persistent Luminescence Zhanjun Li,† Yuanwei Zhang,† Xiang Wu, † Ling Huang, † Dongsheng Li§, Wei Fan,‡ Gang Han*† † Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 (U.S.A) § Materials Sciences, Physical Sciences Division, Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352 ‡Chemical Engineering Department, University of Massachusetts Amherst, Amherst, MA 01003
Contact author: Prof. Gang Han, E-mail: [email protected]
S1
METHODS Materials Ga2O3, Zn(NO3)2·6H2O, Cr(NO3)3·9H2O, ammonium hydroxide, hydrochloride acid, and concentrated nitric acid were all analytical reagents and were used as they were received. Ga(NO3)3 solution was prepared by dissolving Ga2O3 in 1:1 concentrated nitric acid and this was followed by air drying it at 105o C in order to remove excess amounts of nitric acid and to be re-dissolved in deionized water. 1 mol/L Zn(NO3)2, 2 mol/L Ga(NO3)3, 4 mmol/L Cr(NO3)3 were stored as the precursor solutions here. A small amount of nitric acid was used to prevent the hydrolysis of Ga(NO3)3. Polyethyleneimine (PEI) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich and used directly without further purification. The synthesis of ZGC-1
A certain amount of Ga(NO3)3 (2 M), Zn(NO3)2 (1 M), Cr(NO3)3 (4 mM) precursor
solutions were mixed at a predetermined molar ratio. Ammonium hydroxide (28%, wt) was added in a quick manner into the above mentioned mixture as stirring vigorously in order to reach a pH of 9. The metal hydroxide precursor was sealed into a Teflon-lined autoclave and this was followed by the hydrothermal reaction, which occurred at 220o C for 10 hours. These ZGC nanocrystals can be obtained within a wide range of molar ratios in regard to the precursors, the latter of which are influenced by the product size and distribution. In a typical procedure, 2 mmol of Zn(NO3)2, 2 mmol of Ga(NO3)3, 0.004 mmol Cr(NO3)3 are mixed together as vigorously stirred. The total volume was adjusted to 15 mL by adding deionized water. Concentrated ammonium hydroxide (28%) solution (about 1 mL) was added in a rapid manner in order to adjust the pH to be that of 9~9.5 and the white precipitate was formed immediately. After another half hour of stirring, the mixture was transmitted into a Teflon-lined autoclave (25 mL) and sealed. The autoclave was first put in an oven at 220o C for 10 hours and was then naturally cooled until reaching room temperature. The white precipitate obtained after centrifugation was dispersed in 0.01 M HCl, forming a transparent solution. Possible ZnO impurity was able to be removed in this step. Then ZGC nanocrystals were washed by centrifigations after being mixed with excess isopropanol. Finally, ZGC-1 nanocrystals were redispersed in deionized water for further characterization and imaging application. The concentration of ZGC that is used for storage is approximately 4 mg/mL.
S2
For the dispersion in the cell culture medium, 200 µL ZGC-1 storage solution was added into 5 mL of cell culture medium. A clear solution can be obtained after a brief ultrasonic mixing. Polyethylenimine (PEI) (positively charged polymer) can be adsorbed onto the crystal surface of ZGC due to the electrostatic interaction with the negatively charged ZGC nanocrystals, forming PEI modified ZGC (ZGC-PEI). In brief, 1 mL of ZGC stored solution was added to 10 mL of 1 mM of HCl solution, and was stirred for 30 min. 1 mL of PEI solution (20%, wt) was then rapidly added as stirring vigorously. After being diluted with excess isopropanol, The ZGC-PEI can be collected by centrifugation. The ZGC-PEI was redispersed in deionized water during the process of ultrasonic dispersion. ZGC-1 was also able to be modified with BSA. 1 mL of ZGC-1 solution (4 mg/mL) was added into 10 mL of BSA solution (1% by weight) drop by drop under stirring followed by 30 min stirring. BSA modified ZGC-1 (ZGC-BSA) was collected by centrifugation and then redispersed in 10 mL of DI water after a brief ultrasonic dispersion.
The synthesis of ZGC-2 ZGC-2 was synthesized for purposes of comparison according to a Ref 1. In brief, 1 mmol of Zn(NO3)2, 2 mmol of Ga(NO3)3, 0.004 mmol Cr(NO3)3 were mixed together as stirring vigorously. The total volume was adjusted to 10 mL by adding deionized water. The concentrated ammonium hydroxide (28%) solution (about 1 mL) was rapidly added in order to adjust the pH to 7.5. The mixture was sealed in a Teflonlined autoclave at 120o C for 24 hours. The resulting precipitate has no PL properties and was sintered at 750o C via a solid-state-reaction for 5 hours. A small amount of 5 mM NaOH solution was added to the powder product to form a slurry and this was followed by wet grinding for 1 hour with a mortar and pestle. The product was washed by ethanol (90%) and then dried at 60o C. The synthesis of ZGC-3 by using a hydrothermal post-treatment The as-synthesized ZGC-2 was mixed with an ammonium aqueous solution (pH9~9.5) and sealed in an autoclave, the latter of which underwent a hydrothermal procedure at 220o C for 10 hours. The as-synthesized ZGC-3 was centrifuged and dried at 80o C. Imaging A white LED (5000 lumen, CREE-T6) was used as the light source in all of the imaging experiments. For the in
S3
vitro experiment, 50 mg of the ZGC sample was put in one well of a black 96-well-plate. This plate was exposed to the LED for 30 s and then put into the IVIS 100 imaging system in order to detect the PL signal. The in vivo imaging was performed after the subcutaneous injection of the sample dispersion (50 µL, 2 mg/mL) and 30 s of in situ LED excitation. The in vivo simulated deep tissue recharging and imaging was performed by covering the subcutaneous injection site with a 1-cm pork slab. The imaging was performed right after 30 s of in situ LED excitation. Characterization The X-ray powder diffraction (XRD) was performed on a Panalytical X’pert PRO diffractometer that was equipped with Cu Kα radiation (λ = 1.5418 Å). The morphology of the samples was examined using a transmission electron microscope (TEM, Techni) at accelerating voltages of 5 kv and 800 kv. The energydispersive X-ray spectroscopy (EDS) was obtained on the FESEM at an accelerating voltage of 30 kv. High resolution TEM (HRTEM) images were performed using a JEM-1200EX II transmission electron microscope. The spectra and lifetimes of the samples were tested via the usage of powder samples. The photoluminescence spectra and decay curves were measured using a fluorospectrophotometer (FluoroMax-3, HORIBA, USA). The sample in the detection of the spectra and decay curves were dispersed in water, 4 mg/mL. The hydrodynamic size distributions were measured five times for each sample using a Zetasizer (Malvern, 3000HS),
average
results
were
used
as
the
mean
hydrodynamic
particle
size.
S4
Figure S1. TEM images of ZGC synthesized with various Zn/Ga molar ratio, (A) 0.7/2, (B) 1/2, (C) 2/2. Scale bar, 100 nm.
Figure S2. XRDs of ZGC doped with diverse Cr3+ concentration by hydrothermal method. The particle size was also calculated by using Scherrer equation and the full width at half maximum of the diffraction peaks of ZnGa2O4:Cr0.004.
The Scherrer equation can be written as:
where: •
τ is the mean particle size;
•
K = 0.9;
•
λ is the X-ray wavelength;
•
β is the line broadening at half the maximum intensity (FWHM),
•
θ is the Bragg angle.
S5
Figure S3. EDX spectrum of ZGC-1.
Figure S4. Optimization of the doping concentration of Cr (vs Ga) in ZGC synthesized by hydrothermal method.
S6
Figure S5. The disperse stability of ZGC-2 in DI water. (A) Bright field picture before 30min (C) Bright field picture after 30min and (B, D) corresponding luminescence pictures of ZGC-2 in DI-water before and after 30min. Persistent luminescence intensity is expressed in false color units (1 unit = 2×107 photons·s-1·cm-2·sr) for all images.
Figure S6. The disperse stability of ZGC-1 and ZGC-2 in cell culture medium. (A) Digital and (B) corresponding PL images 30 min . Persistent luminescence intensity is expressed in false color units (1 unit = 6.8×106 photons·s-1·cm2 ·sr) for all images.
S7
Figure S7. Hydrodynamic size distribution of ZGC-1 in DI water and cell culture medium.
Figure S8. Hydrodynamic size distribution of ZGC-1 before and after PEI or BSA modification.
S8
Figure S9. FTIR spectra of ZGC-1, ZGC-1/PEI, ZGC-1/BSA. The absorption peak at ~585 cm-1 and 450 cm-1 represent the characteristic of Zn-O and Ga-O vibration, respectively. The peaks at 1018 cm-1 (C-C, stretching), 1382 cm-1 (C-H, bending), 1458 cm-1, 1560 cm-1 and 1628 cm-1 (N-H, bending), 2929 cm-1 and 2849 cm-1 (C-H, stretching), strong band at ~3400 cm-1 (N-H, stretching) represent the characteristics of PEI. The peaks at 1005 cm-1 (C-C, stretching), 1382 cm-1 (C-H, bending), 1535 cm-1 (N-H, bending), 1645 cm-1 (C=O, stretching), 2963 cm-1 (C-H, stretching), strong band at ~3400 cm-1 (N-H and COO-H, stretching) represent the characteristics of BSA.
Figure S10. Decay curves of ZGC-1 excited by various wavelengths.
S9
Figure S11. Analysis of the imaging ability of ZGC-1 and ZGC-2. (A) 50 mg in a black 96-well-plate; (B) in vivo activated imaging after subcutaneous injection (50 µL, 2 mg/mL). Persistent luminescence intensity is expressed in false color units (1 unit = 1×106 photons·s-1·cm-2·sr) for all images. Signal to background ratio is 275 for ZGC-1 and 100 for ZGC-2, respectively.
Figure S12. Digital picture of the pork slab used for deep tissue imaging.
S10
Figure S13. TEM of ZGC-2 by annealing method at 750 oC (A) and ZGC-3 after hydrothermal post-treatment (B). Scale bar, 100 nm.
Figure S14. (A) Proposed Mechanism of persistent luminescence (B) Proposed reasoning for enhancing PL via hydrothermal processing.
S11
Table S1. A comparison of the characteristics for the diverse categories of imaging probes. Persistent luminescence nanoparticles
Upconversion nanoparticles
NIR dyes
NIR dots
Signal-tobackground ratio
275, (this study Fig S11)
310[2]
17[3]
4[4]
Need Excitation resource during imaging
No
Yes
Yes
Yes
Need Modification on commercially available imaging system
No
Yes
No
No
quantum
[1]Maldiney, T.; Bessiere, A.; Seguin, J.; Teston, E.; Sharma, S. K.; Viana, B.; Bos, A. J. J.; Dorenbos, P.; Bessodes, M.; Gourier, D.; Scherman, D.; Richard, C. Nat Mater 2014, 13, 418. [2] Chen, G; Shen, J; Ohulchanskyy, T; Patel, N; Kutikov, A; Li, Z; Song, J; Pandey, R; Agren, H; Prasad, P; Han, G. Acs Nano 2012, 6, 8280. [3] Choi, H; Gibbs, S; Lee, J; Kim, S; Ashitate, Y; Liu, F; Hyun, H; Park, G; Xie, Y; Bae, S; Henary, M; Frangioni, J. Nat Biotechnol. 2013,31,148. [4]Cai, W; Shin, D; Chen, K; Gheysens, O; Cao, Q; Wang, S; Gambhir, S; Chen, X. Nano Lett 2006, 6, 669.
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Direct Aqueous-Phase Synthesis of Sub-10 nm “Luminous Pearls” with Enhanced in Vivo Renewable Near-Infrared Persistent Luminescence Zhanjun Li,† Yuanwei Zhang,† Xiang Wu, † Ling Huang, † Dongsheng Li§, Wei Fan,‡ Gang Han*† † Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605 (U.S.A) § Materials Sciences, Physical Sciences Division, Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352 ‡Chemical Engineering Department, University of Massachusetts Amherst, Amherst, MA 01003
Contact author: Prof. Gang Han, E-mail: [email protected]
S1
METHODS Materials Ga2O3, Zn(NO3)2·6H2O, Cr(NO3)3·9H2O, ammonium hydroxide, hydrochloride acid, and concentrated nitric acid were all analytical reagents and were used as they were received. Ga(NO3)3 solution was prepared by dissolving Ga2O3 in 1:1 concentrated nitric acid and this was followed by air drying it at 105o C in order to remove excess amounts of nitric acid and to be re-dissolved in deionized water. 1 mol/L Zn(NO3)2, 2 mol/L Ga(NO3)3, 4 mmol/L Cr(NO3)3 were stored as the precursor solutions here. A small amount of nitric acid was used to prevent the hydrolysis of Ga(NO3)3. Polyethyleneimine (PEI) and bovine serum albumin (BSA) were purchased from Sigma-Aldrich and used directly without further purification. The synthesis of ZGC-1
A certain amount of Ga(NO3)3 (2 M), Zn(NO3)2 (1 M), Cr(NO3)3 (4 mM) precursor
solutions were mixed at a predetermined molar ratio. Ammonium hydroxide (28%, wt) was added in a quick manner into the above mentioned mixture as stirring vigorously in order to reach a pH of 9. The metal hydroxide precursor was sealed into a Teflon-lined autoclave and this was followed by the hydrothermal reaction, which occurred at 220o C for 10 hours. These ZGC nanocrystals can be obtained within a wide range of molar ratios in regard to the precursors, the latter of which are influenced by the product size and distribution. In a typical procedure, 2 mmol of Zn(NO3)2, 2 mmol of Ga(NO3)3, 0.004 mmol Cr(NO3)3 are mixed together as vigorously stirred. The total volume was adjusted to 15 mL by adding deionized water. Concentrated ammonium hydroxide (28%) solution (about 1 mL) was added in a rapid manner in order to adjust the pH to be that of 9~9.5 and the white precipitate was formed immediately. After another half hour of stirring, the mixture was transmitted into a Teflon-lined autoclave (25 mL) and sealed. The autoclave was first put in an oven at 220o C for 10 hours and was then naturally cooled until reaching room temperature. The white precipitate obtained after centrifugation was dispersed in 0.01 M HCl, forming a transparent solution. Possible ZnO impurity was able to be removed in this step. Then ZGC nanocrystals were washed by centrifigations after being mixed with excess isopropanol. Finally, ZGC-1 nanocrystals were redispersed in deionized water for further characterization and imaging application. The concentration of ZGC that is used for storage is approximately 4 mg/mL.
S2
For the dispersion in the cell culture medium, 200 µL ZGC-1 storage solution was added into 5 mL of cell culture medium. A clear solution can be obtained after a brief ultrasonic mixing. Polyethylenimine (PEI) (positively charged polymer) can be adsorbed onto the crystal surface of ZGC due to the electrostatic interaction with the negatively charged ZGC nanocrystals, forming PEI modified ZGC (ZGC-PEI). In brief, 1 mL of ZGC stored solution was added to 10 mL of 1 mM of HCl solution, and was stirred for 30 min. 1 mL of PEI solution (20%, wt) was then rapidly added as stirring vigorously. After being diluted with excess isopropanol, The ZGC-PEI can be collected by centrifugation. The ZGC-PEI was redispersed in deionized water during the process of ultrasonic dispersion. ZGC-1 was also able to be modified with BSA. 1 mL of ZGC-1 solution (4 mg/mL) was added into 10 mL of BSA solution (1% by weight) drop by drop under stirring followed by 30 min stirring. BSA modified ZGC-1 (ZGC-BSA) was collected by centrifugation and then redispersed in 10 mL of DI water after a brief ultrasonic dispersion.
The synthesis of ZGC-2 ZGC-2 was synthesized for purposes of comparison according to a Ref 1. In brief, 1 mmol of Zn(NO3)2, 2 mmol of Ga(NO3)3, 0.004 mmol Cr(NO3)3 were mixed together as stirring vigorously. The total volume was adjusted to 10 mL by adding deionized water. The concentrated ammonium hydroxide (28%) solution (about 1 mL) was rapidly added in order to adjust the pH to 7.5. The mixture was sealed in a Teflonlined autoclave at 120o C for 24 hours. The resulting precipitate has no PL properties and was sintered at 750o C via a solid-state-reaction for 5 hours. A small amount of 5 mM NaOH solution was added to the powder product to form a slurry and this was followed by wet grinding for 1 hour with a mortar and pestle. The product was washed by ethanol (90%) and then dried at 60o C. The synthesis of ZGC-3 by using a hydrothermal post-treatment The as-synthesized ZGC-2 was mixed with an ammonium aqueous solution (pH9~9.5) and sealed in an autoclave, the latter of which underwent a hydrothermal procedure at 220o C for 10 hours. The as-synthesized ZGC-3 was centrifuged and dried at 80o C. Imaging A white LED (5000 lumen, CREE-T6) was used as the light source in all of the imaging experiments. For the in
S3
vitro experiment, 50 mg of the ZGC sample was put in one well of a black 96-well-plate. This plate was exposed to the LED for 30 s and then put into the IVIS 100 imaging system in order to detect the PL signal. The in vivo imaging was performed after the subcutaneous injection of the sample dispersion (50 µL, 2 mg/mL) and 30 s of in situ LED excitation. The in vivo simulated deep tissue recharging and imaging was performed by covering the subcutaneous injection site with a 1-cm pork slab. The imaging was performed right after 30 s of in situ LED excitation. Characterization The X-ray powder diffraction (XRD) was performed on a Panalytical X’pert PRO diffractometer that was equipped with Cu Kα radiation (λ = 1.5418 Å). The morphology of the samples was examined using a transmission electron microscope (TEM, Techni) at accelerating voltages of 5 kv and 800 kv. The energydispersive X-ray spectroscopy (EDS) was obtained on the FESEM at an accelerating voltage of 30 kv. High resolution TEM (HRTEM) images were performed using a JEM-1200EX II transmission electron microscope. The spectra and lifetimes of the samples were tested via the usage of powder samples. The photoluminescence spectra and decay curves were measured using a fluorospectrophotometer (FluoroMax-3, HORIBA, USA). The sample in the detection of the spectra and decay curves were dispersed in water, 4 mg/mL. The hydrodynamic size distributions were measured five times for each sample using a Zetasizer (Malvern, 3000HS),
average
results
were
used
as
the
mean
hydrodynamic
particle
size.
S4
Figure S1. TEM images of ZGC synthesized with various Zn/Ga molar ratio, (A) 0.7/2, (B) 1/2, (C) 2/2. Scale bar, 100 nm.
Figure S2. XRDs of ZGC doped with diverse Cr3+ concentration by hydrothermal method. The particle size was also calculated by using Scherrer equation and the full width at half maximum of the diffraction peaks of ZnGa2O4:Cr0.004.
The Scherrer equation can be written as:
where: •
τ is the mean particle size;
•
K = 0.9;
•
λ is the X-ray wavelength;
•
β is the line broadening at half the maximum intensity (FWHM),
•
θ is the Bragg angle.
S5
Figure S3. EDX spectrum of ZGC-1.
Figure S4. Optimization of the doping concentration of Cr (vs Ga) in ZGC synthesized by hydrothermal method.
S6
Figure S5. The disperse stability of ZGC-2 in DI water. (A) Bright field picture before 30min (C) Bright field picture after 30min and (B, D) corresponding luminescence pictures of ZGC-2 in DI-water before and after 30min. Persistent luminescence intensity is expressed in false color units (1 unit = 2×107 photons·s-1·cm-2·sr) for all images.
Figure S6. The disperse stability of ZGC-1 and ZGC-2 in cell culture medium. (A) Digital and (B) corresponding PL images 30 min . Persistent luminescence intensity is expressed in false color units (1 unit = 6.8×106 photons·s-1·cm2 ·sr) for all images.
S7
Figure S7. Hydrodynamic size distribution of ZGC-1 in DI water and cell culture medium.
Figure S8. Hydrodynamic size distribution of ZGC-1 before and after PEI or BSA modification.
S8
Figure S9. FTIR spectra of ZGC-1, ZGC-1/PEI, ZGC-1/BSA. The absorption peak at ~585 cm-1 and 450 cm-1 represent the characteristic of Zn-O and Ga-O vibration, respectively. The peaks at 1018 cm-1 (C-C, stretching), 1382 cm-1 (C-H, bending), 1458 cm-1, 1560 cm-1 and 1628 cm-1 (N-H, bending), 2929 cm-1 and 2849 cm-1 (C-H, stretching), strong band at ~3400 cm-1 (N-H, stretching) represent the characteristics of PEI. The peaks at 1005 cm-1 (C-C, stretching), 1382 cm-1 (C-H, bending), 1535 cm-1 (N-H, bending), 1645 cm-1 (C=O, stretching), 2963 cm-1 (C-H, stretching), strong band at ~3400 cm-1 (N-H and COO-H, stretching) represent the characteristics of BSA.
Figure S10. Decay curves of ZGC-1 excited by various wavelengths.
S9
Figure S11. Analysis of the imaging ability of ZGC-1 and ZGC-2. (A) 50 mg in a black 96-well-plate; (B) in vivo activated imaging after subcutaneous injection (50 µL, 2 mg/mL). Persistent luminescence intensity is expressed in false color units (1 unit = 1×106 photons·s-1·cm-2·sr) for all images. Signal to background ratio is 275 for ZGC-1 and 100 for ZGC-2, respectively.
Figure S12. Digital picture of the pork slab used for deep tissue imaging.
S10
Figure S13. TEM of ZGC-2 by annealing method at 750 oC (A) and ZGC-3 after hydrothermal post-treatment (B). Scale bar, 100 nm.
Figure S14. (A) Proposed Mechanism of persistent luminescence (B) Proposed reasoning for enhancing PL via hydrothermal processing.
S11
Table S1. A comparison of the characteristics for the diverse categories of imaging probes. Persistent luminescence nanoparticles
Upconversion nanoparticles
NIR dyes
NIR dots
Signal-tobackground ratio
275, (this study Fig S11)
310[2]
17[3]
4[4]
Need Excitation resource during imaging
No
Yes
Yes
Yes
Need Modification on commercially available imaging system
No
Yes
No
No
quantum
[1]Maldiney, T.; Bessiere, A.; Seguin, J.; Teston, E.; Sharma, S. K.; Viana, B.; Bos, A. J. J.; Dorenbos, P.; Bessodes, M.; Gourier, D.; Scherman, D.; Richard, C. Nat Mater 2014, 13, 418. [2] Chen, G; Shen, J; Ohulchanskyy, T; Patel, N; Kutikov, A; Li, Z; Song, J; Pandey, R; Agren, H; Prasad, P; Han, G. Acs Nano 2012, 6, 8280. [3] Choi, H; Gibbs, S; Lee, J; Kim, S; Ashitate, Y; Liu, F; Hyun, H; Park, G; Xie, Y; Bae, S; Henary, M; Frangioni, J. Nat Biotechnol. 2013,31,148. [4]Cai, W; Shin, D; Chen, K; Gheysens, O; Cao, Q; Wang, S; Gambhir, S; Chen, X. Nano Lett 2006, 6, 669.
S12
