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Imaging
Efficient Radioisotope Energy Transfer by Gold Nanoclusters for Molecular Imaging Olga Volotskova,* Conroy Sun,* Jason H. Stafford, Ai Leen Koh, Xiaowei Ma, Zhen Cheng, Bianxiao Cui, Guillem Pratx, and Lei Xing*
Beta-emitting isotopes Fluorine-18 and Yttrium-90 are tested for their potential to stimulate gold nanoclusters conjugated with blood serum proteins (AuNCs). AuNCs excited by either medical radioisotope are found to be highly effective ionizing radiation energy transfer mediators, suitable for in vivo optical imaging. AuNCs synthesized with protein templates convert beta-decaying radioisotope energy into tissue-penetrating optical signals between 620 and 800 nm. Optical signals are not detected from AuNCs incubated with Technetium-99m, a pure gamma emitter that is used as a control. Optical emission from AuNCs is not proportional to Cerenkov radiation, indicating that the energy transfer between the radionuclide and AuNC is only partially mediated by Cerenkov photons. A direct Coulombic interaction is proposed as a novel and significant mechanism of energy transfer between decaying radionuclides and AuNCs.
1. Introduction Novel clinical molecular imaging techniques seek to fulfill the demand for improved cancer detection and diagnosis by providing physicians with precise information on tumor location, Dr. O. Volotskova, Dr. C. Sun,[+] Dr. J. H. Stafford, Prof. G. Pratx, Prof. L. Xing Department of Radiation Oncology Stanford University Stanford, CA 94305, USA E-mail: [email protected]; [email protected]; [email protected] Dr. A. L. Koh Stanford Nanocharacterization Laboratory Stanford University Stanford, CA 94305, USA Dr. X. Ma, Prof. Z. Cheng Department of Radiology Stanford University Stanford, CA 94305, USA Prof. B. Cui Department of Chemistry Stanford University Stanford, CA 94305, USA [+]Present Address: Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, USA DOI: 10.1002/smll.201500907
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size, metastasis, and other biological characteristics that can affect treatment.[1,2] Nuclear medicine imaging modalities, such as positron emission tomography (PET) and singlephoton emission computed tomography (SPECT) are based on the specific uptake of radiopharmaceuticals by malignant cells and have become integral tools in clinical oncology. Using positron-emitting radiotracers, PET imaging has been demonstrated to provide more accurate information in discriminating between viable tumors, fibrotic scars, and necrotic tissue than purely anatomical imaging modalities such as X-ray CT.[3,4] However, the use of PET is limited by its high cost and low molecular sensitivity for small lesions.[5,6] In addition, PET imaging is only able to detect gamma photons and it is currently impossible to accurately determine the biodistributions of beta-emitting therapeutic radioimmunoconjugates (i.e., Iodine-131/Bexxar and Yttrium-90/Zevalin) administered to patients to treat lymphomas. Therefore, various in vivo optical and hybrid imaging technologies are being investigated to overcome these limitations.[7–10] For example, multimodal imaging based on visualization of tumor and healthy tissue differences in absorption and radioactive decay signal conversion has emerged as a novel approach for tumor imaging.[11] Cerenkov radiation (CR) is a type of luminescence produced when a radioisotope decays and emits charged particles that travel through a medium faster than light in that
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Figure 1. Characterization of BSA-conjugated AuNCs. a) Dynamic light scattering data displaying size distribution of AuNCs at pH ≈7. HR-TEM analysis confirms the existence of gold clusters with interplanar spacing of ≈2.35 Å. b) Absorption and photoemission (AuNC λex 450 nm) at room and physiological temperature. c) Photoemission of AuNC excited in “blue” Cerenkov region (300–500 nm). d) Spectrum of Cerenkov photons emitted per unit wavelength in the water for Emax of 18F and 90Y (from the literature).[38]
medium.[12] This unique phenomenon has recently been observed in living subjects,[13,14] and Cerenkov luminescence imaging (CLI) has demonstrated significant potential for use in clinical applications.[15–17] Detection of Cerenkov light may enable the use of optical imaging equipment to visualize biomarkers via PET radiotracers and the dosimetry of therapeutic radiopharmaceuticals but, a major hurdle in its implementation is that CR is primarily composed of UVblue photons (250–550 nm), which can only penetrate a few millimeters of tissue. A promising strategy is to use energy transfer mediators to convert CR to a longer wavelength signal through Cerenkov radiation energy transfer (CRET) for detection of deep-seated tumors.[18–21] Quantum dots (QDs) have been explored for this purpose but toxicity of QD components may prohibit its clinical implementation.[21] In addition to QDs, nanophosphors (NPs) and gold nanoclusters (AuNCs) have also been shown to produce optical signals under ionizing radiation. [22–24] The physiochemical properties of AuNCs suggest that may be highly efficient mediators of radioisotope energy transfer (RET) suitable for use in the clinic. Nanoclusters are composed of a small number of metal atoms packed into a crystal or quasicrystal structure of a simple polyhedral form with an active surface and distinct electronic properties.[25–27] The nanometer size of these particles determines the behavior of free electrons as the Fermi wavelength of an electron is less than 1 nm. Nanoclusters are too small to have the continuous density of states and support “plasmonic” characteristics, resulting in discrete, quantum-confined electronic transitions.[26–29] AuNCs possess high signal-to-noise ratios and are not susceptible to photobleaching due to their unique physiochemical properties. Furthermore, preliminary studies also suggest that various bioconjugates of AuNCs have low toxicity and favorable biodistributions for in vivo imaging applications.[30,31] In the present study, we demonstrate that AuNCs synthesized as conjugates with bovine serum albumin (BSA) can be used for highly efficient RET via direct–beta (Coulombic interactions) and indirect-beta (CRET) interactions from the small 2015, 11, No. 32, 4002–4008
clinically approved radiopharmaceuticals 2-deoxy-2-(18F) fluoro-d-glucose (18F) and Yttrium-90 (90Y).
2. Results and Discussion 2.1. AuNC Synthesis and Characterization We used a protein-templated synthesis method to produce AuNCs conjugated with BSA as recently reported by Xie et al.[32,33] Serum albumin is the most abundant protein in the circulatory system; it is produced by the liver in mammals with a blood half-life of ≈19 d, accounting for 60% of the total serum protein.[34] Blood serum proteins such as albumin and glycoproteins are widely used as macromolecular carriers because they can be loaded with a variety of anticancer drugs, photosensitizers, and contrast-enhancing materials.[9,35,36] We analyzed purified BSA-conjugated AuNCs by transmission electron microscopy (TEM), dynamic light scattering (DLS), and optical spectroscopy (absorption and photoemission). AuNCs exhibited atomic spacing size of ≈2.35 Å corresponding to the Au (111) plane as determined by high-resolution TEM (HRTEM) (Figure 1a). DLS analysis revealed that cluster size varied in diameter depending on AuNC protein incorporation. Optical absorption bands were observed at ≈2.5 and 3.7 eV (490 and 350 nm) confirming the presence of 25-atom gold clusters in all three conjugates.[25,29,37] A strong emission peak was observed at ≈1.8 eV (≈680–700 nm) for all serum protein conjugates. No changes were observed in the optical characteristics at physiological temperature (37 °C) as compared to room temperature. We also confirmed that AuNCs can be continuously excited in the range ≈350–500 nm, which match the emission spectrum of CR (Figure 1b–d).[38] The emission peak at 1.8 eV (≈680–700 nm) was most intense when AuNCs were excited at ≈2.5 eV (500 nm) (Figure 1). AuNCs were tested for stability at different pH levels and AuNCs were stable in aqueous solvent over a physiologically relevant pH range (pH 5–10). Sample concentration (≈1 × 10−6 m) was
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determined by inductively coupled plasma atomic emission spectroscopy (ICP) measurements of the ionic gold content and atomic weight calculated for the 25 atoms of gold.
background luminescence was present. AuNCs exhibited a strong luminescence emission peak at ≈1.8 eV (Figure 2a,b). This peak corresponds well to the spectral properties of AuNCs due to the 6sp electron transition (600–730 nm). The maximum energy of decaying 90Y (≈2.25 MeV) is ≈4 times higher than 18F (0.633 MeV), thus the number of Cerenkov photons produced per unit wavelength was correspondingly higher (Figure 1d) and its detectable emission extends into the long wavelength region of the optical spectrum.[38] We then verified that optical signal observed in AuNC did not originate from high-energy photons (e.g., 511 keV annihilation photons from 18F). 99mTc is a pure gamma emitter that emits at 140 keV and is similar to clinical diagnostic X-ray sources, below the threshold for Cerenkov radiation, and is expected to have a greater probability of photon interaction than 18F.[24] No difference in the spectral signal was observed for any of the serum-protein conjugated AuNCs, serum proteins only, or ionic gold only in the presence of 99mTc or in wells containing 99mTc alone. This confirmed that beta-decay from 90Y and 18F was down-converted by AuNCs, and that 99mTc produced neither CR nor detectable AuNC luminescence. We also templated AuNCs with either human serum albumin (HSA) and human transferrin (Tf) to show that RET is a unique property of AuNC stabilized with Au–S linkages to a capping agent such as blood serum proteins.[32,33,39,40] HSA- and Tf-conjugated AuNC were evaluated for RET from 18F (Figures S1–S3, Supporting Information). The data confirmed an optical peak at ≈1.8 eV with 2–3-fold increase over water CR background, and that no RET signal can be observed from pure proteins or ionic gold. In order to characterize the threshold values of the RET effect produced by AuNCs, we measured dose- and concentration-dependent luminescence (Figure 3). The data are shown for selected wavelengths in the range of 460–720 nm. We found a strong linear dependence of the RET signal produced by AuNC on the amount of activity starting at 10 µCi for 18F and 1 µCi for 90Y. This difference can again be explained by difference in the beta energy ranges of investigated radioisotopes. Signal luminescence increases with the concentration of the AuNCs, with a lower detection limit of 0.1 × 10−6 m of atomic gold content in both cases for 18F and 90Y. At higher concentrations, the relative signal is less than 1 for wavelengths lower than 600 nm, which is likely due to absorbance of CR by the nanoclusters in this wavelength range.
2.2. RET Emission by AuNCs
2.3. Radioisotope–AuNC Interactions
We next sought to determine if BSA-conjugated AuNCs (1 × 10−6 m) can function as a probe to image decaying radioisotopes by RET (Figure 2). 18F and 90Y were used as excitation sources capable of emitting CR. No change in signal was observed from serum proteins or ionic gold when mixed with either radioisotope. CR produced in water was used as a baseline measurement to compensate for change in activity over the time of data acquisition. Samples with no radioactivity were imaged as a negative control to confirm that no
For 18F, the total radiance produced by AuNCs was always greater than CR alone (open-filter reading; 250–900 nm) by ≈20%–30% (Figure S4, Supporting Information). If AuNC emissions were solely due to excitation from CR, the amount of energy emitted by the AuNCs should be less than the total CR energy absorbed by the system for various energy levels due to energy conservation, and the total radiance produced by the mixture should be less than the radiance from CR alone. Our finding thus suggests the contribution
Figure 2. RET spectrum. Spectral data of controls, i.e., water (H2O), gold chloride (HAuCl4), and BSA only, and BSA-conjugated AuNCs. a,b) AuNC were excited by 18F (120 µCi) and 90Y (20 µCi), respectively, data were recorded in the range 460–720 nm. c) Spectrum measurements of control experiment with γ-emitter 99mTc (300 µCi). d–f) Captions are shown for blue (≈460 nm) and red region of spectrum (≈700 nm). All RET data are taken at 37 °C. Data were normalized to the decay value of radiotracers in the water.
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energy-photons (via gamma-irradiation). Our data suggest that gamma-irradiation is a negligible contributor to AuNC luminescence (Figure 4b). Thus, the luminescence output (LO) of AuNC as a result of interactions with radioisotope can be calculated by the equation LO = A × C × ξ CF + A × C × ξ CR × ξ CRET Direct beta CF − term
Indirect beta CR − term
(1)
= A × C × (ξ CF + ξ CR × ξ CRET )
where A is radioisotope activity (µCi), C is AuNC concentration (µM), ξCF is efficiency of nanocluster’s interaction via Coulomb forces (CF-term) (photons µCi−1 µM−1), ξCR is efficiency of Cerenkov Radiation (CR-term) production by radiotracer (photons µCi−1 µM−1) and ξCRET is quantum efficiency of AuNC’s conversion of CR photons into R-NIR photons (determined as the ratio of total number of CR photons to the total number of CRET red photons). This phenomenological model was compared with experimental results. Assuming that both conversion efficiencies (direct Figure 3. Verification of RET in vitro. a,c) Dose-dependence of 18F and 90Y in the presence of constant amount of BSA-conjugated AuNCs with atomic gold concentration of ≈1 × 10−6 M. beta and indirect beta) are equal and, LO = CF + CR then the terms in light outb,d) Concentration dependence of AuNCs in the presence of a constant amount of 18F (200 put of 18F will be CF( 18 F) ≈ 2 × CR( 18 F) µCi) and 90Y (20µCi). All data taken at 37 °C. and, for 90Y, CF( 90 Y) ≈ CR( 90 Y), given that the beta energy spectrum of 90Y of an additional excitation mechanism. Additionally, open has larger CR range (above water threshold 0.225 MeV, filter imaging showed the presence of weak luminescence in Figure 4a). Since the data were normalized to Cerenkov backAuNCs from pure-gamma-emitting 99mTc (Figure S5, Sup- ground radiation (CR-term), LO( 18 F) / CR( 18 F) ≈ CF / CR + 1 ≈ 3 porting Information) and 90Y integral luminescence showed and LO( 90 Y) / CR( 90 Y) ≈ CF / CR + 1 ≈ 2 . Data in Figure 2a,b that the ratio of RET from AuNCs signal to CR only from show that 90Y increased less than 18F, demonstrating that while water was less than 1 (calculated for 20 and 40 µCi of 90Y). CRET conversion was high in 90Y, the contribution of direct Since 18F emits both sub-Cerenkov betas and gamma pho- beta interactions was significant for 18F. The optimization of the tons in large proportions, this suggests that the unexpected photophysical properties (e.g., increase in quantum yield, better 20%–30% increase in luminescence for 18F is related to direct stability, etc.) will lead to enhancement of RET luminescence. Coulombic excitation of AuNC by beta particles (including those below the Cerenkov threshold), or by high-energy photons as reported by Spinelli et al.[41] Our investigation of 2.4. In Vivo RET Signal Conversion by AuNCs 99mTc (140 keV), a pure gamma emitter, revealed that excitation by high-energy photons is highly inefficient (conversion Finally, we tested the ability of BSA-conjugated AuNCs efficiency
Imaging
Efficient Radioisotope Energy Transfer by Gold Nanoclusters for Molecular Imaging Olga Volotskova,* Conroy Sun,* Jason H. Stafford, Ai Leen Koh, Xiaowei Ma, Zhen Cheng, Bianxiao Cui, Guillem Pratx, and Lei Xing*
Beta-emitting isotopes Fluorine-18 and Yttrium-90 are tested for their potential to stimulate gold nanoclusters conjugated with blood serum proteins (AuNCs). AuNCs excited by either medical radioisotope are found to be highly effective ionizing radiation energy transfer mediators, suitable for in vivo optical imaging. AuNCs synthesized with protein templates convert beta-decaying radioisotope energy into tissue-penetrating optical signals between 620 and 800 nm. Optical signals are not detected from AuNCs incubated with Technetium-99m, a pure gamma emitter that is used as a control. Optical emission from AuNCs is not proportional to Cerenkov radiation, indicating that the energy transfer between the radionuclide and AuNC is only partially mediated by Cerenkov photons. A direct Coulombic interaction is proposed as a novel and significant mechanism of energy transfer between decaying radionuclides and AuNCs.
1. Introduction Novel clinical molecular imaging techniques seek to fulfill the demand for improved cancer detection and diagnosis by providing physicians with precise information on tumor location, Dr. O. Volotskova, Dr. C. Sun,[+] Dr. J. H. Stafford, Prof. G. Pratx, Prof. L. Xing Department of Radiation Oncology Stanford University Stanford, CA 94305, USA E-mail: [email protected]; [email protected]; [email protected] Dr. A. L. Koh Stanford Nanocharacterization Laboratory Stanford University Stanford, CA 94305, USA Dr. X. Ma, Prof. Z. Cheng Department of Radiology Stanford University Stanford, CA 94305, USA Prof. B. Cui Department of Chemistry Stanford University Stanford, CA 94305, USA [+]Present Address: Department of Pharmaceutical Sciences, Oregon State University, Corvallis, OR, USA DOI: 10.1002/smll.201500907
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size, metastasis, and other biological characteristics that can affect treatment.[1,2] Nuclear medicine imaging modalities, such as positron emission tomography (PET) and singlephoton emission computed tomography (SPECT) are based on the specific uptake of radiopharmaceuticals by malignant cells and have become integral tools in clinical oncology. Using positron-emitting radiotracers, PET imaging has been demonstrated to provide more accurate information in discriminating between viable tumors, fibrotic scars, and necrotic tissue than purely anatomical imaging modalities such as X-ray CT.[3,4] However, the use of PET is limited by its high cost and low molecular sensitivity for small lesions.[5,6] In addition, PET imaging is only able to detect gamma photons and it is currently impossible to accurately determine the biodistributions of beta-emitting therapeutic radioimmunoconjugates (i.e., Iodine-131/Bexxar and Yttrium-90/Zevalin) administered to patients to treat lymphomas. Therefore, various in vivo optical and hybrid imaging technologies are being investigated to overcome these limitations.[7–10] For example, multimodal imaging based on visualization of tumor and healthy tissue differences in absorption and radioactive decay signal conversion has emerged as a novel approach for tumor imaging.[11] Cerenkov radiation (CR) is a type of luminescence produced when a radioisotope decays and emits charged particles that travel through a medium faster than light in that
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Figure 1. Characterization of BSA-conjugated AuNCs. a) Dynamic light scattering data displaying size distribution of AuNCs at pH ≈7. HR-TEM analysis confirms the existence of gold clusters with interplanar spacing of ≈2.35 Å. b) Absorption and photoemission (AuNC λex 450 nm) at room and physiological temperature. c) Photoemission of AuNC excited in “blue” Cerenkov region (300–500 nm). d) Spectrum of Cerenkov photons emitted per unit wavelength in the water for Emax of 18F and 90Y (from the literature).[38]
medium.[12] This unique phenomenon has recently been observed in living subjects,[13,14] and Cerenkov luminescence imaging (CLI) has demonstrated significant potential for use in clinical applications.[15–17] Detection of Cerenkov light may enable the use of optical imaging equipment to visualize biomarkers via PET radiotracers and the dosimetry of therapeutic radiopharmaceuticals but, a major hurdle in its implementation is that CR is primarily composed of UVblue photons (250–550 nm), which can only penetrate a few millimeters of tissue. A promising strategy is to use energy transfer mediators to convert CR to a longer wavelength signal through Cerenkov radiation energy transfer (CRET) for detection of deep-seated tumors.[18–21] Quantum dots (QDs) have been explored for this purpose but toxicity of QD components may prohibit its clinical implementation.[21] In addition to QDs, nanophosphors (NPs) and gold nanoclusters (AuNCs) have also been shown to produce optical signals under ionizing radiation. [22–24] The physiochemical properties of AuNCs suggest that may be highly efficient mediators of radioisotope energy transfer (RET) suitable for use in the clinic. Nanoclusters are composed of a small number of metal atoms packed into a crystal or quasicrystal structure of a simple polyhedral form with an active surface and distinct electronic properties.[25–27] The nanometer size of these particles determines the behavior of free electrons as the Fermi wavelength of an electron is less than 1 nm. Nanoclusters are too small to have the continuous density of states and support “plasmonic” characteristics, resulting in discrete, quantum-confined electronic transitions.[26–29] AuNCs possess high signal-to-noise ratios and are not susceptible to photobleaching due to their unique physiochemical properties. Furthermore, preliminary studies also suggest that various bioconjugates of AuNCs have low toxicity and favorable biodistributions for in vivo imaging applications.[30,31] In the present study, we demonstrate that AuNCs synthesized as conjugates with bovine serum albumin (BSA) can be used for highly efficient RET via direct–beta (Coulombic interactions) and indirect-beta (CRET) interactions from the small 2015, 11, No. 32, 4002–4008
clinically approved radiopharmaceuticals 2-deoxy-2-(18F) fluoro-d-glucose (18F) and Yttrium-90 (90Y).
2. Results and Discussion 2.1. AuNC Synthesis and Characterization We used a protein-templated synthesis method to produce AuNCs conjugated with BSA as recently reported by Xie et al.[32,33] Serum albumin is the most abundant protein in the circulatory system; it is produced by the liver in mammals with a blood half-life of ≈19 d, accounting for 60% of the total serum protein.[34] Blood serum proteins such as albumin and glycoproteins are widely used as macromolecular carriers because they can be loaded with a variety of anticancer drugs, photosensitizers, and contrast-enhancing materials.[9,35,36] We analyzed purified BSA-conjugated AuNCs by transmission electron microscopy (TEM), dynamic light scattering (DLS), and optical spectroscopy (absorption and photoemission). AuNCs exhibited atomic spacing size of ≈2.35 Å corresponding to the Au (111) plane as determined by high-resolution TEM (HRTEM) (Figure 1a). DLS analysis revealed that cluster size varied in diameter depending on AuNC protein incorporation. Optical absorption bands were observed at ≈2.5 and 3.7 eV (490 and 350 nm) confirming the presence of 25-atom gold clusters in all three conjugates.[25,29,37] A strong emission peak was observed at ≈1.8 eV (≈680–700 nm) for all serum protein conjugates. No changes were observed in the optical characteristics at physiological temperature (37 °C) as compared to room temperature. We also confirmed that AuNCs can be continuously excited in the range ≈350–500 nm, which match the emission spectrum of CR (Figure 1b–d).[38] The emission peak at 1.8 eV (≈680–700 nm) was most intense when AuNCs were excited at ≈2.5 eV (500 nm) (Figure 1). AuNCs were tested for stability at different pH levels and AuNCs were stable in aqueous solvent over a physiologically relevant pH range (pH 5–10). Sample concentration (≈1 × 10−6 m) was
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determined by inductively coupled plasma atomic emission spectroscopy (ICP) measurements of the ionic gold content and atomic weight calculated for the 25 atoms of gold.
background luminescence was present. AuNCs exhibited a strong luminescence emission peak at ≈1.8 eV (Figure 2a,b). This peak corresponds well to the spectral properties of AuNCs due to the 6sp electron transition (600–730 nm). The maximum energy of decaying 90Y (≈2.25 MeV) is ≈4 times higher than 18F (0.633 MeV), thus the number of Cerenkov photons produced per unit wavelength was correspondingly higher (Figure 1d) and its detectable emission extends into the long wavelength region of the optical spectrum.[38] We then verified that optical signal observed in AuNC did not originate from high-energy photons (e.g., 511 keV annihilation photons from 18F). 99mTc is a pure gamma emitter that emits at 140 keV and is similar to clinical diagnostic X-ray sources, below the threshold for Cerenkov radiation, and is expected to have a greater probability of photon interaction than 18F.[24] No difference in the spectral signal was observed for any of the serum-protein conjugated AuNCs, serum proteins only, or ionic gold only in the presence of 99mTc or in wells containing 99mTc alone. This confirmed that beta-decay from 90Y and 18F was down-converted by AuNCs, and that 99mTc produced neither CR nor detectable AuNC luminescence. We also templated AuNCs with either human serum albumin (HSA) and human transferrin (Tf) to show that RET is a unique property of AuNC stabilized with Au–S linkages to a capping agent such as blood serum proteins.[32,33,39,40] HSA- and Tf-conjugated AuNC were evaluated for RET from 18F (Figures S1–S3, Supporting Information). The data confirmed an optical peak at ≈1.8 eV with 2–3-fold increase over water CR background, and that no RET signal can be observed from pure proteins or ionic gold. In order to characterize the threshold values of the RET effect produced by AuNCs, we measured dose- and concentration-dependent luminescence (Figure 3). The data are shown for selected wavelengths in the range of 460–720 nm. We found a strong linear dependence of the RET signal produced by AuNC on the amount of activity starting at 10 µCi for 18F and 1 µCi for 90Y. This difference can again be explained by difference in the beta energy ranges of investigated radioisotopes. Signal luminescence increases with the concentration of the AuNCs, with a lower detection limit of 0.1 × 10−6 m of atomic gold content in both cases for 18F and 90Y. At higher concentrations, the relative signal is less than 1 for wavelengths lower than 600 nm, which is likely due to absorbance of CR by the nanoclusters in this wavelength range.
2.2. RET Emission by AuNCs
2.3. Radioisotope–AuNC Interactions
We next sought to determine if BSA-conjugated AuNCs (1 × 10−6 m) can function as a probe to image decaying radioisotopes by RET (Figure 2). 18F and 90Y were used as excitation sources capable of emitting CR. No change in signal was observed from serum proteins or ionic gold when mixed with either radioisotope. CR produced in water was used as a baseline measurement to compensate for change in activity over the time of data acquisition. Samples with no radioactivity were imaged as a negative control to confirm that no
For 18F, the total radiance produced by AuNCs was always greater than CR alone (open-filter reading; 250–900 nm) by ≈20%–30% (Figure S4, Supporting Information). If AuNC emissions were solely due to excitation from CR, the amount of energy emitted by the AuNCs should be less than the total CR energy absorbed by the system for various energy levels due to energy conservation, and the total radiance produced by the mixture should be less than the radiance from CR alone. Our finding thus suggests the contribution
Figure 2. RET spectrum. Spectral data of controls, i.e., water (H2O), gold chloride (HAuCl4), and BSA only, and BSA-conjugated AuNCs. a,b) AuNC were excited by 18F (120 µCi) and 90Y (20 µCi), respectively, data were recorded in the range 460–720 nm. c) Spectrum measurements of control experiment with γ-emitter 99mTc (300 µCi). d–f) Captions are shown for blue (≈460 nm) and red region of spectrum (≈700 nm). All RET data are taken at 37 °C. Data were normalized to the decay value of radiotracers in the water.
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energy-photons (via gamma-irradiation). Our data suggest that gamma-irradiation is a negligible contributor to AuNC luminescence (Figure 4b). Thus, the luminescence output (LO) of AuNC as a result of interactions with radioisotope can be calculated by the equation LO = A × C × ξ CF + A × C × ξ CR × ξ CRET Direct beta CF − term
Indirect beta CR − term
(1)
= A × C × (ξ CF + ξ CR × ξ CRET )
where A is radioisotope activity (µCi), C is AuNC concentration (µM), ξCF is efficiency of nanocluster’s interaction via Coulomb forces (CF-term) (photons µCi−1 µM−1), ξCR is efficiency of Cerenkov Radiation (CR-term) production by radiotracer (photons µCi−1 µM−1) and ξCRET is quantum efficiency of AuNC’s conversion of CR photons into R-NIR photons (determined as the ratio of total number of CR photons to the total number of CRET red photons). This phenomenological model was compared with experimental results. Assuming that both conversion efficiencies (direct Figure 3. Verification of RET in vitro. a,c) Dose-dependence of 18F and 90Y in the presence of constant amount of BSA-conjugated AuNCs with atomic gold concentration of ≈1 × 10−6 M. beta and indirect beta) are equal and, LO = CF + CR then the terms in light outb,d) Concentration dependence of AuNCs in the presence of a constant amount of 18F (200 put of 18F will be CF( 18 F) ≈ 2 × CR( 18 F) µCi) and 90Y (20µCi). All data taken at 37 °C. and, for 90Y, CF( 90 Y) ≈ CR( 90 Y), given that the beta energy spectrum of 90Y of an additional excitation mechanism. Additionally, open has larger CR range (above water threshold 0.225 MeV, filter imaging showed the presence of weak luminescence in Figure 4a). Since the data were normalized to Cerenkov backAuNCs from pure-gamma-emitting 99mTc (Figure S5, Sup- ground radiation (CR-term), LO( 18 F) / CR( 18 F) ≈ CF / CR + 1 ≈ 3 porting Information) and 90Y integral luminescence showed and LO( 90 Y) / CR( 90 Y) ≈ CF / CR + 1 ≈ 2 . Data in Figure 2a,b that the ratio of RET from AuNCs signal to CR only from show that 90Y increased less than 18F, demonstrating that while water was less than 1 (calculated for 20 and 40 µCi of 90Y). CRET conversion was high in 90Y, the contribution of direct Since 18F emits both sub-Cerenkov betas and gamma pho- beta interactions was significant for 18F. The optimization of the tons in large proportions, this suggests that the unexpected photophysical properties (e.g., increase in quantum yield, better 20%–30% increase in luminescence for 18F is related to direct stability, etc.) will lead to enhancement of RET luminescence. Coulombic excitation of AuNC by beta particles (including those below the Cerenkov threshold), or by high-energy photons as reported by Spinelli et al.[41] Our investigation of 2.4. In Vivo RET Signal Conversion by AuNCs 99mTc (140 keV), a pure gamma emitter, revealed that excitation by high-energy photons is highly inefficient (conversion Finally, we tested the ability of BSA-conjugated AuNCs efficiency
