Article pubs.acs.org/IECR
Industrial-Scale Synthesis of Intrinsically Radiolabeled 64CuS Nanoparticles for Use in Positron Emission Tomography (PET) Imaging of Cancer Rubel Chakravarty,*,† Sudipta Chakraborty,† Raghumani Singh Ningthoujam,‡ K. V. Vimalnath Nair,† K. Shitaljit Sharma,‡ Anand Ballal,§ Apurav Guleria,∥ Amit Kunwar,∥ Haladhar Dev Sarma,⊥ Rajesh Kumar Vatsa,‡ and Ashutosh Dash† †
Radiopharmaceuticals Division, ‡Chemistry Division, §Molecular Biology Division, ∥Radiation and Photochemistry Division, and Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400 085, India
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ABSTRACT: Synthesis of intrinsically radiolabeled nanoparticles is an emerging concept in cancer theranostics and is expected to play an imperative role in translating nanotechnology research into the nuclear medicine industry. In order to reduce reliance on cyclotron produced 64Cu (t1/2 = 12.7 h, EC 45%, β+ 17.9%, β− 37.1%) and increase global accessibility of this radioisotope for preclinical and clinical investigations, we have explored the feasibility of using neutron-activated 64Cu produced in research reactors for potential use in cancer theranostics. A viable strategy has been developed for production of 64Cu in medium-flux research reactors and its utilization toward industrial-scale (GBq level) synthesis of intrinsically radiolabeled 64CuS nanoparticles (∼30 nm particle size). The synthesis procedure was easily executable in a hot cell equipped with remotely operable gadgets and 64 CuS nanoparticles could be synthesized in a form suitable for clinical administration. The stability of the nanoparticles under physiological conditions was established by detailed in vitro studies in phosphate buffered saline (PBS) and mouse serum media. The biological efficacy of intrinsically radiolabeled 64CuS nanoparticles was studied in C57BL/6 mice bearing melanoma tumors. The results of the biodistribution studies revealed significant tumor uptake (4.64% ± 1.71%ID/g) within 4 h post-injection (where %ID is the percent injected radioactivity dose), with good tumor-to-background contrast. Collectively, the promising results obtained in this study suggest that the concept of intrinsically radiolabeled nanoplatforms can be employed to facilitate widespread utilization of neutron-activated 64Cu in nuclear medicine industry.
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INTRODUCTION
Among various radioisotopes used toward preparation of radiolabeled nanoplatforms, 64Cu (t1/2 = 12.7 h) has attracted significant interest, particularly because of its unique nuclear decay characteristics.4 This radioisotope decays via three processes, namely, electron capture (EC), positron, and beta decays (EC 45%, β+ 17.9%, β− 37.1%), which allows for its utility both in the preparation of probes for PET imaging and also in the development of radiotherapy agents for treatment of various types of cancers.4 Another advantage of the unique nuclear characteristics of 64Cu is that the radioisotope can be produced both in a nuclear reactor as well as in a cyclotron. However, the
Nanomedicine has witnessed unprecedented growth over the past few years, thanks to the development of functionalized nanoplatforms for cancer theranostics. With the rapidly growing interest in using radioisotopes for nanomedicine, a broad spectrum of radiolabeled nanoplatforms have been generated that provide the opportunity for in vivo positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging of tumors for noninvasive visualization of the disease in its early stage and also monitor the efficacy of the therapeutic procedures.1−3 The success of a radiolabeled nanoplatform in cancer theranostics is primarily dependent on the choice of the radioisotope and development of facile and robust strategy for its incorporation into the nanoplatform such that the radiolabeled agent demonstrates high in vitro and in vivo stability suitable for clinical utilization. © 2016 American Chemical Society
Received: Revised: Accepted: Published: 12407
September 3, 2016 November 2, 2016 November 14, 2016 November 14, 2016 DOI: 10.1021/acs.iecr.6b03405 Ind. Eng. Chem. Res. 2016, 55, 12407−12419
Industrial & Engineering Chemistry Research
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majority of the present-generation 64Cu-labeled formulations require high-specific-activity 64Cu in no-carrier-added (NCA) form, which can effectively be produced by 64Ni (p,n) 64Cu reaction in a biomedical cyclotron.5,6 Therefore, the utility of this radioisotope is limited to a few developed countries that have excellent cyclotron facilities for radioisotope production. The requirement of using elaborate procedures for radiochemical separation of NCA 64Cu from the irradiated target, as well as the logistic challenges for supply of this relatively short-lived radioisotope to distant user sites from the limited production facilities, are some of the other issues that makes this otherwise excellent radioisotope less popular in the clinical context. A facile approach for large-scale production of 64Cu is via the thermal neutron capture [63Cu (n,γ) 64Cu] reaction in a research reactor.7 The neutron activation route for the production of 64Cu not only precludes the need for radiochemical separation but also leads to negligible radioactive waste generation, which, in turn, makes the overall process cost-effective. Moreover, because of the presence of several medium-flux research reactors with good geographical distribution throughout the world, this approach would facilitate the availability of 64Cu in several developing countries.8 However, the specific activity of neutron-activated 64 Cu is significantly (>1000 times) lower than that of NCA 64Cu produced in a cyclotron.6,7 Therefore, in order to utilize neutronactivated 64Cu for PET imaging of cancer, it is essential to develop a radiolabeling strategy that is independent of the specific activity of the radioisotope. While using reactor-produced 64Cu, the most widely used radiolabeling strategy employing exogenous chelators would not be suitable for the preparation of clinically relevant doses of the radiolabeled agents with adequate specific activity. In this premise, a prudent approach to utilize low-specific-activity 64 Cu for PET imaging is to synthesize intrinsically radiolabeled nanoparticles using a radioactive precursor (64CuCl2 solution). Adopting this strategy, 64Cu atoms are built inside the crystal lattice of the normal nanocrystals, resulting in high radiochemical stability. Intrinsically radiolabeled 64CuS nanoparticles are one such class of nanoplatforms, which can easily be prepared by metathesis reaction of 64CuCl2 and Na2S.9−11 The simplicity of the procedure allows synthesis of clinically relevant doses of the radioactive 64CuS nanoparticles in an adequately shielded facility with minimum radiation exposure to the personnel involved. An inherent characteristic of CuS nanoparticles is that they can absorb light in the near-infrared (NIR) region and convert it to thermal energy for thermal ablation of tumor cells.9,12 Therefore, in addition to its utility as a probe for PET imaging, such nanoparticles hold promise for use as a dual modality therapeutic agent (radiotherapy due to β− emission of 64Cu and photoablation therapy due to CuS nanoparticles). Herein, we report large-scale synthesis of intrinsically radiolabeled 64CuS nanoparticles using reactor-produced 64Cu for potential utility in cancer theranostics. The suitability of the radioactive nanoplatform as a PET probe was evaluated and its efficacy was established by biodistribution studies in C57BL/6 mice bearing melanoma tumors. To the best of our knowledge, this is the first report on the utility of a reactor-produced radioisotope toward the development of a nanoparticle-based probe for PET imaging of cancer. Our technology allows for the preparation of high radioactive doses of 64CuS nanoparticlesan advance that could allow industrial-scale synthesis of intrinsically radiolabeled nanoplatforms conforming to current Good Manufacturing Practice (cGMP) guidelines.
Article
EXPERIMENTAL SECTION
Materials. Copper oxide (spectroscopic grade, >99.99% pure), which was used as the target material for the production of 64 Cu, was obtained from E. Merck. Sodium sulfide (Na2S·9H2O), cetyltrimethylammonium chloride (CTAC), and methoxy-PEGthiol (SH-PEG; molecular weight = 5000 Da) were purchased from Sigma−Aldrich. All other chemicals were analytical reagent (AR) grade and purchased from established manufacturers. Standard source of 152Eu (t1/2 = 13.6 years) is a routine product of Radiopharmaceuticals Division, Bhabha Atomic Research Centre, and was readily available in our laboratory. Flexible silica plates (coating thickness = 0.25 mm), from J.T. Baker Chemical Company, were used for thin layer chromatography (TLC) studies. Equipment. Dynamic light scattering (DLS) measurements were performed using a Malvern 4800 Autosizer employing a 7132 digital correlator for the determination of hydrodynamic diameter. Zeta-potential measurements were performed using Zetasizer nano series (Malvern Instruments). A transmission electron microscopy (TEM) micrograph was obtained using a Philips CM 200 TEM system for particle size determination. The infrared spectra were recorded in the range of 400−4000 cm−1 on a Fourier transform infrared (FTIR) spectrometer (Bomen Hartman and Braun, MB series). Raman spectral studies were carried out on Seki’s STR300 Raman spectrometer, using an excitation wavelength of 532 nm from a fiber-coupled diodepumped solid-state (DPSS) laser source. The absorption spectrum of CuS nanoparticles was recorded using a ultraviolet−visible−near-infrared (UV-vis-NIR) spectrometer (JASCO, Model V-670). X-ray diffraction (XRD) patterns were recorded on a Phillips Model PW1710 diffractometer with Cu Kα radiation. Relaxation time measurements, surface area, and stability of nanoparticles in different media were determined using a Xigo Nanotools Acorn Area instrument. Thermogravimetric measurements were carried out using a simultaneous thermogravimetric analysis−differential thermal analysis (TGADTA) instrument (SETARAM, Model 92-16.18). The activity of 64Cu was measured using a precalibrated “PTW Curiementor 3” dose calibrator. The presence of radionuclidic impurities in 64Cu was determined by recording γ-ray spectra, using a calibrated high-purity germanium detector (HPGe) detector coupled to a 4K multichannel analyzer (MCA) system (Canberra, Eurisys). All other radioactivity measurements were carried out using a well-type NaI (Tl) scintillation counter (Mucha, Raytest) by utilizing the 511 keV gamma photon of 64 Cu, because of positron-annihilation. Synthesis of CuS Nanoparticles. Synthesis of CuS nanoparticles was performed by adopting the procedure reported by Zhou et al.,12 with some modifications. In brief, 40 μL of CTAC was added to a 10 mL solution of CuCl2 (13.5 mg, 0.1 mmol) and stirred at room temperature for 15 min. To this solution, 0.1 mL of sodium sulfide (1 M) solution was added and stirred vigorously for another 15 min at room temperature. The pale-blue CuCl2 solution turned dark brown immediately upon the addition of sodium sulfide solution. Subsequently, the reaction mixture was heated to 90 °C and stirred for 15 min until a dark green solution was obtained. The mixture was then transferred to dry ice and cooled for 10 min, which resulted in the formation of CTAC-stabilized CuS (CuS-CTAC) nanoparticles to form CuS-PEG nanoparticles. In order to introduce the PEG coating, ∼20 mg of SH-PEG powder (maintaining a CuS:PEG ratio of ∼25:1) was added to 12408
DOI: 10.1021/acs.iecr.6b03405 Ind. Eng. Chem. Res. 2016, 55, 12407−12419
Article
Industrial & Engineering Chemistry Research
Subsequently, MTT (100 μL, 5 mg/mL) was added to each well, and incubated for another 4 h under 5% CO2. Dimethyl sulfoxide (100 μL/well) was then added, and the optical density at 490 nm of each well was recorded. Production and Radiochemical Processing of 64Cu. All experiments involving the use of radioactivity were carried out after thorough planning, in compliance with the ALARA (as low as reasonably achievable) principle. 64Cu was produced via thermal neutron bombardment on a natural CuO target (69.1% in 63Cu). A known amount of target was taken in a quartz ampule. After being placed inside an aluminum can, the ampule was subsequently flame-sealed and irradiated at a thermal neutron flux of ∼1 × 1014 n cm−2 s−1 for 7 days in a Dhruva research reactor at Bhabha Atomic Research Centre, India. At the end of the irradiation, the irradiated targets were cooled for 4 h. Radiochemical processing of target was carried out by opening the irradiation can in a lead-shielded facility that was fitted with remotely operable gadgets. The irradiated target was transferred to the dissolution vessel and dissolved in 5 mL of 1 M suprapure HCl by gentle warming. The resultant solution was evaporated to near dryness and reconstituted in 10 mL of deionized water. The activity of 64CuCl2 solution was determined using a dose calibrator. Industrial-Scale Synthesis of 64CuS Nanoparticles. Industrial-scale synthesis of 64CuS-CTAC and 64CuS-PEG nanoparticles was carried out in a hot cell that was equipped with remotely operable tongs. The synthesis procedure was essentially the same as that reported above for the synthesis of nonradioactive nanoparticles, except that 64CuCl2 solution obtained after radiochemical processing of the irradiated target was used in the procedure. After PEG modification, the radiolabeled formulation was transferred to a 50 kDa centrifugal filter tube and centrifuged at 5000 rpm for 15 min in order to remove unreacted PEG-SH and 64CuCl2. This procedure was repeated once and 64CuS-PEG nanoparticles left on the membrane were redispersed in 10 mL of phosphate buffered saline (PBS) solution. Subsequently, 64CuS-PEG nanoparticles were passed through a 0.22 μm filter, collected in a sterile vial, and subjected to quality control tests. Quality Control of 64CuS Nanoparticles. Radionuclidic Purity. The presence of radionuclidic impurities in 64CuS-CTAC and 64CuS-PEG nanoparticles could be determined from the γspectrum of 64Cu recorded using a HPGe detector coupled to an MCA system. For this purpose, a small aliquot of intrinsically radiolabeled nanoparticles was withdrawn and the γ-spectrum was recorded after appropriate dilution of the radioactivity, so that the dead time of the detector was 99
32.9 (0.89) >99
27.7 (0.75) >99
31.8 (0.86) >99
31.1 (0.84) >99
30.3 (0.82) >99
8.5 (0.23) >99 7.8 (0.21)
10.7 (0.29) >99 9.6 (0.26)
8.9 (0.24) >99 8.1 (0.22)
10.7 (0.29) >99 9.6 (0.26)
10.0 (0.27) >99 8.9 (0.24)
9.6 (0.26) >99 8.5 (0.23)
71.7
78.2
78.0
80.6
76.8
75.2
Natural CuO target was irradiated at a flux of 1 × 1014 n cm−2 s−1 in the Dhruva reactor for 7 days. bYield is calculated, with respect to activity of Cu produced at the end of radiochemical processing.
64
Figure 9. γ-ray spectra of intrinsically radiolabeled 64CuS nanoparticles recorded (A) just after completion of synthesis procedure and (B) after decay for 7 days.
radiolabeled nanoparticles through a PD-10 column (Figure 10B). Biological Purity. Intrinsically radiolabeled 64CuS-PEG nanoparticles, after passing through a 0.22 μm filter, was found to be sterile. The endotoxins in all of the decayed samples tested were found to be 98% intact 64 Cu) in both PBS as well as mouse serum media over a period over a period of 48 h (Figures 10C and 10D). Non-PEGylated 64CuSCTAC nanoparticles were slightly less stable in both mouse serum and PBS media, with ∼5% of 64Cu leaching out of the radioactive nanoparticles over a period of 10 h (Figures 10C and 10D). Thus, PEG modification of 64CuS nanoparticles increases its stability under physiological conditions. Furthermore, it was observed from the NOTA challenge study that the intrinsically radiolabeled 64CuS-CTAC and 64CuS-PEG nanoparticles retained their radiochemical purities to the extent of >95% after 2 h of incubation in 0.05 M NOTA solution at room temperature (Figure 11).
synthesized in one batch is adequate for administration in several patients. Quality Control of 64CuS Nanoparticles. Radionuclidic Purity. The presence of radionuclidic impurities in 64CuS nanoparticles was determined by γ-ray spectrometry, using a calibrated HPGe detector coupled to the MCA system (Figure 9). The γ-ray spectra of radioactive nanoparticles recorded immediately after completion of synthesis procedure only shows the 511 keV annihilation peak and no photopeak corresponding to 65Zn could be seen (Figure 9A). However, when 64CuS nanoparticle samples were decayed for 7 days, the 511 and 1115.5 keV photopeaks corresponding to 65Zn could also be observed in the γ-ray spectra (Figure 9B). The level of 65Zn present in 64CuS nanoparticles was found to be 99%. These results were further corroborated by size exclusion chromatography (SEC), by passing the intrinsically 12415
DOI: 10.1021/acs.iecr.6b03405 Ind. Eng. Chem. Res. 2016, 55, 12407−12419
Article
Industrial & Engineering Chemistry Research
Figure 10. (A) Radio-TLC patterns of intrinsically radiolabeled 64CuS nanoparticles developed in 0.1 M sodium citrate medium; inset shows the radioTLC pattern of uncomplexed 64CuCl2 solution developed in a 0.1 M sodium citrate medium as control. (B) Size exclusion chromatography elution profile of intrinsically radiolabeled 64CuS nanoparticles. (C) In vitro stability of intrinsically radiolabeled 64CuS nanoparticles in a PBS medium over a period of 48 h. (D) In vitro stability of intrinsically radiolabeled 64CuS nanoparticles in a mouse serum medium over a period of 48 h.
from 0.76 ± 0.61 to 4.5 ± 2.2 and 0.03 ± 0.02 to 0.17 ± 0.11, respectively, between the same time points. The increase in tumor-to-background was higher in case of 64CuS-PEG nanoparticles, where, the tumor-to-muscle, tumor-to-blood, tumorto-liver ratios were observed to increase from 4.0 ± 1.1 to 51.4 ± 5.5, 0.5 ± 0.4 to 8.7 ± 2.2, and 0.07 ± 0.03 to 0.71 ± 0.21, respectively, between 1 h to 48 h p.i.
Biodistribution Studies in Tumor-Bearing Mice. The uptakes of 64CuS-CTAC and 64CuS-PEG nanoparticles in different organs/tissues of C57/BL6 mice bearing melanoma tumors (expressed as %ID/g) at different p.i. times are shown in Figure 12. At all time points studied, CuS-CTAC nanoparticles displayed significantly higher uptake than did CuS-PEG nanoparticles in the liver and the spleen, both of which are reticuloendothelial system (RES)-enriched tissues. When CuSPEG nanoparticles were administered, the tumor uptake of 2.9 ± 1.5%ID/g was observed within 1 h p.i., which increased to 7.1 ± 1.7%ID/g at 24 h p.i. Conversely, the tumor uptake observed with non-PEGylated CuS-CTAC nanoparticles was significantly lower (1.6 ± 1.1%ID/g was observed within 1 h p.i., which increased to 3.2 ± 0.7%ID/g at 24 h p.i.). The tumor-to-blood, tumor-to-muscle, and tumor-to-liver ratios of 64CuS-CTAC and 64CuS-PEG nanoparticles at different time points p.i. are shown in Figure 12. For 64CuS-CTAC nanoparticles, the tumor-to-muscle ratio was observed to increase from 2.3 ± 1.2 at 1 h p.i. to 31.8 ± 3.5 at 48 h p.i., while the tumor-to-blood and tumor-to-liver ratios increased
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DISCUSSION Radiolabeled nanoparticles have generated great excitement in the field of nanomedicine.3 Radiolabeling of nanoparticles is generally achieved by four main strategies: (1) complexation reaction of radiometal ions with chelators conjugated on the nanoparticle surface; (2) chelator-free post-synthetic radiolabeling; (3) direct bombardment of nanoparticles via hadronic projectiles; and (4) synthesis of intrinsically radiolabeled nanoparticles using radioactive precursors.3 While using lowspecific-activity radioisotopes (such as neutron-activated 64Cu used in the present study), the utility of the first two approaches for radiolabeling of nanoparticles is ruled out. This is because 12416
DOI: 10.1021/acs.iecr.6b03405 Ind. Eng. Chem. Res. 2016, 55, 12407−12419
Article
Industrial & Engineering Chemistry Research
Figure 11. (A) Radio-TLC patterns of intrinsically radiolabeled 64CuS-CTAC and 64CuS-PEG nanoparticles when challenged with an excess amount of NOTA chelator. (B) Radio-TLC pattern of 64Cu-NOTA, as a control.
It is expected that, for clinical utilization, radioactive nanoparticles would be synthesized in a centralized facility from where it would be transported to various hospital radiopharmacies. Because of the short half-life of 64Cu, there would be significant decay loss during the course of transit. Therefore, it would be essential to prepare high radioactivity doses of nanoparticles to meet the clinical demand while accounting for the decay loss. Various strategies have been reported over the past few years for synthesis of Cu-based nanoparticles.9,11,12,21,22 Among these, the synthesis of intrinsically radiolabeled 64CuS nanoparticles adopting metathesis reaction of 64CuCl2 and Na2S at 95 °C is the most appealing procedure, in view of its simplicity.12 It is pertinent to point out here that the earlier reports on the synthesis of intrinsically radiolabeled 64CuS nanoparticles utilized tracer level of cyclotron-produced NCA 64Cu to prepare small radioactive doses suitable only for preclinical studies.11,12 The present study is the first report on industrial-scale synthesis of 64CuS nanoparticles using reactor-produced low-specific-activity 64Cu. In a typical batch, 7−10 GBq of 64CuS-PEG nanoparticles could be synthesized. The nanoparticle formulation was found to be sterile and apyrogenic and, thus, suitable for human administration. The synthesis procedure reported herein is robust, reproducible, and easily executable with remote handling operations in a shielded hot-cell facility. For PET imaging in human subjects with good contrast, the dose of 64CuS nanoparticles to be administered is expected to be similar [185−259 MBq (5−7 mCi)] to that for conventional 64Cu-based radiopharmaceuticals.23 Therefore, doses suitable for 40−50 patients could be synthesized in a single batch. If successfully translated for clinical use, the synthesis procedure is up-scalable to meet the growing demand of this intrinsically radiolabeled nanoplatform for cancer theranostics. The cytotoxicity of nanoparticles is a cause of serious concern, especially when intended for clinical translation. In vitro cytotoxicity study revealed that CuS-PEG nanoparticles may be useful for clinical studies only at concentrations of
