Dilute lattice doping of Cu-64 into 2D-nanoplate; its impact on radio-labeling efficiency and stability for target selective PET imaging

doi:10.21203/rs.3.rs-1542004/v1

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Dilute lattice doping of Cu-64 into 2D-nanoplate; its impact on radio-labeling efficiency and stability for target selective PET imaging

https://doi.org/10.21203/rs.3.rs-1542004/v1

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Backgound:

Positron emission tomography (PET) in nuclear medicine provides useful information for tumor diagnosis due to its high sensitivity and quantitativeness on biochemical and physiological changes in in-vivo system. Radioisotope-labeled materials develped for PET imaging still have decisive weekness, since they are low in labeling efficiency and labeling stability.

Results

Quintinite nanoplate (⁶⁴Cu-QT-NP) isomorphically substituted with ⁶⁴Cu, as the positron emission tomography (PET) imaging material, was prepared via two-step processes. The ⁶⁴Cu labeling efficiency of 99% was realized, for the first time, by immobilizing the ⁶⁴Cu radioisotope directly in the octahedral site of 2 dimensional (2D) quintinite lattice. Furthermore, the ⁶⁴Cu labeling stability of ⁶⁴Cu-QT-NPs was also achieved more than ~ 99% in various solutions such as saline, phosphate-buffered saline (PBS), and other biological media (mouse and human serums). In in-vivo xenograft mice model, the passive targeting behavior of ⁶⁴Cu-QT-NPs into tumor tissue based on enhanced permeability and retention (EPR) effect was also demonstrated by parenteral administration, and successfully visualized using positron emission tomography (PET) scanner. For enhancing the tumor tissue selectivity, the bovine serum albumin (BSA) was coated on ⁶⁴Cu-QT-NPs to form ⁶⁴Cu-QT-NPs/BSA, resulting in better colloidal stability and longer blood circulation time, which was eventually evidenced by the 2-fold higher tumor uptake rate when intravenousely injected in an animal model.

Conclusions

The present ⁶⁴Cu-QT-NPs/BSA with tumor tissue selectivity could be an advanced nano-device for radio-imaging and diagnosis as well.

Quintinite

Radio-labeled nanoplates

Radio-labeling efficiency and stability

Radio-imaging and diagnostic nano-device

Positron emission tomography

In the last decades, precise diagnosis of disease has been an important issue in medicine. In particular, researches on various imaging materials for early detection of cancer have been carried out to enhance their sensitivity, bioaffinity, and stability in the human body [1–9], At the same time, molecular imaging techniques have been also constantly advancing, and contributing to the development of life and medical sciences. For example, nuclear imaging techniques, including scintigraphy, positron emission tomography (PET), and single-photon emission computed tomography (SPECT) have been extensively studied, since they can provide useful information for tumor diagnosis by tracing biochemical and physiological changes in in-vivo systems thanks to their high sensitivity and quantitativeness without any tissue penetration limit [10, 11].

To obtain nuclear imaging detection, radioisotopes, such as ¹⁸F, ⁶⁴Cu, ⁶⁸Ga, and ^99mTc, were used in combination with a variety of materials including chelators, metal oxides, liposomes, dendrimers, and layered compounds [12–22]. Out of various radioisotopes, ⁶⁴Cu was considered as an useful radioisotope for both PET imaging and targeted radiotherapy due to its long half-life (12.7 h) enough to synthesize a various size of molecular assemblies and nanoparticles for radiopharmaceutical materials, and its decay scheme of β⁺ (18%), β⁻ (38.5%), and electron capture (ε, 43.5%) as shown in Fig S1 [23–24]. The ⁶⁴Cu radionuclide has been commonly produced by proton irradiation in medical cyclotron using ⁶⁴Ni(p, n)⁶⁴Cu nuclear reaction (${}_{28}{}^{64}\text{N}\text{i}+ {}_{1}{}^{1}\text{p} \to {}_{29}{}^{64}\text{C}\text{u}+ {}_{0}{}^{1}\text{n}$), as one of the widely used nuclear reactions due to its high production yield and purity [25–27]. The thus produced ⁶⁴Cu has been further applied to develop a variety of ⁶⁴Cu-labeled complexes and/or nanomaterials for PET imaging [14, 23, 28–33]. Typically, various chelating molecules, such as 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), and 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), were used to bind ⁶⁴Cu radionuclides, and the ⁶⁴Cu-chelators were further conjugated with nanoparticles, and/or targeting moieties such as antibodies, proteins, peptides, and other biologically relevant small molecules [28, 29]. However, they turned out to be quite low in radiolabeling efficiency (Table S1) and chemically unstable in in-vitro and in-vivo due to the week bonding interaction between ⁶⁴Cu and chelators [30–33]. For example, the radiolabeling efficiency of NOTA, known as a hexadentate N₃O₃ chelator, for ⁶⁴Cu was obtained with 65 ~ 70 % [30]. A radiolabeling fficiency of 71.9 % was also reported for ⁴Cu-DOTA conjugated with monoclonal antibody (mAb), where the ⁶⁴Cu radionuclide was made of cross-bridged bicyclic tetraamine of DOTA [31]. To overcome such a weak interaction, some bifunctional chelators were developed, but a harsh condition was required to prepare ⁶⁴Cu-chelators. Apart from that, the labeling stability was also still insufficient as well [28]. For PET applications in in-vivo, it has long been required to develop chemically stable ⁶⁴Cu complexes and/or nanomaterials with externally adsorbed ⁶⁴Cu-derivatives [32, 33].

On the other hand, quintinite ([Mg₂Al(OH)₆]¹⁺[CO₃²⁻]_0.5·1.5H₂O, QT), different from hydrotalcite ([Mg₃Al(OH)₈]¹⁺[CO₃²⁻]_0.5·2H₂O, HT) known as an anionic clay, has attracted a great attention in nanomedicine due to its excellent biocompatibility, controllable drug-loading capacity, enhanced cellular permeability, and tumor targeting property [34–40]. Its lamellar structure comprises of brucite (Mg(OH)₂) layers, where magnesium cations (Mg²⁺) are octahedrally coordinated with six OH⁻ ions. In QT structure, however, positive layer charge can be generated by replacing divalent Mg²⁺ in the brucite lattice with trivalent aluminum cations (Al³⁺), and thus the chemical composition of QT can be described by the general formula of [Mg₂Al(OH)₆]¹⁺[CO₃²⁻]_0.5·nH₂O. If the chemical composition of QT was modified, a series of layered double hydroxide (LDH) phases could be formed with a general formula of [M^2 + _1−xM^3 + _x(OH)₂] ^x+[A^m−]_x/m·nH₂O [41–43], where M²⁺ and M³⁺ are divalent and trivalent metal cations such as Mg²⁺, Ca²⁺, Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺, etc., and Al³⁺, Fe³⁺, Co³⁺, Ga³⁺, etc., and A^m− describes the interlayer anions such as (CO₃)²⁻, (NO₃)¹⁻, Cl¹⁻, etc., which can be easily changed depending upon synthetic conditions. One thing to underline here is that interlayer anions are exchangeable so that various negatively charged biofunctional molecules such as anticancer drugs, DNAs, and RNAs can be immobilized in the interlayer space by simple ion exchange reaction [44–50].

Radioisotopes, like ⁶⁴Cu, ⁵⁷Co, ⁶⁸Ga, etc. with divalent or trivalent cations in aqueous solution, could be isomorphically substituted into the octahedral sites of QT lattice under controlled synthetic conditions. Recently, several attempts have been made to study that the radioisotopes label with QT-NPs for radio-imaging and diagnosis [51, 52]. For example, chelator-free nanoparticles, in which ⁶⁴Cu radioisotopes were adsorbed on the external surface of QT-NPs by simple mixing, have been developed for PET imaging [51]. Thus prepared ⁶⁴Cu adsorbed QT-NPs were, however, determined to be extremely poor in ⁶⁴Cu labeling efficiency of only 59.0%, which were reduced down 16.6% even after bovine serum albumin (BSA) coating on them. This attempt is, therefore, considered as a failure one, due to the week chemical bonding interaction between ⁶⁴Cu radioisotopes and QT-NPs. One more thing to note here is that the physisorbed radioisotope was found to be partly transformed to impurity phase such as ⁶⁴Cu²⁺-hydroxycloride, which fell off easily from the external surface of QT-NPs. If this was applied to a patient, an additional toxicity could be expected due to the fact that a large amount of QT-NPs and toxic impurity should be simultaneously injected in order to meet the required radiolabeling efficiency. It is, therefore, highly required to improve the radiolabeling efficiency and chemical stability of radioisotopes immobilized on QT-NPs without any impurities.

In the present study, we attempted to develop biocompatible and chemically stable ⁶⁴Cu-labeled QT-NPs as follows; (1) ⁶⁴Cu²⁺ ion was isomorphically substituted in the octahedral site of QT lattice via two-step processes, co-precipitation and subsequent hydrothermal treatment, to form ⁶⁴Cu-labeled QT-NPs (⁶⁴Cu-QT-NPs) (Fig. 1a), and then (2) ⁶⁴Cu-QT-NPs were coated with BSA (⁶⁴Cu-QT-NPs/BSA) to enhance the circulation time in blood stream when injected (Fig. 1b). And the radiolabeling stability of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA was compared with that of ⁶⁴Cu-ads-QT-NPs (⁶⁴Cu radioisotope physisorbed on QT-NPs) and ⁶⁴Cu-ads-QT-NPs/BSA (the BSA coated one) as the control groups (Fig. S2). Our isomorphically substituted ⁶⁴Cu samples, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, were investigated not only for the radiolabeling efficiency and the chemical stability of ⁶⁴Cu radioisotope, but also for the tumor targeting and imaging efficiency in xenograft mice model.

Materials

Magnesium nitrate hexahydrate (Mg(NO₃)₂∙6H₂O), aluminum nitrate nonahydrate (Al(NO₃)₃∙9H₂O), sodium carbonate (Na₂CO₃), and sodium hydroxide (NaOH) were purchased from Daejung Chemical and Metals (Republic of Korea). Copper chloride dihydrate (CuCl₂∙2H₂O) and bovine serum albumin (BSA) were provided by Sigma-Aldrich (USA). All the reagents were used without further purification. The radioactive copper isotope (⁶⁴Cu) was produced at the Korea Institute of Radiologic and Medical Sciences (KIRAMS) using a 50 MeV cyclotron (Scantronics) and enriched with nickel isotope (⁶⁴Ni, 99%; Isoflex) as the target material [25] based on the ⁶⁴Ni(p, n)⁶⁴Cu nuclear reaction (${}_{28}{}^{64}\text{N}\text{i}+ {}_{1}{}^{1}\text{p} \to {}_{29}{}^{64}\text{C}\text{u}+ {}_{0}{}^{1}\text{n}$).

Synthesis of the ⁶⁴Cu radioisotope labeled samples, the isomorphically substituted ⁶⁴Cu into QT-NPs, ⁶⁴Cu-QT-NPs and the BSA coated, ⁶⁴Cu-QT-NPs/BSA

All the experiments with ⁶⁴Cu radioisotope including its production were carried out at the KIRAMS according to the safety guideline of Nuclear Safety and Security Commission (NSSC) and Korea Institute of Nuclear Safety (KINS). The unit of radioactivity, the number of spontaneous nuclear disintegrations per unit time, was expressed in becquerels (Bq) in accordance with the international normative ISO 19461-1:2018(en). For doping ⁶⁴Cu radioisotope into QT to form ⁶⁴Cu-QT-NPs, first of all, ⁶⁴Cu radioisotope with a special activity of approximately 142.5 MBq/ng (9.03 GBq/nmol) [53] was produced as a form of ⁶⁴CuCl₂ using a 50 MeV cyclotron as follows [25]; (1) 99% of enriched ⁶⁴Ni (10 mm plating diameter) on gold foil was irradiated by the proton beam in cyclotron with 30–50 µA at 18 MeV for an hour, and then (2) the ⁶⁴Ni target was hydrothermally dissolved in hydrochloric acid (HCl, 6.0 M, 10mL) at 80°C. (3) Finally, ⁶⁴Cu radioisotope was separated from thus dissolved solution using purification process with about 740 MBq/h of production yield by ion exchange resin in plastic case (PEEK, dimension as 1 4 cm). And then, the ⁶⁴CuCl₂ solution (2 mL) with 740 MBq (81.94 pmol of ⁶⁴Cu radioisopes) was evaporated under Ar gas stream and re-dissolved in 200 µL of 0.1M HCl. Thus prepared ⁶⁴CuCl₂ solution (740 MBq) was directly mixed with a solution (1 mL) containing reactant metal salts, Mg²⁺ (1 µmol) and Al³⁺ (0.5 µmol), which was then titrated with an aqueous one of NaOH (50 mM) and Na₂CO₃ (20 mM) under a vigorous stirring condition until pH reached to 10.0, as shown in Fig.1a; the nominal molar ratio for the sample, ⁶⁴Cu-QT-NPs, was 1.0:0.5:0.000082 (Mg:Al:⁶⁴Cu). Thus prepared suspension was aged for 10 min, and then the white precipitate was collected via centrifugation, washed with deionized water, re-dispersed, and further hydrothermally treated at 100°C for 3 h to obtain a desired colloidal suspension of ⁶⁴Cu-QT-NPs. Based on this co-precipitation and subsequent hydrothermal route, the radioisotope ⁶⁴Cu was successfully stabilized in the octahedral site of QT lattice. To enhance the colloidal stability of thus prepared ⁶⁴Cu-QT-NPs, BSA was further coated on them to form ⁶⁴Cu-QT-NPs/BSA simply by mixing a suspension of ⁶⁴Cu-QT-NPs and a solution of 0.5 mL BSA (50 mg/mL) under a vigorous stirring condition for 2 h at room temperature. Half (~370 MBq) of the total ⁶⁴Cu-QT-NPs suspension was used to prepare the BSA coated sample, ⁶⁴Cu-QT-NPs/BSA, due to the limitation of the total amount of ⁶⁴Cu radioisotope used.

In order to evaluate the radiolabeling efficiency by the amount of ⁶⁴Cu radioactivity, 0.5 µmol of ⁶⁴Cu-QT-NPs was used to prepare the samples with different doses of ⁶⁴Cu radioactivity of 74, 185, 370, and 740 MBq, corresponding to 8.19, 20.49, 40.97, and 81.94 pmol of ⁶⁴Cu. In addition, considering the detection range of radioactivity measuring equipment at each experimental step, a reasonable amount of radiolabeled samples was used to evaluate in in-vitro and in-vivo studies as follows; ~ 3.7 MBq (~ 5 nmol of QT, ~ 0.41 pmol of ⁶⁴Cu) of the prepared samples for the radiolabeling stability, ~ 0.37 MBq (~ 0.5 nmol of QT, ~ 0.041 pmol of ⁶⁴Cu) for in-vitro cell study, and ~ 1.11 MBq (~ 1.5 nmol of QT, ~ 0.25 pmol of ⁶⁴Cu) for in-vivo biodistribution study, and ~ 11.1 MBq (~ 15 nmol of QT, ~ 2.5 pmol of ⁶⁴Cu) for in-vivo PET imaging study.

Synthesis of the non-radioactive Cu doped samples, the isomorphically substituted Cu into QT-NPs, Cu-QT-NP and the BSA coated, Cu-QT-NPs/BSA

Since the radioisotope was limitedly produced less than µmol level, only a small amount of radioisotope labeled phases was available. To secure an enough amount of samples for various experiments, we have also prepared nonradioactive samples, Cu-QT-NPs and Cu-QT-NPs/BSA, along with the radioisotope labeled. Cu-QT-NPs with a molar ratio of 0.999:0.5:0.001 (Mg:Al:Cu, Cu²⁺ was doped in the 0.1% of Mg²⁺ sites) were prepared by co-precipitation at room temperature, and subsequent hydrothermal reaction; a mixed solution (100 mL) of Mg(NO₃)₂∙6H₂O (9.99 mmol), Al(NO₃)₃∙9H₂O (5 mmol), and CuCl₂∙2H₂O (0.01 mmol) was titrated with an aqueous solution of NaOH (50 mM) and Na₂CO₃ (20 mM) up to pH = 10.0 under vigorous stirring. The resulting suspension was aged for 10 min and subsequently treated under hydrothermal condition at 100°C for 3 h. And thus prepared Cu-QT-NPs precipitate was mixed with a BSA solution under vigorous stirring for 2 h to form Cu-QT-NPs/BSA.

Characterization

The crystal structure analysis for all the samples was performed by powder X-ray diffractometer (D/Max 2000, Rigaku) with Ni-filtered Cu Kα radiation (λ = 1.5418 Å) from 5° to 70° operating at 40 kV and 30 mA, with a scanning rate of 2°/min. In order to obtain XRD patterns from a very small amount (~ nmol level) of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, each sample was very carefully dropped on the surface of XRD sample holder and slowly dried in a drying oven at 60°C. According to the XRD patterns for those samples, the peak intensity was rather lowered in a higher range of 2 theta angle around 30° ~ 70° due to the peak broadening effect, but the peaks seemed to be better resolved in the 2 theta region of 5° ~ 30°. The morphology and particle size were analyzed with a scanning electron microscope (SEM, JEOL JSM-6700F), and the particle size distribution and zeta potential were also measured on the basis of DLS method with Zetasizer Nano (Malvern); only the nonradioactive samples, Cu-QT-NPs and Cu-QT-NPs/BSA, prepared under the same synthetic condition of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, were analyzed due to the limited amount of the radioisotope labeled samples.

In-vitro ⁶⁴Cu labeling efficiency and labeling stability

To determine the labeling efficiency for all the samples, each suspension was centrifuged using a filter tube (MWCO 10k; Millipore) at 13000 rpm for 30 min. The radioactivity of each precipitate and each supernatant was measured using a radioisotope calibrator (CRC®-55tR, Capintec, Inc., USA). The labeling efficiency (%) was derived from the measured data according to the following Eq. (1);

$$\text{L}\text{a}\text{b}\text{e}\text{l}\text{i}\text{n}\text{g} \text{e}\text{f}\text{f}\text{i}\text{c}\text{i}\text{e}\text{n}\text{c}\text{y}= \frac{\text{R}\text{a}\text{d}\text{i}\text{o}\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{o}\text{f} \text{i}\text{s}\text{o}\text{l}\text{a}\text{t}\text{e}\text{d} \text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}}{\text{R}\text{a}\text{d}\text{i}\text{o}\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{o}\text{f} \text{s}\text{u}\text{p}\text{e}\text{r}\text{n}\text{a}\text{t}\text{a}\text{n}\text{t}\text{s}+\text{R}\text{a}\text{d}\text{i}\text{o}\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{o}\text{f} \text{i}\text{s}\text{o}\text{l}\text{a}\text{t}\text{e}\text{d} \text{p}\text{a}\text{r}\text{t}\text{i}\text{c}\text{l}\text{e}\text{s}}\times 100$$

This procedure was repeated three times to ensure accurate results.

The labeling efficiency and stability of each precipitate were also determined by radio-thin layer chromatography (radio-TLC) (Bioscan, AR-2000) using a 20 mM sodium citrate/50 mM ethylenediaminetetraacetic acid (EDTA) solution (pH = 5.5) as a mobile phase.

The labeling stability study of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA was carried out in various media including saline, PBS, mouse serum, and human serum. The sample suspension (3.7 MBq/100 µL) was separately mixed with each media (100 µL), respectively. The mixtures of suspension and media were incubated at 37°C for 1, 3, 12, and 36 h, respectively. And then the mixture in each filter tube was separated into precipitates and supernatants by the centrifugation at 13000 rpm for 30 min. Finally the radioactivity of isolated precipitates and supernatants was measured using a radioisotope calibrator, and the procedure was repeated three times to ensure accurate results. The labeling stability (%) was also estimated from the Eq. (1) with respect to time.

In-vitro cytotoxicity and cellular uptake behavior of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA

The human breast cancer cell line (MDA-MB-231) was purchased from the American Type Culture Collection (ATCC) and cultured in the Roswell Park Memorial Institute (RPMI) 1640 medium (WelGENE, Republic of Korea) with 10% fetal bovine serum (FBS) and 1% antibiotics (both by Invitrogen, USA) under an atmosphere of 5% CO₂ and 95% air at 37°C.

The cytotoxicity for ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA was measured on the basis of trypan blue exclusion assay; the cells (5 × 10⁵ cells/well) incubated for overnight at 37°C under a 5% CO₂ atmosphere were treated, respectively, by ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA with a radioactivity of 0.37 MBq. After incubation for 0.5, 1, 2, 4, 16, and 24 h, the cells were washed twice with ice-cold PBS and detached with 0.1% trypsin. The detached cells were collected in the media, and 20 µL of cell suspension was diluted with 20 µL of 0.4% trypan blue. Finally, viable cells were counted within the grids on hemacytometer, and their cell viability (%) was calculated as the following Eq. (2);

$$\text{C}\text{e}\text{l}\text{l} \text{v}\text{i}\text{a}\text{b}\text{i}\text{l}\text{i}\text{t}\text{y}=\frac{\text{T}\text{o}\text{t}\text{a}\text{l} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{v}\text{i}\text{a}\text{b}\text{l}\text{e} \text{c}\text{e}\text{l}\text{l}\text{s}}{\text{T}\text{o}\text{t}\text{a}\text{l} \text{n}\text{u}\text{m}\text{b}\text{e}\text{r} \text{o}\text{f} \text{c}\text{e}\text{l}\text{l}\text{s}}\times 100$$

To investigate cellular uptake behavior of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, the MDA-MB-231 cells were seeded onto 24-well plates (1 × 10⁵ cells/well) and incubated at 37°C overnight under a 5% CO₂ atmosphere. 0.37 MBq of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA were treated in each well, and then further incubated for 0.5, 2, 6, and 24 h. And then the cells were washed twice with ice-cold PBS in order to remove the remaining nanoparticles not up-taken by the cells. After that, the cells were detached with 0.1% trypsin and then its radioactivity was measured using a gamma counter (1480 WIZARD, PerkinElmer, USA). The cellular uptake rate for each ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA was represented as the percentage injected dose (%ID).

In-vivo small-animal PET imaging for ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA

All the animal experiments were performed according to the guidelines of the IACUC of the KIRAMS. The tumor-bearing animal models were produced in 6-week-old male BALB/c nude mice by subcutaneous injection of MDA-MB-231 cells (1 × 10⁷) in the right thigh. The tumors were allowed to grow for 4 weeks until their size reached a diameter of 10–15 mm, and then the PET imaging and biodistribution studies were performed on the xenograft mice.

Each radioisotope labeled sample, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA (~ 11.1 MBq/ 100 µL) were tail-vein injected into the tumor-bearing mice (n = 3/group), respectively. After 2, 6, 24 and 48 h, the mice were anesthetized with isoflurane (2%) and placed in a spread prone position to obtain PET images with a small-animal PET scanner (Inveon™, Siemens Preclinical Solutions, USA). The PET scan was performed for 30 min, and the emission data was acquired using 64 detectors in PET scanner under the condition with 350–650 keV of energy window and 3.43 ns of timing window (the difference between the late and early arrival times). All images were reconstructed using a 2-dimensionally ordered-subset expectation maximization (2D-OSEM) algorithm with 4 iterations. For image analysis, the static PET images were displayed in the coronal (or frontal) and transverse (or horizontal, axial) directions; the coronal is the vertical plane that divides the body into ventral and dorsal sections, and the transverse is the plane that divides the body into superior and inferior parts. The three-dimensional regions of interest (ROIs) were manually drawn over the tumor, lung, liver and blood pool using the Inveon Research Workplace software (IRW, Siemens Preclinical Solutions) [54].

In-vivo biodistribution for ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA

Biodistribution studies for ⁶⁴Cu-QT-NPs, and ⁶⁴Cu-QT-NPs/BSA were performed in the MDA-MB-231 xenografts. The 100 µL suspension with a radioactivity of 1.11 MBq for each sample was intravenously injected into the tumor-bearing mice (n = 4/group). After a given period of time from 2 to 48 h, the mice were sacrificed, and their blood, organs, and tumors were separated and weighed. The radioactivity of each organ was also measured with the gamma counter and the results were expressed as the percentage of injected dose per gram of tissue (%ID/g), according to the following Eq. (3);

$$\text{I}\text{n}\text{j}\text{e}\text{c}\text{t}\text{e}\text{d} \text{d}\text{o}\text{s}\text{e} \text{p}\text{e}\text{r} \text{g}\text{r}\text{a}\text{m}= \frac{\text{R}\text{a}\text{d}\text{i}\text{o}\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{o}\text{f} \text{o}\text{r}\text{g}\text{a}\text{n} \text{t}\text{i}\text{s}\text{s}\text{u}\text{e}/\text{I}\text{n}\text{j}\text{e}\text{c}\text{t}\text{e}\text{d} \text{r}\text{a}\text{d}\text{i}\text{o}\text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y}}{\text{W}\text{e}\text{i}\text{g}\text{h}\text{t} \text{o}\text{f} \text{o}\text{r}\text{g}\text{a}\text{n} \text{t}\text{i}\text{s}\text{s}\text{u}\text{e}}\times 100$$

Furthermore, intact ⁶⁴Cu (1.11 MBq) was also injected into the BALB/c normal mice (n = 3) to confirm its biodistribution as a control, and the mice were sacrificed after 3 and 22 h. The organs were separated, weighed and measured its radioactivity to obtain %ID/g.

Physico-chemical characterization

To understand the physicochemical properties between the present radioisotope-labeled samples (⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA), and the nonradioisotope-labeled ones (Cu-QT-NPs and Cu-QT-NPs/BSA), their differences and similarities were systematically analyzed and compared to each other as shown in Fig. 2 and Fig. S3. According to the powder X-ray diffraction (XRD) patterns (Fig. 2a and Fig. S3), all the samples exhibited a series of well-developed (00l) reflections, though the (00l) peaks for the BSA coated samples were slightly broadened along with the weakening of their intensities, due to the presence of BSA molecules between QT-NPs giving rise to a random-orientation of 2D nanoplates [55]. As discussed above, the crystal structure for both the samples of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA showed a 2-dimensional nature as clearly evidenced by the presence of (003) and (006) peaks, indicating the formation of hydrotalcite-like structure (JPCDS no 14–0191). Their morphology and particle size were also confirmed by the SEM studies (Fig. 2b). Though the BSA coating on QT-NPs (⁶⁴Cu-QT-NPs/BSA and Cu-QT-NPs/BSA) gave rise to the round-shaped hexagonal, all the samples were observed to be not much different in terms of their shape and morphology of typical 2D QT-NPs. And their average particle size was determined to be ~ 75 nm. According to the dynamic light scattering (DLS) measurements, the particle size of Cu-QT-NPs was determined to be 75 ± 27 nm (PDI: 0.138) in distilled water (DW), but became aggregated to be 195 ± 115 nm (PDI: 0.347) after coating with BSA to form Cu-QT-NPs/BSA. Since the isoelectric point (IEP) of BSA is at pH = 4.5-5.0 [56], the negatively charged BSA molecules in water (neutral pH) could be integrated onto the positively charged Cu-QT-NPs as confirmed by the reduction in zeta potential (ξ) of Cu-QT-NPs upon BSA coating from 22.7 to 7.22 mV (Table 1). Such a nano-cluster formation of Cu-QT-NPs/BSA (~ 195 nm) can be, therefore, rationalized by the fact that globular BSA with 583 amino acids preferably interact with Cu-QT-NPs by electrostatic interaction (Table 1).

Table 1

Particle size and zeta potential for Cu-QT-NPs and Cu-QT-NPs/BSA
Samples	Particle size	Zeta potential
Cu-QT-NPs	75 nm ± 29 *(0.138)	22.7 mV ± 3.30
Cu-QT-NPs/BSA	195 nm ± 115 *(0.347)	7.22 mV ± 3.21
* PDI: Polydispersity index is shown in parentheses

Labeling efficiency and labeling stability of ⁶⁴Cu radioisotope on QT-NPs

To evaluate radiolabeling efficiency of ⁶⁴Cu radioisotope depending on the amount of radioactivity, ⁶⁴Cu-QT-NPs were first prepared with different doses of radioactivity of 74, 185, 370, and 740 MBq, corresponding to 8.19, 20.49, 40.97, and 81.94 pmol of ⁶⁴Cu, respectively, those which were compared with ⁶⁴Cu-ads-QT-NPs with 185 MBq (20.49 pmol of ⁶⁴Cu). As expected, all the samples, except the physisorbed phase, showed very high labeling efficiencies beyond 99%, regardless of the amount of ⁶⁴Cu radioisotope, as shown in Fig. 3; the labeling efficiency was obtained as 99.22 ± 0.16, 99.93 ± 0.08, 99.04 ± 0.99, and 99.25 ± 0.12%, respectively. On the other hand, the latter, where the ⁶⁴Cu radioisotope was physically adsorbed on the surface of QT-NPs, was measured to be only 63.4 ± 0.99%; the low chemical stability of ⁶⁴Cu-ads-QT-NPs could also be explained by the fact that the ⁶⁴Cu radioisotope was weakly bound with physical adsorption on the external surface of QT-NPs in the form of unknown copper compound, which was found to be copper hydroxychloride, as shown in the Supplementary data Fig. S4. Different from the ⁶⁴Cu-ads-QT-NPs sample, the ⁶⁴Cu radioisotope atoms in the present ⁶⁴Cu-QT-NPs were stabilized in the 2D QT lattice, resulting in high thermodynamic stability thanks to the gain of lattice energy as illustrated in Fig. 1a.

In the present study, the sample ⁶⁴Cu-QT-NPs with 740 MBq was divided into 370 MBq of ⁶⁴Cu-QT-NPs and 370 MBq of ⁶⁴Cu-QT-NPs/BSA (Fig. 1) to explore ⁶⁴Cu labeling efficiency and labeling stability. At first both the samples were centrifuged using filter tubes, and then radioactivities of the isolated precipitates and supernatants were measured with a radioisotope dose calibrator; the labeling efficiencies of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA were determined to be 99.0 ± 0.99 and 99.7 ± 0.42%, respectively (Fig. 4a). It was, therefore, concluded that all the ⁶⁴Cu²⁺ ions were stabilized in the 2D QT lattice without remaining in the synthesis solution. To cross-confirm the labeling efficiency for ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, both the samples were studied on basis of radio-TLC, where the mobile phase was a 20 mM sodium citrate / 50 mM EDTA solution (pH = 5.5). As illustrated in Fig. 4b, the instant thin layer chromatograph (iTLC) paper, dropped with the radioactive sample, was placed into the mobile phase for 10 min. And then the unbound and unstable ⁶⁴Cu radioisotopes were removed by the mobile phase. In the case of free ⁶⁴Cu radioisotope, all the ⁶⁴Cu ions were moved to the solvent front by the mobile phase (Fig. 4c). On the other hand, the radio-TLC peaks of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA were obtained at the origin (starting line) as shown in Fig. 4d and 4e. However, from the radio-TLC results of ⁶⁴Cu-ads-QT-NPs and ⁶⁴Cu-ads-QT-NPs/BSA (Fig. S5), it was found that 49.1% of ⁶⁴Cu radioisotopes adsorbed on the former and 90.8% of them on the latter were detached, due to the fact that the ⁶⁴Cu radioisotopes were weakly bound on the external surface of QT-NPs as expected, and eventually removed by mobile phase easily. It is, therefore, concluded that the exellent chemical stability of ⁶⁴Cu radioisotope in ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA can be ensured by the isomorphical substitution of ⁶⁴Cu into the 2D QT lattice.

In addition, the ⁶⁴Cu labeling stability for each sample was investigated not only in saline and PBS but also in biological media such as mouse serum and human one, and it was found that ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA exhibited very high stability beyond 99% in all media (Fig. 5). Such a high thermodynamic stability could only be explained by the gain of lattice energy, since the ⁶⁴Cu radioisotopes were indeed incorporated into the octahedral sites of the QT lattice (Fig. 1).

In-vitro cytotoxicity and cellular uptake studies

Biocompatibility and toxicity of nanoparticles are one of important factors for their biomedical application. To evaluate the cell viability for QT-NPs containing ⁶⁴Cu radioisotope, both the samples with 0.37 MBq, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, were treated on the MDA-MB-231 cell lines. After incubating them for 24 h, their cell viability was observed to be almost 100%, indicating their excellent biocompatibility and chemical stability (Fig. 6a), which is also well consistent with our previous studies; QT-NPs were extremely low in toxicity as confirmed by both in-vitro and in-vivo experiments, and internalized inside the cells very efficiently via clathrin-mediated endocytosis [42, 43]. In this study, the in-vitro cytotoxicity for the non-radioactive Cu-doped samples, Cu-QT-NPs and Cu-QT-NPs/BSA, was also evaluated in the concentration range of 1–100 µg/mL on the cell culture line of MDA-MB-231 for 24 and 48 h, and was found to be very low in toxicity (Fig. S6).

Furthermore, the in-vitro cellular uptake behavior for the biocompatible ⁶⁴Cu-labeled QT-NPs (⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA) was also studied on the cell culture line of MDA-MB-231 for 24 h. As represented in Fig. 6b, the cellular uptake efficiencies (%dose/cells) of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA were obtained as 23.0 ± 1.36 and 7.9 ± 1.24, respectively, within 30 min, but improved remarkably as 43.3 ± 5.02 and 31.2 ± 2.44, respectively, after 24 h, those which were higher than that of other reported radioisotope labeled samples. In the case of ⁶⁴Cu-DOTA conjugated dendrimers, a very low uptake efficiency of 1.11% was observed after 2 h of incubation time on human nasopharyngeal cancer (KB) cell line [57]. For ⁶⁴Cu-NOTA-PA1 formed by conjugating ⁶⁴Cu-NOTA with pasireotide derivative (PA1), its intercellular uptake efficiency was only 3.67 ± 0.36 and 2.97 ± 0.09% at 2 h on human breast cancer (MCF-7) and human lung cancer (A549) cell lines, respectively [58]. In the case of ⁶⁴Cu-Sur-NGR2,⁶⁴Cu-labeled dimeric NGR (asparagine-glycine-arginine) peptide based on sarcophagine (Sur) cage, its uptake value was observed to be 0.72 ± 0.01 and 1.72 ± 0.24% upon treating on the human fibrosarcoma (HT-1080) cell line at 1 h and 2 h, respectively, after incubation [59]. We, therefore, came up with a conclusion that our designed samples, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, can be a promising radioisotope delivery and imaging technology platform not only because of their high labeling efficiency and excellent chemical stability, but also because of their extremely high biocompatibility and low toxicity.

In-vivo PET imaging and biodistribution studies

To demonstrate the targeting property of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA in in-vivo, the PET imaging and biodistribution studies were carried out on the MDA-MB-231 tumor-bearing xenograft mouse model (Fig. 7 and Fig. 8); they were visualized using a small-animal PET scanner (Inveon™), where the reconstructed images could be made by detecting gamma rays (energy, 511 keV) generated from the decay of ⁶⁴Cu radioisotopes (${}_{29}{}^{64}\text{C}\text{u}\to {}_{28}{}^{64}\text{N}\text{i} + {}_{1}{}^{0}{\beta } \left(\text{p}\text{o}\text{s}\text{i}\text{t}\text{r}\text{o}\text{n}\right)+ {{\nu }}_{\text{e}} \left(\text{n}\text{e}\text{u}\text{t}\text{r}\text{i}\text{n}\text{o}\right)$) [60, 61] as shown in Fig. 7a and 7b. The coronal and transverse PET images were made at various progressive time using a PET scanner, and then the time activity curves for tumors, muscles, liver, and blood were obtained by performing the quantitative region-of-interest (ROI) analysis as shown in Fig. 7c and 7d and Table S2. A prompt and persistent uptake of nanoplates into tumor tissues was achieved in both cases due to the passive targeting effect of QT-NPs (50 to 200 nm) as well described as the enhanced permeability and retention (EPR) effect. Therefore the tumor region (white dot circles and yellow arrow) in PET images becomes brighter for both, but the image of ⁶⁴Cu-QT-NPs/BSA appears to be much brighter than that of ⁶⁴Cu-QT-NPs with respect to the time. As presented in Fig. 8a and Table S2, the ⁶⁴Cu-QT-NPs contents uptaken by tumor tissues and cells were increased gradually from 0.71 ± 0.10, 1.40 ± 0.29, and 2.13 ± 0.25, to 2.43 ± 0.60 %ID/g with respect to the given time (2, 6,24, and 48 h). After BSA coating, however, ⁶⁴Cu-QT-NPs/BSA seemed to be more efficiently uptaken by tumor tissues, since their contents were even more increased from 0.96 ± 0.36 to 1.80 ± 0.20, and 4.53 ± 0.51, to 4.93 ± 0.81 %ID/g with respect to the same period of tie of 2, 6, 24, and 48 h, indicating that the BSA coated sample exhibited 2-fold higher than the uncoated after 24 h from the injection. Such a difference could be explained by the fact that the blood circulation time of ⁶⁴Cu-QT-NPs was surely improved by the BSA coating. In practice, %ID/g of ⁶⁴Cu-QT-NPs in blood pool was observed to be 0.55 ± 0.03, 0.56 ± 0.03, 0.55 ± 0.10, and 0.45 ± 0.02 for 2, 6, 24 and 48 h, respectively, while that of ⁶⁴Cu-QT-NPs/BSA was measured to be 0.70 ± 0.10, 0.93 ± 0.18, 1.25 ± 0.10, and 1.23 ± 0.19, respectively, for the same period of time. This is an evidence that the uptake of ⁶⁴Cu-QT-NPs/BSA in tumor tissues was more efficiently made after BSA coating due to the enhanced blood circulation time. In addition, the tumor-to-liver ratio (%) of ⁶⁴Cu-QT-NPs/BSA (21.86 ± 5.05 %) was also examined to check its targetingfunction, and was found to be 1.7-fold higher than that of ⁶⁴Cu-QT-NPs (12.67 ± 6.38 %) after 48 h from the injection (Fig. 8b).It is surely due to an enhanced colloidal stability of Cu-QT-NPs/BSA (186 ± 38 nm of size distribution in saline, PDI: 0.356) compared to Cu-QT-NPs (585 ± 102 nm of size distribution in saline, PDI: 0.669) as shown in Table S3. From the above results, the particle size of ⁶⁴Cu-QT-NPs/BSA could also be expected to be ~ 200 nm in the blood enabling the enhanced tumor penetration by the EPR effect. And it is, therefore, not that surprising that the BSA coated, ⁶⁴Cu-QT-NPs/BSA, showed 2-fold higher tumor uptake rate than ⁶⁴Cu-QT-NPs.

Even though the present radio-labeled samples, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, could effectively deliver the radioisotopes to tumor tissues, thanks to the passive targeting effect, there were some toxicity issues due to a high uptake rate in the liver. After intravenous administration of inorganic nanoparticles within the size of 10 ~ 200 nm, they were known to be rapidly sequestered from the blood and severely accumulated in reticuloendothelial system (RES) organs including liver, spleen, and lymph nodes [62–64]. Thus the liver uptake and clearance of injected nanoparticles have been usually considered as major concerns for bio-applications. Therefore, the U.S. Food and Drug Administration (FDA) regulates that all injected agents have to be cleared completely in a reasonable period of time [62–64]. According to the present in-vivo biodistribution study for ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA as shown in Fig. 9, the uptake rates into liver for the former (49.1 ± 8.7%ID/g) and the latter (49.5 ± 8.0%ID/g) were determined to be rather high at 2 h after injection, but decreased repectively to 58.0 and 56.4% after 48 h in liver, resulting in 20.6 ± 3.7 and 21.6 ± 3.1%ID/g, respectively. And the uptake rates into spleen for the former (134.1 ± 51.1%ID/g) and the latter (88.9 ± 41.4%ID/g) were also reduced down to 58.9 and 68.2% after 48 h, giving rise to 55.0 ± 31.8 and 28.3 ± 3.6%ID/g, respectively. As well documented [62], inorganic nanoparticles could be mainly trapped in the reticuloendothelial system (RES) organs (liver and spleen) in the beginning, and then significantly decreased over time due to the excretion mechanism by hepatic (bile to feces) pathway. As expected, the present samples, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, were uptaken more into liver and spleen than the other organs (Fig. 7 and Fig. 9). In addition, intact ⁶⁴Cu ions (free ⁶⁴Cu radioisotopes from ⁶⁴CuCl₂) were also uptaken up into liver tissues but usually cleared within 24 h by the renal (kidney) clearance (Fig. S6).

Nevertheless, the liver uptake of ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA over 24 h may be posed its toxicity, which is always one of the major concern for the use of nanoparticles in nanomedicine [63]. However, according to our previous in-vivo liver toxicity results for QT-NPs as summarized in Table S4, the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), as the recognition index of liver toxicity, liver disease, or liver damage, were found to be within the normal ranges (7-227 and 37–329 U/L for ALT and AST, respectively) after 72 h from the intraperitoneal administration of QT-NPs [44], indicating the biocompatibility and non-toxicity of the present samples, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA. Furthermore, the concentrations of Mg and Al ions, the major elements of QT, found in liver tissue for the QT treated animal group were determined to be very similar with those for the QT non-treated one at one week after the final injection (Table S3) [44]. As described above, the uptake rate in liver for ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA was decreased ~ 60% from 2 to 48 h after injection, expecting its clearance further from the body over time. We, therefore, propose that the present biocompatible nanoplates, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, could be the promising new nanoagent for PET imaging.

We were successful in preparing ⁶⁴Cu-QT-NPs based on the co-precipitation and subsequent hydrothermal reaction as the two-step processes. The divalent ⁶⁴Cu radioisotope was isomorphically substituted in the octahedral site of the 2D QT lattice to form ⁶⁴Cu-QT-NPs, which showed excellent labeling efficiency and stability, cellular uptake efficiency and biocompatibility even after the BSA treatment, suggesting its potential as a PET imaging agent. According to the present in-vivo PET imaging experiments, both the radioisotope labeled samples, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, were selectively targeted to the tumor tissues. One more thing to note here is that the latter, the BSA coated, showed 2-fold higher uptake rate in tumor tissues than the former surely due to an enhanced colloidal stability eventually leading to the longer blood circulation when injected, and as a consequence, the better EPR effect. Moreover, the present 2D QT-NPs were determined to be excreted over time by hepatic pathway after intravenous administration. Therefore, the present samples, ⁶⁴Cu-QT-NPs and ⁶⁴Cu-QT-NPs/BSA, can be proposed as the alternative to escape from the toxicity issue of nanoparticles in nanomedicine, thanks to its high labeling efficiency and labeling stability as well as biocompatibility. So we conclude that both phases are surely the promising candidates as radionuclides delivery nano-devices not only for PET imaging but also for radiotherapy as the nanomedicine. Nevertheless, further studies are still required to understand their long-term stability and toxicology in in-vivo, and the enhanced tumor uptake behaviors for the radioisotope doped phases conjugated with some passive and active targeting agents (folate, antigen, etc.) eventually to establish the radioisotope-labeled QT platform technology, which will be made in the very near future.

Acknowledgments

The authors would like to thank Dr. Kyo Chul Lee, Dr. Jung Young Kim, and Mr. Hyun Park in KIRAMS for production of ⁶⁴Cu.

Authors’ contributions

Sairan Eom: Methodology, Investigation, Formal analysis, Visualization, Writing – original draft, Writing - review & editing. Min Hwan Kim: Investigation, Formal analysis. Ranji Yoo: Investigation. Goeun Choi: Investigation, Writing - review & editing, Project administration, Funding acquisition. Joo Hyun Kang: Project administration. Yong Jin Lee: Conceptualization, Supervision, Writing – review. Jin-Ho Choy: Supervision, Project administration, Funding acquisition, Conceptualization, Writing - review & editing.

Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (No. 2020R1I1A2074844), by the NRF grant funded by the Korea government (MSIT) (No. 2020R1F1A1075509), and under the framework of the International Cooperation Program managed by NRF (2017K2A9A2A10013104). This study was also partly supported by a grant from the Korea Institute of Radiological and Medical Sciences (KIRAMS), by the Ministry of Science and ICT (MSIT), the Republic of Korea (No. 50536–2022).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

All experimental procedures involving animals were conducted in strict accordance with the appropriate institutional guidelines for animal research. The protocol was approved by the Committee on the Ethics of Animal Experiments of the KIRAMS (Approval Number: kirams2017-0091 and kirams2019-0076).

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No competing interests reported.

JNanotechnolSupplementary64CuQTNPsProf.Choy.pdf

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Dilute lattice doping of Cu-64 into 2D-nanoplate; its impact on radio-labeling efficiency and stability for target selective PET imaging

Status:

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Abstract

Backgound:

Results

Conclusions

Figures

Background

Methods

Materials

Characterization

Results And Discussion

Physico-chemical characterization

Labeling efficiency and labeling stability of ⁶⁴Cu radioisotope on QT-NPs

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Dilute lattice doping of Cu-64 into 2D-nanoplate; its impact on radio-labeling efficiency and stability for target selective PET imaging

Status:

Version 1

Abstract

Backgound:

Results

Conclusions

Figures

Background

Methods

Materials

Characterization

Results And Discussion

Physico-chemical characterization

Labeling efficiency and labeling stability of 64Cu radioisotope on QT-NPs

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1

Labeling efficiency and labeling stability of ⁶⁴Cu radioisotope on QT-NPs