⁶⁴Cu-labeled melanin nanoparticles for PET/CT and radionuclide therapy of tumor

Abstract

Melanin is a group of natural pigments found in living organism. It can be used for positron emission tomography (PET) imaging due to its inherent chelating ability to radioactive cupric ion. This study was to prepare ⁶⁴Cu-labeled PEGylated melanin nanoparticles (⁶⁴Cu-PEG-MNPs), and to further take advantage of the enhanced permeability and retention (EPR) effect of radiolabeled nanoparticles to realize the integration of tumor diagnosis and treatment. We successfully synthesized PEG-MNPs. Saline and serum stability experiments demonstrated good stability. PET/CT showed high tumor aggregation. Moreover, ⁶⁴Cu-PEG-MNPs resulted in a therapeutic effect on the A431 tumor-bearing mice in the treatment group. The pathological results further confirmed that the therapeutic doses of ⁶⁴Cu-PEG-MNPs cause pathological changes of tumor tissues while showing minimal toxicity to normal tissues. Our data successfully demonstrate the good imaging performance of ⁶⁴Cu-PEG-MNPs on A431 tumors and further proved its therapeutic effect, highlighting a great potential in targeted radionuclide therapy.

With high morbidity and mortality, cancer has become a major cause of death and public health problem in China. According to “Cancer Statistics, 2019”, there exist approximately 1,762,450 new cancer cases and 606,880 cancer deaths in the United States in 2019.¹ So far, surgery, radiotherapy and chemotherapy are still the primary methods of cancer treatment. The goal of cancer therapies is to minimize damage to normal cells while killing tumor cells efficiently. However, traditional chemoradiotherapy damages normal tissues during the process of killing cancer cells.^2,3 Therefore, the development of a new type of targeted radiopharmaceutical and treatment method with small dosage, long-term effects, accurate positioning and less side effects, which enables the radiopharmaceutical to be concentrated to the tumor or even inside the tumor cells, has attracted tremendous attention in the field of cancer radionuclide therapy recently.^4–7

The use of radionuclide-labeled compounds for the treatment of tumor, i.e. radionuclide-targeted anti-tumor therapy, has become an attractive treatment and has been actively explored in pre-clinical research and clinical practice. Compared to external irradiation treatment and chemotherapy, targeted radionuclide therapy can specifically deliver radiation dosages to tumor cells with minimum damage to normal tissues and organs. Radioisotopes that emit Auger electrons, α-particles, and β-particles can be used for radionuclide therapy.^8,9 Some β particle-emitting radioisotopes such as ⁹⁰Y, ¹⁷⁷Lu, ¹⁸⁸Re, ¹³¹I, and ⁶⁴Cu have been studied for tumor-targeted radiotherapy. It has been widely recognized that high-energy β-particle-emitting radioisotopes such as 188Re and 90Y are suitable for treating larger tumors, while medium-low energy emission particles, such as ⁶⁴Cu, ¹⁵³Sm, and 177Lu, are more suitable for treating small or metastatic tumors.^8,10–14

The half-life of ⁶⁴Cu is 12.7 h, and it emits β⁺ (0.653 MeV [17.8%]) and β⁻ (0.579 MeV [38.4%]) electrons. 64Cu is considered to be suitable for both positron emission tomography (PET) and tumor radionuclide therapy.^15,16 It can be easily coordinated with numerous chelators and thus used for radiolabeling various peptides and other biologically related small molecules, monoclonal antibodies, proteins and nanoparticles.^17–20 The moderate half-life of 64Cu renders it feasible for multiple time point imaging study and also makes it suitable for in vivo studies of small molecules as well as larger, slower-removing proteins and nanoparticles. Based on the above advantages, there have been reports on PET and tumor radionuclide therapy using various types of 64Cu-based molecules in the past two decades. For example, studies have shown that 64CuCl₂ PET has a good imaging performance and therapeutic effect on tumors, highlighting the strong potential of ⁶⁴Cu in the treatment of some tumor types.¹⁷

Melanin is a natural pigment widely found in living organisms. It is produced by the oxidation of tyrosine and is often used as a biomarker for the imaging of melanoma.^21,22 In our previous research, a type of small size (11 nm), water-soluble melanin nanoparticles (MNPs) was prepared and displayed tumor accumulation property through the enhanced permeability and retention (EPR) effect.^23,24 It retains many characteristics of melanin and can directly complex with various metal ions without traditional surface modification of chelating agents, which significantly simplifies the preparation process and reduces the heterogeneity of the nanoparticles. MNP has been demonstrated to be used for tumor multimodality imaging, such as photoacoustic imaging (PAI), PET and magnetic resonance imaging (MRI). In addition, MNP is an organic and biodegradable material and exhibits good performance in tumor imaging.²⁴ All these properties make MNPs great potential for clinical transformation. The purpose of the current study was to prepare ⁶⁴Cu-labeled MNPs for PET/CT of tumor and further evaluate its tumor radionuclide therapy effect in the nude mice bearing human epidermoid carcinoma (A431) models.

Methods

Reagents and chemicals

Melanin, amine-PEG₅₀₀₀-amine, sodium hydroxide, hydrochloric acid, and NH₄OH solution were purchased from Sigma-Aldrich. Dulbecco’s modified Eagle medium (DMEM), 10% fetal bovine serum (FBS) and phosphate buffered saline (PBS) were purchased from Gibco. The A431 cell line was obtained from the American Tissue Culture Collection. 64CuCl₂ was purchased from the Department of Medical Physics, University of Wisconsin at Madison. The synthesis of polyethylene glycol modified MNPs (PEG-MNPs) was performed according to previous work.²⁴

⁶⁴Cu²⁺ radiolabeling of PEG-MNPs

Briefly, the 1 mg mL⁻¹ polyethylene glycol modified MNPs (PEG-MNPs) in solution as prepared in our previous study²⁴ was radiolabeled with ⁶⁴Cu²⁺ by addition of 555 MBq (15 mCi) ⁶⁴CuCl₂ in NaOAc (0.1 N, pH 5.5) buffer followed by a 1 h incubation at 40 °C. The reaction was then terminated by adding 15 μL of trifluoroacetic acid (TFA), and the radiolabeled nanoparticles, ⁶⁴Cu-PEG-MNPs, were purified using a PD-10 column.

In vitro radiostability assays

⁶⁴Cu-PEG-MNPs (200 μCi, 7.4MBq) were mixed with 500 μl FBS and placed at 37 °C, or mixed with 500 μl of saline and incubated at room temperature. Samples were taken at 2, 4, 8 and 24 h. Then the radiochemical purity was determined by radio-thin layer chromatography (TLC) to evaluate its in vitro radiostability with acetone as developing agent for TLC.

In vitro cell assays

A431 cells were seeded in 24-well plates at a concentration of 1 × 10⁵ cells/well and grown overnight. Subsequently, ⁶⁴Cu-PEG-MNPs (~2 μCi [74 kBq]/well) were added and incubated at 37 °C for 4 h. Three replicate wells per group and two plates were used. Thereafter, the incubated cells were washed three times with cold PBS solution, and then cells and lysates were collected by the method of trypsinization. The radioactivity of the cells was measured using a gamma counter. The cell uptake value of 64Cu-PEG-MNPs was expressed as percentage of radioactivity bound to the cells divided by the total radioactivity added. All cell binding experiments were repeated three times.

The cellular retention of ⁶⁴Cu-PEG-MNPs (~2 μCi [74 kBq]/well) was determined by incubating A431 cells in binding media for 2 h. After that, the incubation medium was removed and the cells were gently washed with cold PBS three times. Then to each well culture medium was added and incubated for 4 h. Three replicate wells per group were used. Then at each time point, the incubation PBS was taken, and the cells were collected by trypsinization. The radioactivity of the cells was measured by a gamma counter.

Small-animal PET and biodistribution studies

All animal experiments were performed in compliance with the Guidelines for the Care and Use of Research Animals as established by the Stanford University Animal Studies Committee. Small animal PET/CT scans were performed on A431 tumor-bearing mice (n = 4) using a Siemens Inveon microPET/CT scanner. Each nude mouse was injected with 11.1 MBq (300 μCi) of ⁶⁴Cu-PEG-MNPs via tail vein. Inhaled anesthesia was performed on each nude mouse with 2% isoflurane–oxygen gas mixture. Thirty minute static scans were performed at 2, 4, 8 and 24 h after the injection. All images were reconstructed by a two-dimensional ordered subsets expectation maximum (OSEM) algorithm with attenuation and scatter correction.

For biodistribution study, A431 tumor-bearing mice were divided into 4 groups and each mouse was injected with 11.1 MBq (300 μCi) of ⁶⁴Cu-PEG-MNPs. After 2, 4, 8 and 24 h of injection, the nude mice were sacrificed and dissected. Blood, tumor and other major organs (heart, lung, spleen, liver, stomach, kidney, intestine, bone and muscle tissue) were collected and weighed; the radioactivity was measured by the γ counter. The percentage of the injected radioactive dose per gram of tissue (% ID/g) value was calculated and expressed as mean ± standard deviation.

Dosimetry studies

The biodistribution of ⁶⁴Cu-PEG-MNPs was assessed at 2, 4, 8, and 24 h in A431 tumor-bearing mice to evaluate radiation-absorbed doses from 64Cu-PEG-MNPs in tumor and normal organs and tissues according to the literature.²⁵ When estimating the tumor absorbed does, it was assumed that once the radiopharmaceutical is inside the tumor, there is no biological elimination.²⁶ Organ dosimetry was estimated using the OLINDA/EXM software (version 2.0; Hermes Medical Solutions AB).

Radionuclide therapy

A431 tumor-bearing nude mice were used to determine the therapeutic effect of ⁶⁴Cu-PEG-MNPs. Nude mice with tumor diameters of 5–8 mm were randomly divided into treatment group and control groups. The treatment group (n=6) was injected with ~55.5 MBq (1.21±0.14 mCi) ⁶⁴Cu-PEG-MNPs through tail vein to observe treatment effect. The other two groups (n=5 per group) were injected with the same amount (0.3 ml) of PBS and PEG-MNP respectively through the tail vein as controls, and the mice body weight and tumor size were measured every two days. Tumor size was calculated by the following formula: volume = short diameter² × long diameter/2. Nude mice were sacrificed when the tumor diameter exceeded 1.75 cm, or the tumor weight was greater than 10% of body weight, or the tumor was ulcerated, or the animal was dying.

After euthanasia, the liver, kidney and tumor were collected and fixed in 10% formalin. Then the organs of A431 tumor-bearing nude mice were stained with hematoxylin and eosin (H&E). The liver, kidney and tumor of the mice in the treatment group were observed for pathological differences. Section images were taken using an Olympus IX71 microscope.

Statistical analysis

GraphPad Prism 5.0 software was used. The measurement data were expressed as mean ± standard deviation, and multiple sets of data were compared. One-way analysis of variance was used.

Results

⁶⁴Cu radiolabeling and in vitro stability

The radiolabeling process was completed in 1 h. The final product ⁶⁴Cu-PEG-MNPs were purified by PD-10 column to remove free ⁶⁴Cu²⁺, and its radiochemical purity was assessed by radio-TLC, which showed that the radiochemical purity of the labeled nanoparticles was greater than 95% (Figure 1, A).

Figure 1.

(A) Radiochemical purity of ⁶⁴Cu-PEG-MNPs was measured by radio-TLC. (B) In vitro radiostability of ⁶⁴Cu-PEG-MNPs in saline at room temperature and FBS at 37 °C for 2, 4, 8 and 24 h.

Radiostability assay showed that 64Cu-PEG-MNPs were stable in saline (the purity was greater than 95.0% at 24 h measured). And its stability was slightly reduced in FBS but there were still about 90.0% labeled nanoparticles at 24 h incubation at 37 °C (Figure 1, B).

Cell uptake and efflux study

Uptake of ⁶⁴Cu-PEG-MNPs by A431 cells at 0.25 h to 4 h is shown in Figure 2. The maximum uptake value was reached during the 4-h incubation period. The uptake value was 2.72% ± 0.2% for 1 h, 4.91% ± 0.27% for 2 h and 5.78% ± 0.12% for 4 h. ⁶⁴Cu-PEG-MNPs showed good retention in A431 cells. Even after four hours incubation in PBS, only 4.49% of radioactivity release from the A431 cells.

Figure 2.

Time-dependent cell uptake (A) and efflux (B) of ⁶⁴Cu-PEG-MNP in A431 cells.

PET and biodistribution

Static PET/CT images of A431 tumor-bearing mice were collected at 2, 4, 8 and 24 h after tail vein injection of ⁶⁴Cu-PEG-MNPs (n = 4). The coronal and transverse images of the delayed acquisition are shown in Figure 3. Tumors were clearly visible at all time points. Also, the imaging study showed high accumulation of ⁶⁴Cu -PEG-MNPs in the liver, indicating that the main clearance pathway of ⁶⁴Cu -PEG-MNPs is through the hepatobiliary system.

Figure 3.

Representative images of decay corrected coronal (top) and transaxial (bottom) small animal PET/CT images of nude mice bearing A431 tumor at 2, 4, 8 and 24 h after tail vein injection of ⁶⁴Cu-PEG-MNPs (11.1MBq).

In vivo biodistribution study of A431 tumor-bearing mice that injected with ⁶⁴Cu-PEG-MNPs for 2, 4, 8 and 24 h confirmed the PET results (Table 1, Figure 4). The tumor uptake value of ⁶⁴Cu-PEG-MNPs was 7.45 ± 0.50, 11.75 ± 0.49, 13.59±0.69 and 5.62 ± 0.52 % ID/g at 2, 4, 8 and 24 h after injection, respectively. The liver and spleen had relatively high uptake values (liver’s uptake values were all greater than 10% ID/g at 2 h, 4 h, 8 h and 24 h). In addition, in vivo biodistribution results at 24 h post-injection showed tumor/blood and tumor/muscle ratio of 3.60 ± 0.17 and 15.12 ± 0.62, respectively.

Table 1.

Biodistribution of ⁶⁴Cu-PEG-MNPs in A431 tumor-bearing mice (%ID/g, mean ± SD, n = 4).

Organ	2 h	4 h	8 h	24 h
Blood	18.07±0.05	16.77±0.02	19.65±0.58	2.07±0.04
Tumor	7.45±0.50	11.75±0.49	13.59±0.69	5.62±0.52
Lung	2.64±0.36	3.21±0.31	4.52±0.66	1.61±0.35
Spleen	24.30±0.46	23.01±0.24	25.84±0.01	7.34±0.47
Kidney	19.48±0.40	21.68±0.37	22.36±0.59	5.34±0.32
Heart	9.23±0.61	8.51±0.55	10.55±0.92	3.45±0.61
Stomach	6.64±0.47	9.62±0.47	9.69±0.85	0.53±0.46
Liver	30.97±0.53	33.86±0.34	36.10±0.25	16.24±0.64
Muscle	2.09±0.08	1.28±0.02	1.75±0.68	0.49±0.01
Intestine	12.30±0.40	18.63±0.52	20.38±0.56	3.13±0.28
Bone	5.44±0.05	4.84±0.10	6.08±0.61	1.76±0.09
Tumor/blood	0.31±0.03	0.70±0.03	0.69±0.05	3.60±0.17
Tumor/muscle	2.69±0.14	9.17±0.54	7.77±1.85	15.12±0.62

Figure 4.

Decay-corrected biodistribution of ⁶⁴Cu-PEG-MNPs at 2, 4, 8 and 24 h post-injection in A431 tumor bearing mice (mean ± SD, n = 4).

Tumor and major organs dosimetry

The primary and secondary critical normal organs were liver and spleen, with absorbed doses of 0.136 mGy/MBq and 0.0358 mGy/MBq, respectively. The data for other target organs are shown in Table 2. The effective dose is 0.0258 mGy/MBq. For the 2 g tumor a spherical model was used (OLINDA/EXM software) and the absorbed dose was 57.5 mGy/MBq.

Table 2.

Radiation absorbed doses from ⁶⁴Cu-PEG-MNPs.

Organ	mSv/MBq
Adrenals	2.81E-02
Brain	2.27E-02
Breasts	2.07E-02
Gallbladder wall	3.33E-02
LLI wall	2.39E-02
Small intestine	2.98E-02
Stomach wall	2.72E-02
ULI wall	2.63E-02
Heart wall	2.87E-02
Kidneys	6.22E-02
Liver	1.36E-01
Lungs	1.59E-02
Muscle	9.33E-03
Ovaries	2.44E-02
Pancreas	2.81E-02
Red marrow	2.91E-02
Osteogenic cells	6.72E-02
Skin	1.88E-02
Spleen	3.58E-02
Thymus	2.18E-02
Thyroid	2.15E-02
Urinary bladder wall	2.35E-02
Uterus	2.47E-02
Total body	2.63E-02
Effective dose(mSv/MBq)	2.58E-02

Extrapolated radiation dose for a 70 kg female model.

Radionuclide therapy

The tumor size and body weight changes curves are shown in Figure 5. The A431 tumor growth rate of the ⁶⁴Cu-PEG-MNPs treatment group was significantly slower than that of the two control groups (Figures 5, 6). Nude mice initially showed a downward trend in weight and then returned to their original body weight levels after a few days. The H&E staining results of A431 tumor-bearing nude mice showed that there was no significant difference in livers and kidneys between ⁶⁴Cu-PEG-MNPs treatment group and PBS control group. In addition, pathological change in tumor tissue was found for the ⁶⁴Cu-PEG-MNPs treatment group (Figure 7). Tumor necrosis cells in the drug administration group were scattered, nuclei disappeared, and chromatin was loose and fuzzy, suggesting tumor necrosis caused by radiation damage.

Figure 5.

Tumor growth (A) and weight variation curves (B) of the ⁶⁴Cu-PEG-MNPs radionuclide therapy group, the PBS treated group, and PEG-MNPs treated group as controls in A431 tumor models.

Figure 6.

Color image of tumor samples obtained from different groups (PBS and PEG-MNPs treated control groups and ⁶⁴Cu-PEG-MNPs treated group at 1 week post-injection)

Figure 7.

H&E staining of kidneys, livers, tumors from the radionuclide therapy group and the control group.

Discussion

Over the past decades, along with the development of molecular imaging probes, melanin has been used as an effective molecular targeting probe and a primary contrast agent for photoacoustic imaging with its superior light absorption capacity.^27–29 In addition, melanin has a natural property of chelating various metal ions (including Cu²⁺)^23,30–32; therefore, it can be used for chelating radionuclides for imaging. Traditional biomolecular-based nanoplatforms generally require complex chelating functions to incorporate radiometals. MNPs are a bioinspired nanoplatform and easily complex metal ions without surface modification or introduction of chelating groups, making them highly attractive in biomedical applications. In this study, 64Cu was successfully loaded on MNPs without any chelating ligands. And to improve the biocompatibility and the water solubility, and efficiently prevent the formation of metal ion-induced precipitation, polyethylene glycol (PEG) chains were introduced to the MNPs. Moreover, the obtained ⁶⁴Cu-PEG-MNPs exhibited good radiostability in fetal bovine serum at 37 °C at 4 h. At 8 h and 24 h, 90.3% ± 1.27% and 90.05% ± 1.34% of 64Cu-PEG-MNPs remained intact, respectively, suggesting good in vitro stability of the radiolabeled nanoparticles. However, the stability of ⁶⁴Cu-PEG-MNPs may not be as high as commercially available macrocyclic Cu complexes, such as Cu-NOTA, Cu-DOTA and Cu-TETA, etc., which are kinetically more inert.³³ At present, they are widely used for ⁶⁴Cu marker imaging. However, due to the limitations of in vivo stability and biosafety of these drugs, these copper chelates have not been used in clinical practice for the time being yet.^34–36 The ⁶⁴Cu-PEG-MNPs also exhibited good biocompatibility and exhibited rapid and high tumor uptake, extended tumor retention, and high organ ratios of tumor to normal organ, which are all the preconditions for being radiolabeled therapeutic agents and indicate the MNP therapeutic nanoparticles can accumulate in solid tumors through EPR effect.^{37–39 64}Cu-PEG-MNPs exhibited rapid tumor uptake of 7.45±0.50 percentage injected dose per gram (%ID/g) at 2 h after injection in A431 tumor-bearing nude mice and the tumor was clearly visualized by PET which lasted until 8 h. Although the tumor uptake value of ⁶⁴Cu-PEG-MNPs dropped to 5.62 ± 0.52%ID/g at 24 h, it still accumulated relatively high enough in tumor. The long-term retention of PEG-MNPs provides a better choice for dynamic observation and radiotherapy for tumor. However, relying solely on the EPR effect cannot slow down the metabolic rate of ⁶⁴Cu-PEG-MNPs. Active targeting of nanoparticles can further increase accumulation in tumor. The simultaneous use of active and passive targeting will greatly increase the tumor-targeting effect of nanoprobes and provide ideas for further research.

For dosimetry calculation, the OLINDA/MXM was used for specific human models of different weights. Inputting extrapolated data from mice can provide acceptable data for humans. The estimated absorbed dose for a 2 g tumor was 0.057 Gy/MBq with a 55.5 MBq dose injected in the mice and the mean calculated effective dose for the female model was 0.0258 mSv/MBq. Thus the absorbed and effective dose of ⁶⁴Cu-PEG-MNPs in human body can be effectively estimated, and the effective dose is lower than the upper limit of the allowable range.

In addition, we observed high accumulation of ⁶⁴Cu-PEG-MNPs in liver and spleen from PET and in vivo biodistribution results. Since kidney uptake was also seen, it can be concluded that the radiolabeled melanin nanoparticles are cleared through both the hepatobiliary system and kidney-urinary system. This is consistent with the in vivo metabolic distribution characteristics of nanoparticles. Besides, studies have shown that most of the nanoparticles will be deposited in the liver and spleen when the nano-diameter is greater than 6 nm.^40–42

This study also demonstrated that ⁶⁴Cu-PEG-MNPs produced therapeutic effects on A431 tumor-bearing nude mice. Treatment with a single dosage of ~55.5 MBq of ⁶⁴Cu-PEG-MNPs resulted in substantial tumor growth inhibition over the time period of the therapy study. Mice treated with ⁶⁴Cu-PEG-MNPs developed obvious reaction, such as weight loss during the first week after start of treatment. After a few days, however, the mice returned to normal weight and maintained normal growth patterns. For the control group, most of the tumor diameter reached 1.75 cm, the tumor weight was greater than 10% of body weight, or the tumors were ulcerated on the seventh day, then the monitoring was stopped and the mice were sacrificed. But for treatment group, above-mentioned condition didn’t occur until the thirteenth day. Besides, the potential radiation toxicity caused by ⁶⁴Cu-PEG-MNPs needs to be considered. It should be pointed out that the high energy Auger electrons emitted by ⁶⁴Cu may cause damage at the chromosomal and cellular levels, thereby facilitating its application for radionuclide therapy.^43,44 The treatment efficacy of ⁶⁴Cu-PEG-MNPs demonstrated here may be partially caused by this physical property of ⁶⁴Cu as well. Moreover, radioactive cytotoxicity to normal tissues was not observed based on the H&E staining results of the liver and kidney of the nude mice of the treatment group and the control group. Taken together, these results indicate that the use of a therapeutic dose of 64Cu-PEG-MNPs does not result in significant systemic toxicity in the short term.

Our results suggest that the ⁶⁴Cu-PEG-MNP is expected to be a promising radiolabeled nanomaterial for tumor theranostics. However, the findings still have some limitations. Optimal administration timing and therapeutic dose of ⁶⁴Cu-PEG-MNPs were not examined. These issues should be taken into account in future preclinical and clinical studies to achieve a more effective outcome of this therapy. In addition, we chose single human epidermoid carcinoma (A431) tumor-bearing mice model; then the efficacy of the ⁶⁴Cu-PEG-MNPs in the other types of cancer would be warranted by further studies.

Abbreviations:

PET

positron emission tomography

EPR

enhanced permeability and retention

MNPs

melanin nanoparticles

PEG

polyethylene glycol

PBS

phosphate buffered saline

FBS

fetal bovine serum

MRI

magnetic resonance imaging

PAI

photoacoustic imaging

DMEM

Dulbecco’s modified Eagle medium