A Ferritin-Albumin-Cu Nanoparticle that Efficaciously Delivers Copper(II) Ions to a Tumor and Improves the Therapeutic Efficacy of Disulfiram.

The application of disulfiram can be traced back to the 1920s, from when it was utilized to facilitate alcohol abstinence by producing allergic reactions toward alcohol. In previous research, combination of disulfiram and copper ions has demonstrated significant anti-tumor activity. However, both disulfiram and copper ions lack the ability of specific targeting to tumor tissues and may lead to a high risk of side effects, especially for copper ion, which is a kind of toxic heavy metal. Herein, a ferritin-albumin-Cu nanoparticle (FHC NP) was introduced. The nanoparticle was synthesized by first covalently cross-linking ferritin and albumin and then encapsulating the copper ions in the ferritin nanocage. The nanoparticle showed good accumulation in a tumor and when combined with disulfiram exhibited good in vitro selectivity toward cancer cells and better in vivo anti-tumor activity. Herein, the nanoparticle invented here represented a good strategy to efficaciously deliver copper ions into tumors and thus improve the therapeutic efficacy of disulfiram in tumor therapy.

Introduction

Cancer is one of the most malignant diseases all over the world, and the incidence and mortality of cancer are terrifying.[1,2] Despite the rapid progress in cancer therapy lately, cancer is still a primary threatening factor to human survival.[3−5] Herein, demands of new drugs for cancer are urgent.

Disulfiram (DSF) is a small molecule first discovered in the 1920s and has been utilized to facilitate alcohol abstinence by producing allergic reactions toward alcohol. It has been approved by the US Food and Drug Administration (FDA) and used in clinical trials for decades.[6−8] Recent research studies have indicated that when combined with copper(II) ions, disulfiram had significant anti-tumor activity toward multiple types of cancers,[9,10] including breast cancer,[11] non-small lung cancer,[12] and glioblastoma,[13] with a potential mechanism of action revealed in following studies.[14] Briefly, disulfiram first metabolizes to ditiocarb (diethyldithiocarbamate, DTC) in vivo, which then tends to form DTC–metal ion complexes with multiple metal ions.[15] Among these complexes, the DTC–copper complex (bis(diethyldithiocarbamate)–copper (CuET)) shows strong anti-tumor activity by suppressing nuclear protein localization protein 4 (NPL4).[16] Based on this knowledge, oral administration of disulfiram and copper gluconate has been evaluated in clinical trials (NCT03323346 and NCT01777919) for the treatment of metastatic breast cancer and glioblastoma multiform.[17,18] However, oral administration of copper gluconate and disulfiram increases the risk of adverse side effects, including reduction of red blood cells (RBC), white blood cells (WBC), and platelets,[19−21] partially due to a lack of tumor specificity. Moreover, high uptake of copper interferes with iron transport and metabolism and results in anemia and impairment of the immune system.[22] Therefore, a strategy to specifically deliver copper ions to tumor tissues and reduce its in vivo cytotoxicity is desired.

Herein, we developed a carrier-and-deliver system to efficaciously deliver copper ions to tumor tissues. Human ferritin (Fn) protein, which self-assembles into a 24-mer nanocage and possesses the ability to encapsulate metal ions in its cavity,[23−25] was chosen as the carrier module.[26−35] Albumin, which is the most abundant component in serum and is highly uptaken in multiple tumors, was chosen as the deliverer module toward the tumor. The ferritin–albumin–Cu nanoparticle (FHC NP) was thus invented. This nanoparticle enhanced the selectivity toward cancer cells and significantly suppressed tumor proliferation in vivo in combination with disulfiram.

Results and Discussion

Synthesis and Characterization of FHC NPs

First, we chose N-hydroxysuccinimide- and maleimide-terminated polyethylene glycol (NHS-PEG-MAL) as the cross-linker for ferritin (Fn) and human serum albumin (HSA), which reacts with the sulfhydryl group on albumin and primary amine on ferritin. Size exclusion chromatography was adopted to separate the cross-linked product of ferritin and albumin (Fn–HSA). After separation, Fn–HSA was mixed with CuCl2 solution to enable the nanoformulation of copper ions into the cavity of ferritin. After removal of the excess amount of copper ions, Na2CO3 was introduced to the system to react with the copper ions and finally formed the Fn–HSA–Cu nanoparticles (FHC NPs) (Scheme ).

Scheme 1

Synthesis of FHC NPs

Ferritin and HSA were first covalently conjugated by an NHS-PEG-MAL cross-linker. Then, copper ions were encapsulated in the ferritin nanocage to form FHC NPs, which could release copper ions at acidic pH.

After synthesis, characterizations on the FHC NPs were performed. Analytical ultracentrifugation (AUC) revealed that the molecular weight of FHC NPs was 659 kDa, indicating that the ratio of ferritin to albumin was about 1:2 (Figure S1). Dynamic light scattering (DLS) analysis (Figure B) and transmission electron microscopy (TEM) imaging (Figure A) showed that the diameter of FHC NPs was about 12 nm, slightly bigger than ferritin itself (Figures S2 and S3), which was consistent with the attachment of two albumin molecules to ferritin. Then, inductively coupled plasma mass spectrometry (ICP-MS) was utilized to quantify the amount of copper ions encapsulated in FHC NPs (Figure C). Results indicated that each FHC NP encapsulated an average about 100 copper ions. The release curves of copper ions from the nanoparticle were measured in vitro at pH 7.5, 6, and 4.5, each simulating the microenvironment of the blood stream, tumor tissue, or lysosome in vivo (Figure D). Results showed that copper ions are prone to be released from the carrier proteins in an acidic environment, which ensured the safety profile during circulation.

Figure 1

Characterization of FHC NPs. (A) Transmission electron microscopy (TEM) image of FHC NPs. Scale bar: 100 nm. (B) Dynamic light scattering (DLS) analysis of FHC NPs. (C) Inductively coupled plasma mass spectrometry (ICP-MS) analysis of copper contents in FHC NPs. (D) Release curves of copper ions from FHC NPs at different pHs.

In Vitro Cytotoxicity of FHC NPs and DSF in Tumor Cells

FHC NPs were labeled with Cy5.5 and incubated with 4T1 cells to analyze whether FHC NPs could accumulate in cancer cells. Flow cytometry analysis showed that the nanoparticle accumulated in 4T1 cells in a time-dependent manner (Figure A). The fluorescence image also proved that Cy5.5-labeled FHC NPs were uptaken by 4T1 cells after being treated for 6 h (Figure S4). Besides, the concentration of copper ions in 4T1 cells was analyzed by ICP-MS (Figure B). As assumed, the concentration of copper ions within 4T1 cells significantly increased, indicating that FHC NPs delivered and released copper ions in 4T1 cells.

Figure 2

Combination of FHC NPs with DSF selectively killing cancer cells in vitro. (A) Flow cytometry analysis of uptake of FHC NPs by 4T1 cells incubated for different times. (B) Copper concentration in 4T1 cells incubated with FHC NPs for 0, 6, or 24 h as determined by ICP-AES. (C) Viability of multiple cancer cells (4T1, MDA-MB-231, HeLa) and HEK293T cells treated with 300 nM DSF and/or 10 nM FHC NPs. Viability of (D) 4T1 cells and (E) HEK293T cells treated with different concentrations of FHC NPs and DSF. (F) Flow cytometry analysis of apoptotic 4T1 cells treated with FHC NPs and DSF for 24 h using Annexin V-FITC and PI.

Next, we verified the in vitro anti-tumor activity of FHC NPs in combination with DSF. With diverse concentrations of DSF (100, 200, 300, and 600 nM), FHC NPs exhibited significant proliferation inhibition of 4T1 cells after 24 h treatment (Figure D). Compared with the results of 4T1 cells treated with DSF or FHC NPs alone (Figure S5), the combination of FHC NPs and DSF showed a better suppression effect. Moreover, lower inhibition of HEK293T cells was observed (Figure E), indicating potential cancer cell selectivity for FHC NPs and DSF combined therapy. Besides 4T1 cells, similar selectivity toward MDA-MB-231 and HeLa cells over HEK293T cells was also observed when treated with 300 nM DSF and/or 10 nM FHC NPs (Figure C). Meanwhile, no such selectivity was displayed when treated with combination of DSF and CuCl2 (Figure S6). These data demonstrated that the FHC nanoparticle invented here possessed obvious advantages in terms of cancer selectivity.

The mechanism of inhibition of combination of FHC NPs and DSF was determined. Annexin V-FITC and propidium (PI) double staining was performed in 4T1 cells treated with FHC NPs and DSF for 24 h. Flow cytometry results showed that the FHC NPs and DSF-induced proliferation inhibition of tumor cells resulted from the apoptosis pathway (Figure F).

Tumor Targeting of FHC NPs In Vivo

Next, we examined the tumor targeting ability of FHC NPs in vivo. FHC NPs were labeled with Cy5.5 and intravenously injected to 4T1-Luc tumor-bearing mice. Fluorescence images were captured on day 1, 2, 3, 4, 6, and 9 post injection. Good co-localization of Cy5.5 fluorescence and luciferase-enabled bioluminescence was observed, indicating that FHC NPs possessed good tumor targeting ability (Figure A). As comparison, ferritin encapsulating copper ions only showed minimal tumor accumulation (Figure A), which verified the necessity of the albumin module. Quantitative analysis showed that FHC NPs possessed a longer half-life in tumor tissues (Figure B). Major organs of the mice were collected 24 h post injection. Accordingly, FHC NPs accumulated five-fold more than ferritin in the tumor (Figure D). We further analyzed the concentration of copper ions in the tumor tissue by ICP-MS. As designed and assumed, copper concentration of the group treated with FHC NPs was much higher than that of groups treated with saline, ferritin, or albumin (Figure E).

Figure 3

Tumor targeting of FHC NPs. (A) Accumulation of FHC NPs in the 4T1 tumor at various times (bioluminescence imaging showing the location of the tumor). (B) Quantitative analysis of tumor accumulation of FHC NPs and ferritin. (C) Biodistribution of FHC NPs and ferritin in major organs. (D) Quantitative analysis of biodistribution of FHC NPs and ferritin in major organs. (E) Copper concentration in tumor tissues as measured by ICP-MS. Mice were treated with saline, ferritin, albumin, and/or FHC NPs, intravenously for 24 h. Error bars represented ±SD. n = 3.

In Vivo Anti-Tumor Activity of FHC NPs and DSF

A 4T1-Luc tumor-bearing mice xenograft was used to evaluate the anti-tumor activity of combination of DSF and FHC NPs. Mice were randomly divided into four groups when tumor volume reached about 50 mm3. The four groups of mice were treated with saline, DSF, FHC NPs, or DSF and FHC NPs, respectively. The dosage was 10 mg/kg intravenously for FHC NPs and 50 mg/kg orally for DSF (Figure A). Tumor proliferation was verified in two ways. In vivo luciferase-enabled bioluminescence imaging was taken (Figure B), and tumor volumes were calculated by measuring the length and width (Figure C). Significant tumor inhibition was observed in the group treated with both FHC NPs and DSF. Body weights showed no obvious differences among all four groups (Figure D), suggesting that the combination of DSF and FHC NPs had no obvious side toxicity. Major organs were collected after sacrificing of the mice and subjected to hematoxylin–eosin staining. Negligible organ damage was observed (Figure F), which further demonstrated the safety of FHC NPs in combination with DSF. Besides, consistent with the acquired strong anti-tumor activity of combination of FHC NPs and DSF, the survival of the group was greatly extended (Figure E). Because the FHC NPs will accumulate in the spleen of a mouse, so, we further analyzed the concentrations of superoxide dismutase (SOD) and malondialdehyde (MDA) in blood in order to prove its safety. Results indicated that the FHC NPs had no effect on mice (Figure S7). Moreover, the hematoxylin–eosin staining results of brains showed that DSF had no neurotoxicity (Figure S8).

Figure 4

FHC NPs and DSF showing good anti-tumor activity. (A) Schematic representation of the design of animal experiments. Tumor cells were implanted to the right flank of mice 7 days before drug administration. Drugs were administrated to mice at indicated days (1, 4, 7, 10, 13, and 16) with indicated dosage. (B) Representative bioluminescence images of four groups of 4T1-Luc tumor-bearing mice captured at day 1, 4, 7, 10, 13, and 16. (C) Tumor volume curves of four groups of mice. (D) Body weight curves of four groups of mice. (E) Survival of four groups of mice. (F) H&E-stained primary organs collected from four groups of mice after treatment. n = 8. Error bars represented ±SD.

All these data strongly suggested that combination of FHC NPs and DSF was safe and meanwhile effective as anti-tumor agents.

In summary, we developed a carrier-and-deliver system for copper(II) ions using ferritin as the copper ion carrier and albumin as the deliverer module to specifically target the tumor. The three components formed unique FHC NPs with a high content of copper ion loading. FHC NPs specifically delivered copper(II) ions into the tumor and then released them at acidic pH. When combined with DSF, FHC NPs showed good cytotoxicity selectivity toward cancer cells in vitro. Also, when applied to an in vivo tumor xenograft model, the combination of FHC NPs and DSF showed strong anti-tumor activity with a very good safety profile. The FHC NPs developed herein served as a good candidate of anti-tumor agents in combination with DSF, which has long been evaluated in clinical trials.

Materials and Methods

Materials

Ferritin was expressed in Escherichia coli (E. coli) cells and purified by gel filtration. Human serum albumin was purchased from Solarbio. NHS-PEG-MAL, disulfiram, CuCl2, Na2CO3, and all buffers were purchased from Aladdin. DMSO was purchased from Sigma-Aldrich. Cy5.5-NHS was purchased from Lumiprobe. d-Luciferin potassium salt was purchased from Sigma-Aldrich. All Balb/c nude mice were purchased from Charles River. Animal experiments were performed in accordance with the guidelines from the IACUC of Peking University Health Science Center, Beijing, China.

Synthesis of Ferritin–Albumin–Cu Nanoparticles (FHC NPs)

Five milligrams of N-hydroxysuccinimide- and maleimide-terminated polyethylene glycol (NHS-PEG-MAL) and 10 mg of human serum albumin (HSA) were both dissolved in 2 mL of PBS (pH 7.5) and mixed for 40 min at room temperature (RT). In order to remove excess linker molecules and change buffer to PBS buffer (pH 7.5), the reaction mixture was purified with Amicon centrifugal filters (Millipore) with 30 kD cutoff. Then, 4 mg of ferritin was added and reacted with modified HSA for 2 h at RT. The mixture was concentrated again with Amicon centrifugal filters and was purified by size exclusion chromatography with a Superdex 200.

Next, 5 mg of purified Fn–HSA was added to 10 mL of pH 5.5 sodium acetate buffer and incubated with 12 mM CuCl2 solution for 20 min at RT to encapsulate copper ions. Excess copper ions were removed by centrifuging in 10 kDa Millipore at 4000 rpm with dilution for three times with 10 mL of pH 5.5 sodium acetate buffer. Then, the mixture was added to 10 mL of pH 5.5 sodium acetate buffer and mixed with 12 mM Na2CO3. This mixture was heated to 60 °C for 20 min and cooled with ice. Then, FHC NPs were purified by centrifuging in 10 kDa Millipore at 4000 rpm for three times with 10 mL of PBS buffer.

Characterization of FHC NPs

Cu concentration was measured by inductively coupled plasma mass spectrometry (ICP-MS) with a PerkinElmer NexlON 350X. Transmission electron microscopy (TEM) was carried out on a HITACHI-H-7650 microscope. Dynamic light scattering (DLS) was carried out on a Malvern Zetasizer Nano ZS 90.

Cell Culture

Mouse breast cancer cell line (4T1), human cervical carcinoma cell line (HeLa), human breast cancer cell line (MDA-MB-231), human renal epithelial cell line (HEK293T), and 4T1 cells transfected with luciferase (4T1-Luc) were obtained from PerkinElmer. All cell culture-related reagents were purchased from Gibco. All cells were cultured in DMEM culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37 °C under 5% CO2.

Cytotoxicity Determination by MTT Assay

In vitro cytotoxicity of FHC NPs and DSF was evaluated in 4T1, HeLa, MDA-MB-231, and HEK293T cells by the MTT assay. All cells were seeded to 96-well cell culture plates at 1 × 104 cells per well and incubated overnight at 37 °C under 5% CO2. After removing the culture medium, different concentrations of FHC NPs or DSF were added to the fresh culture. The plates were incubated at 37 °C for further 24 h. MTT assay was performed after washing cells with PBS. After 4 h incubation at 37 °C, solution of each well was removed and 150 μL of DMSO was added. The plates were read at 490 nm absorbance within 10 min.

Flow Cytometry

4T1 cells were seeded to 24-well cell culture plates at a density of 5 × 104 cells per well and incubated overnight at 37 °C under 5% CO2. Then, the 4T1 cells were treated with 10 nM Cy5.5-labeled FHC NPs for 1, 3, 6, 9, and 24 h at 37 °C. After treatment, cells were digested and collected for flow cytometry analysis. The fluorescence of 4T1 cells was measured on a BD LSRFortessa.

Copper Concentration Measurement

4T1 cells were seeded to 6-well cell culture at a density of 2 × 105 cells per well and incubated with 10 nM FHC NPs for 0, 6, and 24 h. After removing the culture medium and washing with PBS buffer for three times, 4T1 cells were digested and collected in 1 mL of PBS buffer. Then, the cell count was calculated, and copper concentration was measured by ICP-AES.

Animal and Tumor Model

Balb/c nude female mice of 4–6 weeks old, with the average weight of 20 g, were provided by Peking University Health Science Center. Mice were kept in an SPF animal house and were fed with a standard laboratory diet and tap water ad libitum. Mice were subcutaneously injected with 5 × 105 4T1-Luc cells in 100 μL of PBS to establish a subcutaneous tumor model.

Animal Therapeutic Treatment

Mice were injected with 4T1-Luc cells for a week, and the tumors reached to about 50 mm3. Balb/c nude mice were divided into four groups: control group, Fn–HSA–Cu group, DSF group, and Fn–HSA–Cu+DSF group, with eight mice in each group. These four groups of mice were respectively treated with saline, 10 mg/kg FHC NPs by intravenous injection every 3 days, 50 mg/kg DSF orally every 3 days, and combination of FHC NPs and DSF. FHC NPs, DSF, and saline were injected for six times in the treatment. Tumor size and body weight were measured every 3 days during the treatment. Tumor volume was calculated according to the formula (a × b2)/2, where a and b are the long and short diameter of the tumor.

In Vivo Fluorescence Imaging

When the tumor volume reached about 100 mm3, Cy5.5-labeled FHC NPs were intravenously injected to tumor-bearing mice. Then, the fluorescence image was taken by PerkinElmer IVIS Lumina XRMS Series III optical preclinical in vivo imaging systems. Then, primary organs of mice were harvested. Images of primary organs were taken, and fluorescence intensity was also measured.

Histology Analysis

After treatment, mice were sacrificed for histology analysis. Also, major organs were recovered from the necropsy, fixed with 10% neutral buffered formalin. Then, the organs were sectioned at about 5 μm and deparaffinized with xylene. The slides were subjected to rehydration with different concentrations of alcohol from 100% to 70%. After washing in water, the slides were stained with hematoxylin and eosin (H&E). After staining, sections were dehydrated through increasing the concentration of ethanol and xylene.