Targeted Therapy against Metastatic Melanoma Based on Self‐Assembled Metal‐Phenolic Nanocomplexes Comprised of Green Tea Catechin

Abstract The targeted therapy of metastatic melanoma is an important yet challenging goal that has received only limited attention to date. Herein, green tea polyphenols, (–)‐epigallocatechin‐3‐gallate (EGCG), and lanthanide metal ions (Sm3+) are used as building blocks to engineer self‐assembled SmIII‐EGCG nanocomplexes with synergistically enhanced tumor inhibitory properties. These nanocomplexes have negligible systemic toxic effects on healthy cells but cause a significant reduction in the viability of melanoma cells by efficiently regulating their metabolic pathways. Moreover, the wound‐induced migration of melanoma cells can be efficiently inhibited by SmIII‐EGCG, which is a key criterion for metastatic melanoma therapy. In a mouse melanoma tumor model, SmIII‐EGCG is directly compared with a clinical anticancer drug, 5‐fluorouracil and shows remarkable tumor inhibition. Moreover, the targeted therapy of SmIII‐EGCG is shown to prevent metastatic lung melanoma from spreading to main organs with no adverse side effects on the body weight or organs. These in vivo results demonstrate significant advantages of SmIII‐EGCG over its clinical counterpart. The results suggest that these green tea‐based, self‐assembled nanocomplexes possess all of the key traits of a clinically promising candidate to address the challenges associated with the treatment of advanced stage metastatic melanoma.


Section 1. Preparation of Complexes
In optimal conditions, Sm(III) (Sm(NO 3 ) 3 .6H 2 O) and EGCG in a molar proportion (Sm: EGCG) of 4: 1 were dissolved in EtOH (40 mL) to obtain final concentrations of 4 mmol/L and 1 mmol/L, and the mixture was stirred until the solid was dissolved.
The mixture was adjusted by sodium carbonate solution to pH 7.0 and reacted at 37 °C for 24 h. Then, the complex in the mixture was harvested by centrifugation and washed several times with ethanol, and finally the Sm III -EGCG complex with brown color was obtained after vacuum drying. The as prepared Sm III -EGCG was identified by 1 H NMR spectra (Bruker Avance 500 MHz NMR Spectrometer, USA) analysis. , HUVEC and HLECs cell lines were cultured in DMEM, the B16F10 cell line was maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS), penicillin (100 U/mL) and streptomycin (100 mg/mL). Cells were sustained at 37 °C in a humidified atmosphere with 5% CO 2 .

Section 3. Cell Proliferation Assay
Cell proliferation was analyzed using Cell Counting Assay Kit-8 (CCK-8) (Dojindo Molecular Technologies, Japan). 100 L of cell suspension (5×10 3 cells/well) was dispensed into a 96-well plate and pre-incubated overnight, then exposed to various concentrations of Sm III -EGCG for 24 h. Ten microliters of CCK-8 solution was added to each well. After incubation for 1 h, the absorbance at 450 nm was measured using microplate reader. Experiments were performed at least three times with representative data presented.

Section 4. Cell Apoptosis Detection
Cell apoptosis analysis was performed using Annexin V/PI apopotosis assay kit (KeyGEN Biotech, China). Various concentrations of Sm III -EGCG for 24 h, melanoma cells were collected by centrifugation and resuspended in 500 l of binding buffer. Then, 5 l of Annexin V-FITC was added to the re-suspended cells. After incubation for 5 min on ice in the dark, 1 g of PI was added to the cell suspension.
Apoptotic and necrotic cells were quantified using a FACScan flow cytometer and the Cell Quest pro software (Beckton-Dickinson).

Section 5. Western Blotting
B16F10 cells in 6-well plate were collected and washed with PBS and then lysed with 60 L lysis buffer (Beyotime) for 15 min at 4 °C. The cell supernatant was collected by centrifugation at 12,000 g centrifuged for 10min at 4 °C. Protein concentration was determined using the BCA protein assay kit. The equal amount (30 g) of extract protein was loaded, separated by 15% SDS-PAGE, and transferred to a polyvinylidene difluoride membrane (PVDF, Carlo Erba reagents, Milan, Italy). The transferred membranes were blocked with 5% skim milk in Tris-buffered saline-Tween 20 (TBST: 0.1% Tween 20, 100 mM NaCl and 10 mM Tris-HCl, (pH 7.6)) for 2 h at room temperature. After blocked with 5% milk in the TBST buffer for 1 h, membranes were subsequently probed with appropriate primary antibodies overnight at 4 °C. The membranes were then incubated with HRP-conjugated secondary antibodies and analyzed by using ChemiDoc™-XRS imaging system (Bio-Rad).
Quantifications of relative protein expression were carried out were performed with the Image Lab 3.0 software (Bio-Rad).

Section 6. Transwell Migration Assay
Transwell migration assay was performed to assess the effect of Sm III -EGCG on melanoma cell migration as previously described (24). Wounds were created by scratching the cells using a plastic pipette tip, and any loose cellular debris or detached cells were removed by PBS wash. The medium was replaced with fresh medium 24 h. The wound gap was observed and cells were photographed using phase-contrast microscopy. The gaps of the wounds were observed with phase contrast microscopy and digitally photographed. Each experiment was performed in triplicate.
Seven days following implantation, the mice with tumor sizes > 50 mm 3 were selected and randomly divided into three groups (n = 10 per group), termed the model, Sm III -EGCG and 5-FU groups. These groups were treated with saline, 150 mg/kg Sm III -EGCG and 30 mg/kg 5-FU respectively, the concentrations of which were determined in a preliminary study. Equal volumes of the drugs and vehicle were administered orally to the mice every day for 3 weeks. The body weight and tumor size were measured three times weekly and the tumor volumes were calculated according to the following formula: width 2 × length × 0.5. All experimental animals were treated according to the protocols approved by the Bioethics Committee of West China Center of Medical Sciences, Sichuan University.

Section 8. Experimental Melanoma Metastasis Model
40 male C57Bl/6J mice injected intravenously via the tail vein with 1 × 10 6 B16F10 cells to produce experimental lung metastasis after 1 week of acclimatization.
Intraperitoneal is herein preferred as its enables a maintained bioavailability and monodispersity compared to intravenous injection. Although, ultimately intravenous injection may yield to a final clinical use method, intraperitoneal injections are still used in this work because of the center on fundamental study and clinical fact that many antitumor drugs, including BARF inhibitors, are administered intragastrically.
The mice were randomly divided into four groups (n=10): vehicle group, model group, Sm III -EGCG group and 5-FU (positive vehicle) group. Animals in each group were intraperitoneally injected with Sm III -EGCG 150 mg/kg, 5-FU 30 mg/kg or saline as vehicle once daily. All animals were scarified on the 24th day, followed by a comprehensive visual examination of all organs. Black dots on lung surface were counted and confirmed as melanoma metastases. Mouse tissue samples (heart, liver, spleen and kidney) were fixed in 4% paraformaldehyde and embedded in paraffin.
The tissue samples were processed into sections of 4 m thick sections and the slides were stained with hematoxylin and eosin (H&E) according to standard protocols.

Section 9. Statistical Analysis
Data were expressed as mean ± standard deviation (SD). Data were analyzed with one-way ANOVA using SPSS 16.0. Differences were considered statistically significant when P< 0.05.