A de novo dual-targeting supramolecular self-assembly peptide against pulmonary metastasis of melanoma

Despite recent advances in treatment, overall survival rates for metastatic melanoma, especially those that invade the lungs, continue to be low, with 5-year survival rates of only 3% to 5%. It was recently discovered that Wnt/β-catenin signaling pathways and MAPK/ERK signaling pathways are involved in melanoma metastasis. Methods: Herein, a bifunctional supramolecular peptide termed HBBplus@CA was constructed by a self-assembling RGD-modified MAPK/ERK peptide inhibitor (HBBplus) and a small molecule catenin inhibitor (carnosic acid (CA)). Results: Expectedly, the HBBplus@CA could internalize melanoma cells, accumulate in the tumor-bearing lung, and be biosafe. As designed, HBBplus@CA simultaneously suppressed both Wnt/β-catenin and MAPK/ERK signaling pathways and suppressed melanoma cell proliferation, migration, and invasion in more action than CA or HBBplus monotherapy. More importantly, HBBplus@CA demonstrated potent inhibition of lung metastasis in mice bearing metastatic melanoma of B16F10 and significantly prolonged their survival. Conclusion: In summary, a supramolecular peptide-based strategy was not only developed to suppress pulmonary metastasis of melanoma, but it also renewed efforts to identify cocktail drugs that act on intracellular targets in various human diseases, including cancer.


Materials and methods
Bioinformatics data acquisition and processing GSE19234 series was obtained from the GEO dataset for further correlation analysis and survival analysis, with the GPL570 platform being used to annotate the gene symbol. The duplicated genes were replaced with mean values, and the raw expression matrix was log-transformed. Survival analysis was performed via R software, "survminer" and "survival" packages. The "surv_cutpoint" function determined the cut-off value of MAPK15 and CTNNB1 and therefore transformed them into categorical variables. Convert it to the high and low expression group to draw the KM curve.
Use the gg-scatter function in the ggpubr package to visualize the correlation between MAPK15 and CTNNB1.

Synthesis of HBB plus and HBB plus @CA
HBB plus peptides were synthesized on appropriate resins on a CS bio 336X automated peptide synthesizer using the optimized HBTU activation/DIEA in situ neutralization protocol developed by an HBTU/HOBt protocol for Fmoc-chemistry SPPS. After cleavage and deprotection in a reagent cocktail containing 88% TFA, 5% phenol, 5% H2O, and 2% TIPS, crude products were precipitated with cold ether and purified to homogeneity by preparative C18 reversed-phase HPLC. The molecular masses were ascertained by electrospray ionization mass spectrometry (ESI-MS).
The HBB plus were dispersed in distilled water at a concentration of 1 mg/mL; CA (Energy Chemical, China) was dissolved in DMSO at a concentration of 1mg/10 uL and then dropwise added into the HBB plus solution at the mass rate of 4:1, 2:1, 1:1, 0.5:1, 0.25:1, and 30 min ultrasonic concussion.

Synthesis of HBB and RGDSP-X
HBB (ENFRLLGNVLVCVLA) and RGDSP-X (ANVLNECVPVGRLLL, a control peptide X of a scrambled amino acid sequence designated scrambled-HBB, were chemically synthesized.) sources were obtained from QYAOBIO (Shanghai) Ltd.

Characterization of HBB plus @CA
We combined the GROMACS 2018.3 software package with the MARTINI coarse-grained model (version 2.2) to conduct dynamics simulations on a system composed of 24 peptide molecules and 100 CA molecules in an aqueous solution for 5000 ns.
The morphology structure was observed on Lorenz transmission electron microscopy (TEM), performed on a Talos F200X. EDS elemental mappings were acquired using an FEI Talos  B16F10 and RAW 264.7 cells were maintained in DMEM medium, Jurkat was cultured in RPMI-1640 medium, and the above mediums were supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. All cells were maintained at 37°C in an atmosphere of 5% CO2.

Cell viability analysis
Melanoma cells B16F10 were inoculated uniformly in 96 empty plates according to 2000 cells.
After 24 h, different gradient concentrations of HBB plus @CA or CA, HBB, or RGDSP-X were intervened at 37°C in an atmosphere of 5% CO2 for 72 h. The in vitro cell viability was measured using a standard Alamar blue (Thermo Fisher Scientific, China) assay in the B16F10 cells.

Transwell assays
B16F10 cells were planted in 24-well plates at a density of 1 × 10 5 cells per well. Cells were put into the upper chamber with or without a Matrigel-coated membrane (1:7 dilution, Corning).
B16F10 cells were planted in 24-well plates at a density of 1 × 10 5 cells per well. After incubation of CA, HBB plus , HBB plus @CA, RGDSP-X with a concentration of 30 μM into the lower chamber containing 15% FBS medium for 24h -48h. Cells that migrated or invaded across the membrane were fixed with 4% PFA for 20 min and then stained with 0.1% crystal violet solution for 20 min.
The chamber was repeatedly cleaned with distilled water and captured under a light microscope (Leica).

Scratch wound healing assay
B16F10 cells at 5 × 10 5 cells/well were inoculated into the 6-well plate at 37 ℃ with 5% CO2 for 24 h. After the cells formed a confluent monolayer, a scratch was created in the center of the monolayer with a pipette tip (100 μL). The monolayer membrane was washed with PBS three times, and then 60 μM of CA， HBB plus , RGDSP-X, or HBB plus @CA containing 1% fetal bovine serum was added to each well. The images of cells invading the scratch were captured at indicated time points (0 h, 24 h) with a Leica microscope (Longbase, China), and the pictures were analyzed independently with Image J. All samples were assayed in triplicate.

Cell apoptosis was evaluated by flow cytometric analysis using the FITC Annexin V Apoptosis
Detection Kit (BD Falco, China). Briefly, cells were treated with HBB plus ， HBB plus @CA, and CA for 24 h at a concentration of 40 μM. Cells were harvested, washed twice with cold PBS, and resuspended in 1 × binding buffer at a concentration of 1 × 10 6 cells/mL Cells (1 × 10 5 cells/100 µL) were transferred to a centrifugal tube, followed by adding 5 µL of FITC Annexin V and 5 µL of PI.
After gentle vortexing and a 15 min incubation in the dark at room temperature, 400 µL of 1× binding buffer was added to the tube, and cells were analyzed by FACS.

Cellular uptake analysis
3 × 10 4 B16F10 cells/well, 3 × 10 5 RAW264.7 cells/well, and 3 × 10 5 Jurkat cells/well were seeded into Nunc Glass Bottom Dish at 37 ℃ with 5% CO2 for 24 h. FITC-labeled HBB, HBB plus , and HBB plus CA were incubated with cells for 6 h at a concentration of 30 μM, respectively. After the medium was removed, PBS was used to wash it three times, and 4% formaldehyde was used to fix it for 10 min. 0.2% Triton X-100 destroyed the cell membrane for 10 min. DAPI (Sigma-Aldrich, USA) was used to label the cell nuclei for 5 min. Finally, a super-resolution confocal microscope was used to observe the cell uptake situation.

Transcriptome analyses
B16F10 cells were seeded in 6-well plates and left to cultivate overnight. Then cells were treated with HBB plus and HBB plus @CA, respectively, at a concentration of 60 μM for 24 h. And RNA was isolated using the TRIzol reagent (Ambion, USA). RNA sequencing libraries were constructed using the NEBNext® Ultra RNA Library Prep Kit for Illumina® (NEB England BioLabs). Fragmented and randomly primed 2 × 150 bp paired-end libraries were sequenced using Illumina HiSeq X Ten.
Heat maps and Gene Expression Enrichment Analysis were generated using the Qlucore Omics Explorer 3.2. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA) software.

Mouse study
All mice were purchased from the Laboratory Animal Center of Xi'an Jiaotong University. Animals were housed under standard specific pathogen-free conditions with standard chow and typical light/dark cycles. All experimental procedures involving animals were conducted in accordance with Institution Guidelines and were approved by the Laboratory Animal Center of Xi'an Jiaotong University (approval number: 2021-1735).

Quantification of HBB plus @CA pharmacokinetics
HBB plus @CA labeled with Cy5-SE (MedChemExpress, USA) was injected intravenously at 200 μL (1 mg/mL) into healthy female C57BL/6 mice. The main organs were obtained at 0 h, 0.5 h,1 h, 2 h, 4 h, 6 h, 12 h, 24 h, and 48 h after injection. The mice were euthanized, and blood plasma extracted from the mice was detected and quantified by a microplate reader (excitation: 649 nm, emission: 670 nm); the fluorescence absorbance was analyzed using Origin. All samples were assayed in triplicate.

In vivo Fluorescence Imaging
HBB plus @CA labeled with Cy5-SE was injected intravenously at 200 μL (1 mg/mL) into C57BL/6 mice with tumor-bearing pulmonary metastatic melanoma. The main organs were obtained at 0h, 2 h, 4 h, 6 h, 12 h, and 24 h after injection and were used for detection in the CRI Maestro imaging system in vivo. HBB plus and HBB labeled with Cy5-SE fluorescent dye were injected intravenously at 200 μL (1 mg/mL) into C57BL/6 mice with tumor-bearing pulmonary metastatic melanoma. The main organs were obtained at 12 h and 24 h after injection and were used for detection in the CRI Maestro imaging system in vivo. All samples were assayed in triplicate.

Construct a lung metastasis model of melanoma
The lung metastasis model of melanoma was constructed as follows: B16F10 cells (5 × 10 5 cells per 100 µL) were transplanted into healthy C57BL/6 mice by intravenous injection at 5-6 weeks of age.
After 3 days, the mice were randomly divided into the PBS control group, CA group (4 mg/kg), HBB plus group (4 mg/kg), HBB plus @CA group (4 mg/kg), HBB plus + CA group (4 mg/kg) tail-vein injected, every other day, summed six cycles. The experiment was terminated, and the mice were euthanized. For histological examination, the tumor, liver, kidney, heart, spleen, and lung tissues were fixed with formaldehyde, dehydrated, sliced into 4 μm sections, and subjected to H&E or immunohistochemical staining.

H&E and immunohistochemistry (IHC) staining
Tumor-bearing lungs and primary organ tissues were dissected and immersed in formalin solution.
Then they were paraffin-embedded, sectioned with a 4 μm thick. Nextly, the sections were stained To evaluate immunostaining intensity (I), we used a numeric score ranging from 0 to 3, reflecting