Enhanced Melanoma‐Targeted Therapy by “Fru‐Blocked” Phenyboronic Acid‐Modified Multiphase Antimetastatic Micellar Nanoparticles

Abstract Metastasis remains the main driver of mortality in patients suffering from cancer because of the refractoriness resulting from the multi‐phase metastatic cascade. Herein, a multifunctional self‐delivering PBA‐LMWH‐TOS nanoparticle (PLT NP) is established that acts as both nanocarrier and anti‐metastatic agent with effects on most hematogenous metastases of cancers. The hydrophilic segment (low molecular weight heparin, LMWH) inhibits the interactions between tumor cells and platelets. The hydrophobic segment (d‐α‐tocopheryl succinate, TOS) could inhibit the expression of matrix metalloproteinase‐9 (MMP‐9) in B16F10 cells which is first reported in this article. Surprisingly, even the blank NPs showed excellent anti‐metastatic capacity in three mouse models by acting on different phases of the metastatic cascade. Moreover, the overexpression of sialic acid (SA) residues on tumor cells is implicated in the malignant and metastatic phenotypes of cancers. Thus, these 3‐aminophenylboronic acid (PBA)‐modified doxorubicin (DOX)‐loaded NPs offer an efficient approach for the treatment of both solid melanomas and metastases. Furthermore, a simple pH‐sensitive “Fructose (Fru)‐blocking” coping strategy is established to reduce the NP distribution in normal tissues and distinctly increases the accumulation in melanoma tumors. These micellar NPs consisting of biocompatible materials offer a promising approach for the clinical therapy of highly invasive solid tumors and metastases.

mice for predetermined periods (1,2,4, and 8 h) at 75 rpm and 37 °C. Then, each sample was centrifuged at 1500 rpm for 5 min at each time point. The absorbance of the supernatant was measured at 540 nm using a Varioskan Flash multimode reader (Thermo, USA). PBS and the RBC suspension treated with 0.2% Triton X-100 were used as negative and positive control groups, respectively. The hemolysis percentage was calculated according to the following formula: Hemolysis (%) = (A sample -A PBS / (A Triton-100 -A PBS )) × 100%. 7. In Vitro Cytotoxicity Analysis. The cytotoxicities of different formulations were evaluated in B16F10 cells by the MTT assay. Briefly, B16F10 cells were seeded in 96-well plates (3 × 10 3 cells/well) and incubated for 12 h at 37 °C and in a 5% CO 2 atmosphere. The culture medium was removed, and then, free DOX, LT NPs, PLT NPs, LT/DOX NPs or PLT/DOX NPs were added at a series of concentrations and incubated with the cells for an additional 48 h. Then, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and the plates were incubated for 4 h at 37 °C and in a 5% CO 2 atmosphere. Finally, the culture medium was removed, and the residue was dissolved in 150 μL of DMSO per well. The absorbance of each group was measured by a Varioskan Flash multimode reader (Thermo, USA) at 570 nm. The cell viability (%) was calculated by the following formula: Cell viability (%) = A test -A DMSO / A control -A DMSO × 100.

Cellular Uptake and Intracellular Delivery in B16F10 Cells.
To investigate the cellular internalization of different micelles, uptake assays using SA-overexpressing B16F10 cells and HepG2 cells and SA-underexpressing COS-7 cells as the recipient cells were conducted. We conducted the cell uptake assays in pH 7.4 and pH 6.5 serum-free media. Briefly, B16F10 cells were seeded in 6-well plates (3 × 10 5 per well) and grown overnight before incubation with DiDloaded NPs. After incubation with LT/DiD NPs, PLT/DiD NPs, PLT-Fru/DiD NPs or PLT-SA/DiD NPs in pH 6.5 or pH 7.4 medium for 2 h, the cells were harvested and washed with PBS. After centrifugation (3000 rpm for 3 min), the cells were resuspended in PBS (300 μL), and the mean fluorescence of DiD in individual cells was quantified by a flow cytometer (FL4 675 nm, Beckman Coulter, Inc., USA, Cytomics FC 500).
We also used CLSM to detect the fluorescence signals of different cells treated with different preparations. Three types of cells were cultured on coverslips in 6-well plates (2× 10 5 per well) as described above. To verify the interaction between PBA and SA residues, free SA (500 μM) was pre-incubated with micelle suspensions for 2 h, and the mixture was added to the cells. Meanwhile, PBA (500 μM) was pre-incubated with cells for 1 h prior to the addition of the DOX-loaded NP suspensions to the cells, respectively. The cells were incubated for 2 h, washed with PBS (pH 7.4) for three times, and fixed in 4% (m/v) paraformaldehyde solution for 10 min. Then, the cells were dried under an air stream and stained with 0.5% 4′,6-diamidino-2-phenylindole (DAPI) for 4 min.
To track intracellular NP delivery, B16F10 cells and HepG2 cells were stained with LysoTracker Red DND-99 (E x = 577 nm, E m = 590 nm) 30 min before the end of the cellular uptake assay and then photographed by CLSM using the staining and mounting method described above.

In Vitro Inhibitory Effect on Cell Migration and Invasion. The inhibitory effects of LT/DOX
NPs and PLT/DOX NPs on the migration and invasion of B16F10 cells were evaluated by woundhealing and Transwell assays. For the wound-healing assay, cells were seeded into 6-well plates and allowed to grow to 90-95% confluency. Scratch wounds were generated with a 200-μL pipette tip, and then, the cells were washed twice with PBS to form cell-free wounds. Then, the cells were incubated with blank medium (as a control), free DOX, LT NPs, PLT NPs, LT/DOX NPs or PLT/DOX NPs at predetermined concentrations. After incubation with different formulations, images were obtained at 0 h and 24 h with an inverted microscope. The wound-healing rate was calculated according to the previously described method. For the cell invasion assay, 1 × 10 5 cells were added to the upper chambers of the Transwells (24-well insert; pore size, 8 μm; Corning, USA) coated with Matrigel (BD Biosciences). In this assay, 100 μL of serum-free medium with PBS, LMWH, free DOX, blank LT NPs, PLT NPs (40 μg mL -1 ), LT/DOX NPs or PLT/DOX NPs (at an equivalent dose of 0.5 μg mL -1 DOX) was loaded into the upper chambers, while the lower chambers were filled with 600 μL of culture medium containing 20% FBS as a chemoattractant.
After 48 h of incubation, the migrated or invaded cells were fixed in 4% (m/v) paraformaldehyde solution for 10 min and stained with crystal violet. Finally, the bottoms of the chambers were viewed under an inverted microscope in five predetermined fields. Then, the crystal violet was eluted by a 33% acetic acid solution, and the absorbance was measured at 570 nm using a Varioskan Flash multimode reader.

11.
In vitro MMP-9 Expression Detection. In brief, B16F10 cells were seeded in 6-well plates (1.5 × 10 5 per well) individually and grown overnight before incubating with NPs. Afterwards cells were treated with PBS, LMWH, LT NPs, PLT NPs, LT/DOX NPs or PLT/DOX NPs (at an equivalent dose of 100 μg mL -1 PLT NPs) for 30 h. Then, the culture medium of each group was analyzed using mouse MMP-9 ELISA kit. Moreover, MMP-9 in cells was detected by Western blot assay.
Cells in each well were harvested and mixed with 50 μL cell lysate buffer (Beyotime, China).
Samples were centrifuged at 4°C and 13,000 rpm for 15 min, and the supernatant was collected as Western blot assay samples. Each sample was then separated by 6% SDS-PAGE, and followed by transferring to polyvinylidene difluoride membranes. Finally, samples were incubated with antibody.
HRP-labeled mouse anti-goat secondary antibody was added and measured using a Bio-Rad ChemiDoc MP System.

Adherence of Platelets to Tumor Cells In Vitro and Implantation in Lung Tissue of B16F10 Cells
In Vivo. Platelet-rich plasma (PRP) was obtained from male C57BL/6 mice and washed with PBS and then labeled with 5 μM calcein AM for 20 min using a previously reported method. [2] Meanwhile, the B16F10 cells were seeded in 6-well plates and grown to approximately 80% confluency. Then, the medium was changed, and a total of 1 × 10 6 fluorescein-labeled platelets were added to each well. After treatment with PBS (as a control), free LMWH, LT NPs or PLT NPs, the cells were washed three times with PBS. Finally, the cells were lysed with DMSO, and the fluorescence signal was detected by a fluorospectro-photometer at E x = 490 nm and E m = 515 nm.
To investigate the efficacy of NPs against CTC implantation, C57BL/6 mice were pretreated with PBS, free LMWH, LT NPs or PLT NPs (60 mg kg -1 ) 30 min in advance. Meanwhile, the B16F10 cells were stained with carboxyfluorescein succinimidyl ester (CFSE) (20 μM, 37 °C, 15 min). Then, 2 × 10 5 CFSE-labeled B16F10 cells were injected into mice via the tail vein. The lungs were harvested 30 min after cell injection and frozen section analysis was performed. Cell nuclei were stained with DAPI. The distribution of tumor cells in the lungs was observed by CLSM (the fluorescence signal of CFSE was detected at E x = 496 nm and E m = 516 nm).

In vitro E-cadherin and N-cadherin Expression Detection.
Platelet-rich plasma (PRP) was obtained from male C57BL/6 mice. B16F10 cells were seeded in 6-well plates (1.5 × 10 5 per well) individually and grown overnight before incubating with NPs and platelets. After treated with PBS-, PBS+, LMWH+, LT NPs+, PLT NPs+, PLT/DOX NPs+ (at an equivalent dose of 50 μg mL -1 PLT NPs; + means co-incubation with platelets at 30 minutes after administration) for 30 h, protein samples were prepared and analyzed according to the above method of Western blot assay.
14. In Vivo Tumor-targeting Assay and Metastasis-targeting assay. The B16F10 solid tumor model was generated by inoculating 1 × 10 6 B16F10 cells on the right sides of the backs of male C57BL/6 mice. In vivo imaging and biodistribution experiments were performed on the tumorbearing C57BL/6 male mice on the 13 th day after the injection of B16F10 cells, by which time the tumors had grown to an approximate diameter of 1 cm. The NPs were labeled with DiD via physical entrapment using the method described above for DOX entrapment. Briefly, the LT conjugate or PLT conjugate was added to DiD dissolved in dichloromethane and stirred at 37 °C for 3 h. Then, the mixture was added to five volumes of PBS, sonicated and purified. The tumor-bearing C57BL/6 mice were injected with PBS, LT/DiD NPs, PLT/DiD NPs or PLT-Fru/DiD NPs (NPs : Fru = 1 : 1, w/w, 20 μg DiD per mouse) through the tail vein. Then, the tumor regions were imaged using an in vivo imaging system (IVIS Lumina Series Ⅲ, PerkinElmer, USA) at predetermined time points (1,4,8, and 24 h). The mice were sacrificed at 24 h, and the tumors as well as major organs, including the heart, liver, spleen, lungs, and kidneys, were collected and subjected to ex vivo imaging. Then, the distribution of NPs in the tumor site was observed by CLSM of frozen sections.

Preliminary Safety Evaluation.
To evaluate the preliminary in vivo toxicities of different preparations, healthy male C57BL/6 mice (approximately 5 weeks old, 18-22 g, specific pathogen free (SPF)) were treated with PBS, free DOX, LMWH, LT NPs, PLT NPs and PLT/DOX NPs (at an equivalent dose of 3 mg kg -1 DOX) individually every 2 days for a total of 5 doses. Then, 24 h after the final administration, whole blood was obtained in EDTAK 2 anticoagulative tubes for routine blood examination (Mindray, BC-2800Vet, China), and serum was obtained for the biochemical analysis (TECOM, TC6010L, China).

In Vivo Efficacy of NPs against Lung Metastasis.
Three tumor metastasis mouse models were established to investigate the anti-metastatic ability of blank NPs by injecting three tumor cell lines into mice via the tail vein respectively. Briefly, mice were intravenously injected with PBS, free LMWH, LT NPs or PLT NPs (60 mg kg -1 ) to inhibit the adhesion of platelets in advance, and then, 2 × 10 5 B16F10 cells, 2 × 10 5 CT26 cells or 1 × 10 5 4T1 cells in 100 μL of PBS were injected into the mice via the tail vein after 30 min. The once-daily administration continued for an additional two days. The mice were sacrificed on the 20 th day, and the lungs were collected and imaged. Then, the lung tissues were subjected to H&E staining.
We also tested another dosage regimen to investigate the anti-metastatic effect of DOX-loaded NPs. A total of 2 × 10 5 B16F10 cells in 100 μL of PBS were injected into the C57BL/6 mice via the tail vein to generate a lung metastatic mouse model of melanoma. On the 4 th day, the mice were randomly divided into 6 groups and administered PBS, free DOX, free LMWH, blank LT NPs (60 mg kg -1 ), LT/DOX NPs or PLT/DOX NPs (at an equivalent dose of 2.5 mg kg -1 DOX) every 2 days for a total of 4 times. At day 19, the mice were sacrificed, and the lungs were collected and imaged.
Then, the lung tissues were subjected to H&E staining.

Statistical Analysis.
Data were expressed as the means ± standard deviations (SD). Statistical analysis was performed by one-way ANOVA or Student's t-test. Significant differences between groups are indicated by *p＜0.05, **p＜0.01 and ***p＜0.001. Tables   Table S1. Characterization of NPs conjugated by different amount of LMWH, TOS and PBA. (data represent mean data ± SD, n=3 ).