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.

Keywords: Fru‐blocking; LMWH; antimetastatic nanoparticles; d‐α‐tocopheryl succinate (TOS); metastatic cascade; self‐delivering nanoparticles.

Scheme 1

A) Schematic illustration of the preparation of PLT/ DOX NPs and the “Fru‐blocking” strategy. B,C) Schematic illustration of PLT/DOX NPs targeting a melanoma solid tumor and inhibiting lung metastasis.

Figure 1

Characterization of NPs and investigation of the combination between Fru, SA, and PBA. A) Dynamic light scattering (DLS) size distribution and transmission electron microscopy (TEM) image of PLT NPs. B) Hemolysis ratio (%) and C) photo images of red blood cells cultured with PLT NPs at various concentrations. A hemolysis ratio less than 5% was regarded as no obvious hemolysis. (Means ± SD, n = 3). D) Cumulative DOX release of LT/DOX NPs and PLT/DOX NPs in phosphate buffered saline (PBS) at 37 °C. ((a) Free DOX in pH 7.4 PBS, (b) PLT/DOX NPs in pH 5.0 PBS, (c) PLT/DOX NPs in pH 6.5 PBS, (d) PLT/DOX NPs in pH 7.4 PBS, (e) LT/DOX NPs in pH 7.4 PBS) (means ± SD, n = 3). E) The serum stability of NPs during 24 h incubation with 50% FBS at 37 °C (means ± SD, n = 3). F) The affinity of free PBA to Fru and G) to free SA in pH 7.4 and pH 6.5 PBS and H) the stability testing of PBA‐Fru conjugate at different time points. The concentration of Fru was 20 × 10⁻³ m and the concentration of PBA was 60 × 10⁻⁶ m.

Figure 2

Cellular uptake of B16F10 cells after incubation with different preparations. A) Confocal laser scanning microscopy (CLSM) images of B16F10 cells after incubation with free DOX, LT/DOX NPs, PLT/DOX NPs, PLT‐BA/DOX NPs, or PLT‐SA/DOX NPs for 2 h. The competitive inhibition assay was performed by preincubating with free PBA or free SA for 1 h and observing the DOX channel (red) and DAPI‐stained nucleus channel (blue). The scale bar represents 100 µm. B) The representative histograms of competitive uptake assay analyzed by flow cytometry (FACS). C) Quantitative cellular uptake of B16F10 cells after incubation with LT/DiD NPs, PLT/DiD NPs, PLT‐Fru/DiD NPs, and PLT‐SA/DiD NPs for 2 h at pH 6.5 and pH 7.4, respectively (means ± SD, n = 3, *** indicates p < 0.001). D) CLSM images of B16F10 cells after incubation with PLT/DOX NPs for 2 h, showing LysoTracker‐stained lysosome channel (green), DOX channel (red), and DAPI‐stained nucleus channel (blue). The arrow in the left indicated the signal of DOX in nucleus and the arrow in the right indicated the colocalization of DOX and lysosome. The scale bar represents 10 µm.

Figure 3

Inhibitory effect of NPs on B16F10 cell migration and invasion in vitro and anti‐implantation effect in vivo. A) Images and D) quantitative analysis of invaded B16F10 cells after separate incubation with PBS, LMWH, LT NPs, PLT NPs, LT/DOX NPs, and PLT/DOX NPs for 48 h. The invaded cells were stained with crystal violet (means ± SD, n = 3). *** indicates p < 0.001. B) Images and C) healing rate from the wound healing assay after separate incubation with free DOX, LMWH, LT NPs, PLT NPs, LT/DOX NPs, and PLT/DOX NPs for 24 h (means ± SD, n = 3). * indicates p < 0.05. E) Detection of MMP‐9 in B16F10 cell culture medium by ELISA after separate incubation with PBS, LMWH, LT NPs, PLT NPs, LT/DOX NPs, and PLT/DOX NPs for 30 h (means ± SD, n = 3). *** indicates p < 0.001. F) The fluorescence intensity of platelets adhering to B16F10 cells in vitro. + indicates coincubation with calcein‐AM labeled platelets, – indicates no coincubation with calcein‐AM labeled platelets (means ± SD, n = 3). *** indicates p < 0.001. G) Expression of MMP‐9 in B16F10 cells tested by Western blot after separate incubation with (a) PBS, (b) LMWH, (c) LT NPs, (d) PLT NPs, (e) LT/DOX NPs, and (f) PLT/DOX NPs for 30 h. H) Detection of E‐cadherin and N‐cadherin in B16F10 cell culture medium by Western blot after separate incubation with (a) PBS−, (b) PBS+, (c) LMWH+, (d) LT NPs+, (e) PLT NPs+, and (f) PLT/DOX NPs+ for 30 h, + means coincubation with platelets after administration. J) CLSM images of the frozen sections of lungs. The implanted B16F10 tumor cells were identified by CFSE staining (yellow). Cell nuclei were stained with DAPI (blue). The scale bar indicates 200 µm.

Figure 4

Biodistribution and metastasis targeting capacity of NPs in vivo. A) In vivo images of B16F10 tumor‐bearing mice at 4 h and B) ex vivo images of tumors and organs of tumor‐bearing mice at 24 h after the systemic administration of DiD‐loaded NPs. C) Semiquantitative mean fluorescence intensity results showing the tumor and organ distribution of DiD‐loaded NPs in B16F10 tumor‐bearing mice 24 h after systemic administration (mean ± SD, n = 3). * indicates p < 0.05, and ** indicates p < 0.01. D) In vivo image and average fluorescence intensity semiquantitative results of B16F10 metastasis model mice at 6 h after systemic administration of DiD‐loaded NPs in vivo. *** indicates p < 0.001. E) Ex vivo image and average fluorescence intensity semiquantitative results of lungs from B16F10 metastasis mouse model at 6 h after systemic administration of DiD‐loaded NPs. ** indicates p < 0.01. F) Confocal images of lung sections from B16F10 metastasis model mice at 6 h after systemic administration of DiD‐loaded NPs (green). Cell nuclei were stained with DAPI (blue). M indicates the metastases in lung. The scale bar represents 200 µm. G) The circulation profile of LT NPs, PLT NPs, and PLT‐Fru NPs in the blood. Cy7‐labeled NPs were used for visualization. Blood was drawn from C57BL/6 mice after tail vein injection with different formulations at each time point and imaged under a fluorescence imaging system.

Figure 5

In vivo B16F10 solid tumor treatment. A) Images of B16F10 tumors harvested from C57BL/6 mice administered PBS, free DOX, LT/DOX NPs, PLT/DOX NPs, or PLT‐Fru/DOX NPs (DOX 3 mg kg⁻¹) by intravenous injection. B) Tumor volume changes in B16F10 tumor‐bearing C57BL/6 mice during the respective therapies (means ± SD, n = 5). * indicates p < 0.05,** indicates p < 0.01, *** indicates p < 0.001. C) Weights of the harvested B16F10 tumors (means ± SD, n = 5). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001. D) DOX plasma concentration–time profiles in C57BL/6 mice treated with different DOX formulations (at a dose of 3 mg kg⁻¹ DOX; n = 3). E) Histological analysis of hematoxylin and eosin (H&E) assays and immunohistochemical analysis of MMP‐9 staining for B16F10 tumors. The scale bar indicates 200 µm. The secreted MMP‐9 was stained violet and indicated by black arrows, and the scale bar represents 100 µm.

Figure 6

In vivo antimetastasis treatment. A) Images of lungs, B) the number of B16F10 metastases on the lung surface, and C) histological analysis of H&E assays of lungs from B16F10 metastasis mouse model after pretreatment with PBS, free LMWH, LT NPs, or PLT NPs by intravenous injection (at doses of 60 mg kg⁻¹) (means ± SD, n = 6). ** indicates p < 0.01 and *** indicates p < 0.001. The scale bar indicates 100 µm. D) Images of lungs, E) the number of B16F10 metastases on the lung surface, and F) histological analysis of H&E assays for lungs from B16F10 metastasis mouse model after treatment with PBS, free DOX, free LMWH, LT NPs, LT/DOX NPs, or PLT/DOX NPs by intravenous injection (at equivalent dose of 2.5 mg kg⁻¹ DOX) (means ± SD, n = 5). ** indicates p < 0.01. The scale bar represents 100 µm.