Cooperative coordination-mediated multi-component self-assembly of "all-in-one" nanospike theranostic nano-platform for MRI-guided synergistic therapy against breast cancer

Abstract

Carrier-free multi-component self-assembled nano-systems have attracted widespread attention owing to their easy preparation, high drug-loading efficiency, and excellent therapeutic efficacy. Herein, MnAs-ICG nanospike was generated by self-assembly of indocyanine green (ICG), manganese ions (Mn²⁺), and arsenate (AsO₄ ^3-) based on electrostatic and coordination interactions, effectively integrating the bimodal imaging ability of magnetic resonance imaging (MRI) and fluorescence (FL) imaging-guided synergistic therapy of photothermal/chemo/chemodynamic therapy within an "all-in-one" theranostic nano-platform. The as-prepared MnAs-ICG nanospike had a uniform size, well-defined nanospike morphology, and impressive loading capacities. The MnAs-ICG nanospike exhibited sensitive responsiveness to the acidic tumor microenvironment with morphological transformation and dimensional variability, enabling deep penetration into tumor tissue and on-demand release of functional therapeutic components. In vitro and in vivo results revealed that MnAs-ICG nanospike showed synergistic tumor-killing effect, prolonged blood circulation and increased tumor accumulation compared to their individual components, effectively resulting in synergistic therapy of photothermal/chemo/chemodynamic therapy with excellent anti-tumor effect. Taken together, this new strategy might hold great promise for rationally engineering multifunctional theranostic nano-platforms for breast cancer treatment.

Keywords: Breast cancer; Carrier-free nanodrugs; Deep penetration; Magnetic resonance imaging; Nanospike; Self-assembly; Synergistic therapy; Tumor microenvironment-responsive.

Graphical abstract

Figure 1

Schematic illustration of MnAs nanofiber and MnAs-ICG nanospike preparation, and pH-sensitive morphological transformation-enabled deep penetration and MRI and FL bimodal imaging-guided synergistic therapy.

Figure 2

Characterization of MnAs nanofiber and MnAs-ICG nanospike. (A) TEM and SEM images of MnAs and MnAs-ICG. (B) Hydrodynamic diameter and zeta potential of MnAs and MnAs-ICG. Data are presented as mean ± SD (n = 3). (C) UV–Vis absorption spectra of MnAs, ICG and MnAs-ICG. (D) Elemental mapping and (E) EDS spectrum of MnAs-ICG. Scale bar = 100 nm. (F) MD simulation and (G) interaction energy of MnAs nanofiber and MnAs-ICG nanospike. The covalent interactions were marked by dashed line.

Figure 3

Photothermal conversion and ∙OH generation. (A) Photothermal images and (B) corresponding temperature variation curve of MnAs-ICG with various ICG concentrations under 808-nm laser irradiation. (C) Photothermal stability of ICG and MnAs-ICG undergoing five rounds of laser irradiation. (D) The dynamic stability of ICG and MnAs-ICG at 4 °C, and calculated at 780 and 808 nm for ICG. (E) ESR spectra of ∙OH generation under different conditions.

Figure 4

pH-responsive study. (A) The morphology and (B) size variation of MnAs-ICG at pH 6.2 and 5.0. Data are presented as mean ± SD (n = 3). Scale bar = 200 nm. (C) Cumulative release of ICG from MnAs-ICG at various conditions (pH 7.4, 6.2 or 5.0). (D) CLSM images of 3D tumor spheroids with different treatment. Scale bar = 100 μm ∗P < 0.05, ∗∗P < 0.01.

Figure 5

In vitro cellular uptake study. (A) Schematic illustration of the balance between ROS and GSH. (B) CLSM images of ICG and MnAs-ICG in 4T1 cells of different treatment. Scale bar = 10 μm. (D) CLSM images and (C) its corresponding inhibition rate of ICG and MnAs-ICG internalized by 4T1 cells treated with PBS, amiloride (macropinocytosis inhibitor), chlorpromazine (clathrin inhibitor), dynasore (caveolae/clathrin inhibitor), filipin (caveolae inhibitor) at 37 °C, and PBS at 4 °C (inhibition of ATP-mediated endocytosis). Scale bar = 20 μm. (E) Intracellular ROS detections after PBS, ICG or MnAs-ICG treatment. Scale bar = 50 μm.

Figure 6

In vitro cytotoxicity study. (A) Live/dead staining assay after treatment with PBS, ICG or MnAs-ICG. Scale bar = 100 μm. Cell viability of 4T1 cells after treatment with (B) MnAs or (C) MnAs-ICG at various concentrations. Data are presented as mean ± SD (n = 6). (E) Cell apoptosis by flow cytometry and (D) its quantification after treatment with PBS, ICG or MnAs-ICG with or without laser irradiation. Data are presented as mean ± SD (n = 3). ∗∗P < 0.01.

Figure 7

In vivo pharmacokinetic study, fluorescence, MRI and photothermal imaging. (A) Schematic illustration of in vivo applications of MnAs-ICG nanospike. (B) The pharmacokinetics profiles after ICG or MnAs-ICG treatment. Data are presented as mean ± SD (n = 3). (C) FL images of ICG or MnAs-ICG injected 4T1 tumor-bearing mice. (D) Semi-quantitative FL intensity of ICG around the tumors after ICG or MnAs-ICG treatment. Data are presented as mean ± SD (n = 3). (E) Ex vivo FL images of major organs and tumors at 24-h post-injection. (F) Ex vivo FL intensity of ICG or MnAs-ICG. Data are presented as mean ± SD (n = 3). (G) In vitro MRI images and (H) corresponding r₁ value of MnAs-ICG at various pH solution. (I) In vivo T₁-weighted MRI of mice at selected times after MnAs-ICG injection. (J) Infrared thermal images and (K) corresponding temperature change of 4T1 tumor-bearing mice at 5 h after saline, ICG or MnAs-ICG injection upon 808-nm laser irradiation. The tumor was marked by white dotted circles. ∗P < 0.05, ∗∗P < 0.01.

Figure 8

In vivo pharmacodynamics study. (A) Schematic illustration of the therapeutic protocol. (B) Relative tumor volume, (C) survival ratio and (D) body weight of mice after saline, ICG, ICG + laser, MnAs-ICG or MnAs-ICG + laser treatment. Data are presented as mean ± SD (n = 5). (E) Photographs of mice on Days 1 and 21. The tumor was marked by red dotted circles. (F) H&E and TUNEL staining of tumors. Scale bar = 100 μm. ∗P < 0.05, ∗∗P < 0.01.

Figure 9

H&E staining of major organs after treatment with saline, ICG, ICG + laser, MnAs-ICG or MnAs-ICG + laser. Scale bar = 20 μm.