Oxygen-Generating Biomimetic Nano-Herb System for Synergistic Therapy & Pain Relief in Triple-Negative Breast Cancer via HIF-1α/VEGF Pathway

Abstract

Purpose: This study aims to develop an innovative delivery system, (Q+M/MnOx)@Clip, to enhance the bioavailability and therapeutic efficacy of quercetin both in tumor treatment and pain alleviation.

Methods: The (Q+M/MnOx)@Clip system was evaluated to enhance the release of quercetin, investigate its ability to target cancer cells, alleviate tumor hypoxia, and improve the efficacy of chemodynamic therapy (CDT). Tumor hypoxia markers and immune response activation were assessed, along with the impact on pain relief biomarkers.

Results: (Q+M/MnOx)@Clip successfully mitigated tumor hypoxia, facilitated controlled Q release, and enhanced CDT in vitro and in vivo. The system demonstrated a dual therapeutic effect: anti-tumor immunity and significant cancer pain relief by reducing HIF-1α and VEGF-A levels.

Conclusion: The novel (Q+M/MnOx)@Clip system represents a promising advancement in nanomedicine, improving the bioavailability of quercetin and offering a more effective approach to cancer treatment by downregulation of HIF-1α and VEGF-A. This study demonstrates the potential for combining anti-tumor immunity with pain relief for triple-negative breast cancer therapy.

Keywords: biomimetic; cancer immunotherapy; chemodynamic therapy; oxygen generation; pain relief; quercetin.

Figure 1

Schematic illustration of this system. (A) Synthesis diagram of oxygen-producing biomimetic nano-herb delivery system ((Q+M/MnOx)@Clip). (B) Schematic of (Q+M/MnOx)@Clip for potentiated cancer therapy and pain mitigation in vivo.

Figure 2

The characterization of the nano-herb delivery system. (A) Particle size distribution and (B) TEM of M/MnOx hybrids. (C) Particle size distribution and (D) TEM of (Q+M/MnOx)@Clip (bar = 200 nm). (E) Elemental mapping of (Q+M/MnOx)@Clip (bar = 50 nm). Columnar chart of (F) the particle size and (G) potential diagram of four different nanoformulations. (H) Representative XPS spectra of (Q+M/MnOx)@Clip.

Figure 3

The analysis of protein and stability of this system. (A) SDS-PAGE analysis of protein components (Group-1 stands for 4T1 cells, 2 stands for 4T1 cell membrane, and 3 stands for (Q+M/MnOx)@Clip). (B) WB analysis for CD44, markers of membrane marker protein (lane 1, 4T1 cells; lane 2, 4T1 cell membrane; lane 3, (Q+M/MnOx)@Clip). (C) Representative photomicrograph of the cofusion of cell membrane (CM) and liposome membrane labeled with red (Dil) and green (DiO) fluorescent dyes (bar = 275 µm). (D) Time-dependent colloidal stability of (Q+M/MnOx)@Clip. (E) TEM images of (Q+M/MnOx)@Clip treated in different pH solutions (bar = 200 nm). (F) Cumulative release percentage of quercetin over time. (G) Oxygen-producing capability of (Q+M/MnOx)@Clip under different conditions(mean ± SD, n = 3, ns, P > 0.05, ***, P < 0.001). (H) UV-vis absorption spectra and photo (inset) after MB degradation at different solutions.

Figure 4

The cellular uptake and effects of nanoparticles on tumor cells. (A) In vitro cellular uptake and internalization of fluorescent nanoparticles with and without the cell membrane fusion at different lengths of time (bar = 125 µm). (B) Homologous targeting of (Q+M/MnOx)@Clip for different cells analyzed by FCM and (C) corresponding quantitative analysis. (D) Cytotoxicity and (E) cytocompatibility determined by MTT. (F) Live/dead images of different formulations (green-live, red-dead) (bar = 275 µm). (G) FCM analysis of cell apoptosis. (mean ± SD, n = 3, ns, P > 0.05, *, P < 0.05, **, P < 0.01, ***, P < 0.001).

Figure 5

The system induced the hypoxia alleviation and anti-tumor immune. (A) Intracellular hypoxia status after different treatments was detected by fluorescence microscopy after the cells were stained by the intracellular hypoxia reagent (bar = 125 µm). (B) HIF-1α levels were detected by immunofluorescence (bar = 125 µm). (C) HIF-1α and VEGF-A expression levels were analyzed by WB. (D) VEGF-A in cell supernatants was quantified by ELISA. (E) HMGB1 release in supernatant was analyzed by ELISA. (F) ATP content was determined with ATP Assay Kit. (G) Representative immunofluorescence staining images of CRT expression (green) on the surface of 4T1 cells after various treatments (bar = 125 µm). (H) DC maturation markers (CD80 and CD86) were assessed using FCM. (mean ± SD, n = 3, *, P < 0.05, ***, P < 0.001).

Figure 6

The biodistribution analyses of nanoparticles. (A) Tissue distributions of (Q+M/MnOx)@lip and (Q+M/MnOx)@Clip at indicated time points following i.v. (n = 3). (B) Ex vivo fluorescence imaging and (C) quantification of tumors at 48 h after intravenous injection (mean ± SD, n = 3, **, P < 0.01). (D) Ex vivo fluorescence imaging and (E) quantification of different organs (heart, liver, spleen, lung, and kidney) at 48 h after intravenous injection (mean ± SD, n = 3, ns, P > 0.05). (F) In vitro T1-weighted MRI images of (Q+M/MnOx)@Clip at different concentrations.

Figure 7

(Q+M/MnOx)@Clip inhibited tumor growth in vivo. (A) Schematic of the treatment protocol in mice. (B) Tumor volume curves (mean ± SD, n = 5, ***, P < 0.001 vs the PBS group). (C) Photos of tumors. (D) Tumor weights (mean ± SD, n = 5, *, P < 0.05, **, P < 0.01 vs the PBS group). (E) H&E-stained section of the tumor (bar = 100µm). (F) H&E-stained liver, spleen, kidney, heart, and lung (bar = 100 µm).

Figure 8

Nanoparticles-mediated immune responses in vivo. (A) DC maturation state markers CD86 and CD80 were detected using FCM. (B) FCM analysis of CD8⁺ and (C) CD4⁺ T cell expression profiles in blood. (D) IHC staining of CD4 (bar = 125 µm). (E) IHC staining of CD8 (bar = 125 µm).

Figure 9

Particles reduced the tumor-induced pain. (A) Establishment of cancer pain mouse model. (B) MWT value. (C) TWL value. (D) IF staining of tumor tissues showing VEGF-A expression (green) and cell nuclei stained with DAPI (blue) (bar = 125 µm). (E) HIF-1α and VEGF-A expression levels were analyzed by WB. (F) WB quantification of VEGF-A. (mean ± SD, n = 3, **, P < 0.01, ***, P < 0.001 vs the PBS group).