Carrier-Free Nanomedicine Based on Celastrol and Methotrexate for Synergistic Treatment of Breast Cancer via Folate Targeting

Abstract

Purpose: To address celastrol(Ce)'s efficacy and toxicity challenges in breast cancer, we first developed a carrier-free, self-targeting nanosystem with synergistic anti-tumor action by leveraging methotrexate (MTX)'s intrinsic folate moiety for active tumor targeting.

Methods: Ce-MTX nanoparticles (NPs) were prepared using a solvent precipitation method, with formulation parameters optimized. Characterization included particle size, polydispersity index (PDI), encapsulation efficiency (EE), loading efficiency (LE), and TEM. Drug release was investigated under physiological and tumor-mimetic conditions via a dialysis method. Cellular uptake and in vitro anti-tumor effects were evaluated in A549 and 4T1 cell lines. In vivo, tumor distribution and normal tissue accumulation were analyzed in 4T1 tumor-bearing mice. Anti-tumor efficacy and biosafety were evaluated through tumor growth curves, tumor inhibition rates, body weight changes, organ indices, histological analysis, and serum biochemistry.

Results: The optimized Ce-MTX NPs exhibited a particle size of 90.20 nm, PDI of 0.062, and spherical morphology. The EE and LE were 95.15% and 66.53% for Ce, and 95.74% and 33.6% for MTX, respectively. The NPs demonstrated excellent stability over 7 days. Notably, Ce-MTX NPs exhibited pH-dependent drug release, with accelerated release at pH 5.5. Qualitative and quantitative cellular uptake assays revealed significantly higher uptake of Ce-MTX NPs compared to the free drugs, with enhanced folate receptor-targeting in 4T1 cells. Cytotoxicity assays showed stronger anti-tumor activity of Ce-MTX NPs in 4T1 cells compared to the free drug mixture, thus demonstrating the superior synergistic anti-cancer effects achieved by the nanoparticle formulation. Importantly, in vivo studies confirmed substantial tumor growth inhibition and an excellent biosafety profile.

Conclusion: The carrier-free Ce-MTX NPs demonstrated enhanced stability, tumor targeting, and rapid drug release within tumor cells, significantly improving the efficacy and biosafety of breast tumor treatment. These nanoparticles offer a promising strategy for combined cancer therapy and hold great potential for further development in nanomedicine.

Keywords: breast cancer; carrier-free nanoparticles; celastrol; methotrexate; synergistic antitumor efficacy; tumor targeting.

Figure 1

Schematic diagram of Ce and MTX self-assembly into carrier-free nanoparticles for in vivo targeted distribution.

Figure 2

Characterization and drug release of Ce-MTX NPs (A) Particle size distribution. (B) Zeta potential. (C) TEM analysis at 200 nm. (D) TEM analysis at 100 nm. (E) UV absorption spectra of Ce, MTX, and Ce-MTX NPs. (F) FT-IR spectra of Ce, MTX, and Ce-MTX NPs. (G) Particle size distribution of Ce-MTX NPs incubated for 7 days in ultrapure water, PBS solution, and 10% fetal calf serum-containing 1640 medium. (H) Cumulative drug release profiles of Ce from Ce-MTX NPs at different pH conditions. (I) Cumulative drug release profiles of MTX from Ce-MTX NPs at different pH conditions ( ± s, n = 3).

Figure 3

In vitro experiments of cells uptake (A) CLSM images of 4T1 cells incubated with free ICG, Ce-MTX-ICG NPs and Ce-MTX-ICG NPs+FA for 4h. (B) CLSM images of A549 cells incubated with free ICG, Ce-MTX-ICG NPs and Ce-MTX-ICG NPs+FA for 4h. (C) Fluorescence distribution and average fluorescence intensity of free ICG, Ce-MTX-ICG NPs and Ce-MTX-ICG NPs+FA in 4T1. (D) Fluorescence distribution and average fluorescence intensity of free ICG, Ce-MTX-ICG NPs and Ce-MTX-ICG NPs+FA in A549.(n=3, **P<0.01).

Figure 4

The synergistic effect of Ce and MTX, and the cytotoxicity in vitro. (A) Survival rate of 4T1 cells after treatment with varying concentrations for 24 h. (B) Survival rate of 4T1 cells after treatment with varying concentrations for 48 h. (C) Survival rate of A549 cells after treatment with varying concentrations for 24 h. (D) Survival rate of A549 cells after treatment with varying concentrations for 48 h. (E) IC₅₀ values of free Ce, Ce+MTX mixture, and Ce-MTX NPs in 4T1 and A549 cells. (F) Combination index of Ce+MTX mixture and Ce-MTX NPs in 4T1 cells. (G) Combination index of Ce+MTX mixture and Ce-MTX NPs in A549 cells. (n=3, *P<0.05, **P<0.01, ***P<0.001).

Figure 5

In vivo anti-tumor study of Ce-MTX NPs in 4T1 tumor-bearing mice (A) In vivo and ex vivo NIR fluorescence distribution of major organs and tumors following intravenous injection of free ICG or Ce-MTX-ICG NPs. (B) Body weight changes of mice in each group. (C) Tumor volume changes in each group. (D) Average tumor weight of each group. (E) Tumor images after 15 days of treatment. (F) Tumor inhibition rate in each group. (n=3, *P<0.05, **P<0.01, ***P<0.001).

Figure 6

In vivo safety evaluation of Ce-MTX NPs in 4T1 tumor-bearing mice (A) Organ index of mice. (B) Histopathological sections of organs. (C and D) Serum liver function: ALT and AST levels. (E and F) Serum renal function: BUN and CRE levels. (n=6, *P<0.05, **P<0.01).