Xiaojuan Li,1 Mengxin Zhang,1 Xiaolin Wang,1 Ping Ma,2 Yu Song1 

1School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, 350122, People’s Republic of China; 2Research and Development, Hikma Pharmaceuticals USA Inc., Bedford, OH, USA

Correspondence: Yu Song, School of Pharmacy, Fujian University of Traditional Chinese Medicine, Fuzhou, 350122, People’s Republic of China, Tel/Fax +86 591 22861135, Email songecho@163.com

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: celastrol, methotrexate, carrier-free nanoparticles, tumor targeting, breast cancer, synergistic antitumor efficacy