Objective

Breast cancer is a very common malignancy that may metastasize during treatment. However, the low specificity and high toxicity of some current strategies may cause serious side effects, and the low drug solubility can lead to poor efficacy. Therefore, we designed a 4T1 cancer cell membrane (CCM)-encapsulated celastrol (Cela) nanocrystals (Cela-NCs) biomimetic targeted drug delivery system (CCM/Cela-NCs) for the precise delivery of drugs to primary tumors. The proteins expressed on the CCMs allow homologous targeting and adhesion to tumor cells by the CCM/Cela-NCs as well as the ability to evade immune surveillance.
Methods

We prepared Cela-NCs using an inverse solvent precipitation approach, followed by molecular dynamics simulation. The Cela-NCs were mixed with extracted CCMs, and the CCM/Cela-NCs were synthesized using an extrusion method and subsequently characterized by transmission electron microscopy (TEM), dynamic light scattering (DLS), and SDS-PAGE. In vitro experiments evaluated and compared the anti-tumor effects of Cela, Cela-NCs, and CCM/Cela-NCs, and the effects of CCM/Cela-NCs on 4T1 cell proliferation, migration, and invasion were assessed by MTT, colony-forming assays, and Transwell assays, respectively. We also investigated the cellular uptake of CCM/Cela-NCs and verified the expression of proteins associated with tumor metastasis by western blotting. Tumor volumes and numbers of nodules were assessed in in vivo experiments.
Results

Van der Waals forces were found to dominate the binding between the Cela and the stabilizer. TEM showed that the Cela-NCs and CCM/Cela-NCs were spherical, with the CCM/Cela-NCs having a typical core-shell structure with a particle size of 143.3 nm, slightly larger than that of the Cela-NCs (134.8 nm). The zeta potential of the Cela-NCs was -23.84 mV, while the potential of CCM/Cela-NCs was -26.82 mV after CCM coating, and the membrane proteins expressed on the CCMs were retained in the CCM/Cela-NCs. MTT assays showed that CCM/Cela-NCs killed 4T1 cells effectively without affecting the viability of normal cells, and significantly reduced proliferation, migration, and invasion of 4T1 cells compared with the control group. Uptake of CCM/Cela-NCs by RAW264.7 macrophages was lower than that of Cela-NCs, indicating immune evasion leading to extended circulation, while uptake by 4T1 cells was time- and concentration-dependent. Western blotting showed increased expression of E-cadherin and reduced levels of MMP-9. In an in vivo experiment, we found that CCM/Cela-NCs inhibited tumor growth and reduced the number of lung nodules.
Conclusions

The prepared CCM/Cela-NCs improved the solubility of Cela and showed both homologous targeting to tumor cells and immune evasion. The CCM/Cela-NCs also inhibited tumor growth and metastasis in in vivo experiments. Due to their good stability and biocompatibility, CCM/Cela-NCs thus offer a promising nanodelivery system for the treatment of breast cancer and its lung metastases. We gratefully acknowledged the support from the Guangdong Basic and Applied Basic Research Foundation (2019B1515120043 and 2022A1515012154) and the Key Fields of Biomedicine and Health Foundation of Colleges and Universities in Guangdong Province (2022ZDZX2017).