Abstract: To systematically investigate the capacity of cytomimetic microspheres (CMM) to load different drugs and to evaluate their in vivo behavior, CMM were prepared from 4T1 cells using a freeze-thaw method. The preparation procedure involved freezing in liquid nitrogen for 12 h followed by thawing at 37 °C. Small molecules (doxorubicin and rhodamine B), biomacromolecules (bovine serum albumin and protamine), and nanoparticles (soybean phospholipid-cholesterol liposomes, polyethyleneimine, polyethyleneimine-cinnamaldehyde, and polyethyleneimine-phenylboronic acid) were selected as model drugs. Fluorescence spectrophotometry was employed to evaluate the encapsulation and release capacities of CMM for different types of drugs. The results showed that the drug loadings of CMM for polyethyleneimine-cinnamaldehyde and protamine were (41.09±1.21) and (31.07±0.79) μg/10⁶ cells, respectively, which were significantly higher than those for the other drugs, indicating that CMM are suitable for encapsulating positively charged and hydrophobic drugs. In a mouse model of 4T1 breast cancer lung metastasis, CMM exhibited favorable lung accumulation and inhibited tumor growth. The carrier has a simple preparation procedure and a stable structure, and possesses a certain degree of loading capacity for small molecules, biomacromolecules, and nanoparticles. These findings help deepen the understanding of the drug-loading and release properties of CMM and provide a new strategy for the treatment of tumor lung metastasis.