Development of an innovative method to enhance doxorubicin loading into erythrocyte-derived nanovesicles for breast cancer drug delivery

Here, a novel strategy for doxorubicin (DOX) loading into nano-erythrosomes (NEs) was developed, in which the loading process was adjusted through a combination of co-incubation and osmosis–based methods, along with the establishment of a pH gradient. Additionally, a method for determining the molar concentration of nano-erythrosomes was developed. DOX-loaded nano-erythrosomes (DOX-NEs) were obtained with the desired size (~ 109 nm) and homogeneity (PDI ~ 0.2), demonstrating high drug encapsulation efficiency (81.6%) and DOX concentration (808.0 µg/mL), along with acceptable short-term stability. Flow cytometry analysis showed that 25.2% of CD47 protein (a ‘self’ marker on erythrocytes) was retained on the surface of the nanocarriers. In addition, the cellular uptake of DOX-NEs by tumor cells was comparable to that of free DOX and the commercial formulation Doxil when all formulations were applied at the same DOX concentration (5 µM), with no statistically significant differences observed among the groups. Finally, the percentage of cell survival following treatment with DOX-NEs, Doxil, and free DOX was determined on 4T1 and NIH/3T3 cell lines. In conclusion, DOX-NEs exhibit acceptable physicochemical properties and merit further investigation.