Detection of doxorubicin conjugated to tHBcAg VLNPs separated on sucrose gradients. The absorbance at 495 nm (A495) of samples was measured with a spectrophotometer. (a) Doxorubicin (DOX), and the DOX added to tHBcAg without the conjugation procedure (tHBcAg + DOX) stayed on top of the sucrose gradients. tHBcAg VLNPs (tHBcAg), which served as a negative control, were not detected at A495; (b) DOX-conjugated tHBcAg VLNPs (tHBcAg-DOX), as well as tHBcAg VLNPs conjugated with folic acid (FA) and DOX (FA-tHBcAg-DOX) migrated into the sucrose gradients, and the conjugated DOX was detected at A495.