Dually Active Targeting Nanomedicines Based on a Direct Conjugate of Two Purely Natural Ligands for Potent Chemotherapy of Ovarian Tumors

Actively targeted nanomedicines have promised to revolutionize cancer treatment; however, their clinical translation has been limited by either low targetability, use of unsafe materials, or tedious fabrication. Here, we developed CD44 and folate receptor (FR) dually targeted nanoparticulate doxorubicin (HA/FA-NP-DOX) based on a direct conjugate of two purely natural ligands, hyaluronic acid and folic acid (FA), for safe, highly specific, and potent treatment of ovarian tumors in vivo. HA/FA-NP-DOX had a small size and high DOX loading, wherein the particle size decreased from 115, 93, to 89 nm with increasing degree of substitution of FA from 6.4, 8.5, to 11.1, while increased from 80, 93, to 103 nm with increasing DOX loading from 15.0, 23.1, to 31.4 wt %. Interestingly, HA/FA-NP-DOX exhibited excellent lyophilization redispersibility and long-term storage stability with negligible drug leakage while it released 91% of DOX in 48 h at pH 5.0. Cellular studies corroborated that HA/FA-NP-DOX possessed high selectivity to both CD44 and FR, resulting in strong killing of CD44- and FR-positive SKOV-3 ovarian cancer cells while low toxicity against CD44- and FR-negative L929 fibroblast cells. In vivo studies revealed a long elimination half-life of 5.6 h, an elevated tumor accumulation of 12.0% ID/g, and an effective inhibition of the SKOV-3 ovarian tumor for HA/FA-NP-DOX, leading to significant survival benefits over free DOX·HCl and phosphate-buffered saline controls. These dually targeted nanomedicines are simple and safe, providing a potentially translatable treatment for CD44- and FR-positive malignancies.