Lactoferrin-functionalized PEGylation liposomes loaded with norcantharidin acid for targeted therapy of hepatocellular carcinoma

Norcantharidin (NCTD), an antitumor agent with an increased leukocyte function, has been used for the treatment of hepatocellular carcinoma (HCC) in clinical. However, the clinical application of NCTD is limited due to its inadequate hydrophilicity and lipophilicity, short half-life (t_1/2), as well as adverse effects such as vascular irritation, cardiotoxicity, and nephrotoxicity. Herein, a lactoferrin (Lf) and DSPE-mPEG₂₀₀₀ functionalized liposomes loaded with norcantharidic acid (NCA), an active metabolite of NCTD, was constructed for the targeted therapy of HCC. In this study, blank PEGylated liposomes were prepared using the film hydration method, and the NCA was loaded by calcium acetate active loading method to increase the encapsulation efficiency (EE). Subsequently, lactoferrin was covalently coupled to DSPE-PEG₂₀₀₀-COOH activated by EDC and NHS. In addition, the in vivo pharmacokinetics and pharmacodynamics were investigated in Sprague-Dawley (SD) rats and H22 tumor-bearing BALB/c mice, respectively. As expected, the encapsulation efficiency measurement showed that the encapsulation efficiency of the NCA liposomes was 89.3±1.25 %, and the coupling efficiency of lactoferrin was more than 65.97 %. Additionally, the variations in both the dynamic size and encapsulation efficiency of norcantharidic acid liposomes in long-term storage stability and serum stability studies did not exceed 10 %. Furthermore, the pharmacokinetics and pharmacodynamics results showed that, the NCA-Lips-Lf were able to significantly improve antitumor activity by enhancing tumor-targeting accumulation and prolonging circulation time in the body compared to the sodium demethylcantharidate for injection (Na₂DCA). Notably, the AUC_0-48 and the t_1/2 of NCA-Lips-Lf increased 4.28-time and 5.17-time in comparison to those of NCA-sol, respectively. The tumor inhibition rate of NCA-Lips-Lf (85.29 %) was significantly higher than that of sodium demethylcantharidate for injection (Na₂DCA) (59.13 %), without obvious vascular irritation, cardiotoxicity and nephrotoxicity. In conclusion, NCA-Lips-Lf have the potential to eliminate hepatocellular carcinoma more effectively with fewer side effects than Na₂DCA, which further advances the clinical application of norcantharidin-related drugs.