CD73 Downregulation by EGFR-Targeted Liposomal CD73 siRNA Potentiates Antitumor Effect of Liposomal Doxorubicin (Doxil) in 4T1 Tumor-Bearing Mice

Background Blocking CD73 ectonucleotidase has been proposed as a potential therapeutic approach for cancer treatment. The purpose of the present study was to investigate the antitumor effect of a novel EGFR-Targeted liposomal CD73 siRNA formulation in combination therapy with Doxil in the 4T1 mouse model. Methods CD73 siRNA was encapsulated into nanoliposomes by the ethanol injection method. After preparation, characterization, morphology, and stability evaluation of formulations, the toxicity was measured by MTT assay. Uptake assay and efficiency of the liposomal formulations were investigated on the 4T1 cell line. The liposomal formulation containing CD73 siRNA was targeted with GE11 peptide for in vivo evaluations. Antitumor activity of prepared formulations in combination with Doxil was studied in mice bearing 4T1 metastatic breast cancer cells. Finally, the antitumor efficacy of the formulation in concomitant treatment with Doxil was evaluated in a mouse model of breast cancer. Results The size of prepared liposomal formulations at N/P=16 for the liposomal CD73 siRNA and GE11-lipo CD73 siRNA groups were 89 nm ± 4.4 and 95 nm ± 6.6, respectively. The nanoparticle’s PDI was less than 0.3 and their surface charge was below 10 mV. The results demonstrated that N/P=16 yielded the best encapsulation efficiency which was 94% ± 3. 3. AFM results showed that the liposomes were spherical in shape and were less than 100 nm in size. The results of the MTT assay showed significant toxicity of the liposomes containing CD73 siRNA during the 48-hour cell culture. Real-time PCR and flow cytometry results showed that liposomes containing CD73 siRNA could effectively downregulate CD73 expression. Liposomal formulations were able to significantly downregulate CD73 gene expression, in vivo. However, CD73 downregulation efficiency was significantly higher for targeted form in comparison with non-targeted formulation (P-value <0.01). The combination showed maximum tumor growth delay with remarkable survival improvement compared to the control group. Studying the immune responses in the treatment groups which received doxorubicin, showed decreased number of lymphocytes in the tumor environment. However, this decrease was lower in the combination therapy group. Finally, our results clearly showed that CD73 downregulation increases the activity of CD8+ lymphocytes (INF-ℽ production) and also significantly decreases the Foxp3 in the CD25+ lymphocytes compared to the control group. Conclusion GE11-Lipo CD73 siRNA formulation can efficiently knock down CD73 ectonucleotidase. Also, the efficacy of Doxil is significantly enhanced via the downregulation of CD73 ectonucleotidase.