Blocking CD73 ectonucleotidase has been proposed as a potential therapeutic approach for
cancer treatment. The present study aimed to investigate the antitumor effect of a novel EGFR-Targeted liposomal CD73
siRNA formulation in combination
therapy with
liposomal doxorubicin in the 4T1 mouse model. 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. Following biodistribution analysis, the antitumor activity of prepared formulations in combination with
liposomal doxorubicin was studied in mice bearing 4T1 metastatic
breast cancer cells. Finally, the induction of immune response of formulations in concomitant treatment with
liposomal doxorubicin was evaluated in the tumor microenvironment of a mouse model of
breast cancer. The size of prepared liposomal formulations at N/P = 16 for the liposomal CD73
siRNA and GE11-liposomal 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-h 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 the targeted form compared to the 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 (IFN-ℽ production) and also significantly decreases the Foxp3 in the CD25+ lymphocytes compared to the control group. GE11-Lipo CD73
siRNA formulation can efficiently knockdown CD73 ectonucleotidase. Also, the efficacy of
liposomal doxorubicin is significantly enhanced via the downregulation of CD73 ectonucleotidase.