(T1530-05-27) Co-delivery of siRNA and Chemotherapeutic Drug Using 2C5 Antibody Modified Dendrimer-Based Mixed Micelles for the Treatment of Multidrug Resistant Cancers

Purpose: A major hurdle in chemotherapy is multidrug resistance (MDR) observed in tumors. Downregulation of efflux proteins such as P-glycoprotein (P-gp) using small interfering RNA (siRNA) can be an effective way to minimize the resistance in tumors. In this study, monoclonal antibody 2C5 (mAb 2C5) - PEG_7k-DOPE conjugates were post-inserted into the mixed dendrimer micelles containing generation 4 (G4) polyamidoamine (PAMAM)-PEG_2k-DOPE and PEG_5k-DOPE. The inherent amphiphilic nature of DOPE conjugates causes the copolymers to self-assemble to form a micelle which can encapsulate hydrophobic chemotherapeutic drug in its core. The siRNA electrostatically binds to the cationic charges on the G4 PAMAM dendrimer [1]. The tumor-specific mAb 2C5 on the surface of these nano-preparations resulted in improved tumor targeting [2]. This active targeting to the tumors can cause increase in the drug and siRNA accumulation at the tumor site and thereby minimizing off target effects.

Methods: G4 PAMAM-PEG_2k-DOPE was prepared by conjugating G4 PAMAM with para-nitrophenol (pNP)-PEG_2k-DOPE [3]. To prepare 2C5-PEG_7k-DOPE, mAb 2C5 was mixed with micelles containing pNP-PEG_7k-DOPE and PEG_5k-DOPE in sodium citrate buffer at pH 5.0 and later adjusted the pH to 8.0 with 1 N NaOH. The free 2C5 and pNP were removed by dialysis using 300KDa MWCO membrane[4]. The concentration and immunoactivity of antibody after conjugation were analyzed using bicinchoninic acid (BCA) assay and enzyme-linked immune sorbet assay (ELISA) respectively. The cellular cytotoxicity of the 2C5-conjugated mixed dendrimer micelles loaded with siMDR-1 and doxorubicin (DOX) was evaluated in both MDA-MB-231 and SKOV-3TR resistant cell lines in vitro [5]. Further a xenograft MDA-MB-231 in vivo mice tumor models were performed.

Results: The 2C5-conjugated mixed dendrimer micelles have a uniform size distribution with a slightly positive zeta potential. They show a stable size and zeta potential for 20 days. We evaluated the formulation to see if there are any observable hemolysis and noticed no hemolysis in the samples tested. The morphology of the 2C5 modified micelles was confirmed using TEM imaging. A higher cellular association was observed in the micelles with the 2C5 antibody targeting using fluorescence imaging (DOPE-FITC tagged micelle) in both the cell lines tested. At 30 minutes the 2C5 targeted mixed dendrimer micelles showed high cellular uptake was observed using flow cytometry. We further evaluated the P-gp downregulation using western blot and observed a significant downregulation of P-gp levels in the targeted formulation versus the non-targeted formulation. Cellular cytotoxicity in MDA-MB-231 and SKOV-3TR cell lines were evaluated and we observed a dose response relationship. A higher cytotoxicity is observed in micelles with the highest siRNA concentration. The in vivo tumor growth inhibition study in nude NCG 572 MDA-MB-231 tumor bearing mice showed that the immuno-micelles loaded with siMDR-1 and chemotherapeutics resulted in significantly increased therapeutic efficacy compared to non-modified mixed dendrimer micelles. We evaluated the micelles in both male and female tumor bearing mice.

Conclusion: We have evaluated the 2C5 antibody-modified mixed dendrimer micelles in different cell models, in addition to the models tested in our previous studies. We have successfully established the stability of the formulation in this study and established a 20-day shelf-life. The hemolysis data proves that the formulation is safe for injecting into the mice. Cellular association, internalization and P-gp downregulation studies show the superiority of the 2C5 modified dendrimer micelles.

References: 1. Biswas, S., et al., Lipid modified triblock PAMAM-based nanocarriers for siRNA drug co-delivery. Biomaterials, 2013. 34(4): p. 1289-1301.
2. Elbayoumi, T.A. and V.P. Torchilin, Enhanced cytotoxicity of monoclonal anticancer antibody 2C5-modified doxorubicin-loaded PEGylated liposomes against various tumor cell lines. european journal of pharmaceutical sciences, 2007. 32(3): p. 159-168.
3. Pan, J., et al., Polyamidoamine dendrimers-based nanomedicine for combination therapy with siRNA and chemotherapeutics to overcome multidrug resistance. European Journal of Pharmaceutics and Biopharmaceutics, 2019. 136: p. 18-28.
4. Pan, J., et al., Monoclonal antibody 2C5-modified mixed dendrimer micelles for tumor-targeted codelivery of chemotherapeutics and siRNA. Molecular Pharmaceutics, 2020. 17(5): p. 1638-1647.
5. Yalamarty, S.S.K., et al., Co-Delivery of siRNA and Chemotherapeutic Drug Using 2C5 Antibody-Targeted Dendrimer-Based Mixed Micelles for Multidrug Resistant Cancers. Pharmaceutics, 2022. 14(7): p. 1470.

Disclaimers: Protocol # 19-1035R approved by Northeastern University Institutional Animal Care and Use Committee

Funding: This research was funded by National Institute of Health (NIH), United States [1R01CA200844]. The grant was issued to V. P. Torchilin
Conflict of Interest: NA

Figure 1. TEM images of 2C5-modified mixed dendrimer micelles, A: 10,000x magnification, B: 20,000x magnification.



Figure 2. Cellular association of 2C5 targeted and non-targeted micelles. The figure shows the Hoechst-stained nuclei with FITC labelled micelles along with brightfield and overlay images, A: MDA-MB-231 cells and, B: SKOV-3TR cells for 15 and 30 minutes



Table 1, Stability data of 2C5-modified mixed dendrimer micelles