Chemokine receptor CXCR4 plays an important role in cancer cell invasion and metastasis. Recent findings suggest that anti-VEGF therapies upregulate CXCR4 expression, which contributes to resistance to antiangiogenic therapies. Here, we report the development of novel derivatives of polyethylenimine (PEI) that effectively inhibit CXCR4 while delivering anti-VEGF siRNA. PEI was alkylated with different amounts of a CXCR4-binding cyclam derivative to prepare PEI-C. Modification with the cyclam derivatives resulted in a considerable decrease in cytotoxicity when compared with unmodified PEI. All the PEI-C showed significant CXCR4 antagonism and the ability to inhibit cancer cell invasion. Polyplexes of PEI-C prepared with siVEGF showed effective silencing of the VEGF expression in vitro. In vivo testing in a syngeneic breast cancer model showed promising antitumor and antimetastatic activity of the PEI-C/siVEGF polyplexes. Our data demonstrate the feasibility of using PEI-C as a carrier for simultaneous VEGF silencing and CXCR4 inhibition for enhanced antiangiogenic cancer therapies.

Polycations are promising vectors for the delivery of siRNA therapeutics but they often suffer from toxicity and low in vivo delivery efficacy. This study tests the hypothesis that combining fluorination and bioreducibility of polycations will overcome problems with both the toxicity and delivery efficacy. To test the hypothesis, we synthesized bioreducible (RHB) and nonreducible (NHB) poly(amido amine)s. The RHB were additionally fluorinated using reaction with heptafluorobutyric anhydride to obtain F-RHB. We found that both RHB and F-RHB showed significantly reduced cytotoxicity compared with NHB, which allowed their safe use in a wider range of doses than NHB. All three synthesized polycations formed polyplexes with siRNA. F-RHB achieved the best siRNA silencing efficacy in multiple cell lines in vitro, which was at least in part because of fluorination-induced enhancement of cellular uptake and improved endosomal escape. Lastly, F-RHB showed greatly improved Luc silencing efficacy in tumors in vivo when compared with polyplexes based on RHB, NHB, as well as control poly(ethylenimine) (PEI). This study suggests that combining fluorination with bioreducibility of polycations is a promising strategy to the design of siRNA delivery vectors with improved toxicity and in vivo activity profiles.

The use of small interfering RNA (siRNA) in cancer treatment has been limited by the lack of effective systemic delivery methods. Although synthetic polycations have been widely explored in siRNA delivery, polycation/siRNA polyplexes often suffer from insufficient stability in vivo. Here, rationally designed siRNA delivery systems that meet the requirements for systemic siRNA delivery to distant tumors are reported. The hypothesis that modular design of delivery systems based on poly(amido amine)s that combine fluorination for systemic stability with bioreducibility for easy intracellular siRNA release, and PEGylation for improved safety and colloidal stability will overcome problems with contradicting siRNA delivery demands is tested. PEGylated, fluorinated, and bioreducible copolymers (PEG-PCD-F) with different degree of fluorination are thus synthesized. The fluorinated copolymers readily formed polyplexes with siRNA and achieved greatly improved gene silencing efficacy in multiple cell lines in vitro when compared with nonfluorinated controls. The results show fluorination-induced enhancement of stability, cellular uptake, and endosomal escape of the polyplexes, while exhibiting efficient siRNA release in reducing intracellular environment. PEG-PCD-F polyplexes with siRNA against Bcl2 inhibit breast tumor growth following systemic intravenous administration. The results provide strong evidence of successful combination of bioreducibility with fluorination and PEGylation to achieve systemic siRNA polyplex delivery.

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