Since our in vitro studies established that the HuR-FNP exhibited specificity toward FRα-positive lung tumor cells resulting in antitumor activity, we next conducted pilot in vivo studies to determine if a similar antitumor activity was observed. For this purpose, subcutaneous lung tumor xenografts were established in nude mice by injecting H1299 lung tumor cells expressing the luciferase (luc) marker gene. The expression of luciferase by the tumor cells allows for monitoring tumor growth by noninvasive molecular imaging. When the tumors were about 50–100 mm in size, mice were injected intravenously with HuR-FNP via the tail vein. The HuR siRNA was fluorescently tagged with DY647 dye such that we could noninvasively monitor the nanoparticle biodistribution and accumulation in the tumor and other organs over time using the IVIS Spectrum (PerkinElmer, Waltham, MA) molecular imaging system. As shown in Fig. 17.3a, colocalization of fluorescently tagged HuR siRNA within the luciferase-positive H1299 tumor showed efficient accumulation of the particles in the tumor. The results also revealed that the FNPs were selectively internalized by the tumors compared to the other surrounding organs. From our preliminary study results, we can speculate that the nonspecific uptake of the particles by RES is significantly reduced and that pegylation of the NPs facilitated in reducing the RES-mediated clearance of the NPs.

To evaluate whether the accumulation of HuR-FNP in the tumor caused HuR gene silencing, tumors were harvested and analyzed for HuR protein expression. Western blotting showed that HuR protein expression was significantly reduced in tumors harvested from mice that received HuR-FNP treatment compared to HuR expression in tumor harvested from control mice that did not receive any treatment (Fig. 17.3b). Associated with the reduction in HuR protein expression was the reduction in cyclin D, cyclin E, and COX2 protein expression in the tumors from HuR-FNP-treated mice. Our preliminary in vitro and in vivo study results demonstrated that lung tumor-targeted delivery of HuR siRNA using FNPs suppresses cell proliferation and induces cell cycle arrest by global knockdown of HuR-regulated proteins resulting in an antitumor activity. While our study results are interesting, it is to be noted that additional in vitro and in vivo studies are warranted. In vivo studies that are currently underway in the laboratory are focused on determining HuR-FNP biodistribution, pharmacokinetics, and efficacy. The results of these ongoing studies are expected to be available in the future.