Nanoparticle Delivery of siRNA for the Silencing of TWIST1 in Metastatic Cancer.

摘要

Metatstatic disease is the primary cause of mortality in cancer patients. Cancer cells spreading beyond the primary tumor can ultimately lead to organ failure. Traditional therapeutic approaches (surgery, radiation, and chemotherapy) can be very effective in the treatment of primary solid tumors. However, ease of treatment and survival rates drastically decrease as tumor cells migrate beyond the initial neoplastic lesion. The pathway to metastatic lesions occurs when cells detach from neighboring cells, invade through the basement membrane, enter vessels (blood or lymphatic), travel to distant tissues, and begin to divide in a new location.;There are two processes that occur along the metastatic pathway that are crucial to its advancement. First, cancer cells must undergo a phenotypic alteration to detach from their neighboring cells and become motile. Cancers which are epithelial in origin undergo a process called Epithelial-Mesenchymal Transition (EMT) in which cells change from a stationary, fully adherent cell into a spindle-shaped mobile cell capable of invasive activity. The second crucial process in the metastatic pathway is angiogenesis in which extensions of current blood vessels are formed in and around a growing tumor following signaling from oxygen deprived tumor cells. Angiogenesis is absolutely essential for sustained tumor growth as it allows delivery of oxygen and nutrients to rapidly dividing tumor cells. Furthermore, newly grown vessels allow newly detached cells to enter circulation. Therefore, angiogenesis not only promotes tumor growth it also allows for dissemination of tumor cells that ultimately can become metastatic lesions.;Because EMT and angiogenesis are essential processes of metastasis and tumor growth they have emerged as attractive therapeutic targets. The transcription factor TWIST1 is a known regulator of both EMT and angiogenesis. TWIST1 is highly expressed in early embryogenesis during neural crest cell migration and mesoderm formation in species ranging from flies to humans. TWIST1 is mostly silenced in adult tissues. However, in many cancers TWIST1 is reactivated thus allowing cells to undergo EMT and promote angiogenesis. Therefore, a main goal of this project was to knock down expression of TWIST1 to hinder cellular migration (EMT) and angiogenesis.;RNA interference (RNAi) uses endogenous cellular machinery to silence protein expression at the level of mRNA. Here, two siRNA against TWIST1 (si419 and si494) were designed and tested in two metastatic cancer cell lines (breast and melanoma). Both siRNA were shown to be highly efficient at reducing TWIST1 expression. Chemical modifications were incorporated into si419 to enhance its stability and efficacy in an in vivo environment.;Delivery remains one of the chief obstacles in successful siRNA therapy. Two unique nanoparticle delivery methods were optimized and tested here. First, a polyamidoamine (PAMAM) dendrimer (YTZ3-15) was used to silence TWIST1 in SUM1315 breast cancer cells. YTZ3-15 was complexed with fluorescently labeled siRNA to form a dendriplex. The dendriplex served to protect the siRNA and facilitate its intracellular delivery. In vitro testing revealed significant TWIST1 knock down, TWIST1 target gene knock down, and migration inhibition. In vivo biodistribution testing of YTZ3-15 dendriplexes (with control siRNA) showed substantial accumulation within the orthotopic tumors via intratumoral and intravenous administration.;The second siRNA delivery system used was mesoporous silica nanopartiles (MSN). MSNs are round, ~120 nm, porous silica spheres capable of carrying a variety of surface attachments and modifications to suit specific cargo and targeting needs. For siRNA delivery, a cationic polyethylenimine (PEI) coating as added to the MSNs. Successful siRNA delivery using MSNs was observed using confocal microscopy and resulted in TWIST1 knockdown. Interleukin 8 (IL-8, a contributor to angiogenesis) levels also decreased secondary to TWIST1 knockdown. In vivo a significant decrease in tumor weight was observed following MSN+siRNA IV delivery over a 5 week period, but only with the chemically modified si419H. Collected tumor samples from the mice treated MSN+si419H and MSN+si494 showed significant decreases in TWIST1, the EMT marker Vimentin, and the angiogenesis promoting CCL2. Gross and histopathologic examination of mouse tissues did not reveal any adverse effects associated with MSN+siRNA treatment. There was no difference in number of metastatic lesions among the treatment groups, though this could have been due to experimental design shortcomings.;Taken together these findings suggest that an siRNA approach to TWIST1 silencing via nanoparticle delivery merits further investigation. In particular, there is significant untapped potential with the MSNs. They are capable of not only delivering siRNA, but also can carry chemotherapies within its pores thereby allowing for a double therapeutic approach. Furthermore, cancer cell targeting moieties can also be added to MSNs to allow for increased accumulation within tumors as well as decreased overall drug dosage.