SUMMARY Triple negative breast cancer (TNBC) is a heterogeneous, complex, and aggressive breast cancer subtype. TNBC patients respond poorly to chemotherapy, leading to high mortality rates and a worsening prognosis. RNAi therapeutics, including siRNA and miRNA, have shown tremendous potential for TNBC cancer therapy. Developing a safe targeted delivery system with endosomal avoidance of the payload is crucial in terms of realizing the full potential of RNAi therapeutics and could revolutionize clinical treatment of TNBC. Our group has demonstrated that delivery of anti-miR21 to TNBC can efficiently inhibit TNBC cell proliferation (Shu D, et al. ACS Nano. 2015, 27:9731; Yin H, et al. Shu D. Mol Therapy. 2019, 27:1252). We have also recently reported the use of RNA nanoparticle orientation for decorating exosome surfaces with targeting ligands to deliver siRNA loaded exosomes for TNBC treatment. We proved that exosomes can be utilized as nanocarriers to deliver siRNA to TNBC cancer cells very efficiently to inhibit cancer growth (Pi F, Binzel D et al. Nat. Nanotechnol. 2018;13:82). Preliminary data from the investigation with KB cell models in vitro has shown the mechanism behind the high efficiency of cancer inhibition to be the cytosolic delivery of siRNA via exosomes without endosomal trapping (Zhen Z, et al. J Control Release. 2019,311:43). In this study, we will investigate the targeting and delivery mechanisms of RNAi to TNBC cells by exosomes displayed with TNBC specific ligands for targeted delivery. It’s our goal to select a lead TNBC therapeutic candidate to move towards potential clinical translation. We will combine the targeting and drug delivery capabilities of RNA nanotechnology and exosomes for targeted delivery of RNAi to cell cytosols without endosomal entrapment. We will construct and evaluate the multi-functional exosomal RNA nanoparticle complexes harboring targeting ligands including RNA aptamers (EGFRapt, or alternatively, CD133apt, CD44apt, and nAHRsapt) or chemical ligand (Methotrexate) and tumor suppressing RNAi therapeutics (siRNA, suppressor miRNAs and anti- oncogenic miRNA). We will further investigate the pathways of internalization and intracellular trafficking in addition to investigating whether the payloads in the exosome are delivered to cytosol via fusion or to the endosome via endocytosis. In depth studies of the release and cellular processing of RNAi cargo loaded into exosomes will be completed. We will investigate the PK/PD parameters, delivery mechanisms, antitumor efficacy, and safety of the therapeutic RNA nanoparticles in order to select a lead candidate for preclinical translation. The preclinical studies will give a clear understanding of the viability of exosome-RNA nanoparticle complexes for TNBC therapy and provide data to move towards an Investigational New Drug (IND) application that will facilitate advancement to clinical trials.