Triple-negative breast cancer (TNBC) is an aggressive disease with a short median time from relapse to death. The increased aggressiveness, drug resistance, disease relapse, and metastasis are associated with the presence of stem cells within tumors. Several stem cell markers, such as CD24, CD44, CD133, ALDH1, and ABCG2, have been reported, but their roles in breast cancer tumorigenesis remain unclear. Herein, we apply RNA nanotechnology to deliver anti-microRNA (miRNA) for TNBC therapy. The thermodynamically and chemically stable three-way junction (3WJ) motif was utilized as the scaffold to carry an RNA aptamer binding to CD133 receptor and a locked nuclei acid (LNA) sequence for miRNA21 inhibition. Binding assays revealed the specific uptake of the nanoparticles to breast cancer stem cells (BCSCs) and TNBC cells. Functional assays showed that cancer cell migration was reduced, miR21 expression was inhibited, and downstream tumor suppressor PTEN and PDCD4 expressions were upregulated. In vitro and in vivo studies revealed that these therapeutic RNA nanoparticles did not induce cytokine secretion. Systemic injection of these RNA nanoparticles in animal trial demonstrated high specificity in TNBC tumor targeting and high efficacy for tumor growth inhibition. These results revealed the clinical translation potential of these RNA nanoparticles for TNBC therapy.

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An idea drug carrier, with good binding affinity, selectivity, drug payload capacity, and cellular internalized capability, will greatly improve the efficiency of target delivery. Herein a self-assembled and multivalent DNA nanostructure was developed as drug carrier for efficient and targeted delivery. The DNA structure was similar to that of a centipede, composed of trunk and legs: The trunk was a self-assembled DNA scaffold via hybridization chain reaction (HCR) from two biotinylated hairpin monomers created upon initiation by a trigger DNA, and the legs were biotinylated aptamers conjugated to the trunk via streptavidin-biotin affinity interaction. The long trunk of the "DNA nanocentipede" was loaded with doxorubicin (Dox), and the legs were SMMC-7721 cell-binding aptamers (Zy1) which functioned as targeting moieties to firmly and selectively grasp target cells. The results of agarose gel electrophoresis and fluorescence anisotropy confirmed that Zy1-based DNA nanocentipedes (Zy1-Nces) were successfully constructed. Flow cytometric analyses demonstrated that Zy1-Nces were more effective than free Zy1 in binding affinity and selectivity due to a multivalent effect. Confocal microscopy studies demonstrated that the internalization was highly dependent on the higher valences of DNA nanocentipedes without the loss of selectivity. Meanwhile, Zy1-Nces exhibited high drug-loading capacity and selective drug transport. The results of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed enhanced cellular cytotoxicity of the Dox-loaded Zy1-Nces (Zy1-Nces-Dox) to the target SMMC-7721 cells but not negative control L02 cells. This approach is applicable to prepare drug carriers for other targets by construction of the nanocentipedes with relevant nucleic acid fragments.

Rubber is a fascinating material in both industry and daily life. The development of elastomeric material in nanotechnology is imperative due to its economic and technological potential. By virtue of their distinctive physicochemical properties, nucleic acids have been extensively explored in material science. Phi29 DNA packaging motor contains a 3WJ with three angles of 97°, 125°, and 138°. Here, the rubber-like property of RNA architectures was investigated using optical tweezers and in vivo imaging technologies. The 3WJ 97° interior angle was contracted or stretched to 60°, 90°, and 108° at will to build elegant RNA triangles, squares, pentagons, cubes, tetrahedrons, dendrimers, and prisms. RNA nanoarchitectures was stretchable and shrinkable by optical tweezer with multiple extension and relaxation repeats like a rubber. Comparing to gold and iron nanoparticles with the same size, RNA nanoparticles display stronger cancer-targeting outcomes while less accumulation in healthy organs. Generally, the upper limit of renal excretion is 5.5-nm, however, the 5, 10, and 20-nm RNA nanoparticles passed the renal filtration and resume their original structure identified in urine. These findings solve two previous mysteries: 1) why RNA nanoparticles have unusual high tumor targeting efficiency since their rubber or amoeba-like deformation property enables them to squeeze out of the leaky vasculature to improve EPR effect; 2) why RNA nanoparticles remain nontoxic since they can be rapidly cleared from the body via renal excretion into urine with little accumulation in the body. Considering its controllable shape and size plus its rubber-like property, RNA holds great promises for industrial and biomedical applications especially in cancer therapeutics delivery.

Cancer stem-like cells, which have been described as tumor-initiating cells or tumor-propagating cells, play a crucial role in our fundamental understanding of glioblastoma multiforme (GBM) and its recurrence. GBM is a lethal cancer, characterized by florid vascularization and aberrantly elevated vascular endothelial growth factor (VEGF). VEGF promotes tumorigenesis and angiogenesis of human GBM stem-like cells (GBSCs). However, whether and how VEGF contributes to GBSCs proliferation remain largely uncertain. In this study, human GBSCs were isolated from surgical specimens of glioblastoma and cultured in medium favored for stem cell growth. Neural Colony-Forming Cell Assay and ATP assay were performed to measure GBSC proliferation under normoxia (20% O2) and hypoxia (1% O2). Our observations demonstrate that exogenous VEGF stimulates GBSC proliferation in a dose-dependent manner via VEGF Receptor 2 (VEGFR2); while VEGF Receptor 1 (VEGFR1) has a negative feedback effect on VEGFR2 when cells were exposed to higher concentration of VEGF. These results suggest that suppressing VEGFR2-dependent GBSC proliferation is a potentially therapeutic strategy in GBM.

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The past decades have witnessed the successful transition of several nanotechnology platforms into the clinical trials. However, specific delivery of therapeutics to tumors is hindered by several barriers including cancer recognition and tissue penetration, particle heterogeneity and aggregation, and unfavorable pharmacokinetic profiles such as fast clearance and organ accumulation. With the advent of RNA nanotechnology, a series of RNA nanoparticles have been successfully constructed to overcome many of the aforementioned challenges for in vivo cancer targeting with favorable biodistribution profiles. Compared to other nanodelivery platforms, the physiochemical properties of RNA nanoparticles can be tuned with relative ease for investigating the in vivo behavior of nanoparticles upon systemic injection. The size, shape, and surface chemistry, especially hydrophobic modifications, exert significant impacts on the in vivo fate of RNA nanoparticles. Rationally designed RNA nanoparticles with defined stoichiometry and high homogeneity have been demonstrated to specifically target tumor cells while avoiding accumulation in healthy vital organs after systemic injection. RNA nanoparticles were proven to deliver therapeutics such as siRNA and anti-miRNA to block tumor growth in several animal models. Although the release of anti-miRNA from the RNA nanoparticles has achieved high efficiency of tumor regression in multiple animal models, the efficiency of endosomal escape for siRNA delivery needs further improvement. This review focuses on the advances and perspectives of this promising RNA nanotechnology platform for cancer targeting and therapy.

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