Spherical nucleic acids (SNAs) are highly oriented, well organized, polyvalent structures of nucleic acids conjugated to hollow or solid core nanoparticles. Because they can transfect many tissue and cell types without toxicity, induce minimum immune response, and penetrate various biological barriers (such as the skin, blood-brain barrier, and blood-tumor barrier), they have become versatile tools for the delivery of nucleic acids, drugs, and proteins for various therapeutic purposes. This article describes the unique structures and properties of SNAs and discusses how these properties enable their application in gene regulation, immunomodulation, and drug and protein delivery. It also summarizes current efforts towards clinical translation of SNAs and provides an expert opinion on remaining challenges to be addressed in the path forward to the clinic.

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Educating our immune system via vaccination is an attractive approach to combat infectious diseases. Eliciting antigen specific cytotoxic T cells (CTLs), CD8+ effector T cells, is essential in controlling intracellular infectious diseases such as influenza (Flu), tuberculosis (TB), hepatitis, and HIV/AIDS, as well as tumors. However, vaccination utilizing subunit peptides to elicit a potent CD8+ T cell response with antigenic peptides is typically ineffective due to poor immunogenicity. Here we have engineered a reduction sensitive nanoparticle (NP) based subunit vaccine for intracellular delivery of an antigenic peptide and immunostimulatory adjuvant. We have co-conjugated an antigenic peptide (ovalbumin-derived CTL epitope [OVA257–264: SIINFEKL]) and an immunostimulatory adjuvant (CpG ODNs, TLR9 agonist) to PEG hydrogel NPs via a reduction sensitive linker. Bone-marrow derived dendritic cells (BMDCs) treated with the SIINFEKL conjugated NPs efficiently cross-presented the antigenic peptide via MHC-I surface receptor and induced proliferation of OT-I T cells. CpG ODN-conjugated NPs induced maturation of BMDCs as evidenced by the overexpression of CD80 and CD40 costimulatory receptors. Moreover, codelivery of NP conjugated SIINFEKL and CpG ODN significantly increased the frequency of IFN-γ producing CD8+ effector T cells in mice (~6-fold improvement over soluble antigen and adjuvant). Furthermore, the NP subunit vaccine-induced effector T cells were able to kill up to 90% of the adoptively transferred antigenic peptide-loaded target cell. These results demonstrate that the reduction sensitive NP subunit vaccine elicits a potent CTL response and provide compelling evidence that this approach could be utilized to engineer particulate vaccines to deliver tumor or pathogen associated antigenic peptides to harness the immune system to fight against cancer and infectious diseases.

Introduction MicroRNAs (miRNAs) are short noncoding RNAs whose ability to regulate the expression of multiple genes makes them potentially exciting tools to treat disease. Unfortunately, miRNAs cannot passively enter cells due to their hydrophilicity and negative charge. Here, we report the development of layer-by-layer assembled nanoshells (LbL-NS) as vehicles for efficient intracellular miRNA delivery. Specifically, we developed LbL-NS to deliver the tumor suppressor miR-34a into triple-negative breast cancer (TNBC) cells, and demonstrate that these constructs can safely and effectively regulate the expression of SIRT1 and Bcl-2, two known targets of miR-34a, to decrease cell proliferation. Methods LbL-NS were made by coating negatively charged nanoshells with alternating layers of positive poly-L-lysine (PLL) and negative miRNA, with the outer layer consisting of PLL to facilitate cellular entry and protect the miRNA. Electron microscopy, spectrophotometry, dynamic light scattering, and miRNA release studies were used to characterize LbL-NS. The particles’ ability to enter MDA-MB-231 TNBC cells, inhibit SIRT1 and Bcl-2 expression, and thereby reduce cell proliferation was examined by confocal microscopy, Western blotting, and EdU assays, respectively. Results Each successive coating reversed the nanoparticles’ charge and increased their hydrodynamic diameter, resulting in a final diameter of 208±4 nm and a zeta potential of 53±5 mV. The LbL-NS released ~30% of their miR-34a cargo over 5 days in 1X PBS. Excitingly, LbL-NS carrying miR-34a suppressed SIRT1 and Bcl-2 by 46±3% and 35±3%, respectively, and decreased cell proliferation by 33%. LbL-NS carrying scrambled miRNA did not yield these effects. Conclusion LbL-NS can efficiently deliver miR-34a to TNBC cells to suppress cancer cell growth, warranting their further investigation as tools for miRNA replacement therapy.

Abnormal expression of microRNAs (miRNAs), which are highlyconserved noncoding RNAs that regulate the expression of various genes post transcriptionally to control cellular functions, has been associated with the development of many diseases. In some cases, disease‐promoting miRNAs are upregulated, while in other instances disease‐suppressive miRNAs are downregulated. To alleviate this imbalanced miRNA expression, either antagomiRs or miRNA mimics can be delivered to cells to inhibit or promote miRNA expression, respectively. Unfortunately, the clinical translation of bare antagomiRs and miRNA mimics has been challenging because nucleic acids are susceptible to nuclease degradation, display unfavorable pharmacokinetics, and cannot passively enter cells. This review emphasizes the challenges associated with miRNA mimic delivery and then discusses the design and implementation of polymer nanocarriers to overcome these challenges. Preclinical efforts are summarized, and a forward‐looking perspective on the future clinical translation of polymer nanomaterials as miRNA delivery vehicles is provided. © 2019 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48651.

CpG oligodeoxynucleotides are potent toll-like receptor (TLR) 9 agonists and have shown promise as anticancer agents in preclinical studies and clinical trials. Binding of CpG to TLR9 initiates a cascade of innate and adaptive immune responses, beginning with activation of dendritic cells and resulting in a range of secondary effects that include the secretion of pro-inflammatory cytokines, activation of natural killer cells, and expansion of T cell populations. Recent literature suggests that local delivery of CpG in tumors results in superior antitumor effects as compared to systemic delivery. In this study, we utilized PRINT (particle replication in nonwetting templates) nanoparticles as a vehicle to deliver CpG into murine lungs through orotracheal instillations. In two murine orthotopic metastasis models of non-small-cell lung cancer–344SQ (lung adenocarcinoma) and KAL-LN2E1 (lung squamous carcinoma), local delivery of PRINT-CpG into the lungs effectively promoted substantial tumor regression and also limited systemic toxicities associated with soluble CpG. Furthermore, cured mice were completely resistant to tumor rechallenge. Additionally, nanodelivery showed extended retention of CpG within the lungs as well as prolonged elevation of antitumor cytokines in the lungs, but no elevated levels of proinflammatory cytokines in the serum. These results demonstrate that PRINT-CpG is a potent nanoplatform for local treatment of lung cancer that has collateral therapeutic effects on systemic disease and an encouraging toxicity profile and may have the potential to treat lung metastasis of other cancer types.

Chemosensitizers can improve the therapeutic index of chemotherapy and overcome treatment resistance. Successful translation of chemosensitizers depends on the development of strategies that can preferentially deliver chemosensitizers to tumors while avoiding normal tissue. We hypothesized that nanoparticle (NP) formulation of chemosensitizers can improve their delivery to tumors which can in turn improve their therapeutic index. To demonstrate the proof of principle of this approach, we engineered NP formulations of two chemosensitizers, the PI3-kindase inhibitor wortmanin (Wtmn) and the PARP inhibitor olaparib. NP Wtmn and NP olaparib were evaluated as chemosensitizers using lung cancer cells and breast cancer cells respectively. We found Wtmn to be an efficient chemosensitizer in all tested lung-cancer cell lines reducing tumor cell growth between 20 and 60% compared to drug alone. NP formulation did not decrease its efficacy in vitro. Olaparib showed less consistent chemosensitization as a free drug or in NP formulation. NP Wtmn was further evaluated as a chemosensitizer using mouse models of lung cancer. We found that NP Wtmn is an effective chemosensitizer and more effective than free Wtmn showing a 32% reduction in tumor growth compared to free Wtmn when given with etoposide. Importantly, NP Wtmn was able to sensitize the multi-drug resistant H69AR cells to etoposide. Additionally, the combination of NP Wtmn and etoposide chemotherapy did not significantly increase toxicity. The present study demonstrates the proof of principle of using NP formulation of chemosensitizing drugs to improve the therapeutic index of chemotherapy.