Enhancing RNA Payload and Temperature Stability and Activity with Cationic Peptide-Coated Zinc Oxide Nanoparticles.

The lipid nanoparticle (LNP) mRNA vaccine was first tested through clinic but suffered from relatively low RNA payloads and poor temperature stability. Our lab patented a protamine-coated particle approach for temperature-stabilizing DNA vaccines, translating this successfully to the clinic. In subsequent work, we have characterized RNA interaction and delivery by zinc oxide nanoparticles, filing a patent most recently entitled RNA-stabilizing nanoparticles, similarly utilizing protamine-coated zinc oxide nanoparticles for RNA. Here, we present this data for the first time. Briefly, ZnO, ZnO-protamine, and ZnO-protamine-RNA were characterized by size and zeta potential analyses and the RNA-loaded nanoparticles were visualized by transmission electron microscopy. UV spectroscopic analysis demonstrated up to 95-98% loading efficiency with protamine and approximately 75% loading efficiency with LL37, another cationic antiviral peptide. Elution of the RNA isolated from the particles afforded a calculation in three independent trials where RNA payloads ranged from 18 to 45 µg of RNA per 0.5 mg of coated particles. Circular dichroism (CD) analysis indicated that binding of RNA to ZnO NPs stabilized, enhancing the pattern with a clear dependence on the RNA:ZnO stoichiometry. Enhanced temperature stability was shown by differential scanning calorimetry (DSC), gel electrophoresis, and in vitro mRNA expression analysis. Using poly I:C RNA with a well-defined melting point (64.3 ± 0.32 °C), formation of the ZnO:RNA complex increased the RNA melting point (70.9 ± 0.62 °C). After refrigerated or room-temperature storage or incubation at 30, 40, or 50 °C, RNA comigration with the control RNA was recovered from all samples, exposed to either 14 or 100 nm ZnO, and coated with protamine. Furthermore, the ZnO-protamine-mRNA samples retained significantly higher expression activity when incubated at these elevated temperatures. Finally, the ZnO-protamine-mRNA was functionally active for in vitro translation, in cell extracts, and in cells for expression of GFP, luciferase, and COVID spike protein. These data support further preclinical development of ZnO-protamine-mRNA.

Evidence of Copper Nanoparticles and Poly I:C Modulating Cas9 Interaction and Cleavage of COR (Conserved Omicron RNA).

Conserved omicron RNA (COR) is a 40 base long 99.9% conserved sequence in SARS-CoV-2 Omicron variant, predicted to form a stable stem loop, the targeted cleavage of which can be an ideal next step in controlling the spread of variants. The Cas9 enzyme has been traditionally utilized for gene editing and DNA cleavage. Previously Cas9 has been shown to be capable of RNA editing under certain conditions. Here we investigated the ability of Cas9 to bind to single-stranded conserved omicron RNA (COR) and examined the effect of copper nanoparticles (Cu NPs) and/or polyinosinic-polycytidilic acid (poly I:C) on the RNA cleavage ability of Cas9. The interaction of the Cas9 enzyme and COR with Cu NPs was shown by dynamic light scattering (DLS) and zeta potential measurements and was confirmed by two-dimensional fluorescence difference spectroscopy (2-D FDS). The interaction with and enhanced cleavage of COR by Cas9 in the presence of Cu NPs and poly I:C was shown by agarose gel electrophoresis. These data suggest that Cas9-mediated RNA cleavage may be potentiated at the nanoscale level in the presence of nanoparticles and a secondary RNA component. Further explorations in vitro and in vivo may contribute to the development of a better cellular delivery platform for Cas9.

Interaction of Ras Binding Domain (RBD) by chemotherapeutic zinc oxide nanoparticles: Progress towards RAS pathway protein interference.

Zinc oxide (ZnO) NP is considered as a nanoscale chemotherapeutic. Thus, the drug delivery of this inorganic NP is of considerable importance. Ras mutations are common in cancer and the activation of this signaling pathway is a hallmark in carcinoma, melanoma and many other aggressive malignancies. Thus, here we examined the binding and delivery of Ras binding domain (RBD), a model cancer-relevant protein and effector of Ras by ZnO NP. Shifts in zeta potential in water, PBS, DMEM and DMEM supplemented with FBS supported NP interaction to RBD. Fluorescence quenching of the NP was concentration-dependent for RBD, Stern-Volmer analysis of this data was used to estimate binding strength which was significant for ZnO-RBD (Kd < 10-5). ZnO NP interaction to RBD was further confirmed by pull-down assay demonstrated by SDS-PAGE analysis. The ability of ZnO NP to inhibit 3-D tumor spheroid was demonstrated in HeLa cell spheroids-the ZnO NP breaking apart these structures revealing a significant (>50%) zone of killing as shown by light and fluorescence microscopy after intra-vital staining. ZnO 100 nm was superior to ZnO 14 nm in terms of anticancer activity. When bound to ZnO NP, the anticancer activity of RBD was enhanced. These data indicate the potential diagnostic application or therapeutic activity of RBD-NP complexes in vivo which demands further investigation.

Comparative Molecular Immunological Activity of Physiological Metal Oxide Nanoparticle and its Anticancer Peptide and RNA Complexes.

Currently, there is a great interest in nanoparticle-based vaccine delivery. Recent studies suggest that nanoparticles when introduced into the biological milieu are not simply passive carriers but may also contribute immunological activity themselves or of their own accord. For example there is considerable interest in the biomedical applications of one of the physiologically-based inorganic metal oxide nanoparticle, zinc oxide (ZnO). Indeed zinc oxide (ZnO) NP are now recognized as a nanoscale chemotherapeutic or anticancer nanoparticle (ANP) and several recent reports suggest ZnO NP and/or its complexes with drug and RNA induce a potent antitumor response in immuno-competent mouse models. A variety of cell culture studies have shown that ZnO NP can induce cytokines such as IFN-γ, TNF-α, IL-2, and IL-12 which are known to regulate the tumor microenvironment. Much less work has been done on magnesium oxide (MgO), cobalt oxide (Co3O4), or nickel oxide (NiO); however, despite the fact that these physiologically-based metal oxide NP are reported to functionally load and assemble RNA and protein onto their surface and may thus also be of potential interest as nanovaccine platform. Here we initially compared in vitro immunogenicity of ZnO and Co3O4 NP and their effects on cancer-associated or tolerogenic cytokines. Based on these data we moved ZnO NP forward to testing in the ex vivo splenocyte assay relative to MgO and NiO NP and these data showed significant difference for flow cytometry sorted population for ZnO-NP, relative to NiO and MgO. These data suggesting both molecular and cellular immunogenic activity, a double-stranded anticancer RNA (ACR), polyinosinic:poly cytidylic acid (poly I:C) known to bind ZnO NP; when ZnO-poly I:C was injected into B16F10-BALB/C tumor significantly induced, IL-2 and IL-12 as shown by Cohen's d test. LL37 is an anticancer peptide (ACP) currently in clinical trials as an intratumoral immuno-therapeutic agent against metastatic melanoma. LL37 is known to bind poly I:C where it is thought to compete for receptor binding on the surface of some immune cells, metastatic melanoma and lung cells. Molecular dynamic simulations revealed association of LL37 onto ZnO NP confirmed by gel shift assay. Thus using the well-characterized model human lung cancer model cell line (BEAS-2B), poly I:C RNA, LL37 peptide, or LL37-poly I:C complexes were loaded onto ZnO NP and delivered to BEAS-2B lung cells, and the effect on the main cancer regulating cytokine, IL-6 determined by ELISA. Surprisingly ZnO-LL37, but not ZnO-poly I:C or the more novel tricomplex (ZnO-LL37-poly I:C) significantly suppressed IL-6 by >98-99%. These data support the further evaluation of physiological metal oxide compositions, so-called physiometacomposite (PMC) materials and their formulation with anticancer peptide (ACP) and/or anticancer RNA (ACR) as a potential new class of immuno-therapeutic against melanoma and potentially lung carcinoma or other cancers.

Translating Nanomedicine to Comparative Oncology-the Case for Combining Zinc Oxide Nanomaterials with Nucleic Acid Therapeutic and Protein Delivery for Treating Metastatic Cancer.

The unique anticancer, biochemical, and immunologic properties of nanomaterials are becoming a new tool in biomedical research. Their translation into the clinic promises a new wave of targeted therapies. One nanomaterial of particular interest are zinc oxide (ZnO) nanoparticles (NPs), which has distinct mechanisms of anticancer activity including unique surface, induction of reactive oxygen species, lipid oxidation, pH, and also ionic gradients within cancer cells and the tumor microenvironment. It is recognized that ZnO NPs can serve as a direct enzyme inhibitor. Significantly, ZnO NPs inhibit extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) associated with melanoma progression, drug resistance, and metastasis. Indeed, direct intratumoral injection of ZnO NPs or a complex of ZnO with RNA significantly suppresses ERK and AKT phosphorylation. These data suggest ZnO NPs and their complexes or conjugates with nucleic acid therapeutic or anticancer protein may represent a potential new strategy for the treatment of metastatic melanoma, and potentially other cancers. This review focuses on the anticancer mechanisms of ZnO NPs and what is currently known about its biochemical effects on melanoma, biologic activity, and pharmacokinetics in rodents and its potential for translation into large animal, spontaneously developing models of melanoma and other cancers, which represent models of comparative oncology.