Gene therapy and RNA delivery require a nanoparticle (NP) to stabilize these nucleic acids when administered in vivo. The presence of degradative hydrolytic enzymes within these environments limits the nucleic acids’ pharmacologic activity. This study compared the effects of nanoscale ZnO and MgO in the protection afforded to DNA and RNA from degradation by DNase, serum or tumor homogenate. For double-stranded plasmid DNA degradation by DNase, our results suggest that the presence of MgO NP can protect DNA from DNase digestion at an elevated temperature (65 °C), a biochemical activity not present in ZnO NP-containing samples at any temperature. In this case, intact DNA was remarkably present for MgO NP after ethidium bromide staining and agarose gel electrophoresis where these same stained DNA bands were notably absent for ZnO NP. Anticancer RNA, polyinosinic-polycytidylic acid (poly I:C) is now considered an anti-metastatic RNA targeting agent and as such there is great interest in its delivery by NP. For it to function, the NP must protect it from degradation in serum and the tumor environment. Surprisingly, ZnO NP protected the RNA from degradation in either serum-containing media or melanoma tumor homogenate after gel electrophoretic analysis, whereas the band was much more diminished in the presence of MgO. For both MgO and ZnO NP, buffer-dependent rescue from degradation occurred. These data suggest a fundamental difference in the ability of MgO and ZnO NP to stabilize nucleic acids with implications for DNA and RNA delivery and therapy.

African Swine Fever Virus (ASFV) poses a serious threat to the pork industry worldwide; however, there is no safe vaccine or treatment available. The development of an efficacious subunit vaccine will require the identification of protective antigens. The ASFV pp220 polyprotein is essential for virus structural integrity. This polyprotein is processed to generate p5, p34, p14, p37, and p150 individual proteins. Immunization of pigs with a cocktail of adenoviruses expressing the proteins induced significant IgG, IFN-γ-secreting cells, and cytotoxic T lymphocyte responses. Four predicted SLA-I binding nonamer peptides, namely p34161−169, p37859−867, p1501363−1371, and p1501463−1471, recalled strong IFN-γ+ PBMC and splenocyte responses. Notably, peptide p34161−169 was recognized by PBMCs isolated from 7/10 pigs and by splenocytes isolated from 8/10 pigs. Peptides p37859−867 and p1501363−1371 stimulated recall IFN-γ+ responses in PBMCs and splenocytes isolated from 8/10 pigs, whereas peptide p1501463−1471 recalled responses in PBMCs and splenocytes isolated from 7/10 to 9/10 pigs, respectively. The results demonstrate that the pp220 polyprotein contains multiple epitopes that induce robust immune responses in pigs. Importantly, these epitopes are 100% conserved among different ASFV genotypes and were predicted to bind multiple SLA-I alleles. The outcomes suggest that pp220 is a promising candidate for inclusion in a prototype subunit vaccine.

IntroductionBiomedical applications of nanoparticles (NPs) as enzyme inhibitors have recently come to light. Oxides of metals native to the physiological environment (eg, Fe, Zn, Mg, etc.) are of particular interest—especially the functional consequences of their enzyme interaction.Materials and methodsHere, Fe2O3, zinc oxide (ZnO), magnesium oxide (MgO) and nickel oxide (NiO) NPs are compared to copper (Cu) and boron carbide (B4C) NPs. The functional impact of NP interaction to the model enzyme luciferase is determined by 2-dimensional fluorescence difference spectroscopy (2-D FDS) and 2-dimensional photoluminescence difference spectroscopy (2-D PLDS). By 2-D FDS analysis, the change in maximal intensity and in 2-D FDS area under the curve (AUC) is in the order Cu~B4C>ZnO>NiO>>Fe2O3>MgO. The induced changes in protein conformation are confirmed by tryptic digests and gel electrophoresis.ResultsAnalysis of possible trypsin cleavage sites suggest that cleavage mostly occurs in the range of residues 112–155 and 372–439, giving a major 45 kDa band. By 2-D PLDS, it is found that B4C NPs completely ablate bioluminescence, while Cu and Fe2O3 NPs yield a unique bimodal negative decay rate, −7.67×103 and −3.50×101 relative light units respectively. Cu NPs, in particular, give a remarkable 271% change in enzyme activity. Molecular dynamics simulations in water predicted that the surfaces of metal oxide NPs become capped with metal hydroxide groups under physiological conditions, while the surface of B4C becomes populated with boronic acid or borinic acid groups. These predictions are supported by the experimentally determined zeta potential. Thin layer chromatography patterns further support this conception of the NP surfaces, where stabilizing interactions were in the order ionic>polar>non-polar for the series tested.ConclusionOverall the results suggest that B4C and Cu NP functional dynamics on enzyme biochemistry are unique and should be examined further for potential ramifications on other model, physiological or disease-relevant enzymes.

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