Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions

Citation: Hurst, M. N., & DeLong, R. K. (2016). Two-Dimensional Fluorescence Difference Spectroscopy to Characterize Nanoparticles and their Interactions. Scientific Reports, 6, 9. https://doi.org/10.1038/srep33287Two dimensional fluorescence difference spectroscopy (2D FDS) detects nanoparticle interactions following surface functionalization and biomolecule loading by generating a spectral signature of the fluorescent intensity per excitation and emission wavelengths. Comparing metal oxide nanoparticles revealed a unique spectral signature per material composition. 2D FDS showed to be sensitive to changes in surface properties between ZnO NPs synthesized by different methods. ZnO NP loaded with glycol chitosan, polyacrylic acid (PAA), or methoxy polyethylene glycol (mPEG) exhibited a distinct spectral signature shift. ZnO NP loaded with Torula Yeast RNA (TYRNA)(640 nm), polyinosinic: polycytidylic acid (pIC)(680 nm), or splice switching oligonucleotide (SSO)(650 nm) each revealed a shift in emission. Ras-Binding domain (RBD) at three concentrations (25, 37.5, 50 mu g/mL) showed that fluorescent intensity was inversely related to the concentration of protein loaded. These data support 2D FDS as a novel technique in identifying nanoparticles and their surface interactions as a quality assurance tool

Characterization and performance of nucleic acid nanoparticles combined with protamine and gold

Macromolecular nucleic acids such as DNA vaccines, siRNA, and splice-site switching oligomers (SSO) have vast chemotherapeutic potential. Nanoparticulate biomaterials hold promise for DNA and RNA delivery when a means for binding is identified that retains structure-function and provides stabilization by the nanoparticles. In order to provide these benefits of binding, we combined DNA and RNA with protamine-demonstrating association to gold microparticles by electrophoretic, gel shot, fluorescence, and dynamic laser light spectroscopy (DLLS). A pivotal finding in these studies is that the Au-protamine-DNA conjugates greatly stabilize the DNA; and DNA structure and vaccine activity are maintained even after exposure to physical, chemical, and temperature-accelerated degradation. Specifically, protamine formed nanoparticles when complexed to RNA. These complexes could be detected by gel shift and were probed by high throughput absorbance difference spectroscopy (HTADS). Biological activity of these RNA nanoparticles (RNPs) was demonstrated also by a human tumor cell splice-site switching assay and by siRNA delivery against B-Raf-a key cancer target. Finally, RNA:protamine particles inhibited growth of cultured human tumor cells and bacteria. These data provide new insights into DNA and RNA nanoparticles and prospects for their delivery and chemotherapeutic activity. (C) 2009 Elsevier Ltd. All rights reserved

Sentinel California sea lions provide insight into legacy organochlorine exposure trends and their association with cancer and infectious disease

Background: Organochlorine contaminants (OCs), like polychlorinated biphenyls (PCBs) and dichlorodiphenyltrichloroethanes (DDTs), are widespread marine pollutants resulting from massive historical use and environmental persistence. Exposure to and health effects of these OCs in the marine environment may be examined by studying California sea lions (Zalophus californianus), which are long lived, apex predators capable of accumulating OCs. Methods: We evaluated PCB and DDT levels in 310 sea lions sampled between 1992 and 2007: 204 individuals stranded along the coast of central California, 60 healthy males from Washington State, and 46 healthy females from southern California. Lipid-normalized contaminant concentrations were analyzed using general linear models and logistic regression to ascertain temporal trends; differences between stranded and healthy sea lions; and association of organochlorines with sex, age, and presence of cancer or fatal infectious disease. Results: Concentrations of the contaminants in stranded adults decreased over time in the study period (adjusted for sex, as adult males had higher mean blubber concentrations than adult females and juveniles). Cancer was almost eight and six times more likely in animals with higher summed PCBs and DDTs, compared to those with lower levels (95% CI 5.55-10.51 and 4.54-7.99, respectively). Fatal infectious diseases were similarly seven and five times more likely in animals with higher contaminant burdens (95% CI 4.20-10.89 and 3.27-7.86, respectively). Mean contaminant loads were significantly higher in stranded sea lions than in healthy live captured animals (p b 0.001). Conclusion: Organochlorine contamination has significant associations with health outcomes in California sea lions, raising concerns for humans and other animals eating tainted seafood. While environmental exposure to these organochlorines appears to be decreasing over time based on levels in sea lion tissues, their persistence in the environment and food web for all predators, including humans, and the associated serious health risks, warrant monitoring, possibly through sentinel species like marine mammals

Comparative Functional Dynamics Studies on the Enzyme Nano-bio Interface

Citation: Thomas, S. E., Comer, J., Kim, M. J., Marroquin, S., Murthy, V., Ramani, M., … DeLong, R. K. (2018, August 8). Comparative functional dynamics studies on the enzyme nano-bio interface. https://doi.org/10.2147/IJN.S152222Comparative functional dynamics studies on the enzyme nano-bio interface Spencer E Thomas,1,2,* Jeffrey Comer,1,* Min Jung Kim,1 Shanna Marroquin,1 Vaibhav Murthy,1 Meghana Ramani,1 Tabetha Gaile Hopke,2 Jayden McCall,1 Seong-O Choi,1 Robert K DeLong1 1Nanotechnology Innovation Center of Kansas State, Department of Anatomy and Physiology, College of Veterinary Medicine, Kansas State University, Manhattan, KS, USA; 2Department of Biomedical Science, Missouri State University, Springfield, MO, USA *These authors contributed equally to this work Introduction: Biomedical 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 methods: Here, 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. Results: Analysis 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. Conclusion: Overall 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. Keywords: 2-dimensional fluorescence difference, 2-D FDS, AUC, corrected light intensity emitted, emission wavelength, excitation wavelength, wavelength of max intensity, RL

Characterization and performance of nucleic acid nanoparticles combined with protamine and gold

Macromolecular nucleic acids such as DNA vaccines, siRNA, and splice-site switching oligomers (SSO) have vast chemotherapeutic potential. Nanoparticulate biomaterials hold promise for DNA and RNA delivery when a means for binding is identified that retains structure-function and provides stabilization by the nanoparticles. In order to provide these benefits of binding, we combined DNA and RNA with protamine— demonstrating association to gold microparticles by electrophoretic, gel shot, fluorescence, and dynamic laser light spectroscopy (DLLS). A pivotal finding in these studies is that the Au-protamine-DNA conjugates greatly stabilize the DNA; and DNA structure and vaccine activity are maintained even after exposure to physical, chemical, and temperature-accelerated degradation. Specifically, protamine formed nanoparticles when complexed to RNA. These complexes could be detected by gel shift and were probed by high throughput absorbance difference spectroscopy (HTADS). Biological activity of these RNA nanoparticles (RNPs) was demonstrated also by a human tumor cell splice-site switching assay and by siRNA delivery against B-Raf—a key cancer target. Finally, RNA:protamine particles inhibited growth of cultured human tumor cells and bacteria. These data provide new insights into DNA and RNA nanoparticles and prospects for their delivery and chemotherapeutic activity

Biomolecular Triconjugates Formed between Gold, Protamine, and Nucleic Acid: Comparative Characterization on the Nanoscale

DNA and RNA micro- and nanoparticles are increasingly being used for gene and siRNA drug delivery and a variety of other applications in bionanotechnology. On the nanoscale, these entities represent unique challenges from a physicochemical characterization perspective. Here, nucleic acid conjugates with protamine and gold nanoparticles (GNP) were characterized comparatively in the nanorange of concentration by UV/Vis NanoDrop spectroscopy, fluorimetry, and gel electrophoresis. Given the intense interest in splice-site switching oligomers (SSOs), we utilized a human tumor cell culture system (HeLa pLuc-705), in which SSO-directed splicing repair upregulates luciferase expression, in order to investigate bioactivity of the bionanoconjugates. Process parameters important for bioactivity were investigated, and the bimolecular nanoconjugates were confirmed by shifts in the dynamic laser light scatter (DLLS), UV/Vis spectrum, gel electrophoresis, or sedimentation pattern. The data presented herein may be useful in the future development of pharmaceutical and biotechnology formulations, processes, and analyses concerning protein, DNA, or RNA bionanoconjugates

Characterization of biomolecular nanoconjugates by high-throughput delivery and spectroscopic difference

Nanoparticle conjugates have the potential for delivering siRNA, splice-shifting oligomers or nucleic acid vaccines, and can be applicable to anticancer therapeutics. This article compares tripartite conjugates with gold nanoparticles or synthetic methoxypoly(ethylene glycol)-block-polyamidoamine dendrimers

Covalent Modification of Lipids and Proteins in Rat Hepatocytes, and In Vitro, by Thioacetamide Metabolites

This document is the Accepted Manuscript version of a Published Work that appeared in final form in Chemical Research in Toxicology, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see http://pubs.acs.org/doi/abs/10.1021/tx3001658Thioacetamide (TA) is a well-known hepatotoxin in rats. Acute doses cause centrilobular necrosis and hyperbilirubinemia while chronic administration leads to biliary hyperplasia and cholangiocarcinoma. Its acute toxicity requires its oxidation to a stable S-oxide (TASO) that is oxidized further to a highly reactive S,S-dioxide (TASO2). To explore possible parallels between the metabolism, covalent binding and toxicity of TA and thiobenzamide (TB) we exposed freshly isolated rat hepatocytes to [14C]-TASO or [13C2D3]-TASO. TLC analysis of the cellular lipids showed a single major spot of radioactivity that mass spectral analysis showed to consist of N-acetimidoyl PE lipids having the same side chain composition as the PE fraction from untreated cells; no carbons or hydrogens from TASO were incorporated into the fatty acyl chains. Many cellular proteins contained N-acetyl- or N-acetimidoyl lysine residues in a 3:1 ratio (details to be reported separately). We also oxidized TASO with hydrogen peroxide in the presence of dipalmitoyl phosphatidylenthanolamine (DPPE) or lysozyme. Lysozyme was covalently modified at five of its six lysine side chains; only acetamide-type adducts were formed. DPPE in liposomes also gave only amide-type adducts, even when the reaction was carried out in tetrahydrofuran with only 10% water added. The exclusive formation of N-acetimidoyl PE in hepatocytes means that the concentration or activity of water must be extremely low in the region where TASO2 is formed, whereas at least some of the TASO2 can hydrolyze to acetylsulfinic acid before it reacts with cellular proteins. The requirement for two sequential oxidations to produce a reactive metabolite is unusual, but it is even more unusual that a reactive metabolite would react with water to form a new compound that retains a high degree of chemical reactivity toward biological nucleophiles. The possible contribution of lipid modification to the hepatotoxicity of TA/TASO remains to be determined

Prevalence of dyslipidaemia and associated risk factors in a rural population in south-western Uganda : a community based survey

BACKGROUND: The burden of dyslipidaemia is rising in many low income countries. However, there are few data on the prevalence of, or risk factors for, dyslipidaemia in Africa. METHODS: In 2011, we used the WHO Stepwise approach to collect cardiovascular risk data within a general population cohort in rural south-western Uganda. Dyslipidaemia was defined by high total cholesterol (TC) ≥ 5.2 mmol/L or low high density lipoprotein cholesterol (HDL-C) 6% (men aOR=3.00, 95%CI=1.37-6.59; women aOR=2.74, 95%CI=1.77-4.27). The odds of high TC was also higher among married men, and women with higher education or high BMI. CONCLUSION: Low HDL-C prevalence in this relatively young rural population is high whereas high TC prevalence is low. The consequences of dyslipidaemia in African populations remain unclear and prospective follow-up is required