Publications 2022 - Laboratory of Nanostructures IWC PAN
Nanoformulation Composed of Ellagic Acid and Functionalized Zinc Oxide Nanoparticles Inactivates DNA and RNA Viruses
Khaled AbouAitah, Abdou K. Allayh, Jacek Wojnarowicz, Yasser M. Shaker, Anna Swiderska-Sroda and Witold Lojkowski
The COVID-19 pandemic has strongly impacted life across the globe but no drug therapy has yet been approved for the clinic. In the current study it was presented the inorganic-organic hybrid nanoformulation with potential against DNA and RNA viruses, which could be also implemented in treatment against COVID-19.
This hybrid nanoformulation contained 9,5% wt.% TRP and was loaded with natural prodrug ELG (2,3,7,8-tetrahydroxy-chromeno ﴾5,4,3-cde﴿chrommene-5,10-dione) up to 33,3wt.%, a safe and powerful polyphenol, present in several types of fruits as well as medicinal plants. ELG could serve along as a novel natural agent with efficient antiviral effects against several viruses including human rhinoviruses (HRV2,-3, and -4), HIV-1, herpes simplex virus (HSV), Ebola, influenza etc. The new strategy designed for these experiments, it was using ELG with clinically proven antiviral drugs to produce synergistic effects.
In this study, for the first time, was reported smart design of nanoformulation with ZnO NPs (inorganic material) coated with TRP (organic 3D material) and employed as a core. The core was impregnated with ELG. This constructed nanoformulation was tested for its specific effects to combat DNA viruses and RNA viruses. It were used several viral alternatives like human adenovirus (Ad-7) and herpes virus type 2(HSV-2), human influenza (H1N1), and human coronavirus (HCoV-229E) as models to investigate the antiviral activity against COVID-19 in these research. The reason for testing different viruses was the unique structure of both classes of virus and expected various interactions with the nanoformulation.
As the result of performed study, nanoformulation showed promise as an antiviral agent against H1N1 and HcoV-229E, reflecting the possible implementation in designing nanomedicines against COVID-19.
Antiviral Evaluation was peformed using several viruses and cell lines purchased from ATCC, Manassas,VA, USA. The host cell lines were head and neck cancer cells (hep-2) for propagation of human adenovirus type 7 (Ad-7), green monkey kidney(Vero) cells for propagation of herpes simplex virus type 2 (HSV-2), and clone of Vero(Vero-E6) cells for propagarion of human influenza (H1N1) and human coronavirus 229E (HCoV-229E). The cells were grown in DMEM medium-high glucose containing 10%fetal bovine serum, 0,1%antibiotic/antimycotic solution, and trypsin-EDTA.
Cytotoxicity Assesment in these study was performed with use of B(SRB) colorimetric assay. Hep-2, Vero and Vero-E6 cells were seeded in 96-well culture plates at a density of 2x104 cells/well and it grew for 24h, than samples were diluted in culture medium to the wells and
incubated for another 48h. After that, different steps of preparation (washing with PBS, adding 0,01 ml of cold acetone -70%,v/v to each well, left for 30 min in -20οC; oven dried for 30 min at 60 οC) were performed. Finally was used microplate reader BMG LabTech Gmbh FLUOstar Omega, Ortenberg, Germany to determine optical density (OD) at 540nm. In order to calculate 50% cytotoxic concentration (CC50) was used the GraphPad PRISM(version 5, GraphPad Software, San Diego, CA, USA).
Antiviral Assessment was performed by using the cytopathic inhibition effect CPE to evaluate the antiviral effect.
In order to find out Virucidal Mechanism, the plaque reduction assay was used to explore virusidal potencial. After experiments (untreated viral plaque as the control) viral plaques were fixed and stained. The reduction in formulated plaque HCoV-229 was calculated as the % of inhibition using the formula: viral count (untreated) – viral count (treated)/viral count(untreated)x100.
The nanoformulation preparation was achieved in the steps as shortly described above. To curry out the surface modification of ZnO NPs with few steps functionalization process in order to achive final 3D structure. The full preparation of TRP molecules with the active ester, formed TRP-functionalized NPs (ZnO NPs-NH2-COOH-TRP) resulting tree- dimensional hybrid inorganic-organic NPs that could be used for loading ELG prodrug molecules.
Both TEM and STEM analysis revealed the successful functionalization of ZnO NPs-NH2-COOH with TRP, as well as the possibility of obtaining an inorganic-organic hybrid nanostructures with porosity to assist in ELG impregnation for drug delivery applications.
Particle size measurement of the nanocarrier material is important to consider when constructing any nanoformulations because it governs many functions, such as interactions with the biological entities, affecting their molecular, cellular, and organ levels. As these study concerns tiny biological entities, such as viruses, the size distribution was measured by PTA. In this context PTA allows visualization of NPs, providing the size, count, and concentration measurements. The PTA found varied size distributions following the preparation steps. This observation may be related to improved dispersion stability of particles in aqueous solution, due to the surface functionalization with carboxylic acid groups.
The maximum size distribution was recorded for ELG loaded NPs. Thus, the particle size distribution is modulated by different parameters, such as surface functionalization and drug loading.
For most careful interpretation of results several measurements and characterization of achieved nanoformulation were performed including: Specific Surface Area, Zeta potential measurements, FTIR-ATR Analysis, Thermal and DSC Analysis.
The most important results of these experiments include Antiviral Evaluation.
As general trend, the IC50s varied in response to both treatment and viruses. Interesingly, the findings indicate specific inactivity, depending on whether it was a DNA or RNA virus. In the
case of DNA-based viruses, for HSV-2, the nanoformulation had higher virus inactivation than ZnO NPs and ELG alone. For Ad-7, ELG recorded the lowest IC50. In the case of RNA-based viruses, Zno NPs inhibited viral infection by H1N1 less efficiently, compared with nanoformulation, best inhibition was performed by ELG alone. For HCoV-229E, ELG inhibited much less compared with nanoformulation and the best inhibitor it was ZnO Nps alone. To identify the most effective materials, were calculated a selective index (SI), the CC50/IC50 ratio. The SI confirms the importance of specific inhibition of viral infections.