Project Manager: SR I. Dr. eng. Anamaria ZAHARIA () National Institute for Research & Development in Chemistry and Petrochemistry – ICECHIM, Bucharest

Funding: Executive Unit for the Financing of Higher Education, Research, Development and Innovation (UEFISCDI)

Programme: PN II- Human Resources Programme – Young research teams 2021

Project number: PN-III-P1-1.1-TE-2021-1239

Research domain – Pharmacology and pharmacogenomics (including drug discovery and design, drug delivery and therapy, toxicology)

Financing contract: Nr. 144/13.05.2022

Total funding of the contract: 450.000 RON

Period of project implementation: 13.05.2022 – 12.05.2024 (24 months)

Abstract

SARS-CoV-2 is a new coronavirus type and it is responsible for causing COVID 2019 in humans, with very high contagion rate all over the world. The viral entry of SARS CoV-2 is conferred by the presence of Spike S1 protein on the surface that can direct attachment and enter the plasma membrane of the human cell. The Spike protein through its location is a significant therapeutic target, and targetable using antibodies. Despite recent technological developments, effective and safe therapies are currently not available for treating the infected victims. Thereby, the general objective of the project targets the synthesis of synthetic nanogel antibodies molecularly imprinted with Spike S1 (MIP-SNAs) for recognizing and retaining coronavirus-originated Spike S1 proteins. In this respect, the MIP-SNAs are able to recognize and bond to the Spike S1 proteins, acting as nanogel caps, and thus inhibiting the activity of SARS-CoV-2 antigen to penetrate the human cells. Hence, ANTISPIKE holds significant influence upon the scientific community by new concepts and methodologies for ligand-free delivery systems as MIP-SNAs (short-term impact: scientific ISI papers and communications, and national patent claim) and by opening new research directions associated with the side-benefits of the research (like new immuno-therapies) as long-term impact. Implementation of this project will also bring specific scientific, economic and social benefits at the national and international level.

Objective:

The overall aim of the “ANTISPIKE” project refers to developing original, cost-effective and biocompatible ligand-free nanogel delivery systems that can be used as potential treatment for patients affected by COVID-19.

Estimated ResultsANTISPIKE holds significant influence upon the scientific community by new concepts and methodologies for ligand-free delivery systems as MIP-SNAs: 3 scientific ISI papers sent for publication and 3 scientific communications, one national patent claim, website of project “ANTISPIKE” and by opening new research directions associated with the side-benefits of the research, like new immuno-therapies. Implementation of this project will also bring specific scientific, economic and social benefits at the national and international level.

STAGE 1. TRIALS FOR PREPARING OF THE SYNTHETIC NANOGEL ANTIBODIES (SNAS). – OPTIMIZING THE SPECIFICITY AND SELECTIVITY OF SNA

RESEARCH OBJECTIVES

Act.1.1.- Functionalization of PEG having various molecular weight (Mn=800÷2000 g/mol), by reacting it with acryloyl chloride, as well as the determination of the polydispersity, PD, average number molecular weight, Mn, and functionality, F, of the macromonomers by size exclusion chromatography (HPLC-SEC) and proton nuclear magnetic resonance.

Act 1.2 – Structural characterization of Spike S1 protein by means of Zeta potential, X-Ray Diffraction (XRD) and Fourier transform infrared spectroscopy (FTIR).

Act 1.3. – Reverse Mini-emulsion polymerization (water in oil) of PEGDA functional macromonomers in the presence of coronavirus Spike S1 protein, as a template.

Act 1.4. – Size, size distribution, Zeta-potential (nanogels should be negatively charged to reduce physiological interactions) and PDI determination for the batch ligand-free MIP-SNAs by Dynamic Light Scattering (DLS) and Transmission/Scanning Electron Microscopy (TEM/SEM)

Act 1.5 – Communication and dissemination of results.

EXECUTIVE SUMMARY

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) ­is a new type of coronavirus, responsible for causing the COVID-2019 disease in humans, with a very high contagion rate worldwide. To date, globally, COVID-19 has affected more than 629,370,889 people, leading to more than 6,578,245 deaths by November 8^th, 2022. This project is aimed at obtaining new Spike S1 protein (PSS1)- molecularly imprinted nanogel-type synthetic antibody structures (MIP-SNA), based on polyethylene glycol diacrylate (PEGDA) macromonomers that show high biocompatibility. During stage I/2022, tests were performed regarding the synthesis and characterization of both molecularly imprinted (MIP-SNA) and non-molecularly imprinted (NIP-SNA) nanogel-type synthetic antibodies (SNA) with PSS1, by inverse mini-emulsion polymerization. The laboratory-scale experimental studies regarding the obtaining of SNA and the analysis of the structure and the characteristic composition of SNA have been focused on the following activities: (i) the development of the studies regarding the synthesis conditions of the macromer based on polyethylene glycol (PEG) with acrylate terminal groups; (ii) the physico-chemical characterization of PSS1; and (iii) the development of the studies regarding the synthesis conditions and the characterization of SNA based on polyethylene glycol with acrylate end groups (PEGDA), in the presence of PSS1 used as a template, by inverse mini-emulsion polymerization.

The physico-chemical characterization of the PEGDA-based macromer, PSS1 and of SNA was performed using advanced techniques such as HPLC, FTIR, TGA/DTG, XRD, CD, DLS, SEM, and TEM. Moreover, the characterization of PSS1 was carried out using both circular dichroism (CD) and X-ray diffraction (XRD). XRD results showed peaks with large distances of 183 and 152 Å, characteristic of the interactions of the randomly oriented subunits (random coil structure), these data being consistent with the results of the CD analysis. Regarding the similarity between the FTIR spectra characteristic for NIP-SNA and MIP-SNAext, it could be proven that the chemical structure of MIP-SNA was not modified during the imprinting process (this process is based on non-covalent bonds) and thus confirming the total removal of the PSS1 from MIP-SNA, to obtain artificially created complementary cavities. TGA/DTG investigations confirmed the presence of both macromonomer and protein/emulsifiers in the SNA structure in the case of MIP-SNA. The size of the particles was analyzed using the DLS technique, the measurements indicating sizes ranging from 120 to 220 nm. At the same time, the SEM images revealed the spherical, individual structures of the synthesized particles, and the obtained TEM micrographs indicated the presence of the amyloid fibrillar structure of PSS1 absorbed on the obtained nanoparticles. The results of all these laboratory-scale experimental studies led to the Establishment and Development of the Experimental Model (EM) regarding the obtaining of synthetic nanogel antibodies (SNA).

The degree of achievement of the objectives in the stage 1/2022 was fully achieved and the goals were fulfilled in a proportion of over 100%, the results being in good agreement with the proposed targets: 1 scientific activity report, 1 financial and audit report; the site of the project ANTISPIKE;

the development of the experimental model (EM) for SNA obtaining; participation at 5 international conferences and publishing of 1publish ISI article (with synthesis and characterization methods which were used as starting point and as background for ANTISPIKE project).

DISSEMINATION

           1 Publish ISI Article:1. Zaharia, A.; Gavrila, A.-M.; Caras, I.; Trica, B.; Chiriac, A.-L.; Gifu, C.I.; Neblea, I.E.; Stoica, E.-B.; Dolana, S.V.; Iordache, T.-V. Molecularly Imprinted Ligand-Free Nanogels for Recognizing Bee Venom-Originated Phospholipase A2 Enzyme. Polymers 2022, 14, 4200. https://doi.org/10.3390/polym14194200 (91TE/2018; 144TE/2022).

           5 Scientific Manifestations:

1. Iulia Elena Neblea, Anamaria Zaharia, Andreea Olaru, Mircea Teodorescu, Tanța-Verona Iordache, Elena-Bianca Stoica, Teodor Sandu, Andreea Miron, Andrei Sarbu, Anita-Laura Chiriac, New Innovative Biopolymer-Based Interpenetrated Hydrogels with Potential Antibacterial Activity,22nd Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, ROMANIA – September 7 – 9, 2022. Prez. Orala (144TE/2022; 157/2020)

2. Ana-Mihaela Gavrila, Ana-Lorena Neagu, Petru Epure, Anamaria Zaharia, Catalin Zaharia, Horia Iovu, Andrei Sarbu, Bianca-Elena Stoica, Sorin-Viorel Dolana, Tanta-Verona Iordache, Molecularly Imprinted Polymers for the Detection of Different Hazardous Analytes, 22nd Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, ROMANIA – September 7 – 9, 2022. Poster (144TE/2022; 255PED/2020; PN 19.23.02.01)

3. Verona Iordache, Anamaria Zaharia, Ana-Mihaela Gavrila, Bianca Elena Stoica, Ana Lorena Ciurlica, Marinela Dumitru, Andreea Olaru, Andrei Sarbu, Laura Anita Chiriac, Quaternary Ammonium Functionalized-Clay Microparticles with Bactericidal Activity Applied for Wastewater Treatment, 22nd Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, ROMANIA – September 7 – 9, 2022. Poster (144TE/2022; 135TE/2022; 71/2017)

4. Anamaria Zaharia, Ana-Mihaela Gavrila, Anita-Laura Chiriac, Iulia Elena Neblea, Teodor Sandu, Sorin Dolana, Bogdan Trica, Iuliana Caras, Andrei Sarbu, Tanta-Verona Iordache, Ligand-free targeted delivery nanogels for recognizing Hymenoptera venom-originated PLA2 enzyme, 22nd Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, ROMANIA – September 7 – 9, 2022. Poster (144TE/2022; 91TE/2018; 49PTE/2020)

5. Anita-Laura Chiriac, Iulia Elena Neblea, Ana-Mihaela Gavrila, Anamaria Zaharia, Teodor Sandu, Andreea Miron, Marinela Dumitru, Sorin Dolana, Andrei Sarbu, Tanta-Verona Iordache, Innovative Structures based on Bio-friendly Polymers, 22nd Romanian International Conference on Chemistry and Chemical Engineering, Sinaia, ROMANIA – September 7 – 9, 2022. Poster (144TE/2022; 646PED/2022)