Synthetic nanogel antibodies molecularly imprinted with the Spike S1 protein -ANTISPIKE

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 8th, 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)


STAGE 2. OPTIMIZATION OF SPECIFICITY AND CROSS-REACTIVITY OF LIGAND-FREE MIP-SNAS BY A FEED-BACK PROCESS.


RESEARCH OBJECTIVES

The main objective of the research study was to optimize the specificity and cross-reactivity of ligand-free MIP-SNA through a feed-back process, as well as to verify the reproducibility of synthetic, nanogel-type antibodies (SNA) based on polyethylene glycol with acrylate end groups (PEGDA), in the presence of the Spike S1 protein, used as a template, by inverse mini-emulsion polymerization. Other objectives of the study included:

Act 2.1. –Part 2. Finalization of the studies regarding the reverse mini-emulsion polymerization (water in oil) of PEGDA functional macromonomers in the presence of the Spike S1 protein, used as a template.

Act 2.2. – Part 2. Definitive studies on the 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 2.3. – Using synthesized SNAs, the specificity for the Spike S1 protein will be determined. This step can be considered a Preliminary analysis regarding the evaluation of the performance of the synthesized SNA. This activity consists of rebinding tests of the Spike S1 protein in static conditions to determine the imprinting effect (evidenced by UV-VIS spectroscopy or HPLC). This action refers to Quantifying some critical parameters: the equilibrium concentration of the Spike S1 protein, Qe, the adsorption capacity of the Spike S1 protein, Q (g Spike S1 protein/g MIP-SNA), and the imprinting factor, IF.

Act 2.4. – Determining the efficiency of polymer-protein interactions as well as verifying the specificity of synthetic nanogel antibodies (SNAs) by enzyme-linked immunosorbent assay (ELISA), using antibodies, e.g., primary anti-Spike S1 antibody and secondary antibody, to detect and quantify the retention and binding of the Spike S1 protein.

Act. 2.5. –  Selection of viable MIP-SNAs antibodies after determining the selectivity of the Spike S1 protein against Bat-CoV by immunoadsorption tests with enzyme-labeled antibodies (ELISA).

Act. 2.6. – The MIP-SNA synthesis will b accurately reproduced in several batches according to the optimal recipe.

Act. 2.7. – Reproducibility of MIP-SNA through morphological (TEM/SEM), dimensional characterization an determination of PDI polydispersity index (DLS).

Act 2.8 – Innovation activity and intellectual property protection through patent application

Act 2.9 – Communication activities, drafting/sending for publication of scientific papers in compliance with Open Access conditions.

 


EXECUTIVE SUMMARY

The alarming spread of the SARS-CoV-2 virus made researchers worldwide direct their research in search of advanced therapeutic methods that can prevent or treat the disease of COVID-19 or even act as a complement to classic drug treatments. Drawing inspiration from the body’s natural immune response, an exciting approach to treating this lethal virus has been using Molecularly Imprinted Polymers (MIPs) such as nanohydrogels that can act as synthetic antibodies precisely designed to recognize and bind to specific viral components. In recent years, a substantial increase in the number of specialized papers highlights the potential of MIP to revolutionize modern treatments for COVID-19. The research study under Stage II/2023 had as its primary objective the optimization of the specificity and cross-reactivity of ligand-free MIP-SNA through a feed-back process, as well as the verification of the reproducibility of synthetic, nanogel-type antibodies (SNA) based on polyethylene glycol with acrylate end groups (PEGDA), in the presence of the Spike S1 protein, used as a template, by inverse mini-emulsion polymerization. Thus, the studies on the inverse mini-emulsion polymerization of PEGDA functional macromonomers in the presence of the Spike S1 protein (PSS1) from the coronavirus as a template were completed. The finalization of the investigations regarding the size, the size distribution, and the measurement of the polydispersity index by the DLS technique and the highlighting of the morphological characteristics by SEM and the physico-chemical characteristics by FTIR and TGA/DTG for the MIP/NIP-SNA nanogels were also considered. DLS measurements revealed that the non-imprinted (NIP-SNA) and imprinted nanogels obtained after the washing process (MIP-SNAext) showed similar sizes of about 150-220 nm. In contrast, the imprinted MIP-SNA nanogels showed larger sizes of about 250 nm, thus indicating the presence of Spike S1 protein in the nanoparticle structure. The recorded SEM images revealed a well-defined spherical shape of the particles and the presence of several populations of different sizes, as could also be observed in the DLS analysis. The study continued by determining the efficiency of polymer-protein interactions and verifying the specificity of SNA synthetic antibodies by gel-electrophoresis, spectrophotometric methods (Bradford method), and enzyme-linked immunosorbent assay (ELISA) using antibodies such as primary anti-Spike S1 antibody and antibody secondary. The adsorption capacities and the characteristic imprinting effect of the synthesized MIP-SNA were determined in the specificity tests for PSS1, performed in duplicate, where a high value of the imprinting factor (IF) of 12.8 was observed with an adsorption efficiency of 8% and 15.9 with an adsorption efficiency of 10%. At the same time, the determination of the efficiency of polymer-protein interactions was highlighted with the help of circular dichroism spectroscopy, where the results proved that the secondary structure of PSS1 was not modified or denatured during the imprinting process; this process is based on non-covalent bonds. The verification of the specificity of the nanogel-type synthetic antibodies (SNA) was highlighted by the enzyme-linked immunosorbent assay (ELISA), where the results showed a significantly higher adsorption concentration against PSS1 (59.89%) in relation to the adsorption concentration against BatCoV (29.62%), indicating a preferential/selective adsorption for PSS1 from SARSC-CoV-2 relative to BatCoV. The reproducibility of MIP-SNA consisted of the precise reproduction of the synthesis process of MIP-SNA according to the optimal recipe in several batches, but also through the morphological and dimensional characterization of both the MIP-SNA and the synthesized NIP-SNA, the results confirming the reproducibility analyses. These results encourage the hypothesis of using these systems to create new molecularly imprinted nanogel-type synthetic antibody structures for the recognition and retention of the SPIKE S1 protein from SARS-CoV-2, thus making it possible to block the fusion process between the membranes of SARS-CoV-2 viral cells with those of the host cell. Therefore, the proposed concept is intended for the recognition and retention of PSS1 using molecularly imprinted nanogels, and will represent a completely original solution in the field of nanobiomedicine. The degree of achievement of the objectives within Stage II/2023 was fully achieved, and the objectives were fulfilled in proportion to 100%, the results being consistent with the proposed targets: 1 scientific activity report, 1 financial and audit report; ANTISPIKE project website; development of the functional model (EF) for obtaining SNA; participation in 4 international conferences and the publication of 1 ISI Article (in the WoS red zone, Q1).


DISSEMINATION

Aside from the Development of the Functional Model for obtaining synthetic nanogel antibodies (SNA) molecularly imprinted (MIP-SNA) with the Spike S1 protein, the project also has a strong component in disseminating the results. Thus, methods for obtaining hydrogel structures based on PEGDA, and characterizing hydrogels were used as the background of the ANTISPIKE project No. 144/2022, and were disseminated by: (i). submission of a patent application to OSIM with No. A 2023-00717/21.10.2023 (ii). publication of an article in the Pharmaceutics magazine (in the WoS red area, Q1), with FI=5.4, and (ii) participation in 4 international scientific events (2 oral presentations and 2 as posters).

1 National patent application: Zaharia Anamaria, Neblea Elen Iulia, Sarbu Andrei, Iordache Tanta Verona, Chiriac Anita Laura, Gavrila An Mihaela, Sandu Teodor, Stoica Elena Bianca, Dumitru Marinela- Victoria, Miro Andreea, Dolana Sorin Viorel, Neagu Ana Lorena, Nanogeluri imprentat molecular  cu comportament biomimetic d anticorpi pentru recunoasterea moleculara a proteinei Spike S1 din virusul SAR COV 2 si procedeu de obtinere a nanogelurilor A 2023- 00717/21.10.2023 (TE144/2022 si 15PFE/2023).

1 Published ISI Article:Ana-Lorena Neagu, Anamaria Zaharia, Octavian Dumitru Pavel, Alina Tirsoaga, Iulia Elena Neblea, Sorin Viorel Dolana, Carmen Elena Țebrencu, Tanta-Verona Iordache, Andrei Sarbu, Rodica Zavoianu, Synergistic Sustained Drug Release System based on immobilized Rhamnus Frangula L phytoextract into Layered Double Hydroxide covered by Biocompatible Hydrogel, Pharmaceutics, 2023, 15(7), 1888; https://doi.org/10.3390/pharmaceutics15071888 (646PED, 144TE) (IF=5.4)

4 Scientific Manifestations: 1. Exploratory Workshop Next Chem V ed. Bucuresti, 22-23 May 2023, 5th edition, Bucharest, Romania, Iulia Elena NEBLEA, Anamaria ZAHARIA, Tanța-Verona IORDACHE, Anita Laura Chiriac, Ana-Mihaela GAVRILĂ, Sorin Viorel DOLANA, Andreea MIRON, Ana-Lorena NEAGU, Mircea TEODORESCU, François-Xavier PERRIN, Molecularly Imprinted Nanogels for Spike S1 Protein Recognition (15PFE, 144TE);

2.3rd International Conference on Bioengineering and Polymer Science, Bucharest, June 7-11, 2023 ROMANIA; Anamaria Zaharia, Ana Lorena Neagu, Octavian Dumitru Pavel, Alina Tîrşoaga, Iulia Elena Neblea, Sorin Viorel Dolana, Tanţa Verona Iordache, Rodica Zăvoianu, Andrei Sârbu, Layered Double Hydroxides – Polyethylene Glycol Diacrylate Composite Hydrogels for the Controlled Release of Rhamnus Frangula L Phytoextract, (144TE si 646PED, AQUAMAT);

3. PRIORITIES OF CHEMISTRY FOR A SUSTAINABLE DEVELOPMENT PRIOCHEM – XIXth Edition, 11-13 octombrie 2023; Bucharest; Ana-Lorena Neagu, Anamaria Zaharia, Marinela Victoria Dumitru, Iulia Elena Neblea, Sorin Dolana, Tanta Verona Iordache, Carmen Elena Tebrencu, Rodica Zavoianu, Octavian Dumitru Pavel, Alina Tîrsoaga, Andrei Sarbu, Drug-Release System Based on Biocompatible Hydrogel Charged With Layered Double Hydroxide Containing Encapsulated Rhamnus Frangula L. Phytoextract, prez. Orala (646PED; 144TE);

4. 5th Euro-Mediterranean Conference for Environmental Integration (EMCEI-2023), 2-5 octombrie 2023, Anamaria Zaharia, Iulia Elena Neblea, Anita-Laura Chiriac, Ana-Mihaela Gavrila, Sorin Dolana, Ana Lorena Neagu, Bogdan Trica, Andrei Sarbu, Francois-Xavier Perrin, Tanta-Verona Iordache

STAGE 3  – LABORATORY TECHNOLOGY FOR OBTAINING MIP-SNAS WITHOUT LIGAND. OBJECTIVES OF THE RESEARCH STUDY

The main objective of the research study was to establish the laboratory technology for obtaining molecularly imprinted nano gel-type synthetic antibodies (MIP-SNA) without a ligand based on polyethylene glycol with acrylate end groups (PEGDA). In the current stage, the results obtained to achieve the established objectives will be presented:

Act 3.1. The laboratory technology for the preparation of ligand-free MIP-SNA will be further validated by cyto/biocompatibility studies. In this regard, preliminary cytotoxicity studies will be determined using the L929 cell line of murine fibroblasts

Act 3.2. Communication activities, drafting/sending for publication of scientific papers

EXECUTIVE SUMMARY

The global impact of the SARS-CoV-2 virus that led to the coronavirus pandemic is our time’s defining global health crisis. From this perspective, the COVID-19 pandemic has triggered massive disruptions in all aspects of human life, including education, social interactions, economics, and, last but not least, health. Various strategies have been explored for research and development to combat this virus. Among them, inspired by the body’s natural immune response, one of the promising approaches is the use of molecularly imprinted nanogels under the action of synthetic antibodies. The research study within the framework of Stage 3/2024 had as its primary objective the development of the Laboratory Technology for obtaining synthetic antibodies of the nanogel type (SNA) molecularly imprinted (MIP-SNA) with the Spike S1 protein, based on polyethylene glycol with acrylate terminal groups (PEGDA). Thus, the reproducibility of the recipes for obtaining SNA without ligand was taken into account, following the morphological and dimensional parameters, determining the retention and recognition performance of the Spike S1 protein, and optimizing and validating the technology through preliminary cytotoxicity studies. Thus, the obtained ligand-free SNAs were characterized both from a physical and physicochemical point of view using appropriate techniques and equipment, such as infrared spectroscopy (FTIR), light diffusion technique (DLS), and Transmission Electron Microscopy (TEM). The FTIR spectra of SNA presented the characteristic peaks of the polymer formed by polyethylene glycol and acrylic groups and simultaneously showed the efficiency of the washing/purification procedures. Regarding the size of the particles obtained after the synthesis, the DLS measurements revealed that in the case of the non-imprinted control sample (NIP-SNA) as well as the PSS1-imprinted (MIP-SNA), the sizes remained within a suitable range for the final application (100-220 nm). Before and after the washing-purification procedures, the TEM morphological analysis of the non-imprinted NIP-SNA nanogels and the PSS1-imprinted ones, MIP-SNA, highlighted morphologies with spherical nanoparticles, with sizes between 100-200 nm. The morphology of PSS1-imprinted MIP-SNA nanogels led to a different morpho-structural organization, forming “eye”-like structures – irregular interconnected structures that confirmed the presence of PSS1 on the SNA surface. Considerable differences were also observed between the morphologies of NIP-SNA emulsion, MIP-SNA emulsion, and washed NIP-SNA and extracted MIP-SNA, respectively which explains both the macromer-protein self-assembly phenomenon and the formation of specific nanocavities involved in the molecular recognition process after the extraction of the Spike S1 protein. The adsorption capacities and the characteristic imprinting effect of the synthesized MIP-SNA were determined in the specificity tests for PSS1, performed in duplicate, where a high value of the imprinting factor (IF) of 1.8 was observed with an adsorption efficiency of 51% and 1.48 with an adsorption efficiency of 72%. The analysis of the reproducibility of the properties of nano gels imprinted with PSS1 successfully led to the development of Laboratory Technology for obtaining synthetic antibodies of the molecularly imprinted nano gel type (MIP-SNA) without ligand. To optimize and validate the laboratory technology for obtaining MIP-SNA, the biocompatibility of the synthesized SNA was determined by checking the cytotoxicity using the cell line L929 of murine fibroblasts. The obtained results showed that there is no decrease in cell viability below 90% for any of the tested dilutions in the presence of the supernatants to be tested.

These results encourage the hypothesis of using these systems to create new molecularly imprinted nanogel-type synthetic antibody structures for the recognition and retention of SPIKE S1 protein from SARS-CoV-2, thus making it possible to block the fusion process between SARS-CoV-2 viral cell membranes with those of the host cell. Therefore, the proposed concept is intended for the recognition and retention of PSS1 using molecularly imprinted nanogels and will represent a completely original solution in the field of nanobiomedicine.

The degree of achievement of the objectives within Stage 3/2024 was fully achieved. The goals were fulfilled in proportion to 100%, the results being by the proposed targets: 1 scientific activity report, 1 financial and audit report; ANTISPIKE project website; Laboratory technology (TL) for obtaining SNAs without ligand; the submission for publication of 1 paper to Chemosensors journal (in the red area WoS, Q1), with FI =4.2, 1 Draft Article to be sent for publication using the results obtained in Stage 2/2023 (on the date of submission of the report, the agreement is expected to all co-authors of the article); Media activities within the Radio Romania Cultural show “Science 360”, to promote the ANTISPIKE funded project.


DISSEMINATION

In addition to the Development and Validation of the Laboratory Technology regarding obtaining molecularly imprinted nanogel (SNA) synthetic antibodies (MIP-SNA) with the Spike S1 protein, the project also has a strong component of disseminating the results. Thus, methods of obtaining some structures of molecularly imprinted polymeric nanoparticles to obtain an innovative, versatile, and reusable biosensor regarding the rapid, qualitative, and quantitative detection and monitoring of lipopolysaccharides, as well as the method of quantifying the adsorption capacity as well as determining the specificity of the imprinted particles molecular studies regarding the recognition and binding of the template biomolecule were used as the background of the ANTISPIKE project Nr. 144/2022, and were disseminated by: (i) sending an paper for publication in the Chemosensors journal (in the red area of WoS, Q1), with FI =4.2; (iii). media activities promoting the ANTISPIKE project within the Radio Romania Cultural – Science 360 show from December 14, 2022 (ii). 1 Draft Paper to be sent for publication by May 30, 2024, in the Special Issue entitled “Advance in Molecularly Imprinted Polymers II”, in Polymers using the results obtained in Stage 2/2023.

1 paper sumbmitted for publication : „Lipopolysaccharides detection by sensitive hybrid nanoink based molecularly imprinted polymer printed on plastic electrodes” Ana-Lorena Neagu, Ana-Mihaela Gavrila, Andreea Miron, Iulia Elena Neblea, Teodor Sandu, Catalin Zaharia ,Petru Epure, Horia Iovu, Andrei Sarbu and Tanta-Verona Iordache, ȋn revista Chemosensors (in zona rosie WoS, Q1), cu FI =4.2

1 Draft Paper to be sent by My30, 2024 following an invitation to publish within Special Issue in Polymers „Advance in Molecularly Imprinted Polymers II”, using the results obtained in Stage 2/2023 and being entitled „Molecularly Imprinted Nanogels with Spike S1 Protein as Synthetic Antibodies:  The Summit of Biotechnological Ingenuity”; autori: Iulia Elena Neblea, Tanța-Verona Iordache, Andrei Sarbu, Anita-Laura Chiriac, Ana-Mihaela Gavrila, Bogdan Trica, Iuliana Elena Bîru, Iuliana Caras, Mircea Teodorescu, François-Xavier Perrin, Anamaria Zaharia on the date of submission of the report, the agreement of all co-authors of the paper is expected.