Gold nanoparticles-based nanobiocomposites as artificial immunogens for human and animal immunization, respectively, against SARS-CoV-2

 

PROJECT TITLE: Gold nanoparticles-based nanobiocomposites as artificial immunogens for human and animal immunization, respectively, against SARS-CoV-2

PROJECT ACRONYM: Gold-SARS

FOUNDING SOURCE: Romanian Executive Agency for Higher Education, Research, Development, and Innovation (UEFISCDI) - Exploratory Research Projects - PNCDI III - Program 4 - Fundamental and Frontier Research

PROJECT CODE: Project PN-III-P4-PCE-2021-1081

CONTRACT NUMBER: PCE75/2022

PROJECT DURATION: 31 months (02/06.2022 – 31.12.2024)

PROJECT BUDGET: 1.200.000 lei

PROJECT CONSORTIUM: CO - National Institute of Research and Development for Technical Physics, NIRDTP Iasi

PROJECT SUMMARY: The development of anti-SARS-CoV-2 vaccines is a current problem of humanity, the preparation of specific immunogens against such types of viruses becoming extremely important for the scientific, economic, technological, social, or cultural domains. Although vaccination is one of the most effective healthcare interventions, there are several social, clinical, and economic obstacles to vaccination, including a large number of people reluctant to new vaccines, the side effects of vaccination, the likelihood of varying vaccine effectiveness or poor efficacy in various populations, longevity of the immune protection and access to the vaccine for certain people.

Gold nanoparticles (AuNP) have aroused huge interest in vaccinology, due to their reliable surface functionalization, biocompatibility, size and shape customization, and optical properties. AuNP were exploited in vaccines against bacterial infections, viral infections, cancer, parasite infections etc.

The goal of the project is to provide the vaccine and nutritional supplements industry with an immunogen against SARS-CoV-2 to be used for COVID-19 prophylaxis. The novelty of the project regards the (a) Immunogen preparation; (b) Applications in vaccinology; (c) Solving the old problem of AuNP stability; (d) Solving the problem of ionic binding stability in aqueous solutions with high ionic strength; (e) Preparation of immunizing nutritional supplements and vaccine industry and (f) Social acceptance.

PROJECT OBJECTIVE: The main objective of the project is to prepare a virus-like immunogen based on gold nanoparticles (AuNP) coated with commercially-available recombinant S (spike) proteins for further use in the development of a SARS-CoV-2 vaccine. The resulting vaccine can be used to (1) obtain natural products with immunizing properties (i.e., immunized eggs from vaccinated chickens or lyophilized egg products administered as a nasal spray) that contain anti-SARS-CoV-2 antibodies to provide passive immunization against coronavirus to those who consume such products of animal origin and (2) for active immunization after direct inoculation of such a vaccine in humans. The solution provided should also function as a common platform for other types of vaccines.

EXPECTED RESULTS:

  • A functional anti-SARS-CoV-2 immunogen conditioned as aqueous suspensions of nanometer AuNP-spike bionanocomposites, resembling dimensionally and in surface composition to the coronavirus. Indicator: product
  • A method to stabilize the sodium citrate on the surface of gold nanoparticles. Indicator: method
  • A functional platform based on citrate-coated gold nanoparticles that can be shared in order to prepare a series of very stable magnetic and non-magnetic gold nanoparticles coated, for instance, with ions of aluminum, iron (as proposed in this project), nickel, cobalt, manganese etc. Indicator: method
  • A nature-inspired method to increase the bonding strength between a surface functionalized with carboxyl groups (COO-) and a protein (NH3+ groups). Indicator: method
  • A registered international patent, 3 ISI-indexed articles and an article presented to international conference.

Executive summary of the activities carried out during Stage I of the project (2022)

For the period of the first stage, a series of activities were carried out regarding:
(i) development of the study protocol and implementation of the proposed immunogenic design,
(ii) synthesis and characterization of gold nanoparticles (NP-Au), coated with sodium citrate, having optimized dimensions,
(iii) preparation and characterization of gold nanoparticles, coated with sodium and aluminum citrate (NP-Au-Al), with sizes comparable to those of SARS-CoV-2, as well as
(iv) in vitro biocompatibility testing on two normal cell types (human fibroblasts and Adipose tissue-Derived Stem Cells (ADSC)) and tumor cells (HeLa cells and human osteosarcoma).

The most significant results concern:
(a) the very high biocompatibility of gold nanoparticles coated with sodium citrate (over 130% in the case of STEM cells),
(b) the obtaining of suspensions of gold nanoparticles functionalized with sodium citrate crosslinked with aluminum ions (NP-Au-Al) with dimensions close to those of SARS-CoV-2,
(c) very good biocompatibility of NP-Au-Al nanoparticles (around 100%)
(d) the obtaining of an excellent stability of gold nanoparticles stabilized with sodium aluminum citrate (NP-Au-Al) in a solution with a very high ionic strength (comparable to that of the blood medium).
(e) significant enhancement of surface plasmon resonance of NP-Au-Al nanoparticles.

Synthesis of gold nanoparticles covered with sodium citrate and aluminum with sizes close to those of SARS-CoV-2, which retain their stability in a solution with a very high ionic strength (NaCl, 4M), along with a significant increase in plasmonic resonance (over 20% compared to NP-Au), is a result of particular importance and significance taking into account that this method, which is most often used to obtain gold nanoparticles and which has its roots in the experiments of M. Faraday in the mid-1850s, being later perfected by Turkevich in 1951, always had this shortcoming of instability in media with medium and high ionic strengths, which limited, to some extent, the use of NP-Au in biomedical applications, including in detection and diagnosis, due to the need of increased amounts of surface-active biomolecules (e.g. enzymes and antibodies) to prevent nanoparticles from agglomeration and destabilization, including in the blood environment.

It was also demonstrated the possibility to introduce aluminum ions into the citrate structure, thus creating the premises for an increased response of the body's immune system, knowing that aluminum is an adjuvant often used in the vaccine industry to stimulate the production of specific antibodies. This approach makes the conventional additional introduction of aluminum-based chemical compounds redundant.

Executive summary of the activities carried out during Stage I of the project (2023)

For the period of the first stage, a series of activities were carried out regarding:
(i) synthesis and characterization of gold nanoparticles coated with sodium citrate and iron metal ions (AuNP-Fe) to validate the versatility of the method.
(ii) functionalization of AuNP-Al (mainly) and AuNP-Fe (secondary) with commercially available recombinant spike proteins, with final sizes and surfaces comparable to those of SARS-CoV-2.
(iii) registration of an international patent.
(iv) in vitro biocompatibility testing on two normal cell types (human fibroblasts and Adipose tissue-Derived Stem Cells (ADSCs)) and tumor cells (human osteosarcoma and HeLa cells).

The most significant results concern:
(a) excellent biocompatibility of gold nanoparticles coated with sodium citrate and iron ions,
(b) obtaining suspensions of gold nanoparticles functionalized with sodium citrate cross-linked with iron ions (Au-NP-Fe) with dimensions close to those of SARS-CoV-2,
(c) obtaining a good stability of AuNP-Fe in a solution with a very high ionic strength (comparable to that of the blood environment).
(d) significant enhancement of surface plasmon resonance of AuNP-Fe nanoparticles
(e) very good biocompatibility of AuNP-Al and AuNP-Fe nanoparticles functionalized with spike proteins.
(f) a patent for a green method of obtaining cavitary noble metal nanoparticles, mainly gold and silver.

At this stage of the project, the synthesis of gold nanoparticles coated with iron citrate of sizes close to those of SARS-CoV-2 was achieved. The nanoparticles retain their stability in a solution with increased ionic strength (saline), along with a significant increase in plasmonic resonance (more than 116 % higher than in the case of AuNPs). Although the stability is not as high as in the case of cross-linking with aluminum ions, the result is a special one especially through the significant increase of the surface plasmon resonance. Thus, these nanoparticles can be used in biomedical applications, including detection and diagnosis , especially for the useful signal they can provide.

Also, the surfaces of AuNP-Al and AuNP-Fe nanoparticles were successfully functionalized with spike proteins, the resulting compounds being dimensionally close to SARS-CoV-2. Moreover, nanoparticles functionalized with spike proteins were also tested on laboratory animals (the activity being an additional one).

The registered patent concerned cavitary noble metal nanoparticles (without a core), functionalized on the surface with natural molecules, in order to increase their adsorption surface area. The increase of the adsorption surface area is of great interest in the project because it will allow the increase of the loading capacity of nanoparticles with protein spike.

OBTAINED RESULTS:

ISI-indexed articles

  1. O.-G. Dragos-Pinzaru, G. Buema, D.-D. Herea (autor corespondent), H. Chiriac, N. Lupu, A.E. Minuti, G. Stoian, D. Shore, V.C. Pierre, I. Tabakovic, B.J.H. Stadler, Synthesis and Characterization of Gold-Shell Magnetic Nanowires for Theranostic Applications, Coatings, 12(11):1755 (2022); https://doi.org/10.3390/coatings12111755 (IF - 3.236)
  2. G. Buema, D.-D. Herea, O.-G. Dragos-Pinzaru, Special Issue: Ceramic and Metallic Biomaterials. Application în Medical Sciences, Coatings, 12 (7), 998 (2022); https://doi.org/10.3390/coatings12070998 (IF - 3.236)
  3. Luminita Labusca, Camelia Danceanu, Anca Emanuela Minuti, Dumitru-Daniel Herea (corresponding author) et al. Magnetic nanowires substrate increases adipose-derived mesenchymal cells osteogenesis. Sci Rep, 12, 16698 (2022); https://doi.org/10.1038/s41598-022-21145-z (IF - 4.996)
  4. Adina-Elena Segneanu, Gabriela Vlase, Teodora Alexandra Lukinich-Gruia, Dumitru-Daniel Herea, and Ioan Grozescu, Untargeted Metabolomic Approach of Curcuma longa to Neurodegenerative Phytocarrier System Based on Silver Nanoparticles, Antioxidants, 11, no. 11: 2261 (2022); https://doi.org/10.3390/antiox11112261 (IF - 7.675)
  5. Herea, D.D.; Zară-Dănceanu, C.-M.; Lăbușcă, L.; Minuti, A.-E.; Stavilă, C.; Ababei, G.; Tibu, M.; Grigoraș, M.; Lostun, M.; Stoian, G.; et al. Enhanced Multimodal Effect of Chemotherapy, Hyperthermia and Magneto-Mechanic Actuation of Silver-Coated Magnetite on Cancer Cells. Coatings 2023, 13 (2), 406. https://doi.org/10.3390/coatings13020406 (IF - 3.4)
  6. Segneanu, A.-E.; Vlase, G.; Vlase, T.; Sicoe, C.A.; Ciocalteu, M.V.; Herea, D.D.; Ghirlea, O.-F.; Grozescu, I.; Nanescu, V. Wild-Grown Romanian Helleborus purpurascens Approach to Novel Chitosan Phyto-Nanocarriers—Metabolite Profile and Antioxidant Properties. Plants 2023, 12 (19), 3479. https://doi.org/10.3390/plants12193479 (IF - 4.5)
  7. Segneanu, A.-E.; Vlase, G.; Chirigiu, L.; Herea, D.D.; Pricop, M.-A.; Saracin, P.-A.; Tanasie, Ș.E. Romanian Wild-Growing Armoracia rusticana L.—Untargeted Low-Molecular Metabolomic Approach to a Potential Antitumoral Phyto-Carrier System Based on Kaolinite. Antioxidants 2023, 12, 1268. https://doi.org/10.3390/antiox12061268 (IF - 7)
  8. Segneanu, A.-E.; Trusca, R.; Cepan, C.; Mihailescu, M.; Muntean, C.; Herea, D.D.; Grozescu, I.; Salifoglou, A. Innovative Low-Cost Composite Nanoadsorbents Based on Eggshell Waste for Nickel Removal from Aqueous Media. Nanomaterials 2023, 13, 2572. https://doi.org/10.3390/nano13182572 (IF - 4.5) - Herea, D.D. and Segneanu, A.-E. contributed equally to this work.
  9. Zară-Dănceanu, C.M.; Stavilă, C.; Minuti, A.E.; Lăbușcă, L.; Nastasa, V.; Herea, D.-D.; Malancus, R.-N.; Ghercă, D.; Pasca, S.-A.; Chiriac, H.; et al. Magnetic Nanoemulsions for the Intra-Articular Delivery of Ascorbic Acid and Dexamethasone. Int. J. Mol. Sci. 2023, 24, 11916. https://doi.org/10.3390/ijms241511916 (IF – 5.3)

Book chapter

Dumitru-Daniel Herea, Luminiţa Lăbuşcă, Nicoleta Lupu, Horia Chiriac, Magnetic particles for drug delivery, in Woodhead Publishing Series in Electronic and Optical Materials, Magnetic Sensors and Actuators in Medicine, Editor(s): Horia Chiriac, Nicoleta Lupu, Woodhead Publishing, Elsevier, 2023, Pages 259-304, ISBN 9780128232941, https://doi.org/10.1016/B978-0-12-823294-1.00002-6.

Patent applications

  1. D. D. Herea, N. Lupu, H. Chiriac, G. Stoian, O. G. Dragoș-Pînzaru, G. Buema, C. Stavilă, M. Grigoraș, L. Lăbușcă, C. M. Zară, A. E. Minuti, G. Ababei, D. Gherca
    „Metodă ecologică de preparare de nanoparticule cavitare de metal nobil”
    Nr. de înregistrarea2023 00290 / 09.06.2023
  2. D. D. Herea, N. Lupu, H. Chiriac, G. Stoian, O. G. Dragoș-Pînzaru, G. Buema, C. Stavilă, M. Grigoraș, L. Lăbușcă, C. M. Zară, A. E. Minuti, G. Ababei, D. Gherca
    Ecological method of preparing cavitary nanoparticles of noble metal”
    Patent registration number: PCT/RO2023/000014

CONTACT DETAILS:
Herea Dumitru-Daniel
National Institute of Research and Development for Technical Physics, NIRDTP Iasi
47 Mangeron Boulevard, 700050, Iasi
email: dherea@phys-iasi.ro
Tel: 0232-430.680
Fax: 0232-231.132