Dissemination
Phase 1 (2020) - Summary
In this phase, magnetite nanoparticles were functionalized with eugenol (Fe3O4@E). To demonstrate their nanometric crystallinity and dimension, they were characterized from the physical-chemical points of view. 15 laboratory and clinical strains frequently isolated from cutaneous infections (including biofilm-producing infections of G tubes) were selected. Among these, 9 bacterial strains (3 from laboratory and 6 clinically-isolated, Gram-positive, gram-negative and yeast cells) were chosen, which can adhere and form biofilms on inert support, in standard conditions.
Phase 2 (2021) - Summary
Fe3O4@E nanoparticles were covered with a silica shell in six different concentrations. These were analysed from the physical-chemical and qualitative and quantitative perspectives on S. aureus, E. coli and C. albicans strains. In the case of Fe3O4@E_TEOS_1/2/3 and Fe3O4@E_APTMS_1/2/3 core/shell structures, similar effect on the cellular viability was indicated. No major differences in toxicity were observed between the recepies. The most pronounced toxic effect on CCD-1070Sk cells for both investigated cases, were reported for the smallest concentration. The most pronounced cytotoxic effect on the dermal fibroblast cells was observed after 48 h from the treatment. After the in vitro screening of the core/shell nanoparticles’ cytotoxicity, the optimum concentrations of the components for the surface modification of the original G tubes were determined. The results of this study evidenced that the experiments using the highest silica concentration (TEOS3 si APTMS3) will be studied also as thin films. Both Fe3O4@E and core/shell nanoparticles were synthesized as thin films by MAPLE technique onto glass (initially) and G tube (subsequently) substrates, at different laser fluences (of 200, 300, 400 and 500 mJ/cm2, respectively) to find the optimum parameters which will not alter the stoichiometry of the structures. The highlighting of the growth and multiplication capacity of the selected microorganisms on the obtained coatings was performed both on glass substrates containing the developed experimental recipes, as well as on nanomodified G-tube samples. Both the viability and microorganisms’ multiplication capacity on the G-tube coatings were analyzed and it was observed that all tested coatings showed an inhibitory effect on the analyzed microorganisms. Depending on the fluence used for the film deposition, differences in the adhesion capacity of the microorganisms were observed. Thus, the core/shell coatings deposited at F = 400 mJ/cm2 indicated the most significant inhibitory effect on the adhesion of microorganisms after 24 h of incubation. Analysis of the morphology and proliferation of human cells in the presence of nanomodified G-tube samples was performed. After the determination of the optimal concentration of core/shell nanoparticles and the production of the nanomodified G-tubes, based on the optimal parameters previously determined, investigations were performed in order to evaluate the biocompatibility and biological performance of the final materials. It was demonstrated that changing the surface of G-tubes was absolutely necessary to support the viability of dermal fibroblast cells. After 48 h from seeding, the viability of CCD-1070Sk cells increased significantly when in contact with nanomodified G-tubes, the highest cell viability being observed in the case of the samples with the highest silica concentration, deposited at F = 400 mJ/cm2. The same trend of cell viability was observed even after 5 days of seeding. Moreover, the nanomodified G-tubes supported the proliferation of dermal fibroblasts over time, with a significant increase in cell viability after 5 days of seeding in comparison to 48 h for all analysed samples.
Phase 3 (2022) - Summary
In this phase, fresh core/shell nanostructures based on magnetite-silice shell (starting from known concentrations of APTMS and TEOS) were fabricated. Using a modified co-precipitation protocol to obtain silica shell, a basic solution containing an optimized quantity of APTMS/TEOS was prepared. Considering the IRM results previously reported, we decided to exclude both the fluence of 200 mJ/cm2, when non-uniform (inefficient material transfer), and 500 mJ/cm2, degradation of functional groups, were observed. From the stoichiometric and morphologic points of view, the 400 mJ/cm2 fluence represented the optimum solution to obtain core/shell nanoparticles based on Fe3O4, eugenol and APTMS / TEOS, demonstrating the mandatory characteristics of a coating (i.e., reproductibility, stability, etc.). The as-obtained coatings were therefore demonstrated to inhibit the adhesion, growth and multiplication of tested micro-organisms at the surface of G tubes.
The reported results have indicated that bacterial adhesion and the development of monospecific biofilms were drastically reduced in the presence of the fabricated coatings, depending on the used micro-organisms and the presence of antimicrobial agents. The biofilms investigations at different periods of time demonstrated that, the structuring of the micro-organisms was significantly altered at 24 – 72 h. After 24 h of incubation, biofilm inhibition patterns for Fe3O4@E, APTMS3@E și TEOS3@E of 1 log at S. aureus and E. coli and 1,5 log at C. albicans were revealed.
In general, the tested antimicrobial materials did not accumulate bacteriostatic or bactericidal doses, after 24 - 72 h of incubation, which demonstrated their local antimicrobial effect, dependent on the contact between the antimicrobial cells with the surface of the bioactive coatings. The coatings showed excellent biocompatibility, sustaining human fibroblasts viability, growth and architecture. Moreover, the exposure of whole blood samples to the core/shell Fe3O4@SiO2 nano-systems failed to stimulate cytokine production, highlighting that the novel coating does not exert inflammation. These results demonstrated that, the fabricated coatings did not manifest pro-inflammatory effects.
The proven biocompatibility and lack of pro-inflammatory potential, corroborated with efficient anti-biofilm properties, manifested on Gram-positive and Gram-negative opportunistic bacteria, but also, yeast, should recommend these type of coatings as suitable candidates to be further investigated for the coverage of medical devices.
Brief presentation of the results
The clinical management of patients with critical conditions or pathologies associated with malnutrition and substitution therapy involves the use of the G tube, which represents a necessary alternative for the provision of nutritional support and drug delivery. As a result of the high susceptibility to contamination and microbial colonization phenomena, the G-tubes represent a source of contacting nosocomial infections and the occurrence of infectious complications.
The aim of the project was to develop an experimental model to improve the surface of G tubes by using a bioactive nanosystem based on oxide nanostructures and phytochemicals with antimicrobial effect.
The obtained results confirmed the excellent biocompatibility of the nanostructured coatings (demonstrated by using complementary techniques on dermal fibroblast cell cultures), as well as their antimicrobial (antibiofilm effect maintained for at least 3 days) and anti-inflammatory (non-stimulation of pro-inflammatory cytokine production) effects.
Articles
1. Lazar V., Holban A.M., Curutiu C., Chifiriuc M.C. Modulation of Quorum Sensing and Biofilms in Less Investigated Gram-Negative ESKAPE Pathogens. Front. Microbiol. 12, 676510, 2021. IF = 6.064
2. Marinescu L., Ficai D., Ficai A., Oprea O., Nicoara A.I., Vasile B.S., Boanta L., Marin A., Andronescu E., Holban A.-M. Comparative Antimicrobial Activity of Silver Nanoparticles Obtained by Wet Chemical Reduction and Solvothermal Methods. Int. J. Mol. Sci. 23, 5982, 2022. IF2021 = 6.208
3. Hudiță A., Grumezescu V., Gherasim O., Grumezescu A.M., Dorcioman G., Negut I., Oprea O.C., Vasile B.Ș., Gălățeanu B., Curuțiu C., Holban A.M. MAPLE processed nanostructures for antimicrobial coatings. Int. J. Mol. Sci. 23(23), 15355, 2022. IF2021 = 6.208
Conferences
1. Grumezescu V.; Gherasim, O.; Negut I.; Dorcioman G.; Grumezescu A.M.; Basil H.; Holban A.M. Fe3O4 nanoarchitectures functionalized with eugenol for modulation of virulence and biofilm formation. European Materials Research Society (E-MRS) - VIRTUAL Conference May 31st - June 3rd 2021– ePoster.
2. Holban A.M., Grumezescu A.M., Ditu L.M., Curutiu C., Grumezescu V., Tran V.L., Lazar V., Gheorghe I. Inorganic nanoparticles functionalized with Eugenol to modulate virulence in Pseudomonas aeruginosa clinical isolates. European Congress of Clinical Microbiology & Infectious Diseases (ECCMID) 23 – 26 April 2022, Lisbon, Portugal – Poster
3. Curutiu C., Van L.T., Hagiu I., Popovici A., Marinescu F., Gheorghe I., Ditu L.M., Holban A.-M., Sesan T.E., Lazar V. Antimicrobial activity of essential oils against resistant Pseudomonas aeruginosa strains. 22nd Romanian International Conference on Chemistry and Chemical Engineering (RICCCE) 7 - 9 September 2022, Sinaia, Romania – prezentare orală
4. Grumezescu V., Hudiță A., Gherasim O., Negut I., Dorcioman G., Grumezescu A.M., Ditu L.M., Holban A.M. Core/shell nanosystem for modulation of microbial biofilm formation on G-tube surfaces. European Materials Research Society (E-MRS) VIRTUAL Conference May 30 -June 3, 2022 – ePoster
Patent
1. Grumezescu V., Gherasim O. Rețetă de îmbunătățire a suprafeței dispozitivelor implantabile. (A/00759 / 23.11.2022)