Phase I (2018) Results
(1) Synthesis and characterization of Au-AgNP with antibacterial properties and preparation of AU-AGNP coated with Manan- (initiating activities)
As the main results and conclusions:
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1. The Au-Ag nanoparticles with different metallic proportions were synthesized, using a two-stage working protocol, with the initial synthesis of the Au core and the gradual coating with the Ag being able to produce metallic nanoparticles with controllable Au / Ag ratios.
2. Characterization of the synthesized Au-AgNP properties shows that they have:
- a spherical shape, smooth surface,
- are homogeneously dispersed in aqueous solution and
- have a diameter between 37.84-53nm (adding the Ag coating gradually increasing the size of the nanoparticles).
Hyperspectral microscopy images with spectral 'fingerprints' for the obtained Au and Au-Ag nanoparticles
Atomic Force Microscopy Images of the obtained Au and Au-Ag nanoparticles
Phase II (2019) Results
(2) Synthesis and characterization of Au-AgNP nanoparticles with antibacterial properties and preparation of Mannan-coated Au-AgNP. Bacterial culture testing of antibacterial functions of Mannan-coated Au-AgNP.
The main results and conclusions:
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1. Mannan-coated Au-Ag nanoparticles were synthesized, following a step-by-step protocol that involved mannan carboxylation and finally conjugation with NPs synthesized in project stage 1 of the project.
2. The Au-Ag nanoparticles coated with mannan have been characterized from a physicochemical point of view. The synthesized nanoparticles have:
- a round or ovoid shape
- are smooth
- have an average size of 6.36 -50 nm (2 dimensional populations)
- an Au: Ag ratio of up to 75: 35
- the connection with mannan has been confirmed.
3. Colonies of Mycobacterium tuberculosis expressing fluorescence were obtained in the red zone secondary to the expression of dtTomato protein embedded in the transfer plasmid.
4. The synthesized AuAgMannan nanoparticles have antimicrobial activity in liquid media against the Mycobacterium species tested, and the CMI was established as 5 μg / mL for M abcessus and 10 μg / mL for M tuberculosis and M tuberculosis / bovis subs. goats.
5. The synthesized AuAg-Mannan nanoparticles have as antibacterial mechanism the induction of bacterial membrane lysis, followed by apoptosis / bacteriolysis.
6. The preliminary data of the research and the related bibliographic documentation were processed in the form of an article sent before publication to the International Journal of Nanomedicine (IF = 4,471) [subissions ID 241183].
Hyperspectral microscopy images with spectral 'fingerprints'
Hyperspectral and Fluorescent microscopy images of M. tuberculosis cultures incubated with Au-AgN- Mannan
CLSM images of LIVE/DEAD Mycobacteria incubated with Au-AgNP
TEM images of the Au-Ag NP
Phase III (2020) Results
(3) In vitro quantification of selective internalization and the relationship between dose/particle size and Mannan-coated Au-AgNP toxicity on macrophages. In vitro evaluation of the antimycobacterial function of Mannan-coated Au-AgNP against infected macrophages M tuberculosis.
The main results and conclusions:
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1. Murine peritoneal macrophages, which express antigens specific to this group of cells, have been isolated and characterized.
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2. Mannan-coupled AuAgNPs nanoparticles are internalized at an increased level of macrophages.
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3. Internalization is rapid (starting with 1 hour after exposure) and nanoparticles persist at the intracellular (intramacrophage) level.
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4. Endocytosis is dependent on cell time, being minimal for non-pituitary cell lines (eg HELA).
Fig. 1. "Dark-field" hyperspectral microscopy (CytoViva), together with spectral fingerprints for peritoneal macrophages (A, C) and HeLa cell line (B, D) after exposure to Mannan Au-AgNP (10µg / ml). Image C represents the spectral footprint of internalized Au-AgNP, and image D the spectrum of the cytoplasm of HeLa cells. Obx100, immersion
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6. Au-Ag nanoparticles with reduced cytotoxicity on peritoneal macrophages, indicated by good cell viability, reduced apoptosis and reduced oxidative stress.
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7. Exposure of peritoneal macrophages to mannan-conjugated Au-Ag induces inflammatory activation, indicated by increased expression of p-38 MAPK, c-Jun and SAPK / JNK proteins, and increased IL-1β expression.
Fig. 2. Fig. Peritoneal macrophages exposed to Au-AgNP. C11-BODIPY staining (581/591) for the determination of lipid oxidative stress (lipid peroxidation and intracellular membrane trafficking). Orange, reduced form of C11-BODIPY (A, D, G); Green form, the oxidized form of C11-BODIPY (B, E, H). The right column (C, F, I) overlap the two fluorescence lines. CLSM Images, ob x63, apochrome, immersion.
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8. Infection of macrophages with Mycobactreium tuberculosis (Mtb, strain with attenuated virulence H37Ra) was performed, internalization being rapid (starting with 1 h and well expressed at 6 h after exposure).
Fig. 3. Examination of fluorescent Mycobactreium colonies before infection of macrophages. CLSM, ob x63, apochromatic, immersion.
Fig. 4. Internalization of Mycobacterium tuberculosis in peritoneal macrophages after exposure to Mannan-conjugated Au-Ag NP (6 hours). Red canal - Mycobacterium tuberculosis, green canal - cytoskeleton (filamentous actin). CLSM, ob x63, apochromatic, immersion.
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9. The addition of Mannan to the molecule enhances the antimycobacterial effect of Ag-Au NP.
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10. Au-AgNP and Au-AgNP conjugated with Mannan do not produce complete inhibition of the growth of intrcellular (intramacrophage) mycobacteria.
Fig. 5. "Dark-field" hyperspectral microscopy (CytoViva), together with spectral fingerprints for peritoneal macrophages after exposure to mycobacterial infection and after exposure to Au-AgNP and Au-AgNP Mannan (10µg / ml). The blue arrows indicate the corresponding contrast points for internalized Au-AgNPs. Insect-image C represents the spectral footprint of internalized Au-AgNP. Obx100, immersion.
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11. The addition of Mannan to the molecule enhances the antimycobacterial effect of Ag-Au NP.
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12. Au-AgNP and Au-AgNP conjugated with Mannan do not produce complete inhibition of the growth of intrcellular (intramacrophage) mycobacteria.
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13. One ISI article (IF 5.115) and 1 summary of a paper presented at the conference were published.
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14. There were 3 presentations at scientific symposia (2 as first author at international symposia and 1 as co-author at national symposium) and 2 presentations at Invention Salons.
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15. Three OSIM patents were obtained (which can be found in the WEB of Knowledge) and a patent application was filed with OSIM:
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16. Five national awards were obtained (UEFSCDI, PRECISI-2020 for the published article, PRO INVENT 2020 Gold Medal and three UEFSCDI awards, PRECBVT-2020 for the patents obtained).