Green synthesis, characterization and biofunctionalisation of nanoparticles for medical applications

Abstract

In the presented work, the phytochemicals existent in the aqueous extract of (Hypericum perforatum L.) St. John's wort was harnessed to prepare silver nanoparticles. Many conditions have been tried and changed until we reached the final protocol, through which we obtained the desired nanoparticles in terms of size, shape and effectiveness. The organic compounds present in the St. John's wort plant played an important role in reducing the silver ions in the solution to metallic silver, as well as in protecting the formed silver nanoparticles in nano dimensions and preventing them from growing to millimeter dimensions by forming a protective layer on the surfaces of these nanoparticles and finally maintaining the stability of these formed nanoparticles in colloidal solutions. This green chemistry approach for the preparation of AgNPs is a simple, safe, sustainable, credible and eco-friendly protocol and the resulting silver nanoparticles are considered promising for later application in the treatment of various infectious and non-communicable diseases. green synthesized silver nanoparticles have been characterized using various techniques such as ultraviolet-visible spectroscopy (UV-Vis), dynamic light scattering (DLS), zeta potential, Fourier transform infrared (ATR-FTIR) spectroscopy, X-Ray diffraction (XRD), scanning electron microscope (SEM), transmission electron microscopy (TEM), energy dispersive X-Ray (EDX), nanoparticle tracking analysis (NTA), atomic force microscopy (AFM), thermogravimetric analysis (TGA) and atomic absorption spectroscopy (AAS), and all results proved that biosynthesized silver nanoparticles are spherical in shape, stable in colloidal solution, the size of their particles ranges between 20 to 50 nm, have a face-centered cubic (fcc) and crystalline in nature and on the surfaces of these particles, there is a protective layer consisting of a group of St. John's wort compounds, the percentage of which varies according to the number of washing times. It is known that reducing agents and the chemical composition of nanoparticle surfaces are the most influential factors in determining the activity and toxicity of these nanoparticles later because they affect cellular uptake, biodistribution, penetration into biological barriers and the resulting therapeutic effects. Therefore, the second objective of this study was to identify the organic compounds from the aqueous extract of the St. John’s wort, which is present on the surfaces of silver nanoparticles as a protective agent. To achieve this goal, it was necessary to analyze the plant itself, i.e. develop a protocol in HPLC to separate the components of the extracts for this plant well. The aerial parts of the plant were extracted using 8 different solvents. A simple protocol has been developed to obtain isolated peaks in the HPLC spectrum. Detection was carried out at 260 for phloroglucinols (Hyperforin and derivates), 350 for Flavonols and 590 nm for naphthodianthrones (Hypericins). Various standards were selected for this, which also represent the most important and best-known compounds of St. John's wort and the mass spectrometric analysis in positive ion mode was performed to allow in-line analysis coupled directly to the HPLC system used for the separation of the molecular ions according to mass to charge (m/z). Finally, the major ingredients (Hyperforin, Adhyperforin, Hypericin, Rutin, Quercetin, Quercitrin, Quercitrin-hydrate, Hyperoside, Biapigenin and Chlorogenic acid) have been identified. Total phenolic, antioxidant activity (DPPH and ABTS assays.) and their relationship for different extracts were also presented in this study. In another study, the layer on the surfaces of silver nanoparticles was isolated using a mixture of solvents and following a specific protocol. After that, LC–ESI-Q-TOF–MS/MS analysis was carried out to determine these substances, and they have already been identified, which are 1=Neochlorogenic acid; 2=Hyperoside; 3=Isoquercitrin; 4=l3,II8-biapigenin; 5=Furohyperforin; 6= Hyperforin; 7=Furoadhyperforin; 8=Adhyperforin. Antioxidant activity of the biologically prepared AgNPs was studied using 3 different methods: DPPH, ABTS and SO assays, and the results were very impressive and better than all that was mentioned by other researchers. The antimicrobial effect on about 20 types of microbes (Gram-positive bacteria, Gram-negative bacteria, Pathogenic yeast and leishmaniasis tropica Syrian strain (LT_SYR_24)) has been studied in multiple areas and using different methods. In fact, the results were very excellent compared to antibiotics, silver nitrate, as well as silver nanoparticles prepared by other researchers, as they were mostly lethal at very low concentrations. Anti-cancer activity against 3 types (Hela, Hep G2 and A549 cells) at various concentrations and various exposure times, and the results were very distinctive and promising for use as a future treatment for cancer. After the prepared silver nanoparticles achieved great success in treating different types of cancer cells, the last and most important step was how to modify these particles to be selectivity, that is, when injected in vivo, they go directly to the tumor or cancer cells without affecting healthy cells. Since oligonucleotide-based aptamers (APTs) are excellent ligands for targeting cancer cells, we have already developed a special protocol to conjugate silver nanoparticles prepared in our method with a specific aptamer as a selective targeting part for uptake by A549 cells. Many conditions and factors have been tried to reach a high coupling ratio without affecting the effectiveness of the aptamer. the cytotoxicity of aptamer-conjugated AgNPs against A549 (human non-small cell lung cancer) and BEAS-2B (normal human bronchial epithelial ) were studied using CTB test, cellular uptake, viability staining (using Calcein AM and Propidium Iodide), Quantitation of Apoptosis and Necrosis cells (using Annexin V and Propidium Iodide) and Cellular morphological changes (laser scanning confocal microscope and normal microscope). All results indicated that the effect of aptamer conjugated AgNPs was very large on cancer cells (A549 cells) compared to healthy cells (BEAS2B) at the same or lesser concentrations. This indicates that these nanoparticles exhibited selective binding and internalization to target A549 cells, but not by normal human bronchial epithelium BEAS2B, thus exhibiting high selective specificity.