Green synthesis, characterization and biofunctionalisation of nanoparticles for medical applications

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

Investigation of Cytotoxicity of Biosynthesized Colloidal Nanosilver against Local Leishmania tropica: In Vitro Study

Leishmaniasis is one of the biggest health problems in the world. Traditional therapeutic methods still depend on a small range of products, mostly chemically. However, the treatment with these drugs is expensive and can cause serious adverse effects, and they have inconsistent effectiveness due to the resistance of parasites to these drugs. The treatment of leishmanial disease has always been a challenge for researchers. The development of nanoscale metals such as silver has attracted significant attention in the field of medicine. The unique characteristic features of silver nanoparticles (AgNPs) make them effective antileishmanial agents. In recent years, green nanotechnology has provided the development of green nanoparticle-based treatment methods for Leishmaniasis. Although there are many studies based on green nanoparticles against Leishmania parasites, this is the first study on the antileishmanial effect of biosynthesized AgNPs using an aqueous extract of Eucalyptus camaldulensis leaves (AEECL) as a reducing agent of silver ions. Different parameters such as AgNO3 concentration, AEECL concentration, and reaction time were studied to investigate the optimum factors for the preparation of stable and small-sized silver nanoparticles. The spherical shape of colloidal nanosilver (CN-Ag) was confirmed by atomic force microscope (AFM) and scanning electron microscope (SEM) images with sizes of 27 and 12 nm, respectively. A high density of nanoparticles with a small size of 10 nm has been confirmed from dynamic light scattering (DLS) analysis. The zeta potential value of 23 mV indicated that colloidal silver nanoparticles were stable. The nano-tracker analysis (NTA) showed the Brownian motion of silver nanoparticles with a hydrodynamic diameter of 31 nm. The antioxidant property of CN-Ag was determined using the stable radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. In this study, a significant cytotoxic effect of biosynthesized CN-Ag has been shown against Leishmania tropica parasites at low concentrations (1.25, 2.5, and 3.75 µg/mL). These results could be used as a future alternative drug or could be a supportive treatment for Leishmaniasis

Influence of Polyvinylpyrrolidone Concentration on Properties and Anti-Bacterial Activity of Green Synthesized Silver Nanoparticles

Environmentally green synthesis of stable polyvinyl pyrrolidone (PVP)-capped silver nanoparticles (PVP-AgNPs) was successfully carried out. The present study focused on investigating the influence of adding PVP during the synthesis process on the size, optical properties and antibacterial effect of silver nanoparticles produced. An aqueous extract of Eucalyptus camaldulensis leaves was used as a reducing agent. The effects of different PVP concentrations and reducing time on the synthesis of nanoparticles (NPs) were characterized by UV–Vis spectrophotometry, scanning electron microscopy (SEM), energy dispersive spectrum (EDX), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and nano tracker analysis (NTA). The addition of PVP was studied. The prepared PVP-AgNPs were spherical with an average size of 13 nm. FTIR analysis confirmed that PVP protects AgNPs by a coordination bond between silver nanoparticles and both N and O of PVP. DLS results indicated the good dispersion of silver nanoparticles. PVP-AgNPs were found to be stable for nearly 5 months. Antibacterial studies through the agar well diffusion method confirmed that silver nanoparticles synthesized using PVP had no inhibitor activity toward Gram-positive and Gram-negative bacteria as opposed to silver nanoparticles prepared without adding PVP, which showed a significant antibacterial activity towards some of the tested pathogens

Hypericum perforatum L.-Mediated Green Synthesis of Silver Nanoparticles Exhibiting Antioxidant and Anticancer Activities

This contribution focuses on the green synthesis of silver nanoparticles (AgNPs) with a size < 100 nm for potential medical applications by using silver nitrate solution and Hypericum Perforatum L. (St John’s wort) aqueous extracts. Various synthesis methods were used and compared with regard to their yield and quality of obtained AgNPs. Monodisperse spherical nanoparticles were generated with a size of approximately 20 to 50 nm as elucidated by different techniques (SEM, TEM). XRD measurements showed that metallic silver was formed and the particles possess a face-centered cubic structure (fcc). SEM images and FTIR spectra revealed that the AgNPs are covered by a protective surface layer composed of organic components originating from the plant extract. Ultraviolet-visible spectroscopy, dynamic light scattering, and zeta potential were also measured for biologically synthesized AgNPs. A potential mechanism of reducing silver ions to silver metal and protecting it in the nanoscale form has been proposed based on the obtained results. Moreover, the AgNPs prepared in the present study have been shown to exhibit a high antioxidant activity for 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) radical cation, and super oxide anion radical and 2,2-diphenyl-1-picrylhydrazyl. Synthesized AgNPs showed high cytotoxicity by inhibiting cell viability for Hela, Hep G2, and A549 cells

Identification of Major Constituents of Hypericum perforatum L. Extracts in Syria by Development of a Rapid, Simple, and Reproducible HPLC-ESI-Q-TOF MS Analysis and Their Antioxidant Activities

Hypericum perforatum Linn (St. John’s wort) is a popular and widespread medicine in Syria, which is used for a wide range of conditions, including gastrointestinal diseases, heart disease, skin diseases, and psychological disorders. This widespread use prompted us to identify the main compounds of this plant from Syria that are responsible for its medicinal properties, especially since its components differ between countries according to the nature of the soil, climate, and altitude. This is, to the best of our knowledge, the first report in which St. John’s wort, a plant native to Syria, is extracted using different solvents and its most important compounds are identified. In this study, the dried above-ground parts, i.e., leaves, stem, petals, and flowers, were extracted using different solvents (water, ethanol, methanol, and acetone) and extraction protocols. By increasing the polarity of the solvent, higher yields were obtained, indicating that mainly hydrophobic compounds were extracted. Therefore, we conclude that extraction using the tea method or using a mixture of water and organic solvents resulted in higher yields compared with pure organic solvents or continuous boiling with water for long periods. The obtained extracts were analyzed using high-performance liquid chromatography equipped with a diode array detector (HPLC–DAD), coupled with UV–visible spectrophotometry at a full spectrum (200–800 nm). The HPLC spectra of the extracts were almost identical at three wavelengths (260 nm for phloroglucinols (hyperforin and derivates), 590 nm for naphthodianthrones (hypericins), and 350 nm for other flavonols, flavones, and caffeoylquinic acids), with differences observed only in the intensity of the peaks. This indicates that the same compounds were obtained using different solvents, but in different amounts. Five standards (chlorogenic acid, quercetin, quercitrin hydrate, hyperoside, and hypericin) were used, and a comparison with retention times and ultraviolet (UV) spectra reported in the literature was performed to identify 10 compounds in these extracts: hyperforin, adhyperforin, hypericin, rutin, quercetin, quercitrin, quercitrin hydrate, hyperoside, biapigenin, and chlorogenic acid. Although the European Pharmacopoeia still describes ultraviolet spectroscopy as a method for determining the quantity of Hyperici herba, interference from other metabolites can occur. Combined HPLC–DAD and electrospray ionization–mass spectrometry (LC-ESI-MS) in the positive mode have therefore also been used to confirm the presence of these compounds in the extracts by correlating known masses with the identified masses or through characteristic fragmentation patterns. Total phenolic contents of the extracts were determined by the Folin–Ciocalteu assay, and antioxidant activity was evaluated as free radical scavenging capacity using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) assays. The results indicate that the aqueous extracts prepared by the tea method gave the highest total phenols, while the pure organic solvents gave very low phenols. Also, the extracts that contain the largest amount of phenols gave lower IC50 values or higher antioxidant activity than that of others

Green Synthesis of Silver Nanoparticles Using Hypericum perforatum L. Aqueous Extract with the Evaluation of Its Antibacterial Activity against Clinical and Food Pathogens

The rapid development of nanotechnology and its applications in medicine has provided the perfect solution against a wide range of different microbes, especially antibiotic-resistant ones. In this study, a one-step approach was used in preparing silver nanoparticles (AgNPs) by mixing silver nitrate with hot Hypericum perforatum (St. John’s wort) aqueous extract under high stirring to prevent agglomeration. The formation of silver nanoparticles was monitored by continuous measurement of the surface plasma resonance spectra (UV-VIS). The effect of St. John’s wort aqueous extract on the formation of silver nanoparticles was evaluated and fully characterized by using different physicochemical techniques. The obtained silver nanoparticles were spherical, monodisperse, face-centered cubic (fcc) crystal structures, and the size ranges between 20 to 40 nm. They were covered with a capping layer of organic compounds considered as a nano dimension protective layer that prevents agglomeration and sedimentation. AgNPs revealed antibacterial activity against both tested Gram-positive and Gram-negative bacterial strains causing the formation of 13–32 mm inhibition zones with MIC 6.25–12.5 µg/mL; Escherichia coli strains were resistant to tested AgNPs. The specific growth rate of S. aureus was significantly reduced due to tested AgNPs at concentrations ≥½ MIC. AgNPs did not affect wound migration in fibroblast cell lines compared to control. Our results highlighted the potential use of AgNPs capped with plant extracts in the pharmaceutical and food industries to control bacterial pathogens’ growth; however, further studies are required to confirm their wound healing capability and their health impact must be critically evaluate

Accumulation, Source Identification, and Cancer Risk Assessment of Polycyclic Aromatic Hydrocarbons (PAHs) in Different Jordanian Vegetables

The accumulation of polyaromatic hydrocarbons in plants is considered one of the most serious threats faced by mankind because of their persistence in the environment and their carcinogenic and teratogenic effect on human health. The concentrations of sixteen priority polycyclic aromatic hydrocarbons (16 PAHs) were determined in four types of edible vegetables (tomatoes, zucchini, eggplants, and cucumbers), irrigation water, and agriculture soil, where samples were collected from the Jordan Valley, Jordan. The mean total concentration of 16 PAHs (∑16PAHs) ranged from 10.649 to 21.774 µg kg−1 in vegetables, 28.72 µg kg−1 in soil, and 0.218 µg L−1 in the water samples. The tomato samples posed the highest ∑16PAH concentration level in the vegetables, whereas the zucchini samples had the lowest. Generally, the PAHs with a high molecular weight and four or more benzene rings prevailed among the studied samples. The diagnostic ratios and the principal component analysis (PCA) revealed that the PAH contamination sources in soil and vegetables mainly originated from a pyrogenic origin, traffic emission sources, and biomass combustion. The bioconcentration factors (BCF) for ∑16PAHs have been observed in the order of tomatoes > cucumbers and eggplants > zucchini. A potential cancer risk related to lifetime consumption was revealed based on calculating the incremental lifetime cancer risk of PAHs (ILCR). Therefore, sustainable agricultural practices and avoiding biomass combusting would greatly help in minimizing the potential health risk from dietary exposure to PAHs

Investigation of Cytotoxicity of Biosynthesized Colloidal Nanosilver against Local Leishmania tropica: In Vitro Study

Leishmaniasis is one of the biggest health problems in the world. Traditional therapeutic methods still depend on a small range of products, mostly chemically. However, the treatment with these drugs is expensive and can cause serious adverse effects, and they have inconsistent effectiveness due to the resistance of parasites to these drugs. The treatment of leishmanial disease has always been a challenge for researchers. The development of nanoscale metals such as silver has attracted significant attention in the field of medicine. The unique characteristic features of silver nanoparticles (AgNPs) make them effective antileishmanial agents. In recent years, green nanotechnology has provided the development of green nanoparticle-based treatment methods for Leishmaniasis. Although there are many studies based on green nanoparticles against Leishmania parasites, this is the first study on the antileishmanial effect of biosynthesized AgNPs using an aqueous extract of Eucalyptus camaldulensis leaves (AEECL) as a reducing agent of silver ions. Different parameters such as AgNO3 concentration, AEECL concentration, and reaction time were studied to investigate the optimum factors for the preparation of stable and small-sized silver nanoparticles. The spherical shape of colloidal nanosilver (CN-Ag) was confirmed by atomic force microscope (AFM) and scanning electron microscope (SEM) images with sizes of 27 and 12 nm, respectively. A high density of nanoparticles with a small size of 10 nm has been confirmed from dynamic light scattering (DLS) analysis. The zeta potential value of 23 mV indicated that colloidal silver nanoparticles were stable. The nano-tracker analysis (NTA) showed the Brownian motion of silver nanoparticles with a hydrodynamic diameter of 31 nm. The antioxidant property of CN-Ag was determined using the stable radical 2,2-Diphenyl-1-picrylhydrazyl (DPPH) assay. In this study, a significant cytotoxic effect of biosynthesized CN-Ag has been shown against Leishmania tropica parasites at low concentrations (1.25, 2.5, and 3.75 &micro;g/mL). These results could be used as a future alternative drug or could be a supportive treatment for Leishmaniasis

Influence of Polyvinylpyrrolidone Concentration on Properties and Anti-Bacterial Activity of Green Synthesized Silver Nanoparticles

Environmentally green synthesis of stable polyvinyl pyrrolidone (PVP)-capped silver nanoparticles (PVP-AgNPs) was successfully carried out. The present study focused on investigating the influence of adding PVP during the synthesis process on the size, optical properties and antibacterial effect of silver nanoparticles produced. An aqueous extract of Eucalyptus camaldulensis leaves was used as a reducing agent. The effects of different PVP concentrations and reducing time on the synthesis of nanoparticles (NPs) were characterized by UV&ndash;Vis spectrophotometry, scanning electron microscopy (SEM), energy dispersive spectrum (EDX), Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS) and nano tracker analysis (NTA). The addition of PVP was studied. The prepared PVP-AgNPs were spherical with an average size of 13 nm. FTIR analysis confirmed that PVP protects AgNPs by a coordination bond between silver nanoparticles and both N and O of PVP. DLS results indicated the good dispersion of silver nanoparticles. PVP-AgNPs were found to be stable for nearly 5 months. Antibacterial studies through the agar well diffusion method confirmed that silver nanoparticles synthesized using PVP had no inhibitor activity toward Gram-positive and Gram-negative bacteria as opposed to silver nanoparticles prepared without adding PVP, which showed a significant antibacterial activity towards some of the tested pathogens