Green synthesis and treatment of silver nanoparticles from Leishmania major in Iraq

Leishmaniases are a group of infectious and non-contagious severe parasitic diseases, caused by protozoans of the Leishmania genus. The transmission occurs through blood meal of infected female sand flies of the genera Phlebotomus, that are considered as intermediate hosts of these parasites. The flagellate promastigote form of the parasite is present in the sand fly, and once engulfed by host macrophages, it converts into the aflagellate amastigote form (1,2).

Biological methods of nanoparticles (NPs) synthesis using microorganisms (3,4), enzymes (5), fungus (6), and plants or plant extracts (7,8) have been suggested as possible eco-friendly alternatives to chemical and physical methods. The development of green processes for the synthesis of nanoparticles is evolving into an important branch of nanotechnology especially silver nanoparticles, which have many applications (9-11). Chemical synthesis methods lead to presence of some toxic chemical absorbed on the surface that may have adverse effect in the medical applications. Green synthesis provides advancement over chemical and physical method as it is cost effective, environment friendly, easily scaled up for large scale synthesis and in this method there is no need to use high pressure, energy, temperature and toxic chemicals (12,13). Silver nanoparticles play a profound role in the field of biology and medicine due to their attractive physiochemical properties. Silver products have long been known to have strong inhibitory and bactericidal effects, as well as a broad spectrum of antimicrobial activities, which has been used for centuries to prevent and treat various diseases, most notably infections (14).

The particle size of NPs is play an important role in antimicrobial efficiency (15), so the strongest effect of the smallest sized of NPs showed more than the largest sized (16). The growth of some parasites such as Giardia, Leishmania, Plasmodium, Toxoplasma and insect larva is inhibited with the presence of silver nanoparticles, gold, chitosan, and oxidized metals (17).

How were the silver nanoparticles prepared?

Materials and Methods: Preparation of AgNPs-TA A 5 mL of Tannic Acid (TA) were added on 5 mL of AgNO 3 at 25 °C in conical flask. The mixture was kept under strong magnetic stirring for 5 hours, until a brown colloidal dispersion of AgNPs-TA was seen and kept from light until use.

Characterization of nanoparticles UV-Vis spectra were managed in absorbance mode (range 200-900 nm) with a double beam to check the formation of nanoparticles. The particle size, shape and morphology were evaluated by scanning electron microscopy (SEM) images.

How were the parasites cultured and tested?

Promastigotes preparation Amastigotes of L. major which isolated from human skin lesions were cultured on Novy‐MacNeal‐Nicolle (NNN) medium containing agar (4 mg/100 ml) and defibrinated rabbit blood (10%). After transformation of amastigotes to promastigote‐like forms, the growth of promastigotes was continued in RPMI 1640 culture medium containing 100 μg/ml penicillin,100 μg/ml streptomycin supplemented with 10% fetal calf serum (FCS) (Gibco Co.) in a 28 °C incubator for two weeks. During several passages the parasite suspension was precipitated in a refrigerated centrifuge for 10 minutes at 4500 rpm, and the supernatant was removed. Thus, the parasites achieved a fixed growth phase (stationary‐phase) after 12 days, and were ready for CL injection and induction (18, 19).

Experimental groups The mice infected with L. major were randomly divided into three groups, each consisting of 10 mice as follows:

Group 1 with no treatment, was the control group. Group 2, received AgNPs at a dose of 2 mg/kg under anesthesia (20). Group 3, received AgNPs-TA at a dose of 2 mg/kg under anesthesia.

All above treatment were given to the lesions on days zero, 4, 8, and 12.

The parameters for evaluating treatment efficacy The treatments efficacy were recorded according to the changes in the lesion and spleen parasite burden. The sizes of lesions were measured twice a week. The mice were sacrificed after the first treatment and taken their spleen to determine the number of viable parasites (21). The spleens of the animals were placed into 6‐well plates containing complete RPMI‐1640 medium. After homogenization of the spleen tissue, the content of all wells was completed to 200 microliters with complete RPMI‐1640 medium. All samples were divided in three replicates. The prepared plates were put in incubator at 28 °C for 10 days. At last, the positive and negative results were reported in serial dilutions on the basis of the microscopic observations.

Results: silver nanoparticles against Leishmania major

Results and Discussion: The development of reliable and eco-friendly process for the synthesis of metallic nanoparticles is an important step in the field of application of nanotechnology. Some toxic chemical materials absorbed on the surface that may have side effect in the medical applications when used the chemical synthesis methods. Green synthesis prefer more than the chemical and the physical method as it is cost effective, environment friendly, easily scaled up for large scale synthesis and in this method there is no need to use high pressure, energy, temperature and toxic chemicals. Therefore, the production silver nanoparticles by biological methods of plant extract instead of other toxic methods was the best and most effective (22,23). The absorption bands of some metallic nanoparticles in the visible region were shown due to collective oscillation of electrons in resonance with incident electromagnetic radiation, termed surface plasmon resonance (SPR) band (24). Sixty skin samples of suspected cutaneous leishmaniasis patients were detected for Leishmania amastigotes by microscopic observation out of which, 45 (75%) were positive; however, the NNN culture led to the growth of promastigotes in 42 samples (70%). Also, the results showed that 54 (90%) of samples were positive by PCR method; 60% L.major and 30% L.tropica.

Leishmania major cells when exposed to silver ions showed a distinct and fairly broad UV–Vis absorption band centered at 425 nm because this nanoparticle was well dispersed without aggregation. The appearance of this band, which was assigned to a surface plasmon, is well documented for various metal nanoparticles with sizes ranging from 2 to 100 nm (25). SEM showed the formation of silver nanoparticles with an average size of 35 to 40 nm with inter-particle distance. UV-Vis absorption spectra have been proved to be quite sensitive to the formation of silver colloids because silver nanoparticles exhibit an intense absorption peak due to the surface plasmon excitation (26,27).

The appearance of parasites in the presence of AgNPs-TA suggests some possible mechanisms of action. Previously literature, report that silver attacks bacteria’s cell membrane surface, altering permeability, osmotic equilibrium and thus, metabolic pathways of the cell. Silver nanoparticles may also bind to the DNA, preventing replication (28-32). TA-coated gold and silver nanoparticles SPR bands were red-shifted compared to values reported in literature, 520 and 380-450 nm, respectively (33,34). This bathochromic effect is likely caused by the supramolecular interaction between the nanoparticles and the surface-protecting TA polyphenol, altering the electron transfer energy. Besides shape, size and type of the material, the position of the SPR band is highly dependent on the dielectric constant of the surrounding medium, once it changes the resonance frequency of the electrons onto NMNPs’ surface (35-37).

Conclusion: tannic acid nanoparticles as treatment

Conclusions: Silver nanoparticles synthesized using tannic acid have potential application as an antileishmanial treatment. Furthermore, the use of natural compounds involves to the development of clean, nontoxic, biocompatible and environmentally benign methods to synthesize noble metal nanoparticles.

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Abdulsada A. Rahi 1*, Magda A. Ali 2 and Zaid A. Abdulabbas ( Department of Biology, College of Science, Wasit University, Kut, Iraq.)  (College of Medicine, Wasit University, Kut, Iraq)