Synthesis and characterization of Au nanoparticles for nanomedicine application

Gold nanoparticles AuNPs have proven to be powerful tools in various nanomedicine applications, because of their photo-optical distinctiveness and biocompatibility. Noble metal gold nanoparticles was prepared by pulsed laser ablation method (1064-Nd: YAG with various Laser power from 200 to 800 mJ and 1 Hz frequency) in distil water. The process was characterized using UV-VIS absorption spectroscopy. Morphology and average size of nanoparticles were estimated using AFM and X-ray diffraction (XRD) analysis which show the nature of gold nanoparticles (AuNPs). Antibacterial activity of gold nanoparticles as a function of particles concentration against gram negative bacterium Escherichia coli and gram positive bacterial Staphylococcus aureus was carried out in solid growth media. Gold nanoparticles show high antibacterial activity with zone of inhibition. Antibacterial activities of the synthesized Au nanoparticles were assessed by agar well diffusion method. The stabilized AuNPs exhibited excellent antibacterial sensitivity (12-27 mm) to E. coli and (26-38mm) to Staph aureus.


Introduction
In recent years, nanoparticles have become a promising applicant in place of conventional materials with huge applications in the fields of science and engineering. The unique properties of Au nanoparticles (AuNPs) is due to the higher surface-to-volume ratio and the increased number of atoms at their grain boundaries [1]. AuNPs have unique optical properties in the visible region, because of surface plasmon oscillation of free electrons [2]. Surfase Plasmon Resonance (SPR), the resonance that lay at visible frequency for noble metals as Au and Ag, given those intense colors and interesting optical properties, as reflected are due to their unique interaction with light [3]. Although Au nanoparticles are colloids, the properties at the nano scale differ greatly from their larger bulk Au counterparts [4]. Gold nanoparticles Au NPs are increasingly exploited for the design and development of novel nanomedicines, due to their easiness of synthesis, characterization, and surface functionalization [5]. Synthesize Nanoparticles are generally characterized by their size, shape and disparity. Preliminary characterization of the synthesized NPs is carried out using UV-visible spectroscopy [6]. Two main approaches are used in nanotechnology. In the "top-down" approach, nano-objects are constructed from larger entities without atomiclevel control. In the "bottom-up" approach, materials and devices are built from molecular components which assemble themselves chemically by principles of molecular recognition. ] 7,8]. Nanoparticles can be produced by short pulse laser ablation of a solid target in liquid media. This method gives a unique opportunity to solve the cytotoxic effects reported for the Nanoparticles produced by chemical synthesis [9].
The ability to integrate metal nanoparticles into biological systems with general lack of toxicity has had greatest impact in medicine and biology. Among noble metal particles, have low toxicity and can be readily attached to molecules of biological interest [10]. Different studies demonstrate their excellent antimicrobial properties, with a large spectrumincluding Grampositive and Gram-negative bacteria, including multidrug resistant strains [11,12]. Due to their large surface area to volume ratio and biocompatibility, inorganic nanoparticles are consider as ideal candidates for carrying large amounts of antibiotics with no compromising their activity [13]. The emergence of bacterial resistance to antibiotics and its dissemination, however, are main health problems, leading to treatment drawbacks for a large number of drugs [14]. *Conventional strategies for cancer intervention include chemotherapy, surgery, and radiation therapy. Taking advantage of their unique properties, most studies of gold nanoparticle Au NPs -based cancer therapy have used photo thermal therapy for the destruction of cancer cells or tumor tissue [15]. *The main aim of this research is to prepare gold nanoparticle by pulse laser ablation method for therapy against malignant and benignant diseases. *Different doses of these NPs were utilized to check antibacterial mechanism against tow type bacteria gram positive and gram negative (E.coli and staph aureus ), Anti Cancer therapy.

Experimental work 1. Materials
Circle Bulk of Gold, distal water, Nd-YAG Laser of wavelength (1064 nm). The experiment for antibacterial activity using different bacterial strains.
Escherichia coli, and Staphylococcus aureus, Nutrient media was used for evaluating bacterial growth.

Laser ablation Synthesis of Gold Nanoparticles
Gold Nanoparticles colloid was produced by irradiating a metallic target plate immersed in distilled water at room temp. and place on a rotating sprayer (2 rotation per minute) with pulsed laser beam (department of physics-collage of science -University of Baghdad). The ablation was performed with the (1064 nm) of a Nd-YAG laser (1000 pulses) operating at 1 Hz repetition rate, with a power (200, 360, 500, 660 and 800 mJ). The beam was focused, through a liquid layer, on the surface of the target.

Characteristics Measurements
Structural features of samples (XRD( and the optical features were investigated by UV-Vis spectroscopy. the surface roughness and topography of deposited thin films were studied by (AFM), additionally SEM was employed to confirm the NPs shape, size and particle size distribution. and The nanoparticles were centrifuged and re-dispersed in double distilled water thrice to purify them for FTIR studies.

Antibacterial activity
Antibacterial activities of gold nanoparticles were studied against Gram-negative bacteria (E.Coli) and Gram-positive bacteria (Staph aureus), was used for evaluating antibacterial property of Gold nanoparticles. Antibacterial activity of nanoparticles was evaluated using, standard agar well diffusion method in which gold nanoparticles solution were used. Wells (diameter = 6 mm) were punched in the agar plates and filled with different nanoparticles solutions, samples treated with nanoparticles were spread on nutrient agar plates and after incubation at 37 °C for 24 hrs and measured the diameter of inhibitory zones in mm.

Results and discussion 1. UV-Visible spectroscopy of gold nanoparticles
Optical property of nanoparticles is an important property which gives specific information of shape, size, concentration, agglomeration state, etc., near the surface of the particles. These particles interact with specific wavelength of light and show maximum absorption at a seartin wavelength. The UV-Vis spectra of the synthesized Au nanoparticle are presented in Figs. 1.

AFM analysis of gold NPs
The AFM is a scanning probe microscope describe a profile while maintaining the force or electric current between the tip and sample constant. By scanning the sample line by line and using a calibration file for each scanner a topography image of the surface is reconstructed and can be used in particle imaging and sizing. The size of the gold NPs of samples have range between 40-89 nm Fig. 2.

XRD analysis of gold NPs
The crystal structure of gold NPs is demonstrated by XRD as shown in

FTIR analysis of gold NPs
Inspection other analytical techniques such as FTIR spectroscopy type (The PerkinElmer Spectrum 100 Series FT-IR spectrometer) is used to study the adsorption of organic species on the metal oxides nanoparticles. FTIR spectra were measured at room temperature with the spectrometer using the KBr Pellet technique.   The FTIR spectrum of synthesized gold nanoparticles is shown in Fig.4. peak at 3325.03 cm -1 assigned as N-H stretching of amide (II) band. The peak at 1634.49 cm-1 was O-H stretching vibration of alcohols and phenols. The band located at 1337.42 and 1093.98 cm-1 was because of C-N stretching vibration of aromatic and aliphatic amines, respectively. C-Br stretching vibration was shown in 584.96 cm -1 .

Antibacterial mechanism of gold nanopartecles
Antibacterial activity test of gold nanoparticles were studied against Gram-negative bacteria. Standard strain of Escherichia coli, and Grampositive (Staph aureus) was used for evaluating antibacterial property of Gold nanoparticles. Antibacterial activity of nanoparticles was evaluated using, standard agar well diffusion method in which gold nanoparticles solution were used. The bacterial strain used in test was grown on Broth at 37 ºC. Wells (diameter = 6 mm) were punched in the agar plates and filled with different nanoparticles solutions. Samples treated with nanoparticles were spread on nutrient agar plates and after incubation at 37 °C for 24 hrs the number of CFU were counted.

Conclusion
Gold nanoparticles were successively prepared by laser ablation in water. The energy of laser beam affected on the prepared nanoparticles as the energy increases the size of nanoparticles decreases until they reached their critical size below which the particle not sensitive to the laser energy. As the energy increases above this value the nanoparticles begin to agglomerate again and the size increases. Gold nanoparticles have revolutionized the field of medicine because of its wide spread applications in targeted drug delivery, imaging, diagnosis and therapeutics due to their extremely small size, high surface area, stability, non-cytotoxicity and tunable optical, physical and chemical properties.
It was more influential against Gramnegative bacteria. The antibacterial mechanism of gold NPs against four pathogenic bacteria demonstrated that gold NPs can be the next therapy against this enteric bacterium, the maximum zone of inhibition for Au NPs against E. coli and S. aureus is 27 mm and 38 mm respectively.