THE UTILITY OF GOLD NANOPARTICLES AS AN EFFECTIVE DRUG CARRIER AND ITS ANTIBACTERIAL EFFICACY WITH GEMIFLOXACIN HCL.

A simple and extractive spectrophotometric method was developed for the determination of Gemifloxacin HCl (GEM) in bulk drug and pharmaceutical formulation. The method depends on the reaction between Gemifloxacin and gold nanoparticles (AuNps). The apparent molar absorptivity and Sandell’s sensitivity were found to be 0.1 x 10 4 L mol −1 cm −1 and 0.0038 μg cm -2 respectively. Beer’s law was conformed in the concentration range of 1.5 –38.8 μg mL -1 . The method is very simple and not time consuming, therefore it can be properly applied for the evaluation of Gemifloxacin in dosage forms and also in vitro antibacterial activities of GEM, gold nanoparticles and drug coated gold nanoparticles against several strains of Gram positive and Gram negative organisms. The antibacterial properties of GEM-AuNps complex were higher than that of the pure Gemifloxacin while gold nanoparticles did not produce any antibacterial activity against the bacterial strains. These results recommend that gold nanoparticles could act as an effective drug carrier in drug delivery system. significant type of colorimetric reporter (Brust et al 1995; Mirkin et al 1996; Demers et al 2000; Kim, Johnson and Hupp 2001) formation with AuNps lead to a distinct change in color. In certain new reports; AuNps are used as a detection probe for the anticancer drugs (Selvaraj, Alagar and Hamerton, 2006), however no quantification has been carried out up to now. We report here a plan for drug detection and quantification by gold nanoparticles. The chromatographic methods are time consuming, expensive and require proficiency while, for the routine analysis, a simple, fast and cost effective analytical method is required and favored. UV-spectrophotometric method can be used for routine investigation of raw materials and pharmaceutical formulations (Sharada, Channabasavaraj and Mani , 2014; Menon et al 2012). The impartial of this study is to progress a simple analytical method with a wide detection range for the determination of GEM in bulk and pharmaceutical formulations.

A simple and extractive spectrophotometric method was developed for the determination of Gemifloxacin HCl (GEM) in bulk drug and pharmaceutical formulation. The method depends on the reaction between Gemifloxacin and gold nanoparticles (AuNps). The apparent molar absorptivity and Sandell's sensitivity were found to be 0.1 x 10 4 L mol −1 cm −1 and 0.0038 μg cm -2 respectively. Beer's law was conformed in the concentration range of 1.5 -38.8 μg mL -1 . The method is very simple and not time consuming, therefore it can be properly applied for the evaluation of Gemifloxacin in dosage forms and also in vitro antibacterial activities of GEM, gold nanoparticles and drug coated gold nanoparticles against several strains of Gram positive and Gram negative organisms. The antibacterial properties of GEM-AuNps complex were higher than that of the pure Gemifloxacin while gold nanoparticles did not produce any antibacterial activity against the bacterial strains. These results recommend that gold nanoparticles could act as an effective drug carrier in drug delivery system. ion-pair complex  for tablet formulation. UV spectrophotometric methods, alternatively, provides economic processes, which are less time consuming and more sensitive compared to the RP-HPLC method.
Gold nanoparticles (AuNps) have bio-applications in different areas like drug delivery and sensing (Chen et al 2007;Jalil and Nixon, 1990). Optical properties of AuNps change when they interact with certain molecules, allowing the detection of analytes. Absorption spectra of AuNps change significantly when several particles combine with them (Arulkumar and Sabesan , 2010). AuNps exhibit excellent biocompatibility and unique structural and catalytic attributes which have made them a very attractive material for sensors and electrocatalyst (

Synthesis of gold nanoparticle:-
AuNps were prepared by adding trisodium citrate to a boiling 1 mM HAuCl 4 solution, turning the yellow gold chloride into red wine color (Atta, Galal and Azab, 2012). The solution was stored at 4 °C and a characteristic peak in the UV-vis spectrum at 522 nm for AuNps was obtained.

Preparation of GEM tablets:-
Accurately weighed amount of the finely powdered tablets equivalent to 19.5 mg of the drug salt was dissolved in distilled water, the mixture was agitated by an electrical shaker for about 10 min. This solution was filtered and completed to 50-mL with distilled water. More dilute solutions were prepared whenever required and analyzed by the subsequent procedures.

Details of microbial assay:-
The antibacterial activities study was performed by the standard disc diffusion method against the different standard bacterial strains according to Clinical and Laboratory Standards Institute (CLSI 2013) guidelines. Three to five wellisolated bacterial colonies were touched with a sterile loop from an overnight agar plate culture and moved into 5 mL of brain heart infusion broth. The broth culture was incubated at 37°C with shaking for 4 to 6 hours until it reaches or exceed the turbidity of a 0.5 McFarland standard. This results in a suspension containing approximately 1 × 10 8 CFU/mL. A sterile cotton swab was immersed into the inoculum. The swab was streaked over the whole surface of the Mueller Hinton agar plate and around the rim of the agar. The plates were left for 3-5 minutes. 8μL of AuNps, GEM and the complex were applied respectively to each sterile disk and dried in sterile laminar flow cabinet before used. The disks were placed on the inoculated Mueller Hinton agar plates with sterile forceps. The discs were arranged at 15 mm from edge of the Petri dish and 30 mm from each other to decrease overlapping zones. Inoculated plates were incubated inverted within 15 minutes of disk application at 37 0 C for 24 hours.

Characterization of GEM-AuNps:-
The morphology of AuNps was determined using TEM ( fig 1A). Particles are aggregate in irregular shape with average diameter of ≈ 43 nm. The UV-vis spectrum of GEM, AuNps and GEM-AuNps complex in aqueous solution show absorption maximum at 211, 522 and 650 nm respectively ( fig. 1B). The color change of AuNps from wine red to dark blue on the addition of GEM confirms the interaction between the drug and nanoparticles. Pure drug displays a maxima at 211 nm due to n-π* and π -π * transition of GEM molecule. After the addition of AuNps , the intensity of both bands of GEM and AuNps due to the electrostatic interaction between them and a new peak was observed at 650 nm. The appearance of a new peak is due to the aggregation (Selvaraj and Alagar, 2007) of GEM on AuNps, which resulted in the additional band at longer wavelength. The mechanism of GEM aggregation on AuNps is represented in Schematic 1. The colloidal solution containing citrate-capped AuNps has very intense and characteristic wine red color arising from the ordered electronic motion, which gives the absorption value at 522 nm in the UV-vis. spectrum. After the addition of GEM; a quenching of the characteristic absorption band which follows a shift to the longer wavelength was observed.
1652 Scheme 1:-Schematic diagram of elucidating the aggregation of GEM on gold nanoparticles.

Optimum conditions for drug determination:-Effect of time and temperature:-
The formation of the complex is rapid and the absorbance readings were constant after 10 min and were stable for at least 1hr. Shaking time ranging from 0.5 to 5.0 min did not produce any change in color intensity, and so 1 min shaking time was selected. The influence of temperature on the complex was examined by measuring the absorbance values at different temperatures. It was found that the absorbance was stable up to 35 0 C, therefore all subsequent studies were carried out at room temperature (25± 1 0 C).

Effect of solvent:-
The type of solvent employed affects both wavelength and intensity of the maximum absorption. The effect of distilled water, methanol, dichloromethane, 1,2 dichloroethane , acetone and dioxane at a fixed wavelength was studied. As shown in figure 2, distilled water gives the highest absorbance peak of the complex. 1653

Effect of reagent concentration:-
The effect of reagent was studied by measuring the absorbance of solutions containing a fixed concentration of (GEM) and varied amounts of the reagent. Maximum color intensity of the complex was achieved with 2.0 ml of 1x10 -3 M of reagent solution ( fig 3A).

Stoichiometry of the complex:a) Continuous variation method:-
The composition of complex was established by Job's method of continuous variation (Job, 1928). Continuous variation method of 1.0 x 10 -3 mol L -1 GEM and 1.0 x10 -3 mol L -1 AuNps was performed. A series of solutions were organized in which the total volume of drug and reagent was kept constant at 1 mL, diluted to the volume in 5mL measuring flask with distilled water and the total molar concentration was 2x10 -4 M. The relation between absorbance and mole fraction of reagent is represented in figure 3B, indicating that the composition of the complex of GEM with AuNps was found to be 4:1 complex.

Conditional stability constant of GEM -AuNps complex:-
The conditional stability constant (K f ) of the complex of GEM with AuNps under the experimental conditions described above was calculated from the continuous variation data using the following equation (Sommer, Havel and Kubáň, 1970).
Where A is the maximum absorbance of the complex and A m is the absorbance value when all the drug present is completely associated which corresponding to intersection of the two tangents of the curve. C M is the mole concentration of drug at the maximum absorbance and n is the stoichiometry with which drug is associated with AuNps. The formation constant value (K f ) of complex was calculated to be 1.12x10 19 L/mole.
The standard free energy, ΔG 0 of complexation is related to the formation constant (K f ) by: ΔG 0 = -2.303 R T log K f Where ΔG o is the free energy change of the complex (Kcal.mol -1 ), R is the gas constant (1.987 cal.mol -1 .deg -1 ), T the temperature in Kelvin (273+ 25 0 C) and was found to be 26.08 Kcal mol -1 .  Precision and accuracy:-Three different concentrations of GEM were organized and five replicate determinations were performed using standard addition method. In this method, standard drug was added with three different amounts i.e. 50%, 100% and 150% to known pre-analyzed sample solution. By using the proposed methods the total concentrations were determined. The % recovery of added drug was calculated as: % Recovery = Amount of drug found /Amount of drug added ×100 The results obtained in table 2 prove that the method is precise and accurate. Also the mean calculated relative standard deviation values (RSD) varied from 0.5-0.89, indicating good repeatability of the proposed method.

Robustness and ruggedness:-
The robustness of the method was confirmed by examining the same sample by varying parameters like reagent volume (AuNps) and reaction time. The ruggedness of the proposed method was evaluated by applying the developed procedure to assay of GEM using the same instrument by two different analysts under the same optimized conditions at different days. Since there was no significant difference between the results obtained for recovery ± SD% were 99.92±0.63% and 99.89±0.76% by the two analysts, respectively, the proposed method may be considered rugged.

Application to Pharmaceutical Formulations:-
The proposed method was applied for the analysis of pharmaceutical formulations of GEM as: factive tablets with AuNps. The recovery of the proposed method was presented in table 3. The assay of the pharmaceutical formulations with gold Nps were compared with the reported method (Charan and Satyabrata, 2011). Comparison of the proposed and reported methods was performed concerning the accuracy and precision using the Student's t-and F-tests (Table 4). The results prove that there is no significant difference between the proposed methods and the reported method.  Theoretical values of t-and F-tests at P = 0.05 are 2.306 and 6.39, respectively Microbial activity:-Determination of the antibacterial activity of AuNps, GEM and GEM-AuNps complex was performed by the standard disc diffusion method against the different standard bacterial strains. After incubation for 24 hours, each plate was examined. The diameters of the zones of growth inhibition were measured. As shown in figure 5 and table 5, AuNps disks showed no inhibition zones against the tested bacterial strains. The complex produced higher antibacterial activity than GEM against Staphylococcus aureus, Micrococcus luteus, Escherichia coli, Salmonella typhimurium, Klebsiella pneumonia and Pseudomonas aeruginosa. Drugs coated with AuNps are effective against both Gram positive and Gram negative bacterial organisms, due to the small size of the nanoparticles, large surface area and high penetrating power, therefore more number of GEM molecules can be adsorbed on AuNps surfaces making them as one group improving the antimicrobial activity.