Nano-Coupling of Cephalosporin Antibiotics with Fe 3 O 4 Nanoparticles : Trojan Horse Approach in Antimicrobial Chemotherapy of Infections Caused by Klebsiella spp .

In the present study we had an aim to develop the methods of functionalizing the surface of magnetite nanoparticles with cefotaxime and ceftriaxone antibiotics. The quantitative analysis of the nanostructured cephalosporins was determined by Atom Absorbance Spectroscopy (AAS) and based on the Lambert-Beer law. The engineered nanostructures were tested on gram-negative microorganisms Klebsiella spp., of Enterobacteriaceae, and gram-positive bacteria Staphylococcus aureus, each having multi-drug resistance properties.


Introduction
In past years, the number of nosocomial infections, caused by gram-negative microorganisms, has increased.The microorganisms belonging to the family of Enterobacteriaceae and Pseudomonas have the greatest clinical significance.The bacteria of genera Escherichia, Klebsiella, Proteus, Citrobacter, Enterobacter, and Serratia from the Enterobacteriaceae family are often referred to as causes of postoperative complications, sepsis, and meningitis.Most of Enterobacteriaceae are opportunistic pathogens, since these bacteria are normally (except genus Serratia) obligate or transient representatives of the intestinal microflora and cause infections in immunecompromised patients only under certain conditions [1] [2].
The fundamental requirements for an effective antimicrobial therapy are: high sensitivity of the pathogens towards the antibacterial medicine and the maximum concentration of the drug in the infected zone with minimal side effects.Today the traditional therapy is ineffective due to the great resistance of many bacterial pathogens towards antimicrobial medicines and it makes the treatment of infections intricate.From day to day the number of reports about microorganisms' cultures, which are resistant to antibiotics, is increasing.The fastness to antibiotics is connected with their ability to produce extended spectrum beta-lactamases [3] [4].
Life threatening infections, such as Klebsiella, Escherichia coli or Enterobacter, which belong to the Enterobacteriaceae family and also Staphylococcus aureus, all have a common mechanism of resistance-they are extended spectrum beta-lactamases producers and have developed resistance to the commonly prescribed antibiotics.Intestinal gram-negative bacilli with resistance to cephalosporins were first identified in the mid-80s in Western Europe.Most of these strains (Klebsiella pneumoniae, Escherichia coli) were resistant to all beta-lactam antibiotics, except for carbapenem and cephamycin [5].Genes, in which information about beta-lactamase spread spectrum synthesis is encoded, localize in plasmids that facilitate the dissemination of beta-lactamases spread spectrum among Gram-negative bacteria [6].The study of the nosocomial epidemic of infections, caused by Enterobacteriaceae that produce beta-lactamase spread spectrum, indicates that these strains emerged in response to the intensive use of cephalosporins [7] [8].Therefore, solution of the problem with antibiotic resistance is an urgent global healthcare priority and can potentially prevent thousands of needless deaths around the world.
In this regard, the nanotechnology-based approach will be able to overcome the complications associated with the traditional antimicrobial therapy.The application of magnetite nanoparticles in medicine has attracted a great deal of attention not only for their extraordinary properties, owing to their ultra small size, such as high surface area, enhanced reactivity, biocompatibility, but also because their surface can be functionalized by drugs and these nanostructures can be delivered to targeted zones by the application of an external magnetic field [9]- [11].There are facts that such kind of nanostructure is capable of transporting drugs across cell membranes before release, ensuring that the treatment will only damage bacterial cells.
In this study, we report a simple, one-pot preparation of dispersions of Fe 3 O 4 nanoparticles that bind with cephalosporin antibiotics cefotaxime and ceftriaxone (Figure 1) through a self-assembly process using the method of wet chemical co-precipitation.Carboxylate functional groups of cefotaxime and ceftriaxone as well as amine functionalities are able to bind to the surface of the magnetite nanoparticles and thus stabilize their surface, preventing from further agglomeration.
Materials and methods.All chemicals were used as received.FeCl 3 6H 2 O, FeSO 4 7H 2 O, NH 4 OH (25%), were purchased from Sigma-Aldrich (Taufkirchen, Germany); Acetic Acid solution (33%), phosphate buffer PREP I and II, Crystal Violet Solution were purchased from Merck (Germany) and Nutrient Broth from Biolife (Milano, Italia).Synthesis of Fe 3 O 4 coated by cephalosporin (cefotaxime and ceftriaxone) Fe 3 O 4 @cefotaxime (Fe 3 O 4 @ CTAX) and Fe 3 O 4 @ceftriaxone (Fe 3 O 4 @CTRIX) NPs.Magnetic iron oxide nanoparticles are usually prepared by wet chemical precipitation from aqueous iron salt solutions in alkaline milieu created by using NH 4 OH [12].The formed Fe 3 O 4 nanoparticles (NPs) were stabilized by cefotaxime and ceftriaxone molecules.Originally, FeCl 3 •6H 2 O (0.01 M) and FeSO 4 •7H 2 O (0.006 M) were dissolved in 200 mL of deionized water under the bubbling of gaseous N 2 .Then, 25% aqueous ammonia solution (1.5 M) was dropped into solution with vigorous stirring until the pH of the solution raised to 9.5.When the color of reaction mixture turned black, the aqueous solutions of cephalosporines, taken in excess, were added to reaction mixture.The magnetite NPs coated by corresponding cephalosporines during reaction was separated by strong NdFeB permanent magnet and repeatedly was washed with distilled water.The obtained NPs were dried at ambient conditions and the iron content in the sample was analyzed by atom absorption spectroscopy and performed on Varian SpectrAA 220FS Atomic absorption spectrometer.Samples were prepared by Milestone ETHOS 1 Microwave extraction unit.The UV spectra have been recorded on Spectrophotometer Specord 250 Plus.UV spectra were recorded at 238 nm for standard solutions of cefotaxime with different concentrations in the range of 0.01 -0.1 μg•mL −1  Characterization of structure.X-ray diffraction analysis was performed on Rigaku Mini Flex 600 XRD diffractometer, equipped with CuKα radiation, at ambient.The samples were scanned in the Bragg angle range of 20˚ -70˚ for 2 hours.
The functional groups, present in the powder samples of Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX NPs, were identified by Fourier Transform Infrared (FTIR) spectroscopy.FTIR spectra were recorded on a Varian 3600 FTIR spectrophotometer in KBr tablets.The spectrum was taken in the range of 4000 -400 cm −1 at room temperature.
Scanning Electron Microscope (SEM) and Energy-Dispersive Spectrum (EDS) analysis.SEM and EDS analysis of Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX nanoparticles were taken on Field Emission Scanning Electron Microscope JEOL JSM-7600F at an accelerating voltage of 15.0 kV, SEI regime.EDS of the same samples was performed with an EDAX X-ray energy-dispersive analysis system, attached to the JEOL JSM-7600F transmission electron microscope Determination of antibacterial activity.Antibacterial activity of cephalosporins and Fe 3 O 4 @CTAX, Fe 3 O 4 @CTRIX was tested by diffusion methods on Staphylococcus aureus and Klebsiella spp.[13].For this purpose cephalosporins were taken in amount 30 μkg (indicator disks were purchased by Research-and-Development Center of Pharmacotherapy, 192236 St. Petersburg).Klebsiella spp. was cultivated on Sabouraud medium and Staphylococcus aureus on selective saline agar (cultures were kindly provided by one of the clinical laboratories of Baku).Microbiological tests were performed on Petri dishes.In order to identify the MIC of Fe 3 O 4 @CTAX and Fe 3 O 4 @CTRIX, microdilution method was used.The stock solutions of the substances prepared in distillated water were distributed in multi-well plates.Each well was inoculated with 0.1 mL of microbial suspensions of 0.5 Mc Farland turbidity, prepared from 24h fresh culture.Sterility control wells (nutrient broth) and microbial growth controls (inoculated nutrient broth) were used.The plates were incubated for 24 h at 37˚C [13] [14].In order to measure antibacterial effect on biofilm development, the nutrient broth was deleted; wells were washed with PBS three times and stained by crystal violet 1% for 15 minutes.The biofilm, formed onto the plastic wells, was re-suspended in 30% acetic acid and the intensity of the colored suspension was assayed by measuring the absorbance at 590 nm [15]- [17].All microbiological studies were performed 4 times in order to exclude mistakes and to prove reproducibility.

Results of Experiments
The functionalizing of magnetite nanoparticles surface with cefotaxime and ceftriaxone antibiotics was carried out by wet co-precipitation method.The purity and crystalline properties of the Fe 3 O 4 @cefotaxim (Fe 3 O 4 @CTAX) and Fe 3 O 4 @ceftriaxone (Fe 3 O 4 @CTRIX) were investigated by powder X-ray diffraction (XRD).The XRD patterns are shown in Figure 2. All the reflections can be indexed to Fe 3 O 4 nanoparticles with cubic structure.In XRD peak broadening testifies for the formation of nanocrystals.
The analysis of IR spectra of nanostructures revealed that these compounds bind on the surface of the magnetite nanoparticles via carboxylate, amine, CONH and hydroxyl groups, by non-covalent interactions, depending upon steric necessity and the curvature of the surface.monodisperse with almost uniform size.The average sizes of formed nanoparticles correlate well with data obtained from XRD analysis.The data, obtained from SEM analysis of prepared samples of Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX nanoparticles, are in good compliance with results of XRD analysis.These results revealed that the nanoparticles were homogeneously sized and uniformly shaped.The points, identified in the EDS spectrum Quantitative analysis of cephalosporins' molecules, loaded on magnetic NPs, was performed by UV spectroscopy methods on the basis of Lambert-Beer law and the iron content in NPs samples was determined by AAS method.The UV spectrum of cephalosporin, as it is shown in Figure 7, can be divided into 3 main areas of absorption: intense main maximum absorption region at low wavelengths, consisting of either one, or more maxima ("left shoulder"), and a similar area of absorption at higher wavelengths ("right shoulder"), which is especially characteristic for the UV spectrum of cefotaxime.
The results of measurements through different methods and following calculations are correlating with each other very well and reveal that the concentrations of loaded cephalosporins' molecules in NPs are very close and make 0.18 gr and 0.21 gr of ceftriaxone and cefotaxime in 1 gr of Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX NPs correspondingly.
By the diffusion method it reveals that Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX, unlike usual cephalosporins, have no effect on Staphylococcus aureus (the data not shown).However, Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX have significant antibacterial effect on Klebsiella, whereas pure cephalosporins have not (Figures 8-10).
So, we decided to identify the MIC of nanostructurized cephalosporins only on Klebsiella spp.The MIC of Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX on Klebsiella was 2 µg/mL.According to the spectra, received by spectrophotometry (performed on Spectrometer Specord 250 plus), in case of Klebsiella, Fe 3 O 4 @CTAX exhibited an   inhibitory effect on biofilm development from 0.5 μg/mL and Fe 3 O 4 @CTRIX from 0.25 μg/mL, as it is shown in Figure 11.However, the amount of nanostructurized cephalosporins should not be very large for two reasons.Firstly because cephalosporins are concentration independent antibiotics [20] and the second reason is that NPs in big amount can aggregate and become inactive.

Discussion
The ability of NPs to penetrate through cell membrane of pathogenic microorganism may be forth by various factors, among them the nanoparticles size and engineering the surface of nanoparticles with special signal molecules that make possible for cell to recognize the nanostructure.It is known, microbes are adjusting and developing very fast multi-resistance against to known form of antibiotics, and the speed of working out of new classes of antimicrobial therapeutics is lower than the ability of microbes to develop resistance properties.In the 80 years of last century the majority of hospital strains of microorganisms exhibited high sensitivity to cephalosporins, which determined their high clinical efficacy in infections of different localization.However, in the last decade antimicrobial chemotherapy caused a significant increase in resistance of Gram-negative organisms to cephalosporins, mainly due to the emergence of the ability of production of beta-lactamases of various types and classes by microorganisms [21] [22].Klebsiella genus is extended spectrum beta-lactamases producer, so it destroys the betalactam ring of cephalosporin, but at the same time it synthesizes and secrets siderophores-low molecular weight compounds, in order to improve the availability and absorption of iron [23].Siderophore molecules represent the specific transport system in Gram-negative bacteria.Laden with iron, siderophores' molecules bind to the bacterial wall to move iron into microbial cells.Most probably the prepared nanostructures of and Fe 3 O 4 @CTAX can avoid the destroying of beta-lactam ring of cephalosporins, due to siderophores which can bind the iron-containing nanoparticles, laden with antibiotics, and transport them through microbial cell.
Thus prepared nanostructures play the role of so-called "Trojan horse", who deceives the security system of microbial cells and contributes to dragging the nano-antibiotic into the microbial cell.The absence of antibacterial effect on Staphylococcus aureus of prepared nanostructures Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX is most likely explained by Fe 3 O 4 NPs chelation with CONH group of beta-lactam ring, which is essential for pharmacological activity [24].Staphylococcus aureus is referred to Gram-positive bacteria, which do not posses iron uptake system, based on syderophores.At the same time beta lactam ring of antibiotic is blocked by Fe 3 O 4 NPs and cannot act in its usual way.So, the expanding of antibacterial medicines' assortment by the modification of already existed antibiotics can help to fight against quick development of bacterial resistance.There is a clear advantage of nano-antibiotics that naturally occurring microbes have so far not developed resistance against them.

Conclusion
It can be assumed that the synthesized biocompatible Fe 3 O 4 -based nanostructures have practical importance, since they can be used for targeted drug delivery and controlled release of modified antibiotics.They may also be applied to decrease therapeutic doses of medicines, since the drugs with high dose have few serious side effects.In addition, Fe 3 O 4 @CTRIX and Fe 3 O 4 @CTAX nanostructures can serve as a model for the development of new alternative strategies by applying so called "Trojan horse" principle for improving the bactericidal effect of already used and new developed antibacterial medicines.

Figure 5 (
b) and Figure 6(b), demonstrate the presence of Fe and O as the main elements of the sample and support the data of magnetite nanoparticles formation (the other peaks are corresponding to Cu, C being charac-teristic of the carbon-coated grid).