A Physical-Chemical Study of the Interference of Ceftriaxone Antibiotic with Copper Chloride Salt

The nano-CuCl2.2H2O salt was prepared by the ball milling method. The association parameters for bulk and nano-CuCl2 salts in H2O are estimated at different temperatures using the conductivity method by applying the Fuoss–Shedlovsky equation and it was higher for nano-CuCl2 than bulk CuCl2 salt. The interaction between the cation (Cu2+) and ligand (ceftriaxone) in H2O was determined also by the conductometric method. Two stoichiometric complexes 1/2 and 1/1 (M/L) are estimated and follow the order Kf (1/1) > Kf (1 : 2) and ∆Gf (1/1) > ∆Gf (1/2) for (M : L) (in negative values) indicate the favorable of formation of (1/1) complex compared to the (1 : 2) complex. The Gibbs free energies change was increased in negative signs with increasing the temperature. The antimicrobial activities of CFT, bulk Cu-CFT complex, and nano-Cu-CFT complex were studied on LB agar by the disc diffusion technique against clinical isolates of gram-negative bacteria (Klebsiella pneumonia and Pseudomonas aeruginosa) and Fungi (Candida albicans). It was observed that (CFT) has a higher zone of inhibition and antibacterial activity than that of bulk and nano-Cu-CFT complexes in Klebsiella pneumonia and Pseudomonas aeruginosa (gram-negative bacteria). The nano-Cu-CFT complex showed a higher clear zone of inhibition and antifungal activity against candida than bulk Cu-CFT complex while the absence of the inhibition zone in CFT, so nano-Cu-CFT complex, can be used as an antifungal drug.


Introduction
Nanoparticles (NPs) are a wide class of materials that have a state between bulk and atomic or molecular structures in different shapes of 0D, 1D, 2D, or 3D and have a great scientific interest [1][2][3][4][5]. Bulk materials have constant physical properties, with size larger than one micrometer or micron. Nanoparticles can be used for various applications such as drug delivery purposes [6], diagnostics of cancer therapy, gene delivery purposes, chemical and biological sensing [7], gas sensing [8][9][10], CO 2 capturing [11,12], and other related applications [13][14][15][16][17]. Nanoparticles are often used to photocatalytically break down oil into biodegradable compounds, break down volatile organic pollutants in the air, and clean up carbon tetrachloride pollution in spring water [18,19]. Nanoparticles (NPs) such as gold, silver, platinum, and palladium showed colors with the variation of shape and size and characteristic properties that can be utilized in bioimaging applications [20]. Another application of nanoparticles is the synthesis of photocatalysis S-doped TiO 2 nanoparticles and the study of their photocatalytic, antimicrobial, and antioxidant activities under sunlight illumination [21].
Ceftriaxone [22][23][24][25] is an antibiotic that belongs to a category of medicine referred to as cephalosporin antibiotics, and it treats a variety of bacterial infections by stopping the growth of bacteria variety of bacterial infections (e.g., middle ear, lower tract, skin, and urinary tract), meningitis, gonorrhea, pelvic disease, and joint infections. ermodynamics parameters are an important tool for learning about the spontaneity of a given process at a particular temperature [26,27]. Determination of the formation constant is fundamental for understanding the behavior of the metal cations in the presence of some chelating agent in a solution and is best explained using thermodynamics. A conductivity technique can be used to estimate the interaction between the metal cations and the chelating agent by estimating the thermodynamics parameters of metal-ligand complex formation [28][29][30][31][32].
It is therefore necessary to study the effect of antibiotic ceftriaxone on the properties of copper chloride salt by determining thermodynamics parameters of interaction between them using conductometric techniques to find the extent of benefit of the antibiotic ceftriaxone.

Materials and Solutions.
e purities and sources of the materials used are presented in Table 1. e structure of ceftriaxone is shown in Figure 1.

Apparatus.
e conductance measurements are carried out with a conductometer A JENCO, Vision plus-EC3175 conductance instrument, and connecting with Kottermann ultra-thermostat-4130 (a deviation ±0.01 K) with a cell constant equal unity. e conductivity bridge was calibrated using standard potassium chloride solutions [33].
e Bruker D 8 Advance X-ray diffractometer is a powder XRD instrument used to record X-ray diffraction (XRD) patterns of bulk and nanosamples. e Bruker diffractometer with CuKα anode radiation (λ � 0.1542 nm) as a source is operating at 40 kV and 30 mA. e scanning range of over an angular range was between 4°and 80°A at a temperature of 25°C, and the scan mode was applied with a step width of 0.02°per step and step time of 0.4 s.
IR instrument of the type ermo Scientific Nicolet iS10 FTIR spectrometer is operating in the spectral range of 7800 to 400 cm −1 with a resolution of 4 cm −1 , midinfrared KBr beamsplitter 4000 to 400 cm −1 .
Solid samples can be prepared by grinding about 5 mg of sample mixed with 100 mg of spectroscopic grade KBr. is powder mixture is then compressed into a pellet using a mechanical press between 4 and 8 ton.cm −2 for 2 minutes in the form of 10 mm in diameter disks to form a translucent (http://en.wikipedia.org/wiki/Infrared_spectroscopy%20-% 20cite_note-Har-2). M, 20 mL) was placed in a double jacket glass conductance cell, and the conductance was measured after each addition of the solvent and stirring at a specific temperature.
To calculate the formation constants between CuCl 2 .2H 2 O and a ligand (ceftriaxone) in the solvents, a solution of CuCl 2 .2H 2 O (10 −3 M, 20 mL) was placed in a conductance cell, and the conductance was measured. e ligand (10 −3 M) (ceftriaxone) was added step by step to the conductance cell using a micropipette and the conductance was measured after each addition.

Biological Activity.
e antimicrobial activities of CFT, bulk Cu-CFT complex, and nano-Cu-CFT complex were studied on LB agar by the disc diffusion technique against clinical isolates of gram-negative bacteria (Klebsiella pneumonia and Pseudomonas aeruginosa) and fungi (Candida albicans).
Sterile filter paper discs (6 mm) were individually immersed in dimethyl sulfoxide (DMSO) extract of CFT, bulk Cu-CFT complex, and nano-Cu-CFT complex, and DMSO was used as control. All the discs were dried, placed on the surface of the test bacterial and fungal, and incubated for 18 to 24 h at 37°C. e standard antibiotic used is ceftazidime (30 mg) and finally, the zones of inhibition were examined.

X-Ray Diffraction (XRD).
e X-ray diffraction (XRD) pattern for the bulk and nano-CuCl 2 .2H 2 O salt is shown in Figure 2. e positions of the main peaks and their relative intensities as measured by powder diffraction are listed in Table 2. e mean size of nanocrystals was determined from the diffraction peaks corresponding to the most intensive reflections according to the Joint Committee on Powder Diffraction Standards database. Scherrer's equation was used to determine the average crystallite size for nanoparticles from the XRD diffraction pattern measured [34,35]: where K is Scherrer's constant (about 0.9), λ is the wavelength (λ � 0.154 nm), β is the line broadening at half the maximum intensity in radians, θ is the Bragg angle, and d is the averaged dimension of crystallites in nanometers. Groth assigned CuCl 2 ·2H 2 O to the bipyramidal class of the orthorhombic crystalline system with the axial ratios a : b : c � 0.9179 : 1: 0.4627. Layer line measurements give the identity distances a 0 � 7.38Å, b 0 � 8.04Å, c 0 � 3.72Å. ese lead to the ratios a : b : c � 0.918 : 1: 0.462, in good agreement with the crystallographic data [36]. e mean crystal size (nm) of bulk and nano-CuCl 2 ·2H 2 O salts obtained by XRD are mentioned in Table 2 [37]. A little difference was observed between bulk and nanosalts in peaks other than their intensities. Also, it was found that the salt remains in the crystalline form by converting it to nanoparticles. e main difference between the bulk and nanosalt was in the crystal size, as shown in Table 3.

Infrared Analysis (IR).
Infrared spectra (IR) were used to identify the structure of ceftriaxone (CFT), as shown in Figure 3, and its complexes with bulk and nano-CuCl 2 .2H 2 O salts as their functional groups give rise to characteristic bands in terms of both intensity and position (frequency), as shown in Figures 4 and 5.
It was observed from the IR spectra of ceftriaxone (CFT) ligand that amidic N-H stretching vibrations mean strong intensity bands occurring at 3440 and 3261 cm −1 are due to N-H asymmetric and symmetric stretching, respectively [38]. Amidic C�O stretching vibrations mean a strong intensity band identified at 1649 cm −1 is due to C�O stretching vibrations [38]. Amidic N-H deformation and C-N stretching mean the strong bands observed at 1608, 1537, and 1500 cm −1 are due to amide N-H deformation vibrations [38,39]. C-H stretching vibrations mean the weak bands occurring at 2891 cm −1 are assigned to CH 3 symmetric stretching.
e bands appearing at 2934 cm −1 in the IR spectra are due to CH 3 asymmetric stretching vibrations [40]. C-H deformation vibrations mean the weak bands observed at 822, 804, and 730 cm −1 are allotted as C-H outof-plane deformation vibrations and medium-to-weak intensity bands present at 1104 and 1033 cm −1 are allotted as C-H in plane deformation vibrations. A strong band occurring at 1399 cm −1 is due to the -CH 2 -deformation vibration. Weak bands present at 1243 and 760 cm −1 are due to CH 2 wagging and CH 2 rocking vibrations, respectively [40]. Lactam C�O stretching vibrations mean a strong band observed at 1741 cm −1 in the IR spectrum of ceftriaxone is allotted to be due to C�O stretching vibration [41]. C-O-C stretching vibrations mean strong bands present at 1033 and 1243 cm −1 are assigned as C-O-C symmetric and asymmetric stretching vibrations, respectively [41]. C-S stretching vibrations mean weak bands observed at 646 and 616 cm −1 are due to C-S stretching vibrations [42]. C-N stretching vibrations mean the medium band present at 1285 cm −1 , weak band at 1243 cm −1 , and medium band present at 1185 cm −1 are due to C-N stretching vibrations. C�C and C�N stretching vibrations mean very strong intensity bands present at 1608, 1537, and 1500 cm −1 are assigned to C�C and C�N stretching vibrations [43]. O-H stretching vibrations mean strong intensity bands identified at 3440 and 3261 cm −1 are allotted as O-H stretching vibrations [44]. C-C and C-C-C bending vibrations mean a very weak band occurring at 507 cm −1 in is due to C-C out-of-plane bending vibrations. e weak bands occurring at 646 and 606 cm −1 are due to C-C-C in plane and out of plane deformation vibrations, respectively [45].
In the IR spectra of bulk Cu-CFT complex, as shown in Figure 4, after ceftriaxone coordination to copper ion, the frequencies of the (C�O) lactam shifted from 1741 cm −1 to higher wavenumber 1775 cm −1 , one amidic (C�O) shifted from 1649 to lower wavenumber 1624 cm −1 , and also another amidic (C�O) triazine shifted from 1608 to lower wavenumber 1553 cm −1 . ere are three functional groups participating in the formation of a complex. e increase in the vibrational frequencies of carbonyl groups can be explained by oxygen of lactam and triazine coordinating to Cu(II) indicates the formation of a chelate complex [46,47]. ese intramolecular interactions between oxygen and copper ion result in a more rigid molecular structure around the oxygen and shift of carbonyl (C�O) vibrational frequencies to higher wavenumbers. e frequency of the symmetric stretching mode ] s (COO−) shifts from 1391 to 1398 cm −1 . ese shifts indicate that the carboxylate group (COO), the lactam carbonyl group (C�O), and the oxo group of the triazine ring are involved in the formation of [Cu(CFT)]·3H 2 O. is analysis is in agreement with previous studies where ceftriaxone is described as a polydentate chelating ligand [46,47]. e broadband in the [Cu(CFT)]·     , and ](C�N). A new band appearing at the frequency 468 cm −1 in the complex that is absent in the free ligand is due to ] (Cu-N) stretching vibration also giving strong evidence for the coordination of tertiary nitrogen atom with copper ion [47]. Also, the IR spectra of nano-Cu-CFT complex asym.(NH 2 ) shifted from 3440 to lower wavenumber 3427 cm −1 , carboxylic (-OH) group appeared at wavenumber 2924 cm −1 , carboxylic (C�O) appeared at wavenumber 1742 cm −1 , one amidic (C�O) shifted from 1649 to lower wavenumber 1638 cm −1 and also another amidic (C�O) shifted from 1608 to lower wavenumber 1553 cm −1 , as in Figure 5. is indicates the formation of a complex between CuCl 2 .2H 2 O salt and CFT antibiotic.

Calculation of Association Parameters for Bulk and
where K s and K solv are the specific conductance of the solution and the solvent (distillate H 2 O), respectively, and C is the concentration of the bulk and nano-CuCl 2 ·2H 2 O solutions.
e results obey the Fuoss-Shedlovsky equation and can be applied to obtain the value of limiting molar conductivity (Λ o ) and association constant (K A ) by plotting a graph between 1/ΛS(z) and (CΛS(z)c 2 ± ), which is presented in Figure 6, giving straight line with intercept (1/Λ o ) and slope (K A /Λ 2 o ): A � 1.824 × 10 6 (εT) − 3/2 , where ( e Walden product (Λ o η o ) values were calculated from the values of limiting molar conductance (Λ o ) [54]: e triple ion association constant K 3 can be calculated from e activation energy of the transfer process can be estimated depending on the relation between conductance of ion, ion mobility, and temperature degree, as in the Arrhenius equation: where A is the frequency factor and E a Arrhenius activation energy of the transport process.
By plotting log Λ o versus l/T, as shown in Figure 7, the activation energy of transfer processes values can be calculated from the slope [56].  Tables 4  and 5. Table 4 shows that the limiting molar conductivity (Λ o ) increases with increasing temperature due to the increase in mobility of ions and increasing kinetic energy which increases the separation among the oppositely charged ions while the Walden product (Λ o η) decreases with increasing the temperature due to the decrease in viscosity; also, the association constant (K A ) decreases with increasing the temperature due to the decrease in the association of ions and the increase in mobility of ions; and similarly, the triple ion association constant (K 3 ) decreases with increasing the temperature due to the decrease for the same reason. Table 5 shows the same trend, that is, the limiting molar conductivity (Λ o ) increases with increasing the temperature while the Walden product (Λ o η) decreases with increasing the temperature; also association constant (K A ) decreases with increasing the temperature; and similarly, the triple ion association constant (K 3 ) decreases with increasing the temperature.
By drawing the relation between log K A and 1/T, ΔH A can be calculated from the slope of each line which equals (-ΔH A /2.303 R), as shown in Figure 8. e entropy (ΔS A ) for bulk and nano-CuCl 2 ·2H 2 O salts were calculated by using where (S) is the entropy of the system. e calculated values of (ΔH A ) and (ΔS A ) for bulk and nano-CuCl 2 ·2H 2 O salts are presented in Table 6. It is obvious that the limiting molar conductance (Λ o ) increased as the temperature increased while the dissociation degree decreased as the temperature increased indicating a higher solvation process. e values of the association constant (K A ) and the triple ion association constant (K 3 ) were decreased by increasing the temperature. Gibbs free energies change of association (∆G A ) was decreased with negative signs by increasing the temperature indicating that association is favored with lowering of dielectric constant of solvent mixture. e decrease in the values of activity coefficient, limiting molar conductance, association constant, Gibbs free energy change of association, and Walden product for nano-CuCl 2 ·2H 2 O in comparison to bulk CuCl 2 ·2H 2 O indicates that the association of nano-CuCl 2 ·2H 2 O is greater than bulk CuCl 2 ·2H 2 O salt, due to the high surface to volume ratio of nanoparticles which leads to a greater ability for ion-pair formation.      Figure 9. e formation constants (K f ) for bulk and nano-Cu-CFT complexes were calculated for each type of complexes (1 : 2) and (1 : 1) (M : L) by using

Calculation of Formation Constants for Bulk and
where Λ m is the limiting molar conductance of the bulk and nano-CuCl 2 .2H 2 O alone, Λ obs is the molar conductance of solution during titration, Λ ML is the molar conductance of the complex, and [L] is the CFT concentration. e Gibbs free energies changes of formation (ΔG f ) for each stoichiometric complex were calculated [4] by using e obtained values (K f ) for bulk and nano-Cu-CFT complexes and their calculated ΔG f values are presented in Tables 7 and 8.
By drawing the relation between log K f and 1/T, different lines were obtained indicating the formation of (1 : 2) and (1 : 1) (M : L) stoichiometric complexes, as shown in Figure 10.
From the relation between log K f and 1/T, ΔH f can be calculated for each type of complexes, from the slope of each line which equals (−ΔH f /2.303 R). e entropy (ΔS f ) for bulk and nano-Cu-CFT complexes was calculated for each type of complexes (1 : 2) and (1 : 1) (M : L) by using e calculated values of (ΔH f ) and (ΔS f ) for bulk and nano-Cu-CFT complexes are presented in Tables 9 and 10.
It was observed that inflections at (1 : 2) M/L proportion and (1 : 1) M/L indicating the formation of both stoichiometric complexes in the solutions. ese types of stoichiometric complexes are formed as a result of the interaction of bulk or nano-CuCl 2 with CFT in distillate H 2 O at different temperatures. e complex formation parameters for (1 : 1) complexes are greater than those of (1 : 2) complexes indicating more favorable complexes. Also, the complex formation parameters (K f , ΔG f ) increased by increasing temperatures due to an increase in the kinetic energies. is trend was supported by entropies data which are greater for (1 : 1) M/L complexes than (1 : 2) M/L complexes.

Biological Activity
Many transition metals show arresting biological activity, working as active centers within important bioactive molecules in living systems. Copper (II) plays a significant function in cell metabolism and has proved beneficial in numerous diseases [58][59][60]. Ceftriaxone-metal complexes have both pharmacological and toxicological properties [61]. e interaction between metal ions and ceftriaxone can lead to precipitation resulting in serious adverse drug events [62]. Ceftriaxone complexes have antibacterial properties that can decrease or increase relative to pure ceftriaxone [61]. e antimicrobial activity was estimated based on the size of the inhibition zone formed around discs of bulk CuCl 2 ·2H 2 O, nano-CuCl 2 ·2H 2 O, CFT, and its bulk and nano-Cu complexes on a petri dish with Luria Bertani agar (LB-agar) plates as it measures the compound's efficacy.

Antibacterial Activity.
e antibacterial activities of bulk CuCl 2 .2H 2 O, nano-CuCl 2 ·2H 2 O, bulk and nano-Cu-CFT complexes were compared with the activity of CFT and as presented in Table 11. Bulk CuCl 2 ·2H 2 O provides an excellent antimicrobial activity, and such property is greatly improved when using nano-CuCl 2 .2H 2 O. It was observed also that CFT has a higher zone of inhibition than bulk and nano-Cu-CFT complexes in Klebsiella pneumonia and Pseudomonas aeruginosa (gram-negative bacteria), Figure 11. e antibacterial activity of Cu-CFTcomplexes depends on the bacterial species. e complexes and antibiotics presented inhibition zones of diameters larger than 20 mm showing that they have good activity as bactericides [63]. e antibacterial effect against Staphylococcus aureus is present at the bulk and nano-Cu-CFT complexes (the inhibition zones are 21 and 16 mm, respectively). ese values are lower than the corresponding values for bulk CuCl 2 ·2H 2 O and nano-CuCl 2 ·2H 2 O and ceftriaxone. e data indicate that ceftriaxone ligand is more active than their metal complexes; this may be because the chelating ligands containing N and O donor atoms show wide biological activity through bonding to metal ions [64,65]. However, the synergetic effects of ceftriaxone and Cu ion may play an important function in the inhibition of bacterial growth after the complex decomposition. ese effects are due to the different mechanisms of the action of antibiotics and heavy ions on the bacteria metabolism [66,67].

Antifungal Activity.
e experimental antifungal activity data are presented in Table 11 which indicates that the nano-Cu-CFT complex showed a higher clear zone of inhibition against Candida albicans compared to the bulk Cu-CFT complex while there was an absence of the inhibition zone in CFT. So, the nano-Cu-CFT complex can be used as an antifungal drug, as shown in Figure 12. e zone of inhibition after treatment with bulk and nano-Cu-CFT complex was 8 and 9 mm, respectively. According to the standard criteria for evaluation of the drugs, the antifungal action such as a small zone of inhibition indicates that Candida albicans is stable with respect to CFT. e inhibition zones were completely absent, indicating the resistance of these fungi to CFT as well.

Conclusion
e nano-CuCl 2 ·2H 2 O salt was prepared by ball milling method. e thermodynamic association parameters of both bulk and nano-CuCl 2 ·2H 2 O salts in H 2 O were calculated using the conductometric method by applying the Fuoss-Shedlovsky method at different temperatures. It shows that the association parameters of nano-CuCl 2 ·2H 2 O are greater than bulk CuCl 2 ·2H 2 O salt due to the high surface-to-volume ratio of the nanoparticles which leads to a greater ability for ion-pair formation.
e thermodynamic parameters of complexation between bulk and nano-CuCl 2 ·2H 2 O salts and ceftriaxone antibiotic in H 2 O were calculated from conductance measurement. It was found that the formation of Gibbs free energies change (∆G f ) was decreased in negative signs with increasing temperatures. Two stoichiometric complexes, 1/2 and 1/1 (M/L), are formed with the formation constant and Gibbs free energy of the formed complexes following the order K f (1 : 1) > K f (1 : 2) for (M : L) and ∆G f (1 : 1) > ∆G f (1 : 2) for (M : L) (in negative values) indicates the favorable of formation of (1 : 1) complex compared to (1 : 2) complex. Also, there is a decrease in values of K f and ∆G f in case of using nano-CuCl 2 ·2H 2 O compared to using bulk CuCl 2 ·2H 2 O but in small difference due to the more solvation effect in case of using nanoparticles. It was observed that CFT has a higher zone of inhibition and antibacterial activity than that of bulk and nano-Cu-CFT complexes in Klebsiella pneumonia and Pseudomonas aeruginosa (gram-negative bacteria). e nano-Cu-CFT complex showed a higher clear zone of inhibition and antifungal activity against candida compared to the bulk Cu-CFT complex while there was an absence of the inhibition zone in CFT, so the nano-Cu-CFTcomplex can be used as an antifungal drug.

Data Availability
e data are available on request from the corresponding author.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.

Authors' Contributions
Elsayed M. AbouElleef conceptualized the study, supervised the study, wrote the original draft, reviewed and edited the article, developed the methodology, performed formal Analysis, performed data collection and analysis, performed investigation, performed visualization, and performed project administration. Mowafak M. Mahrouka performed investigation, developed the methodology, and wrote the manuscript. Sherine E. Salem performed data collection and analysis, provided the resources, performed investigation, and developed the methodology.