Silver Nanoparticle Conjugation-Enhanced Antibacterial Efficacy of Clinically Approved Drugs Cephradine and Vildagliptin

This paper sets out to determine whether silver nanoparticles conjugation enhance the antibacterial efficacy of clinically approved drugs. Silver conjugated Cephradine and Vildagliptin were synthesized and thoroughly characterized by ultraviolet visible spectrophotometry (UV-vis), Fourier transform infrared (FT-IR) spectroscopic methods, atomic force microscopy (AFM), and dynamic light scattering (DLS) analysis. Using antibacterial assays, the effects of drugs alone and drugs-conjugated with silver nanoparticles were tested against a variety of Gram-negative and Gram-positive bacteria including neuropathogenic Escherichia coli K1, Pseudomonas aeruginosa, Klebsiella pneumoniae, methicillin-resistant Staphylococcus aureus (MRSA), Bacillus cereus and Streptococcus pyogenes. Cytopathogenicity assays were performed to determine whether pretreatment of bacteria with drugs inhibit bacterial-mediated host cell cytotoxicity. The UV-vis spectra of both silver-drug nanoconjugates showed a characteristic surface plasmon resonance band in the range of 400–450 nm. AFM further confirmed the morphology of nanoparticles and revealed the formation of spherical nanoparticles with size distribution of 30–80 nm. FT-IR analysis demonstrated the involvement of Hydroxyl groups in both drugs in the stabilization of silver nanoparticles. Antibacterial assays showed that silver nanoparticle conjugation enhanced antibacterial potential of both Cephradine and Vildagliptin compared to the drugs alone. Pretreatment of bacteria with drugs inhibited E. coli K1-mediated host cell cytotoxicity. In summary, conjugation with silver nanoparticle enhanced antibacterial effects of clinically approved Cephradine. These findings suggest that modifying and/or repurposing clinically approved drugs using nanotechnology is a feasible approach in our search for effective antibacterial molecules.


Introduction
Infectious diseases are a significant burden on public health, driven largely by socio-economic, environmental and ecological factors [1]. About 15 million of 57 million annual deaths worldwide are estimated to be caused by infectious diseases, principally due to bacterial pathogens [1]. The burden of morbidity and mortality falls most heavily on people in developing countries [2]. Drug-resistant microbes are a major factor causing microbial re-emergence [3]. Among numerous bacteria, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Bacillus species, Klebsiella pneumoniae

Bacterial Cultures
The cultures of six bacterial isolates including neuropathogenic Escherichia coli K1 (a cerebrospinal fluid isolate from a meningitis patient; 018:K1:H7), strain E44, was used in the present study (Malaysian Type Culture Collection 710859), and methicillin-resistant Staphylococcus aureus (MRSA) was used as described previously (Malaysian Type Culture Collection 381123). The MRSA strain was originally derived from the blood cultures, obtained from the Luton & Dunstable Hospital NHS Foundation Trust, Luton, England, UK. Pseudomonas aeruginosa, Klebsiella pneumoniae, Bacillus cereus and Streptococcus pyogenes were obtained from the Microbiology Research Laboratory at Sunway University. Stock cultures were refreshed by subculturing every 15 days on nutrient agar plates and were maintained at 4 • C.

Synthesis of AgNPs Coated with Drugs
Cephradine conjugated silver nanoparticles (Ceph-AgNPs) were synthesized. Briefly, 5 mL (0.1 mM) Cephradine aqueous solution was reacted with 5 mL (0.1 mM) silver nitrate aqueous solution, and the reaction mixture was magnetically stirred for 10 min. Twenty µL of 5 mM freshly prepared Sodium borohydride aqueous solution (NaBH 4 ) was added in the above stirring reaction mixture. The color of solution turned yellow-brown from transparent upon addition of a reducing agent indicating the reduction of silver ions and the formation of Ceph-AgNPs [28,29]. For Vildagliptin-conjugated silver nanoparticles (Vgt-AgNPs), a similar procedure was repeated by optimizing different volume ratio (v/v) of silver solution and drugs. Stable Vgt-AgNPs were obtained at respective v/v of silver to drug at 1:1. The amount of drug loaded on the nanoparticles was also measured. Nanoparticles were centrifuged at 12,000× g for 1 h, supernatant was collected, freeze-dried, and the unloaded drugs was determined by weighing. The results are expressed as the percentage of the drug amount contained in 100 mg of the dried nanoparticle. The percentage of drug loading on nanoparticles was found to be 52% and 68% for Ceph-AgNPs and Vgt-AgNPs, respectively.

Bactericidal Assay
Antibacterial potential of AgNPs, Ceph and Ceph-AgNPs was determined by using bactericidal assay [30]. Briefly, bacterial cultures were adjusted to optical density (OD) of 0.22 at 595 nm using a spectrophotometer (OD 595 = 0.22) which corresponds to 10 8 colony-forming units per mL (C.F.U. mL −1 ). An inoculum of 10 µL of above bacteria culture (equivalent to approximately 10 6 C.F.U.) was incubated with various concentrations of either Ceph-AgNPs, and Vgt-AgNPs in 1.5 mL centrifuge tubes at 37 • C for 2 h. For negative controls untreated bacterial culture were incubated with phosphate buffer saline (PBS). Vildagliptin and Cephradine alone were used as additional controls, while bacteria incubated with 100 µg mL −1 of gentamicin were used as positive control. Next, bacteria were serially diluted and 10 µL of each dilution was plated on nutrient agar plates. These plates were incubated at 37 • C overnight, followed by viable bacterial C.F.U. count.

Cytopathogenicity Assay
Cytopathogenicity assays were performed as described previously [31]. Briefly, E. coli K1 were incubated with various concentrations of Cephradine, Vildagliptin, and their nanoconjugates for 2 h at 37 • C. Next, all test samples were incubated with confluent HeLa monolayers in supplemented medium. Plates were incubated at 37 • C for 24 h in a 5% CO 2 incubator and observed for cytotoxic effects. At the end of this incubation period, the supernatants were collected and cytopathogenicity was detected by measuring lactate dehydrogenase (LDH) release (Cytotoxicity Detection kit) as follows: % cytotoxicity = (sample value − control value)/total LDH release − control value) × 100. Control values were obtained from host cells incubated in RPMI-1640 medium alone. Total LDH release was determined from HeLa cells treated with 1% Triton X-100 for 30 min at 37 • C. The basis of this assay is that cell supernatant containing LDH catalyzes the conversion of lactate to pyruvate, generating reduced form of nicotinamide adenine dinucleotide (NADH) and H + . In the second step, the catalyst (diaphorase, solution from kit) transfers H and H + from NADH and H + to the tetrazolium salt p-iodo-nitrotetrazolium violet (INT), which is reduced to formazan (dye), and absorbance is read at 490 nm.

Characterization of Cephradine and Vildagliptin Coated Silver Nanoparticles
The UV-vis spectra of both silver-drug nanoconjugates showed characteristic surface plasmon resonance band in the range of 400-450 nm. AFM images were recorded to ascertain the morphology of these nanoparticles. The representative topographical images of Ceph-AgNPs and Vgt-AgNPs revealed the formation of spherical nanoparticles (Figure 1a,b). The size distribution of drugs nanoconjugates was determined by DLS (Figure 1c,d). Vgt-AgNPs was found to be a mixture of small and large particle size ranging from 2, 7, and 90 nm with an average size of 33 nm, as compared to Ceph-AgNPs (average size 85 nm). FT-IR analysis demonstrated the involvement of hydroxyl groups in both drugs in the stabilization of silver nanoparticles ( Figure 2).
Antibiotics 2018, 7, 4 of 12 nanoconjugates was determined by DLS (Figure 1c,d). Vgt-AgNPs was found to be a mixture of small and large particle size ranging from 2, 7, and 90 nm with an average size of 33 nm, as compared to Ceph-AgNPs (average size 85 nm). FT-IR analysis demonstrated the involvement of hydroxyl groups in both drugs in the stabilization of silver nanoparticles ( Figure 2).

Cephradine and Vildagliptine Conjugated with AgNPs Exhibited Increased Bactericidal Effects Against E. coli K1 and MRSA Compared with AgNPs and Drugs Alone
Bactericidal assay was performed to determine the effects of drugs conjugated with AgNPs and drugs alone on E. coli K1 and MRSA. The concentrations of samples were adjusted to achieve MIC50 (minimum inhibitory concentration to kill 50% bacteria), which can be observed in MRSA and S. pyogense, and in the rest of bacteria MIC90 (minimum inhibitory concentration to kill 90% bacteria) is

Cephradine and Vildagliptine Conjugated with AgNPs Exhibited Increased Bactericidal Effects against E. coli K1 and MRSA Compared with AgNPs and Drugs Alone
Bactericidal assay was performed to determine the effects of drugs conjugated with AgNPs and drugs alone on E. coli K1 and MRSA. The concentrations of samples were adjusted to achieve MIC50 (minimum inhibitory concentration to kill 50% bacteria), which can be observed in MRSA and S. pyogense, and in the rest of bacteria MIC90 (minimum inhibitory concentration to kill 90% bacteria) is observed at tested concentrations. The results revealed that both drugs conjugated with AgNPs exhibited significant bactericidal effects (p < 0.05 using t-test, two-tailed distribution). In addition, treatment with bare AgNPs alone had limited effects on E. coli K1 and MRSA ( Figure 3). Notably, Ceph-AgNPs showed significant bactericidal effects at 5 and 10 µM compared with drugs alone (p < 0.05). Vildagliptin alone did not show significant bactericidal effects at 5 or 10 µM, while, at 5 and 10 µM, Vgt-AgNPs, bacteria were significantly reduced (from 7.3 × 10 7 to 5.4 × 10 5 ) and (from 8.6 × 10 7 to 4.8 × 10 5 ) respectively for Gram-negative E. coli K1, and (from 1.0 × 10 7 to 4.3 × 10 6 ) (from 9.3 × 10 6 to 2.3 × 10 6 ) at 5 and 10 µM for Gram-positive MRSA (Figure 3).

Cephradine and Vildagliptine Conjugated with AgNPs Exhibited Increased Bactericidal Effects Against P. aeruginosa, K. pneumoniae, B. cereus, S. pyogenes Compared with the Drugs Alone
The results revealed that drugs conjugated with AgNPs exhibited significant bactericidal effects at 1-and 2-µM concentration against P. aeruginosa, K. pneumoniae, B. cereus, S. pyogenes (p < 0.05 using T test and two-tailed distribution). However, at 1 µM and 2 µM concentration, the treatment with AgNPs alone had similar effects on the number of bacteria. Notably, Ceph-AgNPs and Vgt-AgNPs showed significant effects at 1 µM and 2 µM compared with the drugs alone ( Figure 4). ; colonies were determined following incubation with drugs alone, Ag alone, AgNPs conjugated with drugs. Briefly, 1 × 10 6 E. coli K1, MRSA colonies were incubated with drugs and controls at 37 • C for 24 h. Next, colonies were accounted. Both drug-conjugated AgNPs exhibited significant bactericidal effects (p < 0.05 using t-test, two-tailed distribution, as indicated by asterisk). The results are the mean ± standard error of three independent experiments performed in duplicate. The results revealed that drugs conjugated with AgNPs exhibited significant bactericidal effects at 1-and 2-µM concentration against P. aeruginosa, K. pneumoniae, B. cereus, S. pyogenes (p < 0.05 using T test and two-tailed distribution). However, at 1 µM and 2 µM concentration, the treatment with AgNPs alone had similar effects on the number of bacteria. Notably, Ceph-AgNPs and Vgt-AgNPs showed significant effects at 1 µM and 2 µM compared with the drugs alone ( Figure 4).  Bacterial colonies were determined following incubation with Cephradine and Vildagliptine. Briefly, 1 × 10 6 bacteria were incubated with drugs and controls at 37 °C for 24 h. Both drugs-conjugated AgNPs and Ag-NPSalone exhibited significant bactericidal effects (p < 0.05 using t-test, two-tailed distribution, as indicated by asterisk), while as there was no significant effects of drugs alone. The results are the mean ± standard error of three independent experiments performed in duplicate.

Silver Nanoparticle-Conjugated Drugs Inhibited E. coli K1-Mediated Host Cell Cytotoxicity
To determine whether AgNP-conjugated drugs inhibited bacteria-mediated host cell cytotoxicity, assays were performed by incubating 10 6 E. coli K1 with HeLa cells for 24 h. E. coli K1 alone produced 60% host cell death. On the other hand, bacteria pretreated with AgNPs as well as Ceph-AgNPs and Vgt-AgNPs caused minimal host cell damage and host cell cytotoxicity was reduced to less than 15% ( Figure 5). These findings also showed that Ceph-AgNPs and Vgt-AgNPs alone had minimal host cell cytotoxicity.

Figure 5.
Cytopathogenicity assays were performed using E. coli K1 as described in materials and methods. Briefly, bacteria were incubated with test samples for 2 h before putting on HeLa cells monolayer with and without different drugs (10 µM), Cephradine (Ceph) and Vildagliptine (Vgt). Cells were then incubated for 24 h on standard conditions. Untreated bacteria, and bacteria treated with gentamicin were also treated with cells to evaluate their effects on cells cytotoxicity. Triton-X was used to lyse cells before determination of lactate dehydrogenase (LDH) release as marker of cell damage. LDH was determined by using Roche applied sciences LDH kit supplemented with enzyme and buffer. The % cytotoxicity was calculated by formula: % cytotoxicity = (sample value − control value)/total LDH release − control value) × 100. The results are representative of at least three independent experiments performed in duplicates. Asterisk indicates p < 0.05 using t-test, two-tailed distribution. Bacterial colonies were determined following incubation with Cephradine and Vildagliptine. Briefly, 1 × 10 6 bacteria were incubated with drugs and controls at 37 • C for 24 h. Both drugs-conjugated AgNPs and Ag-NPS-alone exhibited significant bactericidal effects (p < 0.05 using t-test, two-tailed distribution, as indicated by asterisk), while as there was no significant effects of drugs alone. The results are the mean ± standard error of three independent experiments performed in duplicate.

Silver Nanoparticle-Conjugated Drugs Inhibited E. coli K1-Mediated Host Cell Cytotoxicity
To determine whether AgNP-conjugated drugs inhibited bacteria-mediated host cell cytotoxicity, assays were performed by incubating 10 6 E. coli K1 with HeLa cells for 24 h. E. coli K1 alone produced 60% host cell death. On the other hand, bacteria pretreated with AgNPs as well as Ceph-AgNPs and Vgt-AgNPs caused minimal host cell damage and host cell cytotoxicity was reduced to less than 15% ( Figure 5). These findings also showed that Ceph-AgNPs and Vgt-AgNPs alone had minimal host cell cytotoxicity.

Silver Nanoparticle-Conjugated Drugs Inhibited E. coli K1-Mediated Host Cell Cytotoxicity
To determine whether AgNP-conjugated drugs inhibited bacteria-mediated host cell cytotoxicity, assays were performed by incubating 10 6 E. coli K1 with HeLa cells for 24 h. E. coli K1 alone produced 60% host cell death. On the other hand, bacteria pretreated with AgNPs as well as Ceph-AgNPs and Vgt-AgNPs caused minimal host cell damage and host cell cytotoxicity was reduced to less than 15% ( Figure 5). These findings also showed that Ceph-AgNPs and Vgt-AgNPs alone had minimal host cell cytotoxicity. Figure 5. Cytopathogenicity assays were performed using E. coli K1 as described in materials and methods. Briefly, bacteria were incubated with test samples for 2 h before putting on HeLa cells monolayer with and without different drugs (10 µM), Cephradine (Ceph) and Vildagliptine (Vgt). Cells were then incubated for 24 h on standard conditions. Untreated bacteria, and bacteria treated with gentamicin were also treated with cells to evaluate their effects on cells cytotoxicity. Triton-X was used to lyse cells before determination of lactate dehydrogenase (LDH) release as marker of cell damage. LDH was determined by using Roche applied sciences LDH kit supplemented with enzyme and buffer. The % cytotoxicity was calculated by formula: % cytotoxicity = (sample value − control value)/total LDH release − control value) × 100. The results are representative of at least three independent experiments performed in duplicates. Asterisk indicates p < 0.05 using t-test, two-tailed distribution. Figure 5. Cytopathogenicity assays were performed using E. coli K1 as described in materials and methods. Briefly, bacteria were incubated with test samples for 2 h before putting on HeLa cells monolayer with and without different drugs (10 µM), Cephradine (Ceph) and Vildagliptine (Vgt). Cells were then incubated for 24 h on standard conditions. Untreated bacteria, and bacteria treated with gentamicin were also treated with cells to evaluate their effects on cells cytotoxicity. Triton-X was used to lyse cells before determination of lactate dehydrogenase (LDH) release as marker of cell damage. LDH was determined by using Roche applied sciences LDH kit supplemented with enzyme and buffer. The % cytotoxicity was calculated by formula: % cytotoxicity = (sample value − control value)/total LDH release − control value) × 100. The results are representative of at least three independent experiments performed in duplicates. Asterisk indicates p < 0.05 using t-test, two-tailed distribution.

Discussion
The lack of development and approval of new and effective antibacterials as well as growing MDR microbes presents a major challenge in our ability to counter bacterial infections [31]. Nanotechnology offers great promise in the field of biomedicines, especially diagnosis and drug delivery. It offers opportunities for therapeutic agent delivery to specific cells and receptors. Nanomaterial-based drug delivery systems have the potential to improve pharmacokinetics and pharmacodynamics of the drugs [32]. The small size of nanoparticles provides them a greater surface area for maximum drug loading as well as high accessibility for specific targets. Recently, various drug-conjugated nanoparticles are being developed against infections caused by resistant microbes [33]. The most common metal carriers for nanoparticle-based drug delivery systems include gold, silver, and iron oxide due to their inertness and biocompatibility [33].
Though the mode of action of silver nanoparticles on the bacteria has been suggested to affect morphological and structural changes in the bacterial cells, the large surface area, provides better uptake by microorganisms [34]. Hence, silver nanoparticles have the ability to anchor to the bacterial cell wall and subsequently penetrate it, thereby causing structural changes leading to increased permeability of the cell membrane and cell death. In addition, the formation of free radicals by the silver nanoparticles have the ability to damage the cell membrane and make it porous resulting in bacterial cell death [35]. The bacterial membrane contains sulfur-containing proteins and the AgNPs interact with these proteins in the cell as well as with the phosphorus containing compounds. When AgNPs enter the bacterial cell, it forms a low molecular weight region in the center of the bacteria to which the bacteria conglomerates thus protecting the DNA from silver ions. Also, it generates reactive oxygen species, which are produced to attack the respiratory chain, cell division, and finally leading to cell death [36].
Silver conjugated Cephradine (mode of action involves binding and inactivation penicillin binding proteins leading to inhibition of peptidoglycan layer and causing cell lysis) and Vildagliptin (DPP4 inhibitor) were synthesized by reducing silver nitrate with sodium borohydride in the presence of drugs. These nanodrug conjugates were characterized by UV-visible spectrophotometry, FT-IR spectroscopy, and AFM. Cephradine and Vildagliptin successfully stabilized AgNPs and displayed surface plasmon resonance band in the range of 400-450 nm. FT-IR analysis showed the interaction of hydroxyl groups of drugs with silver nanoparticles for stabilization. Hence, the mode of stabilization is anticipated to be noncovalent interactions. Ceph-AgNPs and Vgt-AgNPs both were found to be spherical in shape and lie in a range of size distribution from 30-80 nm. Ceph-AgNPs were larger as compared to Vgt-AgNPs.
After characterization, these nanoparticles were subjected to antibacterial assays. Ceph-AgNPs and Vgt-AgNPs showed significant bactericidal effects against Gram-negative E. coli K1, P. aeruginosa, K. pneumonia and Gram-positive MRSA, B. cereus, and S. pyogenes. Nevertheless, these findings show tremendous potential in the development of new antibacterial formulations. As discussed previously, Cephradine is a cephalosporin first-generation antibiotic, which is not very effective against a number of bacteria because the different resistance mechanisms varied from the variation of the penicillin-binding protein, production of β-lactamase, existence of β-lactamase genes on plasmids or on bacterial chromosomes, and efflux pump mechanisms. For instance, MRSA produces abnormal penicillin binding protein 2A, which has low methicillin affinity (mediated through the mecA gene), which is carried on the staphylococcal cassette chromosome mec (SCCmec) by horizontal gene transfer and this results in resistance to Cephradine as an antibiotic [37]. Ceph-AgNPs provide a change in susceptibility of the drug along with, presumably, enhanced bioavailability. On the other hand, Vildagliptin (dipeptidyl peptidase-4 inhibitor) and its silver nanoparticles showed some promising effects as an antibacterial. Furthermore, these nanoparticles significantly and selectively reduced the pathogen-mediated host cell cytotoxicity caused by Gram-negative bacteria.
Drug-loaded nanoparticles enter in the bacteria by endocytosis showing either specific or non-specific type of interactions with cell membrane [38]. The positive charge of AgNPs interacts with lipopolysaccharides of Gram-negative bacteria with more affinity than the cellular wall of Gram-positive bacteria, that possess few sites for interactions, then releases the drugs intracellularly [38]. In previous reports, cephalosporin conjugated with AgNPs also showed enhancement in the antibacterial potency of Ceftriaxone and Cefixime against E. coli and S. pyogenes, respectively [39,40]. In conclusion, Cephradine and Vildagliptin showed bactericidal effects against the six tested bacteria in this study, but their conjugation with AgNPs enhanced their antibacterial efficacy. Moreover, Ceph-AgNPs and Vgt-AgNPs also significantly reduced the host cells cytotoxicity. The exact mechanism of action of these nanoparticles is not precisely understood and it is the subject of future studies along with testing their potential in vivo.