**1. 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*

are important human pathogens contributing to urinary tract infections, neonatal meningitis, gastroenteritis, wound and skin diseases, food poisoning and nosocomial infections [4–10]. The overuse of antibiotics has contributed to bacterial acquisition of drug resistance resulting in reduced efficacy of available drugs.

During the past decade, nanomedicine has shown grea<sup>t</sup> potential due to effectiveness of various nanoconjugates against pathogenic microbes [11]. Nanomaterials have been frequently used as effective coatings to prevent bacterial adhesion to surfaces as well as bactericidal agents [12]. He et al., showed the development of self-defensive and antibacterial adhesion surface coating based on bilayer hydrogel which can promote cell adhesion and proliferation [13,14]. Polymers-based antibacterial agents are also an important class of nanomaterials. Yuan et al. reported various types of hydroxyl-rich cationic derivatives of star-like poly (glycidyl methacrylate) as broad-spectrum antibacterial and antifouling surface coating agents [15]. Antibacterial activity of low molecular weight cationic polymers is shown to affect the membrane permeability and disruption against a broad range of bacteria [16]. In another report, a salivary statherin protein inspired poly(amidoamine) dendrimer is shown to exhibit antibacterial effects as effective coating on hydroxyapatite [17,18]. Similarly, dendrons have been shown as a clicking tool for generating nonleaching antibacterial materials [19]. Metal nanoparticles have been studied extensively because of their unique physicochemical characteristics including catalytic activity, optical properties, electronic properties, antimicrobial activity, and magnetic properties [20]. Among these, silver nanoparticles (AgNPs) have shown growth inhibitory as well as bactericidal effects [21]. The high surface area of AgNPs leads to high antimicrobial activity as compared with the silver metal [22]. With the limited discovery of novel antibacterial agents, a feasible approach is to modify clinically approved drugs to enhance their efficacy and/or drug repurposing to expedite discovery of effective formulation of antibacterial agents.

Cephradine (relative molecular mass 349.406 g mol−1) is a first generation cephalosporins antibiotic drug that is widely used in the treatment of bacterial infections of the urinary and the respiratory tract, as well as ear, skin and soft tissues. It is used against both Gram-positive and Gram-negative bacteria. Its mode of action is inhibition of bacterial cell wall synthesis [23,24]. Vildagliptin (relative molecular mass 303.399 g mol−1) is an antidiabetic drug, which is a small molecule and inhibits dipeptidyl peptidase-4 (DPP4). Vildagliptin has been shown to stimulate insulin secretion and inhibit glucagon secretion in a glucose-dependent manner [25–27]. Here we tested whether conjugation of AgNPs can enhance efficacy of the clinically approved drug, Cephradine.

#### **2. Materials and Methods**

#### *2.1. 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.

#### *2.2. 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 (NaBH4) was added in the above stirring reaction mixture. The color of solution turned yellow-brown from transparent upon addition of a reducing agen<sup>t</sup> 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.

#### *2.3. Characterization of AgNPs-Coated Drugs*

After successful synthesis of nanoconjugates, Ceph-AgNPs and Vgt-AgNPs were subjected to complete analysis via ultraviolet-visible spectrophotometry (UV-vis), Fourier transformation infrared (FT-IR), atomic force microscopy (AFM), and dynamic light scattering (DLS) as described previously [28,29].

## *2.4. 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 (OD595 = 0.22) which corresponds to 10<sup>8</sup> colony-forming units per mL (C.F.U. mL−1). An inoculum of 10 μL of above bacteria culture (equivalent to approximately 10<sup>6</sup> 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−<sup>1</sup> 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.

## *2.5. 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% CO2 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<sup>+</sup> from NADH and H<sup>+</sup> to the tetrazolium salt p-iodo-nitrotetrazolium violet (INT), which is reduced to formazan (dye), and absorbance is read at 490 nm.
