**4. 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 grea<sup>t</sup> promise in the field of biomedicines, especially diagnosis and drug delivery. It offers opportunities for therapeutic agen<sup>t</sup> 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.

**Author Contributions:** Conceptualization, N.A.K., R.B.S., M.R.S.; Methodology, A.M., A.A. and D.A.; Software, A.M., D.A.; Validation, A.A., R.B.S. and N.A.K.; Formal Analysis, A.M. and A.A.; Investigation, A.M.; Resources, N.A.K.; Data Curation A.M., D.A.; Writing—Original Draft Preparation, A.M. and A.A.; Writing—Review & Editing, N.A.K. and R.B.S.; Visualization, M.R.S.; Supervision, R.B.S.; Project Administration, R.B.S.; Funding Acquisition, R.B.S.

**Funding:** This work is supported by University Research Award INT-2017-03 by Sunway University, Malaysia.

**Acknowledgments:** We are thankful to Sunway University for support.

**Conflicts of Interest:** The authors declare that they have no competing interests.
