**3. Results**

#### *3.1. 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).

**Figure 1.** Representative images of atomic force microscopy (AFM) of Ceph-AgNPs (**a**); Vgt-AgNPs (**b**); AFM topographs were recorded on Agilent 5500 instrument used in tapping mode with silicon nitride cantilever. Ceph-AgNPs were found to be 85 nm (**c**); whereas, Vgt-AgNPs with the size distribution of 33 nm as measured by DLS (**d**). Both nanoparticles were spherical and polydisperesed.

**Figure 2.** Representative spectra of FT-IR of Ceph-AgNPs (**a**); Vgt-AgNPs (**b**); shows the presence of hydroxyl, lactam, carboxylic acid and amino groups. However, while compared with drugs alone (**<sup>c</sup>**,**d**), hydroxyl groups peaks showed a shift in wave number which indicates the interaction with silver nanoparticles. Spectra were recorded at Bruker Vector 22 instrument using Potassium bromide (KBr) disc method.

#### *3.2. 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 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<sup>7</sup> to 5.4 × 105) and (from 8.6 × 10<sup>7</sup> to 4.8 × 105) respectively for Gram-negative *E. coli* K1, and (from 1.0 × 10<sup>7</sup> to 4.3 × 106) (from 9.3 × 10<sup>6</sup> to 2.3 × 106) at 5 and 10 μM for Gram-positive MRSA (Figure 3).

**Figure 3.** *E. coli* K1 (**a**); MRSA (**b**); colonies were determined following incubation with drugs alone, Ag alone, AgNPs conjugated with drugs. Briefly, 1 × 10<sup>6</sup> *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.

#### *3.3. 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).

**Figure 4.** *Cont.*

**Figure 4.** *P. aeruginosa* (**a**); *K. pneumoniae* (**b**); *B. cereus* (**c**); *S. pyogenes* (**d**). Bacterial colonies were determined following incubation with Cephradine and Vildagliptine. Briefly, 1 × 10<sup>6</sup> 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.

#### *3.4. 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<sup>6</sup> *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.
