Optimization of spinning conditions

Numerous spinning conditions are required to achieve accepted morphological nanofibers in terms of smooth, beadles, and stretchable nanofiber production [28]. Each composite was fabricated under different voltages, and adapted solution feed rate, as explained in Table (1). Smooth and uniform STG and MER loaded mats were developed using different concentrations of STG (2, 4, and 8 mg/mL) and (5, 7, and 9 mg/mL) of MER. The optimization of conditions for loading drugs on nanofiber scaffolds revealed that the maximum amount of STG that could be loaded onto the PVA was 8 mg/mL, while MER could be loaded up to 9 mg/mL. Based on these results the total amount of both drugs in the combinational mats optimized to be 8 mg/mL as it was observed that increasing the concentrations of solutions affected the feed rate and voltage.

FTIR investigation of different unloaded and loaded electrospun Mats

The nanofibers were characterized by FTIR to analyze their chemical structure, compatibility and incorporation of STG and MER into PVA nanofibrous scaffolds [29]. Figures (1) represents the FTIR absorption spectra of different nanofibers composed of various concentration of STG, MER and combination of both drugs loaded on PVA. This showed that PVA spectrum presents O–H stretching band at 3290 cm− 1, C–H stretching and bending vibrations at 2922 cm− 1 and 1427 cm− 1, as shown in Figure (1I) which was observed in all scaffolds [30]. As shown in Figure (1 A), the characteristic absorption peaks of the pure drug (STG) are due to O = C = O stretching and C—N stretching at 2806 and 1517 cm− 1, respectively [31]. STG loaded-scaffolds obviously showed specific absorption band related to STG at ν 2800 cm− 1 which is assigned to the O = C = O group and C—N group at ν 1517 cm− 1 as revealed in Figure (1B–D). On the other hand, the characteristic absorption peaks of the pure drug (MER) in Figure (1E) are assigned to the C-H stretching and N-O stretching at 2900 cm− 1,1575 cm− 1, respectively [32]. MER loaded-PVA scaffolds obviously showed the specific bands of MER which are C-H absorption band around 2850 cm− 1 while N-O band appeared at1575 cm− 1 displayed in Figure (1E–H). In the combinational ratios illustrated in Figure (1 J–L), the characteristic peak of STG (O = C = O) is prominently observed in the ratio of 1 STG :1 MER and becomes even more intense in the 2 STG :1 MER ratio, indicating an increase in the concentration of STG. This peak disappeared in the 1 STG : 2 MER ratio. Conversely, the C—N peak of STG is present in all combinational scaffolds. N-O peak of MER was shown in all scaffolds. These observations proved the successful incorporation of both STG and MER into PVA nanofiber.

Fig. 1

FTIR spectra of (A) STG, (B) PVA + 2 mg/mL STG, (C) PVA + 4 mg/mL STG, (D) PVA + 8 mg/mL STG, (E) MER, (F) PVA + 5 mg/mL MER, (G) PVA + 7 mg/mL MER, (H) PVA + 9 mg/mL MER, (I) PVA, (J) PVA +(1 STG : 1 MER), (k) PVA +(2 STG : 1 MER), (L) PVA+(1 STG : 2 MER)

XRD analysis

The crystallographic structure of electrospun nanofibrous mats was obtained by X-ray diffraction scan patterns as shown in Figure (2). XRD patterns showed drug embedded into the polymeric nanofibrous scaffolds induced changes in the crystallinity of nanofibers [27]. The prominent peak of PVA at 19.5° was observed in Figure (2I) which is present in all nanofiber scaffolds [30]. The XRD of pure STG and MER showed the fingerprints with sharp observed patterns, indicating its crystalline structure at 2θ 13.8°,18.48°,25°and 12.8°,16.9°,25° respectively revealed in Figure (2 A, E) [33, 34]. It was observed that there was a loss of crystallinity, resulting in an amorphous state at the concentrations of PVA + STG (2 and 4 mg/mL), PVA + MER (5 and 7 mg/mL), and the ratios of PVA+ (1 STG:1 MER) and (2 STG:1 MER). At higher concentrations of STG (8 mg/mL), MER (9 mg/mL), and the ratio of STG/MER (1:2), the materials exhibited a semicrystalline structure. This indicates that the drugs are highly homogenized and completely dispersed and encapsulated within the nanofibrous scaffold. As a result, the crystalline nature of the drugs has been altered [35]. Salim and her coworkers proved that all loaded NFs scaffolds with different concentrations of Brimonidine tartrate (BT) showed halo diffractogram patterns that confirm the complete conversion of BT to an amorphous arrangement and its uniform embedding into PCL/PVP nanofibers scaffolds. It is verified that the absence of intense sharp bands attributed to crystalline drug molecules in the diffractogram of the loaded scaffold validates that the drug is presented in an amorphous state in nanofibers.

Fig. 2

XRD diffractometry patterns of (A) STG, (B) PVA + 2 mg/mL STG, (C) PVA + 4 mg/mL STG, (D) PVA + 8 mg/mL STG, (E) MER, (F) PVA + 5 mg/mL MER, (G) PVA + 7 mg/mL MER, (H) PVA + 9 mg/mL MER, (I) PVA, (J) PVA +(1 STG : 1 MER), (K) PVA +(2 STG : 1 MER), (L) PVA +(1 STG : 2 MER)

Physical morphological features for nanofibrous scaffolds

The prepared nanofibers were characterized by scanning electron microscope; SEM micrographs represented the uniformity, smooth structure and effective encapsulation of the drugs into the fibers, also the average diameter distribution was calculated as observed in Figure (3). The unloaded PVA scaffold revealed that the average diameter of PVA nanofibers was 630 ± 127 nm Figure (3 A). After loading with STG, the average diameter of the nanofibers decreased to 354 ± 81 nm at the highest concentration (Figs. 3B-D). Similarly, for Meropenem-loaded nanofibers, the average diameter decreased to 279 ± 47 nm at the highest concentration Figures (3E-G). In all combinational scaffolds, the diameter was approximately 600 nm Figures (3 H-J), which remained less than that of the plain PVA scaffold. A potential rationale for the reduced diameter of the loaded nanofibers could be an increase in electrical conductivity [35]. This observation aligns with similar findings in Meropenem and colistin-loaded PVA/chitosan nanofibers [23]. For instance, this study has shown that higher concentrations of bioactive agents (antibiotics) can lead to changes in the electrospinning process, resulting in finer fibers due to enhanced jet stretching and reduced viscosity.

Fig. 3

SEM images and fiber diameter histogram of (A) PVA, (B) PVA + 2 mg/mL STG, (C) PVA + 4 mg/mL STG, (D) PVA + 8 mg/mL STG, (E) PVA + 5 mg/mL MER, (F) PVA + 7 mg/mL MER, (G) PVA + 9 mg/mL MER, (H) PVA +(1 STG : 1 MER), (I) PVA +(2 STG : 1 MER), (J) PVA +(1 STG : 2 MER), all captures are at magnification 8000x, scale10µm

UV-visible spectrophotometer

The mean MER content and loading efficiency in various nanofibrous scaffolds were calculated and presented in Table (2). The results showed that MER loading is influenced by the concentration of STG. In the PVA + 9 mg/mL MER scaffold, the loading efficiency was 33%. However, this efficiency doubled in the PVA + (1 STG:1 MER) and PVA + (1 STG:2 MER) formulations, suggesting that STG may play a key role in forming hydrogen bonds with MER through its NH2 group, thereby enhancing MER loading [36]. On the other hand, in the PVA + (2 STG:1 MER) scaffold, the increased amount of STG likely facilitated the formation of intra- and intermolecular hydrogen bonds between STG molecules, which reduced its ability to interact with MER [37, 38].

Table 2 Drug content and loading efficiency on different nanofiber scaffoldsDetermination of minimum inhibitory concentration (MIC) of meropenem and sitagliptin by broth microdilution method

The MIC values of MER and STG were assessed against clinical isolates (n = 10) and two reference strains. For Meropenem, 50% of the isolates (five strains) had an MIC value of 512 µg/mL, three isolates showed an MIC value of 256 µg/mL, one isolate had an MIC value of 16 µg/mL, and one isolate showed an MIC value ˃ 512 µg/mL. Based on CLSI breakpoints, these findings confirm carbapenem resistance in these isolates. The reference strain PAO1 exhibited an MIC value of 1 µg/mL and ATCC 27,853 fall within the quality control range. Regarding Sitagliptin, 70% of the isolates (seven isolates) had an MIC value of 16 mg/mL, three isolates displayed an MIC value of 8 mg/mL while the PAO1 strain showed an MIC value of 16 mg/mL, consistent with findings from previous studies [10, 39]. This is the first report examining the impact of Sitagliptin on CRPA.

Evaluation of synergism (checkerboard microdilution method)

The efficacy of the Meropenem-Sitagliptin combination against all tested strains (n = 11) is depicted in a checkerboard format Figure (4). The addition of Sitagliptin led to a 2-4-fold reduction in the MIC value of Meropenem for all CRPA and PAO1 strains. Based on the FICI values (0.75–1) Table (3), the combination exhibited an additive effect, highlighting the significance of integrating quorum sensing inhibitor sitagliptin with Meropenem. Previous studies demonstrate the QSI activity of STG against the reference strain Pseudomonas aeruginosa PAO1, showing a significant reduction in the expression levels of key QS genes (lasI, lasR, rhlI, rhlR, pqsA, and pqsR) [10]. Additionally, molecular docking studies confirm that STG binds efficiently to the active sites of LasR, QscR, and PqsR, with the highest binding affinity compared to other antidiabetic drugs [10, 13]. Since LasR functions as a global regulator of virulence in P. aeruginosa, its inhibition can impact biofilm development and other pathogenic traits [40]. This may help explain the observed additive effect between STG and meropenem (MER). It may be worthwhile to explore the combination of Sitagliptin with other antibiotic classes as well as other MDR bacteria. This is based on evidence that the same quorum sensing inhibitor can exert varying effects depending on the antibiotic class which is combined with. For example, curcumin, a natural polyphenolic compound with quorum sensing inhibitory activity, demonstrated a synergistic effect when combined with ceftazidime (a cephalosporin), and an additive effect when combined with ciprofloxacin (a fluoroquinolone) against Pseudomonas aeruginosa [41]. Hence, pairing Meropenem with different quorum sensing inhibitors is crucial to identify the most effective synergy in combating high-priority pathogens (CRPA). A previous study investigated the combination of Meropenem with a natural antibiotic-adjuvant molecule, cinnamon bark essential oil, against Klebsiella pneumoniae, demonstrating an additive [42]. To our knowledge, this is the first report to explore the combined effects of Sitagliptin with Meropenem and demonstrate this combinational effect on nanofiber scaffold.

Table 3 FICI values for sitagliptin/meropenem combinations against CRPAFig. 4

Checkerboard showing effect of Sitagliptin-Meropenem combinations against P. aeruginosa strains. The violet color represents growth inhibition (MIC), green color indicates the additive effect with growth inhibition. Blue shows no reduction in the growth (no effect) based on the visible turbidity. The P. aeruginosa strains were treated with fractional concentrations of Meropenem and Sitagliptin in 96-well plate containing MHB for 24 h. The growth based on visible turbidity was used for interpreting the results

Evaluation of the antimicrobial activity of nanofibers by agar disc-diffusion

The results indicate that both the unloaded PVA and (PVA + 8 mg/mL STG) scaffolds exhibited no antibacterial activity against PAO1 and all clinical isolates (n = 10). The (PVA + 9 mg/mL MER) scaffold and the three combination ratios showed no activity against the PR6, PR9, and PR11 strains, while demonstrating varying degrees of significant antibacterial effects against the remaining strains, as illustrated in Figure (5). Among the tested scaffolds, (1 STG: 2 MER) ratio was the best scaffold, since it was the only scaffold that has an inhibition zone of (9.3 mm ± 0.57 and 7.3 mm ± 0.57) for PR4 and PR7, respectively. It also exhibited an inhibition pattern comparable to that of the (PVA + 9 mg/mL MER) scaffold against the other strains. Notably, this effect was achieved using a lower MER concentration (5.3 mg/mL), indicating that STG enhanced the antibacterial performance of MER, particularly against PR4 and PR7, while enabling a reduced MER concentration and maintaining efficacy against the remaining strains. The other combinations, (1 STG:1 MER) and (2 STG:1 MER), showed no antibacterial activity against PR2, PR5, and PR8. However, both demonstrated identical inhibition zones of (29.6 ± 1.5 mm) against PAO1, despite containing different MER concentrations (4 mg/mL and 2.7 mg/mL, respectively), highlighting the contributory role of STG. In contrast, varying inhibition zones were observed against PR1 and PR10: (6.3 ± 0.57 mm and 4.9 ± 0.11 mm) for PR1, and (14.3 ± 0.57 mm and 10.3 ± 0.57 mm) for PR10, respectively. In both ratios, the inhibition zones remained smaller than those achieved with MER alone. The observed variation in the sensitivity of the tested isolates may be attributed to differences in the genetic determinants responsible for carbapenem resistance. In P. aeruginosa, resistance to carbapenems can arise through several mechanisms. These involve non-carbapenemase mechanisms including efflux system (MexAB-OprM) and membrane porins (OprD) as well as carbapenemase-mediated mechanisms comprising enzymes such as Ambler class A (KPC-producing P. aeruginosa) and Ambler class B (e.g., VIM and IMP) [4]. These findings emphasize the potential of STG to potentiate MER’s antibacterial activity while significantly reducing its required concentration, without loss of efficacy. These findings align with the checkerboard assay results, underscoring the promising potential of this combination. The concentrations of STG used in the nanofiber scaffolds were unlikely to affect blood glucose levels; the effective oral dose of STG ranges from 25 mg/mL to 100 mg/mL [43]. In animal studies investigating the topical application of Sitagliptin (eye drops) for treating early stages of diabetic retinopathy, the minimum effective dose was 5 mg/mL twice per day, which exceeded the concentrations utilized in all scaffolds [44].

Fig. 5

Antimicrobial assessment of nanofiber mats against PAO1 and 10 Pseudomonas -resistant clinical isolates. The mats are composed of (A) PVA, (B) PVA + 8 mg/mL STG, (C) PVA + 9 mg/mL MER, (D) PVA +(1 STG: 1 MER), (E) PVA +(2 STG: 1 MER), and (F) PVA +(1 STG : 2 MER). Note: (A) PVA and (B) PVA + 8 mg/mL STG were tested against all strains; however, images are shown only for PAO1 and PR1

Evaluation of the stability of drug loaded nanofiber by disc diffusion

During the fabrication process, MER is dissolved in PVA, and it is known that Meropenem has low stability after reconstitution [45]. The results revealed no statistically significant difference in the activity of the nanofiber scaffolds over a period of three months (P > 0.05), as shown in Table (4). The prepared sheets stored in refrigerator showed good stability over 3 months with the same zone of inhibition obtained directly after fabrication of nanofiber as shown in Figure (6). These results highlighted the potential use of Meropenem nanofiber as a topical drug delivery system and highlighted the necessity of extending the stability study period under various storage conditions. It is worth mentioning that this is the first report confirms the stability of Meropenem loaded on to nanofibrous scaffolds.

Table 4 Comparison of antimicrobial assessment of nanofiber Mats over 3 months against PAO1 and PR1 using Kruskal–WallisFig. 6

Antimicrobial assessment of nanofiber mats over 3 months against PAO1 and PR1: (a) PVA + 9 mg/mL MER, (b) PVA +(1 STG: 1 MER), (c) PVA +(2 STG: 1 MER), (d) PVA +(1 STG : 2 MER)