Electrospinning and antimicrobial properties of PAN-Cu₂O/ZnO nanofibers from green peel extracts

The, PAN was dried at 60 °C in an oven for about 24 h and collected in an airtight reagent bottle for further use. The dried PAN (12 wt%) was dissolved in DMF and stirred at room temperature for 64 h to get a clear solution. This was followed by stirring at 60 °C–70 °C for an additional 1 h to ensure complete dissolution of the polymer in the DMF. The mixture was fed into a 20 ml syringe, fitted into a programmable syringe pump electrospinning system. The PAN mixture was electrospun into a nanofiber at the tip-to-collector distance of 10 cm, at a flow rate of 0.02 ml min⁻¹ and positive potential of 30 kV. All the experimental procedures were carried out under static atmospheric conditions. The fabricated PAN-NFs were collected over aluminium covered metal plate collector, rid of DMF by drying at 100 °C in an oven, and subsequently kept in a cold and dry place for further characterization and application.

About 8 mg of metal oxide (MO) NPs were introduced into 1 ml of DMSO and sonicated for about 2 h 30 min and was added to 5 ml of the prepared DMF-PAN mixture. The mixture was then stirred at room temperature overnight. The resultant mixture was sonicated for another 30 min to ensure smooth surfaced nanofibers. Using the same spinning parameters used to prepare the PAN-NF, the mixture was electrospun into nanofiber composite, and was designated as CPE/MO-NFs.

The inhibitory activities of the prepared nanocomposites against the growth of E. coli are represented in table 4. The OPE/BiONPs resulted in the highest (maximum) inhibition efficiency of 71.22% (MxIE) at the maximum inhibitory concentration of 243 μg ml⁻¹. This performance is also comparable with LPE/BiONPs having a maximum inhibitory efficiency of 66.27% at a very low MIC of 6.8 μg ml⁻¹, and the positive control (ciprofloxacin) with maximum inhibitory efficiency of 72.55%. The minimum inhibition efficiency (MIE) of 19.68% at MIC of 1.9 μg ml⁻¹ was observed for OPE/BiONF against E. coli, followed by LPE/BiONF with MIE of 29.34%. The higher inhibition of these NPs relative to the NFC, against E. coli could be due to the faster access of smaller particle size of the 'free' NPs, thus enhancing the penetration into the microbial cells which could have destroyed the DNA, thus hampered its cell replication, and eventually resulting into cellular death. The antimicrobial activities of the nanomaterials also show inverse relation with their sizes. This trend agrees with earlier reports by Azam [36, 37]. The MIC reported here for CPE/BiONPs, is much lower than that reported by Alekish, Ismail [38], for ZnO-NPs against E. coli. All the NFCs counterparts however demonstrated less than 90% inhibition.

In table 5, the antimicrobial activities of the synthesized nanomaterials against the growth and multiplication of E. faecalis are presented. The LPE/BiONPs resulted in a very high inhibition efficiency (98.29%) against E. faecalis, at a maximum inhibitory concentration (MxIC) of 810 μg ml⁻¹. At a very low concentration of 1.9 μg ml⁻¹, LBiONPs inhibited the growth of E. faecalis (5.94%). At the MxIC of 810 μg ml⁻¹, OPE/BiONPs offered a maximum inhibition of 41.68% and a minimum inhibition concentration (MIC) of 1.9 μg ml⁻¹ against E. faecalis at an efficiency of 16.43%. The PAN-NF also demonstrated a capacity for antimicrobial activity with a maximum inhibitory efficiency of approximately 30% at 810 μg ml⁻¹, and negligible inhibition at the MIC of 1.9 μg ml⁻¹. The LBiONF showed a great improvement in the antibacterial activity of the PAN with more than 80% inhibition at 9 mg ml⁻¹, while the addition of OPE/BiO NPs did not improve the antimicrobial activities of OPE/BiONF against E. faecalis. The synergy between the synthesized LPE/BiONPs and its nanofibre of LPE/BiONF could be responsible for the improved inhibitory activities of the LPE/BiONF composite. The LPE/BiONPs with larger particle size than the OPE/BiONPs, have higher inhibition efficiency against E. faecalis at MIC of 810 μg ml⁻¹. This trend nonetheless, contradicts the earlier reports of Shinde [40], Azam, Ahmed [41]. This, however, is not unlikely, considering the zeta potential values of the NPs. Chang, Lin [42] asserted that particles with more negative zeta potential value, are likely to have lower antimicrobial activities amidst other factors. As expected, the commercial antibiotic had a higher inhibition of E. faecalis at a very low MIC compared to CPE/BiONPs.

The inhibitory activities of the nanomaterials against the growth and multiplication of S. enteritidis are shown in table 6. Most of the designed materials performed relatively better in inhibiting S. enteritidis. The LBiONPs showed maximum inhibitory efficiency of 62.31% at the MxIC of 240 μg ml⁻¹, while the counterpart OPE/BiONPs could not resist the growth of the pathogens for all the range of concentrations investigated. At a very low MxIC of 72.9 μg ml⁻¹, the Pure PAN NF resulted in approximately MxIE of 95% inhibition and MIE of 13.55% (at MIC of 1.9 μg ml⁻¹) to the growth and multiplication of S. enteritidis. Meanwhile, the NFC did better than the NPs but underperformed compared to the inhibitory activities of the pure NF. At the MxIC of 9 mg ml⁻¹ and 243 μg ml⁻¹, the LPE/BiONF and OPE/BiONF offered more than 80% inhibition respectively, to the growth of the target pathogen. Interestingly, at MIC of 2.7 mg ml⁻¹, the LPE/BiONF had 72% inhibition of S. enteritidis. As expected, the positive control, had higher percent inhibition than the prepared nanomaterials. This result correlates with the MIC of silver NPs in the earlier report of Farouk, El-Molla [43], on the antimicrobial activities of silver NPs against antibiotics resistant S. enteritidis strain. Alizadeh-Sani [44] reported a MIC of 3.00 ± 0.21 mg ml⁻¹ for ZnO, and 2.50 ± 0.17 mg ml⁻¹ for TiO₂ against the growth of S. enteritidis. These data put the antimicrobial activities of CPE/BiONPs and their NFCs at a comparative advantage and, could hence have applications in the control of waterborne pathogens.

V. cholerae, is a Gram-negative bacterium and did not show high susceptibility to the nanomaterials prepared in this study as observed in table 7. lBiONPs and the corresponding NF composite only had 64.5% at 243 μg ml⁻¹ and 75% at 2700 μg ml⁻¹ inhibition of V. cholera. Nonetheless, at low MIC (1.9 μg ml⁻¹), there was an inhibitory action of LBiONPs, LBiONF and OBiONF against V. cholera. The positive control as seen in table 7, did inhibit microbial growth more than the prepared materials. The inhibitory effect of metal oxides was also examined against V. cholerae by Raj [45] and Krishnaraj [46] and showed comparable antimicrobial activities with the present work. The data from the antimicrobial activities from the LBiONPs and the corresponding NF showed an advisable application in the control of V. cholerae.

The antimicrobial activities of the OBiONPs substrate with more negative ZP values and smaller particle-sized seem to be better than their counterpart in this study. The LBiONPs offered no resistance to the growth of B. cereus either as NPs and NF composites (see table 8). At the MxIC of 810 μg ml⁻¹, and 79.2 μg ml⁻¹, the OBiONPs and OBiONF counterparts inhibited the growth of the B. cereus by 61.28% and 58.49% respectively. Positive control offered a MxIE of 92.51% at MIC of 3.17 μg ml⁻¹. A similar work by Ansari [47] showed a similar inhibition efficiency at a very low concentration.

The mechanism of the bactericidal effect of nanomaterial against bacteria remains quite unclear [48, 49]. However, this study affirms that the nanomaterials have antimicrobial activities against the public health significant pathogens and was comparable to the conventional antimicrobial agents employed in treating bacterial infections. These agents could have exhibited antimicrobial activities by interfering with the synthesis of the cell walls or impairs pathogens protein and nucleic acid synthesis and replications process or altered the metabolic pathway of pathogen growth and virulence activities [49].

The study by Meghana, Kabra [50] had suggested that the antimicrobial activities of copper oxide nanoparticles are dependent on their oxidation states and could be toxic to E. coli when in their oxidative state. Cu₂O could have promptly deactivated Fumarase A genes, an iron sulphur cluster enzyme thereby binding to the proteins. Therefore, CuO NPs antibacterial activities could be linked to the quick debility in the integrity of cell membranes and the generation of reactive oxygen species (ROS) [51]. It is proposed that within bacterial cells, sulfhydryl reduced Cu(II) ions to cuprous Cu(I) ions, which in turn are responsible to cause oxidative stress through Cu(I)—driven ROS [52]. The discharge of Cu ions from the surface of metallic Cu has also been proposed as the cause of its antimicrobial action [53]. Similarly, copper oxides could leach various copper salts in their microns and nano-dimensioned oxides, leading to bactericidal effects [54].

On the other hand, previous antimicrobial behaviour studies of zinc oxide NPs have revealed that the size, abrasiveness, surface roughness and defects, and oxidative stress play significant functions in the antibacterial activities of nanomaterials [40, 55, 56]. The antimicrobial activity of the NFCs has been attributed to the release of the NPs into the solution of the composite [57] which forms interaction site between the hybrid composite and the pathogen [58] as observed in CPE metal oxides NFCs in this study. Therefore, the integration of the CuO NPs or ZnO NPs and amine functional groups in the mat resulted in bacteriocidal activities against the bacterial pathogen.

Moreover, the CPE/MONPs deposition on the surface of the PAN nanofiber membrane results in an attachment to the cell membrane which allows for permeation into the cell, thereby attacking the genetic materials in the nucleus resulting in cell death. The generation of H₂O₂ from the catalytic process of CPE/MONPs, could have amplified the antibacterial activity against the test pathogens [59, 60]. CPE/MONPs have the ability to neutralize the surface potential of the bacterial cell thereby resulting in the generation of pairs of electron-hole which boosts ROS production that in turn kills the bacteria cell.

Therefore, CPE/MONPs could act as bacteriostatic agents with positive charges against negatively charged bacteria [61]. The resulting strong electrostatic interaction between the positively charged Cu²⁺ or Zn²⁺ions and the electronegative cell wall surface of the microbe could lead to damage in the phospholipid's molecular structure and consequently cause damage to the microbial cell membrane [62, 63]. In summary, the binding of nanoparticles to the surface of the cell surface is likely the most crucial moment that instigates a series of events that could lead to interruption and damage to cellular structures. In this work, however, as in most literature [50], the exact mechanism pathway for the antimicrobial activities of the nanomaterials was not determined, hence, the antimicrobial activities of the nanomaterials are comparable to that of earlier reports by previous authors [40, 58, 64]. However, this study provides information on the antimicrobial potentials of a green synthesized nanomaterials from citrus peel extract.