In order to improve access to the functional locations of PILs, the structure was designed and controlled during the synthesis process so that the structure became porous without the use of a template to have the best form for drug loading and the availability of nanoparticles. Fig. 1 demonstrates the synthetic procedure for PILP-Ag organic-inorganic nanocomposite. In the synthesis of PILP-Ag organic-inorganic nanocomposite, after reaction of benzyl bromide with 1-vinyl imidazole, DVB as a cross-linker, and AIBN as a radical initiator, were added to mentioned mixture and stirred for completely reaction with ILVI-Br. Ethylene glycol was used as green and inexpensive reducing agent with the ability to control the size of silver cations to nanoparticles[30–32]. The placement of silver nanoparticles inside the PIL structure and their integration together give the nanocomposite structure certain characteristics that are listed below:
1) the add new interaction between silver nanoparticle and drug (π-cation, electrostatic interaction, coordination reaction and hydrogen bond) 2) the increase of the hydrophilic property of carrier owing to existence of hydroxyl groups in the mesoporous framework to help to high and appropriate dispersity of carrier in SBF 3) the covalently bonded ionic groups that make the compound steady under various situation, chiefly high temperature, moisture and acidic environment and different pH 4) low using of Il in structure that is economical owing to their cost 5) fabrication of Ag NPs without aggregation owing to anchor of nanoparticle in the pores of the mesoporous framework for the stabilization of Ag NPs.[33–36]
3.1. Characterization of PILP-Ag organic-inorganic nanocomposite
The structure of a) PILP-Ag organic-inorganic nanocomposite and was determined by FT-IR spectroscopy and the results are presented in Fig. 2. Peaks at 2853-3022 cm−1 are attributed to the C-H bonds stretching vibration that were basic characteristic peaks of of the DVB-IM copolymer chain in structure. The peak at 1641 cm−1 is assigned to the bending mode of the -OH group of adsorbed water by hydrogen bonding and the broadband at 3437 cm−1 is due to the remaining hydroxyl groups on the Ag Nps. Furthermore, FT-IR spectrum of PILP-Ag organic-inorganic nanocomposite showed other absorption bonds at 1597-1698, 713-903, 1021-1223, 1358 and 1498 cm−1 that are corresponding to C=C bond in the aromatic rings, unsaturated stretching of C-H, band of the C-N linkage and alkyl stretching of the copolymer, respectively.
In the next step, to prove the successful synthesis, reduction and stabilization of Ag NPs into the voids of the mesoporous polymer, the structural properties were investigated by XRD (Fig. 3) in the range of 2θ = 10°–70° that presence of peaks at 2θ = 38.34, 44.35 and 64.57 can be ascribed to 111, 200 and 220. In addition, the broad XRD peak at a low diffraction angle of 15° to 30° corresponds to the amorphous-state PILP substrate.
The SEM image (Fig. 4) of PILP-Ag organic-inorganic nanocomposite shows the nanocomposites have a homogeneous structure.
The nitrogen adsorption-desorption and BJH pore size distributions results were exposed in Fig. 5. Nitrogen isotherm for synthesized-nanocarrier presented type IV curve with a hysteresis loop of type H1, confirming the presence of uniformity of mesopores. The BET surface area, pore-volume and mean pore diameter of the PILP-Ag organic-inorganic nanocomposite are 548 m2 g−1, 1.17 cm3/g and 2.38 nm, respectively. The availability and abundance of uniform spaces, which are due to the high surface area and the appropriate size of pores, can improve the performance of the drug carrier.
Transmission electron microscopy (TEM) image of the PILP-Ag organic-inorganic nanocomposite indicated that silver nanoparticles are formed and adequately distributed through the polymer lattice. (Fig. 6) and the corresponding particles size distributions of the prepared PILP-Ag organic-inorganic nanocomposite with the 0.8–1.4 nm average diameter. This really protects the PILP from the destruction and leaching of silver NPs throughout the different environments.
3.2. Antibacterial activity of PILP-Ag organic-inorganic nanocomposite
PILP-Ag organic-inorganic nanocomposite were evaluated for antimicrobial activity (Fig. 7). The bacteria selected in this research are classified as pathogenic super bacteria and can be used as appropriate candidates in antibacterial tests. The MIC of PILP-Ag organic-inorganic nanocomposite against Streptococcus aureus ATCC 35668 and Escherichia coli O157 PTCC 1276 were establish to be 2–1024 and 2–1024 µg/mL, respectively. The MBC of PILP-Ag organic-inorganic nanocomposite against the bacteria were found in the range of 2–2048 and 2–2048 µg/mL, respectively. The lowest concentration of PILP-Ag organic-inorganic nanocomposite for MIC test for E. coli. and S. aureus were 256, 256 µg/mL and also, MBC test for E. coli. and S. aureus were 256, 512 µg/mL, respectively, these results show that PILP-Ag organic-inorganic nanocomposite could prevent growth and kill all tested bacterial pathogens. The antibacterial mechanism of the formed nanostructure mainly can be involves the electrostatic interactions of cationic and metallic parts (imidazolium units and silver NPs) with the negatively charged bacterial membrane[39, 40]. However, the hydrophobic part of the nanostructure (PILP) also plays a role in the mechanism and affects the internal and hydrophobic regions of the bacteria that this helps to rupture the cell membrane.
3.3. Cytotoxicity analysis of PILP-Ag organic-inorganic nanocomposite
MTT assay showed that PILP-Ag organic-inorganic nanocomposite with a concentration of 0.001 g and 0.005 g had a less damage on HFF-2 cells, because more than 90% of the treated cells were alive compared to the control group (Fig. 8). Furthermore, there is no significant difference in terms of cell growth and viability between concentrations of 0.001 g and 0.005 g of PILP-Ag organic-inorganic nanocomposite.
However, with increasing the concentration of nanomaterials, cells viability in cells treated was decreased about 25% and 45% with high concentrations of 0.01 g and 0.05 g of PILP-Ag organic-inorganic nanocomposite.
3.4. Drug release behavior
The drug release shows the amount of mass transfer from a solid phase to a broth phase under usual conditions. The principal phase in delivery of drug is the interaction between the nanocarrier of the drug and the buffer solution, which occurs between the carrier and buffer. Hence, kinetics of the drug release are related to the rate of the reaction at the inter-face, the rate of the flow of PBS solution on the path into the inter-face of PBS solution and carrier and the molecular diffusion of the dissolved drug molecules from the inter-face toward the broth solution. Consequently, the MTX release of PILP-Ag organic-inorganic nanocomposite was evaluated at 37°C and pH 7.4 shown in Fig. 9. The drugs were released from PILP-Ag organic-inorganic nanocomposite by exchanging with phosphate anions and capillary forced release from structure at the desired pH in a stable method. In the first 20 h, the most drug releasing levels were regarded to drugs absorbed in the surface of the PILP-Ag organic-inorganic nanocomposite and drugs, which were bonded to the substrate by, π-cation, hydrogen bonds and electrostatic interaction, quickly developing a therapeutic dose. After that, the available and remaining drugs balanced with PILP counter ions and coupling with Ag NPs, in addition to slowing down the release rate, reduce the release rate of the drug and deliver the drug continuously and for a longer constant period. Gradually the release increased and reached its maximum value (96.18%) in 120 hours of release. The slow release rate allows the drug to reach the body over a longer period, thus reducing the number of doses the patient can take.