Acrylonitrile (AN) was purchased from Loba Chemie, Spain. Ethylenediamine (EDA), and hexamethylenediamine (HMDA) were purchased from Across. Benzaldehyde (Bz), 4-hydroxybenzaldehyde (4-HO-Bz) and 2,4-dihydroxybenzaldehyde (2,4-Di-HO-Bz) were purchased from Across. Pipridine was purchased from BDH Chemicals Ltd Poole England. All solvents were used as received without further purification.

Thermal properties were examined using thermogravimetric analysis (TGA) which was carried on TA-Q500 System of TA; samples of 5–10 mg were heated in the temperature range 30–800°C at a scanning rate of 10°C·min^-1 under nitrogen atmosphere. Fourier-transformer infrared (FT-IR) Spectra were recorded using TENSOR 27, Bruker.

Full-stained ultra-thin sections were examined using the transmission electron microscope (JEOL-JEM-100SX, Japan) with beam current = 60 μA, and high voltage of 80 kV.

Polyacrylonitrile (PAN) (II) was prepared in aqueous solution (precipitation polymerization) according to a previously reported procedure [²⁵,²⁶]. PAN (II) was prepared with a redox system in aqueous solution at room temperature with stirring under nitrogen atmosphere. Then, sodium disulfite solution and ferrous sulfate solution were added followed by addition of potassium peroxodisulfate solution. The precipitated polymer (II) was filtered off, washed with distilled water and methanol, respectively (Scheme 1).

Amine-terminated polymers (III & IV) were prepared according to the procedure described by El-Newehy et al.[²⁶] by the reaction of polyacrylonitrile with ethylenediamine (EDA), and hexamethylenediamine (HMDA). In brief: In a 100 mL round-bottomed flask, it was placed an excess amount of the diamine (10-fold) in absolute ethanol, then PAN (II) was added portion-wise over 1 h with stirring under nitrogen. The reaction mixture was stirred at room temperature for 1–2 h and at 70°C for 12 h. The product was filtered, washed with methanol (Scheme 1).

Antimicrobial assessments were carried out at Microbiology Unit, Department of Botany, Faculty of Science, Tanta University, Tanta, Egypt.

The microorganisms include the Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa, and Salmonella typhi, the Gram-positive bacteria Staphylococcus aureus, and the filamentous fungi Aspergillus niger, Aspergillus flavus, yeast form fungi Candida albicans, and Cryptpcoccus neoformans.

Bacteria were maintained on nutrient agar, and the fungi were kept on Sabouroud's agar slopes. All fungi were isolated from patients of Tanta University hospitals, and were authenticated by Assuit Mycology Center, Faculty of Science, Assuit University, Assuit, Egypt. All bacteria were isolated from patients of Tanta University hospitals, and were authenticated by Bacteriology Research Unit, Faculty of Science, Al-Azhar University, Cairo, Egypt.

Cut plug method recorded by Pridham, et.al. [²⁸] was employed to determine the antimicrobial activity of the prepared polymers as following: Freshly prepared spore suspension of different tested microorganisms (0.5 mL of about 10⁶ cells/mL) was mixed with 9.5 mL of melting sterile Sabouraud's dextrose medium (for fungi) or nutrient agar medium (for bacteria) at 45°C, poured on sterile Petri dishes, and left to solidify at room temperature. Regular wells were made in the inoculated agar plates by a sterile cork borer of 0.7 mm diameter. Each well was filled with 20 mg of each tested powder. Three replicates were made for each test, and all plates were incubated at 27°C for 72 h for fungi, and at 32°C for 24 h for bacteria. Then the average diameters of inhibition zones were recorded in millimeters (mm), and compared for all plates.

Half-fold serial dilutions were made for selected polymers in order to prepare concentrations of 6.25, 12.5, 25, 50 and 100 mg/mL in distilled water, zero concentration was considered as a negative control. A previously prepared pure spore suspension of each tested microorganism (0.5 mL of about 10⁶ cells/mL) was mixed with 9.5 mL of each concentration in sterile test tubes, incubated at 27°C for 72 h for fungi, and at 32°C for 24 h for bacteria, then optical density of growth was measured by spectrophotometer (Optima SP-300, Japan) at 620 nm for each incubated mixture, results were represented graphically, and MIC was recorded for each tested material [²⁹].

TEM analyses were carried out in Scanning Electron Microscope Unit, Histology Department, Faculty of Medicine, Tanta University, Tanta 31527, Egypt.

For studying the effect of the antimicrobial agents on the ultra-structure of microbial cells, tested microorganism was cultured on the appropriate liquid nutrition medium to get a cell suspension of 5x10⁶ cells/mL, and then mixed with the selected polymer of the previously recorded MIC. The mixture was incubated overnight on a shaking incubator of 60 rpm at the appropriate temperature. Then the treated mixture was centrifuged at 3000 rpm for 20 min, washed with sterile saline solution, and re-centrifuged to collect the cell pellet in a clean Eppendorf tube [³⁰].

Collected cell pellet was fixed by adding 1 mL of 2.5% glutaraldehyde that was buffered in 0.1 M phosphate buffer saline (PBS) of pH = 7.4 (for fixation of cellular protein content, and to stop all culture reactions), and was cooled at 4°C for 2 h. Fixed sample was washed with 1% osmic acid for 30 min (for fixation of lipid cell content), washed 3 times with PBS (10 min. for each time), and dehydrated in ascending ethanol concentrations (30, 50, 70, 90, and absolute alcohol) for 30 min for each concentration, then dehydrated sample was infiltrated with acetone for 1 h.

For TEM analysis, sample was embedded in araldite 502 resin to build a plastic mold (for complete fixation of all cell contents), that were cut into semi-thin sections in the ultra-cut microtome (LEICA ultracut UCT, Japan), stained with 1% Toluidine blue, examined to confirm the success of sample preparation, then ultra-thin sections were prepared, stained with uranyl acetate, and counter stained with lead citrate [³¹].

Full-stained ultrathin sections were examined, and photographed under the appropriate magnification (at 10000X for larger cells of fungi, or 20000X for smaller cells of bacteria), using the transmission electron microscope (JEOL-JEM-100SX, Japan) with beam current = 60 μA, and high voltage of 80 kV.

Physiological tests were carried out in Microbial physiology lab. For fungal and bacterial measurements, Microbiology Department, Faculty of Science, Al-Azhar University, Cairo, Egypt.

For studying the effect of the antimicrobial agents on the physiological parameters of microbial cells, tested microorganism was cultured on the appropriate liquid nutrition medium to get a cell suspension of 5x10⁶ cells/mL, and then mixed with the tested polymer of the previously recorded MIC. All the mixture was incubated overnight on a shaking incubator of 60 rpm at the appropriate temperature. Then the treated mixture was centrifuged at 3000 rpm for 20 min, washed with sterile distilled water, re-centrifuged to collect the cell pellet in a clean Eppendorf tube; cells were broken for extraction of intracellular content by grinding with glass beads in 1 mL distilled H₂O, filtered, and supernatants were collected, and stored at 4°C for further analysis.

Comassie brilliant blue G-250 dye of 100 mg was dissolved in 50 mL of a mixture of 95% ethanol and 100 mL of 85% phosphoric acid, diluted to 1 L with distilled water, and then filtered. After that, 0.1 mL of previously treated cell extract was mixed with 5 mL of Comassie dye and was shacked well for 5 min, the light absorbance of mixture was recorded at λ = 595 nm. Records of absorbance indicate the concentration of the total soluble cell proteins in tested supernatants using a previously drawn standard curve of light absorbance of known concentration of bovine serum albumin as a standard protein stained and was measured in the same way as the unknown sample [³²].

All minerals and ionic components of the treated cells which were released in the previously treated, and extract of broken cells were stored. Each tested suspension was diluted to 10 mL with distilled water, then the electrode of EC-meter (Sensorex CS200TC lab conductivity sensor, USA) was immersed in the suspension, and the potential of electric current was recorded. The concentration of the total ions present in the tested suspension was indicated by the records of electric current potential, which can be read from a previously prepared standard curve of electric conductivity of known concentration of known electrolyte [³³].

FTIR spectroscopy was used to prove the immobilization of the antimicrobial agents onto amine-terminated PANs (V-X) as shown in (Table 2) and (Figure 1). Characteristic absorption peaks were observed at 1361–1454 cm⁻¹ due to -NH stretching and strong peaks at 2940 cm⁻¹ corresponds to -C-H stretching vibration and at 3555 cm⁻¹ due to = NH stretching. In addition, the spectra of the antimicrobial polymers (V-X) shows many significant changes; a new band was appeared around 1583 cm⁻¹ due to aromatic ring and new band at 3375 and 3414 cm^-1 which is attributed to the –OH stretching vibration, which in conclusion confirm the immobilization of benzaldehyde onto amine-terminated PANs.

The thermogram showed three steps for the degradation of the prepared polymers (II-X) which were performed with a heating rate of 10°C min^-1 under nitrogen atmosphere as shown in (Figure 2) and the data were summarized in (Table 3).

Generally, the first step ranges between room temperature and 150°C for all polymers which may be attributed to the evolution of moisture. The second step of weight loss starts at about 150°C and continues up to 335–400°C due to the degradation of the grafted functional groups. The last step over 350°C up to 800°C which may be due to the degradation of the remainder of polyacrylonitrile chains.

Table 3 summarizes the TGA results of polymers (II-X). The thermal degradation of PAN (II) and amine-terminated PANs (III & IV) was explained in details in our previous work [²⁶]. For the antimicrobial polymers (V-X), the thermogram showed that the onset decomposition temperature was found to be in the range 289-296°C except for polymers (VII and X) were found to be 388 and 342°C, respectively, which may attributed to the forming of hydrogen bond as the number of hydroxyl group increased in the immobilized antimicrobial agent. Moreover, the prepared polymers leave residue around 38–50%.

The obtained data in this study demonstrated a slightly differences in the thermal stabilities between the starting material, PAN and the amine-terminated PANs and an increase in the thermal stability of the antimicrobial polymers (V-X) compared to the starting materials, PAN (II) and the amine-terminated PANs (III & IV) as shown in (Table 3).

The growth-inhibiting effect was quantitatively determined by percentage of the surviving cells (% Optical density) as shown in (Figures 4, 5, 6, 7, 8, 9). The MIC values for these polymers were determined by using the broth dilution method. We considered zero concentration as a negative control. The polymer concentrations ranged from 6.25-100 mg/mL which were obtained by half-fold serial dilutions. Each solution in the series was mixed with 10⁶ cells/mL of each tested microorganism [²⁹].

MIC was recorded at 25 mg/mL for polymers (V & VI), and decreased to 12.5 mg/mL for polymers (VII–X), that possessed higher inhibitory effect on tested microbes due to the increase in space length between the bioactive groups, and the polymer backbone resulting in more released of the inhibitory groups.

Generally, the polymer samples killed 70–75% at MIC concentration the inhibitory effect which was varied according to the polymer microstructure, antimicrobial agent as well as the type of tested microorganisms as shown in (Table 5).

Figures 10 &11 showed the TEM images of antimicrobial effects of the biocidal polymers (V-X) against fungi and pathogenic bacteria. Generally, the treated cells showed totally deformation and exhibited severe destruction. In contrast, the intact cells had a smooth surface with overall intact morphology.

Destructive effects of inhibitory agents against different microorganisms appear greatly causing firstly the rupture of cell wall that lead to the leakage of cytoplasmic contents, and then an observable shrinking of cytoplasmic size will appear. Deeper effects may appear as the coagulation of cell proteins, and precipitation of mineral crystals due to dehydration of cytoplasm. Finally, complete discharge of cytoplasmic contents will occur. Great effects can be easily observed with polymers (VIII & X) while lower effects were recorded for the other tested polymers [³¹].

The observed results from TEM images for the effect of the treated cells were confirmed by the estimation of both total soluble cell proteins concentration (Table 6) and the total cell ionic content (Table 7).

The more lowered protein content, the more effective the polymer in decreasing cellular activity. As shown in (Table 6), the total soluble proteins concentration was decreased to almost 50% or less compared to the control. Polymer (X) gave the highest inhibition of cell protein synthesis, and the lowest effect was recorded for polymer (VI).

In a similar way, the more lowered ion content, the more effective the polymer in leakage of cytoplasmic contents. Polymer (X) gave the highest inhibition of cell protein synthesis, and the lowest effect was recorded for polymers (V & VI) as shown in (Table 7).

¹Benzaldehyde; ²4-Hydroxybezaldehyde; ³2,4-Dihydroxybenzaldehyde.

^*Grafted functional group degradation (150-335°C);^#Grafted functional group degradation (150-400°C).

^a MIC = 25 mg/mL. ^b MIC = 12.5 mg/mL.

* Polymer not Applicable.

* Polymer not Applicable.