Synthesis and characterization of functionalized modified PVC-chitosan as antimicrobial polymeric biomaterial

Amino acetic acid modified poly(vinyl chloride), MPVC, was obtained by chemical modification of PVC using glycine methyl ester. MPVC was used as a precursor to prepare some functionalized MPVC conjugates to be used in biomedical applications. MPVC-Cs was prepared by the chemical reaction of MPVC with chitosan as a natural polymer in absence and presence of epichlorohydrin (Ech) as a crosslinking agent. Further chemical modification was performed by the reaction of MPVC with Cs and salicylic acid in the absence in presence of Ech via one-pot reaction. The chemical structure of the formed MPVC, MPVC-Cs, MPVC-Cs/POH, MPVC-Cs/SA and MPVC-Cs/POH/SA was confirmed by the FTIR spectroscopic analysis, scanning electron microscopy, and thermogravimetric analysis (TGA). The antibacterial activity of the prepared MPVC and its conjugates was investigated against two Gram +ve bacteria (Staphylococcus aurous and Listeria monocytogenes) and (Escherichia coli and Salmonella typhi) as Gram −ve bacteria in addition to the Catondida albicans as yeast. Minimum inhibition concentration (MIC) was also determined for the prepared materials.


Introduction
The medical device sector is a dynamic and growing field in the medical industry that needs great efforts to produce, support and develop new materials. Synthetic biocompatible polymers occupy an important place in the medical industry and a huge number of devices such as prosthetics, catheters, drains, knees, hips, and other items are being manufactured and consumed by patients on daily bases. Polymers used in medical devices include silicone, rubbers, polyurethanes, and vinyl polymers. Many concerns must be taken into consideration during the manufacturing of medical devices for instance the biocompatibility, the chemical and thermal stability, and the mechanical properties of the used synthetic polymer [1]. Polyvinyl chloride (PVC) which is a largely amorphous material, is never processed as a polymer only, therefore, incorporating various additives is necessary for its formulation. These additives include PVC resin, plasticizers, photo, and heat stabilizers, impact modifiers, internal lubricant, pigment, and fillers to make it processible [2]. di-(2-ethylhexyl) phthalate (DEHP) is the most frequently used plasticizer in PVC compounds for the medical devices industry to achieve flexibility. DEHP may migrate from the implanted device to the human body, causing a certain degree of toxicity, inflammation of tissues, and infection in patients [3].
The adhesion of microorganisms to the surfaces of medical devices depends on the interaction of the surface of the biomaterial [4]. Like other polymeric materials, PVC suffers microbial colonization [5,6] which may be due to its hydrophobic nature [7]. Consequently, to obtain an antimicrobial polymeric material, PVC modification is being of important issue to diminish the risk of both infections and cross contamination in industrial processes [8][9][10]. Using biocides agents on PVC is considered one of the possible antibacterial strategies, either through bounding to the surface or by releasing to the surroundings [11][12][13]. Nisin or triclosan antibiotics had been incorporated into PVC products, [14] but silver is still the most used antibacterial agent [15,16]. As the antibacterial agent leaching is undesirable, the alternative is to achieve the functionalized PVC via a nucleophilic substitution reaction. Thiol compounds are used for this purpose because of their high nucleophilicity and low basicity of sulfur [17]. Many organic compounds of antibacterial properties have been incorporated into the main backbone chains of PVC by nucleophilic substitution reaction to obtain antibacterial PVC [18,19].
On the other hand, the biopolymer chitosan (Cs) with its biocompatibility, biodegradability, antimicrobial activity, and many other desired properties is used as a conjugate with synthetic polymers by chemical reactions to achieve antimicrobial polymers. Cs which is characterized by the presence of NH 2 and OH groups as nucleophilic moieties are reactive toward several chemical modifications to obtain modified substrates with the required properties, functions, and applications [20]. As mentioned previously, the free NH 2 groups present at C-2 are more reactive than both OH groups at C-3 and C-6 in the glucopyranose rings of Cs towards electrophile attack. The chemical reaction of NH 2 groups of Cs with aldehydes to obtain the Schiff base derivatives of the corresponding biopolymeric material with a higher antibacterial efficiency against a wide spectrum of both Gram +ve and Gram −ve bacteria [21]. The introduction of heterocyclic rings in the Cs scaffold, which enhances its pharmacological activity, is considered an efficient type of modification. Further modifications of Cs can be attained through the reductive amination reaction by selective introduction of either alkyl or aryl groups at the amino group of Cs [22]. Previous work described the synthesis of novel Cs-PVC conjugates that were functionalized with benzothiazole derivative as an antibacterial agent. Silver nanoparticles (AgNPs) and Ag/TiO 2 NPs are introduced into some of these conjugates to enhance their antibacterial activity against some types of bacteria, two Gram +ve (S. aureus, and L. monocytogenes) bacteria and two Gram −ve (E. coli, and S. typhimurium) ones [23]. This work reports on the chemical modification and the synthesis of some modified PVC conjugates using Cs and salicylic acid as antimicrobial efficient modifiers, in the absence and presence of Ech, to afford PVC as polymeric biomaterial having good antimicrobial properties to be used in the field of biomedical applications.

Materials and methods
All fine chemicals were of analytical grade and purchased from common commercial suppliers. Cs (MW 100-300 kDa, 82% degree of deacetylation). Suspension PVC, with a K value of 70, was obtained from A1-America Company for Petrochemicals, Alexandria, Egypt. Tetrahydrofuran (THF) was distilled over potassium under N 2 . All other solvents were distilled before use.

Preparation of PVC-glycine methyl ester
PVC (6.25 g, 10.0 mmol) was soaked in dry freshly distilled tetrahydrofuran (THF, 50 mL) and left overnight. Glycine methyl ester (4.4 g, 5 mmol) that was dissolved in 10 mL dimethylsulfoxide (DMSO) and triethylamine (2 mL) were added with stirring and heated on a water bath for 6 h at 55 °C. The reaction mixture was poured on ice/methanol to give a pale-yellow precipitate which was separated by filtration and washed with methanol. PVC-glycine methyl ester was finally dried in the oven until constant weight.

Preparation of amino acetic acid modified PVC (MPVC)
PVC-glycine methyl ester (1.78 g, 10 mmol) was mixed with NaOH (0.32 g, 8.0 mmol) in aqueous ethanol (60%) and refluxed for about 4 h. The formed dark yellow solid product was collected by filtration and dried in the oven until constant weight. Scheme 1 represents this modification.

Preparation of Cs modified PVC (MPVC-Cs)
Cs (1.61 g, 10 mmol) was soaked in 50 mL dry DMF and left overnight. MPVC (1.6 g, 10 mmol) dissolved in 20 mL DMF was then added in presence of N,N′dicyclohexylcarbodiimide (DCC) as a catalyst. The mixture was heated at 60 °C for 6 h, cooled, precipitated in cold methanol/water mixture, and then collected by filtration. The product was washed with methanol, acetone, and ether then dried in the oven at 55 °C until constant weight to give a buff solid product.

One-pot synthesis of crosslinked MPVC-Cs (MPVC-Cs/POH)
Cs (1.61 g, 10 mmol) was soaked in 50 mL dry DMF and left overnight. Ech (0.9 mL, 10 mmol) was added with stirring at room temperature and this was followed by addition of MPVC (1.6 g, 10 mmol) that was dissolved in 20 mL DMF in presence of DCC. The reaction mixture was heated at 60 °C for 6 h, cooled, precipitated in cold methanol/water mixture and the solid product was then filtered. The pale-yellow product was washed with methanol, acetone, and ether then dried in the oven at 55 °C until constant weight. Scheme 2 shows the preparation of MPVC-Cs and MPVC-Cs/POH. Cs (1.61 g, 10 mmol) was soaked in 50 mL dry DMF and left overnight. After that, MPVC (1.6 g, 10 mmol) that was dissolved in 20 mL DMF was added which was followed by addition of salicylic acid (0.138 g, 1 mmol). The reaction was carried out in presence of DCC as a catalyst. The reaction mixture was heated at 60 °C for 6 h, cooled, precipitated in cold methanol/water mixture and then the formed solid product was filtered. The product was washed with methanol, acetone, and ether then dried in the oven at 55 °C until constant weight.

One-pot synthesis of salicylic acid functionalized crosslinked MPVC-Cs (MPVC-Cs/ POH/SA)
Cs (1.61 g, 10 mmol) was soaked in 50 mL dry DMF and left overnight. Ech (0.9 mL, 10 mmol) was added with stirring at room temperature. MPVC (1.6 g, 10 mmol) that was previously dissolved in 20 mL DMF was added to the above mixture and this was followed by addition of salicylic acid (0.138 g, 1 mmol). The reaction was carried out in presence of DCC as a catalyst. The reaction mixture was heated at 60 °C for 6 h, cooled, precipitated in cold methanol/water mixture and then filtered to collect the formed pale brown solid product. The product was washed with methanol, acetone, and ether then dried in the oven at 55 °C until constant weight.

Fourier-transform infrared spectroscopy (FTIR)
IR spectral data were recorded on a Shimadzu IR spectrometer (FTIR 8201) using KBr discs at National Research Centre.

Thermogravimetric analysis (TGA)
Thermal stability of tested samples was measured via TGA technique using TGA-50H thermogravimetric analyzer, Shimadzu, Japan. The heating temperature was raised from room temperature up to 600 °C in a platinum pan while the heating rate was 10 °C/min and an inert atmosphere of N 2 flow rate 25 mL/min.

Scanning electron microscopy (SEM)
The morphology of the tested samples was investigated and imaged with SEM (QUANTA FEG 250 ESEM). The dry sample was spread on a double-sided Scheme 3 One-pot reaction of MPVC with Cs, salicylic acid in absence and in presence of Ech conductive adhesive tape, pasted on a metallic stub. Samples were mounted on circular aluminum stubs with double-sided sticky tape. The films were fixed on the surface of the tape.

Antimicrobial activity
The antimicrobial activities of samples under investigation were examined against some targeted pathogenic microorganisms obtained from the American type culture collection (ATCC; Rockville, MD, USA). The tested organisms were Staphylococcus aurous ATCC-47,077 (St.), Listeria monocytogenes ATCC-35,152 (List.), Escherichia coli ATCC-25922 (E. Coli.), Salmonella typhi ATCC-15566 (Salm.) and Candida albicans ATCC-10231 (C. Alb.)Ws\X [24]. The stock cultures of pathogens used in this study were maintained on nutrient agar slants at 4 °C. The Agar well diffusion method was employed to study the antimicrobial activities of tested samples according to the method described previously [25,26]. Reference antibacterial drugs ampicillin and vancomycin were evaluated for their antibacterial and antifungal activities and compared with the investigated samples. Seventy microliters of bacterial and yeast cells (10 6 CFU/mL) of each pathogen were spread on the nutrient agar plates. The wells (6 mm diameter) were dug on the inoculated agar plates and 100 µL of the used samples suspended in DMSO, were added to the wells. The reference antibiotics disks (10 and 30 µg/disk of ampicillin and vancomycin, respectively) were potted onto surface of agar inoculated plates. The plates were allowed to stand at 4 °C for 2 h before incubation to allow for diffusion. The plates were incubated at 37 °C for 24 h except yeast strain that were incubated at 28 °C for 24 h then followed by the measurement of the diameter of the inhibition zone (mm), and three replicates were averaged [27].

Determination of minimum inhibition concentration (MIC)
The MIC calculation of the tested samples was performed according to a slightly modified previous method [28]. In brief, serial dilutions were prepared of samples under investigation dissolved in DMSO. 150 μL of double strength Mueller Hinton broth medium were loaded in each well of the 96 well microtiter plate followed by 150 μL of the twofold appropriate concentration and mix well to obtain the final concentration. Overnight broth cultures of the tested bacterial and yeast strains prepared as an inoculum of 5% (V/V) (OD = 0.5 McFarland standard) was inoculated into the respective wells. For the growth control, the same inoculum size of each test strain was inoculated in wells that did not contain any of the tested samples. DMSO solution was tested as negative control. The plates were statically incubated at 37 °C for 24 h. A 30 μL of resazurin solution (0.18%) was added to each well to act as an electron acceptor and reduce to a pink, red or purple resorufin colored product by active microorganisms (i.e., inhibition of bacterial growth was visible as a dark blue well and the presence of growth was detected by the presence of pink, red or purple color). The MIC was defined as the concentration at which the bacteria and yeast do not show visible growth with respect to the positive control.

Chemistry
Methyl glycine ester was used to prepare amino acetic acid modified PVC (MPVC) as starting polymeric material to be used in preparation of different PVC conjugates for biomedical applications. As shown in the experimental part, PVC was reacted with methyl glycine ester to obtain methyl glycine ester modified PVC which was then hydrolyzed to afford the amino acetic acid modified PVC (MPVC). Modification of the as-prepared amino acetic acid-PVC (MPVC), was performed by the treatment with Cs in presence of DCC as a catalyst to yield MPVC-Cs conjugate which contains amide linker between the two polymeric chains. In another experiment, the treatment of PVC with Cs and Ech under the same ascribed condition yielded MPVC-Cs/POH that possessed propyl-2-hydroxyl moiety as crosslinker. The crosslinking of the Cs chains has been occurred by the reaction of the Ech with the secondary hydroxyl groups of Cs units. Further chemical modification of the amino acetic acid modified PVC (MPVC) via one-pot synthesis using Cs and salicylic acid in the absence and presence of Ech as a crosslinking agent was also carried out to obtain (MPVC-Cs/SA) and (MPVC-Cs/POH/SA), respectively. FTIR spectral data were carried out for the prepared compounds and confirmed their chemical structures. Thermal gravimetric analysis was carried out to determine the thermal stability of these compounds. Scanning electron microscopy (SEM) was also performed to investigate the morphology of the prepared functionalized MPVC-Cs conjugates and the homogeneity of the introduced organic molecules with the polymeric substrate. Antibacterial and antifungal evaluation as well as the minimum inhibition concentration (MIC) of all prepared functionalized MPVC-Cs conjugates was evaluated using Agar well diffusion method.

FTIR spectral data
In this study, the reaction of glycine methyl ester-PVC with Cs in the absence and presence of Ech afforded MPVC-Cs and MPV-Cs/POH, respectively, under the above-described method in Scheme 2. Figure 1a represented the functional group existed in PVC film. The FTIR spectrum showed the peaks at 2922 cm −1 attributed to aliphatic CH and CH 2 groups. The characteristic peaks of PVC appeared at finger print region in IR spectrum between 954 and 610 cm −1 that expressed on the polymeric backbone of PVC included the CH 2 groups and C-Cl groups. The peaks around 742 and 651 cm −1 were assigned to C-Cl vibration band. Figure 1b showed the PVC-methyl glycine ester. It was shown that the peak at 3430 cm −1 is corresponding to the stretching vibration of NH group attached to the PVC backbone chain whereas the peak that appeared at 2922 and 2858 cm −1 is related to the aliphatic CH and CH 2 groups of PVC chain. The characteristic IR band of the carbonyl ester group (C=O) appeared at 1743 cm −1 and the stretching vibration band   Figure 1c represented the characteristic IR bands of amino acetic acid modified PVC (MPVC) at which the carboxylic acid NH and OH group is characterized by the appearance of a stretching vibration broad peak at 3438-3100 cm −1 while the peak that appeared at 1725 cm −1 referred to the acidic carbonyl (C=O) group. The bending IR band at 1427 cm −1 and the stretching vibration at 1251 cm −1 are related to the OH (bending) and C-O (stretching), respectively. Figure 1d represented the FTIR spectrum of asprepared amino acetic acid modified PVC-Cs (MPVC-Cs), the observed broad band at 3438-3100 cm −1 were assigned to (OH) and (NH) stretch, whereas that at 2924 and 2856 cm −1 is attributed to the aliphatic (CH and CH 2 ) groups. The stretching vibration band at 1645 cm −1 represents the amide carbonyl group while the bands observed at 1428 and 1320 cm −1 are attributed to C-H bending. The characteristic peaks that appeared at 1130 and 1093 cm −1 were assigned to C-O and C-C of the glucopyranose ring in Cs. Ech was used as a crosslinking agent to modify the chemical structure of the amino acetic acid modified PVC-Cs (MPVC-Cs) to MPC-Cs/POH. This was followed by a further chemical modification for this polymeric conjugate using salicylic acid in absence and presence of the Ech and their structures were also confirmed by IR spectroscopy and represented in  Figure 2a showed the IR spectrum of MPVC-Cs/POH. It showed a band at 3438-3100 cm −1 which was assigned to the (OH) of propyl-2-hydroxyl moiety (crosslinker). Two bands at 2924 and 2850 cm −1 were assigned to the aliphatic (CH and CH 2 ). The amide carbonyl group (C=O) showed a characteristic band at 1631 cm −1 whereas the two bands at 1428 and 1318 cm −1 are attributed to C-H bending. There are also two bands appeared at 1258 and 1049 cm −1 which may be due to the C-O and C-C of the glucopyranose rings of Cs. Figure 2b represented the IR spectrum of (MPVC-Cs/SA) conjugate. It showed the presence of broad band at 3438-3100 cm −1 that was assigned to the OH and NH functional groups of the polymeric conjugate. There are two other peaks appeared at 2925 and 2850 cm −1 which are corresponding to the aliphatic (CH and CH 2 ). The amide (C=O) groups IR peak appeared at 1631 cm −1 while the two bands at 1428 and 1320 cm −1 are related to the C-H bending. The aromatic (C=C) is characterized by the observed absorption bands at 1410 and 1590 cm −1 whereas the bands appeared at 1100 and 1090 cm −1 are, respectively, due to the C-O and C-C of the glucopyranose ring of Cs. The chemical structure of the polymeric conjugate (MPVC-Cs-SA/POH) is also confirmed by the IR spectrum and is represented by Fig. 2c. The two observed IR peaks that appeared at 3439-3100 cm −1 are assigned to the OH and NH groups, respectively. The aliphatic (CH and CH 2 ) groups are characterized by the bands at 2925 and 2851 cm −1 . The carbonyl (C=O) of the amide group showed an IR peak at 1644 cm −1 while the two bands appeared at 1428 and 1320 cm −1 are correlated to the C-H bending. The C-O and C-C of the glucopyranose ring of Cs are assigned by the two bands present at 1100 and 1090 cm −1 .

Thermogravimetric analysis
Thermal stability of the prepared PVC conjugates was investigated using thermal gravimetric analysis (TGA) presented in Fig. 3. Data of blank PVC was also given for comparison. In Fig. 3, the TGA thermograms demonstrate the thermal degradation of both blank PVC and its prepared conjugates with onset temperature and at different decomposition temperatures. The thermogram shows that Thermal gravimetric analysis of blank PVC and its prepared conjugates the blank PVC starts to decompose at about 180 °C due to the elimination of hydrogen chloride molecules that consequently followed by the formation of conjugated polyenes [29]. A drastic decomposition for PVC occurred until 225 °C with a weight loss of 40%. At elevated temperature, the weight loss of blank PVC reached 70% at 350 °C and this was followed by low rate of degradation with percentages of weight loss 80 and 90% at 450 and 550 °C, respectively. For amino acetic acid modified PVC (MPVC), it was observed that the rate of thermal degradation was slightly affected by the incorporation of the amino acid molecules as a covalently bonded constituent to the backbone chains of the polymer. The partial replacement of the labile chlorine atoms by the amino acid has led to thermal stability for the modified polymer. This stability could be related to the decrease in the rate of liberation HCl from the MPVC chains. Gradual decomposition was noticed for MPVC that starts at 195 °C with 6% weight loss to reach 39% at 250 °C. A lower rate of decomposition occurred until 350 °C with weight loss of 58% that reached 65 and 81% at 450 and 550 °C, respectively. A significant increase in thermal stability of MPVC-Cs was distinct after the introduction of Cs to MPVC chains. This was designated by the elevation of the initial decomposition temperature (IDT), which was nearly at 280 °C. The rate of degradation increased until 350 °C with 38% weight loss, whereas at 450 and 550 °C the weight loss was observed to reach 50 and 69%. The weight loss of MPVC conjugate may be attributed to the depolymerization process of Cs subunits to form low molecular weight fragments [30]. Further chemical modification was performed for MPVC-Cs via using Ech as a crosslinking agent to obtain MPVC-Cs/POH. The crosslinking process occurred to the MPVC-Cs conjugate has led to a relative degree of higher thermal stability, which observed in the form of higher IDT and lower percentages of weight loss at different decomposition temperatures. Thermal degradation of this crosslinked conjugate starts at 320 and at 350 °C while the weight loss was reduced by 20%. The recorded weight loss percentages for this conjugate at 450 and 550 °C was 39 and 65%, respectively. This higher thermal stability may be due to the crosslinking created between the polymeric chains [31]. Another form of chemical modification was carried out by the one-pot reaction of MPVC-Cs with salicylic acid in the absence and presence of Ech as a crosslinking agent. The MPVC-Cs/SA conjugate shows a good thermal stability with respect to the above-mentioned ones. This conjugate exhibits a high IDT (375 °C) and after that temperature, it suffers from thermal degradation until 400 °C with a 30% loss in weight. Further thermal decomposition occurred for the investigated sample with loss in weight of 34 and 48% at 450 and 550 °C, respectively. The crosslinked conjugate, MPVC-Cs/POH/SA showed a slight increase in its IDT, which records 385 °C. The investigated sample lost only 15% of its weight at the decomposition temperature of 400 °C while the weight loss of this conjugate reached 30 and 43% at 450 and 550 °C, respectively. This relative thermal stability of these two conjugates may be due to the formation of something like a network, since the Ech and the modified PVC are present on both sides of the Cs units that may lead to a kind of significant thermal stability.

Scanning electron microscopy (SEM) of functionalized MPVC and its conjugates
The surface morphology of blank PVC, amino acetic acid modified PVC (MPVC), MPVC-Cs, MPVC-Cs/POH and salicylic acid functionalized MPVC in absence and presence of Ech (MPVC-Cs/SA and MPVC-Cs/POH/SA) was examined using SEM and their micrographs are shown in Fig. 4a-f. The SEM image of blank PVC shows a smooth surface with the presence of some holes (Fig. 4a). Figure 4b represents the surface morphology of amino acetic acid modified PVC (MPVC) sample. It shows the rough nature of the surface with the presence of some holes that confirms the chemical reaction of PVC with the amino acetic acid [32]. Figure 4c shows that the surface seems to have a fibrous nature due to the Cs inclusion to the modified PVC matrix while some grooves has appeared on the polymeric surface. Crosslinking of MPVC-Cs with Ech led to some type of aggregation with enlargement to pores between these aggregates (Fig. 4d). The surface appearance of Fig. 4e is characterized by a significant change upon the reaction of MPVC-Cs with salicylic acid with obvious presence of some holes on the surface. Nevertheless, the SEM micrograph of the same conjugate in the presence of Ech shows more roughness surface appearance with enlarged pores and holes due to the displacement between the polymeric chains effected by Ech crosslinking (Fig. 4f).

Antimicrobial activity
Antibacterial activity of all samples under investigation were examined against St. aurous and List. monocytogenes as Gram +ve bacteria plus E. coli and Salm. as gram −ve bacteria in addition to the C. albicans as yeast. Ampicillin and Vancomycin were used as antimicrobial reference drugs while the obtained results are listed in Tables 1, 2. The antimicrobial activity of the investigated prepared MPVC conjugates were used at concentrations of 75 mg/mL and 100 mg/mL. The antibacterial effects were evaluated by measuring the inhibition zone diameter (mm/mg sample) where the large inhibition zone diameter designates a strong antibacterial efficiency of the examined samples. Table 1 shows the antimicrobial activity of PVC, MPVC and its four prepared conjugates. Regarding the antibacterial activity of the amino acetic acid modified PVC (MPVC), it is clearly observed that its inhibitory effect against the tested microorganisms was enhanced by the introduction of the amino acetic acid in the main backbone chains of PVC. MPVC exhibited higher antibacterial inhibitory effect when compared to the blank PVC sample. It shows a good antibacterial efficiency against all tested microorganisms with respect to the two standard antimicrobial drugs. The highest efficiency reached 93% against the two Gram +ve bacterial strains St. aurous and List. monocytogenes with respect to the used reference drugs Ampicillin and Vancomycin, respectively, while it recorded 133 and 80% against C. albicans. The antibacterial inhibitory effect of this MPVC exhibited lower values against the two Gram −ve bacteria but still better than the blank PVC. Incorporation of Cs as natural polymer with its well-known antimicrobial properties [33,34] into the modified PVC led to enhancement of the antibacterial activity of MPVC-Cs conjugate against both Gram +ve and Gram −ve bacterial strains when compared with the reference drugs. The observed significant increase of the inhibitory effect of the MPVC-Cs with respect to either the nonmodified PVC or the MPVC may also be due to the presence of the amide functional group that formed in the PVC chains and the Cs units with their known high antimicrobial activity [35]. On the other hand, the prepared crosslinked modified PVC conjugate, MPVC-Cs/POH revealed lower antibacterial inhibition effect against the two Gram +ve and Gram −ve bacteria but it showed a significant increase in its antifungal activity and this may be due to the presence of Ech that has antifungal activity as stated in previous work [32]. It is well known that either salicylic acid or salicylate derivatives are characterized by their high antimicrobial activities [36,37]. So, the introduction of salicylic acid into the modified PVC was expected to enhance the antimicrobial efficiency of the materials under investigation. Regarding the inhibition effect of the MPVC conjugate containing salicylic acid (MPVC-Cs/ SA), it is clearly noticed that it showed higher antibacterial efficiency against all bacterial strains that in some cases exceeds that of the standard reference antibacterial agents. Similar results were obtained on evaluation of antibacterial activity of the crosslinked MPVC-Cs/POH/SA that shows lower efficiency against the two Gram +ve and Gram −ve bacterial strains and higher antifungal activity. Table 2 illustrates the antimicrobial activity of the all above-mentioned samples with a concentration of 100 mg/mL. The results show the highly significant increase of the antimicrobial activity of the modified PVC (MPVC) and its four prepared conjugates on comparison with the blank PVC using two standard reference drugs. The highest antibacterial efficiency was recorded for MPVC-Cs/SA to reach 143% and 147% against the Gram +ve bacteria, St. aurous and L. monocytogenes, respectively, while this efficiency reached 133% against E. coli. The investigated MPVC conjugates shows also a high antifungal activity especially, in case of the presence of Ech as a crosslinking agent. It could be concluded that the chemical modification of PVC enhanced its antimicrobial properties with. The inclusion of both Cs as a natural polymer and salicylic acid into the modified PVC increased the antimicrobial activity of the conjugates. On the other hand, the crosslinking process of the conjugates using Ech resulted in a relative decrease in their antibacterial activity whereas the antifungal activity was increased. This may be attributed to the antibacterial activity of both Cs and salicylic acid as established before. The crosslinking process of the MPVC-Cs by Ech that led to a slight decrease of the antibacterial activity may be due to the decrease in the number of NH 2 group of Cs which was involved in chemical modifications. Also, the antibacterial efficiency of the conjugate MPVC-Cs/SA was affected by the Ech crosslinking process which may be due to the decrease of both Cs and salicylic acid contents in the conjugate molecules leading to a diminished antibacterial activity. It is also noticed that the presence of Ech in the conjugate molecule shows a high antifungal activity. Figures 5a, b give further illustrations for the attained antibacterial efficiency percentages for the prepared modified PVC-Cs and its conjugates in comparison with the two references antibacterial drugs.

Minimum inhibitory concentration
Minimum Inhibitory Concentration (MIC) of the investigated prepared modified PVC-Cs conjugates was evaluated after utilizing various concentrations of each prepared polymeric material and the least concentration gave inhibition effect against the growth of pathogenic microbes after overnight incubation was reported. MIC results give additional evidence for the high efficiency of the modified PVC conjugates ( Table 3). The results shows that the blank PVC needs concentrations ranges from 106 to 247 mg/mL against the different tested microbes. The results also clearly exhibit that all modified polymeric conjugates have good MIC values and the MPVC-Cs/SA conjugate exhibited the lowest concentrations against the growth of the tested microorganisms and these values lie between 50 and 70 mg/mL for the different prepared conjugates.

Conclusions
Amino acetic acid modified poly(vinyl chloride), MPVC, was prepared via chemical reaction of PVC using glycine methyl ester which was then hydrolyzed to obtain the amino acetic acid modified-PVC, MPVC. Functionalization of MPVC was performed by its reaction with Cs in the absence and presence of Ech (POH) as a crosslinking agent to obtain MPVC-Cs and MPVC-Cs/POH. MPVC was also reacted with Cs, salicylic acid in absence and in presence of Ech via one-pot reaction to obtain further chemically functionalized MPVC, MPVC-Cs/SA and MPVC-Cs/POH/SA, respectively. FTIR spectroscopic analysis confirmed the performance of the chemical modification processes and the inclusion of the different modifiers into the backbone chains of PVC. Thermogravimetric analysis indicates that the modified PVC and its prepared conjugates gained more thermal stability upon chemical modifications with respect to the nonmodified PVC. SEM micrographs of   functionalized MPVC have shown the changes occurred to the surface morphology upon each chemical modification due to the incorporation of the applied modifiers into the backbone chains of PVC as polymeric substrate. All modified PVC samples exhibited antibacterial activity against both Gram +ve and Gram −ve bacteria as well as the applied yeast with different efficiencies according to the incorporated modifier. The inclusion of Cs into the MPVC chains enhanced the antimicrobial activity with respect to the blank PVC and MPVC itself. Further modification using salicylic acid with Cs showed a high significant increase in the antibacterial activity of the conjugate. Ech exhibited a high antifungal activity for the crosslinked samples. All conjugates showed a higher antimicrobial inhibitory effect on applying the 100 mg/mL. PVC-Cs conjugates containing salicylic acid were the most potent of all tested samples as indicated by their lowest MIC. This finding demonstrates that these conjugates could be regarded as good polymeric material candidates that can be used in the manufacturing of biomedical devices.