Conferring materials with antibacterial properties 2

CHAPTER ANTIBACTERIAL AND ADSORPTION CHARACTERISTICS OF ACTIVATED CARBON FUNCTIONALIZED WITH QUATERNARY AMMONIUM MOIETIES 70 Chapter 4.1 Introduction Activated carbon (AC) has been widely used in air pollution control and wastewater treatment to remove various pollutants because of its large surface area and high adsorption capacity (Bansal and Goyal, 2005; Quinlivan et al., 2005). It has diverse applications ranging from filters in gas masks and big ventilation systems (Soares et al., 1995) to water treatment systems of hospital renal haemodialysis care units (Morin, 2000). It has been reported that bacteria attached to carbon particles are highly resistant to disinfection processes due to biofilm formation, which causes the carbon itself to become a source of bacterial contamination (Morin, 2000; Stewart et al., 1990). Hence, it would be advantageous if the AC also possesses antibacterial activity to kill air- or water-borne bacteria. The preparation of antibacterial ACs has been attempted and much effort has been devoted to impregnation with silver or metal oxides (Oya et al., 1993; Park and Jang, 2003; Tamai et al., 2001; Wang et al., 1998; Zhang et al., 2004) in the AC. Though silver and metal oxides have attractive antibacterial activities, their primary shortcoming is that the particles are easily washed out since they are just deposited on the surface of the AC (Wang et al., 1998; Zhang et al., 2004). Furthermore, with increasing silver content the specific surface area of the carbon decreases greatly, resulting in reduced adsorption capability (Wang et al., 1998). A number of quaternary ammonium compounds are known to exhibit good bactericidal properties (Kenawy et al., 1998; Thorsteinsson et al., 2003). At the same time, we and other groups have reported that antibacterial properties can be confered on surfaces of substrates by the covalent attachment of quaternary ammonium with resultant bactericidal activities similar to that of free quaternary ammoniums (Cen et al., 2003; Gottenbos et al., 2002; Hu et al., 2005; Tiller et al., 2001). In this approach, 71 Chapter the antibacterial agents will not leach out from the surface, hence providing long term effectiveness. In this part of the thesis, we describe how two types of quaternary ammonium compounds can be covalently attached to the surface of the AC. The surface chemical compositions of the modified ACs were analyzed by XPS and the characteristics and morphologies of the AC were investigated using surface area and pore size analysis, and SEM. The antibacterial properties of the functionalized AC against Gram-negative E. coli and Gram-positive Staphylococcus aureus (S. aureus) were evaluated. Since the surface functionalization process employed entails changing the nature of the surface of the AC, the issue of whether this results in compromising the adsorption capacity of the AC needs to be addressed. As such, phenol adsorption by the AC before and after the functionalization process was also assessed. 72 Chapter 4.2 Experimental Materials and reagents A granular AC (20-40 mesh) purchased from Aldrich was used as the starting material. 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride (QAS) was obtained from Dow Corning. Poly(4-vinyl pyridine) (PVP) (Mw of 160,000 g/mol), hexylbromide, phenol and all other chemicals and solvents were from Aldrich. Preparation of functionalized ACs The AC was thoroughly washed with water until the washing liquid attained a constant pH of 6.2. It was then dried at 120 oC for 12h before being subjected to the functionalization process. An overall scheme of the functionalization process is shown in Figure 4.1. In a typical experiment, 5.0 g of AC was added to concentrated HNO3 (69 wt% HNO3) at a ratio of g/10 ml to achieve oxidation of its surface. The mixture was stirred for 10 h under reflux. The oxidized carbon was washed with water until no further change in pH could be detected and then dried under vacuum for 12 h at 60 oC , resulting in 4.4 g of oxidized AC, AC-COOH. In the next step, the surface carboxylic acid groups were allowed to react with thionyl chloride to generate acid chloride groups. In a typical experiment, g of dry AC-COOH was dispersed in 20 ml of thionyl chloride and the mixture was stirred at room temperature for h. The solid was then separated by filtration and washed with anhydrous CHCl3. Subsequently it was dried under vacuum at room temperature for h, obtaining AC-COCl (2.8 g). Two types of quaternary ammonium compounds, QAS and poly(vinyl-Nhexylpyridinium bromide), respectively were used to functionalize the as-prepared carbon surface. For the attachment of QAS, g of dry AC-COCl were allowed to react 73 Chapter with 20 ml glycol in the presence of triethylamine. The suspension was stirred at room temperature for 10 h, and the resulting solid was separated and washed with dry THF and CH2Cl2. After repeated washings, the solid was dried overnight under vacuum, obtaining AC-OH (1.8g). Subsequently 1.5 g of AC-OH was reacted with 40 ml of 10 wt% QAS in water at 80 oC for 24 h. QAS possesses a silyl group, which can be covalently bound to AC-OH surface (Gottenbos et al., 2002). The resulting solid was washed with water and dried in vacuum. This functionalized AC will be denoted as Q-AC in the subsequent discussion. For the binding of hexyl-PVP, g of dry AC-COCl and g of 3-bromopropylamine hydrobromide were reacted in 20 ml dry CH2Cl2 in the presence of triethylamine. The resulting solid (AC-Br) was washed and dried in the same manner as mentioned above. Subsequently, 1.5 g of dry AC-Br was placed in a solution of g of PVP in 60 ml of nitromethane/hexylbromide (10:1, v/v). The reaction mixture was stirred at 75 oC for 24 h, and the resulting solid was washed with acetone and methanol followed by drying under vacuum. Under the conditions used, only a few pyridine groups of the PVP chain are alkylated by the Br of 3-bromopropylamine, with the majority being alkylated by hexylbromide (Tiller et al., 2002). This functionalized AC will be denoted as P-AC in the subsequent discussion. 74 Chapter SOCl2 HNO3 COCl COOH reflux OHCH2CH2OH AC H2NCH2CH2CH2Br O CH3O CH3O Si HN-C3H6-Br OH O CH3 + CH2-CH2-CH2-N hexyl-PVP derivatization C18H37 CH3 CH3O O Si - Br CH3 OR CH2-CH2-CH2-N RO HN-C3H6 + N + C18H37 CH3 N + - C6H13 Br Q-AC P-AC RO R = H or Si RO CH3 + CH2-CH2-CH2-N C18H37 CH3 Figure 4.1 Schematic representation of the two routes for the functionalization of AC with quaternary ammonium groups. Materials and surface characterizations FTIR and XPS analysis was carried out as described in Section 3.1.2. The surface morphology of the carbons was visualized using a FE-SEM (JEOL, JSM-6700F). The specific surface area (SBET) was determined using a NOVA 3000 BET Analyzer (Quanta-Chrome) according to the Brunauer-Emmett-Teller (BET) method. Before the adsorption isotherms were obtained, the activated carbon samples were purged with pure nitrogen gas overnight at a temperature of 80 °C to remove any contaminant and moisture that may have been present in the samples. The pore volumes (V) were obtained from the volumes of nitrogen adsorbed at a relative pressure (p/p0, where p0 is 75 Chapter the saturation pressure) of 0.99. The pore size distribution was obtained by applying the density-functional-theory (DFT) method. The amount of N+ immobilized on the ACs was determined by the modified dye interaction method as described in Section 3.1.2. The long-term stability of the quaternary groups on Q-AC and P-AC was assessed using the water abrasion test. 1g of the functionalized AC was stirred in 30 ml of water at 300 rpm and at room temperature. After specific time intervals, the AC was filtered. About 0.1g of the AC was removed each time, dried overnight under vacuum and the N+ concentration was determined as described above. The water abrasion experiment was continued using the remaining AC under the same conditions. Determination of antibacterial activity of functionalized ACs E. coli and S. aureus were cultivated as described in Section 3.1.2 and assays of antibacterial activities were carried out as described in Section 3.2.2. Antibacterial efficacy in repeated applications was investigated using the same 100 mg of functionalized ACs in consecutive antibacterial assays against E. coli. After one batch, the particles were recovered using a filter. The particles were shaken in water for 30 and then used for the next batch of assay. Adsorption of phenol in aqueous solution Experiments for the determination of the adsorption isotherms of phenol were carried out as follows. A known mass of the AC was mixed with aqueous solutions of phenol of different initial concentrations without adding any chemical to control the pH. The 76 Chapter suspensions were shaken at 30 oC for days and the phenol solution was then filtered (Nevskaia et al., 2004). Preliminary kinetic experiments indicated that adsorption equilibrium was reached in less than days for the ACs. The concentration of phenol was analyzed using a UV-visible spectrophotometer (UV-1601, Shimadzu, Japan) at a wavelength of 270 nm. In order to reduce measurement errors, the UV absorption intensity of each equilibrium solution sample was measured three times and the average value was used to calculate the equilibrium concentration based on a standard calibration curve. The adsorbed quantity was determined as the difference between the initial amount of phenol in the solution and the remaining amount after equilibration. The amount adsorbed at equilibrium, qe (mg/g), was calculated using the following equation: qe = (C − Ce)V W where C0 and Ce are the initial and equilibrium concentrations of phenol (mg/L), respectively, V is the volume of the solution and W is the mass of the carbon, qe (mg/g) is the amount adsorbed at equilibrium concentration Ce (mg/L). The adsorption equilibrium data were fitted to the Langmuir equation: qe = q max bCe + bCe where qmax is the maximum adsorption capacity and b is the Langmuir constant. qmax can be obtained by linear regression of (Ce/qe) against Ce. 77 Chapter 4.3 Results and Discussion Functionalization of AC The first step in the functionalization process involves the treatment of the AC with concentrated HNO3 to prepare the AC-COOH sample. The effects of this treatment can be seen from the FTIR results in Figure 4.2. A significant increase in the absorbance in the range of 3200-3600 cm-1 indicates an increase in hydroxy groups, which may be due to surface carboxylic groups. The appearance of a sharp absorption band at 1380 cm-1 indicates an abundance of carboxyl-carbonate structures (Moreno-Castilla et al., 1995; Biniak et al., 1997; Shim et al., 2001; Tamai et al., 2006). These features clearly show that the oxidation of the AC by HNO3 generates a large number of carboxyl groups. The carboxyl groups were then activated by thionyl chloride for the subsequent reaction to attach the quaternary ammonium groups. Transmittance (%) (a) (b) 1380 cm 1000 1500 -1 2000 2500 3000 3500 -1 Wavenumber (cm ) Figure 4.2 FTIR spectra of (a) AC and (b) AC-COOH. The success of the covalent attachment of the two types of quaternary ammonium compounds on AC can be ascertained by comparing the XPS spectra before and after 78 Chapter the functionalization. Figure 4.3(a-c) shows the XPS wide scan of AC, Q-AC and P-AC, respectively. The corresponding N 1s core-level spectra of these ACs are shown in Figure 4.3(d-f). The N 1s peak component is not discernible in the wide scan and core-level spectra of AC (Figure 4.3(a) and (d)). In the case of Q-AC (Figure 4.3(b)) the appearance of a strong N 1s signal at a binding energy of 400 eV, Si 2p signal at 100 eV and Cl 2p signal at 200 eV is consistent with the presence of the QAS (structure given in Figure 4.1). The N 1s core level spectrum of Q-AC (Figure 4.3(e)) has a predominant peak at 401.7 eV attributable to the positively charged nitrogen (N+). In the case of P-AC, the wide scan indicates the presence of N and Br (at 70 eV). However, the wide scan does not offer conclusive evidence for the presence of the desired poly(vinyl-N-pyridinium bromide) since the reaction of AC-COCl with 3-bromopropylamine would also result in the presence of N and Br on the surface of the AC (see Figure 4.1). This issue was further clarified from the N 1s and Br 3d core-level spectra. The N 1s core-level spectrum (Figure 4.3(f)) can be deconvoluted into a predominant N+ peak at 401.7 eV and an additional peak at a binding energy of 398.5 eV due to =NH- of the pyridine groups which were not quaternized. As the probing depth of the XPS technique is < 10nm, the peak attributed to -NH- of 3-bromopropylamine is not readily discernible due to coverage by the PVP. The degree of N-alkylation of the pyridine groups on the P-AC, as calculated from the [N+]/[N] ratio, is 85%, ie 15% of N introduced on the AC by PVP was not quaternized during reaction with hexylbromide. In the Br 3d core-level spectrum (Figure not shown), the presence of a doublet (Br 3d 5/2 and Br 3d3/2) at 67.5 and 68.6 eV attributable to Br- 79 Chapter GENios). The results were expressed as percentages relative to the results obtained with the non-toxic control. The differences in the cytotoxicity results obtained from the pristine and functionalized SS substrates were analyzed statistically using the two sample t-test. The differences observed between samples were considered significant for P < 0.05. 99 Chapter 5.3 Results and Discussion Layer-by-layer surface functionalization The changes in the water contact angle of the functionalized surface during the course of the layer-by-layer deposition of PAA and q-PEI are shown in Figure 5.1. The contact angles reported are calculated from the mean value of three substrates and for each substrate, measurements were carried out at three or more surface locations. The pristine SS has a contact angle of 50º. The contact angle of the adsorbed q-PEI layer was determined to be 70º and upon formation of the PAA/q-PEI bilayer, the contact angle decreased to 30º. As shown in Figure 5.1, the fluctuations of contact angle of the multilayers follow the q-PEI and PAA adsorption cycles closely. This obvious alternating trend in the contact angle data verified the progressive buildup of the multilayers. Similar results were obtained from the PAA/q-PEI-Ag multilayers, with the angle of the q-PEI-Ag layer being 66º. Contact Angle (degrees) 70 60 50 40 30 20 10 11 12 Number of Layers Figure 5.1 Variation in water contact angle of PAA/q-PEI multilayers as a function of the number of layers deposited on stainless steel. Even numbers represent films with PAA as the outermost layer whereas odd numbers denote q-PEI as the outermost layer. 100 Chapter XPS analysis was used to investigate the chemical nature of the functionalized surface. The XPS wide-scan spectrum of the pristine SS (Figure 5.2(a)) shows the presence of carbon and oxygen and iron. In Figure 5.2(b), the presence of N (400 eV) and Ag (370 eV) peaks in the wide scan of PAA/q-PEI-Ag can be clearly seen indicating that silver has been successfully complexed with q-PEI. The peaks at 368.0 eV(3d5/2) and 374.0 eV (3d3/2) in the Ag 3d core-level spectrum (Figure 5.2(c)) attributable to the Ag0 species show that Ag+ ions from the solution have been reduced to the metallic state. (a) C 1s O 1s Fe 2p Intensity (arb. units) (b) Ag 3d N 1s 200 400 600 (c) Ag 3d5/2 364 368 372 Ag 3d3/2 376 380 Binding Energy (eV) Figure 5.2 XPS wide scan spectrum (a) of pristine stainless steel, wide scan (b) and Ag 3d core-level spectra (c) of stainless steel after bilayers of PAA/q-PEI-Ag deposition. The presence of silver on the PEM is also confirmed by SEM-EDX (JEOL JSM 6700F) analysis. The micrographs indicate a uniform distribution of silver nanoparticles with a 101 Chapter mean diameter of 16 nm on the functionalized SS surface (Figure 5.3). Figure 5.3 Scanning electron micrographs of the PAA/q-PEI-Ag The surface composition of the SS before and after functionalization, as determined from XPS analysis, is shown in Table 4.1. The N/C ratio of the PAA/q-PEI calculated from the sensitivity factor corrected spectral area ratio is 0.11, which is lower than the N/C ratio of PEI due to the contribution of the hexylbromide and PAA to the C signal. The N+/N ratio is 0.42 and 0.36 in the PAA/q-PEI and PAA/q-PEI-Ag functionalized substrates respectively, indicating that about 40% of the nitrogen have been quaternized in the hexylbromide alkylation process. The Ag/N mole ratio on the surface of the PAA/q-PEI-Ag functionalized SS is about 0.1. Table 4.1 Surface composition of stainless steel before and after functionalization as determined from XPS analysis N/C Ag/N N+/N - - PAA/q-PEI 0.11 - 0.42 PAA/q-PEI-Ag 0.08 0.11 0.36 Substrate Stainless steel 102 Chapter Antibacterial characterization The antibacterial efficacy of the functionalized SS was investigated by a comparison of the number of viable cells in the E. coli or S. aureus suspension after being in contact with the different substrates. From Figure 5.4(a), it can be seen that with the pristine SS, the number of viable cells in the suspension decreased by < 10% after h. This relatively minor reduction may have resulted from natural death. With the PAA/q-PEI functionalized SS, the viable cell number decreased by about one order of magnitude after h. This reduction in the number of viable cells is significantly slower than that observed in our earlier work on cloth functionalized with N-hexyl pyridine groups where 99.9% of the E. coli cells were killed after h (Cen et al., 2004). Although the mechanism of the antibacterial activity of immobilized quaternary ammonium groups is not entirely clear, it has been hypothesized that these immobilized moieties disrupt the cytoplasmic membrane integrity to cause cell death, similar to the mechanism of free biocides (Tiller et al., 2001). Thus the q-PEI would be expected to have similar antibacterial property as the N-alkyl pyridine groups (Lin et al., 2002). However, the limited mobility of q-PEI chains confined in the PEM matrix of the present work, as compared to the 4-vinyl pyridine chains directly graft-copolymerized on the surface of the cloth in our earlier work, may reduce its biocidal activity. Moreover, due to the higher surface area of the latter, the surface concentration of the biocidal moieties can also be expected to be higher. 103 Chapter Log (viable cell number/ml) Pristine SS PAAc/q-PEI functionalized SS PAAc/q-PEI-Ag functionalized SS (a) Contact Time (h) Log (viable cell number/ml) Pristine SS PAAc/q-PEI functionalized SS PAAc/q-PEI-Ag functionalized SS (b) Contact Time (h) Figure 5.4 Number of viable E. coli (a) and S. aureus (b) cells in PBS at 37oC as a function of time in contact with the different substrates. The cell number was determined by surface-spread method. 104 Chapter With the presence of silver in the PEM, the antibacterial efficacy is significantly enhanced (Figure 5.4(a)). After h, the viable cell number decreased by two orders of magnitude and viable cells decreased to 30 cells/ml after h in contact with the PAA/q-PEI-Ag functionalized SS, ie 99.97% of the cells are no longer viable. The present work confirms the antibacterial activity of the silver nanoparticles reported earlier against Gram-negative E. coli (Jeon et al., 2003; Sondi and Salopek-Sondi, 2004). While the mechanism of the interaction between the silver nanoparticles and the E. coli is still unresolved, it has been shown for the contact of silver nanoparticles with E. coli results in the formation of “pits” in the cell membrane leading to increase in permeability and death of the cell (Sondi and Salopek-Sondi, 2004). Comparing the results in Figure 5.4(b) with those in Figure 5.4(a), both PAA/q-PEI and PAA/q-PEI-Ag PEM are even more efficient against the Gram-positive S. aureus. With the PAA/q-PEI functionalized SS, 96.8% of the S. aureus cells are no longer viable after h, while with the PAA/q-PEI-Ag functionalized SS, 99.99% of the cells are not viable after the same period. This result is consistent with a recent report that immobilized quaternary ammonium compounds are less bactericidal against Gram-negative bacteria such as E. coli than staphylococci (Gottenbos et al., 2002). This difference in antibacterial efficacy is postulated to be the result of the different cell membrane structures between the E. coli and S. aureus bacteria. The multilayered cell envelope structure of Gram-negative bacteria would be more resistant to access by the bactericidal moieties to the inner membrane of the organism. Bacterial viability assay on surfaces If the surface of the implanted materials is bacteriostatic, a single surviving borderline 105 Chapter resistant bacterium may be able to recolonize the surface leading to implant infections (Schierholz et al., 1998). Hence, it is necessary to elucidate whether bacterial cells which adhered on the substrate for a period of time would remain viable when released into a fresh medium. To confirm that the bacteria were indeed killed and not just bound to the surface, the substrates after immersion in a PBS suspension of 107 cells/ml for h were transferred to a 3.1% yeast broth and then cultured at 37 °C for a period of time under constant shaking (100 rpm). During this time, the adherent cells which were not killed by the antibacterial surface would start to multiply and seed out daughter cells. The bacterial growth was monitored by measuring the absorbance of the medium at 600 nm. The results obtained using S. aureus are shown in Figure 5.5. From this Figure, it can be seen that the optical density of the medium with the pristine SS, increases rapidly with culturing time, reaching a value of about 0.8 after 36 h. For the PAA/q-PEI and PAA/q-PEI-Ag functionalized substrates, the optical densities of the medium remain at for the first 12 h. For the former, the optical density shows a slow rate of increase after 12 h, but for the latter, the optical density remains at even after 36 h. Similar results were obtained for E. coli. These results indicate that there was no significant number of viable cells adherent on the PAA/q-PEI-Ag functionalized substrate after its transfer from the bacterial suspension. 106 Chapter Pristine SS PAAc/q-PEI functionalized SS PAAc/q-PEI-Ag functionalized SS OD600 0.8 0.4 0.0 12 24 36 Time (h) Figure 5.5 Optical densities (at 600 nm) of broth containing S. aureus after different periods in contact with the substrates, which were first immersed in an S. aureus suspension of 107 cells/ml for 2h before transferring to the broth. The adhesion of viable and dead bacteria on the surface of the substrates was further observed via staining of the substrates after immersion in the bacterial suspension of 108 cells/ml for h at 37 oC. Figure 5.6 shows the fluorescence microscopy images of the various substrates viewed under green and red filters. These results were obtained using S. aureus. The presence of many viable cells (stained green) can be seen on the pristine SS surface (Figure 5.6(a)), while at the same time, there were hardly any dead cells (image under red filter not shown). For the PAA/q-PEI functionalized substrate, 107 Chapter (a) (b) 100 μm (c) (d) Figure 5.6 Fluorescence microscopy images of pristine stainless steel under green filter (a), PAA/q-PEI functionalized stainless steel under red filter (b) and green filter (c), PAA/q-PEI-Ag functionalized stainless steel under red filter (d), after immersion in a PBS suspension of 108 cells/ml of S. aureus for h. there were both viable and dead cells as shown in Figure 5.6(b) and Figure 5.6(c), although the number of viable cells is significantly less than that of the dead cells. For the PAA/q-PEI-Ag functionalized substrate, there are very few cells and all of them are stained red (Figure 5.6(d)). The total number of adherent cells on this surface is very much less than that shown in Figure 5.6(a) and no cells can be seen under the green filter (image not shown). The experiments using E. coli gave similar results. It should be mentioned that the PAA/q-PEI and PAA/q-PEI-Ag functionalized surfaces are more 108 Chapter hydrophobic than the pristine SS surface (Figure 5.1), and it has been reported that surface hydrophobicity would enhance bacteria attachment (Reid et al., 1993). However, the results in Figure 5.6 show a clear decrease in the number of adherent cells on the functionalized substrates. This may indicate that the dead cells are easily detached during rinsing of the substrates with water after their removal from the bacterial suspension. From the above results, it can be concluded that the bacterial cells adhere and grow on the pristine SS surface and upon release from the substrate into the medium, these cells continue to reproduce. While the quaternary ammonium groups on the PAA/q-PEI functionalized surface cannot inhibit bacteria growth completely, with the added silver nanoparticles on the PAA/q-PEI-Ag surface, cell adhesion decreases dramatically and no viable cells can be seen. Cytotoxicity assay Since the ultimate objective of our work is to confer antibacterial properties to implanted materials, the issue of cytotoxicity of the functionalized surface and its degradation products has to be addressed. The results of the MTT cytotoxicity assay using 3T3 mouse fibroblasts are shown in Figure 5.7. These cells were selected for this assay as they are substrate-dependent, nonspecific cell lines (Armitage et al., 2003). Figure 5.7 shows that there is no statistical difference in cytotoxicity among the pristine SS, PAA/q-PEI and PAA/q-PEI-Ag functionalized SS (P > 0.05). It should also be noted that the results obtained with these pristine and functionalized SS substrates are not significantly different from that of the non-toxic control (growth culture medium). It has been reported earlier that depending on the PEI and the compounds used for modifying the PEI, certain derivatives of PEI are even less cytotoxic than the 109 Chapter parent PEI (Thomas and Klibanov, 2002). Since the results in the present work indicate that the materials themselves not induce cytotoxic effects in the 3T3 mouse fibroblast system, this functionalization technique shows good promise for further development for biomedical applications. Cell Viability, % of Non-toxic Control 120 100 80 60 40 20 h wt o Gr m iu d e m e tin s i Pr EI -P q A/ PA g 00 -1 I-A X E n -P /q ito r A T PA Figure 5.7 Cytotoxicity of the pristine and functionalized stainless steel substrates relative to the non-toxic control (growth culture medium). Triton X-100 serves as the toxic control. Stability of polyelectrolyte multilayer film Most conventional antibacterial agents act by diffusing into the cell and disrupting the essential cell functions and when this type of compound is used for surface treatment, the antibacterial agents must be released from the surface matrix. As highlighted in Section 2.2, the fact that the antibacterial agents are free to leave the surface implies that the functionalized material would only have a limited period of effectiveness. 110 Chapter Another associated potentially serious problem is that the compound released into the environment may increase drug resistance throughout the microbial realm. In the present work, the stability of the quaternary ammonium groups and the entrapped silver nanoparticles in the PEM matrix was assessed. Contact angle measurement of the PAA/q-PEI functionalized SS after 21 days immersion in PBS at 37 °C showed that the PEM remained stable. Moreover, the N+/N and N/C XPS elemental ratios of the “aged” substrate are within 96% and 93%, respectively, of the original values. The antibacterial efficacy of this “aged” PAA/q-PEI functionalized SS is also not significantly different from that of the freshly prepared sample (No significant difference in OD value can be observed before and after aging). On the other hand, there is a loss of silver from the PEM matrix of the PAA/q-PEI-Ag functionalized substrates during immersion in PBS, as shown in Figure 5.8(a). The amount of silver initially entrapped in the PEM was about 25 μg/cm2. As can be seen from Figure 5.8(a), the amount of silver decreases steadily with immersion time and after 21 days of immersion in PBS, only 30% of the initial amount remains in the PEM matrix. The loss of the silver nanoparticles is expected to decrease the antibacterial efficacy of the surface. This is confirmed by Figure 5.9(a), which shows that the “aged” PAA/q-PEI-Ag functionalized substrate with only 30% of the initial silver content can no longer kill all the S. aureus cells on its surface and the surviving cells will reproduce when released into the culture medium. In contrast, there was no increase in optical density of the medium even after 36 h when the substrate was freshly functionalized (Figure 5.5). It should be noted (from a comparison of Figure 5.9(a) and Figure 5.5) that this “aged” PAA/q-PEI-Ag functionalized substrates with 30% of its original silver is still more efficient in killing the bacteria than the 111 Chapter PAA/q-PEI functionalized substrates. 100 80 Ag Released, % (a) 60 40 20 (b) 0 10 15 20 25 Time (days) Figure 5.8 Release of Ag from (a) PAA/q-PEI-Ag functionalized stainless steel and (b) PAA/q-PEI-Ag functionalized stainless steel after heating at 120 oC for h in vacuum. The substrates were immersed in PBS at 37oC with stirring at 100 rpm. In order to inhibit the loss of silver nanoparticles from the PEM matrix, the substrates after functionalization with PAA/q-PEI-Ag were heated to 120 °C for h in vacuum. During the heating process, amide bond formation between the functional groups of PEI and PAA would occur (Mamedov et al., 2002), which complements the intrinsic ionic crosslinking of the PEM. With this thermal treatment, less than 8% of the silver in the PAA/q-PEI-Ag functionalized substrate is lost after 21 days immersion in PBS (Figure 5.8(b)). This thermally treated PAA/q-PEI-Ag after 21 days immersion in PBS retains a high antibacterial efficacy, similar to that of the freshly prepared substrate. (comparing Figure 5.9(b) with Figure 5.5). No increase in the optical density of the medium can be detected after 36 h. 112 Chapter OD600 0.8 Pristine SS 0.4 (a) 0.0 (b) 12 24 36 Time (h) Figure 5.9 Optical densities (at 600 nm) of broth containing S. aureus after different periods in contact with “aged” PAA/q-PEI-Ag functionalized substrates: (a) aging carried out by immersing the substrate in PBS for 21 days at 37 oC, (b) aging was carried out as in (a) but after the substrate has been heated at 120 oC for h in vacuum. The “aged” substrates were first immersed in an S. aureus suspension of 107 cells/ml for 2h before transferring to the broth. The results obtained with pristine stainless steel are represented here for comparison. 113 Chapter 5.4 Conclusions Polyelectrolyte multilayers built up by the alternate deposition of q-PEI or q-PEI-Ag complex and PAA were used to functionalize stainless steel substrates. The successful build-up of the PEM was confirmed by static contact angle measurements and XPS analysis. Effective inhibition of E. coli and S. aureus growth on the surface of functionalized films was achieved using the PAA/q-PEI-Ag functionalized SS while partial inhibition of bacterial growth was achieved with the PAA/q-PEI functionalized SS. The MTT assay showed that there is no significant difference in cytotoxicity between the pristine and PEM functionalized SS, and the non-toxic control. The PAA/q-PEI matrix was stable even after 21 days in PBS and the loss of silver nanoparticles from the PAA/q-PEI-Ag matrix can be prevented by heating the functionalized substrates at 120 oC for h in the vacuum, which results in amide bond formation in the PEM. These results from the present work show that the layer-by-layer method for constructing a matrix containing quaternary ammonium groups and silver nanoparticles provides an attractive means for imparting antibacterial properties to SS for potential biomedical applications. 114 [...]... of implanted materials is to kill the bacteria during their initial attachment rather than trying to remove them once they have adhered This may be achieved by surface functionalization with quaternary ammonium groups (Tiller et al., 20 01; Gottenbos et al., 20 02; Cen et al., 20 03), silver (Gray et al., 20 03; Jeon et al., 20 03; Ho et al., 20 04) and chitosan (Huh et al., 20 01; Mi et al., 20 02) In the present... (μmol/g) a 0 32 18 SBET (m2/g) b 574 3 52 527 V (cm3/g) 0.51 0 .24 0.43 qmax (mg/g) 71 46 65 As determined by fluorescein staining BET surface area The antibacterial efficacy of Q-AC and P-AC in repeated applications against E coli was also investigated The results for P-AC in Figure 4.6 show a slight decrease in efficacy after 10 repeats of antibacterial experiments Similar results were obtained with Q-AC... found that pyridine groups N-alkylated with C6 showed the highest killing efficacy for S aureus and E coli, followed by those with C3 and C4 chains while the C8-C16 chains are significantly less effective (Lin et al., 20 02; Tiller et al., 20 01) However, it has been reported that for soluble quaternary ammonium compounds such as QAS, the antibacterial effects increase with the length of the alkyl moieties... aqueous solution (2 mg/ml) of PAA for 15 min and followed by rinsing with water for 1 min and drying with nitrogen Multilayers were obtained by repeating this cycle Five bilayers with the exception of the first PEI layer were built up For PAA/q-PEI-Ag multilayer film, the substrates were dipped in an aqueous solution containing the q-PEI (2 mg/ml) and AgNO3 (20 0 ppm) (pH adjusted to 7.0 with 0.1 M HNO3)... higher antibacterial activity against S aureus as compared to E coli For Q-AC, no cells remain viable after 3 h while for P-AC no cells remain viable after 2 h This result is consistent with a recent report by Tiller et al (20 02) that an immobilized quaternary ammonium compound is less bactericidal against Gram-negative bacteria such as E coli than Gram-positive S aureus This difference in antibacterial. .. recover its antibacterial ability on being washed with ethanol However, if the quaternary ammonium is in the range of 880-1170 μmol/g, its antibacterial activity can only be recovered upon treatment of the functionalized glass with alkaline solutions due to the stronger electrostatic interaction with the dead cells (Nakagawa et al., 1984) In this work, the N+ concentration of Q-AC and P-AC is 32 and 18... capacity 0.030 AC Q-AC P-AC 0. 020 3 dV/dD (cm /nm/g) 0. 025 0.015 0.010 0.005 0.000 0 1 2 3 4 5 6 Pore size (nm) Figure 4.8 Pore size distributions of AC, Q-AC and P-AC 89 Chapter 4 4.4 Conclusions Two different surface functionalization routes for attaching quaternary ammonium groups to AC to achieve antibacterial properties were demonstrated The AC was first treated with HNO3 and then activated by... would be more resistant to access by the bactericidal moieties to the inner 82 Chapter 4 Log (viable cell number/ml) 8 (a) 6 4 2 0 0 1 2 3 Contact Time (h) Log (viable cell number/ml) 8 (b) 6 4 2 0 0 1 2 3 Contact Time (h) Figure 4.5 Viable E coli cell number (a) and viable S aureus cell number (b) as a function of time in contact with the different substrates: Control (■), AC (●), Q-AC ( ), and P-AC (▲)... for 20 min, rinsed thoroughly with water and blown dry with purified nitrogen Quaternized PEI was synthesized according to the method reported in the literature (Johnson and Klotz, 1974; Noding and Heitz, 1998; Thomas and Klibanov, 20 02) PEI, obtained as a 50% aqueous solution, was lyophilized before being subjected to the chemical reactions A mixture of 1 g of PEI and excess (10ml) hexylbromide in 20 ... Q-AC (b) and P-AC (c) 81 Chapter 4 Antibacterial effect of functionalized ACs The antibacterial activities of ACs before and after functionalization were investigated by contacting the ACs with bacterial cells in suspension In each antibacterial experiment, 100 mg of ACs was incubated with 30 ml of the bacterial suspension The results of the experiments conducted with suspensions containing 107 cells/ml . functionalization of AC with quaternary ammonium groups. COOH HNO 3 SOCl 2 COCl OHCH 2 CH 2 OH O OH CH 3 O Si CH 2 -CH 2 -CH 2 -N + C 18 H 37 CH 3 CH 3 CH 3 O CH 3 O O Si OR RO CH 2 -CH 2 -CH 2 -N + C 18 H 37 CH 3 CH 3 Si CH 2 -CH 2 -CH 2 -N + C 18 H 37 CH 3 CH 3 RO RO H 2 NCH 2 CH 2 CH 2 Br HN-C 3 H 6 -Br C 6 H 13 O HN-C 3 H 6 Br - Br - N + N + reflux R. COOH HNO 3 SOCl 2 COCl OHCH 2 CH 2 OH O OH CH 3 O Si CH 2 -CH 2 -CH 2 -N + C 18 H 37 CH 3 CH 3 CH 3 O CH 3 O O Si OR RO CH 2 -CH 2 -CH 2 -N + C 18 H 37 CH 3 CH 3 Si CH 2 -CH 2 -CH 2 -N + C 18 H 37 CH 3 CH 3 RO RO H 2 NCH 2 CH 2 CH 2 Br HN-C 3 H 6 -Br C 6 H 13 O HN-C 3 H 6 Br - Br - N + N + reflux R. quaternary ammonium with resultant bactericidal activities similar to that of free quaternary ammoniums (Cen et al., 20 03; Gottenbos et al., 20 02; Hu et al., 20 05; Tiller et al., 20 01). In this