On the other hand, the presence of water as a nonsolvent in the solutions used for casting films influenced the AFM images presented in Figure 7; a higher water content decreased

Fig. 6. 2D and 3D AFM images with 2 60 x 60 mμ scanned areas of the PSF-DMOA films obtained from DMF/MeOH solution: (a, a') 100/0; (b, b') 75/25; (c, c') 50/50; (d, d') 45/55

of pores Area Depth Diameter Length Mean

Pore characteristics Surface roughness

width Sa rms nhh

Solvent mixtures

Number

to the 2D AFM images (Ioan et al., 2011)

the thermodynamic quality of the DMF/water solvent mixtures.

solvent mixtures, respectively. According to the AFM images, increasing the nonsolvent content in the casting solutions favores modification of surface morphology.

Fig. 4. 2D and 3D AFM images with 2 20x20 mμ scanned areas of the PSF-DMEA films obtained from DMF/MeOH solutions: (a, a') - 100/0; (b, b') - 75/25; (c, c') – 50/50; (d, d') – 25/75

Thus, Figure 4 and Table 6 show that average surface roughness attains a maximum value at 50/50 DMF/MeOH, and favores the appearance of the smallest number of pores with highest depth values. Also, the area, diameter, length and mean width increase with increasing the nonsolvent content. It should be noted that the thermodynamic quality of the solvent mixtures over the studied domain increases with the addition of nonsolvent, at approximately 50/50 DMF/MeOH becoming constant, while the preferential adsorption of nonsolvent takes a maximum value, according to literature data (Filimon et al., 2010). In addition, the presence of water as a nonsolvent in the solutions used for casting films influenced the AFM images presented in Figure 5; a higher water content decreases the thermodynamic quality of the DMF/water solvent mixtures so that, at 50/50 DMF/water, a minimum value of surface roughness and a maximum number of pores with minimum values of area, depth, diameter, length and mean width, are observed.

Fig. 5. 2D and 3D AFM images with 2 20x20 mμ scanned areas of the PSF-DMEA films obtained from DMF/water solutions: (a, a') - 75/25; (b, b') – 50/50; (c, c') – 40/60

Figure 6 plot also two- and three-dimensional structures of PSF–DMOA films prepared with DMF/MeOH solvent mixtures of various compositions (100/0, 75/25, 50/50, and 45/55). According to these images, increasing the nonsolvent content in the casting solutions favores modification of the surface morphology. Thus, the average surface roughness decreased with increasing MeOH content and favores increases in the pore number and pore characteristics (area, depth, diameter, and mean width) according to Table 6. The thermodynamic quality of solvent mixtures over the studied domain increases with the addition of the nonsolvent (Filimon et al. 2010; Ioan et al., 2011a).

solvent mixtures, respectively. According to the AFM images, increasing the nonsolvent

Fig. 4. 2D and 3D AFM images with 2 20x20 mμ scanned areas of the PSF-DMEA films obtained from DMF/MeOH solutions: (a, a') - 100/0; (b, b') - 75/25; (c, c') – 50/50; (d, d') –

values of area, depth, diameter, length and mean width, are observed.

addition of the nonsolvent (Filimon et al. 2010; Ioan et al., 2011a).

Fig. 5. 2D and 3D AFM images with 2 20x20 mμ scanned areas of the PSF-DMEA films obtained from DMF/water solutions: (a, a') - 75/25; (b, b') – 50/50; (c, c') – 40/60

Figure 6 plot also two- and three-dimensional structures of PSF–DMOA films prepared with DMF/MeOH solvent mixtures of various compositions (100/0, 75/25, 50/50, and 45/55). According to these images, increasing the nonsolvent content in the casting solutions favores modification of the surface morphology. Thus, the average surface roughness decreased with increasing MeOH content and favores increases in the pore number and pore characteristics (area, depth, diameter, and mean width) according to Table 6. The thermodynamic quality of solvent mixtures over the studied domain increases with the

Thus, Figure 4 and Table 6 show that average surface roughness attains a maximum value at 50/50 DMF/MeOH, and favores the appearance of the smallest number of pores with highest depth values. Also, the area, diameter, length and mean width increase with increasing the nonsolvent content. It should be noted that the thermodynamic quality of the solvent mixtures over the studied domain increases with the addition of nonsolvent, at approximately 50/50 DMF/MeOH becoming constant, while the preferential adsorption of nonsolvent takes a maximum value, according to literature data (Filimon et al., 2010). In addition, the presence of water as a nonsolvent in the solutions used for casting films influenced the AFM images presented in Figure 5; a higher water content decreases the thermodynamic quality of the DMF/water solvent mixtures so that, at 50/50 DMF/water, a minimum value of surface roughness and a maximum number of pores with minimum

25/75

content in the casting solutions favores modification of surface morphology.

Table 6. Pore characteristics, including number of pores, area ( 2 μm ), depth (nm), diameter ( μm ), length ( μm ), and mean width ( μm ), and surface roughness paramaters, including average roughness (Sa, nm), root mean square roughness (rms, nm), and nodule average height from the histogram (nhh, nm) of PSF-DMEA and PSF-DMOA films prepared from solutions in DMF/MeOH and DMF/water, with 2 20 x 20 mμ scanned areas, corresponding to the 2D AFM images (Ioan et al., 2011)

On the other hand, the presence of water as a nonsolvent in the solutions used for casting films influenced the AFM images presented in Figure 7; a higher water content decreased the thermodynamic quality of the DMF/water solvent mixtures.

Fig. 6. 2D and 3D AFM images with 2 60 x 60 mμ scanned areas of the PSF-DMOA films obtained from DMF/MeOH solution: (a, a') 100/0; (b, b') 75/25; (c, c') 50/50; (d, d') 45/55

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 259

Pore characteristics (including area, volume, depth, and diameter) and surface roughness parameters (including root-mean-square roughness (rms), and nodules height from the height profile (nhp) from profile analysis, and nodules average height, from the histogram (nhh)), are presented in Table 7. The dimensions of pores increase and their depth decreases with increasing the substitution degrees, so that, for the PS-DOPO-3 sample, the dimensions

On the other hand, AFM studies support the conclusion that the increasing density of bulky phosphorus pendant groups from the side chain and decreases roughness and, implicitly, confirm poor adhesion of the modified polysulfone films. Also, the bulky phosphorus pendant groups, found in position 1\* for samples PS-DOPO-1 and PS-DOPO-2, and in positions 1\* and 2\* for sample PS-DOPO-3, modify the rigidity and hydrophobicity, and determine different forms of entanglement in polymer solutions, influencing membrane

Surface tension parameters of quaternized polysulfones are calculated by the geometric mean method (GM) (equation (1)) (Kälble et al., 1969; Owens et al., 1969) and the acid/base method (LW/AB) (equation (2)) (van Oss et al, 1988a; van Oss et al, 1988b). In this context, the surface tension data of the doubly-distilled water (W), ethylene glycol (EG), glycerol (G) and formamide (FA) used in the calculations of the surface tension parameters of PSFQ are

p

where θ is the contact angle determined for test liquids, subscripts "lv" and "sv" denote the interfacial tension between liquid-vapor and surface-vapor, respectively, while superscripts "p" and "d" denote the polar and disperse components, respectively, of total surface

sv sv sv 2 + − γ = ⋅ γ ⋅ γ , superscript "LW/AB" indicates the total surface tension, and also,

superscript "AB" and "LW" represent the polar component obtained from the electron-

Literature (Della Volpe et al., 2004) shows that an improper utilization of three liquids without dispersive liquids, or with liquids prevalently basic or prevalently acidic, strongly increases the ill-conditioning of the system. In addition, the contact angles should be measured with a liquid whose surface tension is higher than the anticipated solid surface

; d p sv sv sv γ = γ + γ (1)

; LW/AB LW AB

+γ , interactions, and the disperse component,

sv sv sv γ = γ + γ (2)

lv p lv d sv d sv d lv lv

( ) LW LW sv lv sv lv sv lv

2 1 cos +− −+ + θ= ⋅ γ ⋅ γ + γ ⋅ γ + γ ⋅ γ

+ θγ γ ⋅ = γ ⋅ + γ γ γ

exceed the limit of the AFM apparatus.

morphology and AFM images.

presented in Table 8.

tension, sv γ .

where AB

donor, sv

respectively.

tension (Kwok et al., 2000).

**4. Surface tension parameters**

1 cos 2

lv

−γ , and the electron-acceptor, sv

γ

Therefore, for a 60/40 DMF/water mixture, the average surface roughness, number of pores, and pore depth are maximum with a minimum area (Table 6). It can be assumed that the specific interactions with the mixed solvents employed in this study modify the PSF– DMOA solubility and determine the modification of the solution properties.

Fig. 7. 2D and 3D AFM images with 2 20 x 20 mμ scanned areas of the PSF-DMOA films obtained from DMF/water solution: (a, a') 75/25; (b, b') 60/40; (c, c') 50/50

In the same context, from AFM images (Figure 8) one can see that the bulky phosphorus pendant groups in PSF-DOPO determine the formation of domains with pores in an approximately continuous matrix (Ioan et al., 2011b).

Fig. 8. 2D and 3D-AFM images for PS-DOPO-1 (a, a') and PS-DOPO-2 (b, b')

\* From scanning electron microscopy, according to previous data (Petreus *et al*., 2010)

Table 7. Pore characteristics including area ( μ μ m x m ), volume ( μ μ m x m x nm ), depth ( μm ), and diameter ( μm ), and surface roughness parameters including root-mean-square roughness (rms, nm), and nodules height from the height profile (nhp, μm ) and nodules average height from the histogram (nhh, μm ) of polysulfone with bulky phosphorus pendant groups membranes obtained from AFM investigations

Pore characteristics (including area, volume, depth, and diameter) and surface roughness parameters (including root-mean-square roughness (rms), and nodules height from the height profile (nhp) from profile analysis, and nodules average height, from the histogram (nhh)), are presented in Table 7. The dimensions of pores increase and their depth decreases with increasing the substitution degrees, so that, for the PS-DOPO-3 sample, the dimensions exceed the limit of the AFM apparatus.

On the other hand, AFM studies support the conclusion that the increasing density of bulky phosphorus pendant groups from the side chain and decreases roughness and, implicitly, confirm poor adhesion of the modified polysulfone films. Also, the bulky phosphorus pendant groups, found in position 1\* for samples PS-DOPO-1 and PS-DOPO-2, and in positions 1\* and 2\* for sample PS-DOPO-3, modify the rigidity and hydrophobicity, and determine different forms of entanglement in polymer solutions, influencing membrane morphology and AFM images.

#### **4. Surface tension parameters**

258 A Search for Antibacterial Agents

Therefore, for a 60/40 DMF/water mixture, the average surface roughness, number of pores, and pore depth are maximum with a minimum area (Table 6). It can be assumed that the specific interactions with the mixed solvents employed in this study modify the PSF–

Fig. 7. 2D and 3D AFM images with 2 20 x 20 mμ scanned areas of the PSF-DMOA films

In the same context, from AFM images (Figure 8) one can see that the bulky phosphorus pendant groups in PSF-DOPO determine the formation of domains with pores in an

Pore characteristics Surface roughness

6.00\* 343.11 0.70 1.30

Area Volume Depth Diameter rms nhp nhh

PS-DOPO-1 23.25 14.39 1.51 5.02 363.16 0.45 1.40

Table 7. Pore characteristics including area ( μ μ m x m ), volume ( μ μ m x m x nm ), depth ( μm ), and diameter ( μm ), and surface roughness parameters including root-mean-square roughness (rms, nm), and nodules height from the height profile (nhp, μm ) and nodules average height from the histogram (nhh, μm ) of polysulfone with bulky phosphorus

obtained from DMF/water solution: (a, a') 75/25; (b, b') 60/40; (c, c') 50/50

Fig. 8. 2D and 3D-AFM images for PS-DOPO-1 (a, a') and PS-DOPO-2 (b, b')

\* From scanning electron microscopy, according to previous data (Petreus *et al*., 2010)

PS-DOPO-2 28.96 15.19 0.95 7.61

PS-DOPO-3 25.00\*

pendant groups membranes obtained from AFM investigations

approximately continuous matrix (Ioan et al., 2011b).

Samples

DMOA solubility and determine the modification of the solution properties.

Surface tension parameters of quaternized polysulfones are calculated by the geometric mean method (GM) (equation (1)) (Kälble et al., 1969; Owens et al., 1969) and the acid/base method (LW/AB) (equation (2)) (van Oss et al, 1988a; van Oss et al, 1988b). In this context, the surface tension data of the doubly-distilled water (W), ethylene glycol (EG), glycerol (G) and formamide (FA) used in the calculations of the surface tension parameters of PSFQ are presented in Table 8.

$$\frac{1+\cos\Theta}{2} \cdot \frac{\gamma\_{\rm lv}}{\sqrt{\gamma\_{\rm lv}^{\rm d}}} = \sqrt{\gamma\_{\rm sv}^{\rm p}} \cdot \sqrt{\frac{\gamma\_{\rm lv}^{\rm p}}{\gamma\_{\rm lv}^{\rm d}}} + \sqrt{\gamma\_{\rm sv}^{\rm d}} \; ; \; \gamma\_{\rm sv} = \gamma\_{\rm sv}^{\rm d} + \gamma\_{\rm sv}^{\rm p} \tag{1}$$

where θ is the contact angle determined for test liquids, subscripts "lv" and "sv" denote the interfacial tension between liquid-vapor and surface-vapor, respectively, while superscripts "p" and "d" denote the polar and disperse components, respectively, of total surface tension, sv γ .

$$\mathbf{1} + \cos \theta = \frac{2}{\gamma\_{\rm lv}} \cdot \left( \sqrt{\gamma\_{\rm sv}^{\rm LIV} \cdot \gamma\_{\rm lv}^{\rm LIV}} + \sqrt{\gamma\_{\rm sv}^{+} \cdot \gamma\_{\rm lv}^{-}} + \sqrt{\gamma\_{\rm sv}^{-} \cdot \gamma\_{\rm lv}^{+}} \right); \ \gamma\_{\rm sv}^{\rm LIV/AB} = \gamma\_{\rm sv}^{\rm LIV} + \gamma\_{\rm sv}^{\rm AB} \tag{2}$$

where AB sv sv sv 2 + − γ = ⋅ γ ⋅ γ , superscript "LW/AB" indicates the total surface tension, and also, superscript "AB" and "LW" represent the polar component obtained from the electrondonor, sv −γ , and the electron-acceptor, sv +γ , interactions, and the disperse component, respectively.

Literature (Della Volpe et al., 2004) shows that an improper utilization of three liquids without dispersive liquids, or with liquids prevalently basic or prevalently acidic, strongly increases the ill-conditioning of the system. In addition, the contact angles should be measured with a liquid whose surface tension is higher than the anticipated solid surface tension (Kwok et al., 2000).

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 261

+γ . Thus, the functional groups -CH2Cl attached by chloromethylation process increase the polarity for chloromethylated polysulfones with a substitution degree DS>1; also, the N, Ndimethylethanolamine side groups introduced by the quaternization process increase the polarity. The change is moderate for chloromethylated polysulfone with DS>1, with

GM method LW/AB method

PSF 17.81 11.21 28.49 18.77 9.34 8.74 28.10 33.23 CMPSF1 16.13 12.32 28.45 21.27 7.46 11.43 28.73 25.97 CMPSF2 13.23 17.56 30.79 16.85 12.38 17.02 29.23 42.35 CMPSF3 12.66 19.43 32.09 15.79 14.14 17.56 29.93 47.23 PSFQ1 8.12 29.88 38.00 12.70 19.29 27.24 31.98 60.31 PSFQ2 8.04 31.11 39.15 8.15 42.56 29.98 43.71 97.38 PSFQ3 8.08 31.20 39.18 7.82 56.82 49.34 64.74 82.70 Table 10. Surface tension parameters (mN/m) for polysulfone (PSF) film , chloromethylated polysulfone films, (CMPSF1, CMPSF2 and CMPSF3) prepared from solutions in chloroform, and quaternized polysulfone films (PSFQ1, PSFQ2 and PSFQ3) prepared from solutions in

Moreower, the surface tension parameters of quaternized polysulfones PSF-DMEA, and PSF-DMOA, with surface properties of water, methylene iodide, 1-brom-naphtalin test liquids from Table 8, and the contact angles measured between these solvents and

Solvent mixtures Contact angle

Table 11. Contact angle (°) of different liquids on films prepared from solutions of PSF-

DMEA and PSF-DMOA in DMF/MeOH and DMF/water

PSF-DMEA 100/0 DMF/MeOH 71 28 17 75/25 DMF/MeOH 70 31 22 50/50 DMF/MeOH 61 30 20 25/75 DMF/MeOH 63 31 24 75/25 DMF/water 59 35 21 50/50 DMF/water 60 33 18 40/60 DMF/water 56 33 16 PSF-DMOA 100/0 DMF/MeOH 72 32 15 75/25 DMF/MeOH 78 32 16 50/50 DMF/MeOH 58 28 16 45/55 DMF/MeOH 77 35 20 75/25 DMF/water 66 34 21 60/40 DMF/water 65 36 25

quaternized polysulfone films from Table 11, are presented in Table 12.

sv γ AB

sv γ sv

−γ sv

W MI 1-Bn

+γ LW/AB sv γ

sv γ sv γ LW

−γ , smaller than the electron acceptor ones,

polar term, with the electron donor interactions, sv

d sv γ p

sv

methanol

different chloride content.

Samples

Table 9 shows the contact angle values between water, ethylene glycol, glycerol or formamide and PSF, CMPSF and PSFQ membranes. The surface tension of PSF evidences the lowest hydrophilicity, induced by the aromatic rings connected by one carbon and two methyl groups, oxygen elements, and sulfonic groups, while chloromethylation of PSF with the functional group -CH2Cl increases hydrophilicity (see the values of surface tension for PSF and CMPSF in Table 10). Moreover, the results indicate that the PSFQ membranes are the most hydrophilic ones from the studied samples (lowest water contact angle), due to the N,Ndimethylethanolamine hydrophilic side groups. Hence, it is observed that total surface tension, sv γ or LW/AB sv γ , and the polar component, p sv γ or AB sv γ , increase with the degree of substitution of CMPSF and with the quaternization degree of the ammonium groups for PSFQ samples.

Table 8. Surface tension parameters (mN/m) of the liquids used for contact angle measurements: total surface tension, lv γ , disperse component of surface tension, d lv γ , polar component of surface tension, p lv γ , electron-donor contribution on polar component, lv −γ , and electron-acceptor contribution on polar component, lv +γ

Table 9. Contact angle of different probe liquids (in °)

Therewith, the relative ratio of the polar component to the total surface tension ranges from approx. 39% for PSF and 43% for CMPSF1, with the substitution degree DS<1, to 57-61% for CMPSF, with the substitution degree DS>1, and to 79% for PSFQ. The apolar component, d sv γ , decreases from PSF to CMPSF and PSFQ. The total surface tensions of PSF and CMPSF1, where the substitution degree DS<1, are dominated by the apolar component, while the total surface tension of CMPSF2, CMPSF3, (DS>1) and PSFQ are dominated by the

Table 9 shows the contact angle values between water, ethylene glycol, glycerol or formamide and PSF, CMPSF and PSFQ membranes. The surface tension of PSF evidences the lowest hydrophilicity, induced by the aromatic rings connected by one carbon and two methyl groups, oxygen elements, and sulfonic groups, while chloromethylation of PSF with the functional group -CH2Cl increases hydrophilicity (see the values of surface tension for PSF and CMPSF in Table 10). Moreover, the results indicate that the PSFQ membranes are the most hydrophilic ones from the studied samples (lowest water contact angle), due to the N,Ndimethylethanolamine hydrophilic side groups.  Surface tension parameters (mN/m) of the liquids used for contact angle measurements: total surface tension, lv γ , disperse component of surface tension, d

Polymers W G FA EG

Therewith, the relative ratio of the polar component to the total surface tension ranges from approx. 39% for PSF and 43% for CMPSF1, with the substitution degree DS<1, to 57-61% for CMPSF, with the substitution degree DS>1, and to 79% for PSFQ. The apolar component,

sv γ , decreases from PSF to CMPSF and PSFQ. The total surface tensions of PSF and CMPSF1, where the substitution degree DS<1, are dominated by the apolar component, while the total surface tension of CMPSF2, CMPSF3, (DS>1) and PSFQ are dominated by the

PSF 79 70 65 60 CMPSF1 78 72 66 57 CMPSF2 73 69 65 54 CMPSF3 71 68 64 52 PSFQ1 64 63 64 51 PSFQ2 64 56 69 48 PSFQ3 32 28 32 29

of CMPSF and with the quaternization degree of the ammonium groups for PSFQ samples.

sv γ , increase with the degree of substitution

lv γ lv

−γ lv +γ

lv γ , polar

−γ ,

lv γ p

lv γ , electron-donor contribution on polar component, lv

+γ

sv γ or LW/AB

component of surface tension, p

d

sv γ , and the polar component, p

Test liquids lv γ d

and electron-acceptor contribution on polar component, lv

Solvents

Table 9. Contact angle of different probe liquids (in °)

polar term, with the electron donor interactions, sv −γ , smaller than the electron acceptor ones, sv +γ . Thus, the functional groups -CH2Cl attached by chloromethylation process increase the polarity for chloromethylated polysulfones with a substitution degree DS>1; also, the N, Ndimethylethanolamine side groups introduced by the quaternization process increase the polarity. The change is moderate for chloromethylated polysulfone with DS>1, with different chloride content.

Table 10. Surface tension parameters (mN/m) for polysulfone (PSF) film , chloromethylated polysulfone films, (CMPSF1, CMPSF2 and CMPSF3) prepared from solutions in chloroform, and quaternized polysulfone films (PSFQ1, PSFQ2 and PSFQ3) prepared from solutions in methanol

Moreower, the surface tension parameters of quaternized polysulfones PSF-DMEA, and PSF-DMOA, with surface properties of water, methylene iodide, 1-brom-naphtalin test liquids from Table 8, and the contact angles measured between these solvents and quaternized polysulfone films from Table 11, are presented in Table 12.

Table 11. Contact angle (°) of different liquids on films prepared from solutions of PSF-DMEA and PSF-DMOA in DMF/MeOH and DMF/water

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 263

The results reflect the capacity of the N-dimethylethylammonium chloride or Ndimethyloctylammonium chloride pendant groups to determine the acceptor or donor

In the same context, the substitution degrees of polysulfones with bulky phosphorus pendant groups enface the specific properties. Presence of substituted groups in position 1\* for samples PS-DOPO-1 and PS-DOPO-2 and in positions 1\* and 2\* for sample PS-DOPO-3 influence *e.g*. the rheological properties, leading to modification of slopes as a function of viscosity *vs.* substitution degrees, concentration and temperature (Ioan et al., 2011b). This behavior is assigned to the modification of cohesive energy and molar volume for the studied samples. The Arrhenius equation evidenced an increased of activation energy and flow activation entropy, in the following order: PS-DOPO-1 < PS-DOPO-2 < PS-DOPO-3. This implies a higher energy barrier for the movement of an element of the fluid and a more rigid structure for the PS-DOPO3 polymer chain and, as well as, a strong variation with concentration of the activation energy and flow activation entropy. Thus, different forms of the entanglement in polymer solutions are generated by the specific molecular rearrangement of the polysulfone with bulky phosphorus pendant groups with different substitution degrees, at different concentrations and temperatures. Moreover, the viscoelastic characteristics showed that all samples exhibit the behavior characteristic of an elastic gel at high frequencies, when G' > G'' and where both moduli are dependent on frequency. These features are characteristic for a physical gel network, in which the gelation process of phosphorus-modified polysulfones is influenced by intramolecular interactions, depending on the substitution degrees and by intermolecular attractions that depend on

> Samples W F EG MI PS-DOPO-1 77 56 59 43 PS-DOPO-2 78 52 49 21 PS-DOPO-3 82 46 31 ≈ 0

> > Surface tension parameters

sv γ sv

+γ sv

−γ LW/AB sv γ

GM method LW/AB method

PS-DOPO-1 33.18 4.80 37.98 34.23 3.99 0.557 7.15 37.22 PS-DOPO-2 44.62 2.34 46.96 39.20 1.508 0.112 5.063 40.710 PS-DOPO-3 52.85 1.14 53.99 48.85 1.378 0.358 1.327 50.228 Table 14. Surface tension parameters (mN/m) for membranes of PS-DOPO with different

sv γ AB

Table 13. Contact angle of different probe liquids (water (W), formamide (F),

ethylene glycol (EG) and methylene iodide (MI)) in (°) with polysulfones with bulky

sv γ sv γ LW

+γ , for PSF-DMOA, caused by the inductive phenomena from alkyl radical.

−γ , are smaller than the electron acceptor

−γ , exceed the electron acceptor

Furthermore, the electron donor interactions, sv

+γ , for PSF-DMEA, and electron donor interactions, sv

character of the polar terms, generated by these inductive phenomena.

ones, sv

interactions, sv

solution concentration.

phosphorus pendant groups

Samples

d sv γ p

substitution degrees, prepared from solutions in DMF

These parameters are influenced by the solvent/nonsolvent composition from which the films are prepared (Albu et al., 2011; Ioan et al. 2011a). Some studies have reported that the chain shape of a polymer in solution could affect the morphology of the polymer in bulk. In this context, the conformations of both PSF-DMEA and PSF-DMOA are affected by the charged groups from different alkyl radicals of the studied quaternized samples, and also by the compositition of the solvent mixtures. For the PSF-DMEA in the DMF/MeOH system, one may observe that the polymer coil dimension decreases with increasing the DMF content in the 0.25–1 volume fraction domain; below a 0.25 volume fraction of DMF, the polymer precipitates. For the same polymer, but in a DMF/water solvent mixture, the dimensions increase with increasing the DMF content, starting from approximately the same volume fraction of DMF. The PSF-DMOA coil dimensions possess maximum values in DMF/MeOH and DMF/water, around 0.6 and 0.8 DMF volume fractions, respectively. For volume fractions of DMF below 0.25 in DMF/MeOH and 0.5 in DMF/water, the PSF-DMOA precipitates due to the nature of the alkyl radicals and content of nonsolvent in the system. Also, the values of intrinsic viscosity are higher for PSF-DMOA - with bulky carbon atoms in the alkyl side chain, compared with PSF-DMEA, where the alkyl side chain possesses two carbon atoms. Therefore, for a given composition of the DMF/MeOH and DMF/water solvent mixtures, one of the components is preferentially adsorbed by the quaternized polysulfone molecules in the direction of a thermodynamically most effective mixture (Filimon et al., 2010). These aspects influence the surface properties of the polymer. PSF-DMEA films possess low polar surface tension parameters, but slightly higher than those for PSF-DMOA. The hydrophobic character is given by the ethyl radical from the Ndimethylethylammonium chloride pendant group and by the octyl radical from the Ndimethyloctylammonium chloride pendant group, respectively.

Table 12. Surface tension parameters (mN/m) for quaternized polysulfone films PSF-DMEA and PSF-DMOA prepared from solutions in DMF/MeOH and DMF/water

These parameters are influenced by the solvent/nonsolvent composition from which the films are prepared (Albu et al., 2011; Ioan et al. 2011a). Some studies have reported that the chain shape of a polymer in solution could affect the morphology of the polymer in bulk. In this context, the conformations of both PSF-DMEA and PSF-DMOA are affected by the charged groups from different alkyl radicals of the studied quaternized samples, and also by the compositition of the solvent mixtures. For the PSF-DMEA in the DMF/MeOH system, one may observe that the polymer coil dimension decreases with increasing the DMF content in the 0.25–1 volume fraction domain; below a 0.25 volume fraction of DMF, the polymer precipitates. For the same polymer, but in a DMF/water solvent mixture, the dimensions increase with increasing the DMF content, starting from approximately the same volume fraction of DMF. The PSF-DMOA coil dimensions possess maximum values in DMF/MeOH and DMF/water, around 0.6 and 0.8 DMF volume fractions, respectively. For volume fractions of DMF below 0.25 in DMF/MeOH and 0.5 in DMF/water, the PSF-DMOA precipitates due to the nature of the alkyl radicals and content of nonsolvent in the system. Also, the values of intrinsic viscosity are higher for PSF-DMOA - with bulky carbon atoms in the alkyl side chain, compared with PSF-DMEA, where the alkyl side chain possesses two carbon atoms. Therefore, for a given composition of the DMF/MeOH and DMF/water solvent mixtures, one of the components is preferentially adsorbed by the quaternized polysulfone molecules in the direction of a thermodynamically most effective mixture (Filimon et al., 2010). These aspects influence the surface properties of the polymer. PSF-DMEA films possess low polar surface tension parameters, but slightly higher than those for PSF-DMOA.  Surface tension parameters (mN/m) for quaternized polysulfone films PSF-DMEA

sv γ sv

+γ sv

−γ AB

sv γ LW/AB sv γ

dimethyloctylammonium chloride pendant group, respectively.

sv γ sv γ LW

and PSF-DMOA prepared from solutions in DMF/MeOH and DMF/water

Solvent mixtures

d sv γ p Furthermore, the electron donor interactions, sv −γ , are smaller than the electron acceptor ones, sv +γ , for PSF-DMEA, and electron donor interactions, sv −γ , exceed the electron acceptor interactions, sv +γ , for PSF-DMOA, caused by the inductive phenomena from alkyl radical. The results reflect the capacity of the N-dimethylethylammonium chloride or Ndimethyloctylammonium chloride pendant groups to determine the acceptor or donor character of the polar terms, generated by these inductive phenomena.

In the same context, the substitution degrees of polysulfones with bulky phosphorus pendant groups enface the specific properties. Presence of substituted groups in position 1\* for samples PS-DOPO-1 and PS-DOPO-2 and in positions 1\* and 2\* for sample PS-DOPO-3 influence *e.g*. the rheological properties, leading to modification of slopes as a function of viscosity *vs.* substitution degrees, concentration and temperature (Ioan et al., 2011b). This behavior is assigned to the modification of cohesive energy and molar volume for the studied samples. The Arrhenius equation evidenced an increased of activation energy and flow activation entropy, in the following order: PS-DOPO-1 < PS-DOPO-2 < PS-DOPO-3. This implies a higher energy barrier for the movement of an element of the fluid and a more rigid structure for the PS-DOPO3 polymer chain and, as well as, a strong variation with concentration of the activation energy and flow activation entropy. Thus, different forms of the entanglement in polymer solutions are generated by the specific molecular rearrangement of the polysulfone with bulky phosphorus pendant groups with different substitution degrees, at different concentrations and temperatures. Moreover, the viscoelastic characteristics showed that all samples exhibit the behavior characteristic of an elastic gel at high frequencies, when G' > G'' and where both moduli are dependent on frequency. These features are characteristic for a physical gel network, in which the gelation process of phosphorus-modified polysulfones is influenced by intramolecular interactions, depending on the substitution degrees and by intermolecular attractions that depend on solution concentration.

Table 13. Contact angle of different probe liquids (water (W), formamide (F), ethylene glycol (EG) and methylene iodide (MI)) in (°) with polysulfones with bulky phosphorus pendant groups

Table 14. Surface tension parameters (mN/m) for membranes of PS-DOPO with different substitution degrees, prepared from solutions in DMF

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 265

these wettability depends on the surphace morphology of samples prepared from different composition of DMF/MeOH and DMF/water solvent mixtures (see Table 6). Also, a small difference appears between PS-DOPO samples with different substitution degrees. Thus, one can say that increasing the substitution degrees lead to a slight increase in

Fig. 9. Surface free energy *vs.* water contact angle for quaternized polysulfones with

Fig. 10. Surface free energy vs. water contact angle for polysulfones with bulky

adhesion, Wa , and the work of water cohesion, Wc (equation (7)).

The interfacial free energy, GM ΔGsws evaluated from solid-liquid interfacial tension, sl γ , has negative values (see Figure 11 - for PSFQ, Table 15 - for PSF-DMEA and PSF-DMOA, and Table 16 – for PSF-DOPO), indicating an attraction between the two polymer surfaces, s, immersed in water, w; hence, these materials are considered rather hydrophobic (Dourado

In addition, the hydrophobicity of these polymers is described by the work of spreading of water, Ws , over the surface, which represents the difference between the work of water

hydrophobicity.

N,N-dimethylethanolamine

phosphorus pendant groups

et al., 1998; van Oss & Giese, 1995).

On the other hand, according to Table 13 and, finally, Table 14, all phosphorus-modified polysulfones show low polar surface tension parameters, p sv γ or AB sv γ , which decrease with increasing the phosphorus pendant groups, and which manifest bring lower contributions to the total surface tension parameters. Furthermore, the electron donor interactions, sv −γ exceed the electro n acceptor interactions, sv +γ , considering electron-donor capacity of P=O group. These results show that the studied samples evidence high hydrophobicity, which increases by increasing the substitution degrees. The maximum hydrophobicity of the polysulfones with bulky phosphorus pendant groups would be advantageous, *e.g*., for dielectric performance, since a low water absorption causes a significant decrease in the dielectric constant and, implicitly, low adhesion to different interfaces.

#### **5. Surface and interfacial free energy of functionalized polysulfones**

The effect of different radicals of functionalized polysulfones and of the history of the films formed from solutions on surface properties are analyzed by surface free energy, ΔGw expressing the balance between surface hydrophobicity and hydrophilicity (equation (3)) (Faibish et al., 2002; Rankl et al., 2003), by interfacial free energy between two particles of functionalized polysulfones in water phase, GM ΔGsws (equations (4) and (5) or (6)), and by the work of spreading of water, Ws (equation (7)).

$$
\Delta \mathbf{G}\_{\rm w} = -\gamma\_{\rm lv} \cdot \left( 1 + \cos \theta\_{\rm water} \right) \tag{3}
$$

where lv γ is given in Tables 8 and water θ is give in Table 9, 11 and 13 for functionalized polysulfones with N,N-dimethylethanolamine (PSFQ), N,N-dimethylethylamine and N,Ndimethyloctylamine (PSF-DMEA and PSF-DMOA), and polysulfones with bulky phosphorus pendant groups (DOPO), respectively.

$$
\Delta \mathbf{G}\_{\rm sws}^{\rm GM} \text{ (or } \Delta \mathbf{G}\_{\rm sws}^{\rm LW/BAB} \text{ )}=-2 \cdot \gamma\_{\rm sl} \text{ (or } \gamma\_{\rm sl}^{\rm LW/AB} \text{)}\tag{4}
$$

$$\gamma\_{\rm s1} = \left(\sqrt{\gamma\_{\rm lv}^{\rm p}} - \sqrt{\gamma\_{\rm sv}^{\rm p}}\right)^2 + \left(\sqrt{\gamma\_{\rm lv}^{\rm d}} - \sqrt{\gamma\_{\rm sv}^{\rm d}}\right)^2 \tag{5}$$

$$\gamma\_{\rm sl}^{\rm LW/AB} = \left(\sqrt{\gamma\_{\rm sv}^{\rm d}} - \sqrt{\gamma\_{\rm lv}^{\rm d}}\right)^2 + 2 \cdot \left(\sqrt{\gamma\_{\rm sv}^{+} \cdot \gamma\_{\rm sv}^{-}} + \sqrt{\gamma\_{\rm lv}^{+} \cdot \gamma\_{\rm lv}^{-}} - \sqrt{\gamma\_{\rm sv}^{+} \cdot \gamma\_{\rm lv}^{-}} - \sqrt{\gamma\_{\rm sv}^{-} \cdot \gamma\_{\rm lv}^{+}}\right) \tag{6}$$

$$\mathbf{W\_s} = \mathbf{W\_a} - \mathbf{W\_c} = \mathbf{2} \cdot \left[ \left( \boldsymbol{\gamma}\_{\rm sv}^{\rm LIV} \cdot \boldsymbol{\gamma}\_{\rm lv}^{\rm d} \right)^{1/2} + \left( \boldsymbol{\gamma}\_{\rm sv}^{+} \cdot \boldsymbol{\gamma}\_{\rm lv}^{-} \right)^{1/2} + \left( \boldsymbol{\gamma}\_{\rm sv}^{-} \cdot \boldsymbol{\gamma}\_{\rm lv}^{+} \right)^{1/2} \right] - 2 \cdot \boldsymbol{\gamma}\_{\rm lv} \tag{7}$$

Generally, the literature (Faibish et al., 2002; van Oss, 1994) mentions that for 2 Δ <− G 113 mJ/m w , the polymer can be considered more hydrophilic while, when 2 Δ >− G 113 mJ/m w , it should be considered more hydrophobic. It is observed from Figure 9 that the surface free energy for PSF and CMPSF samples possesses low wettability . Moreover, Figures 9, 10 and Table 15 show that all functionalized polysulfones are characterized by small wettability, except for PSFQ3, where the hydrophylicity is higher. On the other hand, N,N-dimethyloctylamine groups determine a higher wettability as N,Ndimethylethylamine group (coresponding to PSF-DMOA and PSF-DMEA, respectively) and

On the other hand, according to Table 13 and, finally, Table 14, all phosphorus-modified

increasing the phosphorus pendant groups, and which manifest bring lower contributions to the total surface tension parameters. Furthermore, the electron donor interactions, sv

group. These results show that the studied samples evidence high hydrophobicity, which increases by increasing the substitution degrees. The maximum hydrophobicity of the polysulfones with bulky phosphorus pendant groups would be advantageous, *e.g*., for dielectric performance, since a low water absorption causes a significant decrease in the

The effect of different radicals of functionalized polysulfones and of the history of the films formed from solutions on surface properties are analyzed by surface free energy, ΔGw expressing the balance between surface hydrophobicity and hydrophilicity (equation (3)) (Faibish et al., 2002; Rankl et al., 2003), by interfacial free energy between two particles of functionalized polysulfones in water phase, GM ΔGsws (equations (4) and (5) or (6)), and by the

where lv γ is given in Tables 8 and water θ is give in Table 9, 11 and 13 for functionalized polysulfones with N,N-dimethylethanolamine (PSFQ), N,N-dimethylethylamine and N,Ndimethyloctylamine (PSF-DMEA and PSF-DMOA), and polysulfones with bulky

sv γ or AB

Δ =− G (1 cos ) w lv water γ ⋅+ θ (3)

(5)

GM LW/BAB LW/AB G (or G ) 2 (or ) sws sws sl sl Δ Δ =− ⋅γ γ (4)

( ) 2 2

p p d d sl lv sv lv sv γ= γ −γ + γ −γ

( ) ( ) 2

Generally, the literature (Faibish et al., 2002; van Oss, 1994) mentions that for 2 Δ <− G 113 mJ/m w , the polymer can be considered more hydrophilic while, when 2 Δ >− G 113 mJ/m w , it should be considered more hydrophobic. It is observed from Figure 9 that the surface free energy for PSF and CMPSF samples possesses low wettability . Moreover, Figures 9, 10 and Table 15 show that all functionalized polysulfones are characterized by small wettability, except for PSFQ3, where the hydrophylicity is higher. On the other hand, N,N-dimethyloctylamine groups determine a higher wettability as N,Ndimethylethylamine group (coresponding to PSF-DMOA and PSF-DMEA, respectively) and

sl sv lv sv sv lv lv sv lv sv lv 2 + − +− + − − + γ = γ − γ + ⋅ γ ⋅ γ + γ ⋅ γ − γ ⋅ γ − γ ⋅ γ (6)

LW d 1/2 1/2 1/2 W W W 2 [( ) ( ) ( ) ] 2 s a c sv lv sv lv sv lv lv +− −+ = − =⋅ γ ⋅ γ + γ ⋅ γ + γ ⋅ γ − ⋅ γ (7)

+γ , considering electron-donor capacity of P=O

sv γ , which decrease with

−γ

polysulfones show low polar surface tension parameters, p

dielectric constant and, implicitly, low adhesion to different interfaces.

**5. Surface and interfacial free energy of functionalized polysulfones**

exceed the electro n acceptor interactions, sv

work of spreading of water, Ws (equation (7)).

phosphorus pendant groups (DOPO), respectively.

LW/AB d d

these wettability depends on the surphace morphology of samples prepared from different composition of DMF/MeOH and DMF/water solvent mixtures (see Table 6). Also, a small difference appears between PS-DOPO samples with different substitution degrees. Thus, one can say that increasing the substitution degrees lead to a slight increase in hydrophobicity.

Fig. 9. Surface free energy *vs.* water contact angle for quaternized polysulfones with N,N-dimethylethanolamine

Fig. 10. Surface free energy vs. water contact angle for polysulfones with bulky phosphorus pendant groups

The interfacial free energy, GM ΔGsws evaluated from solid-liquid interfacial tension, sl γ , has negative values (see Figure 11 - for PSFQ, Table 15 - for PSF-DMEA and PSF-DMOA, and Table 16 – for PSF-DOPO), indicating an attraction between the two polymer surfaces, s, immersed in water, w; hence, these materials are considered rather hydrophobic (Dourado et al., 1998; van Oss & Giese, 1995).

In addition, the hydrophobicity of these polymers is described by the work of spreading of water, Ws , over the surface, which represents the difference between the work of water adhesion, Wa , and the work of water cohesion, Wc (equation (7)).

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 267

Fig. 12. Work of spreading of water, red blood cells and platelets over the surface of PSFQ1,

Fig. 13.  Work of spreading of water, of red blood cells and of platelets over the surface of

PSF-DOPO1, PSF-DOPO2 and PSF-DOPO3 films

DMEA and PSF-DMOA films prepared in DMF/MeOH and DMF/water

PSFQ2 and PSFQ3 films

Table 15. Water interfacial tensions and surface free energy for PSF-DMEA and PSF-DMOA films prepared in different DMF/MeOH and DMF/water, and interfacial free energy between two particles of quaternized polysulfones in water phase

Fig. 11. Water interfacial tensions and surface free energy for polysulfone, chloromethylated polysulfones, and quaternized polysulfones films prepared in DMF/MeOH and DMF/water

Table 16. Solid-water interfacial tensions, sl γ , and interfacial free energy, GM ΔGsws , from the geometrical mean method (GM) and solid-water interfacial tensions, LW/AB sl γ and interfacial free energy, LW/AB ΔGsws , from the acid/base method (LW/AB), and the work of spreading of water, Ws , over the polymer surface

PSF-DMEA

100/0 25.95 26.92 -67 -95.30 -51.90 -53.84 75/25 24.26 32.06 -68 -87.94 -48.52 -64.12 50/50 18.47 37.15 -75 -111.38 -36.94 -74.30 45/55 - 29.91 - -89.18 - -59.82 25/75 19.15 - -74 - -38.30 -

75/25 16.50 21.66 -77 -102.41 -32.30 -43.32 60/40 - 20.08 - -103.57 - -40.16 50/50 17.60 25.30 -76 -97.70 -35.20 -50.60 40/60 15.57 - -79 - -31.14 - Table 15. Water interfacial tensions and surface free energy for PSF-DMEA and PSF-DMOA films prepared in different DMF/MeOH and DMF/water, and interfacial free energy

Fig. 11. Water interfacial tensions and surface free energy for polysulfone, chloromethylated

PS-DOPO-1 25.69 21.84 -51.38 -43.69 -56.42 PS-DOPO-2 35.54 28.93 -71.08 -57.86 -61.03 PS-DOPO-3 43.65 40.09 -87.30 -80.17 -62.66 Table 16. Solid-water interfacial tensions, sl γ , and interfacial free energy, GM ΔGsws , from the

interfacial free energy, LW/AB ΔGsws , from the acid/base method (LW/AB), and the work of

sl γ GM ΔGsws LW/AB ΔGsws Ws

sl γ and

polysulfones, and quaternized polysulfones films prepared in DMF/MeOH and

geometrical mean method (GM) and solid-water interfacial tensions, LW/AB

Samples sl γ LW/AB

spreading of water, Ws , over the polymer surface

sl γ ΔGw GM ΔGsws DMF/MeOH

> PSF-DMOA

PSF-DMEA

PSF-DMOA

Solvent mixtures

DMF/water

PSF-DMEA

PSF-DMOA

DMF/water

between two particles of quaternized polysulfones in water phase

Fig. 12. Work of spreading of water, red blood cells and platelets over the surface of PSFQ1, PSFQ2 and PSFQ3 films

Fig. 13. Work of spreading of water, red blood cells and platelets over the surface of PSF-DMEA and PSF-DMOA films prepared in DMF/MeOH and DMF/water

Fig. 14. Work of spreading of water, of red blood cells and of platelets over the surface of PSF-DOPO1, PSF-DOPO2 and PSF-DOPO3 films

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 269

platelets - which have a significant role in thrombus formation (Reitsma et al., 2007). In this context, adhesion of the red blood cells and cohesion of platelets to surface films must be discussed in correlation with future specific biomedical applications. These results seem to be applicable for evaluating bacterial adhesion to the surfaces, and could be subsequently employed for studying possible implanted induced infections, or for obtaining

The literature shows that quaternary ammonium salts represent one of the most popular types of antimicrobial agents (Hazziza-Laskar et al., 2005; Xu et al., 2006; Yu et al., 2007; Merianos, 1991). Their biological activity, depending on their structure and physicochemical properties, affects the interaction with the cytoplasmic membrane of bacteria and

The antimicrobial activity of quaternized polysulfones with various ionic chlorine contents, in DMSO, at different concentrations, is investigated against *Escherichia coli* (*E. coli*) and *Staphylococcus aureus* (*S. aureus*). As shown in Figure 15 and Table 17, these polymers inhibit the growth of microorganisms, the inhibition becoming stronger with increasing the polycationic nature of the modified polysulfone and with the polymer solution concentration. Also, for all solutions, the inhibition is intense compared to DMSO, which is

Fig. 15. Antimicrobial screening tests: (a) PSFQ1 in DMSO, c = 1.08 g/L, against *Escherichia coli*; (b) PSFQ2 in DMSO, c = 1.10 g/L, against *Escherichia coli*; (c) PSFQ1 in DMSO, c = 1.08 g/L, against *Staphylococcus aureus*; (d) PSFQ2 in DMSO, c = 1.10 g/L, against *Staphylococcus aureus*. In each figure, the inhibition area on the right side is recorded for DMSO as a control

semipermeable membranes.

influences cell metabolism.

used as a control sample.

sample

**7. Antimicrobial activity assessments**

According to the negative values of the interfacial free energy of all polysulfone samples, the work of spreading of water, W , takes negative values, caused by the hydrophobic s,w surfaces, where the work of water adhesion is low, comparatively with the work of cohesion; at the same time, be noticed that adhesion and implicitly work of spreading of water, Ws , increases with increasing substitution degree for PSFQ samples ( W < s,w,PSFQ1 W < s,w,PSFQ2 W ), is lower for PSF-DMOA than for PSF-DMEA, and decreases with s,w,PSFQ3 increasing of substitution degrees for PSF-DOPO ( Ws,w,PS DOPO1 − < Ws,w,PS DOPO2 − < Ws,w,PS DOPO3 − ).

## **6.  To analyze the possibilities of using the functionalized polysulfones in biomedical applications, and for establishing its compatibility with blood, equation (7) is used, where W and s,rbc W describe the work of spreading of s,p red blood cells and platelets (Vijayanand et al., 2005); when blood is exposed to a biomaterial surface, adhesion of cells occurs and the extent of adhesion decides the life of the implanted biomaterials; thus, cellular adhesion to biomaterial surfaces could activate coagulation and the immunological cascades. Therefore, cellular adhesion has a direct bearing on the thrombogenicity and immunogenicity of a biomaterial, and thus dictates its blood compatibility. The work of adhesion of the red blood cells can be considered as a parameter for characterizing biomaterials *versus* cell adhesion. The materials which exhibit a lower work of adhesion would lead to a lower extent of cell adhesion than those with a higher work of adhesion. Considering the surface energy parameters ( lv γ , d lv γ , lv +γ , lv −γ ) given in Table 8 for red blood cells and platelets, the work of spreading of blood cells and platelets is estimated by equation (7), with surface tension parameters listed in Table 10 for PSFQ, Table 12 for PSF-DMEA and PSF-DMOA, and in Table 14 for PSF-DOPO.

Figures 12, 13 and 14 show positive values for the work of spreading of red blood cells, W , and negative values for the wo s,rbc rk of spreading of platelets, W , suggesting a s,p higher work of adhesion comparatively with that of cohesion for the red blood cells, but a smaller work of adhesion comparatively with the one of cohesion for platelets. Among these polysulfones, the work of spreading of red blood cells, W exhibits a negative value for s,rbc the PSFQ1, generated by lower hydrophobicity, and a highest value for the PSF-DMEA, characterized by high disperse parameter of surface tension.

These results suggest that the exposure of platelets to functionalized polysulfone films determines an increase of platelets cohesion, and that a good hydrophobicity can be correlated with a good adhesion of the red blood cells on the surface of the polysulfone films.

In summary, both red blood cells and platelets are extremely important in deciding the blood compatibility of a material. Moreover, it is known that adhesion of the red blood cells onto a surface, *e.g*. modified polysulfones, requires knowledge of the interactions with the vascular components. Thus, endothelial glycocalyx along with the mucopolysaccharides adsorbed to the endothelial surface of the vascular endothelium reject clotting factors and platelets - which have a significant role in thrombus formation (Reitsma et al., 2007). In this context, adhesion of the red blood cells and cohesion of platelets to surface films must be discussed in correlation with future specific biomedical applications. These results seem to be applicable for evaluating bacterial adhesion to the surfaces, and could be subsequently employed for studying possible implanted induced infections, or for obtaining semipermeable membranes.

## **7. Antimicrobial activity assessments**

268 A Search for Antibacterial Agents

According to the negative values of the interfacial free energy of all polysulfone samples, the work of spreading of water, W , takes negative values, caused by the hydrophobic s,w surfaces, where the work of water adhesion is low, comparatively with the work of cohesion; at the same time, be noticed that adhesion and implicitly work of spreading of water, Ws , increases with increasing substitution degree for PSFQ samples ( W < s,w,PSFQ1 W < s,w,PSFQ2 W ), is lower for PSF-DMOA than for PSF-DMEA, and decreases with s,w,PSFQ3 increasing of substitution degrees for PSF-DOPO ( Ws,w,PS DOPO1 − < Ws,w,PS DOPO2 − <

Blood compatibility is dictated by the manner in which their surfaces interact with blood constituents, like red blood cells and platelets. To analyze the possibilities of using the functionalized polysulfones in biomedical applications, and for establishing its compatibility with blood, equation (7) is used, where W and s,rbc W describe the work of spreading of s,p red blood cells and platelets (Vijayanand et al., 2005); when blood is exposed to a biomaterial surface, adhesion of cells occurs and the extent of adhesion decides the life of the implanted biomaterials; thus, cellular adhesion to biomaterial surfaces could activate coagulation and the immunological cascades. Therefore, cellular adhesion has a direct bearing on the thrombogenicity and immunogenicity of a biomaterial, and thus dictates its blood compatibility. The work of adhesion of the red blood cells can be considered as a parameter for characterizing biomaterials *versus* cell adhesion. The materials which exhibit a lower work of adhesion would lead to a lower extent of cell adhesion than those with a

higher work of adhesion. Considering the surface energy parameters ( lv γ , d

PSFQ, Table 12 for PSF-DMEA and PSF-DMOA, and in Table 14 for PSF-DOPO.

characterized by high disperse parameter of surface tension.

given in Table 8 for red blood cells and platelets, the work of spreading of blood cells and platelets is estimated by equation (7), with surface tension parameters listed in Table 10 for

Figures 12, 13 and 14 show positive values for the work of spreading of red blood cells, W , and negative values for the wo s,rbc rk of spreading of platelets, W , suggesting a s,p higher work of adhesion comparatively with that of cohesion for the red blood cells, but a smaller work of adhesion comparatively with the one of cohesion for platelets. Among these polysulfones, the work of spreading of red blood cells, W exhibits a negative value for s,rbc the PSFQ1, generated by lower hydrophobicity, and a highest value for the PSF-DMEA,

These results suggest that the exposure of platelets to functionalized polysulfone films determines an increase of platelets cohesion, and that a good hydrophobicity can be correlated with a good adhesion of the red blood cells on the surface of the polysulfone

In summary, both red blood cells and platelets are extremely important in deciding the blood compatibility of a material. Moreover, it is known that adhesion of the red blood cells onto a surface, *e.g*. modified polysulfones, requires knowledge of the interactions with the vascular components. Thus, endothelial glycocalyx along with the mucopolysaccharides adsorbed to the endothelial surface of the vascular endothelium reject clotting factors and

lv γ , lv

+γ , lv −γ )

Ws,w,PS DOPO3 − ).

films.

**6. Blood - functionalized polysulfone interactions**

The literature shows that quaternary ammonium salts represent one of the most popular types of antimicrobial agents (Hazziza-Laskar et al., 2005; Xu et al., 2006; Yu et al., 2007; Merianos, 1991). Their biological activity, depending on their structure and physicochemical properties, affects the interaction with the cytoplasmic membrane of bacteria and influences cell metabolism.

The antimicrobial activity of quaternized polysulfones with various ionic chlorine contents, in DMSO, at different concentrations, is investigated against *Escherichia coli* (*E. coli*) and *Staphylococcus aureus* (*S. aureus*). As shown in Figure 15 and Table 17, these polymers inhibit the growth of microorganisms, the inhibition becoming stronger with increasing the polycationic nature of the modified polysulfone and with the polymer solution concentration. Also, for all solutions, the inhibition is intense compared to DMSO, which is used as a control sample.

Fig. 15. Antimicrobial screening tests: (a) PSFQ1 in DMSO, c = 1.08 g/L, against *Escherichia coli*; (b) PSFQ2 in DMSO, c = 1.10 g/L, against *Escherichia coli*; (c) PSFQ1 in DMSO, c = 1.08 g/L, against *Staphylococcus aureus*; (d) PSFQ2 in DMSO, c = 1.10 g/L, against *Staphylococcus aureus*. In each figure, the inhibition area on the right side is recorded for DMSO as a control sample

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 271

character of the polar terms, and capacity of P-H bond of 9,10-dihydro-oxa-10 phosphophenanthrene-10-oxide to determine the donor character of the polar terms, caused

The AFM images show that surface morphology is characterized by roughness and nodules formations, depending on the composition of solvent/nonsolvent mixtures, including the characteristics of polysulfones and the thermodynamic quality of the solvents. Moreover,

On the other hand, the antimicrobial activity of polysulfones with quaternary ammonium groups is considered to be one of the important properties that are directly related to new possible applications. In this study, adhesion of *Escherichia coli* and *Staphylococcus aureus* cells to PSFQ is investigated. These polymers inhibit the growth of microorganisms, with inhibition becoming stronger with the increasing polycationic nature of the modified polysulfone and the concentration of the polymer solution. Moreover, the adhesion of the relatively hydrophilic *E. coli* to hydrophilic quaternized polysulfone is higher than the

These results are useful in investigations on specific biomedical applications, including evaluation of bacterial adhesion to the surfaces, and utilization of modified polysulfones as

Albu, R.M.; Avram, E.; Stoica, I.; Ioanid, E.G.; Popovici, D. & Ioan, S. (2011). Surface

Barikani, M. & Mehdipour–Ataei, S. (2000). Synthesis, Characterization and Thermal

Della Volpe, C.; Maniglio, D.; Brugnara, M.; Siboni, S. & Morra, M. (2004). The Solid Surface

Properties and Compatibility with Blood of New Quaternized Polysulfones. *Journal of Biomaterials and Nanobiotechnology*, Vol.2, No.2, (April 2011), pp. 114-123, ISSN

Properties of Novel Arylene Sulfone Ether Polyimides and Polyamides. *Journal of Polymer Science, Part A: Polymer Chemistry*, Vol.38, No.9, (May 2000), pp. 1487-1492,

Free Energy Calculation: in Defense of the Multicomponent Approach. *Journal of*

by the inductive phenomena of different pendant groups.

platelets on the surface of the polysulfone films;

by high disperse parameter of surface tension.

adhesion of the hydrophobic *S. aureus* cells.

semipermeable membranes.

2158-7027

ISSN 1099-0518

**9. References**

the results suggest that:

Table 17. Antimicrobial activity expressed by the diameter of the inhibition zone (mm) of PSFQ1 and PSFQ2 in DMSO at two concentrations and of DMSO used as a control sample against *Escherichia coli* and *Staphylococcus aureus*

The cationic modified polysulfones with quaternary ammonium group interfere with the bacterial metabolism by electrostatic stacking at the cell surface of the bacteria (Xu et al., 2006). This conclusion is evaluated in terms of the diameter of the inhibition zone presented in Table 17. The results show that the bacterial activity of the tested compounds is dependent on the microorganism nature. Thus, the *E. coli* is found be much more sensitive to the investigated polymers than the *S. aureus*.

On the other hand, the differences in the composition of the cell wall of Gram-negative (*E. coli*) and Gram-positive (*S. aureus*) bacteria cause different resistance to killing by antimicrobial agents. It is known that the component of Gram-positive bacteria cell walls is peptidoglycan, whereas the major constituents of Gram-negative bacteria cell walls are peptidoglycan, together with other membranes, such as lipopolysaccharides and proteins. These components of the cell walls generate the hydrophilic character of *E. coli* bacteria and the hydrophobic character of *S. aureus*. Thus, the different inhibiting effects of PSFQ1 and PSFQ2 on the growth of the tested *E. coli* and *S. aureus* bacteria may be due to different antimicrobial activities.

Therefore, all these aspects indicate that the antimicrobial activity depends not only on the substituent groups of the quaternized polysulfones but also on the hydrophilic or hydrophobic character of the bacteria, which generated different interactions of the quaternary ammonium salt groups with the bacterial cell membrane. In particular, the adhesion of the relatively hydrophilic *E. coli* to the hydrophilic quaternized polysulfones is higher than the adhesion of hydrophobic *S. aureus* cells.

## **8. Conclusion**

New quaternized polysulfones, prepared by quaternization of chloromethylated polysulfone with N,N-dimethylethanolamine, N,N-dimethylethylamine, N,Ndimethyloctylamine and also polysulfones with bulky phosphorus pendant groups, obtained by chemical modification of the chloromethylated polysulfones by reacting the chloromethyl group with the P-H bond of 9,10-dihydro-oxa-10- phosphophenanthrene-10 oxide are investigated to obtain information on their hydrophilic/hydrophobic properties and blood compatibility. The history of the formed films, prepared by a dry-cast process in pure solvents or in different solvent/nonsolvent mixtures, influenced the surface tension parameters, surface and interfacial free energy and the work of spreading of water, maintaining the surfaces hydrophobic characteristics of polysulfones. On the other hand, the results reflect the capacity of N-dimethylethanolammonium chloride pendant group to determine the acceptor character of the polar terms, capacity of N-dimethylethylammonium or N-dimethyloctylammonium chloride pendant groups to determine the acceptor or donor character of the polar terms, and capacity of P-H bond of 9,10-dihydro-oxa-10 phosphophenanthrene-10-oxide to determine the donor character of the polar terms, caused by the inductive phenomena of different pendant groups.

The AFM images show that surface morphology is characterized by roughness and nodules formations, depending on the composition of solvent/nonsolvent mixtures, including the characteristics of polysulfones and the thermodynamic quality of the solvents. Moreover, the results suggest that:

On the other hand, the antimicrobial activity of polysulfones with quaternary ammonium groups is considered to be one of the important properties that are directly related to new possible applications. In this study, adhesion of *Escherichia coli* and *Staphylococcus aureus* cells to PSFQ is investigated. These polymers inhibit the growth of microorganisms, with inhibition becoming stronger with the increasing polycationic nature of the modified polysulfone and the concentration of the polymer solution. Moreover, the adhesion of the relatively hydrophilic *E. coli* to hydrophilic quaternized polysulfone is higher than the adhesion of the hydrophobic *S. aureus* cells.

These results are useful in investigations on specific biomedical applications, including evaluation of bacterial adhesion to the surfaces, and utilization of modified polysulfones as semipermeable membranes.

## **9. References**

270 A Search for Antibacterial Agents

Microorganism PSFQ1 PSFQ2 Control

against *Escherichia coli* and *Staphylococcus aureus*

to the investigated polymers than the *S. aureus*.

higher than the adhesion of hydrophobic *S. aureus* cells.

antimicrobial activities.

**8. Conclusion**

*E. coli* 13 19 22 23 12 *S. aureus* 11 13 15 18 10 Table 17. Antimicrobial activity expressed by the diameter of the inhibition zone (mm) of PSFQ1 and PSFQ2 in DMSO at two concentrations and of DMSO used as a control sample

The cationic modified polysulfones with quaternary ammonium group interfere with the bacterial metabolism by electrostatic stacking at the cell surface of the bacteria (Xu et al., 2006). This conclusion is evaluated in terms of the diameter of the inhibition zone presented in Table 17. The results show that the bacterial activity of the tested compounds is dependent on the microorganism nature. Thus, the *E. coli* is found be much more sensitive

On the other hand, the differences in the composition of the cell wall of Gram-negative (*E. coli*) and Gram-positive (*S. aureus*) bacteria cause different resistance to killing by antimicrobial agents. It is known that the component of Gram-positive bacteria cell walls is peptidoglycan, whereas the major constituents of Gram-negative bacteria cell walls are peptidoglycan, together with other membranes, such as lipopolysaccharides and proteins. These components of the cell walls generate the hydrophilic character of *E. coli* bacteria and the hydrophobic character of *S. aureus*. Thus, the different inhibiting effects of PSFQ1 and PSFQ2 on the growth of the tested *E. coli* and *S. aureus* bacteria may be due to different

Therefore, all these aspects indicate that the antimicrobial activity depends not only on the substituent groups of the quaternized polysulfones but also on the hydrophilic or hydrophobic character of the bacteria, which generated different interactions of the quaternary ammonium salt groups with the bacterial cell membrane. In particular, the adhesion of the relatively hydrophilic *E. coli* to the hydrophilic quaternized polysulfones is

New quaternized polysulfones, prepared by quaternization of chloromethylated polysulfone with N,N-dimethylethanolamine, N,N-dimethylethylamine, N,Ndimethyloctylamine and also polysulfones with bulky phosphorus pendant groups, obtained by chemical modification of the chloromethylated polysulfones by reacting the chloromethyl group with the P-H bond of 9,10-dihydro-oxa-10- phosphophenanthrene-10 oxide are investigated to obtain information on their hydrophilic/hydrophobic properties and blood compatibility. The history of the formed films, prepared by a dry-cast process in pure solvents or in different solvent/nonsolvent mixtures, influenced the surface tension parameters, surface and interfacial free energy and the work of spreading of water, maintaining the surfaces hydrophobic characteristics of polysulfones. On the other hand, the results reflect the capacity of N-dimethylethanolammonium chloride pendant group to determine the acceptor character of the polar terms, capacity of N-dimethylethylammonium or N-dimethyloctylammonium chloride pendant groups to determine the acceptor or donor

0.52 g/L 1.08 g/L 0.52 g/L 1.1 g/L (DMSO)

Biocompatibility and Antimicrobial Activity of Some Quaternized Polysulfones 273

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**14**

*USA*

**The Design of Bacteria Strain Selective**

**Incorporation of Unnatural Amino Acids**

*of the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,*

An intensive international research effort is currently underway to develop new classes of compounds that exhibit novel mechanisms of antibacterial activity (Bush, 2004) (Klevens et al., 2007) due to the dramatic and continued evolution of drug resistant strains of bacteria (Hartl, 2000) (Shlaes, Projan, & Edwards, 2004) (Y. Huang, Huang, & Chen, 2010) (Godballe, Nilsson, Petersen, & Jenssen, 2011) (J. Song, 2008) (Findlay, Zhanel, & Schweizer, 2010). Both natural and synthetic antimicrobial peptides (AMPs) because of their novel mechanisms of antibiotic activity, coupled with the inherent difficulty for bacteria to develop resistance to them (Hartl, 2000) (Shlaes et al., 2004) (Y. Huang et al., 2010) (Godballe et al., 2011) (J. Song, 2008) (Findlay et al., 2010) exhibit a very high potential as new therapeutic agents. Several AMPs are currently in development as topical antibiotics

AMPs have evolved as a host defense mechanism against invading micro-organisms in most organisms (Hartl, 2000) (Shlaes et al., 2004) (Y. Huang et al., 2010) (Godballe et al., 2011) (J. Song, 2008) (Findlay et al., 2010) . In addition to antibacterial properties AMPs are believed to be key players in innate immunity (Devine & Hancock, 2002; Y. M. Song et al., 2005). AMPs are normally highly positively charged (+3 to +9) small peptides consisting of 5-50

**1. Introduction**

(Zhang & Falla, 2009).

\* Corresponding Author

**Antimicrobial Peptides Based on the**

Antoine H. Srouji3,Jayendra B. Bhonsl4, Richard Borschel5,

*Walter Reed Army Institute of Research, Silver Spring, Maryland,*

*Walter Reed Army Institute of Research, Silver Spring, Maryland,*

Amanda L. Russell1, David Klapper2,

Allen Mueller5 and Rickey P. Hicks1,\*

*Science and Technology Building, Greenville, 2Peptide Core Facility in the School of Medicine*

*5Division of Bacterial and Rickettsial Diseases,*

*3Synthetic Proteomics, Carlsbad, CA, 4Division of Experimental Therapeutics,*

*1Department of Chemistry, East Carolina University,*

## **The Design of Bacteria Strain Selective Antimicrobial Peptides Based on the Incorporation of Unnatural Amino Acids**

Amanda L. Russell1, David Klapper2, Antoine H. Srouji3,Jayendra B. Bhonsl4, Richard Borschel5, Allen Mueller5 and Rickey P. Hicks1,\* *1Department of Chemistry, East Carolina University, Science and Technology Building, Greenville, 2Peptide Core Facility in the School of Medicine of the University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, 3Synthetic Proteomics, Carlsbad, CA, 4Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Silver Spring, Maryland, 5Division of Bacterial and Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, Maryland, USA*

## **1. Introduction**

274 A Search for Antibacterial Agents

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An intensive international research effort is currently underway to develop new classes of compounds that exhibit novel mechanisms of antibacterial activity (Bush, 2004) (Klevens et al., 2007) due to the dramatic and continued evolution of drug resistant strains of bacteria (Hartl, 2000) (Shlaes, Projan, & Edwards, 2004) (Y. Huang, Huang, & Chen, 2010) (Godballe, Nilsson, Petersen, & Jenssen, 2011) (J. Song, 2008) (Findlay, Zhanel, & Schweizer, 2010). Both natural and synthetic antimicrobial peptides (AMPs) because of their novel mechanisms of antibiotic activity, coupled with the inherent difficulty for bacteria to develop resistance to them (Hartl, 2000) (Shlaes et al., 2004) (Y. Huang et al., 2010) (Godballe et al., 2011) (J. Song, 2008) (Findlay et al., 2010) exhibit a very high potential as new therapeutic agents. Several AMPs are currently in development as topical antibiotics (Zhang & Falla, 2009).

AMPs have evolved as a host defense mechanism against invading micro-organisms in most organisms (Hartl, 2000) (Shlaes et al., 2004) (Y. Huang et al., 2010) (Godballe et al., 2011) (J. Song, 2008) (Findlay et al., 2010) . In addition to antibacterial properties AMPs are believed to be key players in innate immunity (Devine & Hancock, 2002; Y. M. Song et al., 2005).  The basic skeleton of the new analogs containing six Tic-Oic dipeptide units and the

In this report the in vitro antibacterial activity of a new series of AMPs incorporating six Tic-Oic dipeptide units as well as four additional Spacers, A, B, C and D on either side of the intervening hydrophobic and charged residues. These spacers define the overall conformational flexibility of the peptide backbone. A fifth Spacer, E, which defines the distance between the polypeptide backbone and the positively charged side chain amine group. Spacer E is involved in determining the overall surface charge density of the peptide as well as defining the distance between the membrane surface and the polypeptide backbone (Hicks et al., 2007). By varying the number of -CH2- groups in Spacer E from 1 to 4 in the side chain of the basic residues, the distance between the positive charge and the peptide backbone will decrease resulting in a) less side chain flexibility during binding –this flexibility is more important in the binding with zwitterionic lipids than with anionic lipids; and b) the positive charge density will reside closer to the peptide backbone. The additional Tic-Oic dipeptide units and spacers were incorporated in hopes of improving the organism

amino acid residues (Hancock, 1998). These peptides are amphipathic molecules with welldefined hydrophobic and hydrophilic regions (Toke, 2005; Zasloff, 2002). As of January 2009, more than 1330 (Wang, Li, & Wang, 2009) natural and synthetic antimicrobial peptides have been characterized. These peptides have exhibited, a diversity of biological activity (Khandelia, 2008).

The inherent selectivity of AMP's for prokaryotic vs eukaryotic cells is believed to be derived from the difference in the chemical compositions of their respective membranes (Dennison, Wallace, Harris, & Phoenix, 2005). Hancock and co-workers (Powers & Hancock, 2003) have extended this hypothesis to propose that the differences in membrane composition between different strains of bacteria are responsible for the diversity in the potency and selectivity exhibited by a particular AMP against different strains of bacteria. Bacterial cells contain a high percentage of negatively charged phospholipids while mammalian cells contain a much higher concentration of zwitterionic phospholipids (Papo & Shai, 2003) (Zhang & Falla, 2009) (Findlay et al., 2010) (Godballe et al., 2011) (Y. Huang et al., 2010) (Yeaman & Yount, 2003) (Leontiadou, Mark, & Marrink, 2006) (Y. Huang et al., 2010) (Hancock & Lehrer, 1998). Other differences also exist including membrane composition (sterols, lipopolysaccharide, peptidoglycan etc) (Yeaman & Yount, 2003), structure, and transmembrane potential and polarizability. These differences are in part responsible for the observed selectivity of some AMPs for bacterial vs. mammalian cells.(Hancock & Lehrer, 1998; Yeaman & Yount, 2003) Melo and co-workers have characterized the physical properties of bacterial membranes that are conducive for AMP interactions (Melo, Ferre, & Castanho, 2009).

The membranes surrounding different types of bacteria are also different. The lipid bilayer of Gram positive bacteria is covered by a porous layer of peptidoglycan, while the structure of Gram negative bacteria is more complex with two lipid membranes containing lipopolysaccharides and porins (Dennison et al., 2005; Giangaspero, Sandri, & Tossi, 2001). There is a developing preponderance of evidence in the literature supporting the concept that the selectivity and potency of a specific AMP is determined in a large measure by the chemical composition of the target membrane.(Powers & Hancock, 2003; Yeaman & Yount, 2003)

In our laboratory we have focused on developing antimicrobial peptides that contain unnatural amino acids to control the conformational and physicochemical properties (Hicks, Bhonsle, Venugopal, Koser, & Magill, 2007) of the resulting peptide. Our original skeletal design of an unnatural AMP incorporated placement of three L-Tic-L-Oic dipeptide units into the polypeptide backbone to induce an ordered structure onto the peptide (Hicks et al., 2007). The use of the conformationally restrained amino acids (Tic an Oic) reduces the local flexibility of the peptide backbone and thus reduces the total conformational space that may be sampled by the peptide during lipid binding. The AMPs developed in our laboratory, the Tic-Oic units are connected via an amino acid spacer with defined properties of charge and hydrophobicity resulting in peptides with well-defined physiochemical properties while maintaining sufficient conformational flexibility to allow the peptide to adopt different conformations on interacting with membranes of different chemical composition (Hicks et al., 2007; Russell et al., 2010; Venugopal et al., 2010). The basic skeleton of the AMPs under investigation is given in Fig. 1. The amino acid sequences for the AMPs used in this investigation are given in Table 1.

amino acid residues (Hancock, 1998). These peptides are amphipathic molecules with welldefined hydrophobic and hydrophilic regions (Toke, 2005; Zasloff, 2002). As of January 2009, more than 1330 (Wang, Li, & Wang, 2009) natural and synthetic antimicrobial peptides have been characterized. These peptides have exhibited, a diversity of biological activity

The inherent selectivity of AMP's for prokaryotic vs eukaryotic cells is believed to be derived from the difference in the chemical compositions of their respective membranes (Dennison, Wallace, Harris, & Phoenix, 2005). Hancock and co-workers (Powers & Hancock, 2003) have extended this hypothesis to propose that the differences in membrane composition between different strains of bacteria are responsible for the diversity in the potency and selectivity exhibited by a particular AMP against different strains of bacteria. Bacterial cells contain a high percentage of negatively charged phospholipids while mammalian cells contain a much higher concentration of zwitterionic phospholipids (Papo & Shai, 2003) (Zhang & Falla, 2009) (Findlay et al., 2010) (Godballe et al., 2011) (Y. Huang et al., 2010) (Yeaman & Yount, 2003) (Leontiadou, Mark, & Marrink, 2006) (Y. Huang et al., 2010) (Hancock & Lehrer, 1998). Other differences also exist including membrane composition (sterols, lipopolysaccharide, peptidoglycan etc) (Yeaman & Yount, 2003), structure, and transmembrane potential and polarizability. These differences are in part responsible for the observed selectivity of some AMPs for bacterial vs. mammalian cells.(Hancock & Lehrer, 1998; Yeaman & Yount, 2003) Melo and co-workers have characterized the physical properties of bacterial membranes that are conducive for AMP

The membranes surrounding different types of bacteria are also different. The lipid bilayer of Gram positive bacteria is covered by a porous layer of peptidoglycan, while the structure of Gram negative bacteria is more complex with two lipid membranes containing lipopolysaccharides and porins (Dennison et al., 2005; Giangaspero, Sandri, & Tossi, 2001). There is a developing preponderance of evidence in the literature supporting the concept that the selectivity and potency of a specific AMP is determined in a large measure by the chemical composition of the target membrane.(Powers & Hancock, 2003; Yeaman & Yount,

In our laboratory we have focused on developing antimicrobial peptides that contain unnatural amino acids to control the conformational and physicochemical properties (Hicks, Bhonsle, Venugopal, Koser, & Magill, 2007) of the resulting peptide. Our original skeletal design of an unnatural AMP incorporated placement of three L-Tic-L-Oic dipeptide units into the polypeptide backbone to induce an ordered structure onto the peptide (Hicks et al., 2007). The use of the conformationally restrained amino acids (Tic an Oic) reduces the local flexibility of the peptide backbone and thus reduces the total conformational space that may be sampled by the peptide during lipid binding. The AMPs developed in our laboratory, the Tic-Oic units are connected via an amino acid spacer with defined properties of charge and hydrophobicity resulting in peptides with well-defined physiochemical properties while maintaining sufficient conformational flexibility to allow the peptide to adopt different conformations on interacting with membranes of different chemical composition (Hicks et al., 2007; Russell et al., 2010; Venugopal et al., 2010). The basic skeleton of the AMPs under investigation is given in Fig. 1. The amino acid sequences for the AMPs used in this

(Khandelia, 2008).

2003)

interactions (Melo, Ferre, & Castanho, 2009).

investigation are given in Table 1.

hydrophobic residue

Fig. 1. The basic skeleton of the new analogs containing six Tic-Oic dipeptide units and the five SPACER residues.

In this report the in vitro antibacterial activity of a new series of AMPs incorporating six Tic-Oic dipeptide units as well as four additional Spacers, A, B, C and D on either side of the intervening hydrophobic and charged residues. These spacers define the overall conformational flexibility of the peptide backbone. A fifth Spacer, E, which defines the distance between the polypeptide backbone and the positively charged side chain amine group. Spacer E is involved in determining the overall surface charge density of the peptide as well as defining the distance between the membrane surface and the polypeptide backbone (Hicks et al., 2007). By varying the number of -CH2- groups in Spacer E from 1 to 4 in the side chain of the basic residues, the distance between the positive charge and the peptide backbone will decrease resulting in a) less side chain flexibility during binding –this flexibility is more important in the binding with zwitterionic lipids than with anionic lipids; and b) the positive charge density will reside closer to the peptide backbone. The additional Tic-Oic dipeptide units and spacers were incorporated in hopes of improving the organism selectivity of these AMPs.

The Design of Bacteria Strain

**Compound Number**

**Bacteria Strain**

*Acinetobacter baumanii*

*Acinetobacter baumanii*

*Staphylococcus aureus* ATCC

*Brucella melitensis*

*Bacillus anthracis*

*Francisella tularensis*

*Burkholderia*

**2.1 Spacers A and C**

dramatically.

strains and Gram Negative Select Agents.

terminal side of the Phe) as listed in Table 3.

Selective Antimicrobial Peptides Based on the Incorporation of Unnatural Amino Acids 279

ATCC 19606 **40.5 9.30 9.77** 186 162 **4.50** 146.00 139 146 144 139

WRAIR 162 150 156 186 162 144 146.00 139 146 144 139

33591(MRSA) 162 150 156 186 162 **17.90** 146.00 139 146 144 139

16M 162 150 156 186 162 144 **73** 139 146 144 139

AMES **40.5** 150 156 186 162 **8.97 0.50** 139 146 **2.20** 139

SCHUS4 **81** 150 156 186 162 144 146.00 139 146 144 139

*mallei* 162 150 156 **84 81** 144 146 139 146 144 139

In this series of analogs, Spacer A is the residue preceding each internal Lys residues (Nterminal side of the Lys) and Spacer C is the residue preceding each internal Phe residue (N-

Compound **22** exhibited good in vitro activity against Acinetobacter baumanii ATCC 19606, and Bacillus anthracis AMES and moderate activity against Francisella tularensis SCHUS4 and poor activity against the other bacteria strains. Replacement of the Lys-Arg residues at the C-terminus with four Lys residues in compound **74** changes in vitro activity

Table 2. In Vitro MIC values (μM) for the compounds understudy against drug resistant

**22 70** *71* **72 73 74 75 76 77 78 79**

Compound Number/ Amino Acid Sequence

Table 1. Amino acid sequences of the compounds understudy

## **2. Biological activity**

Antimicrobial peptides (AMP) have evolved in almost every class of living organism as a defense mechanism against invading micro-organisms (Yeaman & Yount, 2003) (Dennison et al., 2005). The exact mechanism of lipid-induced cytotoxicity of these peptides is currently debated in the literature (Powers & Hancock, 2003) with AMPs divided into two mechanistic classes, membrane-disruptors and non-membrane-disruptors (Powers & Hancock, 2003) (Brogden, 2005). However, there is a general agreement that the first target for either membrane-disruptor or non-membrane-disruptor AMPs is the negatively charged membranes of bacterial cells (Powers & Hancock, 2003). There are several different structural classes of membrane-disruptor AMPs but, for the purpose of this investigation we focused on linear amphipathic helical peptides. In many cases these peptides exhibit characteristics of a random coil conformation in aqueous or in organic solvents, however on binding to micelles or membranes they adopt an ordered amphipathic secondary structure (Hicks et al., 2003). This structural class of membrane-disruptors can be divided into two sub-classifications based on biological activity: 1) cell selective and 2) non-selective (Y. M. Song et al., 2005). As the name implies cell selective AMPs exhibit potent activity against bacterial cells while remaining inactive against mammalian cells, non-selective AMPs exhibit activity against bacterial as well as mammalian cells. We have previously reported the synthesis and biological evaluation of a series of potent (low μM to nm in vitro activity, and low acute toxicity 125 mg/kg in mice) bacteria selective membrane disruptors based on the incorporation of three Tic-Oic dipeptide units (Hicks et al., 2007) (Venugopal et al., 2010) (Bhonsle, Venugopal, Huddler, Magill, & Hicks, 2007). The in vitro antibacterial activity exhibited by the analogs containing six dipeptide units against seven strains of bacteria is given in Table 2. The in vitro antibacterial activity will be discussed as a function of the Spacers. The activity of Spacers A and C will be discussed together as will Spacers B and D. Spacer E will be discussed separately.

Table 2. In Vitro MIC values (μM) for the compounds understudy against drug resistant strains and Gram Negative Select Agents.

## **2.1 Spacers A and C**

278 A Search for Antibacterial Agents

**71** H2N-Orn-L-Tic-Oic-Orn-Tic-Oic-F-Tic-Oic-Orn-Tic-Oic-F-Tic-Oic-Orn-Tic-Oic-Orn-Orn-Orn-Orn-

**72** H2N-Dpr-L-Tic-Oic-Dpr-Tic-Oic-F-Tic-Oic-Dpr-Tic-Oic-F-Tic-Oic-Dpr-Tic-Oic-Dpr-Dpr-Dpr-

**73** H2N-Dab-L-Tic-Oic-Dab-Tic-Oic-F-Tic-Oic-Dab-Tic-Oic-F-Tic-Oic-Dab-Tic-Oic-Dab-Dab-Dab-

Antimicrobial peptides (AMP) have evolved in almost every class of living organism as a defense mechanism against invading micro-organisms (Yeaman & Yount, 2003) (Dennison et al., 2005). The exact mechanism of lipid-induced cytotoxicity of these peptides is currently debated in the literature (Powers & Hancock, 2003) with AMPs divided into two mechanistic classes, membrane-disruptors and non-membrane-disruptors (Powers & Hancock, 2003) (Brogden, 2005). However, there is a general agreement that the first target for either membrane-disruptor or non-membrane-disruptor AMPs is the negatively charged membranes of bacterial cells (Powers & Hancock, 2003). There are several different structural classes of membrane-disruptor AMPs but, for the purpose of this investigation we focused on linear amphipathic helical peptides. In many cases these peptides exhibit characteristics of a random coil conformation in aqueous or in organic solvents, however on binding to micelles or membranes they adopt an ordered amphipathic secondary structure (Hicks et al., 2003). This structural class of membrane-disruptors can be divided into two sub-classifications based on biological activity: 1) cell selective and 2) non-selective (Y. M. Song et al., 2005). As the name implies cell selective AMPs exhibit potent activity against bacterial cells while remaining inactive against mammalian cells, non-selective AMPs exhibit activity against bacterial as well as mammalian cells. We have previously reported the synthesis and biological evaluation of a series of potent (low μM to nm in vitro activity, and low acute toxicity 125 mg/kg in mice) bacteria selective membrane disruptors based on the incorporation of three Tic-Oic dipeptide units (Hicks et al., 2007) (Venugopal et al., 2010) (Bhonsle, Venugopal, Huddler, Magill, & Hicks, 2007). The in vitro antibacterial activity exhibited by the analogs containing six dipeptide units against seven strains of bacteria is given in Table 2. The in vitro antibacterial activity will be discussed as a function of the Spacers. The activity of Spacers A and C will be discussed together as will Spacers B and D.

 H2N-KL-Tic-Oic-GK-Tic-Oic-F-Tic-Oic-GK-Tic-Oic-F-Tic-Oic-GK-Tic-Oic-KKKK-CONH2 H2N-KL-Tic-Oic-K-Tic-Oic-GF-Tic-Oic-K-Tic-Oic-GF-Tic-Oic-K-Tic-Oic-KKKK-CONH2 H2N-KL-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-Oic-KKKK-CONH2 H2N-KL-Tic-Oic-K-Tic-Oic-FG-Tic-Oic-K-Tic-Oic-FG-Tic-Oic-K-Tic-Oic-KKKK-CONH2 H2N-KL-Tic-Oic-KG-Tic-Oic-F-Tic-Oic-KG-Tic-Oic-F-Tic-Oic-KG-Tic-Oic-KKKK-CONH2 H2N-KL-Tic-Oic-KG-Tic-Oic-FG-Tic-Oic-KG-Tic-Oic-FG-Tic-Oic-KG-Tic-Oic-KKKK-CONH2

Table 1. Amino acid sequences of the compounds understudy

**22** H2N-KL-Tic-Oic-K-Tic-Oic-F-Tic-Oic-K-Tic-Oic-F-Tic-Oic-K-Tic-Oic-KR-CONH2 **70** H2N-KL-Tic-Oic-K-Tic-Oic-F-Tic-Oic-K-Tic-Oic-F-Tic-Oic-K-Tic-Oic-KKKK-CONH2

Compound Number/ Amino Acid Sequence

CONH2

Dpr-CONH2

Dab-CONH2

**2. Biological activity**

Spacer E will be discussed separately.

In this series of analogs, Spacer A is the residue preceding each internal Lys residues (Nterminal side of the Lys) and Spacer C is the residue preceding each internal Phe residue (Nterminal side of the Phe) as listed in Table 3.

Compound **22** exhibited good in vitro activity against Acinetobacter baumanii ATCC 19606, and Bacillus anthracis AMES and moderate activity against Francisella tularensis SCHUS4 and poor activity against the other bacteria strains. Replacement of the Lys-Arg residues at the C-terminus with four Lys residues in compound **74** changes in vitro activity dramatically.

The Design of Bacteria Strain

Table 5. Spacer E

**3.1 Calcein leakage studies**

time period.

19606 as compared to compound **74**.

Selective Antimicrobial Peptides Based on the Incorporation of Unnatural Amino Acids 281

anthracis AMES, while maintaining the activity against Acinetobacter baumanii ATCC

Replacement of the Lys residues with either Dpr residues (three carbon less) or Dab (two carbons less) residues in compounds **72** and **73** respectively dramatically reduces the activity against all of the bacteria strains except Burkholderia mallei. Clearly, the length of the side chain of Spacer E, and the resulting charge density, plays a major role in determining

The observed differences in bacteria strain selectivity for these compounds are believed to be derived directly from the conformational and physicochemical properties presented by these AMPs to the surface of the bacteria membrane. Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides to simple membrane models for bacterial and mammalian cells. LUVs and SUVs consisting of 1- Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) were selected as a simple model for the zwitterionic membranes of mammalian cells and membrane models consisting of (4:1) 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) / **1-**Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt)) (POPG) were selected as a simple model

for the anionic membranes of bacteria cells (Bringezu et al., 2007) Hicks, 2007 #19}.

It is well documented that AMPs containing three Tic-Oic dipeptide units exhibited potent and selective antibacterial activity (Hicks et al., 2007; Venugopal et al., 2010) **and t**hese AMPs were designed to be members of the general class of AMPs known as membrane disruptors (Shlaes et al., 2004) (Kamysz, 2005) (Zhang, Harris, & Falla, 2005) (Toke, 2005) (Godballe et al., 2011) (Ryge, Frimodt-Moller, & Hansen, 2008). Calcein leakage studies clearly indicated that the analogs containing three Tic-Oic dipeptides units are in fact membrane disruptors (Russell et al., 2010). The first step in this investigation was to confirm that the analogs containing six Tic-Oic dipeptide units are also membrane disruptors. Peptide induced calcein leakage from LUVs monitored through fluorescence, is a well documented technique for probing AMP activity to confirm membrane disruption (Andrushchenko, Aarabi, Nquyen, Prenner, & Vogel, 2008) (Medina, Chapman, Bolender, & Plesniak, 2002). Peptides at a concentration of 4μM were introduced to solutions of either POPC or 4:1 POPC/POPG LUVs containing encapsulated 70 mM calcein (Figure 2) and the resulting induced fluorescence due to leakage of calcein was monitored over a 90 minute

**Compound Number Spacer E 71** Orn

 **72** Dpr  **73** Dab **74** Lys

**3. Characterization of peptide lipid interactions**

organism potency and selectivity against these seven strains of bacteria.

Table 3. Spacers A and C

The activity against Acinetobacter baumanii ATCC 19606 and Staphylococcus aureus ATCC 33591(MRSA) are increased by a factor of 9, and the activity against Bacillus anthracis AMES, is increased by a factor of 5, compared to compound **22**. Deleting Spacer A (Gly residue) of compound **74** and introducing Spacer C (Gly residue) in Compound **75** again dramatically changes the activity compared to both compounds **22** and **74**. The activity against Acinetobacter baumanii ATCC 19606 is now very poor. However compound **75** exhibited the highest activity against Bacillus anthracis AMES (80 fold increase over compound **74**) and Brucella melitensis 16M. Activity against the other bacteria strains was very poor. Incorporating both Spacers B and C (both Gly residues) in compound **76** reduced the activity to a level that this compound is no longer therapeutically useful. Removing both Spacers in compound **70** resulted in a compound selective for Acinetobacter baumanii ATCC 19606 and inactive against the remaining bacteria strains. This data indicates that combined use of Spacer A and C coupled with increasing the density of the positive charge at the C-terminus of the peptide produces organism selectivity.

#### **2.2 Spacers B and D**

In this series of analogs Spacers B is the residue following each internal Lys residues (Cterminal side of the Lys) and Spacer D is the residue following each internal Phe residues (C-terminal side of the Phe) as listed in Table 4.

Table 4. Spacers D and E

Incorporation of Spacers D (Gly residues in both cases) in compounds **77** and **79** reduced to the activity of these two compounds against all seven strains of bacteria to poor making these compounds no longer therapeutically useful. Incorporation of Spacer B (Gly residue) only in compound **78** exhibited selective and very good activity against Bacillus anthracis AMES.

#### **2.3 Spacer E**

In this series of analogs, Spacer E replaces the charged Lys residues with charged residues with progressively shorter side chains as shown in Table 5. Replacement of the Lys residues with Orn residues (one less carbon atom in the side chain) in compound **71** dramatically reduces the activity against Staphylococcus aureus ATCC 33591(MRSA) and Bacillus anthracis AMES, while maintaining the activity against Acinetobacter baumanii ATCC 19606 as compared to compound **74**.

Table 5. Spacer E

280 A Search for Antibacterial Agents

**Compound number Spacer A Spacer C**

The activity against Acinetobacter baumanii ATCC 19606 and Staphylococcus aureus ATCC 33591(MRSA) are increased by a factor of 9, and the activity against Bacillus anthracis AMES, is increased by a factor of 5, compared to compound **22**. Deleting Spacer A (Gly residue) of compound **74** and introducing Spacer C (Gly residue) in Compound **75** again dramatically changes the activity compared to both compounds **22** and **74**. The activity against Acinetobacter baumanii ATCC 19606 is now very poor. However compound **75** exhibited the highest activity against Bacillus anthracis AMES (80 fold increase over compound **74**) and Brucella melitensis 16M. Activity against the other bacteria strains was very poor. Incorporating both Spacers B and C (both Gly residues) in compound **76** reduced the activity to a level that this compound is no longer therapeutically useful. Removing both Spacers in compound **70** resulted in a compound selective for Acinetobacter baumanii ATCC 19606 and inactive against the remaining bacteria strains. This data indicates that combined use of Spacer A and C coupled with increasing the density of the positive charge

In this series of analogs Spacers B is the residue following each internal Lys residues (Cterminal side of the Lys) and Spacer D is the residue following each internal Phe residues

**Compound Number Spacer B Spacer D**

Incorporation of Spacers D (Gly residues in both cases) in compounds **77** and **79** reduced to the activity of these two compounds against all seven strains of bacteria to poor making these compounds no longer therapeutically useful. Incorporation of Spacer B (Gly residue) only in compound **78** exhibited selective and very good activity against Bacillus anthracis

In this series of analogs, Spacer E replaces the charged Lys residues with charged residues with progressively shorter side chains as shown in Table 5. Replacement of the Lys residues with Orn residues (one less carbon atom in the side chain) in compound **71** dramatically reduces the activity against Staphylococcus aureus ATCC 33591(MRSA) and Bacillus

 **77** none Gly  **78** Gly none  **79** Gly Gly

 22 Gly none **70** none none **74** Gly none **75** none Gly **76** Gly Gly

at the C-terminus of the peptide produces organism selectivity.

(C-terminal side of the Phe) as listed in Table 4.

Table 3. Spacers A and C

**2.2 Spacers B and D**

Table 4. Spacers D and E

AMES.

**2.3 Spacer E**

Replacement of the Lys residues with either Dpr residues (three carbon less) or Dab (two carbons less) residues in compounds **72** and **73** respectively dramatically reduces the activity against all of the bacteria strains except Burkholderia mallei. Clearly, the length of the side chain of Spacer E, and the resulting charge density, plays a major role in determining organism potency and selectivity against these seven strains of bacteria.

## **3. Characterization of peptide lipid interactions**

The observed differences in bacteria strain selectivity for these compounds are believed to be derived directly from the conformational and physicochemical properties presented by these AMPs to the surface of the bacteria membrane. Circular Dichroism (CD), isothermal calorimetry (ITC) and calcein fluorescence leakage experiments were conducted to provide insight into the mechanisms of binding of a series of antimicrobial peptides to simple membrane models for bacterial and mammalian cells. LUVs and SUVs consisting of 1- Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) were selected as a simple model for the zwitterionic membranes of mammalian cells and membrane models consisting of (4:1) 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (POPC) / **1-**Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt)) (POPG) were selected as a simple model for the anionic membranes of bacteria cells (Bringezu et al., 2007) Hicks, 2007 #19}.

## **3.1 Calcein leakage studies**

It is well documented that AMPs containing three Tic-Oic dipeptide units exhibited potent and selective antibacterial activity (Hicks et al., 2007; Venugopal et al., 2010) **and t**hese AMPs were designed to be members of the general class of AMPs known as membrane disruptors (Shlaes et al., 2004) (Kamysz, 2005) (Zhang, Harris, & Falla, 2005) (Toke, 2005) (Godballe et al., 2011) (Ryge, Frimodt-Moller, & Hansen, 2008). Calcein leakage studies clearly indicated that the analogs containing three Tic-Oic dipeptides units are in fact membrane disruptors (Russell et al., 2010). The first step in this investigation was to confirm that the analogs containing six Tic-Oic dipeptide units are also membrane disruptors. Peptide induced calcein leakage from LUVs monitored through fluorescence, is a well documented technique for probing AMP activity to confirm membrane disruption (Andrushchenko, Aarabi, Nquyen, Prenner, & Vogel, 2008) (Medina, Chapman, Bolender, & Plesniak, 2002). Peptides at a concentration of 4μM were introduced to solutions of either POPC or 4:1 POPC/POPG LUVs containing encapsulated 70 mM calcein (Figure 2) and the resulting induced fluorescence due to leakage of calcein was monitored over a 90 minute time period.

The Design of Bacteria Strain

Selective Antimicrobial Peptides Based on the Incorporation of Unnatural Amino Acids 283

POPC LUVs, inducing calcein leakage, in a non linear concentration dependent manner (Russell et al., 2010). However, compound **23** induces calcein leakage from 4:1 POPC/POPG LUVs in a concentration dependent manner. This data indicates the analogs containing three Tic-Oic dipeptide units interact with zwitterionic POPC LUV and anionic 4:1 POPC/POPG LUVs via different mechanisms (Russell et al., 2010). This data also indicates that the incorporation of three additional Tic-Oic dipeptide units, and thus increasing the overall

length of the peptide, changes the mechanism of binding with both types of LUVs.

 Fig. 3. The calcein leakage induced by compound **23** as a function of peptide concentration

The corresponding experiment using compound **74** were conducted. Compound 74 at various concentrations (4 – 20 μM) was introduced to solutions of either POPC or 4:1 POPC/POPG LUVs and their induced fluorescence, due to leakage of calcein was monitored over a 90 minute time period. It can be seen in Fig. 4, that compound **74** interacts with POPC LUVs, and 4:1 POPC/POPG LUVs in a totally concentration independent manner. In fact almost 100% calcien leakage was observed at all concentrations. This suggests that the analogs containing six Tic-Oic dipeptide units bind to the surface of the LUV and cause the LUV to lysis or break down in some fashion. After causing this disruption of the surface of the LUVs these peptides most likely then separate from the

Circular dichroism (CD) spectroscopy is very sensitive and its use to monitor conformational changes in peptides and proteins is well documented (Glattli, Daura, Seebach, & van Gunsteren, 2002), (Ladokhin, Selsted, & White, 1999) (Ladokhin, Vidal, & White, 2010). Traditionally, SUVs have been employed almost exclusively to investigate the binding of peptides and proteins with lipids in CD studies in order to minimize the contribution of light scattering on the spectra (Ladokhin et al., 2010) (C. H. Huang, 1969).

In the analysis of CD data, it must be noted that the spectrum of a peptide represents the

& Woody, 2000). This is particularly true when liposomes are used as there are several different peptide-liposome interactions possible depending on the lipid to peptide ratio.

(Berova, Nakanishi,

from LEFT) POPC LUVs and RIGHT) 4:1 POPC/POPG LUVs

"disrupted" LUV and bind to another LUV and repeat the process.

linear combination of a number of different conformers (Glattli et al., 2002)-

**3.2 Circular dichroism studies**

Fig. 2. Induced leakage of calcein from TOP) POPC LUVs and BOTTOM) 4:1 POPC/POPG.

The induced calcein leakage data indicates that all AMPs in this investigation interact in a very similar fashion with both zwitterionic (POPC) and anionic (4:1 POPC/POPG) LUVs. This data also strongly suggests that the mechanism of action of these AMP involves some type of membrane disruption. However, this data doesn't explain the observed organism selectivity. The observed induced calcein induce leakage behavior of the analogs containing six Tic-Oic dipeptide units is very different from that observed for the corresponding three Tic-Oic dipeptide containing analogs. The analog containing three Tic-Oic dipeptide units corresponding to compound **74** has the amino acid sequence: Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH2 (compound **23**) (Russell et al., 2010). For compound **23** different concentrations of peptide (4 – 20 μM) were introduced to solutions of either POPC or 4:1 POPC/POPG LUVs and their induced fluorescence, due to leakage of calcein was monitored over a 90 minute time period. It can be seen in Fig. 3, compound **23** interacts with POPC LUVs, inducing calcein leakage, in a non linear concentration dependent manner (Russell et al., 2010). However, compound **23** induces calcein leakage from 4:1 POPC/POPG LUVs in a concentration dependent manner. This data indicates the analogs containing three Tic-Oic dipeptide units interact with zwitterionic POPC LUV and anionic 4:1 POPC/POPG LUVs via different mechanisms (Russell et al., 2010). This data also indicates that the incorporation of three additional Tic-Oic dipeptide units, and thus increasing the overall length of the peptide, changes the mechanism of binding with both types of LUVs.

Fig. 3. The calcein leakage induced by compound **23** as a function of peptide concentration from LEFT) POPC LUVs and RIGHT) 4:1 POPC/POPG LUVs

The corresponding experiment using compound **74** were conducted. Compound 74 at various concentrations (4 – 20 μM) was introduced to solutions of either POPC or 4:1 POPC/POPG LUVs and their induced fluorescence, due to leakage of calcein was monitored over a 90 minute time period. It can be seen in Fig. 4, that compound **74** interacts with POPC LUVs, and 4:1 POPC/POPG LUVs in a totally concentration independent manner. In fact almost 100% calcien leakage was observed at all concentrations. This suggests that the analogs containing six Tic-Oic dipeptide units bind to the surface of the LUV and cause the LUV to lysis or break down in some fashion. After causing this disruption of the surface of the LUVs these peptides most likely then separate from the "disrupted" LUV and bind to another LUV and repeat the process.

### **3.2 Circular dichroism studies**

282 A Search for Antibacterial Agents

Fig. 2. Induced leakage of calcein from TOP) POPC LUVs and BOTTOM) 4:1 POPC/POPG. The induced calcein leakage data indicates that all AMPs in this investigation interact in a very similar fashion with both zwitterionic (POPC) and anionic (4:1 POPC/POPG) LUVs. This data also strongly suggests that the mechanism of action of these AMP involves some type of membrane disruption. However, this data doesn't explain the observed organism selectivity. The observed induced calcein induce leakage behavior of the analogs containing six Tic-Oic dipeptide units is very different from that observed for the corresponding three Tic-Oic dipeptide containing analogs. The analog containing three Tic-Oic dipeptide units corresponding to compound **74** has the amino acid sequence: Ac-GF-Tic-Oic-GK-Tic-Oic-GF-Tic-Oic-GK-Tic-KKKK-CONH2 (compound **23**) (Russell et al., 2010). For compound **23** different concentrations of peptide (4 – 20 μM) were introduced to solutions of either POPC or 4:1 POPC/POPG LUVs and their induced fluorescence, due to leakage of calcein was monitored over a 90 minute time period. It can be seen in Fig. 3, compound **23** interacts with

Circular dichroism (CD) spectroscopy is very sensitive and its use to monitor conformational changes in peptides and proteins is well documented (Glattli, Daura, Seebach, & van Gunsteren, 2002), (Ladokhin, Selsted, & White, 1999) (Ladokhin, Vidal, & White, 2010). Traditionally, SUVs have been employed almost exclusively to investigate the binding of peptides and proteins with lipids in CD studies in order to minimize the contribution of light scattering on the spectra (Ladokhin et al., 2010) (C. H. Huang, 1969).

In the analysis of CD data, it must be noted that the spectrum of a peptide represents the linear combination of a number of different conformers (Glattli et al., 2002)- (Berova, Nakanishi, & Woody, 2000). This is particularly true when liposomes are used as there are several different peptide-liposome interactions possible depending on the lipid to peptide ratio.

The Design of Bacteria Strain

structures adopted by compound **70**.

of spectra.

pH = 6.8.

**3.2.2 CD spectra in the presence of POPC SUVs**

Selective Antimicrobial Peptides Based on the Incorporation of Unnatural Amino Acids 285

The CD spectra of compounds **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in the presence of 1.75 mM POPC SUVs are shown in Fig. 6. This figure illustrates the diversity of conformations adopted by these compounds on binding to POPC SUVs. The CD spectra of these compounds in the presence of POPC SUVs are different in most cases from the corresponding spectra in buffer. This indicates that these compounds are interacting with the POPC SUVs via some mechanism. This is consistent with the very strong interactions observed with POPC LUVs in the induced calcein leakage studies. From this figure it is difficult to characterize the conformational difference associated with the variations in the five Spacers. To accomplish analysis, these CD spectra will be divided into smaller sub sets

The first sub set of CD spectra to be discussed will be those of compounds **70**, **74**, **75**, and **76**  which contain Spacers A or C, or both. The CD spectra of these four compounds, given in Fig. 7, are different indicating that these compounds adopt different conformations on binding to POPC SUVs. This observation is critical in explaining possible organism selectivity. Since this POPC SUVs are a model for mammalian cells, this data suggests that these compounds would adopt different conformations on binding to red blood cells. From a therapeutic perspective this information may allow for the development of analogs with reduced toxicity. The CD spectrum of compound **75** exhibits a maxima at approximately 195 nm and double minima at approximately 220 and 205 nm which would suggest the presence of α-helical conformers (Ladokhin et al., 1999) (Fuchs et al., 2006). While the CD spectra of compounds **74** and **76** exhibit double minima at approximately 220 and 205 nm without the maxima at 195 nm suggesting these compound adopt a mixture of β-turn and β-sheet like conformers on binding to POPC SUVs (Ladokhin et al., 1999) (Fuchs et al., 2006). The CD spectrum of compound **70** exhibits a maxima at approximately 195 nm, however no negative minima is observed. It is very difficult to qualitatively characterize the possible secondary

Fig. 6. Far-UV Circular Dichroism spectra of 100 μM solutions of compounds **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in the presence of 1.75 mM POPC SUV in 40 mM phosphate buffer,

Fig. 4. The calcein leakage induced by compound **74** as a function of peptide concentration from LEFT) POPC LUVs and RIGHT) 4:1 POPC/POPG LUVs

Changes in the intensity or shape of the CD spectrum of a peptide in the presence of a liposome indicate that the peptide is adopting different conformations on interacting with that particular liposome as compared to another environment such as a buffer. (Berova et al., 2000; Glattli et al., 2002). Due to the high percentage of unnatural amino acids incorporated into the peptide under investigation here, no quantitative estimation of secondary structural features are possible. Therefore, analysis of CD spectra will be limited, at best, to qualitative comparisons highlighting differences in the spectra of these compounds with references to possible secondary structural features.

#### **3.2.1 CD spectra in buffer**

The CD spectra of 100 μM solutions of compounds **22**, **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in 40 mM phosphate buffer, pH = 6.8 are given in Fig. 5. As can be seen in Fig. 5, these peptide adopt a variety of different conformations varying from random coil to possible β-sheet like. However, overall these peptides are unordered in buffer only.

Fig. 5. Far-UV Circular Dichroism spectra of 100 μM solutions of compounds **22**, **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in 40 mM phosphate buffer, pH = 6.8.

#### **3.2.2 CD spectra in the presence of POPC SUVs**

284 A Search for Antibacterial Agents

Fig. 4. The calcein leakage induced by compound **74** as a function of peptide concentration

Changes in the intensity or shape of the CD spectrum of a peptide in the presence of a liposome indicate that the peptide is adopting different conformations on interacting with that particular liposome as compared to another environment such as a buffer. (Berova et al., 2000; Glattli et al., 2002). Due to the high percentage of unnatural amino acids incorporated into the peptide under investigation here, no quantitative estimation of secondary structural features are possible. Therefore, analysis of CD spectra will be limited, at best, to qualitative comparisons highlighting differences in the spectra of these

The CD spectra of 100 μM solutions of compounds **22**, **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in 40 mM phosphate buffer, pH = 6.8 are given in Fig. 5. As can be seen in Fig. 5, these peptide adopt a variety of different conformations varying from random coil to possible

Fig. 5. Far-UV Circular Dichroism spectra of 100 μM solutions of compounds **22**, **70, 71, 72,**

**73, 74, 75, 76, 77, 78** and **79** in 40 mM phosphate buffer, pH = 6.8.

from LEFT) POPC LUVs and RIGHT) 4:1 POPC/POPG LUVs

compounds with references to possible secondary structural features.

β-sheet like. However, overall these peptides are unordered in buffer only.

**3.2.1 CD spectra in buffer**

The CD spectra of compounds **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in the presence of 1.75 mM POPC SUVs are shown in Fig. 6. This figure illustrates the diversity of conformations adopted by these compounds on binding to POPC SUVs. The CD spectra of these compounds in the presence of POPC SUVs are different in most cases from the corresponding spectra in buffer. This indicates that these compounds are interacting with the POPC SUVs via some mechanism. This is consistent with the very strong interactions observed with POPC LUVs in the induced calcein leakage studies. From this figure it is difficult to characterize the conformational difference associated with the variations in the five Spacers. To accomplish analysis, these CD spectra will be divided into smaller sub sets of spectra.

The first sub set of CD spectra to be discussed will be those of compounds **70**, **74**, **75**, and **76**  which contain Spacers A or C, or both. The CD spectra of these four compounds, given in Fig. 7, are different indicating that these compounds adopt different conformations on binding to POPC SUVs. This observation is critical in explaining possible organism selectivity. Since this POPC SUVs are a model for mammalian cells, this data suggests that these compounds would adopt different conformations on binding to red blood cells. From a therapeutic perspective this information may allow for the development of analogs with reduced toxicity. The CD spectrum of compound **75** exhibits a maxima at approximately 195 nm and double minima at approximately 220 and 205 nm which would suggest the presence of α-helical conformers (Ladokhin et al., 1999) (Fuchs et al., 2006). While the CD spectra of compounds **74** and **76** exhibit double minima at approximately 220 and 205 nm without the maxima at 195 nm suggesting these compound adopt a mixture of β-turn and β-sheet like conformers on binding to POPC SUVs (Ladokhin et al., 1999) (Fuchs et al., 2006). The CD spectrum of compound **70** exhibits a maxima at approximately 195 nm, however no negative minima is observed. It is very difficult to qualitatively characterize the possible secondary structures adopted by compound **70**.

Fig. 6. Far-UV Circular Dichroism spectra of 100 μM solutions of compounds **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in the presence of 1.75 mM POPC SUV in 40 mM phosphate buffer, pH = 6.8.

The Design of Bacteria Strain

binding to POPC SUVs.

Selective Antimicrobial Peptides Based on the Incorporation of Unnatural Amino Acids 287

The last sub set of CD spectra to be discussed in the presence of 1.75 mM POPC SUVs are those of compounds **71**, **72**, **73**, and **74** (Fig. 9) which contain Spacer E. Compounds **71**, and **74** exhibit double minima at approximately 220 and 205 nm without the maxima at 195 nm suggesting these compound adopt a mixture of β-turn and β-sheet like conformers on binding to POPC SUVs (Ladokhin et al., 1999) (Fuchs et al., 2006). While the CD spectrum of compound **72** exhibits characteristics of a random coil conformation. The CD spectrum of compound **73** exhibited a maxima at approximately 195 nm and a double minima at approximately 220 and 205 nm which would suggest the presence of α-helical conformers (Ladokhin et al., 1999) (Fuchs et al., 2006). This data clearly indicates that the length of Spacer E and the resulting location of the positive charge density relative to the peptide's backbone plays a major role in determining the conformations adopted by these peptides on

Fig. 9. Far-UV Circular Dichroism spectra of 100 μM solutions of compounds **71, 72, 73,** and

The CD spectra of compounds **22**, **70, 71, 72, 73, 74, 75, 76, 77, 78** and **79** in the presence of 1.75 mM 4:1 POPC/POPG SUVs are shown in Fig. 10. This figure illustrates the diversity of conformations adopted by these compounds binding to 4:1 POPC/POPG SUVs. The CD spectra of these compounds in the presence of 4:1 POPC/POPG SUVs are different in most cases from the corresponding spectra in buffer. This indicates that these compounds are interacting with the 4:1 POPC/POPG SUVs. From this figure it is difficult to characterize the conformational difference associated with the variations in the five Spacers. To accomplish this analysis, these CD spectra will be divided into smaller sub sets of spectra. As will become evident from the analysis of the sub sets of spectra, many of these compounds adopt

The first sub set of spectra to be discussed will be focused on Spacers A and C, consisting of compounds **70**, **74**, **75**, and **76**. The CD spectra (Fig. 11) of these four compounds are different indicating that these compounds adopt different conformations on binding to 4:1 POPC/POPG SUVs. The differences in the CD spectra for these compounds binding to 4:1

**74** in the presence of 1.75 mM POPC SUV in 40 mM phosphate buffer, pH = 6.8.

different conformation on binding to POPC and 4:1 POPC/POPG SUVs.

**3.2.3 CD spectra in the presence of 4:1 POPC/POPG SUVs**