Antibacterial activity, thermal stability and ab initio study of copolymer containing sulfobetaine and carboxybetaine groups

Dimethyl sulfoxide (DMSO) is used as a reference in antibacterial study procured from Loba Chemie, Mumbai, India. Bacterial strains have been obtained from Department of Zoology, Banaras Hindu University, Varanasi, India.

Thermogravimetric analyses and differential scanning calorimetry (TGA/DTA and DSC) were performed using Perkin Elmer, Diamond TGA/DTA at 10.00 °C min⁻¹.

All computer simulations have been performed using Gauss 09 software [39]. Various parameters such as geometry optimization, energy calculations, frequency calculations, intrinsic reaction coefficient (IRC) were simulated using HF method. The geometry optimization of the compounds has been performed at HF/3-21 G default level of theory. The frequency calculations were performed via (DFT)/B3LYP 6-31G*(d) level.

The synthesis of carbo and sulfo derivatives of polyimine: Poly (N-phenylene N'imino pentyl) imminium propane sulfonate (PGS), Poly (N-phenylene N'imino pentyl) imminium butane carboxylate (PGC) are carried out by following the same procedure as reported earlier [38]. Equimolar solutions of each p-phenylene diamine and glutaraldehyde (5 mmol) in DMF were refluxed at 80 °C for 1 h. Then 15 mmol of 1,3-propane sultone/ϒ-butyrolactone were added to the mixture and refluxed at 60 °C for 3 h to obtain the respective sulfobetaine and carbobetaine derivatives.

The antibacterial property of synthesized sulfo and carbo derivatives PGS and PGC has been studied. DMSO is used as a reference. The synthesized polymers were screened for antibacterial property against S. aureus (ATCC 25923) and P. aeuroginosa (ATCC 27853) bacterial strains by disc diffusion method and zone of inhibition of the two compounds were compared. The sterilized agar plates were prepared and standard inoculums was introduced on the surface. The filter paper disc of 6.25 mm diameter was sterilized at 140 °C for an hour. The sterile discs were soaked in known concentration of test compounds (suspension of 100 mg ml⁻¹ in DMSO) and positioned in agar medium. The inverted plates were incubated for 24 h at 37 °C. All the measurements were done in triplicate. MIC (minimum inhibitory concentration) and MBC (minimum bactericidal concentration) were analyzed by broth dilution method.

Synthesis of zwitterionic polymers is carried out here in anticipation of purposeful modification of the properties of their constituents and preparation of novel polymer materials with desired behaviour. Sulfobetaine and carbobetaine polymers are synthetically cheaper than phosphobetaine analogues and also easier to prepare. P-phenylene diamine reacts with dialdehyde to form poly-Schiff base where the nitrogen centres of imine groups are quaternized by sulfoalkylating agent 1,3 propane sultone or carboxylating agent ϒ-butyrolactone to give respective PGS and PGC as mentioned. The basic characterization of the two compounds is established elsewhere [38]. Sulfobetaine and carbobetaine polyelectrolytes were found to be highly blood-compatible or in general biocompatible, hence these could be better candidates for use as biocompatible materials [40–42].

The optimized geometries of PGS and PGC are shown in figure 1. The degrees of freedom of PGC and PGS have been found 117 and 114 respectively. Both the systems belong to C1 point group crystal structure.

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Thermodynamic parameters for PGC and PGS were computed based on statistical thermodynamics at room temperature. Here all thermodynamic parameters are computed by considering a monomeric unit of the polymer. Computed thermodynamic parameters (self consistent field (SCF) energy, heat capacity, zero point vibrational energy, entropy, enthalpy, Gibbs free energy, activation energy E_a and dipole moments) are given in table 1.

The computed dipole moments of PGC and PGS are 16.31D and 23.71D, respectively. High values of dipole moments of both monomeric molecules exhibit the nature of zwitterionic molecules while delocalization of charge centres over carboxyl group lessens the dipole moment to a lower value than sulfo analogue. The computed value of SCF energy of PGS and PGC are  −1340.97 hartree and 907.79 hartree, respectively. Similarly, computed zero point vibrational energies of PGS and PGC are found to be 218.75 kcal monomol⁻¹ and 224.59 kcal monomol⁻¹ and corresponding vibrational frequencies are also computed (table 2). These frequencies are comparable to reported values in experimental FTIR data for monomeric units [38]. Using DFT method, computed vibrational frequencies for larger molecules were found to be closer to experimental data proving the efficacy of DFT method over HF; heat capacity of PGC is much higher (70.92 cal/monomol/K) than that of PGS (47.36 cal/monomol/K); similarly entropy calculated for PGS is 110.81 cal/monomol/K and that for PGC is slightly higher (140.71 cal/monomol/K; PGC shows enthalpy of 832.86 Kcal monomol⁻¹ whereas PGS has 814.75 Kcal monomol⁻¹; Gibbs free energy for PGS is 781.73 Kcal monomol⁻¹ and for PGS it is slightly higher (790.28 kcal monomol⁻¹). Activation energy E_a for the PGS monomer unit (169.42 kcal monomol⁻¹) is higher than PGC monomer unit (149.91 kcal monomol⁻¹).

The experimental thermal analysis data of PGS and PGC (reported earlier in [38]) are reproduced here in order to correlate the antibacterial activities shown by polymers. From thermal analysis data, it can be concluded that polysulfobetaine PGS is more resistant to temperature than polycarboxybetaine PGC. Strong hydrophilic nature of imino carbobetaine is evident by loss of 70% water held by it to its original mass and this loss of water is an endothermic process. The carboxyl group itself may be responsible for binding water molecules and the other may be the entrapment of polar water molecules between the chains of polymer in the lamellar structure of the polybetaine [38]. DFT data shows higher values of entropy and enthalpy of PGC thereby suggesting loss of carbo group at lower temperature as compared to PGS. Further to be added higher is total energy predicted by DFT that supports the thermal analysis of the two compounds under study. The exact polymeric chain degradation pattern of such compounds are not certain but the probable mechanism is the loss of quaternized sulfopropyl imminium centres by Hoffman elimination of β-hydrogen [38, 43, 44] and thereby eliminating CH₂=CH–CH₂–$\text{SO}_{3}^{-}$ group in PGS and CH₂=CH–CH₂–COO⁻ in PGC leaving behind the neutral polyimine chain which are quite resistant to thermal degradation, thus leaving behind some residual mass even after  >800 °C. The probable mechanisms are shown in figure 2.

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Figure 3(a) shows zone inhibition of PGS for S. aureus and P. aeuroginosa which is found to be 10  ±  1.15 mm for both whereas in case of PGC it is found to be 9  ±  0.58 mm and 10  ±  0.58 mm, respectively. The molecular weight of PGS polymeric chain is higher than PGC, therefore on comparing the zone of inhibition of the two, the difference of chain length between different polymers may be considered as the polymer with higher molecular weight that may diffuse slowly in agar. The evaluation of MIC and MBC helps in determining the antibiotic efficiency of the synthesized materials. The growth of S. aureus and P. aeuroginosa are inhibited in the presence of PGS and PGC at every serial dilution viz., 50 mg ml⁻¹, 25 mg ml⁻¹, 12.5 mg ml⁻¹, 6.25 mg ml⁻¹, 3.12 mg ml⁻¹ and 1.56 mg ml⁻¹ in broth culture. After prolonged incubation of more than 48 h, very small vegetative growth is seen at concentration of 3.125 mg ml⁻¹. Further, at a very low concentration of 1.56 mg ml⁻¹, good vegetative growth is seen in broth culture. The minimum concentration of the polymer required to inhibit both kinds of bacterial growth is 1.56 µg ml⁻¹ for PGC and PGS. Minimum concentration to completely kill the bacteria for PGS is 6.25 µg ml⁻¹ whereas MBC for PGC is 12.50 µg ml⁻¹. Results suggest that both polymers confirm broad spectrum antibacterial activity and PGS is more effective towards killing bacteria at lower concentration as compared to PGC. MBC is four-fold higher than the corresponding MIC result in case of PGS whereas MBC is eight-fold higher than the corresponding MIC in case of PGC. In various studies of zwitterionic materials for their antibacterial or antibioadherent studies, it is observed that there is a difference in the adhesion of bacteria to these materials. It is also propounded that this difference in adhesion mechanism may depend on surface charges of organisms as well as the ability of zwitterionic head groups to bind water which will produce different extent of hydration of the surface. As data suggest, carbobetaine (PGC) was much more hydrophilic than PGS, which could be correlated to higher MBC and MIC shown by PGC in comparison to PGS.

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Sulfobetaine headgroup is found to contain only eight water molecules per sulfobetaine group reported somewhere else [45]. In another study, Kitano et al have observed that zwitterionic materials in general and caroxybetaines in particular, disturbed the native hydrogen-bonded structure the least, in comparison to the other polycationic or polyanionic systems [40]. Hence, the antibacterial activity of zwitterionic materials would be governed by extent of hydrophilicity, orientation and arrangement of water molecules around zwitterionic headgroups, as well as on surface charge of polymeric material as well of organism under study. Additionally, antibacterial studies of various charged materials have been propounded to be caused by electrostatic interactions between the organism cell surface and the charged polymeric material, so more the electrostatic interaction, the more antibacterial activity. PGS, in general has higher chain length (n  =  27) as determined by conductometric titration [38] than PGC (n  =  22), thus longer chain length will offer more zwitterionic head groups to enhance the electrostatic interactions, thus justifying lower MIC and MBC for PGS. Further, this experimental data could be supported by DFT study showing high dipole moment of PGS and PGC. Moreover imine groups have shown good antibacterial property in literature [46–48].