Annotation of the results obtained in 2022
The aim of the research was to obtain thin films from polymers and interpolyelectrolyte complexes (IPECs) on various surfaces and study their antimicrobial activity. Cationic polydiallyldimethylammonium chloride (PDADMAC) and anionic sodium polyacrylate (PaNa) were used. Mixing of PDADMAC and PaNa aqueous solutions resulted in the formation of IPECs stabilized by multiple ionic bonds between the ionic groups of both polymers. Positively charged IPEC particles were obtained at the ratio of molar concentrations of anionic and cationic groups [-]/[+] = 0.2 (IPEC-0.2) and 0.4 (IPEC-0.4). IPEC dispersions showed stability against aggregation for at least two weeks after preparation. To form coatings, solutions of PDADMAC, IPEC-0.2, and IPEC-0.4 were deposited onto the surface of glass, foil, textile, and plastic and dried to a constant weight. For better visualization, a dye, Rhodamine 6G, was added to the solutions. On the glass and foil surfaces, uniformly colored pale pink coatings were obtained. The textile acquired a uniform pale pink color without visible film formation on the surface. On the hydrophobic plastic, the drops did not spread. Measurement of the contact angle for aqueous polymer formulations on a polystyrene surface showed a decrease in its value in the row of PDADMAC – IPEC-0.2 – IPEC-0.4, which reflected an increase in the content of non-polar segments in the macromolecules. Addition of Silwet surfactant to the aqueous polymer formulations improved wettability of a hydrophobic surface. Complete spreading of aqueous PDADMAC/IPEC-0.2/IPEC-0.4 formulations on the plastic surface occurred at a concentration of 0.01/0.006/0.003 wt.% Silwet, respectively. For an optimal treatment of hydrophilic surfaces, the recommended dose of polymer formulations is of 5×10-2 mL/cm2, for the impregnation of cotton textiles it is of 1.7×10-2 mL/cm2. For a treatment of hydrophobic surfaces, it is of 5×10-2 mL/cm2 with Silwet addition. Stability of polymer coatings on glass to washing with water was evaluated by successive application of bi-distilled water on the surface followed by the sample drying. After 4-5 washings, no more than 2 wt.% of the applied polymer remained on the glasses. Coatings morphology was studied by scanning electron microscopy technique. On the surface of the PDADMAC coatings, there were NaCl crystals from the buffer solution. On the IPEC coatings, there were much more crystals; additional salt came as small counterions released into the solution during the IPEC formation. Double washing of the coatings with water led to a complete salt removal. After deposition of the IPEC-0.4 suspension on the textile, the original cellular structure of the textile was preserved. IPEC was adsorbed on the surface of the fibers and formed a thin coating, which smoothed the relief of the initial surface. Thickness of the ultimately washed-out PDADMAC coating evaluated by atomic force microscopy method was of 17–20 nm. In contrast to the PDADMAC coating, the IPEC-0.4 coating had a porous structure with an average pore depth of 8 nm. Antimicrobial activity of the coatings on the glass surface was tested using a standard technique, which included applying a cell aliquot onto the polymer coating, incubation the cells on the coating for 15 or 30 minutes, washing the cells with water onto the substrate (nutrient medium) and counting grown colonies. Gram-negative bacteria Pseudomonas aeruginosa and Gram-positive bacteria Staphylococcus aureus were used as the microorganisms. The antimicrobial effect of the coatings after a 15-minutes incubation reduced with the increase in the cell amount in the aliquot deposited. S. aureus cells were the most sensitive: for a 200-cell aliquot, the content of survived cells on the cationic coatings did not exceed 5%. The IPEC-0.4 coating proved to be the most active; for all aliquots percentage of the survived S. aureus cells was less than 2%. P. aeruginosa cells showed greater stability. An increase in the incubation time from 15 to 30 minutes reduced the number of the survived cells by 1.5-10 times. The interaction of PDADMAC and IPEC with liposomes synthesized from neutral dioleoylphosphocholine (PC) and anionic dioleoylphosphoserine (PS), [PC]/[PS] = 8/2, was studied. Addition of the cationic polymer formulations first led to a gradual decrease in the liposome surface charge down to its complete neutralization. Then the liposome surface acquired a positive charge. The molar concentration of cationic PDADMAC units, at which the liposome charge was neutralized, in the row of PDADMAC, IPEC-0.2 and IPEC-0.4 was equal to 2.8×10-4, 3.3×10-4 and 3.9×10-4 M, respectively. In addition to the linear anionic PaNa, anionic latex of carboxylated styrene butadiene (C-SB) was used to obtain aqueous IPEC formulations and coatings. Addition of a PDADMAC solution to the suspension of C-SB particles resulted in electrostatic adsorption of the polycation on the latex microspheres. The composition of the complexes was described by a z = [+]/[-] ratio, where the first component is the molar concentration of cationic PDADMAC units, and the second is the molar concentration of the surface anionic groups of the latex particles. At z < 0.5 and z > 1.2 the solutions remained homogeneous due to the stabilizing negative or positive charge of the latex particles with the adsorbed polymer. The electrically neutral complex was formed at z = 0.82; the maximum polycation adsorption was achieved at z = 1.5, while positively charged IPEC particles with electrophoretic mobility of +2 (μm/s)/(V/cm) were registered in the solution. At z > 1.5 positively charged polycomplex particles and a free polycation were found in the solution. Addition of 3.6 wt.% PDADMAC to the latex (z =1.5) led to a positively charged IPEC and, at the same time, had almost no effect on the mechanical properties of the resulting films. At a higher content of PDADMAC, the film became two-component with a positively charged IPEC and a free polycation. An increase in the content of PDADMAC up to 50 wt. % resulted in the film with a 20-fold increased elasticity modulus, a 2-fold increased strength, and a 2-fold decreased limiting deformation of the films. Film coatings from the original latex and from the positively charged PDADMAC/C-SB complex showed a high stability to water treatment. Two-component coatings with 7.7 wt.% of PDADMAC lost ~67% of the polycation during a 7-fold repeated washing procedure. Polymer formulations based on positively charged IPEC PDADMAC/C-SB show antimicrobial properties which increase with elevating free polycation content in the coating. The coatings with 7.7 wt.% PDADMAC caused a 100% death of Gram-positive bacteria within 5 minutes incubation. All Gram-negative Escherichia coli bacteria were killed within 15 minutes, and more than 90% of Gram-negative P. aeruginosa bacteria died within 30 minutes incubation. A washing the polycation excess had no effect on the bactericidal activity of the coatings against Gram-positive bacteria, but significantly reduced the bactericidal activity against Gram-negative bacteria. An order of magnitude increases in the number of applied P. aeruginosa cells resulted in a 10-fold increase in the content of survived cells. To obtain polymer coatings, polymers of natural origin were also used cationic quaternized ethoxylate hydroxyethylcellulose and anionic sodium alginate. These polymers form positively charged water-soluble IPECs at a molar ratio of units of anionic and cationic polymers [-]/[+] < 0.8 and negatively charged water-soluble IPECs at [-]/[+] > 2. Polycomplexes do not dissociate to the initial components up to 0.2 M NaCl. IPEC formation is accompanied by mutual neutralizing the charges of both polyelectrolytes, which leads to a decrease in the size of macromolecular coils and a progressive decrease in the solution viscosity.

 

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Annotation of the results obtained in 2023
In order to obtain coatings, nonstoichiometric interpolyelectrolyte complexes (NIPECs) were used, formed by oppositely charged linear synthetic polyelectrolytes: anionic hydrolyzed polyacrylonitrile (HIPAN) - a copolymer of acrylic acid and acrylamide, and cationic poly-N,N-diallyl-N,N-dimethylammonium chloride (PDADMAC). The composition of the mixture was expressed as a Z=[+]/[-] molar ratio, where [+] is the concentration of the polycation quaternary amino groups and [-] is the concentration of the polyanion carboxylic groups. In the NIPEC solutions with Z=0.2 and 0.4 (“anionic” NIPEC), particles with electrophoretic mobility (EPM) of -3.2 and -2.8 (mkm/s)/(V/cm) were recorded, respectively, and an average hydrodynamic diameter of 195 and 205 nm. NIPEC particles with Z=2.5 and 5.0 (“cationic” NIPEC) were characterized by EPM values of +3.6 and +2.4 (mkm/s)/(V/cm), respectively, and an average hydrodynamic diameter of 198 and 300 nm. In order to obtain coatings, NIPECs from oppositely charged polysaccharides were also used: anionic sodium alginate and cationic quaternized hydroxyethylcellulose ethoxylate. A low molecular weight biocide, 4-hexylresorcinol (HR), was dissolved in an organic solvent (acetone or ethyl alcohol) and the HR solution was mixed with aqueous NIPEC solutions/suspension. As a result, weakly opalescent homogeneous suspensions of ternary NIPEC-HR complexes were obtained. The homogeneous suspensions of ternary complexes remained stable for 3-6 months at 5 °C. Polymer coatings were prepared using two methods. According to the first, aqueous suspensions of NIPEC HIPAN-PDADMAC with/without HR were applied onto a glass plate or Parafilm plastic film and dried to constant weight. Following the second method, the plate or film was immersed for 2 minutes in an aqueous suspension of NIPEC HIPAN-PDADMAC with/without HR. The plates/films were removed from the solution and the samples were dried as described above. Uniform film coatings were obtained on the glass surface. When drying the NIPEC formulation without HR on the plastic surface, a non-uniform film formed. The introduction of HR into a polymer suspension ensured the affinity of the polymer formulation for the hydrophobic surface and its good adhesion to the plastic. In order to study the stability of coatings in an aqueous environment, an aqueous polymer formulation was evenly distributed on the glass surface. The glasses with the applied formulations were dried to constant weight, after which the coatings were washed with distilled water. After 4-5 washed cycles, no more than 2% of the initial HIPAN-HR formulation remained on the glasses. The use of NIPEC led to an increase in the water resistance of the formulation: after 6 washes, up to 15% of the initial NIPEC-HR formulation remained on the glass. Deposition of aqueous solutions of cationic NIPEC alginate-cellulose over Petri dishes made of glass or polystyrene led to the formation of transparent homogeneous films that were easily dissolved in water. The ternary complex NIPEC-HR formed a coating on the surface of glass and polystyrene with a uniform distribution of white inclusions with size of 1-5 mm. After adding water, the coating swelled only slightly but remained on the surface. Each 2 min washing of the NIPEC HIPAN-PDADMAC-HR coatings, preformed on the Parafilm, with water resulted in the loss of 2.7% HR. The morphology of polymer coatings was studied with scanning electron microscopy. At 0.1 wt.% HR content, the NIPEC HIPAN-PDADMAC-HR coatings on the glass were characterized by a homogeneous surface. An increase in the HR content up to 0.5 wt.% led to the appearance of round-shaped inclusions in the polymer matrix, whose size varied from 40 to 1,400 nm. In the tensile curve for the original Parafilm, three sections can be distinguished : a straight section up to a strain of 1%, a peak in the strain region of 3%, and a subsequent decrease in stress reaching a plateau and a rupture of the sample at a strain of about 400%. Applying the ternary NIPEC HIPAN-PDADMAC-HR complex coating to the surface of Parafilm did not have a noticeable effect on its mechanical properties. The interaction of NIPEC with anionic lipid vesicles (liposomes) simulated the interaction of polycomplexes with the cell membrane. The internal volume of liposomes was loaded with a 1 M NaCl solution. After adding anionic NIPEC HIPAN-PDADMAC and ternary NIPEC HIPAN-PDADMAC-HR to the liposome suspension, a gradual increase in the conductivity of the suspension was recorded, which indicated the destruction of the liposomal membrane. Microbiological studies were carried out using gram-negative bacteria, gram-positive bacteria and yeast. The survival of microorganisms in solutions in the presence of polymer formulations was assessed using two standard parameters – minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). Anionic polymer formulations – individual HIPAN and sodium alginate and anionic NIPEC HIPAN-PDADMAC were non-toxic for all studied microorganisms, while PDADMAC and cationic NIPEC HIPAN-PDADMAC showed antimicrobial activity. Cationic cellulose showed no toxicity to all microorganisms tested. The polymer formulation of cationic NIPEC alginate-cellulose also did not exhibit antimicrobial activity. Ternary cationic NIPEC alginate-cellulose-HR inhibited the growth of all microorganisms. In most cases, the MIC and MBC values of the ternary complex were comparable to the corresponding values for individual HR. The antibacterial activity of the cationic NIPEC HIPAN-PDADMAC-HR ternary complexes increased with increasing HR content in the formulation and the proportion of free positively charged PDADMAC units in the polycomplex. Quantitative assessment of the cytotoxicity of polymer coatings via counting grown cell colonies showed that coatings from the ternary complex NIPEC HIPAN-PDADMAC-HR caused cell death. Incubation of cell suspensions on coatings, obtained on the glass surface, for 45 minutes caused 99% cell death of all test microorganisms. The antibacterial activity of the ternary complexes based on cationic NIPECs was higher compared to anionic ones and increased with increasing the number of free positively charged PDADMAC units in NIPECs. A significant portion of microorganisms was removed from the cationic polymer coating during a 3-minute treatment with water. Increasing the duration of water treatment led to decreasing the number of cells on the surface down to 20-30% in the case of bacteria and 60% for yeast. Desorption of cells of all test microorganisms from the surface of the PDADMAC coating was associated with the dissolution of the cationic coating when treated with water. This facilitated desorption of the cells, which left the surface being complexed with PDADMAC. The viability of microbial cells on the surface was assessed by staining them using a set of Leave/Dead fluorescent dyes. Cells applied to control glass without a polymer coating retained the ability to divide. Cells on the PDADMAC coatings died in 1 hour after application. The same technique allowed to record the death of all cells which remained on the polymer coating after washing with water. The viability of washed cells was analyzed using a traditional method – via counting the number of grown cell colonies. All cell coatings washed from the PDADMAC coating were nonviable. Thus, the study allowed to estimate the number of cells washed away during a standard procedure for testing cell survival on a biocidal coating and to determine the viability of cells washed away and remaining on the coating.

 

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