Copolymerization
The polysulfones based on 2,2-diallyl-1,1,3,3-tetraethylguanidiniumchloride, tris(diethylamino)diallylaminophosphonium tetrafluoroborate and chloride have been obtained by free radical polymerization according to the methods described in our publications [36, 37].
Copolymerization of guanidinium and diallylaminophosphonium salts with sulfur dioxide proceeds via complexation resulting in obtaining of alternating copolymers of equimolar composition independent on the monomer ratio in the initial mixture and reaction conditions.
The structure of the polysulfones obtained was investigated by 13C NMR spectroscopy. AGC and diallylaminophosphonium salts copolymerize with SO2, both double bonds participating with formation of cis-, trans-stereoisomeric pyrrolidinium structures in a cyclolinear polymer chain (Table 1S in Supplementary data).
The polysulfones obtained are light powders. They are soluble due to intramolecular cyclization of AGC and diallylaminophosphonium salts during formation of polymer chains and the absence of intermolecular crosslinks. All polysulfones are soluble in methanol, DMSO, dimethylformamide; polysulfones of AGC and DAAP-Cl are also soluble in water.
The molecular weights of AGC, DAAP-Cl and DAAP-BF4 polysulfones are 9000, 10900 and 17800 respectively.
Synthesis of AgNCs
We used our novel polysulfones as stabilizing agents in the synthesis of silver nanoparticles. Synthesis of AgNCs was conducted by the reduction of silver ions from AgNO3 with NaBH4 in aqueous (or alcoholic) solution of polysulfones. The nanocomposites obtained are dark brown powders. The content of silver in the composites was found to be in the range from 4 to 25 wt%. The ratio of silver nitrate, reducing agent and copolymer significantly affects the silver concentration in nanocomposites.
The representative IR spectra of poly(AGC-SO2), poly(DAAP-Cl-SO2) and poly(DAAP-BF4-SO2) and their AgNCs are presented in Fig. 1. The formation of nanocomposites is accompanied by a slight change in the chemical structure of the polymer matrix. In IR spectrum of nanocomposite based on poly(AGC-SO2) increase of the band at 1299 cm− 1 that belongs to the SO2 vibrations and the weak shift of this band to 1304 cm− 1 can be noticed (compare IR spectra 1 and 2 in Fig. 1(A)). In IR spectrum of nanocomposite based on poly(DAAP-Cl-SO2) the increase of the shoulder band at 1301 cm− 1 corresponding to SO2 vibrations and the weak shift of this band to 1309 cm− 1 can be noticed (compare IR spectra 1 and 2 in Fig. 1(B)). In IR spectrum of nanocomposite based on poly(DAAP-BF4-SO2) the poorly marked shift of the band at 1300 cm− 1 to 1302 cm− 1 corresponding to SO2 vibrations can be noticed (compare IR spectra 1 and 2 in Fig. 1(С)). But no other changes are observed. This means the involvement of O and S atoms of polysulfones into interaction with silver nanoparticles.
UV–Vis absorption spectra have been proved to be quite sensitive to the formation of silver colloids since silver has the highest efficiency of plasmon resonance [39]. In general, the surface plasmon resonance peak is located between 400 and 450 nm for silver particles that are smaller than 100 nm [40]. The location and shape of the absorption peak are strongly dependent on the particle size, surrounding matrix material and dielectric medium. The peak width depends on the particle size distribution and, in addition, its height corresponds to the concentration of the silver nanoparticles.
In the UV spectra of aqueous or alcoholic solutions of the nanocomposites obtained, there are the characteristic plasmon absorption bands with a maximum in the range of 393–396 nm (Fig. 2). The position of the absorption spectra indicated a narrow size distribution without the aggregation. These peaks are shifted toward red on decreasing the polysulfone concentration that is clearly demonstrated for nanocomposite based on poly(DAAP-Cl-SO2) in Fig. 1S in Supplementary data.
The general trend is that an increase in average size of the primary particles results in a shift of the absorption peak towards higher wavelengths [41]. The aggregation of silver nanoparticles leads to a decrease in the intensity of the peak at about 400 nm and also results in a long tail at the long-wavelength side of the peak [42]. In our experiments for nanocomposites based on poly(AGC-SO2) and poly(DAAP-Cl-SO2) only one absorption peak around 395 nm was observed, which is mainly attributable to primary dipolar excitation. The low intensity of the peak at about 400 nm and the appearance of an implicit peak around 430 nm in the UV spectrum of the poly(DAAP-BF4-SO2) based nanocomposite solution has been well correlated with the presence of a small fraction of microaggregates consisting of several particles [43]. However, this effect is minor because our spectra in Fig. 2 are different from the absorption features attributed to multiple polarization [44] and dipole-dipole interaction [45, 46] in large aggregates. This is further confirmed by microscopy results.
SEM results prove the obtaining of AgNCs with regular narrow-dispersed distribution of silver nanoparticles in polymer matrices. Silver nanoparticles of spherical and nearly spherical shape are formed. To obtain size distributions of silver nanoparticles, approximately 200 particles were counted and then combined into histograms. SEM micrographs of silver nanoparticles synthesized using polysulfones and the corresponding histograms of the size distribution are shown in Fig. 3. In our experiments the average sizes of silver nanoparticles were 12, 16 and 18 nm for poly(AGC-SO2), poly(DAAP-Cl-SO2) and poly(DAAP-BF4-SO2) respectively.
The XRD spectra of AgNCs on the basis of poly(AGC-SO2), poly(DAAP-BF4-SO2) and poly(DAAP-Cl-SO2) are shown in Fig. 4. Four main diffraction peaks were observed at around 38°, 44°, 65° and 78° under the diffraction angle 2θ = 10–80° and can be indexed to the (1 1 1), (2 0 0), (2 2 0) and (3 1 1) planes which corresponded to the four faces of silver cube crystal according to the standard specimen [JCPDS file № 04-0783], indicating the silver exited the cube crystal. The crystal diffraction peaks were dilated obviously because of the effect of the nanometer particles. The average crystallite sizes of silver nanoparticles were estimated using Scherrer’s equation [47] from the peak width of (1 1 1) reflection plane and were found to be 12.5, 16.7 and 18.4 nm for poly(AGC-SO2), poly(DAAP-Cl-SO2) and poly(DAAP-BF4-SO2) respectively. It should be noticed that XRD data concerning the size of silver nanoparticles are consistent with SEM results.
These experimental results are in a reasonable good agreement with DLS measurements (Fig. 2S in Supplementary data), where the sizes of silver nanoparticles were 9.9, 17.6 and 19.1 nm for poly(AGC-SO2), poly(DAAP-Cl-SO2) and poly(DAAP-BF4-SO2) respectively.
Importantly, even after three months, the aqueous dispersions of AgNCs displayed UV spectral characteristics of spherical silver nanoparticles, confirming the colloidal stability and uniformity of the silver hydrosol, as distinctly demonstrated for nanocomposite based on poly(DAAP-Cl-SO2) in Fig. 3S in Supplementary data.
The antimicrobial activity of silver nanoparticles is size dependent [10]. Silver nanoparticles should be small enough to pass through the cell membrane. Therefore, the influence of molecular weight of copolymer, the polysulfone concentration and Ag+ concentration on the size of Ag nanoparticles was investigated (Table 2S, 3S and 4S in Supplementary data). Tables illustrated that when molecular weight of the polysulfone, the polysulfone concentration and the Ag+ concentration increased, the average size of particles decreased for all systems. Polysulfones affect the molecular motion of reduced silver and subsequently limit the aggregation of nanoparticles. Therefore, we can prepare nanocomposites containing silver nanoparticles with convenient and controlled size.
Antimicrobial activity of nanocomposites
Our previous studies of antimicrobial activity showed that polysulfones exhibit pronounced bactericidal effect [48, 49] (Table 1). The polysulfone concentration of 7.8–31.2 µg/mL ensured 100 % reduction of Staphylococcus aureus and Micrococcus luteus. The poly(DAAP-Cl-SO2) and poly(DAAP-BF4-SO2) at concentration of 62.5 µg/mL and poly(AGC-SO2) at concentration of 500 µg/mL inhibited 100% Escherichia coli.
Table 1
Antimicrobial activity of polysulfones [42, 43] and their silver nanocomposites
N | Test cultures | Minimal bacteriostatic concentration (MBsC), µg/ml |
---|
poly(AGC-SO2) | nano poly(AGC-SO2) | poly(DAAP-BF4-SO2) | nano poly(DAAP-BF4-SO2) | poly(DAAP-Cl-SO2) | nano poly(DAAP-Cl-SO2) |
---|
1 | Escherichia coli, АТСС 25922 | 500 | 62.5 | 62.5 | 31.2 | 62.5 | 31.2 |
2 | Staphylococcus aureus, АТСС 25923 | 7.8 | 3.9 | 15.6 | 7.8 | 7.8 | 7.8 |
3 | Micrococcus luteus, NCIMB 196 | 31.2 | 15.6 | 15.6 | 7.8 | 7.8 | 3.9 |
4 | Staphylococcus epidermidis 33 | 31.2 | 7.8 | 15.6 | 3.9 | 3.9 | 3.9 |
5 | Staphylococcus epidermidis, АТСС 29887 | 500 | 125 | 62.5 | 31.2 | 62.5 | 62.5 |
6 | Salmonella spp. | 1000 | 62.5 | 125.0 | 125.0 | 125.0 | 62.5 |
7 | Bacillus subtilis, ATCC 6633 | 500 | 125 | 31.2 | 15.6 | 62.5 | 31.2 |
8 | Pseudomonas aeruginosa, ATCC 27853 | 500 | 62.5 | 31.2 | 15.6 | 31.2 | 15.6 |
The antimicrobial activity of AgNCs with respect to Gram positive and Gram negative bacteria was also determined (Table 1). We can see that nanocomposites have a high activity against both Gram positive and Gram negative microflora. It is seen that biocide effect of new AgNCs is generally higher as compared to initial polysulfones. Minimum bacteriostatic concentrations of nanocomposites based on polyphosphonium salts and poly(AGC-SO2) against S. epidermidis 33 are equal to 3.9 and 7.8 µg/mL respectively. All nanocomposites at concentration of 3.9–15.6 µg/mL inhibited 100% Micrococcus luteus and Staphylococcus aureus. The poly(DAAP-Cl-SO2) and poly(DAAP-BF4-SO2) nanocomposite concentration of 31.2 µg/mL ensured 100% reduction of Escherichia coli.
Effect of polysulfones and their nanocomposites on biofilm formation
More than 99% of bacteria exist in natural ecosystems not in the form of freely floating cells, but in the form of biofilms attached to the substrate. The microflora of the biofilm is more resistant to the effects of adverse physical, chemical and biological factors compared to plankton cells. Microorganisms form biofilms on any biotic and abiotic surfaces, which creates great problems in medical practice and in various fields of economic activity. Therefore, the study of the effect of antimicrobial compounds on bacterial biofilms is relevant.
To study the effect of antimicrobial compounds on biofilms, water-soluble poly(AGC-SO2) and poly(DAAP-Cl-SO2) and their AgNCs were selected.
Figure 5 illustrates the effects of poly(AGC-SO2) and poly(DAAP-Cl-SO2) and their silver nanocomposites on the biofilm formation evaluated by the absorbance of crystal violet at A570 for bacteria S. epidermidis 33 and Escherichia coli.
The noticeable decrease of the bacterial mass in the S. epidermidis 33 biofilm detected as A570 absorbance was observed when nanocomposite concentration was > 3.9 µg/mL (Fig. 5A). The use of the nanocomposite based on poly(AGC-SO2) in the concentration of 31.2 µg/mL makes it possible to completely prevent the formation of S. epidermidis 33 biofilm.
As is seen from Fig. 5B, the use of poly(DAAP-Cl-SO2) and its silver nanocomposite does not prevent the Escherichia coli biofilm formation and furthermore stimulates this process. The use of silver nanocomposite based on poly(AGC-SO2) at the concentration above 250 µg/mL noticeably prevents the formation of Escherichia coli biofilms.
Effect of polysulfones and their nanocomposites on the developed biofilms
Biofilm formation was carried out on glass slides, and cells were grown in the stationary conditions without shaking. After the biofilm formation during 24 h, the cultured liquid containing planktonic cells was removed and a fresh medium containing tested compounds was added.
Figure 6 illustrates the effects of poly(AGC-SO2) and poly(DAAP-Cl-SO2) and their AgNCs on the developed biofilms for bacteria S. epidermidis 33 and Escherichia coli. Analysis of biofilms with crystal violet staining showed that it is possible to destruct by 30 % the developed S. еpidermidis 33 biofilms (within 24 h) using the poly(DAAP-Cl-SO2) and its silver nanocomposite (Fig. 6A). At concentration of 125 µg/mL for poly(AGC-SO2) and of 62.5 µg/mL for its silver nanocomposite the biomass of the S. еpidermidis 33 biofilms was reduced by half. The use of the AgNCs based on the poly(AGC-SO2) at the concentration above 500 µg/mL makes it possible to almost completely destruct the developed S. еpidermidis 33 biofilm.
Figure 6B shows that poly(DAAP-Cl-SO2) and its nanocomposite at a concentration of more than 600–700 µg/mL destroy Escherichia coli biofilms and vice versa, at lower concentrations, they stimulate further biofilm formation. A similar effect is observed for poly(AGC-SO2). This polysulfone at a concentration of more than 1000 µg/mL destroys the Escherichia coli biofilms, at lower concentrations there is an accelerated process of biofilm formation. At concentration of 1000 µg/mL for poly(AGC-SO2) silver nanocomposite the biomass of the Escherichia coli biofilm was reduced by half.
Toxicity of AgNCs
Our results indicate a high antimicrobial effect of silver nanocomposites on pathogenic bacteria and, as a result, a beneficial impact on human health. However, the silver nanoparticles may cause adverse effects. Therefore, the investigation of biocides is impossible without their toxicity testing.
Polysulfones and their silver nanocomposites were found to be nontoxic (the LD50 values were more 1000 mg/kg) and therefore could be used for medical purposes.
With increased exposure of silver nanoparticles to human beings, their biocompatibility requires further research in terms of cytotoxicity.
Cytotoxicity of AgNCs with respect to cell lines, namely the Bronchial carcinoma (А549), Rhabdomyosarcoma (RD), Melanoma (MS) and Human Embryonic Kidney (HEK293) was evaluated in vitro by MTT-test (Table 2). After 72 h cultivation with the AgNCs at the 100 − 1.56 µM concentration, cell viability was evaluated. The cell viability in control wells was not less than 95%. In the wells containing 5.35 µM of AgNC based on poly(DAAP-BF4-SO2), 21.28 µM of AgNC based on poly(DAAP-Cl-SO2) and 28.73 µM of AgNC based on poly(AGC-SO2), 50% cell viability of the MS line cells is observed. 50% of the RD line cells was not survived in the wells containing 12.17 µM and 21.28 µM of AgNCs based on poly(DAAP-BF4-SO2) and poly(DAAP-Cl-SO2) respectively. In the wells containing 23.81 µM of AgNC based on poly(DAAP-BF4-SO2) and 40.37 µM of AgNC based on poly(DAAP-Cl-SO2), 50% cell viability of the A549 line cells is observed. The AgNC based on poly(AGC-SO2) had exhibited lower activity with respect to A549 and RD line cells. An important property of nanocomposites is the absence of a cytotoxic effect with respect to pseudonormal HEK293 cell line.
Table 2
Cytotoxic activity of polysulfones and AgNCs
Culture | IC50 ,µM |
---|
Camptothecin | Doxorubicin | poly(AGC-SO2) | nano poly(AGC-SO2) | poly(DAAP-BF4-SO2) | nano poly(DAAP-BF4-SO2) | poly(DAAP-Cl-SO2) | nano poly(DAAP-Cl-SO2) |
---|
Rhabdomyosarcoma RD | 1.72 ± 0.37 | 1.28 ± 0.03 | > 200 | > 200 | 148.3 ± 4.70 | 12.17 ± 1.31 | 97.16 ± 2,26 | 17.89 ± 1.65 |
Bronchial carcinoma А549 | 1.31 ± 0.03 | 2.04 ± 0.22 | > 200 | > 200 | > 200 | 23.81 ± 0.47 | > 200 | 40.37 ± 0.93 |
Melanoma MS | 0.77 ± 0.34 | 1.29 ± 0.16 | > 200 | 28.73 ± 1.01 | 131.5 ± 0.85 | 5.35 ± 1.25 | > 200 | 21.28 ± 1.01 |
Human embryonic kidney Hek293 | 1.61 ± 1.08 | 0.44 ± 0.04 | > 200 | 115.2 ± 6.68 | > 200 | 77.87 ± 10.23 | > 200 | 84.85 ± 4.75 |