Danica Z. Zmejkoski*a,
Zoran M. Markovića,
Nemanja M. Zdravkovićb,
Dijana D. Trišić
c,
Milica D. Budimira,
Sanja B. Kuzmana,
Natalia O. Kozyrovska
d,
Iryna V. Orlovskad,
Nikol Bugárová
e,
Đorđe Ž. Petrovića,
Mária Kováčováe,
Angela Kleinová
e,
Zdeno Špitalskýe,
Vladimir B. Pavlovićf and
Biljana M. Todorović Marković*a
aVinča Institute of Nuclear Sciences, National Institute of the Republic of Serbia, University of Belgrade, P.O.B. 522, 11001 Belgrade, Serbia. E-mail: danica@vinca.rs; biljatod@vinca.rs; biljatod@vin.bg.ac.rs; Tel: +381 113408582
bScientific Veterinary Institute of Serbia, Janisa Janulisa 14, 11107 Belgrade, Serbia
cFaculty of Dental Medicine, University of Belgrade, Dr. Subotića 8, 11000 Belgrade, Serbia
dInstitute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150, Zabolotnogo Str., Kyiv, 03143, Ukraine
ePolymer Institute, Slovak Academy of Sciences, Dúbravska cestá 9, 84541 Bratislava, Slovakia
fFaculty of Agriculture, University of Belgrade, Nemanjina 6, 11080 Belgrade-Zemun, Serbia
First published on 24th February 2021
Therapy of bacterial urinary tract infections (UTIs) and catheter associated urinary tract infections (CAUTIs) is still a great challenge because of the resistance of bacteria to nowadays used antibiotics and encrustation of catheters. Bacterial cellulose (BC) as a biocompatible material with a high porosity allows incorporation of different materials in its three dimensional network structure. In this work a low molecular weight chitosan (Chi) polymer is incorporated in BC with different concentrations. Different characterization techniques are used to investigate structural and optical properties of these composites. Radical scavenging activity test shows moderate antioxidant activity of these biocompatible composites whereas in vitro release test shows that 13.3% of chitosan is released after 72 h. Antibacterial testing of BC–Chi composites conducted on Gram-positive and Gram-negative bacteria causing UTIs and CAUTIs (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and encrustation (Proteus mirabilis) show bactericidal effect. The morphology analysis of bacteria after the application of BC–Chi shows that they are flattened with a rough surface, with a tendency to agglomerate and with decreased length and width. All obtained results show that BC–Chi composites might be considered as potential biomedical agents in treatment of UTIs and CAUTIs and as a urinary catheter coating in encrustation prevention.
Therefore, the aim of this research was to find an adequate and potential antibacterial agent that will be particularly effective against P. mirabilis, E. coli and P. aeruginosa. One of the possibilities for UTIs and CAUTIs treatment is the usage of polysaccharides and bacterial cellulose composites. Polysaccharides have diverse biological functions and their low toxicity, biocompatibility and high biodegradability are the properties that make them promising biomaterials.3 Among them are chitin, chitosan, alginate, cellulose and cellulose based antimicrobial materials used in research of cosmetics, drug delivery, food packaging, tissue engineering and wound healing.4–7
Bacterial cellulose (BC) is a natural 3D network polymer synthesized by different acetic acid- and other bacteria, mainly by Gluconacetobacter xylinus (formerly Acetobacter xylinum). In comparison with plant cellulose, BC is devoid of hemicellulose and lignin, has higher degree of polymerization and crystallinity (water content up to 99%), insoluble in water and most of organic solvents, melting point of 467 °C, good sorption of liquids, high wet strength, high chemical purity and as biocompatible material,8 it is interesting material in developing of wide range of biomedical applications.9 BC itself does not have antibacterial activity but combined with organic and inorganic materials with antibacterial activity could be used as potential biomedical agent.10
Chitosan (Chi) is linear polysaccharide provided by deacetylation of chitin with 40–50% alkaline solution. Depending of degree of deacetylation and molecular weight chitosan has different physical properties and different antimicrobial effect.11 Since it was found that low molecular weight Chi (Chi oligosaccharides higher than 10 kDa) has better antimicrobial inhibition in comparison to high molecular weight Chi (more than 250 kDa),12 in this study was used low molecular weight Chi (50–190 kDa) to test its effect on bacterial strains the most present in UTIs and CAUTIs. Lin et al.13 produced BC–Chi composites by immersing BC in Chi followed by freeze-drying. These composites showed significant growth inhibition against E. coli and S. aureus. Jia et al.14 prepared composites of Chi chloride and BC by in situ method and showed their moderate bacteriostatic activity against E. coli. Furthermore, the inhibition effect of bacteria growth was significantly enhanced by increasing the Chi content in the composites. Wahid et al.15 developed BC–Chi based semi-interpenetrating hydrogels with improved mechanical and antibacterial properties. The authors found that antibacterial properties of these hydrogels depended significantly on the ratio of BC to Chi.
Since hydrogels coatings increases hydrophilicity and establish a barrier to inhibit nonspecific bacteria protein adsorption,16 it is proposed that BC as hydrogel and good carrier of antimicrobial materials17 might be promising agent in UTIs and CAUTIs. So far, the hydrogel catheter coatings are combined with inorganic, synthetic materials and antibiotics which may cause immunogenicity or simple no effect because of the resistance to antibiotics in the coating.16 The antibacterial hydrogel catheter coatings with natural materials is promising strategy in this field.
In this work, the new designed composite hydrogels of BC and low molecular weight synthetic polymer Chi (BC–Chi) were prepared and studied in details on structural, chemical and biomedical properties. Since the urinary catheter hydrogel coatings are proposed as successful reducer of encrustation, in comparison to pure silicon,18 one of the idea was to incorporate Chi in BC matrix as potential coating for urinary catheters. Different tests have been conducted to check Chi release from BC, antioxidant activity of this composite, biocompatibility as well as its antibacterial potentials against different bacterial strains. The bacterial morphology after the application of control BC and BC–Chi composites was presented by atomic force microscopy. Since antibacterial tests included strains which cause UTIs, CAUTIs and encrustation, this composite is proposed to be potential formulation suitable for application as treatment or prevention of UTIs, CAUTIs and encrustation.
The low molecular weight chitosan (Chi, 50–190 kDa, 75–85% deacetylated, Aldrich, Germany) dissolved in 1% acetic acid (ZORKA Pharma-HEMIJA d.o.o., Serbia) mechanically stirred at room temperature for 3 h was used in preparation of BC–Chi composites. The composites named BC–Chi0.2 and BC–Chi2 were prepared by immersing of BC into 0.2% and 2% Chi solutions for 48 h, respectively. In order to remove the excess of solution the samples were lightly dashed on filter paper and depending of the characterization analysis samples were used as previously described or dried, on air (50 °C for 2 h) or lyophilized (−30 °C for 12 h at 10 mTorr, Mini Fast 680 laboratory freeze-dryer, Edwards Ltd, UK).
For AFM images of bacteria after the application of control BC and BC–Chi composite samples, it was conducted a pilot try-out to determine the appropriate way of cell fixation (hot air, in ethanol, glutaraldehyde and Gram stain). The most suitable method was hot air drying and fixation, where the morphology of cells after the application of control BC were as typical. Surface roughness, diameters and height of control BC, composites BC–Chi0.2 and BC–Chi2 as well as height distribution, length and width of bacteria were calculated by Gwyddion software.
Xc (%) = (Acr/(Acr + Aam)) × 100 | (1) |
Drug loading (%) = ((mt − m0)/m0) × 100 | (2) |
In order to test the statistical differences between the data of Chi loading in BC–Chi0.2 and BC–Chi2 it was used Student t-test.
RSA (%) = (Ac − ABC–Chi)/Ac × 100 | (3) |
For assessment of mitochondrial activity, BC, BC–Chi0.2 and BC–Chi2 samples were prepared as described earlier. Prepared discs were placed in 96-well plates, and DMEM/F12 supplemented with 2% ABAM was added to the wells and incubated in humidified 5% CO2 atmosphere at 37 °C for 24 h. Medium was discarded, 5000 HGC cells per well were seeded onto discs in freshly prepared growth medium, and incubated. After 24 h medium was discarded, and medium containing 0.5 mg mL−1 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St. Louis, USA) was added to each well, and incubated for 4 hours, as previous described by Castiglioni et al.22 After incubation, supernatant was discarded, and dimethyl sulfoxide (Sigma-Aldrich, St. Louis, USA) was added to each well. Plate was placed on shaker for 20 minutes, on 250 rpm, in dark, at 37 °C. Extracted coloured solutions were transferred into new 96-well plate. Optical density was measured at 540 nm using microplate reader RT-2100c (Rayto, China). Six wells without discs were used as internal control of experiment. Percentage of mitochondrial activity was calculated as difference to the control group (BC).
After the drying process (air dried and lyophilized, AFM and SEM respectively) samples were used for characterization. In Fig. 2 are shown top view AFM images and SEM micrographs of control BC and BC–Chi0.2 and BC–Chi2 composites.
The surface morphology of control BC and BC–Chi0.2 showed fibrous texture, whereas BC–Chi2 had granular structure. The control BC with smooth texture was consisted of the interconnected network of fairly uniform in width BC fibers organized into filaments around 120 nm in diameter. Lin et al.23 described that cellulose polymer had a network like structure consisted of glucose chains forming fibers are further aggregating in ribbons (3 nm), and these ribbons, aggregating in filaments (30–100 nm). The BC–Chi composites exhibited rough texture with average surface roughness 26.04 nm and 0.8 nm, for BC–Chi0.2 and BC–Chi2, respectively. The control BC showed the highest average roughness of 36.04 nm. The SEM images showed a Chi concentration-dependent decrease in the amount of pores in the composite samples. BC–Chi2 is almost completely covered by Chi and lacked in pores.
Control BC | Binding energy (eV) | Atomic (%) | BC–Chi2 | Binding energy (eV) | Atomic (%) |
---|---|---|---|---|---|
O 1s | 533.0 | 36.8 | O 1s | 532.4 | 29.4 |
C 1s | 286.8 | 59.5 | C 1s | 286–0 | 62.5 |
N 1s | 400.2 | 3.7 | N 1s | 399.4 | 8.1 |
As can be seen from Fig. 3a, C 1s, O 1s and N 1s peaks are detected in the survey XPS spectra. After Chi incorporation in the BC the content of carbon increases for 3 Atomic% but the content of nitrogen increases for 4.4 Atomic%. The higher nitrogen content after Chi incorporation in BC indicates that this macromolecule is predominantly adhered to the surface fibrils of BC due to high viscosity.24
The Raman spectra of BC and BC–Chi2 composites are presented in Fig. 3b. After incorporation of Chi in BC the most representative peaks of BC are reduced (374, 1095, 1119, 1332, 1375 cm−1).25 The Chi characteristic peaks are overlapped with BC peaks (895, 1262, 1346, 1377, 1420 cm−1).26 There are small upshifts of these peaks (3–5 cm−1) compared to pure Chi. The Chi characteristic peaks in BC–Chi0.2 sample could not be identified due to small Chi concentration and overlapping with characteristic Raman BC peaks.
Fig. 3c presents FTIR spectra of BC, Chi powder and BC–Chi samples. In the FTIR spectra of BC we detected all peaks characteristic for BC: 3347 cm−1 (O–H vibrations), 2897 cm−1 (C–H vibrations), 1652 cm−1 stem from water molecules in the amorphous region, 1427 cm−1 (C–H vibrations), 1168 cm−1 (vibration from the β-anomeric link) and 1080 cm−1 (C–O–C stretching vibrations).24 As for peaks refers to Chi powder we detected the following peaks: 3358 cm−1 (O–H vibrations), 2928 and 2880 cm−1 (C–H stretching vibrations), 1652 cm−1 (CO vibrations), 1597 cm−1 (N–H vibrations), 1383 cm−1 (C–H vibrations), 1159 cm−1 and 1082 cm−1 (C–O–C vibrations).
In the FTIR spectra of BC–Chi0.2 and BC–Chi2 composites can be observed the peaks at: 3346 and 3340 cm−1 (O–H vibrations), 2897 and 2892 cm−1 (C–H vibrations), 1658 and 1653 cm−1 (CO vibrations), 1563 cm−1 (N–H vibrations), 1156 and 1162 cm−1, 1055 and 1062 cm−1 (C–O–C stretching vibrations).26,27 The characteristic peak for Chi powder sample at 1597 cm−1 is down shifted for BC–Chi2 sample at 1563 cm−1. As for BC–Chi0.2 sample this peak exists but its intensity is low.28
XRD patterns of control BC, Chi powder, BC–Chi0.2 and BC–Chi2 composite samples are shown in Fig. 3d. Chi powder shows three peaks at 10.1°, 19.8° and 40.6° (blue curve). The control BC, BC–Chi0.2 and BC–Chi2 samples were amorphous or with disordered-crystalline phase but showed peaks at 14.2°, 16.7° and 22.5°, corresponding to the (1–10), (200) and (110) Miller indices of microbial cellulose.29 The absence of Chi characteristic peaks at 10.1° and 19.8° in BC–Chi0.2 composite sample (green curve) suggests that crystalline form of Chi is converted into amorphous form and that Chi is incorporated in BC matrix. As for BC–Chi2 sample (red curve), it can be observed the absence of peak at 10.1° but peak at 19.8° can be observed as a shoulder of BC peak (designated with black arrow in Fig. 3d – red curve). The amorphous structure of composites is essential and provides more solubility, stability and it is strong associated with drug release rate (in detail Section In vitro release of Chi from BC–Chi composites). Since antimicrobial agents require water for activity, improving the water solubility of Chi by chemical modification (alkylation, acylation, quaternization, metallization…) its biomedical application is widened.30 The Chi crystallinity depends on the degree of deacetylation.31 Namely, Chi chains with higher degree of deacetylation are more compact thus enables hydrogen bond formation, which is affected by the content of glucosamine groups. The degree of crystallinity (Xc, in percentage) for Chi powder, control BC, BC–Chi0.2 and BC–Chi2 composite samples were 99.0, 62.6, 68.6 and 68.9%, respectively. Thus the Chi incorporation into BC matrix contributed to slight improvement of BC crystallinity and showed concentration-dependent trend. The values of full width at half maximum (FWHM) for Chi powder, control BC, BC–Chi0.2 and BC–Chi2 composite samples are presented in Table 2.
2θ | FWHM | |
---|---|---|
Chi powder | 10.1 | 2.89 |
19.8 | 4.75 | |
40.6 | 7.38 | |
Control BC | 14.2 | 1.74 |
16.7 | 0.92 | |
22.5 | 2.17 | |
BC–Chi0.2 | 14.2 | 1.67 |
16.7 | 1.03 | |
22.5 | 2.27 | |
BC–Chi2 | 14.2 | 1.62 |
16.7 | 0.79 | |
22.5 | 2.68 |
The broadened characteristic peak of BC at 22.5° for BC–Chi2 sample (red curve in Fig. 3d) indicates the successful incorporation of Chi into BC fibrous matrix.
By conducting different techniques to check chemical composition of control BC, BC–Chi0.2 and BC–Chi2 composites we established that Chi was found not only on the composite surface but also in the interior of BC matrix.
![]() | ||
Fig. 4 (a) Release profile of Chi from the BC–Chi composites; (b) Chi release data fitted to Korsmeyer–Peppas mathematical kinetic model. |
From all obtained results it is obvious that Chi was constantly released in small amount during the 72 h and that release of Chi is partially directed by diffusion from BC dependent on swellabillity of hydrogels. Insolubility of Chi in PBS and electrostatic interaction with BC might be the reason of very slow Chi releasing. Cabañas-Romero et al.32 showed also very small amount of Chi release (5%) from BC–Chi composites after 96 h incubation in aqueous medium. The release of Chi depends of its molecular weight and the pH of solution.33 The authors showed that low molecular weight Chi leakages more from composites and in solution with pH < 7.4 the releasing profile is much higher than in alkaline condition, which is in correspondence to the solubility of Chi in acidic medium. Since, pH of urine in UTIs and CAUTIs is in range of 8.5 to 9, BC–Chi composites with slow Chi release makes it suitable for maintaining antimicrobial capacity. In further studies, it will be monitored for how long Chi can diffuse from hydrogel.
From these results it is clear that even control sample, BC itself shows low antioxidant activity, but in comparison to composite samples it is obvious that Chi shows significant role in DPPH˙ scavenging activity (Fig. 5). Concentration dependent antioxidant activity of BC–Chi0.2 and BC–Chi2 composites show peaks at 3rd and 6th day with RSA values 17% and 36%, respectively. Since the composite samples were immersed in DPPH˙ solution during the whole experiment and monitoring time of 9 days, the hydrogels swelled and depending of the Chi incorporated and/or covering the BC in composites this ability was more or less rapid. In the case of BC–Chi0.2 where the BC filaments were not totally covered by Chi (in correspondence with Fig. 2b and e), the swelling of hydrogel was faster and the maximum antioxidant activity was detected on 3rd day. The composite BC–Chi2, totally covered with Chi (in correspondence with Fig. 2c and f), swelled slower and the maximum radical scavenging activity was observed on 6th day. In many studies is already confirmed antioxidant effect of Chi,34 but for BC it was only cited in study Schönfelder et al.35 and described as little antioxidant capacity against ROS. Recently, Yin et al.36 reported weak antioxidant activity of the BC–Chi composites. But, Cabañas-Romero et al.32 claim that BC–Chi paper has very good antioxidant property due to mainly amino groups residue from Chi and secondarily to OH groups of Chi that have the capacity to scavenge radicals. Since UTIs cause oxidative stress, increase lipid peroxidation level, and lead to insufficiency of antioxidant enzymes37 the BC–Chi composites may be consider as biomedical agent in UTIs treatment.
![]() | ||
Fig. 6 Mitochondrial activity 24 h after direct cell seeding on BC, BC–Chi0.2, and BC–Chi2 samples. One-way ANOVA revealed no statistical differences between groups, p < 0.05. |
Bacteria | BC–Chi | Control BC | Genta, ng μL−1 | |
---|---|---|---|---|
0.2% | 2% | |||
MIC/MBCb | MIC/MBC | MIC/MBC | MIC/MBC | |
a “+” the effect was observed; “−” the effect was not observed.b MIC – minimum inhibitory concentration; MBC – minimum bactericidal concentration. | ||||
S. aureus | +/+a | +/+ | −/− | 0.156/0.156 |
E. coli | +/+ | +/+ | −/− | 0.313/0.625 |
K. pneumoniae | +/+ | +/+ | −/− | 5/5 |
P. mirabilis | +/+ | +/+ | −/− | 0.625/0.625 |
P. aeruginosa | +/+ | +/+ | −/− | 0.625/0.625 |
E. faecalis | +/+ | +/+ | −/− | 5/10 |
Str. β hemo | +/+ | +/+ | −/− | 2.5/5 |
In other studies, it was also reported good antibacterial potentials against S. aureus and E. coli36 and against P. aeruginosa and yeast Candida albicans.33
Fig. 7 presents AFM images of P. mirabilis and P. aeruginosa morphology treated with BC (control) samples and BC–Chi2 composite samples. It can be seen that both strains after the application of BC–Chi2 composites were with rough surface and agglomerated. The height distribution of P. mirabilis control and BC–Chi2 treated was almost similar, 0.18 μm and 0.22 μm, respectively. In the case of P. aeruginosa after the BC–Chi2 treatment the height distribution was decreased to 0.04 μm (for control samples the height distribution was 0.36 μm). This confirmed that after the treatment with BC–Chi2 composites P. aeruginosa was flattened. Also, both bacterial strains were shrunk after the application of composites. The length and width of P. mirabilis was decreased after the BC–Chi2 composite application (control length and width 1.92 μm and 1.15 μm; after the BC–Chi2 application length and width 0.78 μm and 0.38 μm). The length of P. aeruginosa treated with BC–Chi2 composite was also decreased to 1.14 μm (control 1.65 μm), while the width was almost similar 0.79 μm (control 0.75 μm). From all the obtained results, this new BC–Chi composites might be considered as coating for urinary catheters for prevention of encrustation in CAUTIs, since it was showed bactericidal effect on strain P. mirabilis, the strain responsible for encrustation. In the review43 authors cited the potential coatings for urinary catheters and discussed their effectiveness in preventing biofilm forming.
![]() | ||
Fig. 7 Top view AFM images of P. mirabilis and P. aeruginosa before (a and c) and after (b and d) the application of BC–Chi2 composite. |
The mechanism how BC–Chi composite acts against these bacteria might be by binding to the negatively charged bacterial cell wall disrupting cell membrane and resulting in leakage of intracellular components and cell death, which is in correlation with already published results.44
The composites show strong antibacterial activity against strains the most found in patients with bacterial urinary tract infections, where bactericidal effect is achieved on all other tested strains. The most challenging results are bactericidal effect on P. mirabilis, the strain responsible for encrustation in catheter associated urinary tract infections, as well as on the most frequent strains in urinary tract infections E. coli and P. aeruginosa. The AFM images of bacterial morphology after the application of BC–Chi composites show their alternation, where they are shrunk, flattened and agglomerated. The putative mechanisms of the Chi antibacterial activity are hidden in the interaction of the BC–Chi composites with the bacterial outer membrane, as well as they are associated with a number of factors that act in an orderly independent fashion. These BC–Chi composites show very good RSA activity with the peak at 36% after the 6th day of observation. In in vitro conditions, chitosan is constantly released in small amount during the 72 h and that release is partially directed by diffusion from BC-dependent swellabillity of hydrogels. Obtained results indicate high potential of these composites for successful elimination of bacteria and their promising usage in UTIs and CAUTIs treatments, as well as urinary catheter coating in prevention of encrustation.
This journal is © The Royal Society of Chemistry 2021 |