Vladimíra
Vosmanská
*a,
Kateřina
Kolářová
a,
Silvie
Rimpelová
b,
Zdeňka
Kolská
c and
Václav
Švorčík
a
aDepartment of Solid State Engineering, Institute of Chemical Technology, Technická 5, 166 28 Praha 6, Dejvice, Czech Republic. E-mail: vosmansv@vscht.cz; Fax: +420 220 444 330; Tel: +420 220 445 142
bDepartment of Biochemistry and Microbiology, Institute of Chemical Technology Prague, Technická 5, 166 28 Praha 6, Dejvice, Czech Republic. E-mail: rimpelos@vscht.cz; Fax: +420 220 444 330; Tel: +420 220 445 207
cFaculty of Science, J. E. Purkyně University, Moskevská 54, 400 96 Ústí nad Labem, Czech Republic. E-mail: zdenka.kolska@ujep.cz; Fax: +420 220 444 330; Tel: +420 475 2831 44
First published on 4th February 2015
The treatment of wounds often deals with bacterial infections which complicate healing. Our aim was to prepare cellulose wound dressings with antibacterial properties. A cellulose dressing was exposed to argon plasma discharge, impregnated with chitosan and then silver chloride particles were precipitated in situ on the dressing's surface. The effect of plasma treatment on both the chitosan impregnation and silver chloride precipitation was studied, together with the antibacterial properties of the prepared dressings. The materials were characterized by optical microscopy, scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), goniometry, absorption atomic spectroscopy (AAS) and zeta potential measurement. The antibacterial potency of the dressings was assessed using environmental bacterial strains of Escherichia coli and Staphylococcus epidermidis. Plasma treatment had a positive effect on both chitosan impregnation and the silver chloride precipitation. The antibacterial tests performed on these wound dressings exhibited growth prevention of the two representative strains of Gram-positive and Gram-negative bacteria. These results are of medical interest.
Cellulose and chitosan structure similarity induces high affinity between both polymers. Cellulose has anionic character and chitosan is readily adsorbed onto fibres by electrostatic interaction.6 More probable intermolecular bonds are based on H-bonds and van der Waals forces. Irreversible adsorption of chitosan onto cellulose is a process predominantly driven by non-electrostatic attraction occurring preferably at low pH, in which its amino groups are charged. For irreversible binding, carboxyl and aldehyde groups present on cellulose are anchoring sites for amino groups of chitosan.7 Ionic or covalent bonds between cellulose and chitosan can be formed under special conditions, one of them is plasma treatment.8
Surface plasma treatment has been used in medicine for modification or sterilization of various materials for a long time. It has been widely used for contact lenses, intraocular lenses, vascular grafts, catheters, filters for blood elements separation and others.9–11 The plasma treatment improves polymer–polymer adhesion.12 More specifically, argon plasma treatment causes ablation and hydrophilization of the treated materials.13 Free radicals are generated during the process which is followed by the cross-linking reactions and reactions of non-saturated species.11 Argon, as an inert gas, is often used to modify and reorganize chemical groups which are already present on the surface.14 This involves breaking old bonds and forming new ones. It was also observed that argon plasma can introduce oxygen onto the surface.15,16
Silver is well-known for its strong antibacterial properties, and is often exploited in topical pharmaceutical products. It is used in the form of Ag nanoparticles, Ag+ ions or in a bound form as a part of a compound. Cytotoxicity issues of the Ag nanoparticles are still somewhat controversial. The Ag+ ions are suspected to be toxic for mammalian cells.17–20 For our wound dressing, silver in the form of insoluble inorganic salt, silver chloride (AgCl), was used. Antimicrobial activity of AgCl can be as significant as the Ag+ ions.21 Silver cations can be bound by chelation on amine groups of chitosan in near neutral solutions. However, the binding mechanism of Ag+ by chitosan is pH-dependent; the amino groups get easily protonated in acidic environment, thus turning the chelation into the electrostatic attraction of anions.22
Since there is a constant need for antibacterial wound dressings, we modified a standard cellulose non-woven wound dressing functionally combining three types of modifications that are considered to be antibacterial – plasma treatment, chitosan impregnation and addition of silver. The aim was to prepare a cellulose-based wound dressing with antibacterial properties, a wound dressing that would not release antimicrobial agens and that might be able to promote healing. The positive effect of chitosan in wound healing promotion was first reported in 1978.23 This work was based on the assumptions that argon plasma treatment oxidizes cellulose, as shown in our previous works24,25 and that the aldehyde and carboxyl groups of cellulose, both original and newly formed after the plasma treatment, would serve as anchoring sites for chitosan.7 The presence of chitosan should then positively influence binding of silver onto the dressing, since chitosan is able to chelate metals and silver has higher attraction to nitrogen than to oxygen.6,26 In another words, one modification step should advantageously influence the next one. Silver in the form of AgCl precipitate was chosen purposely because of the sufficient antibacterial activity and lower toxicity than Ag+ ions.21 Moreover, AgCl is poorly soluble in water, so we supposed to get firmly bonded AgCl precipitate on the wound dressing that would not be released into the aqueous environment.
Surface morphology of the materials and visualization of the AgCl particles was studied by scanning electron microscopy (SEM), device VEGA 3 LMU, TESCAN (Czech Republic), voltage 20 eV. The samples were not sputter coated prior to the measurement. Size of the AgCl particles was evaluated from the SEM images by the NIS-Elements software using statistically significant number of measurements. Optical microscope (LEXT OLS 3100, Olympus, Tokyo, Japan) was used to make images of the cellulose fibres impregnated with chitosan.
Static contact angle experiment was done to visualize the difference in the surface polarity of the samples using conventional goniometry. The measurement was performed at room temperature. Automatic pipette was used to make water drops, 10 μL in volume. The contact angle was calculated by the three point method,31 performed on See System device (Surface Energy Evaluation system, Advex Instruments, Czech Republic).
Electrokinetic analysis was used to examine the changes in chemistry and surface charge at solid–liquid interface. Determination of zeta potential was carried out on SurPASS Instrument (Anton Paar, Austria). Samples were studied in a cylindrical cell in contact with electrolyte (0.001 mol dm−3 KCl) at pH equal to 6.6 and room temperature. The samples were measured four times at constant pH with relative error of 5%. Streaming potential method was used to determine the zeta potential and the Fairbrother–Mastin equation was applied for the calculations.32
For determination of the amount of silver released into the aqueous solution, atomic absorption spectra of the aqueous extracts were recorded. Spectrometer AA880 (Varian Medical Systems, Inc., Palo Alto, CA) with flame atomization was used to determine the total amount of silver in the aqueous extracts. The samples that underwent the precipitation of AgCl were analysed, 1 cm2 of each sample was emerged into 10 ml of deionized water for 24 h and then the extracts were measured. The detection limit of the spectrometer was 0.03 mg dm−3 and the detected amounts of silver in the aqueous extracts were at the edge of the detection limit.
For drip test, the cultures were serially diluted with fresh saline solution (PS, 0.9% NaCl, w/v). Samples of the tested materials were cut with a die of 1 cm in diameter. These discs were immersed in 2 ml of PS and inoculated with 1.1 × 104 of colony forming units (CFU) of E. coli and 2.2 × 104 CFU of S. epidermidis. In parallel, E. coli and S. epidermidis incubated only in pure PS were used as controls. The samples were incubated statically at 24 °C for 1 h. Aliquots of 25 μL were dripped on pre-dried LB agar plates. The plates were incubated at 24 °C (E. coli) and 37 °C (S. epidermidis) for 24 h, and then the number of CFU was counted. Each sample was prepared in a triplicate by inoculating three separate discs. Similarly to Kolářová et al.24
Disc test, also called disc diffusion method, consisted in assessment of inhibition zone formed around the samples possessing antibacterial activity, similarly to Bauer et al.33 The overnight bacterial inocula were diluted to achieve final OD600 equal to 2. Bacteria were equally spread on pre-dried LB agar plates. The tested samples, 1 cm in diameter, were placed on the plates with the spread inocula. In parallel, E. coli and S. epidermidis plates without samples were used as positive controls. The plates were incubated statically for 24 h at 24 °C (E. coli) and 37 °C (S. epidermidis). The diameters of the inhibition zones were documented after 24 h growth and then evaluated using ImageJ 1.43 software (NIH, USA). Each sample was prepared in a triplicate using three separate discs.
Sample | Binding energy (eV) | O/C ratio | ||||
---|---|---|---|---|---|---|
284.7 | 532.4 | 400.1 | 367.7373.7 | 199.6 | ||
Element composition (at.%) | ||||||
C (1s) | O (1s) | N (1s) | Ag (3d3/2, 3d5/2) | Cl (2s) | ||
a Below detection limit. | ||||||
Pristine cellulose (theoretical) | 54.5 | 45.5 | — | — | — | 0.83 |
Pristine cellulose | 75.2 | 24.8 | — | — | — | 0.33 |
Cellulose/plasma | 69.5 | 30.5 | — | — | — | 0.44 |
Cellulose/chitosan | 69.5 | 29.6 | 0.9 | — | — | 0.46 |
Cellulose/plasma/chitosan | 64.8 | 34.2 | 1.0 | — | — | 0.53 |
Cellulose/AgCl | 69.2 | 30.7 | — | 0.05 | a | 0.44 |
Cellulose/plasma/AgCl | 66.3 | 33.5 | — | 0.09 | 0.08 | 0.51 |
Cellulose/chitosan/AgCl | 70.5 | 28.5 | 0.7 | 0.11 | 0.11 | 0.40 |
Cellulose/plasma/chitosan/AgCl | 67.8 | 30.9 | 1.0 | 0.11 | 0.15 | 0.46 |
As for the reaction mechanism itself, the binding of chitosan to cellulose is presumed to go via the reaction between primary amino groups of chitosan and aldehyde and/or carboxyl groups of cellulose. The reaction of amino group and carboxyl group results in a C–N amide bond40 meanwhile the reaction with aldehyde group leads to the formation of Schiff's base with imine bond.7,41 Pristine cellulose molecule has aldehyde end-groups on C1 and hydroxyl groups on C2, C3 and C6 and no carboxyl groups. During the plasma treatment, the aldehyde groups can undergo oxidation to carboxyl groups and the hydroxyl groups can be oxidized to aldehyde groups or even further to carboxyl groups.40 Argon plasma treatment leads to such oxidation, however the reaction is not selective.24 As was reported before, even the trace amounts of aldehyde groups in cellulose play an important role in a cross-linking to chitosan, which was demonstrated on the composite film formation.41 The composite films derived from the carbonyl-free cellulose had high degree of swelling and low strength in comparison with that from cellulose containing carbonyl groups which was caused by the formation cross-linking composed of Schiff's base bonds and more complex ones originating from the Schiff's base.41
As was stated in the previous section (see plasma treatment), plasma treatment caused increase in the content of C–O–/C–OH and CO/O–C–O and decrease of O
C–O functional groups, for details see our previous work,24 and these carboxyl and aldehyde groups were responsible for anchoring of chitosan and the increase of the amount of bounded chitosan on the samples after the plasma treatment. Taking into account the aforementioned, it seems to be likely, that after the plasma treatment chitosan was bound irreversibly on cellulose. It should be noted that cellulose fibres (including wound dressings) are usually weakly acidic due to the pre-treatments such as scouring and bleaching allowing effective chitosan adsorption as well.42
Another phenomenon connected to the chitosan impregnation was the change of polarity of the impregnated samples. The impregnated samples were significantly slowly soaking water in the AgCl precipitation step. Therefore we decided to investigate the wettability of the surface by standard goniometry. Pristine cellulose was hydrophilic (Fig. 3A), the water droplet soaked into the fibres immediately and the contact angle was 0°, meanwhile chitosan impregnated sample was hydrophobic (Fig. 3B) and the water droplet remained on the fibre surface, resulting in a contact angle value of 110 ± 5°. When the samples were treated with plasma and then impregnated with chitosan, they were again hydrophilic and the water droplet soaked into the fibres immediately, the contact angle was 0°. For the cellulose/chitosan/AgCl samples and the cellulose/plasma/chitosan/AgCl samples similar trend was observed. The cellulose/chitosan/AgCl samples absorbed the water droplet immediately, the contact angle was 0°, meanwhile on the cellulose/plasma/chitosan/AgCl samples the water droplet remained, resulting in a contact angle value of 95 ± 5°. Our results show that the plasma pre-treatment positively affected wettability of the chitosan impregnated samples.
![]() | ||
Fig. 3 Images of the water droplet on the surface of (A) cellulose/plasma/chitosan and (B) cellulose/chitosan surface. |
The difference in the surface wettability was closely connected to the way of binding of chitosan to cellulose. In the work of Hosokawa et al.41 was documented that a composite film of chitosan and carbonyl-free cellulose had a high swelling degree which was not affected with the increase of carboxyl groups. On the other hand, the swelling degree decreased with the increasing content of carbonyl groups in cellulose. In conclusion, the reduction of swelling degree enhances the cross-linking structures in the film. These cross-linkings are composed of Shiff's base bonds and more complex ones originating from Schiff's base.41 Based on these findings, the low surface wettability of the cellulose/chitosan (110 ± 5°) and cellulose/chitosan/AgCl (95 ± 5°) samples was caused by the binding of chitosan to the aldehyde groups via Schiff's base and the following formation of the cross-linkings. This was further supported by the XPS analysis of pristine and plasma treated cellulose. Deconvolution of the carbon peak showed that the amount of CO/O–C–O groups decreased significantly after the plasma treatment; from 40.7 at.% to 21.8 at.%. On the contrary, the amount of the O
C–O groups increased after the plasma treatment, from 5.9 at.% to 9.9 at.%. Such high content of aldehyde groups in the pristine cellulose and low content of carboxyl groups ensured cross-linking with chitosan and thus low surface wettability. The plasma treated samples did not have such amount of binding sites for the formation of Schiff's base and cross-linkings so the resulting wettability was thus affected by the formation of amide bonds.
The overall water absorption estimated by gravimetry was not comprised in this study, since we studied that in detail our previous work.24
To support the above mentioned, the changes introduced by the plasma treatment and chitosan impregnation were studied by the zeta potential measurement. The electrokinetic potential (zeta potential) at the interface of sample surface and liquid is caused, among others, by ionization or dissociation of surface functional groups.43 The negative charge of the cellulose samples resulted from the adsorption of chloride and hydroxide ions from the KCl water solution during the measurement.44 The positive charge (meaning the positive increment to the overall zeta potential) was caused by the presence of chitosan amino groups that were ionized (–NH3+) and therefore the surface was less negatively (more positively) charged than cellulose without impregnation. Furthermore, the dissociation of acidic or basic groups during the zeta potential measurement is considered to be equivalent to the adsorption of hydroxide or hydroxonium ions.45
In the case of the chitosan impregnated samples, they showed less negatively (more positively) charged surface than pristine cellulose (Table 2). The highest zeta potential, −4.0 mV, was observed for samples treated by plasma and then impregnated with chitosan. Pristine cellulose had the zeta potential of −18.0 mV, meanwhile the zeta potential of plasma treated cellulose was more negative, which was caused by the oxidation documented by the XPS measurement (Table 1). The difference between the cellulose/plasma/chitosan samples and the cellulose/chitosan samples was interesting, the higher zeta potential of the cellulose/plasma/chitosan samples depended on the amount of positively charged groups on the surface. Therefore we assumed that there was more chitosan bound on the surface of the samples that were treated by plasma discharge prior to the impregnation. The XPS results confirmed our hypothesis. In addition, Zemljič et al. published that the oxygen plasma treatment of cellulose improved adsorption of chitosan onto cellulosic fabric, which is consistent with our results.39,42
Sample | Zeta potential (mV) |
---|---|
Pristine cellulose | −18.0 |
Cellulose/plasma | −25.0 |
Cellulose/chitosan | −8.5 |
Cellulose/plasma/chitosan | −4.0 |
![]() | ||
Fig. 4 SEM images of the samples with in situ prepared AgCl particles. (A) cellulose/AgCl, (B) cellulose/plasma/AgCl, (C) cellulose/chitosan/AgCl and (D) cellulose/plasma/chitosan/AgCl. |
Silver has high affinity for nitrogen, lower for oxygen and much lower for carbon.26 Nitrogen atoms have free electron doublets that are able to react with silver cations, because they have lone electron pairs in orbitals. Oxygen is able to attract the silver via electrostatic interactions, but the attraction is weaker than in the case of nitrogen.28,29 In the case of cellulose, silver ions are attracted to hydroxyl and ether groups. In the case of chitosan, amine groups in chitosan are strongly attractive for silver cations. In another words, the affinity of silver to nitrogen prevailed over the affinity to oxygen. However, it must be pointed out that the uptake mechanism of silver cations by amine groups is pH-dependent; the amine groups get easily protonated (–NH3+) in acidic environment, thus turning the chelation of cations into the electrostatic attraction of anions.22 Therefore it was important to rinse the chitosan impregnated samples in distilled water because the impregnation was done under acidic conditions.
Comparing the cellulose/AgCl and cellulose/plasma/AgCl samples, more silver was found in the plasma treated samples. This was related to the fact that plasma exposure led to oxidation, more oxygen functional groups were situated on the sample surface and this oxygen attracted the silver. In the case of cellulose/AgCl and cellulose/chitosan/AgCl, up to twice more silver was found in the cellulose/chitosan/AgCl samples, for the result see Table 1. The difference was caused by the presence of nitrogen in chitosan that attracted and chelated the silver.
We also wondered whether the silver was irreversibly bounded on the wound dressing or not, therefore we performed a leaking test. An aqueous extracts of the samples with AgCl were analyzed and the AAS results showed that leaking of AgCl from the samples was considerably low, owing to the fact that AgCl is poorly soluble in water. AgCl had to be securely bounded on the samples. Results from the AAS were 0.054 mg l−1 for cellulose/AgCl, 0.031 mg l−1 for cellulose/plasma/AgCl, 0.056 mg l−1 for cellulose/chitosan/AgCl and 0.075 mg l−1 for cellulose/plasma/chitosan/AgCl. The standard error of the AAS measurement was 0–10% and the detection limit for silver was 0.03 mg l−1. The results were only slightly above the detection limit and on the edge of the standard error of the measurement, thus concluding that the release of silver from the samples to the aqueous environment was negligible.
For the results of the drip test performed on S. epidermidis see Fig. 6. The results were particularly interesting, since the growth of S. epidermidis was not completely reduced and the difference between the samples could be clearly observed. Generally, the growth of S. epidermidis was diminished from 50 up to 75% for all samples within 24 h, in comparison to the control. More specifically, the samples containing silver showed better reduction of the growth than the samples without silver, however the cellulose/chitosan and cellulose/plasma/chitosan samples showed sufficiently high growth reduction as well, up to 50%. Concerning the antibacterial effect of chitosan on S. epidermidis, the data were consistent with the literature.2,3 When the samples were arranged in pairs, interesting results were observed. The cellulose/chitosan and cellulose/plasma/chitosan samples showed very similar result, the growth reduction of S. epidermidis being a little bit lower for the samples that were treated by argon plasma prior to the chitosan impregnation. This was in agreement with our prior study that plasma treated cellulose underwent structure degradation and slightly supported the bacterial growth on the samples.24 This could be well observed for the pair cellulose/AgCl and cellulose/plasma/AgCl, where the difference in the bacterial growth is more pronounced and even better observed for the pair cellulose/plasma/chitosan and cellulose/plasma/AgCl that showed that plasma pre-treatment lowered the antibacterial efficiency of AgCl if compared to the pair cellulose/chitosan and cellulose/AgCl.
As regards the efficiency of antibacterial properties, it is evident that AgCl reduced the bacterial growth more than chitosan, see the bars in Fig. 6. However, the situation gets interesting when it comes to the combination of both chitosan and AgCl. When the cellulose/chitosan, cellulose/AgCl samples and cellulose/chitosan/AgCl samples were compared, it was evident that there was a trend of rising efficiency against bacterial growth. The same trend was observed in the order of the cellulose/plasma/chitosan, cellulose/plasma/AgCl and cellulose/plasma/chitosan/AgCl samples. Based on these results, the combination of chitosan and AgCl treatment increased the antibacterial efficiency against S. epidermidis. As regards the meaning of plasma treatment, the cellulose/chitosan/AgCl samples showed the highest reduction of the bacterial growth, but their surface exhibited impaired wettability (Fig. 3), whereas with the use of plasma pre-treatment the cellulose/plasma/chitosan/AgCl samples exhibited good wettability and kept very good antibacterial activity.
When comparing the drip test for E. coli and S. epidermidis, our results were consistent with the work of Xia et al., that chitosan generally showed stronger bactericidal effect on Gram-positive bacteria than Gram-negative bacteria.3
In the disc test, inhibition zones were observed around the samples with AgCl and no inhibition zones around other samples. For typical inhibition zone see Fig. 7 and for the summarized data see Table 3. The best results were observed for the cellulose/plasma/chitosan/AgCl samples, which is also consistent with the XPS results (Table 1). The highest amount of antibacterially active silver was detected in these samples. Even though the area of the zones of inhibition was not as big as was expected, it was still very good, most probably owing to the poor solubility of AgCl in water, thus not diffusing to the LB-agar medium. The plasma treated samples showed better results than the non-treated ones; the inhibition zone of cellulose/plasma/AgCl was larger than for cellulose/AgCl and the same trend was observed for cellulose/plasma/chitosan/AgCl and cellulose/chitosan/AgCl. The antibacterial effect of the samples impregnated only with chitosan (and no AgCl) was not observed, although it should have resulted in the elimination of the bacterial growth.46,47 The most probable explanation is that the concentration of chitosan on the samples' surface was not sufficient for the disc test and did not reach the minimum inhibitory concentration,47,48 even though the antibacterial activity was first proven in the drip test (Fig. 5 and 6). For both bacterial strains, E. coli and S. epidermidis, was experienced the same result.
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Fig. 7 The zone of inhibition (clear ring around the sample) induced by the cellulose/plasma/chitosan/AgCl sample on E. coli. |
Sample | Zone of inhibition (cm) | Radius (cm) | Area (cm2) |
---|---|---|---|
Cellulose/chitosan | 0 | 0 | 0 |
Cellulose/plasma/chitosan | 0 | 0 | 0 |
cellulose/AgCl | 0.13 | 0.63 | 1.25 |
Cellulose/plasma/AgCl | 0.17 | 0.67 | 1.41 |
Cellulose/chitosan/AgCl | 0.20 | 0.70 | 1.54 |
Cellulose/plasma/chitosan/AgCl | 0.22 | 0.72 | 1.63 |
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