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Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine
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Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine Ives Bernardelli de Mattos a , Sebastian P. Nischwitz b,c , Alexandru-Cristian Tuca b , Florian Groeber-Becker a , Martin Funk d , Thomas Birngruber e , Selma I. Mautner b,e , Lars-Peter Kamolz b,c, Judith C.J. Holzer b,c, * a
Fraunhofer Institute for Silicate Research ISC, Translational Center Regenerative Therapies, Würzburg, Germany Division of Plastic, Aesthetic and Reconstructive Surgery, Department of Surgery, Medical University of Graz, Austria c Joanneum Research Forschungsgesellschaft mbH, COREMED — Cooperative Centre for Regenerative Medicine, Graz, Austria d QRSKIN GmbH, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany e Joanneum Research Forschungsgesellschaft mbH, HEALTH Institute for Biomedicine and Health Sciences, Graz, Austria b
article info
abstract
Article history:
Background: Bacterial nanocellulose (BNC) is considered a promising carrier for various substances and novel
Accepted 2 October 2019
approaches using BNC in combination with antiseptics are well documented. However, the difference in the
Available online xxx
molecular weight of these molecules influences their uptake by and release from BNC. Analysing the diffusion of standard molecules with different weight, e.g. dextrans, offers the possibility to investigate the mobility of various molecules. We aimed to test the use of BNC regarding uptake and release of different standard molecules as well
Keywords: Bacterial nanocellulose Antiseptic uptake Antiseptic release Octenidine Povidone-iodine Antimicrobial wound dressing
as two commercially available antiseptics for possible applications in future wound dressings. Material and methods: Diffusion profiles, uptake and release of three FITC-dextran molecules differing in weight as well as octenidine (Octenisept1 ) and povidone-iodine (Betaisodona1 )-based antiseptics were tested with BNC-based wound dressings. Furthermore, the antiseptic efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested. Results: Uptake and release capacity for FITC-dextran molecules showed a molecular weight-dependent mobility from BNC into an agarose gel. The loading capacity of BNC was also inversely proportional to the molecular weight of the antiseptics. The release test for octenidine showed a sustained and prolonged delivery into a solid matrix, whereas povidone-iodine was released faster. Both antiseptic solutions combined with BNC showed a good dose-dependent efficacy against S. aureus. Conclusion: Results obtained from the mobility of FITC-dextran molecules in the BNC matrix could open possible applications for the combination of BNC with other molecules for medical applications. Combination of both tested antiseptics with BNC showed to be an efficient approach to control bacterial infections. © 2019 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
* Corresponding author at: Division of Plastic, Aesthetic and Reconstructive Surgery, Department of Surgery, Medical University of Graz, Auenbruggerplatz 29, 8036 Graz, Austria. E-mail address: [email protected] (J.C.J. Holzer). https://doi.org/10.1016/j.burns.2019.10.006 0305-4179/© 2019 Elsevier Ltd and ISBI. All rights reserved.
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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An efficient healing process of burn injuries depends on intricate biological mechanisms [1 3]. Wound dressings have to meet many requirements in order to support the healing processes optimally. Currently, the development progress of novel wound dressing is very well documented [4 12]. Characteristics such as being non-adhesive, reducing the number of dressing changes [13], offering an antimicrobial activity, allowing routine monitoring of biomarkers [14], providing a controlled moisture balance on the wound-dressing interface [15 17], are just some of the requirements that such dressings should comprise. Notwithstanding this challenge, a product with those characteristics would be very important to shorten hospitalization time, reduce the number of painful dressing changes and avoid infections, therefore increase the quality, reduce morbidity and mortality, and ameliorate the treatment for patients, apart from a possible cost reduction [18]. Regarding the capacity of preventing infections, a source for the continuous release of antiseptic molecules to control the growth of microorganisms is needed. Some antiseptics, such as Octenisept1 and Betaisodona1 , are already largely used in the routine of burn treatment centres [19,20]. In order to combine some of those above-mentioned characteristics, a biomaterial produced by certain bacteria such as Komagataeibacter sp., Agrobacterium sp., Rhizobium sp., Sarcina sp. [21] arise as a suitable candidate [22 26]. Combining wound dressings based on bacterial nanocellulose (BNC) with different antiseptics were also prior subject of investigation [21 28], however the contribution of differences in the molecular weight of active molecules of antiseptics (PHMB = 186 Da; octenidine = 624 Da; and povidone-iodine up to 40,000 Da) in the uptake and release from the BNC has not yet been addressed systematically. Thus, dextran molecules just differing in weight decorated with fluorescent dyes can be used as a tracking system to study their uptake and release kinetics [29]. In the present study, the uptake and release capacity of BNC loaded with fluorescein isothioeyanate labelled dextran (FITC-dextran) molecules of different weights were analysed. Further, the diffusion rates of fluorescent molecules into a solid matrix to mimic a wound bed was performed. The uptake and release capacity of BNC was also tested using two antiseptic solutions containing povidone-iodine and octenidine dihydrochloride in comparison to commercially available formulations of these antiseptics. In addition, the antiseptic efficacy against Staphylococcus aureus using BNC loaded with the aforementioned antibacterial solutions was tested.
2.
Material and methods 1
Epicitehydro 10 10 cm sheets (QRSkin GmbH, Germany; Ref-No. 800003-M02B) were used as the BNC matrix for the experiments. 10 kDa (FD10S-100MG, FITC-labelled dextran molecules lot#SLBN7802 V), 70 kDa (FD70S-100MG, lot#SLBQ4377 V) and were obtained from 2000 kDa (FD2000S-100MG, lot# SLBL5816 V) SIGMA-ALDRICH Chemie GmbH (Germany). A commercially available octenidine-containing antiseptic was used as a solution (Octenisept1 ; 0.1% octenidine dihydrochlorid = 624 Da) and as a gel formulation (Octenisept1 Gel; 0.05% octenidine dihydrochlorid) both obtained from Schülke & Mayr GmbH, Germany. Betaisodona1 solution (Mundipharma GmbH, Germany) was used as source of povidone-iodine (10%; up to 40 kDa) in solution for the diffusion tests and Betaisodona1 Wundgaze (Mundipharma GmbH, Germany) as wound dressing.
2.1.
xxx
Uptake of FTIC-dextran molecules in BNC sheets
The uptake of FITC-dextran molecules with different molecular weight was performed by incubating three BNC sheets in a 10 10 cm square petri dish covered with 100 mL solution of 0.025 mg/mL of FITC-dextran in three different weights (10 kDa, 70 kDa and 2000 kDa). The incubation was performed at room temperature under light-protected conditions. After an incubation period of either 10, 20, 30, 60 and 120 min, two punches with 8 mm in diameter were extracted from each BNC sheet and placed in membrane spin columns (200 mm pore size) and centrifuged 10 min at 10,000 rpm. Any retained content in the spin columns was eluted in 4% sodium dodecyl sulphate (SDS) solution and centrifuged a second time. The collected samples were pooled together and analysed by fluorescence spectrophotometry.
2.2.
Uptake of antiseptics in BNC sheets
In order to emulate the clinical emergency setting, two BNC sheets were put in a steel kidney dish and covered with 200 mL of Octenisept1 or Betaisodona1 solution. After 10, 20, 30, 60 and 120 min incubation at room temperature, two 8 mm diameter punch samples were taken from each BNC sheet, centrifuged with the same method as described for the FITC-dextran molecules and analysed by UV/VIS spectrophotometry.
2.3. Quantification of FITC-dextran molecules and antiseptic solutions Ultraviolet/Visible (UV/VIS) spectrophotometry analysis was chosen to quantify the concentrations of the antiseptic molecules. For both Octenisept 1 (281 nm) and Betaisodona1 (293 nm) solution a UV-readable 96 well plate (CLS3635 SIGMA Corning1 UV-Transparent Microplates) was used. To quantify the uptake and release of FITC-dextran molecules in BNC sheets, the excitation/emission spectrum peaks for the fluorophore (495 nm and 519 nm respectively) were measured using a Corning1 96 Well Black Polystyrene Microplate (CLS3603 SIGMA). All the spectrophotometry readings were performed using an Infinite1 200 PRO (TECAN Trading AG, Switzerland).
2.4. Release of the FITC-dextran molecules and the antiseptic solutions 1% agarose gels were prepared with a 50 mL volume in 10 10 cm square petri dishes. The gels acted as receptors for the release of molecules from loaded BNC sheets. For loading, BNC sheets were immersed for 120 min in one of the five solutions: FITC-dextran molecules (10 kDa, 70 kDa and 2000 kDa), Octenisept1 and Betaisodona1 . 21 punches (8 mm) were taken from each of the loaded BNC sheets and arranged on the surface of the gels together with 3 punches from unloaded BNC sheets as negative control resulting in a total of 24 punches on each gel. Betaisodona1 loaded punches were incubated on the gel under light-protected conditions to avoid oxidation and thus colour loss. Incubation and release of the FITC-dextran molecules were performed without exposure to light, to avoid fluorescent bleaching. At 30 min, 1, 2, 4, 24 and 48 h of incubation time, three BNC punches from each solution were taken off the gels to measure the retained content of each solution. 3 loaded punches with no contact with the gel were also analysed and considered as t =0 min. All these collected punches were immediately placed in membrane spin columns (200 mm pore size) and centrifuged 10 min at 10,000 rpm. Any retained solution on the column was eluted with a 4% SDS solution and centrifuged again. The collected antiseptic solutions
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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were measured by UV/VIS spectrophotometry. Fluorescent samples were analysed through fluorescent spectrometry.
3.
2.5. Diffusion profiles of the FITC-dextran molecules and Betaisodona1
3.1. Uptake, release and diffusion profile of the FITC-dextran molecules
In order to visualize the diffusion profile of the FITC-dextran molecules and Betaisodona1 into a wound-emulating agarose matrix, 100 mL of a 1% agarose gel was prepared in a 10 10 cm square petri dish. BNC sheets were incubated overnight with each of the FITC-dextran molecule solutions (10 kDa, 70 kDa and 2000 kDa) and 120 min with the Betaisodona1 solution. 8 mm punches of each BNC sheet loaded with FITC-dextran were placed on the agarose for 2 h and 24 h. A longitudinal section of the agarose gel was performed using a scalpel and pictures of the FTIC-dextran profile was taken using a FluorChem Q (Cell Biosciences acquired from Biozym GmbH) imaging system. 8 mm punches of the BNC sheet loaded with Betaisodona1 solution were placed on the agarose for 1, 3, 6, 12 and 24 h. To compare the diffusion profile of Betaisodona1 solution to the Betaisodona1 Wundgaze formulation, 8 mm punches of the commercially available Betaisodona1 Wundgaze were also placed on the agarose gel at the same time points. Due to its strong brown colour, the diffusion of the povidoneiodine-based antiseptic was possible without the addition of a tracking molecule, therefore profiles for Betaisodona1 were analysed through standard photography.
Both the 10 kDa and 70 kDa FITC-dextran molecules were taken up efficiently (Fig. 1A) with about 65% respectively 82% uptake already after 30 min and a similar maximum uptake of 97% and 96% after 120 min. In comparison, the 2000 kDa molecule showed an uptake of 69% after 30 min but an overall maximum uptake of only 65%. The release of the FITC-dextran molecules showed more differences than the uptake profiles (Fig. 1B). 13% of the 10 kDa FITC-dextran molecules were retained after 48 h, releasing 87% of its content, compared to about 39% of the 70 kDa after the same period, releasing only 61%. The 2000 kDa molecules showed a long and sustained release of about 10% (24 h) into the agarose gel resulting in a final retained concentration of 60%. A similar pattern of release from the BNC sheets was observed when the FITC-dextran molecules diffusion into the agarose gel was visualized. Especially the 10 kDa FITC-dextran molecules showed good mobility reaching a larger and deeper area in the agarose matrix, while the 2000 kDa showed a reduced mobility, only reaching areas close to the surface even after 24 h of contact (Fig. 1C).
3.2. 2.6.
Uptake and release of Octenisept1 and Betaisodona1
Efficacy of the loaded antiseptics against S. aureus 1
1
Efficacy of both Octenisept and Betaisodona loaded BNC sheets was tested using a modified version of the method [30,31] used by prior works [25]. In short, an adapted standardized bacterial disk diffusion assay for S. aureus (ATCC 6538) was performed, where cells were diluted to a concentration of 105 cfu/mL and cultured on Müller-Hinton agar medium. BNC sheets were loaded with the two antiseptic solutions for 10, 30 and 120 min. 4 mm punch samples were then extracted in duplicates from each BNC sheet and placed on the surface of the agar plates. 4 mm punches of Betaisodona1 Wundgaze were used as a positive control for the BNC sheets loaded with Betaisodona1 . 70 mL of Octenisept1 gel was used as positive control for the BNC sheets loaded with Octenisept1 . Unloaded 4 mm BNC punches were used as negative control. The agar plates containing the bacteria as well as the BNC punches were incubated overnight at 37 C. Images acquired from the agar plates were analysed using the software ImageJ in order to measure the pixel number and hence estimate the area of zone of inhibition (ZOI) produced by each treatment.
2.7.
Results
Octenisept1 showed a good uptake rate already reaching 50% after 10 min and about 70% after 30 min. Complete equilibration was obtained after 120 min. Compared to Octenisept1 , the uptake of Betaisodona1 was far slower reaching only about 30% after 30 min and a maximum of 65% after 120 min (Fig. 2A). 100% indicates the possible maximum uptake of the antiseptics in the BNC sheets, which is usually around 80% of its content because of the pre-existing water content of the BNC sheets. Compared to previously tested antiseptics [27] the uptake of Octenisept1 was much higher whereas Betaisodona1 showed the lowest uptake concentrations of all (Fig. 3). Regarding the release of the antiseptic solutions from the BNC sheets, contrary observations were made in comparison to the uptake of the antiseptic solutions. After 3, 24 and 48 h considerably less Betaisodona1 was retained in the BNC punches compared to the values achieved for Octenisept1 at the same time points (Fig. 2B). After 48 h, only 1% of Betaisodona1 was retained, indicating that nearly all of its content had been released, whereas 18% of Octenisept1 was still left in the punches after the same time.
Statistical analysis 3.3.
The data acquired by the UV/VIS spectrophotometer and the fluorescent spectrometer were automatically transferred into Excel data sheets. The statistical analysis was performed by using Microsoft Excel 2016 (Microsoft Corporation, United States). The diagrams were made using the same programme. The figures were made using Inkscape (Inkscape Community, United States), the software ImageJ was used to measure the number of pixels and calculate the zone of inhibition in the images of the adapted standardized bacterial disk diffusion assay.
Diffusion profile of Betaisodona1
Betaisodona1 solution was released from the BNC sheets efficiently over 24 h (Fig. 4 bottom rows) while the release of the commercially available Betaisodona1 Wundgaze was no longer visually detectable after more than 6 h (Fig. 4 top rows).
3.4.
Antimicrobial effects in the agar-diffusion-assay
The ZOI increased distinctly with the incubation time used to load the BNC with Octenisept1 , with the highest values achieved between 30 and 120 min (Fig. 5A and B). The incubation time of 10 min yielded similar results as the positive control (commercially available Octenisept1 gel). The ZOI obtained for Betaisodona1 after loading into BNC (Fig. 5C and D) was also time dependent. Inhibition capacity was always higher compared Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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Fig. 1 – Uptake and release capacity for bacterial nanocellulose (BNC) loaded with 10 kDa, 70 kDa and 2000 kDa fluorescein isothioeyanate labelled dextran (FITC-dextran) molecules; A — Percent uptake relative to the maximum absolute loading capacity of BNC (100%). * marks the significant difference (p < 0.05) of the uptake of 2000 kDa FITC-dextran in comparison to the 10 kDa and 70 kDa FITC-dextrans. B — Relative amount of FITC-dextran molecules remaining in BNC punches relative to the starting concentration of each molecule. * marks the significant difference (p < 0.05) in the release of the FITCdextran molecules in comparison to all the other molecules. C — Diffusion profile of the FITC-dextran molecules on agarose gel. Longitudinal section of the agarose gel after contact with 8 mm BNC loaded punches for 2 h and 24 h. Values are shown as mean SD.
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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to the positive control (Wundgaze). At each time point, the mean calculated values for the inhibition area were much higher compared to the BNC punches loaded with Octenisept1 showing higher efficacy of Betaisodona1 against S. aureus. Even though the ZOI for Octenisept1 was lower in comparison to Betaisodona1 , the loaded concentration in the punches were far higher than the necessary effective minimal bactericidal concentration after 24 h of incubation (MBC24) for methicillin-resistant S. aureus (Table 1). Already after 30 min both tested antiseptics showed higher concentrations than the needed MCB24 similar to previously tested antiseptics [27].
4.
Discussion
We assessed the uptake and release capacity of BNC for molecules and two antiseptics with different molecular weight and compared the diffusion rates into a solid matrix mimicking a wound bed. In addition, the antiseptic efficacy of BNC loaded with the antiseptic solutions was tested against S. aureus.
Fig. 2 – Uptake and release capacity for bacterial nanocellulose (BNC) loaded with Octenisept1 and Betaisodona1 ; A — Percent uptake based on the possible maximum loading capacity for Octenisept1 (110%) and for Betaisodona1 (100%). B — Relative amount of Octenisept1 and Betaisodona1 retained in BNC punches relative to the starting concentration of each solution. Values are shown as mean SD.
Fig. 3 – Uptake comparison of antiseptic solutions. Bars (mean SD) indicate the maximum uptake (in percent) in bacterial nanocellulose (BNC) sheets of Octenisept1 and Betaisodona1 compared to previously tested LAVANID1 2 and Prontosan1 [27].
In prior experiments, we made the observation that lighter molecules not necessarily achieved better uptake or release kinetics in BNC. Therefore, the usage of, pure molecules with an accurately defined molecular weight would give more insight in the uptake and release abilities of BNC. The uptake of lighter FITC-dextran molecules into the BNC sheets was much faster in comparison to heavier molecules due to the difference in molecular weight which has already been described for the diffusion into aqueous solutions [29]. The release tests and diffusion profiles showed that the mobility of the molecules through the BNC/ agarose gel interface was also inversely proportional to the weight of the molecules. Lighter molecules are loaded and released from BNC faster than high molecular weight molecules, which in return are delivered more continuously for extended periods of time. This is an interesting feature of BNC for potential applications as a transdermal delivery system [32] for heavier molecules such as hyaluronic acids (5 kDa 20,000 kDa) [33], albumin (66.5 kDa) [34,35], peptides which improve transdermal delivery (29 kDa) [36] or antibodies (e.g. 150 kDa for IgG) [37]. Octenidine (624 Da) had an uptake and release behaviour similar to the two lighter FITC-dextran molecules as well as previously tested PHMB (186 Da) [27]. Preliminary tests have shown that the used BNC sheets are usually able to take up around 80% of any solution because of their pre-existing water content. BNC sheets loaded with Octenisept1 showed an unexpected swelling capacity, which was 30% higher than the other antiseptics tested such as Prontosan1 , LAVANID1 2 or Betaisodona1 reaching results of more than 100%. Thanks to pioneering works such as the ones published by Wiegand et al. and Moritz et al. [21,28], analysis of the release capacity of the tested antiseptics through BNC fleeces have already been focus of research. The continuous release curve observed for Octenisept1 can be beneficial for wound treatment applications because it achieves steady concentrations, in concordance of results prior published [28], over the entire treatment period when a dressing is placed on the wound. Comparing all tested antiseptic solutions, including our previously published work [27], good uptake rates were achieved for all antiseptics. However, Octenisept1 achieved even better uptake rates than PHMB-
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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Fig. 4 – Diffusion profile for Betaisodona1 Wundgaze and Betaisodona1 loaded bacterial nanocellulose (BNC) punches. Top row: Betaisodona1 Wundgaze punches, third row: Betaisodona1 loaded BNC punches. Colour of punches indicates remaining content. Second and bottom row: Diffusion area on agarose gel after removal of 8 mm punches after 1, 3, 6, 12 and 24 h of incubation.
containing antiseptics although having a greater molecular weight in comparison. The formulation of Octenisept1 might be the reason for this difference in uptake rates, since in our previous published results also different loading capacities were observed for PHMB based antiseptics with two different formulations (Prontosan1 and LAVANID 21 ). Based on the results for the 10 and 70 kDa FITC-dextran molecules, we higher uptake levels for the expected Betaisodona1 solution considering the similar weight of 40 kDa when povidone-iodine molecules form chains. However, the release of Betaisodona1 was even faster between 3 and 24 h relative to the lighter tested FITC-dextran molecules. Similar pace was described for the same molecule in the literature but using different experimenting settings [21]. This might be due to the unknown extent of chain forming of povidoneiodine. The actual molecular weight could be smaller than expected or the oxidation of povidone-iodine, which is responsible for the loss of colour over time, could have influenced the UV/VIS spectrophotometry analysis resulting in falsely low retention rates. Another characteristic to be considered regarding the difference on the results for the FITC-dextran molecules and the antiseptics is the presence of excipients, as considered for octenidine as well, that might influence its uptake and release kinetics in BNC. Using BNC sheets to release Betaisodona1 solution allowed us to achieve an almost 6 times higher concentration of povidone-iodine compared to Betaisodona1 Wundgaze punch of the same size. Betaisodona1 Wundgaze released most of its content in the first 3 h while the BNC sheet loaded with the Betaisodona1 solution continuously released the povidone-iodine for much longer periods of up to 24 h. The efficacy of Octenisept1 loaded BNC sheets against S. aureus was comparable for every incubation time tested relative to the Octenisept1 gel. Even after a short period of incubation (10 min), the inhibition by the Octenisept1 loaded BNC sheet was similar to the gel
formulation, although the applied absolute amount of octenidine in the gel as positive control was, approximately, three times higher (11,9 ng for the former and 35,4 ng for the latter). The antibacterial efficacy achieved by Betaisodona1 loaded BNC sheets was also superior to Betaisodona1 Wundgaze. The inhibition area achieved after a short incubation period (10 min) was more than 1.5 times larger than the positive control with Betaisodona1 Wundgaze. The higher efficacy achieved by the combination of BNC and the antiseptics could become a useful tool against methicillin-resistant S. aureus (MRSA) when compared to the minimal bactericidal concentration needed to kill the bacteria after 24 h (MBC24). Since, even after very short loading period, the BNC was able to carry enough antiseptic concentrations exceeding the MBC24 reported in the literature [38,39]. Although, further tests aiming this capacity is still in progress. Based on the achieved results, we consider that the combination of the tested antiseptics and the BNC wound dressings could have a benefit in clinical practise. Since some BNC-based wound dressing could be left on a wound for up to 7 days, the combination with an antimicrobial agent would enforce this reduced need of dressing changes, which would be done every two to three days in a standard procedure. Fewer changes would mean less interference with the wound healing that could otherwise potentially lead to the removal of the newly produced layer of keratinocytes [40,41]. In addition, the combined set would also reduce the number of wound irrigations needed. Since positive pressure irrigation is highly controversial whether or not it leads to further tissue damage or displacement of contamination [42,43]. Furthermore, a continuous antiseptic release as shown in this study would have a positive effect on fragile tissue, especially in combination with the ability of BNC to also absorb excess exudate of the wound.
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Fig. 5 – Adapted standardized bacterial disk diffusion assay for Staphylococcus aureus using bacterial nanocellulose (BNC) loaded with Octenisept1 (top) and Betaisodona1 (bottom). Octenisept1 gel and Betaisodona1 Wundgaze were used as positive controls. Unloaded BNC punches were used as negative control. A and C — Images of the zone of inhibition achieved by punches loaded with Octenisept1 /Betaisodona1 for different periods (10, 30 and 120 min). The dotted line delineates the area where Octenisept1 gel was applied. B and D — Quantification of the inhibition area based on the number of pixels of the image (excluding the area in direct contact with the 4 mm punch or with the Octenisept1 gel/Betaisodona1 Wundgaze). Inside the bars are the concentration of octenidine contained in each treatment. * marks the significant difference to the positive control, p < 0.05. For Octenisept1 the difference to positive control at 120 min was not significant (p = 0.050245). Neg. — negative control; Pos. — positive control.
Further experiments will be performed focusing on in-vivo studies analysing the influence of the prolonged release of antiseptics on the healing process.
5.
Conclusion
The utilization of FITC-dextrans with different molecular weights for testing the uptake and release in BNC is useful to investigate the molecular weight
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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Table 1 – Concentration of the loaded antiseptic in the bacterial nanocellulose (BNC) sheets in comparison to the minimal bactericidal concentration (MBC) against methicillin-resistant Staphylococcus aureus (S. aureus MRSA). Concentration (mg/L) of Octenisept1 and Betaisodona1 in BNC sheets after 30, 60 and 120 min incubation compared with previously published results for LAVANID1 2, Prontosan1 [27]. Last column shows the MBC24 for methicillinresistant S. aureus (MRSA). PHMB — Polyhexanide. Antiseptics LAVANID1 2 (PHMB) [27] Prontosan1 (PHMB) [27] Octenisept1 (octenidine) Betaisodona1 (povidone-iodine)
Stock solution
30 min
60 min
120 min
400 1000 1000 100,000
110 260 360 10,000
190 530 610 20,000
230 760 800 50,000
dependent mobility of molecules into and out of BNC matrices. The obtained results can support further tests combining BNC with other high weight molecules with medical applications, such as hyaluronic acid, albumin, or even antibodies. Results of kinetic achieved for the povidone-iodine solution in comparison to the one achieved with FITC-dextran molecules also showed that molecular weight is most likely not the only factor influencing the uptake/release of the molecule. Specific formulations of commercially available antiseptics might have a considerable influence. The steady concentration release for the octenidine antiseptic loaded into the BNC matrix highlight the versatility of this wound dressing. Therefore, novel combinations of BNC and antiseptics or other therapeutics should be tested regarding uptake, release and efficacy prior to any clinical use.
6.
Limitations
All tests were performed in vitro although tested antiseptics are already available on the market. The excipients present in the analysed antiseptics must be considered for the obtained results on the uptake and release in order to establish a correlation between the kinetics of the FITC-dextran molecules and the active molecules, since the aim of the work was not to analyse the active molecule alone but rather the commercially available formulation. The results of the release experiments of the antiseptics may not be directly transferred into clinical practice without consideration. The use of an agar plate is not a comparable receiving medium to an actual wound bed. These results do not give final information how the release kinetics would look like if the antiseptics were to be released onto an actual wound. Additionally, in every wound the presence of exudate might occur which would interact with the BNC as well as the antiseptic solution. This interaction was not considered in the present study and will be assessed in future works. To confirm the superior antimicrobial efficacy of the loaded BNC relative to the commercially available formulations (Octenisept1 gel and Betaisodona1 Wundgaze) results should be further tested in a clinical trial to determine the in-vivo efficacy.
Conflict of interests Martin Funk is an employee of QRSkin GmbH whose product epicitehydro was used for the experiments.
MBC24 for MRSA 1.0 [44] 0.375 3.0 [39,44] 1024 [44]
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Available online at www.sciencedirect.com
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Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine Ives Bernardelli de Mattos a , Sebastian P. Nischwitz b,c , Alexandru-Cristian Tuca b , Florian Groeber-Becker a , Martin Funk d , Thomas Birngruber e , Selma I. Mautner b,e , Lars-Peter Kamolz b,c, Judith C.J. Holzer b,c, * a
Fraunhofer Institute for Silicate Research ISC, Translational Center Regenerative Therapies, Würzburg, Germany Division of Plastic, Aesthetic and Reconstructive Surgery, Department of Surgery, Medical University of Graz, Austria c Joanneum Research Forschungsgesellschaft mbH, COREMED — Cooperative Centre for Regenerative Medicine, Graz, Austria d QRSKIN GmbH, Friedrich-Bergius-Ring 15, 97076 Würzburg, Germany e Joanneum Research Forschungsgesellschaft mbH, HEALTH Institute for Biomedicine and Health Sciences, Graz, Austria b
article info
abstract
Article history:
Background: Bacterial nanocellulose (BNC) is considered a promising carrier for various substances and novel
Accepted 2 October 2019
approaches using BNC in combination with antiseptics are well documented. However, the difference in the
Available online xxx
molecular weight of these molecules influences their uptake by and release from BNC. Analysing the diffusion of standard molecules with different weight, e.g. dextrans, offers the possibility to investigate the mobility of various molecules. We aimed to test the use of BNC regarding uptake and release of different standard molecules as well
Keywords: Bacterial nanocellulose Antiseptic uptake Antiseptic release Octenidine Povidone-iodine Antimicrobial wound dressing
as two commercially available antiseptics for possible applications in future wound dressings. Material and methods: Diffusion profiles, uptake and release of three FITC-dextran molecules differing in weight as well as octenidine (Octenisept1 ) and povidone-iodine (Betaisodona1 )-based antiseptics were tested with BNC-based wound dressings. Furthermore, the antiseptic efficacy of BNC in combination with antiseptics against Staphylococcus aureus was tested. Results: Uptake and release capacity for FITC-dextran molecules showed a molecular weight-dependent mobility from BNC into an agarose gel. The loading capacity of BNC was also inversely proportional to the molecular weight of the antiseptics. The release test for octenidine showed a sustained and prolonged delivery into a solid matrix, whereas povidone-iodine was released faster. Both antiseptic solutions combined with BNC showed a good dose-dependent efficacy against S. aureus. Conclusion: Results obtained from the mobility of FITC-dextran molecules in the BNC matrix could open possible applications for the combination of BNC with other molecules for medical applications. Combination of both tested antiseptics with BNC showed to be an efficient approach to control bacterial infections. © 2019 Elsevier Ltd and ISBI. All rights reserved.
1.
Introduction
* Corresponding author at: Division of Plastic, Aesthetic and Reconstructive Surgery, Department of Surgery, Medical University of Graz, Auenbruggerplatz 29, 8036 Graz, Austria. E-mail address: [email protected] (J.C.J. Holzer). https://doi.org/10.1016/j.burns.2019.10.006 0305-4179/© 2019 Elsevier Ltd and ISBI. All rights reserved.
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An efficient healing process of burn injuries depends on intricate biological mechanisms [1 3]. Wound dressings have to meet many requirements in order to support the healing processes optimally. Currently, the development progress of novel wound dressing is very well documented [4 12]. Characteristics such as being non-adhesive, reducing the number of dressing changes [13], offering an antimicrobial activity, allowing routine monitoring of biomarkers [14], providing a controlled moisture balance on the wound-dressing interface [15 17], are just some of the requirements that such dressings should comprise. Notwithstanding this challenge, a product with those characteristics would be very important to shorten hospitalization time, reduce the number of painful dressing changes and avoid infections, therefore increase the quality, reduce morbidity and mortality, and ameliorate the treatment for patients, apart from a possible cost reduction [18]. Regarding the capacity of preventing infections, a source for the continuous release of antiseptic molecules to control the growth of microorganisms is needed. Some antiseptics, such as Octenisept1 and Betaisodona1 , are already largely used in the routine of burn treatment centres [19,20]. In order to combine some of those above-mentioned characteristics, a biomaterial produced by certain bacteria such as Komagataeibacter sp., Agrobacterium sp., Rhizobium sp., Sarcina sp. [21] arise as a suitable candidate [22 26]. Combining wound dressings based on bacterial nanocellulose (BNC) with different antiseptics were also prior subject of investigation [21 28], however the contribution of differences in the molecular weight of active molecules of antiseptics (PHMB = 186 Da; octenidine = 624 Da; and povidone-iodine up to 40,000 Da) in the uptake and release from the BNC has not yet been addressed systematically. Thus, dextran molecules just differing in weight decorated with fluorescent dyes can be used as a tracking system to study their uptake and release kinetics [29]. In the present study, the uptake and release capacity of BNC loaded with fluorescein isothioeyanate labelled dextran (FITC-dextran) molecules of different weights were analysed. Further, the diffusion rates of fluorescent molecules into a solid matrix to mimic a wound bed was performed. The uptake and release capacity of BNC was also tested using two antiseptic solutions containing povidone-iodine and octenidine dihydrochloride in comparison to commercially available formulations of these antiseptics. In addition, the antiseptic efficacy against Staphylococcus aureus using BNC loaded with the aforementioned antibacterial solutions was tested.
2.
Material and methods 1
Epicitehydro 10 10 cm sheets (QRSkin GmbH, Germany; Ref-No. 800003-M02B) were used as the BNC matrix for the experiments. 10 kDa (FD10S-100MG, FITC-labelled dextran molecules lot#SLBN7802 V), 70 kDa (FD70S-100MG, lot#SLBQ4377 V) and were obtained from 2000 kDa (FD2000S-100MG, lot# SLBL5816 V) SIGMA-ALDRICH Chemie GmbH (Germany). A commercially available octenidine-containing antiseptic was used as a solution (Octenisept1 ; 0.1% octenidine dihydrochlorid = 624 Da) and as a gel formulation (Octenisept1 Gel; 0.05% octenidine dihydrochlorid) both obtained from Schülke & Mayr GmbH, Germany. Betaisodona1 solution (Mundipharma GmbH, Germany) was used as source of povidone-iodine (10%; up to 40 kDa) in solution for the diffusion tests and Betaisodona1 Wundgaze (Mundipharma GmbH, Germany) as wound dressing.
2.1.
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Uptake of FTIC-dextran molecules in BNC sheets
The uptake of FITC-dextran molecules with different molecular weight was performed by incubating three BNC sheets in a 10 10 cm square petri dish covered with 100 mL solution of 0.025 mg/mL of FITC-dextran in three different weights (10 kDa, 70 kDa and 2000 kDa). The incubation was performed at room temperature under light-protected conditions. After an incubation period of either 10, 20, 30, 60 and 120 min, two punches with 8 mm in diameter were extracted from each BNC sheet and placed in membrane spin columns (200 mm pore size) and centrifuged 10 min at 10,000 rpm. Any retained content in the spin columns was eluted in 4% sodium dodecyl sulphate (SDS) solution and centrifuged a second time. The collected samples were pooled together and analysed by fluorescence spectrophotometry.
2.2.
Uptake of antiseptics in BNC sheets
In order to emulate the clinical emergency setting, two BNC sheets were put in a steel kidney dish and covered with 200 mL of Octenisept1 or Betaisodona1 solution. After 10, 20, 30, 60 and 120 min incubation at room temperature, two 8 mm diameter punch samples were taken from each BNC sheet, centrifuged with the same method as described for the FITC-dextran molecules and analysed by UV/VIS spectrophotometry.
2.3. Quantification of FITC-dextran molecules and antiseptic solutions Ultraviolet/Visible (UV/VIS) spectrophotometry analysis was chosen to quantify the concentrations of the antiseptic molecules. For both Octenisept 1 (281 nm) and Betaisodona1 (293 nm) solution a UV-readable 96 well plate (CLS3635 SIGMA Corning1 UV-Transparent Microplates) was used. To quantify the uptake and release of FITC-dextran molecules in BNC sheets, the excitation/emission spectrum peaks for the fluorophore (495 nm and 519 nm respectively) were measured using a Corning1 96 Well Black Polystyrene Microplate (CLS3603 SIGMA). All the spectrophotometry readings were performed using an Infinite1 200 PRO (TECAN Trading AG, Switzerland).
2.4. Release of the FITC-dextran molecules and the antiseptic solutions 1% agarose gels were prepared with a 50 mL volume in 10 10 cm square petri dishes. The gels acted as receptors for the release of molecules from loaded BNC sheets. For loading, BNC sheets were immersed for 120 min in one of the five solutions: FITC-dextran molecules (10 kDa, 70 kDa and 2000 kDa), Octenisept1 and Betaisodona1 . 21 punches (8 mm) were taken from each of the loaded BNC sheets and arranged on the surface of the gels together with 3 punches from unloaded BNC sheets as negative control resulting in a total of 24 punches on each gel. Betaisodona1 loaded punches were incubated on the gel under light-protected conditions to avoid oxidation and thus colour loss. Incubation and release of the FITC-dextran molecules were performed without exposure to light, to avoid fluorescent bleaching. At 30 min, 1, 2, 4, 24 and 48 h of incubation time, three BNC punches from each solution were taken off the gels to measure the retained content of each solution. 3 loaded punches with no contact with the gel were also analysed and considered as t =0 min. All these collected punches were immediately placed in membrane spin columns (200 mm pore size) and centrifuged 10 min at 10,000 rpm. Any retained solution on the column was eluted with a 4% SDS solution and centrifuged again. The collected antiseptic solutions
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were measured by UV/VIS spectrophotometry. Fluorescent samples were analysed through fluorescent spectrometry.
3.
2.5. Diffusion profiles of the FITC-dextran molecules and Betaisodona1
3.1. Uptake, release and diffusion profile of the FITC-dextran molecules
In order to visualize the diffusion profile of the FITC-dextran molecules and Betaisodona1 into a wound-emulating agarose matrix, 100 mL of a 1% agarose gel was prepared in a 10 10 cm square petri dish. BNC sheets were incubated overnight with each of the FITC-dextran molecule solutions (10 kDa, 70 kDa and 2000 kDa) and 120 min with the Betaisodona1 solution. 8 mm punches of each BNC sheet loaded with FITC-dextran were placed on the agarose for 2 h and 24 h. A longitudinal section of the agarose gel was performed using a scalpel and pictures of the FTIC-dextran profile was taken using a FluorChem Q (Cell Biosciences acquired from Biozym GmbH) imaging system. 8 mm punches of the BNC sheet loaded with Betaisodona1 solution were placed on the agarose for 1, 3, 6, 12 and 24 h. To compare the diffusion profile of Betaisodona1 solution to the Betaisodona1 Wundgaze formulation, 8 mm punches of the commercially available Betaisodona1 Wundgaze were also placed on the agarose gel at the same time points. Due to its strong brown colour, the diffusion of the povidoneiodine-based antiseptic was possible without the addition of a tracking molecule, therefore profiles for Betaisodona1 were analysed through standard photography.
Both the 10 kDa and 70 kDa FITC-dextran molecules were taken up efficiently (Fig. 1A) with about 65% respectively 82% uptake already after 30 min and a similar maximum uptake of 97% and 96% after 120 min. In comparison, the 2000 kDa molecule showed an uptake of 69% after 30 min but an overall maximum uptake of only 65%. The release of the FITC-dextran molecules showed more differences than the uptake profiles (Fig. 1B). 13% of the 10 kDa FITC-dextran molecules were retained after 48 h, releasing 87% of its content, compared to about 39% of the 70 kDa after the same period, releasing only 61%. The 2000 kDa molecules showed a long and sustained release of about 10% (24 h) into the agarose gel resulting in a final retained concentration of 60%. A similar pattern of release from the BNC sheets was observed when the FITC-dextran molecules diffusion into the agarose gel was visualized. Especially the 10 kDa FITC-dextran molecules showed good mobility reaching a larger and deeper area in the agarose matrix, while the 2000 kDa showed a reduced mobility, only reaching areas close to the surface even after 24 h of contact (Fig. 1C).
3.2. 2.6.
Uptake and release of Octenisept1 and Betaisodona1
Efficacy of the loaded antiseptics against S. aureus 1
1
Efficacy of both Octenisept and Betaisodona loaded BNC sheets was tested using a modified version of the method [30,31] used by prior works [25]. In short, an adapted standardized bacterial disk diffusion assay for S. aureus (ATCC 6538) was performed, where cells were diluted to a concentration of 105 cfu/mL and cultured on Müller-Hinton agar medium. BNC sheets were loaded with the two antiseptic solutions for 10, 30 and 120 min. 4 mm punch samples were then extracted in duplicates from each BNC sheet and placed on the surface of the agar plates. 4 mm punches of Betaisodona1 Wundgaze were used as a positive control for the BNC sheets loaded with Betaisodona1 . 70 mL of Octenisept1 gel was used as positive control for the BNC sheets loaded with Octenisept1 . Unloaded 4 mm BNC punches were used as negative control. The agar plates containing the bacteria as well as the BNC punches were incubated overnight at 37 C. Images acquired from the agar plates were analysed using the software ImageJ in order to measure the pixel number and hence estimate the area of zone of inhibition (ZOI) produced by each treatment.
2.7.
Results
Octenisept1 showed a good uptake rate already reaching 50% after 10 min and about 70% after 30 min. Complete equilibration was obtained after 120 min. Compared to Octenisept1 , the uptake of Betaisodona1 was far slower reaching only about 30% after 30 min and a maximum of 65% after 120 min (Fig. 2A). 100% indicates the possible maximum uptake of the antiseptics in the BNC sheets, which is usually around 80% of its content because of the pre-existing water content of the BNC sheets. Compared to previously tested antiseptics [27] the uptake of Octenisept1 was much higher whereas Betaisodona1 showed the lowest uptake concentrations of all (Fig. 3). Regarding the release of the antiseptic solutions from the BNC sheets, contrary observations were made in comparison to the uptake of the antiseptic solutions. After 3, 24 and 48 h considerably less Betaisodona1 was retained in the BNC punches compared to the values achieved for Octenisept1 at the same time points (Fig. 2B). After 48 h, only 1% of Betaisodona1 was retained, indicating that nearly all of its content had been released, whereas 18% of Octenisept1 was still left in the punches after the same time.
Statistical analysis 3.3.
The data acquired by the UV/VIS spectrophotometer and the fluorescent spectrometer were automatically transferred into Excel data sheets. The statistical analysis was performed by using Microsoft Excel 2016 (Microsoft Corporation, United States). The diagrams were made using the same programme. The figures were made using Inkscape (Inkscape Community, United States), the software ImageJ was used to measure the number of pixels and calculate the zone of inhibition in the images of the adapted standardized bacterial disk diffusion assay.
Diffusion profile of Betaisodona1
Betaisodona1 solution was released from the BNC sheets efficiently over 24 h (Fig. 4 bottom rows) while the release of the commercially available Betaisodona1 Wundgaze was no longer visually detectable after more than 6 h (Fig. 4 top rows).
3.4.
Antimicrobial effects in the agar-diffusion-assay
The ZOI increased distinctly with the incubation time used to load the BNC with Octenisept1 , with the highest values achieved between 30 and 120 min (Fig. 5A and B). The incubation time of 10 min yielded similar results as the positive control (commercially available Octenisept1 gel). The ZOI obtained for Betaisodona1 after loading into BNC (Fig. 5C and D) was also time dependent. Inhibition capacity was always higher compared Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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Fig. 1 – Uptake and release capacity for bacterial nanocellulose (BNC) loaded with 10 kDa, 70 kDa and 2000 kDa fluorescein isothioeyanate labelled dextran (FITC-dextran) molecules; A — Percent uptake relative to the maximum absolute loading capacity of BNC (100%). * marks the significant difference (p < 0.05) of the uptake of 2000 kDa FITC-dextran in comparison to the 10 kDa and 70 kDa FITC-dextrans. B — Relative amount of FITC-dextran molecules remaining in BNC punches relative to the starting concentration of each molecule. * marks the significant difference (p < 0.05) in the release of the FITCdextran molecules in comparison to all the other molecules. C — Diffusion profile of the FITC-dextran molecules on agarose gel. Longitudinal section of the agarose gel after contact with 8 mm BNC loaded punches for 2 h and 24 h. Values are shown as mean SD.
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to the positive control (Wundgaze). At each time point, the mean calculated values for the inhibition area were much higher compared to the BNC punches loaded with Octenisept1 showing higher efficacy of Betaisodona1 against S. aureus. Even though the ZOI for Octenisept1 was lower in comparison to Betaisodona1 , the loaded concentration in the punches were far higher than the necessary effective minimal bactericidal concentration after 24 h of incubation (MBC24) for methicillin-resistant S. aureus (Table 1). Already after 30 min both tested antiseptics showed higher concentrations than the needed MCB24 similar to previously tested antiseptics [27].
4.
Discussion
We assessed the uptake and release capacity of BNC for molecules and two antiseptics with different molecular weight and compared the diffusion rates into a solid matrix mimicking a wound bed. In addition, the antiseptic efficacy of BNC loaded with the antiseptic solutions was tested against S. aureus.
Fig. 2 – Uptake and release capacity for bacterial nanocellulose (BNC) loaded with Octenisept1 and Betaisodona1 ; A — Percent uptake based on the possible maximum loading capacity for Octenisept1 (110%) and for Betaisodona1 (100%). B — Relative amount of Octenisept1 and Betaisodona1 retained in BNC punches relative to the starting concentration of each solution. Values are shown as mean SD.
Fig. 3 – Uptake comparison of antiseptic solutions. Bars (mean SD) indicate the maximum uptake (in percent) in bacterial nanocellulose (BNC) sheets of Octenisept1 and Betaisodona1 compared to previously tested LAVANID1 2 and Prontosan1 [27].
In prior experiments, we made the observation that lighter molecules not necessarily achieved better uptake or release kinetics in BNC. Therefore, the usage of, pure molecules with an accurately defined molecular weight would give more insight in the uptake and release abilities of BNC. The uptake of lighter FITC-dextran molecules into the BNC sheets was much faster in comparison to heavier molecules due to the difference in molecular weight which has already been described for the diffusion into aqueous solutions [29]. The release tests and diffusion profiles showed that the mobility of the molecules through the BNC/ agarose gel interface was also inversely proportional to the weight of the molecules. Lighter molecules are loaded and released from BNC faster than high molecular weight molecules, which in return are delivered more continuously for extended periods of time. This is an interesting feature of BNC for potential applications as a transdermal delivery system [32] for heavier molecules such as hyaluronic acids (5 kDa 20,000 kDa) [33], albumin (66.5 kDa) [34,35], peptides which improve transdermal delivery (29 kDa) [36] or antibodies (e.g. 150 kDa for IgG) [37]. Octenidine (624 Da) had an uptake and release behaviour similar to the two lighter FITC-dextran molecules as well as previously tested PHMB (186 Da) [27]. Preliminary tests have shown that the used BNC sheets are usually able to take up around 80% of any solution because of their pre-existing water content. BNC sheets loaded with Octenisept1 showed an unexpected swelling capacity, which was 30% higher than the other antiseptics tested such as Prontosan1 , LAVANID1 2 or Betaisodona1 reaching results of more than 100%. Thanks to pioneering works such as the ones published by Wiegand et al. and Moritz et al. [21,28], analysis of the release capacity of the tested antiseptics through BNC fleeces have already been focus of research. The continuous release curve observed for Octenisept1 can be beneficial for wound treatment applications because it achieves steady concentrations, in concordance of results prior published [28], over the entire treatment period when a dressing is placed on the wound. Comparing all tested antiseptic solutions, including our previously published work [27], good uptake rates were achieved for all antiseptics. However, Octenisept1 achieved even better uptake rates than PHMB-
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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Fig. 4 – Diffusion profile for Betaisodona1 Wundgaze and Betaisodona1 loaded bacterial nanocellulose (BNC) punches. Top row: Betaisodona1 Wundgaze punches, third row: Betaisodona1 loaded BNC punches. Colour of punches indicates remaining content. Second and bottom row: Diffusion area on agarose gel after removal of 8 mm punches after 1, 3, 6, 12 and 24 h of incubation.
containing antiseptics although having a greater molecular weight in comparison. The formulation of Octenisept1 might be the reason for this difference in uptake rates, since in our previous published results also different loading capacities were observed for PHMB based antiseptics with two different formulations (Prontosan1 and LAVANID 21 ). Based on the results for the 10 and 70 kDa FITC-dextran molecules, we higher uptake levels for the expected Betaisodona1 solution considering the similar weight of 40 kDa when povidone-iodine molecules form chains. However, the release of Betaisodona1 was even faster between 3 and 24 h relative to the lighter tested FITC-dextran molecules. Similar pace was described for the same molecule in the literature but using different experimenting settings [21]. This might be due to the unknown extent of chain forming of povidoneiodine. The actual molecular weight could be smaller than expected or the oxidation of povidone-iodine, which is responsible for the loss of colour over time, could have influenced the UV/VIS spectrophotometry analysis resulting in falsely low retention rates. Another characteristic to be considered regarding the difference on the results for the FITC-dextran molecules and the antiseptics is the presence of excipients, as considered for octenidine as well, that might influence its uptake and release kinetics in BNC. Using BNC sheets to release Betaisodona1 solution allowed us to achieve an almost 6 times higher concentration of povidone-iodine compared to Betaisodona1 Wundgaze punch of the same size. Betaisodona1 Wundgaze released most of its content in the first 3 h while the BNC sheet loaded with the Betaisodona1 solution continuously released the povidone-iodine for much longer periods of up to 24 h. The efficacy of Octenisept1 loaded BNC sheets against S. aureus was comparable for every incubation time tested relative to the Octenisept1 gel. Even after a short period of incubation (10 min), the inhibition by the Octenisept1 loaded BNC sheet was similar to the gel
formulation, although the applied absolute amount of octenidine in the gel as positive control was, approximately, three times higher (11,9 ng for the former and 35,4 ng for the latter). The antibacterial efficacy achieved by Betaisodona1 loaded BNC sheets was also superior to Betaisodona1 Wundgaze. The inhibition area achieved after a short incubation period (10 min) was more than 1.5 times larger than the positive control with Betaisodona1 Wundgaze. The higher efficacy achieved by the combination of BNC and the antiseptics could become a useful tool against methicillin-resistant S. aureus (MRSA) when compared to the minimal bactericidal concentration needed to kill the bacteria after 24 h (MBC24). Since, even after very short loading period, the BNC was able to carry enough antiseptic concentrations exceeding the MBC24 reported in the literature [38,39]. Although, further tests aiming this capacity is still in progress. Based on the achieved results, we consider that the combination of the tested antiseptics and the BNC wound dressings could have a benefit in clinical practise. Since some BNC-based wound dressing could be left on a wound for up to 7 days, the combination with an antimicrobial agent would enforce this reduced need of dressing changes, which would be done every two to three days in a standard procedure. Fewer changes would mean less interference with the wound healing that could otherwise potentially lead to the removal of the newly produced layer of keratinocytes [40,41]. In addition, the combined set would also reduce the number of wound irrigations needed. Since positive pressure irrigation is highly controversial whether or not it leads to further tissue damage or displacement of contamination [42,43]. Furthermore, a continuous antiseptic release as shown in this study would have a positive effect on fragile tissue, especially in combination with the ability of BNC to also absorb excess exudate of the wound.
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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Fig. 5 – Adapted standardized bacterial disk diffusion assay for Staphylococcus aureus using bacterial nanocellulose (BNC) loaded with Octenisept1 (top) and Betaisodona1 (bottom). Octenisept1 gel and Betaisodona1 Wundgaze were used as positive controls. Unloaded BNC punches were used as negative control. A and C — Images of the zone of inhibition achieved by punches loaded with Octenisept1 /Betaisodona1 for different periods (10, 30 and 120 min). The dotted line delineates the area where Octenisept1 gel was applied. B and D — Quantification of the inhibition area based on the number of pixels of the image (excluding the area in direct contact with the 4 mm punch or with the Octenisept1 gel/Betaisodona1 Wundgaze). Inside the bars are the concentration of octenidine contained in each treatment. * marks the significant difference to the positive control, p < 0.05. For Octenisept1 the difference to positive control at 120 min was not significant (p = 0.050245). Neg. — negative control; Pos. — positive control.
Further experiments will be performed focusing on in-vivo studies analysing the influence of the prolonged release of antiseptics on the healing process.
5.
Conclusion
The utilization of FITC-dextrans with different molecular weights for testing the uptake and release in BNC is useful to investigate the molecular weight
Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006
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Table 1 – Concentration of the loaded antiseptic in the bacterial nanocellulose (BNC) sheets in comparison to the minimal bactericidal concentration (MBC) against methicillin-resistant Staphylococcus aureus (S. aureus MRSA). Concentration (mg/L) of Octenisept1 and Betaisodona1 in BNC sheets after 30, 60 and 120 min incubation compared with previously published results for LAVANID1 2, Prontosan1 [27]. Last column shows the MBC24 for methicillinresistant S. aureus (MRSA). PHMB — Polyhexanide. Antiseptics LAVANID1 2 (PHMB) [27] Prontosan1 (PHMB) [27] Octenisept1 (octenidine) Betaisodona1 (povidone-iodine)
Stock solution
30 min
60 min
120 min
400 1000 1000 100,000
110 260 360 10,000
190 530 610 20,000
230 760 800 50,000
dependent mobility of molecules into and out of BNC matrices. The obtained results can support further tests combining BNC with other high weight molecules with medical applications, such as hyaluronic acid, albumin, or even antibodies. Results of kinetic achieved for the povidone-iodine solution in comparison to the one achieved with FITC-dextran molecules also showed that molecular weight is most likely not the only factor influencing the uptake/release of the molecule. Specific formulations of commercially available antiseptics might have a considerable influence. The steady concentration release for the octenidine antiseptic loaded into the BNC matrix highlight the versatility of this wound dressing. Therefore, novel combinations of BNC and antiseptics or other therapeutics should be tested regarding uptake, release and efficacy prior to any clinical use.
6.
Limitations
All tests were performed in vitro although tested antiseptics are already available on the market. The excipients present in the analysed antiseptics must be considered for the obtained results on the uptake and release in order to establish a correlation between the kinetics of the FITC-dextran molecules and the active molecules, since the aim of the work was not to analyse the active molecule alone but rather the commercially available formulation. The results of the release experiments of the antiseptics may not be directly transferred into clinical practice without consideration. The use of an agar plate is not a comparable receiving medium to an actual wound bed. These results do not give final information how the release kinetics would look like if the antiseptics were to be released onto an actual wound. Additionally, in every wound the presence of exudate might occur which would interact with the BNC as well as the antiseptic solution. This interaction was not considered in the present study and will be assessed in future works. To confirm the superior antimicrobial efficacy of the loaded BNC relative to the commercially available formulations (Octenisept1 gel and Betaisodona1 Wundgaze) results should be further tested in a clinical trial to determine the in-vivo efficacy.
Conflict of interests Martin Funk is an employee of QRSkin GmbH whose product epicitehydro was used for the experiments.
MBC24 for MRSA 1.0 [44] 0.375 3.0 [39,44] 1024 [44]
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Please cite this article in press as: I. Bernardelli de Mattos, et al., Delivery of antiseptic solutions by a bacterial cellulose wound dressing: Uptake, release and antibacterial efficacy of octenidine and povidone-iodine, Burns (2019), https://doi.org/10.1016/j.burns.2019.10.006