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Nanofibrous membranes as smart wound dressings that release antibiotics when an injury is infected
Journal Pre-proof Nanofibrous membranes as smart wound dressings that release antibiotics when an injury is infected G. Rivero, M. Meuter, A. Pepe, G. Guevara, A.R. Boccaccini, G.A. Abraham
PII:
S0927-7757(19)31310-X
DOI:
https://doi.org/10.1016/j.colsurfa.2019.124313
Reference:
COLSUA 124313
To appear in:
Colloids and Surfaces A: Physicochemical and Engineering Aspects
Received Date:
2 October 2019
Revised Date:
28 November 2019
Accepted Date:
2 December 2019
Please cite this article as: Rivero G, Meuter M, Pepe A, Guevara G, Boccaccini AR, Abraham GA, Nanofibrous membranes as smart wound dressings that release antibiotics when an injury is infected, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2019), doi: https://doi.org/10.1016/j.colsurfa.2019.124313
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Nanofibrous membranes as smart wound dressings that release antibiotics when an injury is infected
G. Rivero1,* [email protected], M. Meuter3, A. Pepe2, G. Guevara2, A.R. Boccaccini3, G.A. Abraham1 1
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Instituto de Investigaciones en Ciencia y Tecnología de Materiales, INTEMA (UNMdP-CONICET),Av. Juan B. Justo 4302, Mar del Plata B7608FDQ, Buenos Aires, Argentina. 2 Instituto de Investigaciones Biológicas IIB (UNMdP-CONICET),Funes 3250, B7602AYJ, Mar del Plata,7600 Buenos Aires, Argentina. 3 Institute of Biomaterials, Department of Materials Science and Engineering, University ofErlangen-Nuremberg, 91058 Erlangen, Germany
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* Correspondence: Tel.: +54-223-4816600
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Graphical abstract
Abstract
Simple and coaxial electrospinning were used for the preparation of nanofibers containing antibiotic. The use of a pH-sensitive polymer provided the membranes with a
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selective releasing capability, as a function of the environmental pH. Considering that pH increases as a consequence of local wound infection, the acidity changes of the wound could act as a trigger for the onset of antibiotic treatment in an autonomous way.
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In this work, electrospun membranes capable of releasing nitrofurazone when there is a change in the acidity of the environment were designed and prepared. The electrospun nanomaterials were fabricated with polymers with selective solubility at pH values greater than 7 so that the active agent is released only in this condition. The use of these membranes as wound dressings would selectively trigger an antibiotic treatment
according to the physiological pH of the wound, i.e. only when there are natural signs of infection. The behavior of the membranes was exhaustively studied in different pH media, as well as the membranes integrity and morphology. The nitrofurazone release profiles were measured in-vitro and the antibiotic capability was tested against bacteria.These pH-sensitive nanofibrous drug delivery carriers are proposed as smart wound dressings with remarkable potential for enlarging the therapeutic benefits during medical treatments, according to the physiological conditions of the damaged skin
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tissues, especially for burns or chronic wounds.
Introduction
The self-healing ability of the skin, the largest organ of the body, allows keeping its functionality as protective barrier, thermoregulator, fluid homeostasis, immunoactive
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defender, sensorial interface, D vitamin producer, etc. However, this functionality may
not be naturally restored when serious burns or chronic injuries occur, as those produced in patients with diabetic ulcers. In these cases, fungi and/or bacteria may
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seriously complicate the healing process. The scope of the problem becomes global when considering that 25% of the 425 million of diabetics in the world have a risk of
developing chronic, non-cicatrizing foot ulcers. This disease seriously deteriorates their
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life quality and it is the cause of 85% of foot amputations, leading to enormous costs and medical efforts [1].
Many efforts have been done for providing membranes with antibacterial properties.
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Recently, novel antimicrobial coatings based on chlorine, silver nanoparticles or ketoprofen were applied to cotton fabrics [2,3] and cellulose [4]. However, the use of antibiotics in wounds is often contraindicated unless there is an infection. Moreover, the diagnosis of infections is particularly ambiguous in chronic wounds where classic
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clinical signs can be affected by other pathologies of the disease. In suspect of infection, the time spent in the performance of swabs, biopsies and microbiological tests may delay the treatment. There are a few recent complex analytical/instrumental techniques
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for the early identification of bacteria by using metabolites release or very specific biomarkers that may alert of the presence of an infection[52]. Some new advances in biomedical sensors have reported the incorporation of electrochemical devices in wound dressings for monitoring pre-marked antibodies [63]. However, there are no devices
capable of detecting natural non-specific physiological indicators of infections that could trigger an antibiotic treatment in an autonomous way. The pH of the physiological human skin is in a range between 4.0-6.0 [74] but pHchanges can influence the activity of chemical reactions, i.e. the enzymatic activity of microorganisms. Indeed, one of the main functions of the human skin includes acting as a barrier to pathogenic microorganisms due to its acidic milieu.In wounds, pH is a dynamic factor which varies rapidly in the different phases of wound healing. Due to
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disorders in the physical balance between tissue reformation and degradation in wounds and preponderance of catabolic processes, pH-changes from acidic to alkaline milieu occur [85]. These disorders increase the chances of infections of the wound by bacteria
which can result in stagnation of the healing process holding the wound in the
inflammatory phase of healing and forming a chronic wound [96]. In chronic wounds, the pH reaches a plateau in the range of 7-8 during stagnation of the healing process.
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The formation of a biofilm allows the isolation of bacteria from the human immune
system, increasing then the risk of microbialresistance against antibacterial agents, and thus the chance of systemic infection [107].
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In this context, the ability of a material to response to an external stimulus in an
autonomous way is a substantial advantage for the treatment of certain pathologies in the biomedical field. In this work, electrospun membranes capable of releasing
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antibiotic when there is a change in the environmental acidity were designed and studied. The nanomaterials were fabricated with pH-responsive polymers that could dissolve at pH values greater than 7, releasing an active agent in this condition. The use
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of these membranes as wound dressings would selectively trigger an antibiotic treatment in an autonomous way, according to the physiological pH of the wound, i.e. only when there are natural signs of infection.
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Beyond the passive role of conventional wound dressings, the nanostructure of electrospun membranes is ideal for using in this application [107]. The interconnected porosity allows a good absorption of exudates and water, and a humid environment
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favors cicatrization and fluids exchange with the milieu. They are adaptable, nonadherent physical barriers with vast surface area that can host active agents. Recently,composite nanofibrouspolymeric membranes with antimicrobial properties were proposed for wound dressing applications, based on silveror drug-blended nanoparticles loaded inside the fibers [11,12]. In this work, the use of biocompatible
and biodegradable polymers with pH selective dissolution allowed to modulate the release of an active agent. Eudragit® S100 (ES100) is a synthetic anionic copolymer based on methacrylic and methylmethacrylic acids with specific pH-dependent dissolution properties. Specifically, ES100 dissolves at pH ≥ 7, leading to non-toxic degradation products excretable through the kidneys [138]. A wide range of Eudragit® formulations are available with different commercial forms, commonly used as coatings for controlled and targeted drug release. Although manufacturers report the typical
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specifications for tablets coating, the release of active agents incorporated to other materials or structures depends also on the size (e.g. thickness), complexity and surface properties.
Nanofibrous membranes were produced via cutting-edge electrohydrodynamic techniques in single and coaxial mode, using ES100 polymeric solutions loaded with
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nitrofurazone, a bactericide and fungicide used against skin infections.In this work, the
pH changes of the wound are proposed to act as a trigger for the onset of antibiotic treatment in an autonomous way. The optimization of the compositional and processing
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conditions in combination with a detailed study on the fibrous membrane architecture were required in order to fine tune the agent release while preserving the structural integrity of the mat. The behavior of the membranes in different pH mediawas
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exhaustively studied.Ideally, the pH-dependent dissolution of ES100 when pH≥7 would tune the release of the antibiotic, only if the local acidity decreases in the wound, i.e. solely when there is an actual risk of infection. These pH-sensitive nanofibrousdrug
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delivery carriers are proposed as smart wound dressings with remarkable potential for enlarging the therapeutic benefits during medical treatments, according to the physiological conditions of the damaged skin tissues, especially for burns or chronic
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wounds.
Materials and methods
Eudragit® S100 was gently provided by EvonikRöhm GmbH (Darmstadt, Germany).
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Acetone, ethanol andN,N-dimethylformamide (DMF) were purchased from Cicarelli (San Lorenzo, Argentina). Nitrofurazone was supplied by Saporiti (Buenos Aires, Argentina). All reagents were used without further purification. Different solutions of 15% w/v ES100 were prepared with different solvents. Further compositional details of the prepared systems are listed in Table 1.
Phosphate buffer solutions (PBS) of different pH values were prepared using different ratios of solutions of monosodium dihydrogen orthophosphate and disodium hydrogenphosphate purchased from Anedra, in order to have a range of buffers with pH values of 5.5; 6.0; 6.5; 7.0; 7.5 and 8.0. The polymer solutions were electrospun in a YFLOW® 2.2.D-350 equipment using single or coaxial nozzles and an aluminum flat collector located 18 cm away. All
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electrospinning experiments were performed under ambient conditions, with temperature of 20±3°C and relative humidity of 54±6%. The flow rate was adjusted between 0.1 and 0.7 mL/h and the voltage varied between 8 and 11kV. For the
membranes fabricated with a coaxial nozzle (CN05), a flow rate of 1 mL/h with 15kV were used for the external nozzle. After processing, all nanofibrous membranes were dried and stored protected from light until usage.
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Membranes thickness was measured with a Mitutoyo® constant force caliper. Scanning electron microscopy (SEM, Jeol JSM-6460) was performed after sputtering with gold/palladium. The surface morphology was inspected on the original membranes
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andalsoon membranes immersed in each pH media for 1 min and then dried for 2 hours. Image-Pro Plus®software was used for measuring the fiber diameters distribution, with
at least 100 measurements per image. Attenuated total reflectance- Fourier transform
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infrared spectroscopy (ATR-FTIR) was performed in a Thermo Scientific Nicolet 6700 equip.
Contact angles were measured in a Ramé-hart goniometer using 50 μl droplets of
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MilliQ water or PBS solutions. Measurements were performed per triplicate and followed in time for at least 10 minutes. Differential Scanning Calorimetry (DSC) was performed in a Perkin-Elmer calorimeter(Pyris 1) from ambient temperature to 300°C at 10°C/min under nitrogen atmosphere. Thermogravimetric Analyses (TGA) were
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performed in Shimadzu TGA-50 from 20 to 800°C at 10°C/min in nitrogen atmosphere. For the nitrofurazone in vitro release tests, 1.2±0.2 mg of EDN05 and CN05 membranes were immersed in 25ml of the six different pH media and incubated at 37°C with orbital
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platform shaker at150rpm. Aliquots of 1 mLwere extracted from each media at different times and nitrofurazone was quantified by UV spectroscopy with calibration curves for each pH medium, using the characteristic peak of the drug at 365 nm. In order to keep the volume constant, 1 mLof the same fresh pH solution was replenished after each measurement. Measurements were conducted in triplicate in all cases.
For the growth rate measurements, 10 l of Escherichia coli DE3 (Novagen) culture was grown in LB medium (10 g/LBacto-tryptone(Aldrich), 5 g/L of yeast extract (Aldrich) and 10 g/L of NaCl (Aldrich) at 37 ºC overnight. The growth rate of E. coli was measuredby incubating 3 mL of LB with 20 μl of the previous culture with or without the different membranes. The acidity of each media wasadjusted with 0.1 M Na2HPO4 / NaH2PO4 buffer (Aldrich) at different pHvalues: 5.5, 6.0, 6.5 and 7.0. The optical density (OD) of the cultures was recorded at 540 nm every 1 h, during8 h, using
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a Hitachi Radio Beam U-1900 spectrophotometer.
Results and discussion
Electrospun membranes were successfully obtained with 15% w/v ES100 solutions. The
use of different solvents led to significant differences,both in the processing stability (Fig. 1a) as in the morphology of the resulting fibers, as observed in the SEM images
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(Fig. 1b). The Taylor cones of A and E systems showed large instabilities, probably due to the high volatility of acetone and ethanol. The resulting fibers were very thin and
ribbon-like shaped, respectively, while beads and defects were observedin membrane D.
angle (A: 105°±3°, E: 96°±2°, D: 119°±5°).
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The differences in the fiber architecture led to slight differences in the water contact
The solvent mixture ED resulted optimal, and it was maintained for the nitrofurazone-
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loaded systems. Continuous, smooth and uniform nanofibrous membranes were obtained. The water contact angle in ED systems (104°±5°) was enlarged in the loaded systems (120°±2°), but the fibers morphology and average diameter (around 500 nm)
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did not vary appreciably with the incorporation of nitrofurazone. As expected, the fibers fabricated with a coaxial nozzle (CN05) showed a larger size distribution, due to the
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additional sheath of polymer surrounding the loaded core of the fibers (Fig.1c).
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Fig.1) a) Images of the Taylor cone during the electrospinning process, and b) SEM
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images of the resulting membranesof the different drug-free systems. Scale bar: 50 m (upper raw) and 5 m (bottom raw) c) SEM images and fiber size distribution of the
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loaded systems.
Due to the low nitrafurazone cargo, the thermal properties of the membranes were not modified, and only certain weak peaks assigned to nitrofurazone could be detected in
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the FTIR-ATR spectrumof EDN05 around 1350 and 1580 cm-1 (not shown). Eudragit S100 ® was designed as an organic coating for gastro-intestinal targeting, given its insolubility at the acidic media of the stomach during digestion time. However, these features respond to a film coating-based system for tablets, where the polymer
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amount depends on the application and surface area of the substrate, as reported by the manufacturer. Given that thenanofibers high surface-area-to-volume ratio could favor a closer contact or the solvent with the polymer, a detailed study was performed for the
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evaluation of the prepared nanofibrous membranes behavior.Nanofibers prepared with ES100 and other compounds [14-179-12] were reported specifically for oral delivery, where gastrointestinal conditions were simulated, i.e. nanofibers were sequentially exposed to pH 1 (2h), 6.8 (4h) and 7.4. However, the re-evaluation of the dissolution
profiles in various pH media is mandatory if the polymer is used in a completely different morphological structure and final application. The nanofibrous structures weredeteriorated when the membranes were immersed for one minute in basic pH media (Fig.2). Nanofibers completely disappeared in EDN05, but they gradually agglomerated and preserved certain remaining morphology in CN05. This difference could be attributed to the extra layer of polymer in CN05, that could cause a delay in the dissolution. The contact angles of different PBS droplets with acidic pHs applied on the surface of EDN05 and CN05 revealed no changes in time, while the
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droplets with pH≥7 were progressively absorbed, as evidencedby the changes of the
contact angles in time (Fig.3). It should be considered that in real applications, dressings
would not be either totally immersed nor in contact with one single droplet of exudates.
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An intermediate real behavior could be expected for these materials applied in wounds.
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Fig.2) SEM images of the membranes a) EDN05 and b) CN05, after one minute of
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immersion in PBS of different pH values.
Fig.3) Relative contact angle of EDN05 with adroplet of different PBS pH values.
The lossof the nanofibrous structure due tothe pH-responsive polymer dissolution is in
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accordance with the fast in vitro release of nitrofurazone from both EDN05 and CN05 membranes in the media with pH≥7 (Fig.4a). The drug was totally released in less than15 minutes in EDN05, and after 45 minutes in CN05 membranes. Although ES100
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is reported to be insoluble in acidic mediawhen used as pills coatings, a significant release of nitrofurazonefrom the nanofibers was detected in media with pH 5.5, 6.0 and 6.5. Membranes composed of simple nanofibers released most of the nitrofurazone in just 2 hours. This behavior could be a consequence of the location of part of the drug directly at or near the fibers surface, in accordance with the ATR-FTIR and contact
angle results. However, it is possible that alow percentage of remaining drug kept trapped within the disintegrated structure.The strategy of enlarging the diffusion sheath of the fibers led to a very different release profile for CN05. In this case, less than 20% of nitrofurazone was released in the first 2 hourswhile a similar amount of remaining
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drug was measured after around 4 days.
Fig.4) Nitrofurazone release profiles in PBS for EDN05 and CN05 membranes ata) pH≥7 and b) pH<7.
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The antibacterial behavior of all membranes was studied against E. coli in different pH media. The bacterial growth of the samples incubated in presence of ED membranes (without NFZ) was normalin all the acidity range. Bacteria started growing
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exponentially until reaching a stationary plateau. No significant increase in optical density was observed due to the dissolution of the membrane itself, in absence of bacteria at any pH (Fig. 5). Thus, the changes in the optical density valuescould be
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directly correlated with the modifications in the number of cells in the culture.
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Fig. 5) Growth rate curves: A: pH 5.5; B: pH 6.0; C: pH 6.5; D: pH 7.0. Note: B-
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corresponds to LB medium with EDN05 membrane without bacteria.
The curves of the systems containing antibiotic-loaded membranes showed significant
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differences with the drug-free membranes, and between each other. A lower OD value of the plateau can be attributed to a minor number of bacteria in the culture as a consequence of the antibiotic effect.In accordance with the obtained in-vitro nitrofurazone release profiles, the systems with EDN05 and CN05 reached significantly
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different values of optical density at pH 5.5 and 6.0 (Fig. 5A and 5B). This disparity in the bacterial growth decrease is linked to the different nanofiber architectures, and the consequent different drug releasing kinetics. However, at pH 6.5 and 7.0, the OD values
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of the loaded membranes were lower than the ED control system, but similar between each other (Fig. 5C and 5C). In these media, the polymer dissolution led to a faster and similar antibiotic release and action. This behavior is assumed to be comparablefor
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higher pH values (test not performed).
Conclusions
Nitrofurazone-loaded nanofibrous membranes were successfully prepared by simple and coaxial electrospinning using pH-responsive polymers. The in-vitro drug release profiles showed significant differences with respect to the pH media. Albeit not
completely negligible, the nitrofurazone release in EDN05 is mostly hindered in the usual pH range of the healthy skin. However, a large, fast but relatively gradual release was detected when pH>7, in accordance with the detected loose of structure because of the pH-selective dissolution of ES100. Membranes fabricated with a coaxial nozzle succeeded to delay the nitrofurazone release because of the additional diffusion layer. Significant differences in the antibiotic activity of the membranes according to their architecture were correlated against E.coli.
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In practice, an increase in the pH of wounds is an indicator of infection and a drug treatment is actually required. This fact provides the prepared nanofibrous membranes with a high potential as pH-responsive wound dressing.
Declaration of interests
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The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper.
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Author contributions
G. Rivero conceived the idea and designed the analysis. She also wrote the paper and performed part of the experimental tests regarding the membranes processing and characterization.
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M. Meuter performed the experiments related with the preparation of some membranes and their characterization.
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A. Pepe and M.G. Guevara performed the experiments related to the antimicrobial tests and collaborated with the results critical evaluation.
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G. Abraham and A. Boccaccini provided critical feedback and helped shape the research, analysis and manuscript.
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Acknowledgements
The authors thank CONICET(grant PIP 153/2017) and I.DEAR Project (Argentina-Germany) for partial funding of this work.
References [1] Atlas International Diabetes Federation 2018. [2] Banerjee, S.L.; Potluri, P.; Singha, N. Antimicrobial cotton fibre coated with UV cured colloidal natural rubber latex: A sustainable material. Colloids and Surfaces A 566 (2019) 176-187. [3] Pan, N.; Liu, Y.; Ren, X.; Huang, T. Fabrication of cotton fabrics through in-situ
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reduction of polymeric N-halamine modified graphene oxide with enhanced ultravioletblocking, self-cleaning, and highly efficient, and monitorable antibacterial properties. Colloids and Surfaces A 555(2018) 765-771.
[4]Sun, B.; Zhang, Y.; Li, W.; Xu, X.; Zhang, H.; Zhao, Y.; Lin, J.; Sun, D.Facile synthesis and light-induced antibacterial activity of ketoprofen functionalized bacterial cellulose membranes.Colloids and Surfaces A 568(2019) 231-238.
Smart bandages technologies, 6 (2016) Academic Press, 195-227.
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[25] Davis, J.; McAlister, A. Sensors for Detecting and Combating Wound Infection, in:
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M, Wolfbeis, OS, Landthaler, M, Babilas, P. Luminescent dual sensors reveal
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[58] Schneider, L. A.; Korber, A.; Grabbe, S.; Dissemond, J. Influence of pH on woundhealing: a new perspective for wound-therapy? Archives of dermatological research 298, 9 (2007) 413-420.
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[69]Percival, S. L.; McCarty, S.; Hunt, J.; Woods, E. The effects of pH on wound healing, biofilms, and antimicrobial efficacy. Wound Repair and Regeneration 22, 2
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[710]Abrigo, M.; McArthur, S.; Kingshott, P. Electrospun nanofibers as dressings for
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chronic wounds care: advances, challenges, and future prospects. Macromolecular Bioscience 14, 6 (2014) 772-792. [11]Artürk, A; Taygun, M.E.; Güler, F.K.; Goller, G.; Küҫükbayrak.Fabrication of antibacterial polyvinylalcohol nanocomposite mats with soluble starch coated silver
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nanoparticles. Colloids and Surfaces A: Physicochemical and Engineering Aspects 562,5 (2019) 255-262. [12] Han, C.; Cai, N..; Chan, V.; Liu, M.; Feng, X.; Yu, F. Enhanced drug delivery, mechanical properties and antimicrobial activities in poly(lactic acid) nanofiber with
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[161] Xu, Q.; Zhang, N.; Qin, W.; Liu, J.; Jia, Z.; Liu, H. Preparation, In Vitro and In Vivo Evaluation of Budesonide Loaded Core/Shell Nanofibers as Oral Colonic Drug Delivery System. J. Nanosci. Nanotechnol. 13, 1 (2013) 149-156.
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Table 1. Composition of the solutions used for electrospinning. ES100 concentration (%w/v) Sheath solution 2:1 v/v 7.5 ethanol/DMF Solvent
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Solvent
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ES100 Nitrofurazone concentration concentration (%w/v) (%w/v) Core solution E Ethanol 15 D DMF 15 A Acetone 15 ED 2:1 v/v ethanol/DMF 15 ED1 4:1 v/v ethanol/DMF 15 EDN05 2:1 v/v ethanol/DMF 15 0.5 CN05 2:1 v/v ethanol/DMF 15 0.5 Code
PII:
S0927-7757(19)31310-X
DOI:
https://doi.org/10.1016/j.colsurfa.2019.124313
Reference:
COLSUA 124313
To appear in:
Colloids and Surfaces A: Physicochemical and Engineering Aspects
Received Date:
2 October 2019
Revised Date:
28 November 2019
Accepted Date:
2 December 2019
Please cite this article as: Rivero G, Meuter M, Pepe A, Guevara G, Boccaccini AR, Abraham GA, Nanofibrous membranes as smart wound dressings that release antibiotics when an injury is infected, Colloids and Surfaces A: Physicochemical and Engineering Aspects (2019), doi: https://doi.org/10.1016/j.colsurfa.2019.124313
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.
Nanofibrous membranes as smart wound dressings that release antibiotics when an injury is infected
G. Rivero1,* [email protected], M. Meuter3, A. Pepe2, G. Guevara2, A.R. Boccaccini3, G.A. Abraham1 1
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Instituto de Investigaciones en Ciencia y Tecnología de Materiales, INTEMA (UNMdP-CONICET),Av. Juan B. Justo 4302, Mar del Plata B7608FDQ, Buenos Aires, Argentina. 2 Instituto de Investigaciones Biológicas IIB (UNMdP-CONICET),Funes 3250, B7602AYJ, Mar del Plata,7600 Buenos Aires, Argentina. 3 Institute of Biomaterials, Department of Materials Science and Engineering, University ofErlangen-Nuremberg, 91058 Erlangen, Germany
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* Correspondence: Tel.: +54-223-4816600
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Graphical abstract
Abstract
Simple and coaxial electrospinning were used for the preparation of nanofibers containing antibiotic. The use of a pH-sensitive polymer provided the membranes with a
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selective releasing capability, as a function of the environmental pH. Considering that pH increases as a consequence of local wound infection, the acidity changes of the wound could act as a trigger for the onset of antibiotic treatment in an autonomous way.
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In this work, electrospun membranes capable of releasing nitrofurazone when there is a change in the acidity of the environment were designed and prepared. The electrospun nanomaterials were fabricated with polymers with selective solubility at pH values greater than 7 so that the active agent is released only in this condition. The use of these membranes as wound dressings would selectively trigger an antibiotic treatment
according to the physiological pH of the wound, i.e. only when there are natural signs of infection. The behavior of the membranes was exhaustively studied in different pH media, as well as the membranes integrity and morphology. The nitrofurazone release profiles were measured in-vitro and the antibiotic capability was tested against bacteria.These pH-sensitive nanofibrous drug delivery carriers are proposed as smart wound dressings with remarkable potential for enlarging the therapeutic benefits during medical treatments, according to the physiological conditions of the damaged skin
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tissues, especially for burns or chronic wounds.
Introduction
The self-healing ability of the skin, the largest organ of the body, allows keeping its functionality as protective barrier, thermoregulator, fluid homeostasis, immunoactive
-p
defender, sensorial interface, D vitamin producer, etc. However, this functionality may
not be naturally restored when serious burns or chronic injuries occur, as those produced in patients with diabetic ulcers. In these cases, fungi and/or bacteria may
re
seriously complicate the healing process. The scope of the problem becomes global when considering that 25% of the 425 million of diabetics in the world have a risk of
developing chronic, non-cicatrizing foot ulcers. This disease seriously deteriorates their
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life quality and it is the cause of 85% of foot amputations, leading to enormous costs and medical efforts [1].
Many efforts have been done for providing membranes with antibacterial properties.
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Recently, novel antimicrobial coatings based on chlorine, silver nanoparticles or ketoprofen were applied to cotton fabrics [2,3] and cellulose [4]. However, the use of antibiotics in wounds is often contraindicated unless there is an infection. Moreover, the diagnosis of infections is particularly ambiguous in chronic wounds where classic
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clinical signs can be affected by other pathologies of the disease. In suspect of infection, the time spent in the performance of swabs, biopsies and microbiological tests may delay the treatment. There are a few recent complex analytical/instrumental techniques
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for the early identification of bacteria by using metabolites release or very specific biomarkers that may alert of the presence of an infection[52]. Some new advances in biomedical sensors have reported the incorporation of electrochemical devices in wound dressings for monitoring pre-marked antibodies [63]. However, there are no devices
capable of detecting natural non-specific physiological indicators of infections that could trigger an antibiotic treatment in an autonomous way. The pH of the physiological human skin is in a range between 4.0-6.0 [74] but pHchanges can influence the activity of chemical reactions, i.e. the enzymatic activity of microorganisms. Indeed, one of the main functions of the human skin includes acting as a barrier to pathogenic microorganisms due to its acidic milieu.In wounds, pH is a dynamic factor which varies rapidly in the different phases of wound healing. Due to
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disorders in the physical balance between tissue reformation and degradation in wounds and preponderance of catabolic processes, pH-changes from acidic to alkaline milieu occur [85]. These disorders increase the chances of infections of the wound by bacteria
which can result in stagnation of the healing process holding the wound in the
inflammatory phase of healing and forming a chronic wound [96]. In chronic wounds, the pH reaches a plateau in the range of 7-8 during stagnation of the healing process.
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The formation of a biofilm allows the isolation of bacteria from the human immune
system, increasing then the risk of microbialresistance against antibacterial agents, and thus the chance of systemic infection [107].
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In this context, the ability of a material to response to an external stimulus in an
autonomous way is a substantial advantage for the treatment of certain pathologies in the biomedical field. In this work, electrospun membranes capable of releasing
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antibiotic when there is a change in the environmental acidity were designed and studied. The nanomaterials were fabricated with pH-responsive polymers that could dissolve at pH values greater than 7, releasing an active agent in this condition. The use
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of these membranes as wound dressings would selectively trigger an antibiotic treatment in an autonomous way, according to the physiological pH of the wound, i.e. only when there are natural signs of infection.
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Beyond the passive role of conventional wound dressings, the nanostructure of electrospun membranes is ideal for using in this application [107]. The interconnected porosity allows a good absorption of exudates and water, and a humid environment
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favors cicatrization and fluids exchange with the milieu. They are adaptable, nonadherent physical barriers with vast surface area that can host active agents. Recently,composite nanofibrouspolymeric membranes with antimicrobial properties were proposed for wound dressing applications, based on silveror drug-blended nanoparticles loaded inside the fibers [11,12]. In this work, the use of biocompatible
and biodegradable polymers with pH selective dissolution allowed to modulate the release of an active agent. Eudragit® S100 (ES100) is a synthetic anionic copolymer based on methacrylic and methylmethacrylic acids with specific pH-dependent dissolution properties. Specifically, ES100 dissolves at pH ≥ 7, leading to non-toxic degradation products excretable through the kidneys [138]. A wide range of Eudragit® formulations are available with different commercial forms, commonly used as coatings for controlled and targeted drug release. Although manufacturers report the typical
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specifications for tablets coating, the release of active agents incorporated to other materials or structures depends also on the size (e.g. thickness), complexity and surface properties.
Nanofibrous membranes were produced via cutting-edge electrohydrodynamic techniques in single and coaxial mode, using ES100 polymeric solutions loaded with
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nitrofurazone, a bactericide and fungicide used against skin infections.In this work, the
pH changes of the wound are proposed to act as a trigger for the onset of antibiotic treatment in an autonomous way. The optimization of the compositional and processing
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conditions in combination with a detailed study on the fibrous membrane architecture were required in order to fine tune the agent release while preserving the structural integrity of the mat. The behavior of the membranes in different pH mediawas
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exhaustively studied.Ideally, the pH-dependent dissolution of ES100 when pH≥7 would tune the release of the antibiotic, only if the local acidity decreases in the wound, i.e. solely when there is an actual risk of infection. These pH-sensitive nanofibrousdrug
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delivery carriers are proposed as smart wound dressings with remarkable potential for enlarging the therapeutic benefits during medical treatments, according to the physiological conditions of the damaged skin tissues, especially for burns or chronic
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wounds.
Materials and methods
Eudragit® S100 was gently provided by EvonikRöhm GmbH (Darmstadt, Germany).
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Acetone, ethanol andN,N-dimethylformamide (DMF) were purchased from Cicarelli (San Lorenzo, Argentina). Nitrofurazone was supplied by Saporiti (Buenos Aires, Argentina). All reagents were used without further purification. Different solutions of 15% w/v ES100 were prepared with different solvents. Further compositional details of the prepared systems are listed in Table 1.
Phosphate buffer solutions (PBS) of different pH values were prepared using different ratios of solutions of monosodium dihydrogen orthophosphate and disodium hydrogenphosphate purchased from Anedra, in order to have a range of buffers with pH values of 5.5; 6.0; 6.5; 7.0; 7.5 and 8.0. The polymer solutions were electrospun in a YFLOW® 2.2.D-350 equipment using single or coaxial nozzles and an aluminum flat collector located 18 cm away. All
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electrospinning experiments were performed under ambient conditions, with temperature of 20±3°C and relative humidity of 54±6%. The flow rate was adjusted between 0.1 and 0.7 mL/h and the voltage varied between 8 and 11kV. For the
membranes fabricated with a coaxial nozzle (CN05), a flow rate of 1 mL/h with 15kV were used for the external nozzle. After processing, all nanofibrous membranes were dried and stored protected from light until usage.
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Membranes thickness was measured with a Mitutoyo® constant force caliper. Scanning electron microscopy (SEM, Jeol JSM-6460) was performed after sputtering with gold/palladium. The surface morphology was inspected on the original membranes
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andalsoon membranes immersed in each pH media for 1 min and then dried for 2 hours. Image-Pro Plus®software was used for measuring the fiber diameters distribution, with
at least 100 measurements per image. Attenuated total reflectance- Fourier transform
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infrared spectroscopy (ATR-FTIR) was performed in a Thermo Scientific Nicolet 6700 equip.
Contact angles were measured in a Ramé-hart goniometer using 50 μl droplets of
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MilliQ water or PBS solutions. Measurements were performed per triplicate and followed in time for at least 10 minutes. Differential Scanning Calorimetry (DSC) was performed in a Perkin-Elmer calorimeter(Pyris 1) from ambient temperature to 300°C at 10°C/min under nitrogen atmosphere. Thermogravimetric Analyses (TGA) were
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performed in Shimadzu TGA-50 from 20 to 800°C at 10°C/min in nitrogen atmosphere. For the nitrofurazone in vitro release tests, 1.2±0.2 mg of EDN05 and CN05 membranes were immersed in 25ml of the six different pH media and incubated at 37°C with orbital
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platform shaker at150rpm. Aliquots of 1 mLwere extracted from each media at different times and nitrofurazone was quantified by UV spectroscopy with calibration curves for each pH medium, using the characteristic peak of the drug at 365 nm. In order to keep the volume constant, 1 mLof the same fresh pH solution was replenished after each measurement. Measurements were conducted in triplicate in all cases.
For the growth rate measurements, 10 l of Escherichia coli DE3 (Novagen) culture was grown in LB medium (10 g/LBacto-tryptone(Aldrich), 5 g/L of yeast extract (Aldrich) and 10 g/L of NaCl (Aldrich) at 37 ºC overnight. The growth rate of E. coli was measuredby incubating 3 mL of LB with 20 μl of the previous culture with or without the different membranes. The acidity of each media wasadjusted with 0.1 M Na2HPO4 / NaH2PO4 buffer (Aldrich) at different pHvalues: 5.5, 6.0, 6.5 and 7.0. The optical density (OD) of the cultures was recorded at 540 nm every 1 h, during8 h, using
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a Hitachi Radio Beam U-1900 spectrophotometer.
Results and discussion
Electrospun membranes were successfully obtained with 15% w/v ES100 solutions. The
use of different solvents led to significant differences,both in the processing stability (Fig. 1a) as in the morphology of the resulting fibers, as observed in the SEM images
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(Fig. 1b). The Taylor cones of A and E systems showed large instabilities, probably due to the high volatility of acetone and ethanol. The resulting fibers were very thin and
ribbon-like shaped, respectively, while beads and defects were observedin membrane D.
angle (A: 105°±3°, E: 96°±2°, D: 119°±5°).
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The differences in the fiber architecture led to slight differences in the water contact
The solvent mixture ED resulted optimal, and it was maintained for the nitrofurazone-
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loaded systems. Continuous, smooth and uniform nanofibrous membranes were obtained. The water contact angle in ED systems (104°±5°) was enlarged in the loaded systems (120°±2°), but the fibers morphology and average diameter (around 500 nm)
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did not vary appreciably with the incorporation of nitrofurazone. As expected, the fibers fabricated with a coaxial nozzle (CN05) showed a larger size distribution, due to the
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additional sheath of polymer surrounding the loaded core of the fibers (Fig.1c).
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Fig.1) a) Images of the Taylor cone during the electrospinning process, and b) SEM
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images of the resulting membranesof the different drug-free systems. Scale bar: 50 m (upper raw) and 5 m (bottom raw) c) SEM images and fiber size distribution of the
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loaded systems.
Due to the low nitrafurazone cargo, the thermal properties of the membranes were not modified, and only certain weak peaks assigned to nitrofurazone could be detected in
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the FTIR-ATR spectrumof EDN05 around 1350 and 1580 cm-1 (not shown). Eudragit S100 ® was designed as an organic coating for gastro-intestinal targeting, given its insolubility at the acidic media of the stomach during digestion time. However, these features respond to a film coating-based system for tablets, where the polymer
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amount depends on the application and surface area of the substrate, as reported by the manufacturer. Given that thenanofibers high surface-area-to-volume ratio could favor a closer contact or the solvent with the polymer, a detailed study was performed for the
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evaluation of the prepared nanofibrous membranes behavior.Nanofibers prepared with ES100 and other compounds [14-179-12] were reported specifically for oral delivery, where gastrointestinal conditions were simulated, i.e. nanofibers were sequentially exposed to pH 1 (2h), 6.8 (4h) and 7.4. However, the re-evaluation of the dissolution
profiles in various pH media is mandatory if the polymer is used in a completely different morphological structure and final application. The nanofibrous structures weredeteriorated when the membranes were immersed for one minute in basic pH media (Fig.2). Nanofibers completely disappeared in EDN05, but they gradually agglomerated and preserved certain remaining morphology in CN05. This difference could be attributed to the extra layer of polymer in CN05, that could cause a delay in the dissolution. The contact angles of different PBS droplets with acidic pHs applied on the surface of EDN05 and CN05 revealed no changes in time, while the
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droplets with pH≥7 were progressively absorbed, as evidencedby the changes of the
contact angles in time (Fig.3). It should be considered that in real applications, dressings
would not be either totally immersed nor in contact with one single droplet of exudates.
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An intermediate real behavior could be expected for these materials applied in wounds.
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Fig.2) SEM images of the membranes a) EDN05 and b) CN05, after one minute of
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immersion in PBS of different pH values.
Fig.3) Relative contact angle of EDN05 with adroplet of different PBS pH values.
The lossof the nanofibrous structure due tothe pH-responsive polymer dissolution is in
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accordance with the fast in vitro release of nitrofurazone from both EDN05 and CN05 membranes in the media with pH≥7 (Fig.4a). The drug was totally released in less than15 minutes in EDN05, and after 45 minutes in CN05 membranes. Although ES100
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is reported to be insoluble in acidic mediawhen used as pills coatings, a significant release of nitrofurazonefrom the nanofibers was detected in media with pH 5.5, 6.0 and 6.5. Membranes composed of simple nanofibers released most of the nitrofurazone in just 2 hours. This behavior could be a consequence of the location of part of the drug directly at or near the fibers surface, in accordance with the ATR-FTIR and contact
angle results. However, it is possible that alow percentage of remaining drug kept trapped within the disintegrated structure.The strategy of enlarging the diffusion sheath of the fibers led to a very different release profile for CN05. In this case, less than 20% of nitrofurazone was released in the first 2 hourswhile a similar amount of remaining
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drug was measured after around 4 days.
Fig.4) Nitrofurazone release profiles in PBS for EDN05 and CN05 membranes ata) pH≥7 and b) pH<7.
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The antibacterial behavior of all membranes was studied against E. coli in different pH media. The bacterial growth of the samples incubated in presence of ED membranes (without NFZ) was normalin all the acidity range. Bacteria started growing
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exponentially until reaching a stationary plateau. No significant increase in optical density was observed due to the dissolution of the membrane itself, in absence of bacteria at any pH (Fig. 5). Thus, the changes in the optical density valuescould be
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directly correlated with the modifications in the number of cells in the culture.
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Fig. 5) Growth rate curves: A: pH 5.5; B: pH 6.0; C: pH 6.5; D: pH 7.0. Note: B-
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corresponds to LB medium with EDN05 membrane without bacteria.
The curves of the systems containing antibiotic-loaded membranes showed significant
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differences with the drug-free membranes, and between each other. A lower OD value of the plateau can be attributed to a minor number of bacteria in the culture as a consequence of the antibiotic effect.In accordance with the obtained in-vitro nitrofurazone release profiles, the systems with EDN05 and CN05 reached significantly
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different values of optical density at pH 5.5 and 6.0 (Fig. 5A and 5B). This disparity in the bacterial growth decrease is linked to the different nanofiber architectures, and the consequent different drug releasing kinetics. However, at pH 6.5 and 7.0, the OD values
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of the loaded membranes were lower than the ED control system, but similar between each other (Fig. 5C and 5C). In these media, the polymer dissolution led to a faster and similar antibiotic release and action. This behavior is assumed to be comparablefor
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higher pH values (test not performed).
Conclusions
Nitrofurazone-loaded nanofibrous membranes were successfully prepared by simple and coaxial electrospinning using pH-responsive polymers. The in-vitro drug release profiles showed significant differences with respect to the pH media. Albeit not
completely negligible, the nitrofurazone release in EDN05 is mostly hindered in the usual pH range of the healthy skin. However, a large, fast but relatively gradual release was detected when pH>7, in accordance with the detected loose of structure because of the pH-selective dissolution of ES100. Membranes fabricated with a coaxial nozzle succeeded to delay the nitrofurazone release because of the additional diffusion layer. Significant differences in the antibiotic activity of the membranes according to their architecture were correlated against E.coli.
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In practice, an increase in the pH of wounds is an indicator of infection and a drug treatment is actually required. This fact provides the prepared nanofibrous membranes with a high potential as pH-responsive wound dressing.
Declaration of interests
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The authors declare that they have no known competing financialinterestsor personal relationships that could have appeared to influence the work reported in this paper.
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Author contributions
G. Rivero conceived the idea and designed the analysis. She also wrote the paper and performed part of the experimental tests regarding the membranes processing and characterization.
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M. Meuter performed the experiments related with the preparation of some membranes and their characterization.
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A. Pepe and M.G. Guevara performed the experiments related to the antimicrobial tests and collaborated with the results critical evaluation.
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G. Abraham and A. Boccaccini provided critical feedback and helped shape the research, analysis and manuscript.
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Acknowledgements
The authors thank CONICET(grant PIP 153/2017) and I.DEAR Project (Argentina-Germany) for partial funding of this work.
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Table 1. Composition of the solutions used for electrospinning. ES100 concentration (%w/v) Sheath solution 2:1 v/v 7.5 ethanol/DMF Solvent
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Solvent
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ES100 Nitrofurazone concentration concentration (%w/v) (%w/v) Core solution E Ethanol 15 D DMF 15 A Acetone 15 ED 2:1 v/v ethanol/DMF 15 ED1 4:1 v/v ethanol/DMF 15 EDN05 2:1 v/v ethanol/DMF 15 0.5 CN05 2:1 v/v ethanol/DMF 15 0.5 Code