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Antimicrobial Properties of Electrically Formed Elastomeric Polyurethane–Copper Oxide Nanocomposites for Medical and Dental Applications
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Antimicrobial Properties of Electrically Formed Elastomeric Polyurethane–Copper Oxide Nanocomposites for Medical and Dental Applications Z. Ahmad,* M. A. Vargas-Reus,† R. Bakhshi,‡ F. Ryan,‡ G. G. Ren,§ F. Oktar,} and R. P. Allaker† Contents 88 90 90 90 91 91 94 94 97 98 98
1. Introduction 2. Materials and Methods 2.1. Materials 2.2. Methods 3. Results and Discussion 3.1. Antimicrobial fiber and film preparation 3.2. Structural analysis 3.3. Antimicrobial testing using MRSA 4. Concluding Remarks and Future Work Acknowledgments References
Abstract With the rapidly advancing field of nanotechnology having an impact in several areas interfacing life and physical sciences, the potential applications of nanoparticles as antimicrobial agents have been realized and offer great opportunities in addressing several viral and bacterial outbreak issues. Polyurethanes (PUs) are a diverse class of polymeric materials which also have applications in several areas of biomedical science ranging from blood contact devices to * School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Institute of Dentistry, London, United Kingdom { Department of Mechanical Engineering, University College London, London, United Kingdom } School of Engineering and Technology, University of Hertfordshire, Hatfield, United Kingdom } Nanotechnology and Biomaterials Application & Research Centre, Marmara University, Istanbul, Turkey {
Methods in Enzymology, Volume 509 ISSN 0076-6879, DOI: 10.1016/B978-0-12-391858-1.00005-8
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2012 Elsevier Inc. All rights reserved.
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implantable dental technologies. In this report, copper oxide (CuO) nanoparticles (mean size 50 nm) are embedded into a PU matrix via two electrical fabrication processes. To elucidate the antimicrobial activity, a range of different loading compositions of CuO within the PU matrix (0%, 1%, 5%, and 10% w/w) are electrospun to form thin porous films (thickness <10 mm). After washing, the films are tested for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA). Significant reduction of populations was demonstrated with 10% w/w CuO over a 4-h period. This approach demonstrates the potential of generating tailored antimicrobial structures for a host of applications, such as designer filters, patterned coatings, breathable fabrics, adhesive films (as opposed to sutures), and mechanically supporting structures.
1. Introduction Nanoscaled particles have diverse applications ranging from medical device coatings to drug delivery carriers (De Jong and Borm, 2008; Dickinson et al., 2011). More recently, and in a timely manner, their applications as antimicrobial agents have also been realized especially with metal and metal oxide particle systems (Borkow et al., 2010; Ren et al., 2009). The antimicrobial properties of copper (Cu) and its oxide (CuO) have been known for centuries (on the macroscale) and with current advances in technology; selected textile and material composite applications have demonstrated their potential as antimicrobial agents on the micro- and nanometer scales (Ruparelia et al., 2008; Zhang et al., 2006). Other types of metallic nanoparticles demonstrating such properties include gold and silver (Perni et al., 2009; Ruparelia et al., 2008); however, cost remains an important factor when considering the scale-up potential and the broad range of potential applications. Polyurethanes (PUs) have several applications as biomaterials including coatings, blood bags, catheters, heart valves, dental fillers, protective clothing, and even tissue-engineering constructs (Bertoldi et al., 2010; Luo et al., 2010; Sui et al., 2010). The mechanical, degradation, and biostability properties of these materials can be controlled by carefully selecting the various segments which constitute toward the polymeric backbone (Zdrahala and Zdrahala, 1999). They have also found several applications as antimicrobial materials and in many instances utilize an active agent, which is capable of killing bacteria or viruses (Ghosh, 2005). Numerous devices serving as filters, adhesive films, fillers, eluting structures, and coatings require a polymeric matrix to be impregnated with such components or secondary materials which possess the functional characteristics, for example, antimicrobial properties. Depending on the intended function, the polymeric matrix can contribute toward a mechanism for the release of active agents, the mechanical properties of the device, and the localization of the active agent at a specific point. In such cases, the
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loading volume of the active agent (e.g., possessing antimicrobial properties) must be sufficient to demonstrate the desired effect. In addition to this, polymers with elastomeric properties have potential uses in applications requiring considerable material or device flexibility and mechanical interaction. These properties are found in PUs with high molecular weights (> 50 kD). Various antimicrobial applications require a simple thin-film coating of polymer-active agent, which is sufficient in delivering the desired functionality; however, the ability to control the deposition rate of films (and subsequently, coating thickness) provides a method to estimate the optimal thickness for such structures. There is also a need to control the porosity of films, which is highly desirable in specific biomaterial applications, for example, in the case of biosensors or in the development of breathable fabrics, that is, filters for masks. Also, it is well established that rough and patterned surfaces provide an enhanced topography for cell adhesion (Hoffman-Kim et al., 2010), which can be achieved using advanced material fabrication and processing techniques. All of these properties can be achieved using a fibrous coating methodology. Hence, utilizing a fiberforming process or fabricating method will combine all these benefits, which can be readily coupled to intrinsic (e.g., antimicrobial) properties of the selected materials. There are numerous ways to generate fibrous structures, which are rapidly finding increasing applications in several areas of biomedical science and engineering. One such method is electrospinning (Ahmad et al., 2009; Pham et al., 2006), which has been used to prepare biomaterial technologies in drug delivery, tissue engineering, and implantable devices (Almodovar and Kipper, 2011). The process has several advantages, one of which is the ability to utilize coarse processing needles to generate a high volume of ultrafine spun fibers. As the process takes place at ambient temperature, there is no heat-induced change to the chemical structure of the active agent or polymer, and the fibrous structures can be deposited directly onto a device or ready-to-deploy substrate. The method requires a flowing medium, which is perfused into an electrically conducting needle. A controllable electric field is generated, and at the optimized voltage window, fibers can be spun ranging from a few micrometers down to the nanometer scale (Xie et al., 2010). Alternatively, patterns comprising the same material can be deposited in an ordered fashion using a direct write process, which also makes use of the electric field to generate a liquid writing-tip at the exit of the nozzle (Ahmad et al., 2010). In both cases, the key controlling parameters are the applied voltage and flow rate, both of which have a direct impact on the size and structure of individual fiber morphologies. The deposition time is also a controlling parameter, but reflects the dimensions of the bulk structures, that is, film thickness and porosity. This method combines biocompatible PU polymer and CuO nanoparticles to prepare a series of active composite solution blends, although a
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whole host of other nanoparticles can be selected and used in a similar fashion. Selected solutions are spun and result in dry fibrous mats. Here, PU–CuO composite mats (porous and elastomeric) are assessed for their antibacterial properties, using the epidemic methicillin-resistant strain of Staphylococcus aureus (EMRSA) 16. The activated porous elastomeric mats have a host of potential applications, which can reduce, control, or prevent various problems associated with microbial outbreaks such as MRSA. The same materials can also be plotted into patterned structures that are identical to those currently being used in several branches of biomedical science.
2. Materials and Methods 2.1. Materials PU (poly [4,40 -methylenebis (phenyl-isocyanate)-alt-1,4-butanediol/polytetrahydrofuran]) elastomer is purchased from Sigma-Aldrich Company (Poole, UK). CuO nanoparticles are supplied by Intrinsiq Materials (Ren et al., 2009); with a mean size of 50 nm. The solvents dimethyl formamide (DMF, 99%) and tetrahydrofuran (THF, 99%) are purchased from SigmaAldrich Company. Ethanol (99%) and microscopic glass slides are purchased from VWR (Poole, UK). For the studies reported here, the MRSA epidemic strain is kindly provided by the Department of Infectious Disease Epidemiology, Imperial College.
2.2. Methods 2.2.1. Solution preparation An initial stock polymer solution is prepared by suspending PU (10% w/w) in a cosolvent solution comprised of DMF and THF (30:70, respectively). The stock solution is sealed and allowed to stir mechanically at ambient temperature for 5 h. From this, four separate solutions are prepared by dissolving known quantities of CuO (0%, 1%, 5%, and 10% w/w) into individual containers (sealed), which are allowed to stir mechanically for 30 min and then utilized in the electrospinning process. 2.2.2. Fiber, film, and pattern generation For fiber and film generation, individual solutions are used directly after being mechanically stirred. Solutions are infused into the processing needle via silicon tubing. Individual solutions are then introduced into a conducting needle (inner diameter of 330 mm) using a precision Harvard pump. An electrical field is generated using a Glassman high-voltage power supply. After preliminary testing of variables (applied voltage 0–30 kV, flow rate 10–30 ml/min, and collecting distance 20–100 mm), the correct processing
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parameters are determined. Samples are collected on microscopic glass slides directly below the processing orifice and the collection distance and deposition time are recorded. Patterned structures are generated using a printing method developed earlier (Ahmad et al., 2009). Samples to be assessed for surface analysis and antimicrobial testing are washed with ethanol and then water. They are then allowed to stand alone to dry (24 h). The PU polymer used in this study is stable to degradation from ethanol and water. 2.2.3. Microscopy and elemental analysis (EDX) CuO nanoparticles are characterized using transmission electron microscopy ( JEOL 100-CX microscope) to determine the particle size. Electrospun fibers and directly deposited (writing) composite tracks are initially screened using a Nikon Eclipse optical microscope (ME600). Selected samples are then analyzed using a JEOL JSM-6301F scanning electron microscope (SEM) and are also characterized using an inbuilt elemental analysis (EDX) method. For this, samples are coated using a carbon and gold coating-sputtering device. Microscopy is carried out at an accelerating voltage of 5 kV. Other selected samples are also analyzed using an atomic force microscope (AFM) to observe the surface of the various fibers impregnated with different CuO concentrations. 2.2.4. Bacterial testing EMRSA 16 is grown overnight in Tryptone Soya Broth (TSB, Oxoid). Optical density of the culture is adjusted to 0.1 (l ¼ 540 nm) with phosphate buffered saline (PBS). For this particular strain, it is found that this provides an approximate bacterial concentration of 108 colony-forming units per ml (cfu/ml). Each test slide is placed in a 50-ml Corning tube and filled with 45 ml of PBS þ 550 ml of the OD adjusted bacterial culture. These are then placed into a shaker incubator at 200-rpm (37 C). Twenty microliters of each sample is taken at 0, 1, 2, 3, and 4 h, and serial dilutions are made with PBS. The dilutions are plated on Tryptone Soya Agar (TSA, Oxoid) and incubated overnight at 37 C for 24 h. After this period, colonies are counted. The experiment is performed in triplicate with a different set of slides on each occasion.
3. Results and Discussion 3.1. Antimicrobial fiber and film preparation The electrospinning process is a versatile method, which can generate porous and dense films depending on the processing conditions. As it is a time-dependent deposition process, the thickness of films or membranes can also be controlled. In the process deployed to generate elastomeric films,
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the electric field is capable of spinning fibers on the micrometer scale from relatively coarse processing needles, in this instance 330 mm. The process, as shown in Fig. 5.1A, proceeds from conventional dripping (low voltage) to jet formation (progressive higher voltages), and at the optimal voltage, fibers are spun resulting from the jet apex (Kim and Dunn, 2010). In such processes, the solvent is lost rapidly due to various bending and whipping motions (Kim and Dunn, 2010). The CuO particles in the composite solutions (Pu–CuO) have a mean size of 50 nm (Fig. 5.1B) which can A
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Figure 5.1 Fiber and thin-film (CuO) preparation. (A) Process method for spinning, (B) transmission electron micrograph of CuO nanoparticles alone, (C) micrograph of highly porous thin fibrous film on glass slide, (D) micrograph of dense film on glass slide, (E) micrograph of elastomeric fibrous coating on glass slide edge—complete cover, (F) micrograph of mechanically peeled elastomeric film, (G) process method for writing and an array or antimicrobial tracks at (H) low magnification, and (I) high magnification. (Scale bars: (B) ¼ 50 nm, (C) ¼ 20 mm, (D) ¼ 20 mm, (E) ¼ 10 mm, (F) ¼ 20 mm, (H) ¼ 200 mm, and (I) ¼ 30 mm.).
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be easily accommodated in micrometer-scaled fibers. The optimal electrospinning conditions are set at 20-ml/min (flow rate of media being introduced into the processing needle) and the applied voltage is variable between 10 and 13 kV. The deposition distance is another important factor as it permits greater time for any remaining residual solvent to evaporate. For example, collecting the spun fibers (CuO 10% w/w, in PU) under these optimal conditions and a working deposition distance of 150 mm (for 30 s) results in fiber morphologies that are well defined (Fig. 5.1C). This morphology is capable of providing antimicrobial properties for biomaterials requiring “breathable” and “interaction” characteristics, where gaseous exchange, moisture flow, and rough surface topography are made possible by fibers and interconnected fibers resulting in a porous meshwork. Generally at reduced deposition distances, for example, 20 mm, the effects of residual solvent become apparent and there is trivial merging of fibers at adjoining points (Fig. 5.1D). Here, the structures maintain their fibrous morphologies albeit not as distinctly as those afforded with the greater deposition distance. The antimicrobial fiber size obtained under these generating parameters is in the range of 1–3 mm and the various micrographs also suggest that the fibers have a smooth surface. Setting the sample collecting processing parameters (collecting distance at 150 mm, deposition time of 60 s), followed by washing with ethanol (to sterilize and remove any impurities or microscopic solvent residue), a microscopic glass slide (Fig. 5.1E), or any other medical device, can be coated on the surface, around edges and curves. Using these conditions, the deposited fibers provide intrinsic properties to the active base material (CuO). The polymeric material is elastomeric (Fig. 5.1F) which transpires into spun films that have been removed mechanically (peeled). The elastic deformation of these films is clearly evident once detached from the glass surface. These films can therefore potentially serve as filters, masks, and fillers. They may also have potential as controlled release or eluting devices, whereby antimicrobial agents are secreted (by diffusion or degradation) over a designated time period based on polymeric properties. The electrical writing method (Fig. 5.1G) can generate ordered structures and can be used to deposit any pattern or parametric image onto a surface. It offers greater control on deposition and spatial arrangement. Hence, designer filters, grids, topographies, coatings, and antimicrobial spacers can be deposited at specific locations on or to fit inside an object. To demonstrate this, the 10% w/w (CuO) polymer solution was used for fabrication and for this a reduced flow rate of 15 ml/min was deployed at an applied voltage range of 10–13 kV. While the composite structures are ordered (Fig. 5.1H), the distance in between each thread can be varied by simplistic data entry into the uploading program for the plotter head. This can be further multifaceted by patterned structures using arches, angles, and also in three-dimensional (3D) formats, which would enable the movement
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of the plotter over a 3D object. A closer inspection of these structures reveals that these patterns are 30 mm in width.
3.2. Structural analysis Conventional electron micrographs (SEM) show that the fibrous polymer surface is smooth but the location of CuO in the PU fibers, after processing and washing, is not clear. Using various analysis techniques in addition to imaging indicates how well the CuO particles are dispersed in the polymeric matrix. Figure 5.2A1–A4 and B1–B4 show contrast-based images that indicate the location of CuO particles. At lower magnifications these appear as white dots scattered throughout fibers and there is an increase in these dots as the CuO loading is increased. High-magnification contrast imaging also reveals that the increase in the CuO concentration leads to appreciable particle agglomeration within the fibers, which appear as clusters rather than specs. The trend in loading volume is also supported by elemental analysis (Fig. 5.2C1–C4). However, from these graphs, it is clear that the presence of Cu is relatively low when compared to various other elements contributing toward the overall composite composition. Elemental mapping (Fig. 5.2D1– D4) confirms the presence of CuO-agglomerated clusters, some of which can also be found in the 1% w/w fibers, although at a reduced frequency. Figure 5.2D4 shows an inset of how the distribution of CuO particles appear at the fiber edge and on the glass surface interface, which confirms CuO particle entrapment in the PU matrix. Figure 5.2E1–E4 shows AFM images of all types of fibers prepared. The surfaces appear smooth and there is little difference between the fibers prepared from varying compositions. There is little evidence to suggest any fracture. The morphology at overlapping points demonstrate an increase in height, also supporting how the fibers were formed and collected at an increased deposition distance (150 mm) during the electrospinning process.
3.3. Antimicrobial testing using MRSA Figure 5.3 shows the antimicrobial properties of the various coated slides against EMRSA 16. Although bacterial survival with the control slides (polymer free) was not 100%, the reduction was minimal over the 4-h test period. There was a clear relationship between the CuO concentration used in the matrix and the survival rates of MRSA; the highest concentration (10% w/w) reduced survival rates to below 50% after 1 h and close to 10% after 2 h. The 1% CuO concentration coatings reached this level of survival ( 10%) after 4 h. It was also found that the PU fibers without any CuO demonstrated antimicrobial properties, although, after 4 h the survival rates were only down to 35% which is appreciably higher than with the 1% CuO (10%)-containing structures. A clear reduction in bacterial survival
Sample
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Figure 5.2 Fiber analysis on various electrospun compositions. Showing (A1–A4) low magnification contrast imaging—electron microscopy, (B1–B4) high magnification contrast imaging—electron microscopy, (C1–C4) element analysis, (D1–D4) element mapping, and (E1–E4) atomic force microscopy. (Scale bars: A1 ¼ 10 mm, B1 ¼ 1 mm, D1 ¼ 1 mm, A2 ¼ 10 mm, B2 ¼ 5 mm, D2 ¼ 1 mm, A3 ¼ 10 mm, B3 ¼ 5 mm, D3 ¼ 1 mm, A4 ¼ 10 mm, B4 ¼ 5 mm, D4 ¼ 1 mm.)
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EMRSA 16
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Figure 5.3 Survival of EMRSA 16 with respect to time when exposed to microscope slides coated with polyurethane blended with CuO nanoparticles at different concentrations (0%, 1%, 5%, and 10%). The survival of the bacterium in PBS (microscope slide free) was considered to be 100%. Standard deviation bars are shown (n ¼ 3).
with time was observed and a dose–response was noted. ANOVA analysis did not show any significant differences in the antimicrobial potential between PU alone and when CuO nanoparticles were incorporated at 1% and 5%. However, when this percentage was increased to 10%, significant differences (p < 0.05) were found with respect to PU alone in every case, and also when compared to the 1% and 5% slides at 2, 3, and 4 h. With 10% CuO slides, at the 2-h time point the bacterial survival had decreased to 10%, virtually the entire bacterial population was killed by 4 h. The antimicrobial properties shown by the PU polymer alone need to be investigated further as such characteristics have been reported with other polymeric systems (Kawahara et al., 2009). These polymers are segmented and various constituents can be altered to observe any changes to their antimicrobial properties. As they are used extensively in biomaterial applications in medicine, this would provide excellent opportunities in developing standalone antimicrobial polymer devices. Although the fibrous film coatings are washed with ethanol and water to eliminate any remaining residual solvent, the bulk of which is evaporated during the spinning process, this may still be a contributing factor toward the antimicrobial effects observed, and also needs to be investigated further. The antimicrobial activity is thought to be contact-based inhibition, as the polymer is stable from hydrolytic degradation and this would be potentially
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Spin Elastomeric polymer biomaterial and active component
Medical device coatings Breathable fabrics
Electrical Process
Random filters
Tissue engineering structures
e.g., Antibacterial
Write
Ordered filters and meshworks
Active patches
Orthopaedic coatings
Patterning for enhanced fixation
Figure 5.4 Potential applications of randomly and directly deposited elastomeric structures which combine a polymeric matrix and an active agent (i.e., CuO nanoparticles) system. Structures are formed using variations in electric field deposition.
useful in material contact devices (fillers, coatings, filters, masks, etc.) with the ability to also directly spray onto surfaces. If the mechanism is release based, then this also suggests a sustained release profile over 4 h and applications in eluting devices (timely purifiers) are possible. For both of these mechanisms, altering the active agent or the polymer-degradation properties can further highlight the potential. There is also the prospect to encapsulate (Ahmad et al., 2008, 2009) structures (core shell) with such materials using a two-tier inhibition mechanism that includes release and contact. These potential applications are highlighted in Fig. 5.4, where some of these areas are currently under detailed investigation for their potential application.
4. Concluding Remarks and Future Work At this stage, there is evidence to suggest that the content of CuO particles in the PU fibrous matrix has an effect on the level of MRSA inhibition. With the ability to control the pore size and film thickness, the direct deposition onto devices, the type of active particle in use, the type of polymer in use, there is huge potential of such materials and agents in several biomedical applications as afore mentioned. With these preliminary findings in mind we hope to focus our attention on the mechanism of action, the properties of electrospun fibers, and their ability in inhibiting pathogens such as MRSA. We will also aim to develop a series of novel antimicrobial structures and develop a greater understanding into their inhibitory action, which in the current climate poses a huge burden on the healthcare remit.
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ACKNOWLEDGMENTS The authors would like to thank the Archaeology Department at University College London (UCL) for the use of their electron microscope facilities. They would also like to thank Kings College London (KCL) for the use of their atomic force microscope (AFM). The Royal Society is also gratefully acknowledged for their help in providing equipment. Finally they would like to thank The Department of Infectious Disease Epidemiology (DIDE), Imperial College, who provided the MRSA epidemic strain.
REFERENCES Ahmad, Z., Zhang, H. R., Farook, U., Edirisinghe, M., Stride, E., and Columbo, P. (2008). Generation of multi-layered structures for biomedical applications using a novel trineedle co-axial device and electrohydrodynamic flow. J. R. Soc. Interface 5, 1255–1261. Ahmad, Z., Nangrejo, M., Edirisinghe, M., Stride, E., Columbo, P., and Zhang, H. (2009). Engineering a material for biomedical applications with electric field assisted processing. Appl. Phys. A Mater. Sci. Process 97, 31–37. Ahmad, Z., Rasekh, M., and Edirisinghe, M. (2010). Electrohydrodynamic direct writing of biomedical polymers and composites. Macromol. Mater. Eng. 295, 315–319. Almodovar, J. And, and Kipper, M. J. (2011). Coating electrospun chitosan nanofibers with polyelectrolyte multilayers using the polysaccharides heparin and N, N, N-trimethyl chitosan. Macromol. Biosci. 11, 72–76. Bertoldi, S., Fare`, S., Denegri, M., Rossi, M., Haugen, H. J., Parolini, O. And, and Tanzi, M. C. (2010). Ability of polyurethane foams to support placenta-derived cell adhesion and osteogenic differentiation: Preliminary results. J. Mater. Sci. Mater. Med. 21, 1005–1011. Borkow, G., Zhou, S. S., Page, T., and Gabbay, J. (2010). A novel anti-influenza copper oxide containing respiratory face mask. PLoS One 5, e11295. De Jong, W. H., and Borm, P. J. (2008). Drug delivery and nanoparticles: Applications and hazards. Int. J. Nanomedicine 3, 133–149. Dickinson, L. E., Kusuma, S., and Gerecht, S. (2011). Reconstructing the differentiation niche of embryonic stem cells using biomaterials. Macromol. Biosci. 11, 36–49. Ghosh, S. (2005). Antibacterial effects of silver doped polyethyleneglycol based polyamidoamine side chain dendritic polyurethane. J. Macromol. Sci. Pure Appl. Chem. 42, 765–770. Hoffman-Kim, D., Mitchel, J. A., and Bellamkonda, R. V. (2010). Topography, cell response and nerve regeneration. Annu. Rev. Biomed. Eng. 12, 203–231. Kawahara, T., Takeuchi, Y., Wei, G., Shirai, K., Yamauchi, T., and Tsubokawa, N. (2009). Preparation of antibacterial polymer-grafted silica nanoparticle and surface properties of composites filled with the silica. Polym. J. 41, 744–751. Kim, O. V., and Dunn, P. F. (2010). Controlled production of droplets by in-flight electrospinning. Langmuir 26, 15807–15813. Luo, J., Deng, Y., and Sun, Y. (2010). Antimicrobial activity and biocompatibility of polyurethane-iodine complexes. J. Bioact. Compat. Polym. 25, 185–206. Perni, S., Piccirillo, C., Pratten, J., Prokopovich, P., Chrzanowski, W., Parkin, I. P., and Wilson, M. (2009). The antimicrobial properties of light-activated polymers containing methylene blue and gold nanoparticles. Biomaterials 30, 89–93. Pham, Q. P., Sharma, U., and Mikos, A. G. (2006). Electrospinning of polymeric nanofibers for tissue engineering applications: A review. Tissue Eng. 5, 1197–1211. Ren, G., Hu, D., Cheng, E., Vargas-Reus, M. A., Reip, P., and Allaker, R. P. (2009). Characterisation of copper oxide nanoparticles for antimicrobial applications. Int. J. Antimicrob. Agents 33, 587–590.
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F I V E
Antimicrobial Properties of Electrically Formed Elastomeric Polyurethane–Copper Oxide Nanocomposites for Medical and Dental Applications Z. Ahmad,* M. A. Vargas-Reus,† R. Bakhshi,‡ F. Ryan,‡ G. G. Ren,§ F. Oktar,} and R. P. Allaker† Contents 88 90 90 90 91 91 94 94 97 98 98
1. Introduction 2. Materials and Methods 2.1. Materials 2.2. Methods 3. Results and Discussion 3.1. Antimicrobial fiber and film preparation 3.2. Structural analysis 3.3. Antimicrobial testing using MRSA 4. Concluding Remarks and Future Work Acknowledgments References
Abstract With the rapidly advancing field of nanotechnology having an impact in several areas interfacing life and physical sciences, the potential applications of nanoparticles as antimicrobial agents have been realized and offer great opportunities in addressing several viral and bacterial outbreak issues. Polyurethanes (PUs) are a diverse class of polymeric materials which also have applications in several areas of biomedical science ranging from blood contact devices to * School of Pharmacy and Biomedical Sciences, University of Portsmouth, Portsmouth, United Kingdom Queen Mary University of London, Barts and The London School of Medicine and Dentistry, Institute of Dentistry, London, United Kingdom { Department of Mechanical Engineering, University College London, London, United Kingdom } School of Engineering and Technology, University of Hertfordshire, Hatfield, United Kingdom } Nanotechnology and Biomaterials Application & Research Centre, Marmara University, Istanbul, Turkey {
Methods in Enzymology, Volume 509 ISSN 0076-6879, DOI: 10.1016/B978-0-12-391858-1.00005-8
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2012 Elsevier Inc. All rights reserved.
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implantable dental technologies. In this report, copper oxide (CuO) nanoparticles (mean size 50 nm) are embedded into a PU matrix via two electrical fabrication processes. To elucidate the antimicrobial activity, a range of different loading compositions of CuO within the PU matrix (0%, 1%, 5%, and 10% w/w) are electrospun to form thin porous films (thickness <10 mm). After washing, the films are tested for their antimicrobial properties against methicillin-resistant Staphylococcus aureus (MRSA). Significant reduction of populations was demonstrated with 10% w/w CuO over a 4-h period. This approach demonstrates the potential of generating tailored antimicrobial structures for a host of applications, such as designer filters, patterned coatings, breathable fabrics, adhesive films (as opposed to sutures), and mechanically supporting structures.
1. Introduction Nanoscaled particles have diverse applications ranging from medical device coatings to drug delivery carriers (De Jong and Borm, 2008; Dickinson et al., 2011). More recently, and in a timely manner, their applications as antimicrobial agents have also been realized especially with metal and metal oxide particle systems (Borkow et al., 2010; Ren et al., 2009). The antimicrobial properties of copper (Cu) and its oxide (CuO) have been known for centuries (on the macroscale) and with current advances in technology; selected textile and material composite applications have demonstrated their potential as antimicrobial agents on the micro- and nanometer scales (Ruparelia et al., 2008; Zhang et al., 2006). Other types of metallic nanoparticles demonstrating such properties include gold and silver (Perni et al., 2009; Ruparelia et al., 2008); however, cost remains an important factor when considering the scale-up potential and the broad range of potential applications. Polyurethanes (PUs) have several applications as biomaterials including coatings, blood bags, catheters, heart valves, dental fillers, protective clothing, and even tissue-engineering constructs (Bertoldi et al., 2010; Luo et al., 2010; Sui et al., 2010). The mechanical, degradation, and biostability properties of these materials can be controlled by carefully selecting the various segments which constitute toward the polymeric backbone (Zdrahala and Zdrahala, 1999). They have also found several applications as antimicrobial materials and in many instances utilize an active agent, which is capable of killing bacteria or viruses (Ghosh, 2005). Numerous devices serving as filters, adhesive films, fillers, eluting structures, and coatings require a polymeric matrix to be impregnated with such components or secondary materials which possess the functional characteristics, for example, antimicrobial properties. Depending on the intended function, the polymeric matrix can contribute toward a mechanism for the release of active agents, the mechanical properties of the device, and the localization of the active agent at a specific point. In such cases, the
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loading volume of the active agent (e.g., possessing antimicrobial properties) must be sufficient to demonstrate the desired effect. In addition to this, polymers with elastomeric properties have potential uses in applications requiring considerable material or device flexibility and mechanical interaction. These properties are found in PUs with high molecular weights (> 50 kD). Various antimicrobial applications require a simple thin-film coating of polymer-active agent, which is sufficient in delivering the desired functionality; however, the ability to control the deposition rate of films (and subsequently, coating thickness) provides a method to estimate the optimal thickness for such structures. There is also a need to control the porosity of films, which is highly desirable in specific biomaterial applications, for example, in the case of biosensors or in the development of breathable fabrics, that is, filters for masks. Also, it is well established that rough and patterned surfaces provide an enhanced topography for cell adhesion (Hoffman-Kim et al., 2010), which can be achieved using advanced material fabrication and processing techniques. All of these properties can be achieved using a fibrous coating methodology. Hence, utilizing a fiberforming process or fabricating method will combine all these benefits, which can be readily coupled to intrinsic (e.g., antimicrobial) properties of the selected materials. There are numerous ways to generate fibrous structures, which are rapidly finding increasing applications in several areas of biomedical science and engineering. One such method is electrospinning (Ahmad et al., 2009; Pham et al., 2006), which has been used to prepare biomaterial technologies in drug delivery, tissue engineering, and implantable devices (Almodovar and Kipper, 2011). The process has several advantages, one of which is the ability to utilize coarse processing needles to generate a high volume of ultrafine spun fibers. As the process takes place at ambient temperature, there is no heat-induced change to the chemical structure of the active agent or polymer, and the fibrous structures can be deposited directly onto a device or ready-to-deploy substrate. The method requires a flowing medium, which is perfused into an electrically conducting needle. A controllable electric field is generated, and at the optimized voltage window, fibers can be spun ranging from a few micrometers down to the nanometer scale (Xie et al., 2010). Alternatively, patterns comprising the same material can be deposited in an ordered fashion using a direct write process, which also makes use of the electric field to generate a liquid writing-tip at the exit of the nozzle (Ahmad et al., 2010). In both cases, the key controlling parameters are the applied voltage and flow rate, both of which have a direct impact on the size and structure of individual fiber morphologies. The deposition time is also a controlling parameter, but reflects the dimensions of the bulk structures, that is, film thickness and porosity. This method combines biocompatible PU polymer and CuO nanoparticles to prepare a series of active composite solution blends, although a
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whole host of other nanoparticles can be selected and used in a similar fashion. Selected solutions are spun and result in dry fibrous mats. Here, PU–CuO composite mats (porous and elastomeric) are assessed for their antibacterial properties, using the epidemic methicillin-resistant strain of Staphylococcus aureus (EMRSA) 16. The activated porous elastomeric mats have a host of potential applications, which can reduce, control, or prevent various problems associated with microbial outbreaks such as MRSA. The same materials can also be plotted into patterned structures that are identical to those currently being used in several branches of biomedical science.
2. Materials and Methods 2.1. Materials PU (poly [4,40 -methylenebis (phenyl-isocyanate)-alt-1,4-butanediol/polytetrahydrofuran]) elastomer is purchased from Sigma-Aldrich Company (Poole, UK). CuO nanoparticles are supplied by Intrinsiq Materials (Ren et al., 2009); with a mean size of 50 nm. The solvents dimethyl formamide (DMF, 99%) and tetrahydrofuran (THF, 99%) are purchased from SigmaAldrich Company. Ethanol (99%) and microscopic glass slides are purchased from VWR (Poole, UK). For the studies reported here, the MRSA epidemic strain is kindly provided by the Department of Infectious Disease Epidemiology, Imperial College.
2.2. Methods 2.2.1. Solution preparation An initial stock polymer solution is prepared by suspending PU (10% w/w) in a cosolvent solution comprised of DMF and THF (30:70, respectively). The stock solution is sealed and allowed to stir mechanically at ambient temperature for 5 h. From this, four separate solutions are prepared by dissolving known quantities of CuO (0%, 1%, 5%, and 10% w/w) into individual containers (sealed), which are allowed to stir mechanically for 30 min and then utilized in the electrospinning process. 2.2.2. Fiber, film, and pattern generation For fiber and film generation, individual solutions are used directly after being mechanically stirred. Solutions are infused into the processing needle via silicon tubing. Individual solutions are then introduced into a conducting needle (inner diameter of 330 mm) using a precision Harvard pump. An electrical field is generated using a Glassman high-voltage power supply. After preliminary testing of variables (applied voltage 0–30 kV, flow rate 10–30 ml/min, and collecting distance 20–100 mm), the correct processing
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parameters are determined. Samples are collected on microscopic glass slides directly below the processing orifice and the collection distance and deposition time are recorded. Patterned structures are generated using a printing method developed earlier (Ahmad et al., 2009). Samples to be assessed for surface analysis and antimicrobial testing are washed with ethanol and then water. They are then allowed to stand alone to dry (24 h). The PU polymer used in this study is stable to degradation from ethanol and water. 2.2.3. Microscopy and elemental analysis (EDX) CuO nanoparticles are characterized using transmission electron microscopy ( JEOL 100-CX microscope) to determine the particle size. Electrospun fibers and directly deposited (writing) composite tracks are initially screened using a Nikon Eclipse optical microscope (ME600). Selected samples are then analyzed using a JEOL JSM-6301F scanning electron microscope (SEM) and are also characterized using an inbuilt elemental analysis (EDX) method. For this, samples are coated using a carbon and gold coating-sputtering device. Microscopy is carried out at an accelerating voltage of 5 kV. Other selected samples are also analyzed using an atomic force microscope (AFM) to observe the surface of the various fibers impregnated with different CuO concentrations. 2.2.4. Bacterial testing EMRSA 16 is grown overnight in Tryptone Soya Broth (TSB, Oxoid). Optical density of the culture is adjusted to 0.1 (l ¼ 540 nm) with phosphate buffered saline (PBS). For this particular strain, it is found that this provides an approximate bacterial concentration of 108 colony-forming units per ml (cfu/ml). Each test slide is placed in a 50-ml Corning tube and filled with 45 ml of PBS þ 550 ml of the OD adjusted bacterial culture. These are then placed into a shaker incubator at 200-rpm (37 C). Twenty microliters of each sample is taken at 0, 1, 2, 3, and 4 h, and serial dilutions are made with PBS. The dilutions are plated on Tryptone Soya Agar (TSA, Oxoid) and incubated overnight at 37 C for 24 h. After this period, colonies are counted. The experiment is performed in triplicate with a different set of slides on each occasion.
3. Results and Discussion 3.1. Antimicrobial fiber and film preparation The electrospinning process is a versatile method, which can generate porous and dense films depending on the processing conditions. As it is a time-dependent deposition process, the thickness of films or membranes can also be controlled. In the process deployed to generate elastomeric films,
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the electric field is capable of spinning fibers on the micrometer scale from relatively coarse processing needles, in this instance 330 mm. The process, as shown in Fig. 5.1A, proceeds from conventional dripping (low voltage) to jet formation (progressive higher voltages), and at the optimal voltage, fibers are spun resulting from the jet apex (Kim and Dunn, 2010). In such processes, the solvent is lost rapidly due to various bending and whipping motions (Kim and Dunn, 2010). The CuO particles in the composite solutions (Pu–CuO) have a mean size of 50 nm (Fig. 5.1B) which can A
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Figure 5.1 Fiber and thin-film (CuO) preparation. (A) Process method for spinning, (B) transmission electron micrograph of CuO nanoparticles alone, (C) micrograph of highly porous thin fibrous film on glass slide, (D) micrograph of dense film on glass slide, (E) micrograph of elastomeric fibrous coating on glass slide edge—complete cover, (F) micrograph of mechanically peeled elastomeric film, (G) process method for writing and an array or antimicrobial tracks at (H) low magnification, and (I) high magnification. (Scale bars: (B) ¼ 50 nm, (C) ¼ 20 mm, (D) ¼ 20 mm, (E) ¼ 10 mm, (F) ¼ 20 mm, (H) ¼ 200 mm, and (I) ¼ 30 mm.).
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be easily accommodated in micrometer-scaled fibers. The optimal electrospinning conditions are set at 20-ml/min (flow rate of media being introduced into the processing needle) and the applied voltage is variable between 10 and 13 kV. The deposition distance is another important factor as it permits greater time for any remaining residual solvent to evaporate. For example, collecting the spun fibers (CuO 10% w/w, in PU) under these optimal conditions and a working deposition distance of 150 mm (for 30 s) results in fiber morphologies that are well defined (Fig. 5.1C). This morphology is capable of providing antimicrobial properties for biomaterials requiring “breathable” and “interaction” characteristics, where gaseous exchange, moisture flow, and rough surface topography are made possible by fibers and interconnected fibers resulting in a porous meshwork. Generally at reduced deposition distances, for example, 20 mm, the effects of residual solvent become apparent and there is trivial merging of fibers at adjoining points (Fig. 5.1D). Here, the structures maintain their fibrous morphologies albeit not as distinctly as those afforded with the greater deposition distance. The antimicrobial fiber size obtained under these generating parameters is in the range of 1–3 mm and the various micrographs also suggest that the fibers have a smooth surface. Setting the sample collecting processing parameters (collecting distance at 150 mm, deposition time of 60 s), followed by washing with ethanol (to sterilize and remove any impurities or microscopic solvent residue), a microscopic glass slide (Fig. 5.1E), or any other medical device, can be coated on the surface, around edges and curves. Using these conditions, the deposited fibers provide intrinsic properties to the active base material (CuO). The polymeric material is elastomeric (Fig. 5.1F) which transpires into spun films that have been removed mechanically (peeled). The elastic deformation of these films is clearly evident once detached from the glass surface. These films can therefore potentially serve as filters, masks, and fillers. They may also have potential as controlled release or eluting devices, whereby antimicrobial agents are secreted (by diffusion or degradation) over a designated time period based on polymeric properties. The electrical writing method (Fig. 5.1G) can generate ordered structures and can be used to deposit any pattern or parametric image onto a surface. It offers greater control on deposition and spatial arrangement. Hence, designer filters, grids, topographies, coatings, and antimicrobial spacers can be deposited at specific locations on or to fit inside an object. To demonstrate this, the 10% w/w (CuO) polymer solution was used for fabrication and for this a reduced flow rate of 15 ml/min was deployed at an applied voltage range of 10–13 kV. While the composite structures are ordered (Fig. 5.1H), the distance in between each thread can be varied by simplistic data entry into the uploading program for the plotter head. This can be further multifaceted by patterned structures using arches, angles, and also in three-dimensional (3D) formats, which would enable the movement
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of the plotter over a 3D object. A closer inspection of these structures reveals that these patterns are 30 mm in width.
3.2. Structural analysis Conventional electron micrographs (SEM) show that the fibrous polymer surface is smooth but the location of CuO in the PU fibers, after processing and washing, is not clear. Using various analysis techniques in addition to imaging indicates how well the CuO particles are dispersed in the polymeric matrix. Figure 5.2A1–A4 and B1–B4 show contrast-based images that indicate the location of CuO particles. At lower magnifications these appear as white dots scattered throughout fibers and there is an increase in these dots as the CuO loading is increased. High-magnification contrast imaging also reveals that the increase in the CuO concentration leads to appreciable particle agglomeration within the fibers, which appear as clusters rather than specs. The trend in loading volume is also supported by elemental analysis (Fig. 5.2C1–C4). However, from these graphs, it is clear that the presence of Cu is relatively low when compared to various other elements contributing toward the overall composite composition. Elemental mapping (Fig. 5.2D1– D4) confirms the presence of CuO-agglomerated clusters, some of which can also be found in the 1% w/w fibers, although at a reduced frequency. Figure 5.2D4 shows an inset of how the distribution of CuO particles appear at the fiber edge and on the glass surface interface, which confirms CuO particle entrapment in the PU matrix. Figure 5.2E1–E4 shows AFM images of all types of fibers prepared. The surfaces appear smooth and there is little difference between the fibers prepared from varying compositions. There is little evidence to suggest any fracture. The morphology at overlapping points demonstrate an increase in height, also supporting how the fibers were formed and collected at an increased deposition distance (150 mm) during the electrospinning process.
3.3. Antimicrobial testing using MRSA Figure 5.3 shows the antimicrobial properties of the various coated slides against EMRSA 16. Although bacterial survival with the control slides (polymer free) was not 100%, the reduction was minimal over the 4-h test period. There was a clear relationship between the CuO concentration used in the matrix and the survival rates of MRSA; the highest concentration (10% w/w) reduced survival rates to below 50% after 1 h and close to 10% after 2 h. The 1% CuO concentration coatings reached this level of survival ( 10%) after 4 h. It was also found that the PU fibers without any CuO demonstrated antimicrobial properties, although, after 4 h the survival rates were only down to 35% which is appreciably higher than with the 1% CuO (10%)-containing structures. A clear reduction in bacterial survival
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Figure 5.2 Fiber analysis on various electrospun compositions. Showing (A1–A4) low magnification contrast imaging—electron microscopy, (B1–B4) high magnification contrast imaging—electron microscopy, (C1–C4) element analysis, (D1–D4) element mapping, and (E1–E4) atomic force microscopy. (Scale bars: A1 ¼ 10 mm, B1 ¼ 1 mm, D1 ¼ 1 mm, A2 ¼ 10 mm, B2 ¼ 5 mm, D2 ¼ 1 mm, A3 ¼ 10 mm, B3 ¼ 5 mm, D3 ¼ 1 mm, A4 ¼ 10 mm, B4 ¼ 5 mm, D4 ¼ 1 mm.)
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Figure 5.3 Survival of EMRSA 16 with respect to time when exposed to microscope slides coated with polyurethane blended with CuO nanoparticles at different concentrations (0%, 1%, 5%, and 10%). The survival of the bacterium in PBS (microscope slide free) was considered to be 100%. Standard deviation bars are shown (n ¼ 3).
with time was observed and a dose–response was noted. ANOVA analysis did not show any significant differences in the antimicrobial potential between PU alone and when CuO nanoparticles were incorporated at 1% and 5%. However, when this percentage was increased to 10%, significant differences (p < 0.05) were found with respect to PU alone in every case, and also when compared to the 1% and 5% slides at 2, 3, and 4 h. With 10% CuO slides, at the 2-h time point the bacterial survival had decreased to 10%, virtually the entire bacterial population was killed by 4 h. The antimicrobial properties shown by the PU polymer alone need to be investigated further as such characteristics have been reported with other polymeric systems (Kawahara et al., 2009). These polymers are segmented and various constituents can be altered to observe any changes to their antimicrobial properties. As they are used extensively in biomaterial applications in medicine, this would provide excellent opportunities in developing standalone antimicrobial polymer devices. Although the fibrous film coatings are washed with ethanol and water to eliminate any remaining residual solvent, the bulk of which is evaporated during the spinning process, this may still be a contributing factor toward the antimicrobial effects observed, and also needs to be investigated further. The antimicrobial activity is thought to be contact-based inhibition, as the polymer is stable from hydrolytic degradation and this would be potentially
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Spin Elastomeric polymer biomaterial and active component
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Figure 5.4 Potential applications of randomly and directly deposited elastomeric structures which combine a polymeric matrix and an active agent (i.e., CuO nanoparticles) system. Structures are formed using variations in electric field deposition.
useful in material contact devices (fillers, coatings, filters, masks, etc.) with the ability to also directly spray onto surfaces. If the mechanism is release based, then this also suggests a sustained release profile over 4 h and applications in eluting devices (timely purifiers) are possible. For both of these mechanisms, altering the active agent or the polymer-degradation properties can further highlight the potential. There is also the prospect to encapsulate (Ahmad et al., 2008, 2009) structures (core shell) with such materials using a two-tier inhibition mechanism that includes release and contact. These potential applications are highlighted in Fig. 5.4, where some of these areas are currently under detailed investigation for their potential application.
4. Concluding Remarks and Future Work At this stage, there is evidence to suggest that the content of CuO particles in the PU fibrous matrix has an effect on the level of MRSA inhibition. With the ability to control the pore size and film thickness, the direct deposition onto devices, the type of active particle in use, the type of polymer in use, there is huge potential of such materials and agents in several biomedical applications as afore mentioned. With these preliminary findings in mind we hope to focus our attention on the mechanism of action, the properties of electrospun fibers, and their ability in inhibiting pathogens such as MRSA. We will also aim to develop a series of novel antimicrobial structures and develop a greater understanding into their inhibitory action, which in the current climate poses a huge burden on the healthcare remit.
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ACKNOWLEDGMENTS The authors would like to thank the Archaeology Department at University College London (UCL) for the use of their electron microscope facilities. They would also like to thank Kings College London (KCL) for the use of their atomic force microscope (AFM). The Royal Society is also gratefully acknowledged for their help in providing equipment. Finally they would like to thank The Department of Infectious Disease Epidemiology (DIDE), Imperial College, who provided the MRSA epidemic strain.
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