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Plasmon-based biofilm inhibition on surgical implants Romain Quidant, Ignacio de Miguel, Irene Prieto, Arantxa Albornoz, Vanesa Sanz, Christine Weis, and Pau Turon Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00187 • Publication Date (Web): 12 Mar 2019 Downloaded from http://pubs.acs.org on March 13, 2019
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Plasmon-based biofilm inhibition on surgical implants Ignacio de Miguel1, Irene Prieto2, Arantxa Albornoz1, Vanesa Sanz2, Christine Weis2, Pau Turon2, Romain Quidant1,3 1ICFO-Institut
de Ciències Fotòniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels,
Spain 2
B. Braun Surgical, S.A., Department of Research and Development, 08191 Rubí, Barcelona, Spain
3
ICREA -Institució Catalana de Recerca i Estudis Avançats, 08010 Barcelona, Spain
The insertion of an implant in the body of a patient raises the risk of a posterior infection and formation of a biofilm, which can have critical consequences on the patient health and be associated to a high sanitary cost. While antibacterial agents can be used to prevent the infection, such a strategy is time-limited and causes bacteria resistance. As an alternative to biochemical approaches, we propose here to use light-induced local hyperthermia with plasmonic nanoparticles. This strategy is implemented on surgical meshes, extensively used in the context of hernia repairing, one of the most common general surgeries. Surgical meshes were homogeneously coated with gold nanorods designed to efficiently convert near-infrared light into heat. The modified mesh was exposed to a biofilm of Staphylococcus aureus (S. aureus) bacteria before being treated with a train of light pulses. We systematically study how the illumination parameters, namely fluence, peak intensity and pulse length, influence the elimination of attached bacteria. Additionally, fluorescence confocal microscopy provides us some insight on the mechanism involved in the degradation of the biofilm. This proof-of-principle study opens a new set of opportunities for the development of novel disinfection approaches combining light and nanotechnology. Keywords: Nanotechnology, Plasmonics, Biofilm, Surgical implants, Disinfection Medical devices have been extensively used in most fields of medicine for diagnostic and therapeutic procedures. However, the risks associated with their use are not negligible, especially when they are intended to be implanted inside the body for long periods. One of the most relevant issue is bacterial colonization of the medical device after surgery that has a significant impact on both the patient health and the costs related to the treatment of the infection. Such complications are frequently linked with bacteria that contaminate surgical wounds during surgery or during the time of hospitalization resulting in a so-called nosocomial infection. In the United States, approximately 2 million nosocomial infections cost nearly $11 billion annually.1 Those infections are difficult to handle as bacteria (e.g. Methicillin-resistant Staphylococcus aureus (MRSA) and Vancomycin-resistant Enterococcus (VRE)) are becoming more resistant to classical antibiotic therapies, particularly when the devices are colonized by bacteria developing a biofilm.2 Microorganisms once adhered to the implant surface are able to protect themselves by producing an extracellular matrix, a substance rich in exopolysaccharides. The combination of the bacteria colonizing the surface with such a polysaccharide shelter is what confers the biofilm its resistance to the immune
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system.3 A recent study published by the European Antimicrobial Resistance Surveillance Network (EARS-Net) estimates more than 30.000 deaths in Europe attributable to infections with antibiotic-resistant bacteria in 2015.4 Hernia repair is one of the most common procedures in general surgery, with an estimated 20 million operations performed annually worldwide.5 The concept of tension-free repair by using synthetic meshes was introduced more than 50 years ago and is nowadays the standard procedure.6 The benefits of mesh repair over the traditional repair procedures with sutures include a significant reduction in the incidence of hernia recurrence. Mesh infection is a potentially devastating complication, which has a high patient morbidity. The clinically reported incidences of meshrelated infection after hernia repair surgery are in the range within 1–8%, depending on the surgical technique, and up to 6-8% for open incisional hernia repair.7 The contamination of implantable surgical meshes most likely occurs by inoculation with a few microorganisms from the patient´s skin or mucous membranes during surgery. These microorganisms once embedded in the biofilm layer become significantly more resistant to antimicrobial agents and chemical therapies as they are able to synchronize themselves to resist their effects.3 When this clinical complication occurs, the treatment may require additional surgical procedures, including the removal of the mesh.8 The most used strategies to prevent implant contamination are the use of aseptic protocols during surgery and the prophylactic administration of antibiotics via the systemic route or local antibiotic irrigation of the surgical site. The use of post-operative antibiotic therapy was the first choice to fight against infections in general, but is currently of greatest concern because of already developed antibiotic resistant strains, like Methicillin-resistant Staphylococcus aureus, which is frequently the responsible strain for nosocomial infections, especially for mesh infections.9 Moreover, further strategies were developed to fight biofilms: i) modifying the interactions between biomaterials and bacteria by giving anti-adhesive properties to the implant surface itself (i.e. by using cationic charges, hydrophobic interactions or surface modifications intended to repel the microorganism and avoid their attachment)10 and ii) the use of antibacterial agents like antibiotics, antiseptics or metals like silver, mostly incorporated in implant coatings.3 By releasing the antibacterial agent from the coating the bacterial adhesion to the implant surface is reduced, nevertheless, this release strategy is time-limited.11 Taken into consideration that the possibility of a mesh-related infection is occurring weeks or even months after the surgery, a timely unlimited antibacterial approach would be more appropriate. Recent advances in Nanotechnology can provide solutions to current limitations. A variety of non-organic nanoparticles have been evaluated for their potential application as antimicrobial agents on implantable devices. 12 For instance, catheters were functionalized with silver nanoparticles, which upon oxidation release Ag+ ions and are able to disrupt the bacteria membrane, bind to the DNA and thereby inhibit protein synthesis.13 Owing to their unique optical, chemical and biological properties, gold nanoparticles (GNPs) are increasingly used in the biomedical field 14. In particular, GNPs interact very efficiently with light at their Localized Surface Plasmon (LSP) resonance 15. While part of the interacting light is elastically scattered, the remaining part is absorbed by the metal and eventually dissipated to the surrounding in the form of heat 16. This capability of GNPs to locally deliver heat upon illumination
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has been thoroughly investigated and is currently exploited for hyperthermia-based therapies in cancer and other diseases 17-19. Here, we investigate the use of plasmon-enabled targeted hyperthermia in the context of disinfection, to prevent bacterial contamination on surgical implants. The surface of a surgical mesh was chemically modified to anchor gold nanorods (GNRs) tuned to be resonant at 800nm, inside the first transparency window of biological tissues (650-1100nm)20 where the lower absorption of water, hemoglobin and deoxyhemoglobin enables a deeper penetration of light. Upon illumination, under harmless conditions, matching the LSP resonance of the GNRs, the local temperature increase damages the biofilm. Because this damage is caused by physical means, the microorganisms cannot adapt to the treatment and create resistance. In practice, NIR irradiation could be performed repeatedly after the mesh implantation and as many times as necessary. In this paper, a protocol to ensure homogenous and robust GNR coating of the mesh surface is first presented. Subsequently, we characterize the light to heat conversion efficiency as a function of the GNR concentration. Finally, we systematically study how the illumination parameters, such as pulse intensity and duration, affect the bacteria adhesion and destruction. Similar to cells, mesophilic bacteria viability can be altered by increasing the temperature above 45 ºC. Previous studies demonstrated that light-to-heat conversion in plasmonic nanoparticles could be used to harm bacteria immobilized on a flat glass substrate 21-22. The extension of this concept to biofilm prevention on 3D surgical implants raises several major challenges. First, one needs to ensure homogeneous immobilization of plasmonic nanoparticles at a non-flat surface and its long-term stability, in order to avoid uncontrolled release of particles in the body. Another important question is related to how localized heating at the implant surface can alter the integrity of the biofilm, which can be several hundred of micrometers thick. Last but not least, the relevance of such an approach is conditioned by a minimum antibacterial efficiency in realistic conditions. To illustrate the capability of plasmon-enabled hyperthermia in biofilm prevention on surgical implants, we chose to work with a polymer surgical mesh from B. Braun Surgical, S.A. (Optilene® Mesh LP), widely used for hernia repair. Optilene® Mesh LP meshes are lightweight (36 g/m2) large pore (1 mm pore) meshes constituted of polypropylene monofilaments of 100 µm diameter. The experimental sequence we follow is schematized in figure 1. The mesh surface is first chemically modified to enable robust and homogeneous immobilization of GNR. The modified mesh is subsequently inoculated with S. aureus, until the formation of a biofilm. Decontaminating treatment itself consists of a train of light pulses from a filtered Intense Pulsed Light (IPL). Finally, the treated mesh is processed for Colony-Forming Units (CFU) counting and confocal fluorescence microscopy to study the impact of the treatment on the biofilm.
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Figure 1 – Schematic view of plasmon-enabled biofilm prevention on surgical meshes. The mesh surface is functionalized to enable robust and homogeneous GNR coverage. The modified mesh is then inoculated with S. aureus to induce the formation of a biofilm. Treatment is performed by a train of light pulses from a filtered IPL (7501200nm), covering the main localized plasmon resonance of the GNRs. Biofilm viability study and CFU counting are finally used to evaluate the treatment efficiency. Characterization of the modified meshes – The main steps of the mesh conjugation with GNRs are schematized in Figure 2.a while experimental details can be found in the experimental section. Figure 2.b shows the results of Au contents and surface coverage density (GNR/µm2) considering a molecular weight per GNR of 73 MDa. The fixation yield exceeds 90% and starts decreasing for a surface coverage greater than 225 GNR/µm2. Additional data about the grafting yield and surface coverage are presented in the SI (Figure S1). The distribution of immobilized GNRs was evaluated by Scanning Electron Microscopy (SEM) showing a good homogeneity and little or no GNR aggregation (Figure 3.a). Subsequently, we evaluate the ability of the surface of the modified meshes to heat up upon illumination with a CW 810-nm laser source delivering a fixed intensity of 0.435 W/cm2. The samples were irradiated during 30 seconds to ensure reaching thermal equilibrium, and the maximum temperature measured using a calibrated IR camera. It is important to underline that since measurements are performed in air, temperatures attained at equilibrium greatly exceed the ones expected in a more realistic setting involving pulsed illumination and contact with a thermal bath (either tissue in vivo or tissue phantom in vitro) dissipating the accumulated heat, as considered hereafter. Figure 3.b plots the maximum temperature increase as a function of the GNR coverage, reaching a plateau for concentrations greater than 300 GNR/µm2. While interparticle nearfield interaction inducing a broadening and redshift of the LSP resonance is excluded, this saturation is attributed to the regime where the absorption crosssection of adjacent nanorods (2450 nm2) starts to overlap.
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Figure 2 – (a) Chemical activation of the mesh surface and citrate-stabilized GNR anchoring (b) GNR density at the amino-activated mesh surface for different concentrations of citrate-stabilized GNRs.
Based on these results, we chose to work with a density of 250 GNRs/µm2 which ensures close to optimum light-toheat conversion while minimizing the amount of gold, which eventually determines the production cost. To assess the stability of the GNR-modified meshes, they were stored in PBS at 37 ˚C. After 12 months, their heating ability was not substantially altered. Additionally, their gold contents measured by ICP-MS remained greater than 95% of the initial values, indicating negligible release of GNRs (Figure S2 of SI).
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Antibiofilm activity – At this stage, we evaluate the ability of GNR-modified mesh to alter the biofilm integrity upon NIR illumination. For this purpose, a 250 GNR/µm2 mesh was immersed during 24 hours in PBS containing 106 CFU/mL of Staphylococcus aureus (S. aureus). Figure 4 shows SEM micrographs of the contaminated mesh surface. Following the protocol described in the method section, the contaminated meshes were immersed into an agar sandwich and treated with a train of light pulses from an Intense Pulsed Light (IPL) source. The illumination area was 7.5 cm2 and emission spectrum ranging from 750 to 1200 nm, covering the main absorption peak of the anchored GNRs (spectrum shown in figure S3 of SI). Each treatment includes 20 consecutive pulses separated by a 4 seconds rest interval. For a given light fluency, the energy was delivered to the modified mesh under different conditions; either in the form of a short and intense pulse or using a lower peak intensity over a longer time. Following treatment, meshes were recovered, washed softly with PBS/Tween 80 to remove unattached bacteria without extracting the biofilm. Subsequently, all remaining bacteria from the mesh samples were extracted in Peptone (0.1%) Tween 80 (1%) water by treatment on an ultrasonic bath (4 minutes) and 1 hour stirring. The extracted medium was seeded in tryptone soy agar (TSA) at different dilutions and incubated at 37 °C. CFU counting was performed after 24 h.
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Figure 4 – (a) SEM micrographs of the S. aureus biofilm formed at the mesh surface. (b) Summary of the bacteria counting after treatment under different conditions.
Figure 4.b summarizes the CFU results obtained for the different illumination conditions. The reduction in the number of bacteria is expressed in logarithmic decrease of the attached bacteria population with respect to a contaminated sample without light treatment. For convenience, we also indicate the corresponding percentage. In this table, the red color is used to highlight the conditions under which the antibiofilm efficiency is lower than 90 % (below 1 logarithm), orange color shows an efficiency between 90-99 % (between 1 and 2 logarithms) while green indicates an efficiency greater than 99 % (above two logarithms), which stands as a relevant threshold in microbiology. For all other illumination conditions, no significant decrease of the bacteria population was observed.
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Data show that more efficacy is obtained with short and intense pulses. This can be interpreted with simple thermodynamic considerations. Upon illumination, the modified mesh thread dissipates heat away with a rate which is determined by the thermal conductivity of its surrounding which remains constant, independently of the pulse parameters. Because under pulsed illumination steady state is not reached, for the shortest pulses, the heating rate of the system is greater than its cooling rate leading to higher surface temperatures. 14 J/cm2
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Figure 5 – Fluorescence confocal microscopy at the mesh surface. Fluorescence labelling with green (alive) and red (dead) dyes enables us to identify the number of bacteria still adhered to the mesh after treatment and which portion of them are dead. For the sake of clarity, both merged fluorescent and bright field images (a-c) and pure fluorescent maps (d-f) are presented. While the first column is associated to the control experiment in presence of NRs without light treatment, the second and third column correspond to two different fluences at fixed pulse length (300 ms), 14 J/cm2 and 15 J/cm2, respectively. (g) Proportion of alive and dead bacteria at the mesh surface (normalized by control without illumination). While previous data provide information about the number of alive bacteria remaining at the mesh surface after treatment, we are interested in getting further insight about the actual inhibition mechanism. To gain a microscopic perspective on the problem, fluorescence confocal microscopy imaging was performed on treated meshes, after soft washing in PBS/Tween 80. Filmtracer™ LIVE/DEAD™ Biofilm viability kit was used to enable differentiate between alive and dead bacteria at the mesh surface (Figure 5). For reference, we realized a control experiment in which the GNR-modified mesh was not treated by light. To assess the effect of the illumination conditions, our experiment includes two different fluences at fixed pulse duration (300ms), 14 J/cm2 and 15 J/cm2, respectively. The meshes treated under the lowest fluency show a significant number of bacteria at the mesh surface, however, most of these bacteria are dead. Conversely, by further increasing the delivered energy, bacteria loose adherence, leaving only few adhered bacteria. From our data, we can conclude that the main mechanism behind biofilm elimination is its peel off from the mesh, most probably resulting from the denaturation of adhesive exopolysaccharides. It is hypothesized that in an in vivo setting, treatment will result in less resistance of the biofilm to complementary treatments. Furthermore, biofilm bacteria become planktonic cells, and hence should recover their sensitivity to antibiotic
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therapy and to the immune system response minimizing self-defense mechanisms such as insusceptibility to antimicrobial substances, resistance plasmids exchange and immune response inhibition.
Conclusion - Controlled local delivery of heat based on the interaction of light with resonant plasmonic nanoparticles represents a promising strategy to prevent the formation of bacterial biofilm at the surface of surgical implants. In this in vitro study we have demonstrated that surgical polymer meshes decorated with GNRs enable, upon suitable illumination conditions, to alter the integrity of a biofilm formed at their surface. Our approach was designed to operate with NIR light, in the so-called transparency window of tissues, making it compatible with an in vivo setting. In the latter, light would be delivered either from outside if the mesh sits less than one centimeter from the skin surface or through a minor incision. Also, because the temperature increment is spatially confined to the modified mesh surface, we do not foresee significant damage to surrounding healthy tissues. Yet, the transition to in vivo raises several open questions. While gold is known to have high biocompatibility, we aim at studying the longterm acceptance of surrounding tissues to inserted meshes. While the considered illumination conditions were chosen to be compatible with in vivo operation, like in cosmetics (identical conditions are indeed widely used in the context of hair removal) 23 it is here important to account for other factors like patient pain and skin injuries threshold that would depend on the patient phototype. Our data show that by adapting the pulse duration, there is room to account for these parameters while not compromising the treatment efficacy. EXPERIMENTAL METHODS Citrate stabilized GNRs preparation Gold Chloride trihydrate (HAuCl4), Cetyl trimmonium bromide (CTAB), silver nitrate (AgNO3), ascorbic acid, sodium borohydride (NaBH4), polystyrene sulfonate (PSS, Mw 70000) and sodium citrate were purchased from Sigma-Aldrich and used without further purification. GNRs preparation having LSPR absorbance around 810 nm (13nm × 49nm) were prepared accordingly to Al Sayed et al. 24. Briefly a seed was prepared in a Teflon vessel by mixing 5 mL of HAuCl4 0.5 mM with 5 mL of CTAB (200 mM). The mixture was maintained at 30 °C under stirring and added of 0.6 mL of NaBH4 10 mM. The resulting fair brown suspension was kept at 30 °C for 3 hours before use. For the GNRs growth, a mixture 50 mL of HAuCl4 (1 mM) and 50 mL of CTAB (200 mM) was maintained under stirring at 30 °C. The mixture was added 2 mL of AgNO3 (4 mM) and 1 mL of Ascorbic acid (78.9 mM), sequentially, in portions of 0.1 mL each 5 minutes. The resulting reddish GNRs suspension was maintained 2 hours at 30 °C. Exchange of CTAB by citrate as GNRs stabilizing agent was `performed as describe by Wei et al.25 Briefly, 40 mL of the GNRs suspension were centrifuged at 14000 rpm over 25 minutes and 95% (38 mL) of the supernatant were eliminated. The supernatant was replaced by 38 mL of a PSS Mw70000 solution at 1.5 mg/mL. This operation was repeated twice. After the second centrifuge cycle, the PSS solution was replaced by a 10 mM solution of sodium citrate and 3 more centrifugation cycles were performed. The final GNRs suspension in sodium citrate was kept at room temperature to be used for the conjugation with the activated mesh. Figure S3 show the LSPR absorption spectrum of the so prepared citrate stabilized GNRs. Plasma activation and GNR conjugation of the meshes – Surgical meshes (Optilene© Mesh LP) were provided by Bbraun Medical S.A. Meshes were plasma-activated in a NANO plasma system (40 kHz, 100 W) from Diener Electronic GmbH (Germany) before dipping them in pure ethylene diamine to introduce free amino functions at their surface. Oxygen plasma activation of polypropylene causes surface oxidation and crosslinking. Oxygen reacts immediately with the plasma-treated surface leading to a consistent amount of free O- radical groups. The free
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radical sites react with the ethylene diamine generating the desired anchoring bonds.26-27 The activated meshes are washed sequentially with HCl 1 M, water with 10 % of ethanol and finally with pure water. Conjugation of GNRs to the activated mesh— 500 mg of amino-activated polypropylene meshes were incubated in 20 mL of citrate stabilized GNRs in 20 mM citrate buffer at pH 6.5 at different concentrations of GNRs. Incubation was carried out during 4 hours at room temperature. The meshes were accurately weighted to calculate the different ratios gold/mesh and the resulting Au contents attached to the mesh surface was determined by ICP-MS analysis. Modified meshes were then packed into a porous peel pouch packaging and sterilized by ethylene oxide for their use in Biofilm removal experiments. Antibiofilm test using Filtered IPL – Specimens of 0.5 x 1.5 cm were cut from the conjugated sample and the control mesh (Optilene© Mesh LP, pure polypropylene). The specimens were introduced in a 10 mL phosphate buffer solution with of Staphylococcus aureus at 105-106 CFU/mL concentration, and incubated for 18 hours at 37 °C for the biofilm formation. After a softly cleaning with Peptone water 0.1% + Tween 80 1%, they are dried. Each specimen was placed separately on a Tryptone Soy Agar (TSA) plate and covered with more semi-liquid TSA to form an agar sandwich. The pieces of control mesh were disposed on another plate in the same way. The upper agar layer measured a thickness of 4-5 mm. A battery of treatments using different fluences (12, 13, 14, 15, 16 J/cm2) given at different times (40, 100, 300 ms) were tested on the specimens using a Ros’s IPL Belvet Premium IPL device, with filtered light at 755 nm (spectral range 755-1200 nm). One of the specimens was reserved as a Positive Control, with no IPL treatment. After the IPL treatment, each piece was removed from the agar sandwich, softly cleaned and introduced in a test tube with 3 mL of Peptone water 0.1 % + Tween 80 1 % for their biofilm removal. The test tubes were submitted to sonication for 4 minutes and agitation for 1-2 hours at 350 rpm. Serial dilutions were prepared from the removal solutions and sowed on TSA plates, recounted after 24 hours of incubation at 37 ºC. Live/Dead bacteria evaluation by confocal fluorescence microscopy – After soft washing in PBS/Tween 80. Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit (Molecular Probes, Invitrogen) supplied by Thermofisher was used to assess the respective proportion of dead and alive bacteria at the surface of treated mesh. The meshes were incubated 2minutes in the dye mixture before being washed with water. The samples were mounted on microscope slides and observed at two different wavelengths (520nm and 635nm). Live bacteria were observed in green (SYTO 9 dye) and dead bacteria observed in red (propidium iodide dye). Acknowledgments The authors acknowledge financial support from Fundació Privada Cellex, the CERCA programme and the Spanish Ministry of Economy and Competitiveness, through the “Severo Ochoa” Programme for Centres of Excellence in R&D. REFERENCES (1) Schierholz, J.M.; Beuth, J. J. Hosp Infect. 2001, 49,(2), 87-93 (2) Dupont, H. Int. J. Infect. Dis. 2007, 11, (1), S1-S6. (3) Perez-Köhler, B.; Bayon, Y.; Bellon, J.M. Surg. Infect., 2016, 17, (2), 124-137 (4) Cassini, A.; Diaz Högberg, L.; Plachouras, D.; Quattrocchi, A.; Hoxha, A.; Simonsen, G.S.; Colomb-Cotinat, M.; Kretzschmar, M.E.; Devleesschauwer, B.; Cecchini, M.; Quakrim, D.A.; Oliveira, T.C.; Struelens, M.J.; Suetens, C.; Monnet, D.L.; and the Burden of AMR Collaborative Group. Lancet Infect Dis. 2019,19, (1), 56-66 (5) Bay-Nielsen, M.; Kehlet, H.; Strand, L. Lancet, 2001, 358, 1124-1128 (6) Usher, F.C. Archives of Surgery, 1962, 84, 325-328
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