Silver nanoparticles and polymeric medical devices: a new approach to prevention of infection?

Franck Furno1,2, Kelly S. Morley2, Ben Wong2, Barry L. Sharp3, Polly L. Arnold2, Steven M. Howdle2, Roger Bayston1,*, Paul D. Brown4, Peter D. Winship3 and Helen J. Reid3

1 Biomaterials-Related Infection Group, School of Medical and Surgical Sciences, University of Nottingham, Nottingham NG7 2UH; 2 School of Chemistry, University of Nottingham, Nottingham NG7 2RD; 3 Department of Chemistry, University of Loughborough, Loughborough LE11 3TU; 4 School of Mechanical, Materials and Manufacturing Engineering, University of Nottingham, Nottingham NG7 2RD, UK

Received 19 May 2004; returned 13 July 2004; revised 24 August 2004; accepted 27 September 2004


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives: Implantable devices are major risk factors for hospital-acquired infection. Biomaterials coated with silver oxide or silver alloy have all been used in attempts to reduce infection, in most cases with controversial or disappointing clinical results. We have developed a completely new approach using supercritical carbon dioxide to impregnate silicone with nanoparticulate silver metal. This study aimed to evaluate the impregnated polymer for antimicrobial activity.

Methods: After impregnation the nature of the impregnation was determined by transmission electron microscopy. Two series of polymer discs were then tested, one washed in deionized water and the other unwashed. In each series, half of the discs were coated with a plasma protein conditioning film. The serial plate transfer test was used as a screen for persisting activity. Bacterial adherence to the polymers and the rate of kill, and effect on planktonic bacteria were measured by chemiluminescence and viable counts. Release rates of silver ions from the polymers in the presence and absence of plasma was measured using inductively coupled plasma mass spectrometry (ICP-MS).

Results: Tests for antimicrobial activity under various conditions showed mixed results, explained by the modes and rates of release of silver ions. While washing removed much of the initial activity there was continued release of silver ions. Unexpectedly, this was not blocked by conditioning film.

Conclusions: The methodology allows for the first time silver impregnation (as opposed to coating) of medical polymers and promises to lead to an antimicrobial biomaterial whose activity is not restricted by increasing antibiotic resistance.

Keywords: antimicrobial biomaterials , implantable devices , medical polymers


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Infection is a well-recognized complication of implantable devices, of which a wide variety is now in use. The devices can be categorized according to the nature and risk of infection.1 While Category I devices are totally implanted and are at risk mainly at insertion, Category II devices are incompletely implanted and have periods of risk extending throughout their use. Central venous catheters (CVC), wound drains and catheters for continuous ambulatory peritoneal dialysis (CAPD) are examples. Approximately 3 million CVC are inserted each year in the USA, resulting in 850 000 infections, almost 20% of them serious2 with a mortality rate of up to 28%.3 An essential event in initiation of biomaterials-related infection (BRI) is microbial adhesion to the device, or more often to the patient-derived glycoprotein coating (conditioning film) which begins to be deposited immediately on implantation in most devices.4,5 Once adhesion has occurred, proliferation leads to the development of a biofilm, which is insusceptible to most therapeutic agents at achievable concentrations.68 Device removal is often required in order to ensure eradication of infection and to avoid relapse. Several approaches to prevention of BRI involving surface coatings have been proposed. Many, such as surface modification by gas plasma, appear to reduce microbial adhesion in vitro but are ineffective in vivo, probably because of obliteration by conditioning film.9 Silver has long enjoyed a reputation for antimicrobial properties, and has therefore been seen as a candidate for coating of medical devices. While various coatings, using silver salts or ion beam implantation of metallic silver, have been devised they have shown disappointing clinical results1013 or even further complications.14 Possible reasons for this are obliteration or inactivation of the silver coating by blood plasma, and the lack of durable activity inherent in coatings. True impregnation of polymers15 with antimicrobials has led to only one clinical application so far, but this has demonstrated the superiority of impregnation over coating.16 Hitherto, silver could not be impregnated into biomaterials. Attempts to admix silver metal or salts into biomaterials have failed to improve antimicrobial activity and have risked impaired mechanical properties. In an attempt to address these shortcomings while taking advantage of the broad antimicrobial spectrum of silver, we have devised a process whereby nanoparticles of silver metal can be impregnated into medical polymers. This paper reports studies on their release and antimicrobial activity in vitro.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
Biomaterial

Medical grade unfilled silicone sheet 0.45 mm thick (Dow Corning Ltd, Meriden, UK) was cut into discs of 15 mm diameter.

Impregnation process

The impregnation was carried out using a novel method.17 Briefly, organic complexes of silver were synthesized in anaerobic conditions. A solution of the organometallic precursors dissolved in supercritical carbon dioxide (scCO2) was then used to swell and permeate the polymer at 4000 psi, 40°C, for 24 h. For this stage, the silicone discs were placed in a 10 mL stainless steel high-pressure autoclave (Thar Technologies, Pittsburgh, PA, USA) itself placed inside a metal heating block and attached to a transducer and a thermocouple to monitor the pressure and temperature. The scCO2 was then vented and the polymers were exposed to H2 gas (1500 psi, 40°C, 24 h) in order to decompose the organometallic precursors, leading to a homogeneous distribution of nanoparticles of silver (10–100 nm) in the polymer (Figure 1).18,19 The residues of the organic precursors were then removed completely by flushing with scCO2. This was checked carefully by thermogravimetric analysis of the final polymer composite. The formation of silver nanoparticles in the silicone matrix was confirmed by detailed analyses using transmission electron microscopy (Figure 1). The overall loading, particle size and particle distribution of the silver nanoparticles was controlled by careful moderation of the supercritical fluid conditions.18,19



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Figure 1. Transmission electron microscopy image of nanoparticulate silver (dark, electron-dense particles) dispersed in silicone. The associated diffraction pattern (inset) has been indexed against standards to confirm that the particles are metallic silver. Note the extremely small size of the silver particles.

 
Test bacterium

A clinical isolate of Staphylococcus epidermidis (F22) whose adherence and biofilm characteristics were known was kept at –20°C in cryoprotectant until use. To resuscitate, 20 mL of tryptone soya broth (TSB; Oxoid, Basingstoke, UK) was inoculated and incubated overnight at 37°C. One drop of this was then inoculated into 20 mL of fresh TSB and incubated with shaking at 37°C for 4 h to ensure expression of adhesins in early–mid log phase.20 The cells were then washed in PBS and resuspended in 2% TSB (found by experiment to maintain viability without proliferation in these test conditions) to 1 x 108 cfu/mL, corresponding to A490 0.7–0.8.

Application of conditioning film

One series of discs was washed after impregnation. This consisted of immersion in sterile deionized water for 1 h. A second series remained unwashed. Both sets of discs were immersed in 50% human plasma (National Blood Authority, Sheffield, UK) for 30 min at 37°C after which excess plasma was rinsed away by gentle immersion three times in sterile water. In an additional series, discs were immersed in plasma for 1 h.

Serial plate transfer test (SPTT)

Four sets of discs were tested: washed and unwashed after impregnation, and with or without 30 min conditioning film; 20 mL ISA plates (Oxoid, Basingstoke, UK) were inoculated with S. epidermidis F22 using a rotary inoculator and the discs placed centrally on each plate. After overnight incubation, any inhibition zones were recorded (diameter minus disc diameter, mm) and the discs were transferred to fresh inoculated plates, taking care to present the same surface to the agar. The process was continued until no more inhibition was seen.21

Quantitative adherence

As with the SPTT, four sets of discs were tested. They were immersed in a suspension of S. epidermidis F22 at A490 for 1 h and after rinsing to remove non-adhered bacteria they were sonicated at 50 Hz/10 min (Ultrawave, Cardiff, UK). Triplicates of 125 µL of the sonicate were added to the wells of chemiluminescence trays (Zeptogen Ltd, Middlesex, UK) and read in a luminometer (Berthold Technologies GmbH, Bad-Wildbad, Germany) using a Lumitech Vialite kit (BioWhittaker Ltd, Berks, UK). Plate viable counts were also carried out.

Planktonic killing

Groups of discs as above were immersed in a suspension of S. epidermidis F22 at A490 0.7–0.8 for 1, 2, 3, 4 and 5 h at 37°C and the suspensions assayed by chemiluminescence and plate counting. An additional two groups of discs, one washed and the other unwashed, were plasma-coated for 1 h before testing.

Rate of kill

Triplicates of washed and unwashed discs with or without conditioning film were immersed as above in a suspension of S. epidermidis F22 at A490 0.7–0.8 for 1 h. They were then rinsed and immersed in 2% TSB for 2, 3, 4 and 5 h at 37°C and after rinsing to remove non-adhered bacteria they were sonicated. The sonicates were assayed by chemiluminescence and plate counting.

Release of silver ions

The four groups of discs as above were thoroughly washed in sterile deionized water, then immersed in 5 mL of either sterile deionized water or 50% human plasma for 1 day at room temperature. The next day they were removed, blotted free of excess fluid, and transferred to a fresh 5 mL of the same elutant. The process was continued for 5 days. The suspending fluids from days 3, 4 and 5 were then analysed for silver by inductively coupled plasma mass spectrometry (ICP-MS: VG PQ ExCell, Winsford, UK). While the water samples remained clear, the plasma samples contained amorphous material that in some cases assumed a web-like appearance. Unused suspending fluids were also analysed as controls. A 100 mL aliquot of each of the deionized water samples was diluted 1000-fold before ICP-MS analysis. Then 1 mL of each plasma sample was centrifuged (2500 rpm, 5 min), and 100 µL of the resulting supernatant was removed and diluted 1000-fold before analysis by ICP-MS. This ensured the absence of any particulate matter in the plasma samples that were analysed. Standard solutions of inorganic silver salts were also included.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
SPTT

The results may be presented as two sets of data, one referring to discs washed after processing, and the other to those left unwashed. In the case of the unwashed discs, zones of inhibition were seen for 10 days, decreasing from a mean of 8.5 mm on day 1 to 3 mm on day 5, showing clear evidence of persisting, diffusible activity (Figure 2). No residual bacteria were seen under the discs after removal, but further studies were not carried out to detect them. This activity remained unchanged in the presence of a conditioning film, that is in those discs which had been immersed in plasma before being applied to the plate. Tests for residual organic complexes that could have accounted for this were negative. However, when the second set of discs were washed after impregnation, all the diffusible inhibitory activity was extinguished and no zones were seen, even on day 1. No inhibition zones were seen with plain unprocessed silicone discs or with those which had undergone supercritical fluid processing but without silver.



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Figure 2. Serial plate transfer test (SPTT) clearly showing a 15 mm diameter zone of growth inhibition (arrow, circled) surrounding the silicone disc with silver nanoparticles (unwashed) on the third day of the experiment (b), compared with the control (a).

 
Adherence

Again, there was a difference between washed and unwashed discs. In the unwashed series, no viable bacteria were found after 1 h of exposure, confirmed by chemiluminescence (data not shown). The control unprocessed discs, with or without a conditioning film, showed 1 x 107 cfu/mL, whereas the washed impregnated discs, again irrespective of a conditioning film, showed a 4 log reduction to 1x103 cfu/mL.

Killing of adhered bacteria

A series of the above discs, after exposure to bacterial suspension for 1 h, were rinsed and suspended in 2% TSB. Triplicates were removed and sonicated at 2, 3, 4 and 5 h. The results in Table 1 show again that bacteria attached to unwashed discs were all killed within 1 h, irrespective of conditioning film. The washed discs showed an initial reduction compared with controls of 4 logs but the numbers of viable attached bacteria gradually increased by 2–3 logs over 5 h. A slight difference of 1 log was seen when a conditioning film was present.


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Table 1. Rate of killing of adhered bacteria

 
Killing of planktonic bacteria

The results are shown in Figure 3. A clear difference was seen between washed and unwashed discs. While the former showed no effect on planktonic S. epidermidis, in the unwashed discs there was a progressive decline in viability as shown by chemiluminescence over the 5 h test period. This was confirmed by viable counts (data not shown). The presence of plasma coating slowed the rate of killing, and this was more evident when plasma coating was applied for 1 h.



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Figure 3. Chemiluminescence assays showing the antimicrobial effect of silicone impregnated with silver nanoparticles. Results are expressed as percentage of viable planktonic bacteria at time zero. Column 1, unwashed discs, no conditioning film (CF); column 2, unwashed discs, 30 min CF; column 3, unwashed discs, 1 h CF; column 4, washed discs, no CF. Control (unimpregnated) discs had no effect on numbers of viable planktonic bacteria. The unwashed silver nanoparticle-loaded discs showed a dramatic and sustained activity over 5 h. The rate of reduction of viability was reduced with a 30 min conditioning film, and even more with a 1 h film. The washed discs showed no significant antimicrobial activity against planktonic bacteria over the test period. The controls remained consistently negative (i.e. no activity) over the duration of the experiment (data not shown). All tests were repeated nine times.

 
Release of silver ions

The impregnated materials used in this part of the work had first been washed as described above. From the data generated by this analysis, calibration curves were plotted for both 107Ag and 109Ag isotopes. Using these curves and the equations associated with them, the concentrations of Ag isotopes in the deionized water and plasma samples were calculated and are shown in Table 2. Very little Ag ion was released into the water samples (<0.5 ppm). A greater quantity of Ag ions was released into the plasma samples with the bulk (4 ppm) being removed during the first 3 days. A significant amount of silver ions was still being released into the plasma on days 4 and 5 (~0.859/0.854 and 0.568/0.562 ppm, respectively). The precipitated material seen in the plasma series was also analysed for silver content, and concentrations were found to exceed the range covered by the standard curve. However, an approximate value of 70–90 ppm was obtained. Further analysis of this material could not be completed but initial results from a Biuret test suggested that it was proteinaceous.


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Table 2. Comparison of the mean Ag isotope concentrations, each assayed in triplicate by inductively coupled plasma mass spectrometry, in water and plasma extracts

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
The method used here to introduce silver into silicone elastomer has been shown to result in distribution of nanoparticles of metallic silver throughout the polymer, and is the only method known to us of impregnation of polymers with silver. However, the results show that much of the antimicrobial activity was removed by washing the impregnated discs. This clearly shows that a significant level of surface (or near surface) deposited silver nanoparticles is contributing to an initial ‘burst’ effect—one which is abolished if the samples are carefully washed before use. A variety of tests was used in order to explore several possible modes of action. The serial plate transfer test was originally formulated to test biomaterials containing antimicrobials that could readily diffuse through agar to produce a zone of nhibition. This is not usually the case with silver, and we were surprised to find zones of inhibition against S. epidermidis that suggested a diffusible antimicrobial effect. However, these were abolished by washing the discs. Killing of bacteria adhered to the silicone was complete within 1 h but again this was diminished by washing. The fact that it was not completely abolished by washing suggests that while there appears to be a high surface concentration of silver initially, there is also a much slower release of silver from nanoparticles buried deeper into the polymer matrix. This would probably not be detected by the SPTT method, but would show on the adherence studies. The data presented in Table 1 clearly demonstrate that there is an effect of the silver from the matrix in the washed samples, demonstrating release of silver from below the polymer surface. The killing of planktonic S. epidermidis was again abolished by washing, but the cultures with unwashed discs showed a progressive decline in viable bacteria over 5 h, again indicating high surface activity. Clearly, if there were residues of silver precursor compound, or organic ligands, these might also have an antimicrobial effect. However, thermogravimetric analyses of a series of the silicone materials after careful and exhaustive extraction using supercritical carbon dioxide demonstrated that all such residues had been removed in the processing. That the antimicrobial effect was due to silver is supported by the ICP-MS results, which were accumulated over a much longer period of 5 days. The ICP-MS analyses were carried out on washed discs, yet they clearly showed release of silver over the test period. Interestingly, this occurred most strikingly in the presence of plasma, reaching a peak at 3 days. This finding should be seen in conjunction with the results of tests on discs bearing a plasma conditioning film: while this appeared to slow the release of silver ions, as shown by comparison of the effect of 30 min and 1 h conditioning films on planktonic killing results (Figure 3), the silver ions were clearly able to penetrate the conditioning film. This finding is interesting as silver is known to have a high avidity for protein, and the presence of a protein conditioning film, or any extraneous plasma protein, has previously been assumed to inactivate any silver ions released.22 The finding has obvious clinical implications. The surfaces of implanted devices rapidly become coated with glycoproteins from tissue and plasma, with the possible exception of the inner surface of central venous catheters, and this has been cited as one of the main reasons for clinical failure of silver-coated devices.22 However, in the case of impregnation, we have now demonstrated that there is both a depot effect and a diffusion pressure available to ‘push’ the silver ions through the conditioning film. In order to do this, there must be enough silver ions available over a sufficient period to exceed those lost to protein binding. Indeed, this is one of the objectives of impregnation. The precipitate found during the ICP-MS analysis was likely to be plasma proteins complexed with released silver, and this was supported by the finding of extremely high silver concentrations in the precipitated material.

However, another clinically important advantage of impregnation is the protection of both inner and outer surfaces of catheters against bacterial colonization.23 This has been shown to be crucial in clinical trials of efficacy.24 These two advantages of impregnation, that is the continued release of silver ions in antimicrobial concentrations even in the presence of a conditioning film, and the ability to protect both inner and outer surfaces of catheters, could be expected from the use of this novel method to impregnate polymeric biomaterials with silver. A further advantage is the known wide antimicrobial spectrum of activity of silver.

The data presented here clearly demonstrate that supercritical fluid impregnation of silver precursor followed by controlled decomposition and extraction leads to a distribution of silver throughout the silicone matrix. The impregnation process also yields a significant surface coating of silver which is easily removed by simple washing. However, underlying this is a sustained release which shows great promise, and provides significant scope for optimization of these release kinetics.


    Acknowledgements
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was supported by grants from the Engineering and Physical Sciences Research Council, The Medical Research Council, The European Community and the Wade Charitable Trust. S.M.H is a Royal Society Wolfson Research Merit Award holder.


    Footnotes
 
* Corresponding author. Email: roger.bayston{at}nottingham.ac.uk


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 Acknowledgements
 References
 
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