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
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Abstract |
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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
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Introduction |
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Methods |
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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 (10100 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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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 earlymid 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 0.70.8.
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 0.70.8 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 0.70.8 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.
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Results |
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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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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 23 logs over 5 h. A slight difference of 1 log was seen when a conditioning film was present.
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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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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 7090 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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Discussion |
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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.
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Acknowledgements |
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Footnotes |
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References |
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