Departments of 1 Orthopaedic Surgery/VU University Medical Center, PO Box 7057; 2 Oral Cell Biology/ACTA; 3 Oral Biochemistry/ACTA, 1007 MB Amsterdam, The Netherlands
Received 11 February 2003; returned 6 May 2003; revised 13 August 2003; accepted 13 August 2003
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Abstract |
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Methods: We combined six calcium phosphate cements and six granule-types with 5 mg/g hLF1-11 and measured its availability and release in vitro from cements (7 days) and granules (3 days). The integrity and antimicrobial activity of the hLF1-11 that was released during the first 24 h were measured, using mass spectrometry, and a killing assay on methicillin-resistant Staphylococcus aureus (MRSA).
Results: Most of the cements showed burst release followed by low-level continuous release, whereas the coated granules showed high burst release for 24 h. After release the peptide was active (in nine of 12 materials) and intact.
Conclusions: Different release profiles may be obtained by choosing the appropriate carrier, which supports the feasibility of biodegradable carriers releasing AMPs against resistant infections.
Keywords: bone infections, human lactoferrin, biodegradable, carriers
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Introduction |
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We aimed to address this increasing problem by combining an antimicrobial peptide (AMP) of human origin (hLF1-11) with biodegradable carrierswhich obviates the need for operative removaland analysing its availability and release. These carriers, which consist of (a combination of) calcium-phosphate ceramics, e.g. tricalcium phosphate, Ca3(PO4)2, are slowly replaced by ingrowing bone after implantation.2 AMPs form a novel class of antimicrobial agents of natural origin that have been identified in virtually all forms of life as part of the antimicrobial defence system. These positively charged peptides kill by forming pores in the negatively charged bacterial cell-membrane and targeting intracellular organelles, without development of resistance.3 Moreover, they seem to have an immuno-modulating effect, killing microorganisms at lower concentrations in vivo (ng/mL) than in vitro (µg/mL).4,5
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Materials and methods |
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hLF1-11-peptide (GRRRRSVQWCA, 1375 Da) was manufactured by solid-phase peptide synthesis using Fmoc (9-fluorenyl-methoxycarbonyl) chemistry as described previously.6 Reanalysis of peak fractions by reversed phase HPLC resulted in one major peak revealing at least 90% purity. The authenticity was confirmed by electrospray-ionization quadrupole-time-of-flight mass-spectrometry (Q-TOF MS, Micromass Inc., Manchester, UK). Thermal stability in solution, adhesion to polystyrene and solubility were tested as described previously.7
Release experiment
After mixing cement powder and liquid containing 5 mg hLF1-11 per gram of powder (liquid/powder ratio according to the manufacturer), cylindrical specimens hardened overnight at 37°C in 6 x 5 mm moulds. The cements were: Biobon (Biomet Merck Biomaterials, Darmstadt, Germany), Calcibon (Biomet Merck Biomaterials), Biofil, (experimental, DePuy CMW, Blackpool, UK), Bonesource (Stryker-Leibinger, Freiburg, Germany), Chronos Inject, (experimental, Mathys, Bettlach, Switzerland) and Norian SRS (Mathys).
Granules were immersed in 1.0 mL of dH2O containing 5 mg of hLF1-11 per gram of material and lyophilized. After removal of the granules, the residual hLF1-11 in the vessel was measured. The granules were: Bonesave (Stryker-Leibinger), Biosorb (Science for Biomaterials, Lourdes, France), Allogran-R (Orthos, Bristol, UK), Vitoss (Orthovita Malvern, PA, USA), Cerasorb (Curasan, Kleinostheim, Germany) and Bicalphos (Medtronic, Memphis, TN, USA).
Specimens were immersed in 500 µL of dH2O and kept in sealed polystyrene 48-well plates (Costar) at room temperature on a shaking device (180 rpm). The water was replaced at regular intervals: 30, 90 and 180 min on day 1 and then 24 hourly for 7 days (cements) or 3 days (granules) and stored at 20°C.
After production and after release, three specimens per group were finely ground and suspended in 5 mL dH2O containing 1 M NaCl and the initial and residual hLF1-11 were determined. The hLF1-11 remaining in the vessel after lyophilization of the granules was also determined.
The hLF1-11 concentration was measured using a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA) and read at 540 nm (Bio-Rad) (hLF1-11 serial dilutions were used as a reference, the detection limit was 2.5 µg). Accuracy of the assay was calculated as 6.5% (mean error of true value), precision 3.5% (coefficient of variance). Control samples without hLF1-11 were used for background correction. The authenticity of the hLF1-11 of the first day release samples was analysed by Q-TOF MS and sequencing of the 1375 Da peak in one of the samples.
Antimicrobial activity
Samples of 500 µL release medium (taken after 24 h) were lyophilized (without carrier material) and 106 cfu of an MRSA clinical isolate (ATCC BAA-811) in PBS containing 0.01% Brain Heart Infusion was added. After 60 min at 37°C, these were plated on blood agar (n = 6), and colonies were counted after 18 h at 37°C. The percentage of killing was calculated: [1 (cfu in sample/cfu in control)] x 100% (>90% was considered active). Samples without hLF1-11 were used as controls, samples from the first day were used because these all contained adequate amounts of peptide.
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Results |
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Discussion |
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Using methods described by Kühn, adapted for biodegradable specimens, we determined the available hLF1-11 in samples before and after release.9 Not all hLF1-11 added to cement was available for extraction, even after fine grinding and adding high salt concentrations to decrease charge-dependent binding. This suggests that part of the hLF1-11 strongly binds to the hardening cement leading to sequestration and unavailability for release, but it might still become available after osteoclastic resorption.
A variable amount of hLF1-11 attached to the granules during lyophilization, and the rest was detected in the coating vessel. High-porosity granules (Vitoss) bound most hLF1-11 indicating that the surface area of the carrier material might determine the loading capacity.
Thus, two different release profiles were observed in this study, which support a two-phase explanation of the release process. The initial burst-release is predominantly determined by the release of the drug from the surface of the carrier; the second phase shows the more gradual diffusion of the drug from deeper layers, determined by the porosity of the carrier.9 The hLF1-11 released in the first day was active in nine of the 12 materials. Of these, Chronos was the highest releasing cement-type and Vitoss the highest releasing granule-type (Table 1). The amount of hLF1-11 released may be relevant in vivo, as Nibbering and co-workers demonstrated that hLF1-11 concentrations as low as 0.1 ng/mL kill MRSA in vivo.
Limitations of this study can be found in the extrapolation of the in vitro results to in vivo concentrations in bone and surrounding tissues. Instead of using simulated body fluid as the release medium, the experiment was carried out using small volumes of water. This allowed the use of a simple protein assay for the detection of hLF1-11. Disadvantages of this method are: (i) pH and ionic content were different from the in vivo situation, (ii) proteases that could degrade the hLF1-11 were not present, (iii) any positive interaction of AMP with the adaptive immune system could not be included.5 The strength of in vitro release studies of this kind lies in a qualitative comparison of different carrier materials, allowing the identification of the most suitable materials for in vivo testing. In spite of the in vitro shortcomings, a positive correlation between in vivo and in vitro results has been reported by several authors.10
To conclude, in nine of the 12 combinations, basic requirements for designing a drug delivery system (controlled release of an active substance) have been met.8 At present, investigations using animal models to study the in vivo efficacy of AMP-releasing drug delivery systems are under way.
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Acknowledgements |
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Footnotes |
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References |
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