2007 Abstract : 4

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Author(s): *Scott P. Noel, Warren O. Haggard, Joel D. Bumgardner; Memphis, TN

Title: Elution and activity characteristics of amikacin- and daptomycin-loaded chitosan films: A preliminary investigation.

Purpose: The purpose of this study was to investigate the in vitro characteristics of an antibiotic-loadedchitosan film. Amikacin (Bedford Labs) and daptomycin (Cubist) were incorporated into a biocompatible, biodegradable chitosan film and drug release and

Methods:

  • Elution tests:Chitosan films were prepared by dissolving 2.0 grams of chitosan (AgraTech), 80-81 % deacetylation, into 98.0 milliters (ml) of 1 (V/V) % of a lactic/acetic acid mixture. 100 milligrams of antibiotic were pre-dissolved into 50 ml of acid before being added to the chitosan solution. The chitosan solution was stirred for four hours and allowed to de-gas overnight. The chitosan solution was cast into two glass Petri dishes and placed into a 37oC convection oven for a drying period of 12 hours. After drying, the films were removed from the glass dishes and prepared for elution testing. For elution tests, 5/8¡¨ discs were punched from the chitosan films. These discs were weighed in order to obtain a theoretical amount of antibiotic present. The discs were placed into 50 ml of 1x Phosphate Buffered Saline solution (PBS) in glass vials. The vials were placed into a 37oC agitated water bath. One ml aliquots were taken at designated timepoints and stored in a laboratory freezer until testing occurred. Time points were 1, 3, 6, 24, and 72 hours. The aliquots were tested for antibiotic concentration using a fluorescence polarization immunoassay technique via TDxFLx instrument (Abbott Labs) for amikacin and HPLC (Varian) for daptomycin.
  • ¡E Activity tests:Activity of the antibiotic-containing eluates was tested by performing a series of turbidity assays. The assays allowed for quantitative measurement of antibiotic activity against S. aureus. 200 £gl of each eluate was added to tubes containing 1.8 ml of Mueller-Hinton II broth with a 50 £gg/ml CaCl2 supplement. Tubes were inoculated with 20 £gl of S. aureus and placed in a 37oC incubator for 24 hours. Absorbance measurements were taken and recorded after incubation at a wavelength of 530 nm (A530) on a spectrophotometer. Blanks were used to zero the spectrophotometer (1.8 ml of MHII broth and 200 £gl of PBS) and a percent inhibition determination was made when comparing eluate absorbance measurements to positive c

Results: Significant elution of both amikacin and daptomycin occurred after the first hour in PBS solution.Amikacin release was determined to be 24.66 £gg/ml, or 85.68% of initial antibiotic load. The total recovery percentage for amikacin was found to be 96.23% with a final cumulative release concentration of 30.2 £gg/ml. The total % release is seen in figure 1. Daptomycin displayed similar results, also having substantial release after 1 hour with a concentration of 16.05 £gg/ml being released, or 61.97% of initial antibiotic load. Final cumulative release was found to be 28.05 £gg/ml with the total release percentage at 88.28%. The turbidity assay showed that the antibiotics being released were active against S. sureus. The eluates containing amikacin had an %inhibition of 85.13% when compared to the positive control after 1 hour. After 72 hours, the %inhibition rose to 96.91%. The eluates containing daptomycin had a %inhibition of 99.32% when compared to the positive control after 1 hour. This percentage increased to 99.43% after the duration of the study (72 hours). Figure 2 shows the results of the turbidity study in regards to % inhibition of S. aureus growth.

Discussion: Chitosan is a well-studied biocompatible material that can be used to deliver therapeuticagents in vivo. The localized delivery of antibiotics is an emerging area of study that could offer the benefit of high local concentrations of drug while avoiding the problems associated with whole-body dosing. This study characterized the in vitro release of two different antibiotics eluting from a chitosan film. Both amikacin and daptomycin eluted from the films in a desirable manner and the eluates were found to be active against S. aureus. Near-complete recovery of drug in a film that degraded over the time was found in this in vitro study. Chitosan films are currently available in a non-loaded form to be used as a hemostatic device. Incorporating antibiotics which are active against harmful bacteria such as S. aureus into such films could offer a potential aid in the treatment of musculoskeletal infections. Future studies for this approach to localized drug delivery include various antibiotic combinations as well as extensive activity evaluations. The results presented in this study offer insight to the potential use of degradable chitosan films as a carrier vehicle for antibiotics in musculoskeletal applications.