Development of EDTA Crosslinked BetaCyclodextrin for Use in
Development of EDTA Crosslinked Beta-Cyclodextrin for Use in Drug-Eluting Cardiovascular Stents 1 2 3 Grace Burkhart , Kathleen Young , Nathan Mu , and Horst A. von 2 Recum 1 Department of Chemistry, Valparaiso University, Valparaiso, IN 2 Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 3 University School, Cleveland, OH Particle Characterization • The particles were synthesized, then analyzed with Scanning Electron Microscopy (SEM) and Fourier Transformed Infrared Spectroscopy (FTIR) to characterize the particles by morphology and composition. A. Figure 4. SEM images of EDTA-BCD hydrate polymer particles. A. ) 1: 6 BCD hydrate: EDTA particles. The crystal, oblong shape indicates that polymer was formed from the starting reagents. B. ) 1: 10 BCD hydrate: EDTA particles. B. Figure 1. Illustration of the process of stenting arteries and in-stent restenosis. • of EDTA and the structure of EDTA once it has been chelated with cobalt. 2 Objective Develop EDTA-crosslinked BCD particles which can be refilled with drug and are also capable of extended drug release. • Use particles to develop a coating for cardiovascular stents for use in preventing the onset of late in-stent restenosis. • Materials and Methods Materials Methods • EDTA-polymerized BCD particles were synthesized using a previously published method. 3 EDTA in its salt form was used. Two forms of BCD were used, the hydrate and the prepolymer form. 3. Figure Structure of BCD and EDTA crosslinked into a polymer. 3 • Sirolimus, a drug used in treating in-stent restenosis, was loaded into the particles by mixing particles in solution of sirolimus dissolved in DMF for three days, washing particles in DI water to remove excess drug, then lyophilized particles for three days. • The drug loaded particles were placed in a solution of 1 X PBS or 0. 1% TWEEN/1 X PBS to test the drug release kinetics. Aliquots of solution were taken out daily to determine amount of drug eluted over time. • Drug activity of the eluted drug from the particles was analyzed using a scratch migration assay. • FTIR data showed the particles successfully polymerized because the spectrums of the polymers are divergent from the spectra of the BCD by itself. A. Figure 7. Chart of EDS showing elemental composition on the surface of cobalt chromium stents. Increased amount of carbon on the polymer coated stent confirmed adhesion between stent and polymer. 35 30 25 20 Uncoated Stent 15 EDTA Coated Stent 10 EDTA-BCD Coated Stent 5 0 Co Ni Cr Mo C Elements • To test adhesion between polymer particles and stent surfaces, cobalt chromium stents were submersed overnight, washed, then run with Energy Dispersive Spectroscopy (EDS) to determine surface composition. A. B. Figure 5. FTIR spectrums for EDTA-polymerized particles. A. ) Comparison of BCD hydrate with EDTA-BCD hydrate polymers. The spectrum of the polymers were identical, but differed from the plain BCD hydrate, meaning the polymers had indeed formed as expected. B. ) Comparison of EDTA-dextran polymers to EDTA and plan dextran. The polymers have a similar spectrum to the EDTA, indicating that EDTA may be overpowering the spectrum. Drug Release Study Comparison of Sirolimus Release Cumulative Concentration of All Particles Over Time 2000 1800 1600 Concentration (ug/m. L) Drug-eluting stents (DES) have been developed to prevent restenosis. However, the drug release window for current DES is too short to adequately prevent late in-stent restenosis. 1 Beta-cyclodextrin (BCD) is a molecule capable of extended drug release once polymerized. It be polymerized by Ethylenediaminetetraacetic acid (EDTA), a heavy metal chelator. EDTA can chelate with cobalt in cobalt chromium Figure 2. Structure stents, providing a strong bond between polymer and stent. • EDS Elemental Composition of Coated Stents Wt. % In-stent restenosis, the narrowing of arteries once a stent has already been implanted, is a major consequence of stenting cardiac arteries. • Results and Discussion (ctd. ) Results and Discussion Introduction 1400 1200 1000 800 600 400 200 0 0 100 200 300 400 500 600 Time (hrs) Dextran Salt n=6 in 0. 1% Tween/1 X PBS Dextran Salt n=10 in 0. 1% Tween/1 X PBS BCD Prepolymer Salt n=6 in 1 X PBS BCD Prepolymer Salt n=10 in 0. 1% Tween/1 X PBS BCD Hydrate Salt n=6 in 0. 1% Tween/1 X PBS BCD Hydrate Salt n=10 in 0. 1% Tween/1 X PBS BCD Hydrate Salt n=6 in 1 X PBS BCD Hydrate Salt n=10 in 1 X PBS 700 800 Figure 6. Chart of cumulative concentration of sirolimus released from EDTA-crosslinked BCD particles versus time in order to study the drug release kinetics of the polymers. Notable drug release occurred for the dextran and hydrate polymers. • Both the EDTA-dextran and EDTA-BCD hydrate polymers showed promising levels of extended drug release. Dextran vs. BCD structure, and then monomer vs. prepolymer (concentration of CDs). Epichlorohydrin-linked particles are amorphous though? ? ? Thoughts for paper maybe • After 100 hrs, the EDTA-BCD prepolymers did not show an increase in the amount of drug released, meaning this polymer was not successful at drug release for an extended period of time, and the study was stopped early. Figure 8. Images of scratch migration assay to determine eluted drug activity. A. ) Outline of scratch made in Endothelial cells after one day. B. ) Outline of scratch in cells after two days. The significant reduction indicated strong migration of cells. Conclusions • EDTA-polymerized Beta-Cyclodextrin particles were synthesized for use in developing a coating for cardiovascualar stents. • The particles were capable of extended drug release of sirolimus. (affinity) • Characterization of particles showed successful polymerization, as well as ability to bind to cobalt chromium stents. Future work: • Complete release studies and scratch migration assays on drug eluted from polymer particles. • Further assess activity of eluted drug with a cell migration assay. • Continue to develop the particles into a coating for cobalt chromium cardiovascular stents. Acknowledgements The authors would like to thank NSF Bio-Inspired Materials and Systems REU for funding this work. References 1. G. G. Stefanini, D. R. Holmes, Drug-Eluting Coronary-Artery Stents, New England Journal of Medicine. 368 (2013) 254– 265. doi: 10. 1056/nejmra 1210816. 2. K. Rekab, C. Lepeytre, F. Goettmann, M. Dunand, C. Guillard, J. -M. Herrmann, Degradation of a cobalt(II)–EDTA complex by photocatalysis and H 2 O 2/UV-C. Application to nuclear wastes containing 60 Co, Journal of Radioanalytical and Nuclear Chemistry. 303 (2014) 131– 137. doi: 10. 1007/s 10967 -014 -3311 -y. 3. V. Venuti, B. Rossi, F. Damico, A. Mele, F. Castiglione, C. Punta, et al. , Combining Raman and infrared spectroscopy as a powerful tool for the structural elucidation of cyclodextrin-based polymeric hydrogels, Physical Chemistry Chemical Physics. 17 (2015) 10274– 10282. doi: 10. 1039/c 5 cp 00607 d.
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