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Benedikt Deuringer,1 Carmen Härdtner,2 Katja Krebs,2 Ralf Thomann,3 Martin Holzer,1 Ingo Hilgendorf,2,4,* Regine Süss1,* 1Department of Pharmaceutical Technology and Biopharmacy, Institute of Pharmaceutical Sciences, University of Freiburg, Freiburg, 79104, Germany; 2Department of Cardiology and Angiology, University Heart Center Freiburg-Bad Krozingen and Faculty of Medicine, University of Freiburg, Freiburg, 79106, Germany; 3FMF Materials Research Center, University of Freiburg, Freiburg, 79104, Germany; 4Institute for Experimental Cardiovascular Medicine, University Heart Center Freiburg-Bad Krozingen and Faculty of Medicine, University of Freiburg, Freiburg, 79110, Germany*These authors contributed equally to this workCorrespondence: Benedikt Deuringer, Pharmaceutical Technology and Biopharmacy, Sonnenstraße 5, Freiburg, 79104, Germany, Tel +49 761 203 6329, Fax +49 761 203 6326, Email bdeuringer@pharmazeutischetechnologie.dePurpose: The conventional techniques for the preparation of reconstituted high-density lipoprotein (rHDL) are hampered by long process times, the need for large amounts of starting material, and harsh preparation conditions. Here, we present a novel rHDL preparation method to overcome these challenges. Furthermore, we propose a dual mode of action for rHDL loaded with the immunosuppressant drug everolimus (Eve-rHDL) in the context of atherosclerosis and cardiovascular disease.Methods: We use dual centrifugation for rHDL nanoparticle preparation and characterize the physicochemical properties by NS-TEM, N-PAGE, DLS, AF4, and HPLC. In addition, we determine the biological efficacy in human and murine cell culture with regard to cellular uptake, cholesterol efflux, and proliferation.Results: We confirm the characteristic particle size of 10 nm, discoidal morphology, and chemical composition of the rHDL preparations and identify dual centrifugation as an ideal method for cost-effective aseptic rHDL manufacturing. rHDL can be prepared in approx. 1.5 h with batch sizes as little as 89 μL. Moreover, we demonstrate the cholesterol efflux capacity and anti-proliferative activity of Eve-rHDL in vitro. The anti-proliferative effects were comparable to free Eve, thus confirming the suitability of rHDL as a capable drug delivery vehicle.Conclusion: Eve-rHDL shows great efficacy in vitro and may further be employed to target atherosclerotic plaques in vivo. Highly effective anti-atherosclerotic therapy might be feasible by reducing both inflammatory- and lipid burden of the plaques. Dual centrifugation is an ideal technique for the efficient application of the rHDL platform in cardiovascular disease and beyond.Keywords: atherosclerosis, cholesterol efflux, drug delivery, everolimus, proliferation, protein-conjugate |
