Everolimus-Loaded Reconstituted High-Density Lipoprotein Prepared by a Novel Dual Centrifugation Approach for Anti-Atherosclerotic Therapy.

Abstract

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. 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. 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. 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.