Subnormothermic Oxygenated Machine Perfusion (24 h) in DCD Kidney Transplantation.

Background: Ex vivo kidney perfusion is an evolving platform that demonstrates promise in preserving and rehabilitating the kidney grafts. Despite this, there is little consensus on the optimal perfusion conditions. Hypothermic perfusion offers limited functional assessment, whereas normothermic perfusion requires a more complex mechanical system and perfusate. Subnormothermic machine perfusion (SNMP) has the potential to combine the advantages of both approaches but has undergone limited investigation. Therefore, the present study sought to determine the suitability of SNMP for extended kidney preservation. Methods: SNMP at 22-25 °C was performed on a portable device for 24 h with porcine kidneys. Graft assessment included measurement of mechanical parameters and biochemical analysis of the perfusate using point-of-care tests. To investigate the viability of kidneys preserved by SNMP, porcine kidney autotransplants were performed in a donation after circulatory death (DCD) model. SNMP was also compared with static cold storage (SCS). Finally, follow-up experiments were conducted in a subset of human kidneys to test the translational significance of findings in porcine kidneys. Results: In the perfusion-only cohort, porcine kidneys all displayed successful perfusion for 24 h by SNMP, evidenced by stable mechanical parameters and biological markers of graft function. Furthermore, in the transplant cohort, DCD grafts with 30 min of warm ischemic injury demonstrated superior posttransplant graft function when preserved by SNMP in comparison with SCS. Finally, human kidneys that underwent 24-h perfusion exhibited stable functional and biological parameters consistent with observations in porcine organs. Conclusions: These observations demonstrate the suitability and cross-species generalizability of subnormothermic machine perfusion to maintain stable kidney perfusion and provide foundational evidence for improved posttransplant graft function of DCD kidneys after SNMP compared with SCS.

Development of ionic liquid-coated PLGA nanoparticles for applications in intravenous drug delivery.

Polymeric nanoparticles (NPs) are a promising platform for medical applications in drug delivery. However, their use as drug carriers is limited by biological (e.g., immunological) barriers after intravenous administration. Ionic liquids (ILs), formed from bulky asymmetric cations and anions, have a wide variety of physical internal and external interfacing properties. When assembled on polymeric NPs as biomaterial coatings, these external-interfacing properties can be tuned to extend their circulation half-life when intravenously injected, as well as drive biodistribution to sites of interest for selective organ accumulation. In our work, we are particularly interested in optimizing IL coatings to enable red blood cell hitchhiking in whole blood. In this protocol, we describe the preparation and physicochemical and biological characterization of choline carboxylate IL-coated polymeric NPs. The procedure is divided into five stages: (1) synthesis and characterization of choline-based ILs (1 week); (2) bare poly(lactic-co-glycolic acid) (50:50, acid terminated) Resomer 504H (PLGA) NP assembly, modified from previously established protocols, with dye encapsulation (7 h); (3) modification of the bare particles with IL coating (3 h); (4) physicochemical characterization of both PLGA and IL-PLGA NPs by dynamic light scattering, 1H nuclear magnetic resonance spectroscopy, and transmission electron microscopy (1 week); (5) ex vivo evaluation of intravenous biocompatibility (including serum-protein resistance and hemolysis) and red blood cell hitchhiking in whole BALB/c mouse blood via fluorescence-activated cell sorting (1 week). With practice and technique refinement, this protocol is accessible to late-stage graduate students and early-stage postdoctoral scientists.

Selective Near-Infrared Blood Detection Driven by Ionic Liquid-Dye-Albumin Nanointeractions.

Due to its abundance in blood, a great deal of research has been undertaken to develop efficient biosensors for serum albumin and provide insight into the interactions that take place between these biosensing molecules and the protein. Near-infrared (NIR, >700 nm) organic dyes have been shown to be effective biosensors of serum albumin, but their effectiveness is diminished in whole blood. Herein, it is shown that an NIR sulfonate indolizine-donor-based squaraine dye, SO3SQ, can be strengthened as a biosensor of albumin through the addition of biocompatible ionic liquids (ILs). Specifically, the IL choline glycolate (1:1), at a concentration of 160 mM, results in the enhanced fluorescence emission ("switch-on") of the dye in the presence of blood. The origin of the fluorescence enhancement was investigated via methods, including DLS, ITC, and molecular dynamics. Further, fluorescence measurements were conducted to see the impact the dye-IL system had on the fluorescence of the tryptophan residue of human serum albumin (HSA), as well as to determine its apparent association constants in relation to albumin. Circular dichroism (CD) spectroscopy was used to provide evidence that the dye-IL system does not alter the secondary structures of albumin or DNA. Our results suggest that the enhanced fluorescence of the dye in the presence of IL and blood is due to diversification of binding sites in albumin, controlled by the interaction of the IL-dye-albumin complex.

Selective Blood Cell Hitchhiking in Whole Blood with Ionic Liquid-Coated PLGA Nanoparticles to Redirect Biodistribution After Intravenous Injection.

Less than 5% of intravenously-injected nanoparticles (NPs) reach destined sites in the body due to opsonization and immune-based clearance in vascular circulation. By hitchhiking in situ onto specific blood components post-injection, NPs can selectively target tissue sites for unprecedentedly high drug delivery rates. Choline carboxylate ionic liquids (ILs) are biocompatible liquid salts <100X composed of bulky asymmetric cations and anions. This class of ILs has been previously shown to significantly extend circulation time and redirect biodistribution in BALB/c mice post-IV injection via hitchhiking on red blood cell (RBC) membranes. Herein, we synthesized & screened 60 choline carboxylic acid-based ILs to coat PLGA NPs and present the impact of structurally engineering the coordinated anion identity to selectively interface and hitchhike lymphocytes, monocytes, granulocytes, platelets, and RBCs in whole mouse blood for in situ targeted drug delivery. Furthermore, we find this nanoparticle platform to be biocompatible (non-cytotoxic), translate to human whole blood by resisting serum uptake and maintaining modest hitchhiking, and also significantly extend circulation retention over 24 hours in BALB/c healthy adult mice after IV injection. Because of their altered circulation profiles, we additionally observe dramatically different organ accumulation profiles compared to bare PLGA NPs. This study establishes an initial breakthrough platform for a modular and transformative targeting technology to hitchhike onto blood components with high efficacy and safety in the bloodstream post-IV administration.