SBIR Phase I: Developing Nanopores to Facilitate Delivery of Intracellular Cryoprotectants for Biopreservation at Low Temperature

This SBIR Phase I project aims to develop a cutting-edge nanotechnology that will greatly enhance biological preservation of regenerative medicines such as stem cells, complex tissues, and organs. Such a technology has the potential to changing regenerative healthcare forever. It would change biobanking for on-demand cells and tissues and improve mass trauma care and advanced personalized medical procedures. Biopreservation is required in regenerative medicine at nearly all levels in the acquisition of source material, isolation, storage and shipment of a final product to patient. Yet, critically, the field lacks the ability to safely and efficiently preserve these tissues and medicines severely limiting product shelf life. Nowhere is the absence of a biobanking technology more palpable than organ transplantation, where the time window between donor and recipient (4-7 hours) is not enough to properly match donations, screen for pathogens, or transport distances. More people will die from premature organ failure than cancer. Enabling the United States to safely bank organs at subzero temperature will significantly enhance national healthcare. The US faces strong commercial and competitiveness reasons to invest in all facets of regenerative medicine, including organ therapies. Cryopreservation solutions would indirectly enable significant savings to the healthcare system, the patient, and healthcare insurance companies with the cost savings from regenerative medicine treatments estimated to be nearly $250 billion per year in the U.S. The cytotoxicity of current biopreservation techniques is largely associated with inefficient cryoprotective agent and water delivery across the cell membrane during cooling leading to irreparable cell damage from ice formation. The goal of this project is to establish a fundamentally different approach to cryoprotective agent optimization by developing first-in-the-field bioinspired nanopores as transmembrane mega highways to facilitate safe and efficient intracellular delivery and removal of cryoprotective agents during cryopreservation. Past research has demonstrated the reliability of constructing well-defined nanotubular assemblies via the enforced stacking of shape-persistent macrocycles based on the interplay of multiple hydrogen-bonding, dipole-dipole, and aromatic pi-pi stacking interactions and their self-insertion into lipid bilayers. These rationally designed organic nanopores will serve as selective transmembrane channels when protein channels malfunction at or below 3 °C. As a result, the cell's exposure time to reach ice-free cryopreservation temperature will be significantly reduced. Post-preservation cell yield and viability will be greatly improved by reducing intracellular ice formation. Upon rewarming, these organic nanopores will facilitate rapid removal of the cryoprotective agents. At physiological temperature, the nanopores will seal off, and be washed out from the system resulting in low toxicity. Nanopore function and effect will be examined using liposome-based glucose transport, cell-based toxicity and cell-based cryopreservation assays.