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

SBIR一期项目旨在开发一种尖端纳米技术，该技术将极大地增强再生药物（如干细胞、复杂组织和器官）的生物保存。这种技术有可能永远改变再生医疗保健。它将改变按需细胞和组织的生物银行，改善大规模创伤护理和先进的个性化医疗程序。在再生医学中，几乎所有层面都需要生物保存，包括获取源材料、分离、储存和将最终产品运送到患者手中。然而，关键的是，该领域缺乏安全有效地保存这些组织和药物的能力，严重限制了产品的保质期。没有什么比器官移植更明显地缺乏生物库技术了，在器官移植中，供体和受体之间的时间窗口（4-7小时）不足以正确匹配供体、筛查病原体或运输距离。死于器官过早衰竭的人比死于癌症的人要多。使美国能够安全地储存零度以下的器官，将大大提高国民医疗保健水平。美国有强大的商业和竞争力理由投资再生医学的各个方面，包括器官治疗。冷冻保存解决方案将间接地为医疗保健系统、患者和医疗保险公司节省大量成本，据估计，美国每年再生医学治疗的成本将节省近2500亿美元。目前生物保存技术的细胞毒性主要与冷却过程中低温保护剂效率低下和水在细胞膜上的输送有关，导致冰形成对细胞造成不可修复的损害。该项目的目标是建立一种完全不同的方法来优化冷冻保护剂，通过开发首个领域的生物启发纳米孔作为跨膜高速公路，以促进在冷冻保存过程中安全有效地在细胞内递送和去除冷冻保护剂。过去的研究已经证明，基于多个氢键、偶极子-偶极子和芳香pi-pi堆叠相互作用及其自插入到脂质双层中的相互作用，通过强制堆叠形状持久的大环，构建定义良好的纳米管组件是可靠的。当蛋白质通道在3°C或低于3°C时发生故障时，这些合理设计的有机纳米孔将作为选择性跨膜通道。因此，细胞达到无冰低温保存温度的暴露时间将大大减少。通过减少细胞内冰的形成，保存后的细胞产量和活力将大大提高。在重新加热后，这些有机纳米孔将有助于快速去除冷冻保护剂。在生理温度下，纳米孔将被封闭，并从系统中冲洗出来，从而产生低毒性。纳米孔的功能和作用将通过基于脂质体的葡萄糖转运、基于细胞的毒性和基于细胞的冷冻保存试验来检验。

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.