Injectable Hydrogels to Protect Transplanted Cells from Hypoxia

项目摘要 PAR-18-206：保护移植细胞免受缺氧的可注射水凝胶 通过直接局部注射的细胞移植是许多再生医学疗法的有前途的策略; 然而，无论临床适应症如何，这种策略的治疗潜力都受到了极大的限制 由于细胞输送效率低和移植细胞的长期存活率低。我们最近设计了一个 可注射水凝胶，其通过在注射期间提供（1）机械屏蔽来改善细胞递送 防止细胞膜破裂的过程，（2）体内快速凝胶化以将细胞定位在预期的递送位置 位点，和（3）促进移植细胞扩散和迁移到宿主中的细胞粘附配体 组织.在脊髓损伤（SCI）的临床前模型中，使用这种水凝胶移植雪旺细胞（SC） 导致成功的细胞递送显著增加，这与改善的治疗效果相关。 结果。然而，移植细胞的长期存活率差仍然是一个未满足的挑战， 缺氧的宿主环境因此，我们提出了两个正交生物材料设计的发展 策略（目标1中的生物力学策略和目标2中的生物化学策略）来创建可注射的 水凝胶，改善移植细胞的输送和促进缺氧长期生存。这些材料， 名为SHIELD（用于注射封装和长期递送的剪切稀化水凝胶）的产品完全 化学定义，以方便未来的FDA研究。作为概念验证，SHIELD将在 SCI的临床前模型，其中已知移植的SC疗法遭受显著的缺氧细胞损伤。 死亡在目的1中，我们评估了基质力学可以改变促生存分泌组的假设， 包裹的细胞，从而产生可溶性的自分泌信号，提高缺氧生存。细胞将被 封装在具有一定硬度的SHIELD材料中，在常氧和低氧条件下培养 （分别为5%和1%O2），并评估活力、增殖、神经营养因子分泌和生长 因子和细胞坏死（亲环素A和胞衬蛋白分解产物）和凋亡（半胱天冬酶-3）的标志物 和TUNEL）。作为一种平行的方法，在目标2中，我们评估了持续的局部递送 可以通过设计稳定的脂质囊泡贮库来获得促存活因子， 交联到我们的可注射水凝胶中。多层脂质胶囊通过双层间共价键稳定， 交联，并且交联度用于调节释放速率。因此，这种模块化设计 该策略可用于独立控制多种促存活因子的递送动力学。 将按照目标1评价包封细胞。在目标3中，我们在临床前大鼠中验证了我们的体外研究结果 颈挫伤性脊髓损伤SC移植模型。SC存活和分布，天然组织反应， 将评估神经再生和功能性前肢恢复。总而言之，由于 基于细胞的再生医学疗法取决于移植细胞的存活， 直接解决缺氧引起的细胞死亡可以显著改善临床结果。

Project Summary PAR-18-206: Injectable Hydrogels to Protect Transplanted Cells from Hypoxia Cell transplantation by direct local injection is a promising strategy for many regenerative medicine therapies; however, regardless of clinical indication, the therapeutic potential of this strategy has been drastically limited by inefficient cell delivery and poor long-term survival of transplanted cells. We have recently designed an injectable hydrogel that improves cell delivery by providing (1) mechanical shielding during the injection process to prevent cell membrane rupture, (2) rapid gelation in vivo to localize cells at the intended delivery site, and (3) cell-adhesive ligands that promote the spreading and migration of transplanted cells into the host tissue. In a preclinical model of spinal cord injury (SCI), use of this hydrogel to transplant Schwann cells (SCs) resulted in a significant increase in successful cell delivery, which correlated with improved therapeutic outcomes. However, poor long-term survival of transplanted cells continues to be an unmet challenge due to the hypoxic host environment. Therefore, we propose the development of two orthogonal biomaterial design strategies (a biomechanical strategy in Aim 1 and a biochemical strategy in Aim 2) to create injectable hydrogels that improve transplanted cell delivery and promote long-term survival in hypoxia. These materials, named SHIELD (Shear-thinning Hydrogels for Injectable Encapsulation and Long-term Delivery) are fully chemically defined to facilitate future FDA studies. As a proof of concept, SHIELD will be evaluated in a preclinical model of SCI, where transplanted SC therapies are known to suffer from significant hypoxic cell death. In Aim 1, we evaluate the hypothesis that matrix mechanics can alter the pro-survival secretome of encapsulated cells, thereby creating soluble, autocrine signals that improve hypoxic survival. Cells will be encapsulated in SHIELD materials with a range of stiffness, cultured under normoxic and hypoxic conditions (5% and 1% O2, respectively), and assessed for viability, proliferation, secretion of neurotrophins and growth factors, and markers of cell necrosis (cyclophilin A and fodrin breakdown product) and apoptosis (caspase-3 and TUNEL). As a parallel approach, in Aim 2, we evaluate the hypothesis that sustained, localized delivery of pro-survival factors can be achieved through the design of stabilized, lipid-vesicle depots that physically crosslink into our injectable hydrogel. The multi-lamellar lipid capsules are stabilized by inter-bilayer covalent crosslinking, and the degree of crosslinking is used to tune the release rate. Thus, this modular design strategy can be used to independently control the delivery kinetics of multiple pro-survival factors. Encapsulated cells will be evaluated as in Aim 1. In Aim 3, we validate our in vitro findings in a preclinical rat model of cervical, contusive SCI with SC transplantation. SC survival and distribution, native tissue response, neuro-regeneration, and functional forelimb recovery will be assessed. In summary, because the success of cell-based regenerative medicine therapies hinges on the survival of transplanted cells, technologies that directly address cell death by hypoxia can significantly improve clinical outcomes.