Electrospun fiber-mediated delivery of neurotrophin-3 mRNA for neural tissue engineering applications
Electrospun fiber-mediated delivery of neurotrophin-3 mRNA for neural tissue engineering applications
复制标题
DOI：
10.1016/j.actbio.2022.11.025
复制
发表时间：
2022-12-30
影响因子：
9.7
通讯作者：
Gilbert，Ryan J.
中科院分区：
文献类型：
--
作者：
Puhl，Devan L.;Funnell，Jessica L.;Gilbert，Ryan J.
对齐的电纺纤维提供地形线索和局部治疗递送以促进稳健的外周神经再生。mRNA递送能够瞬时表达促进轴突再生的所需蛋白质。然而，没有先前的工作从电纺纤维递送mRNA用于外周神经再生应用。在这里，我们开发了第一个对齐的静电纺丝纤维，以提供假尿苷修饰的神经营养素-3（NT-3）mRNA（NT-3 mRNA）的原代雪旺细胞和评估NT-3的分泌和生物活性。我们首先静电纺丝排列的聚（L-乳酸）（PLLA）纤维，并将它们与阴离子底物葡聚糖硫酸钠盐（DSS）或聚（3，4-二羟基-L-苯丙氨酸）（pDOPA）。然后将含有与JetMESSENGER®复合的JNNT-3 mRNA的阳离子脂质复合物固定到纤维上，导致从所有研究的纤维组（PLLA+mRNA、0.5DSS 4 h +mRNA和2 pDOPA 4 h +mRNA）中可检测的JNNT-3 mRNA释放28天。与对照PLLA纤维相比，2 pDOPA 4 h +mRNA组显著增加了21天的许旺细胞NT-3分泌（p< 0.001-0.05），并且与1µ g Bolus +mRNA和3µ g Bolus +mRNA组的推注mRNA递送相比，许旺细胞NT-3分泌平均增加≥ 2倍。与对照PLLA纤维相比，2 pDOPA 4 h +mRNA纤维支持雪旺细胞分泌NT-3，其水平显著增加背根神经节（DRG）神经突延伸44%（p< 0.0001）和神经突面积64%（p< 0.001）。数据显示，2 pDOPA 4 h +mRNA纤维增强了雪旺细胞促进DRG神经突生长的能力，证明了该平台改善外周神经再生的潜在能力。Statement of significance排列的电纺纤维通过提供结构支持和引导线索来增强轴突再生，但进一步的治疗刺激是必要的，以改善功能结果。mRNA递送能够瞬时表达治疗性蛋白质，但实现局部持续递送仍然具有挑战性。以前的工作表明，从电纺纤维传递遗传物质可以改善再生;然而，mRNA的传递尚未被探索。在这里，我们研究mRNA的交付从对齐的静电纺丝纤维，以提高神经突生长。我们表明，将NT-3 mRNA/JetMESSENGER®脂质复合物固定到用pDOPA官能化的对齐的电纺纤维上能够局部、持续地将NT-3 mRNA递送到许旺细胞，增加许旺细胞分泌NT-3并增强DRG神经突生长。这项研究显示了电纺纤维介导的mRNA递送平台用于神经组织工程的潜在益处。
Aligned electrospun fibers provide topographical cues and local therapeutic delivery to facilitate robust peripheral nerve regeneration. mRNA delivery enables transient expression of desired proteins that promote axonal regeneration. However, no prior work delivers mRNA from electrospun fibers for peripheral nerve regeneration applications. Here, we developed the first aligned electrospun fibers to deliver pseudouridine-modified (Ψ) neurotrophin-3 (NT-3) mRNA (ΨNT-3mRNA) to primary Schwann cells and assessed NT-3 secretion and bioactivity. We first electrospun aligned poly(L-lactic acid) (PLLA) fibers and coated them with the anionic substrates dextran sulfate sodium salt (DSS) or poly(3,4-dihydroxy-L-phenylalanine) (pDOPA). Cationic lipoplexes containing ΨNT-3mRNA complexed to JetMESSENGER® were then immobilized to the fibers, resulting in detectable ΨNT-3mRNA release for 28 days from all fiber groups investigated (PLLA+mRNA, 0.5DSS4h+mRNA, and 2pDOPA4h+mRNA). The 2pDOPA4h+mRNA group significantly increased Schwann cell secretion of NT-3 for 21 days compared to control PLLA fibers (p< 0.001-0.05) and, on average, increased Schwann cell secretion of NT-3 by ≥ 2-fold compared to bolus mRNA delivery from the 1µgBolus+mRNA and 3µgBolus+mRNA groups. The 2pDOPA4h+mRNA fibers supported Schwann cell secretion of NT-3 at levels that significantly increased dorsal root ganglia (DRG) neurite extension by 44% (p< 0.0001) and neurite area by 64% (p< 0.001) compared to control PLLA fibers. The data show that the 2pDOPA4h+mRNA fibers enhance the ability of Schwann cells to promote neurite growth from DRG, demonstrating this platform's potential capability to improve peripheral nerve regeneration.Statement of significanceAligned electrospun fibers enhance axonal regeneration by providing structural support and guidance cues, but further therapeutic stimulation is necessary to improve functional outcomes. mRNA delivery enables the transient expression of therapeutic proteins, yet achieving local, sustained delivery remains challenging. Previous work shows that genetic material delivery from electrospun fibers improves regeneration; however, mRNA delivery has not been explored. Here, we examine mRNA delivery from aligned electrospun fibers to enhance neurite outgrowth. We show that immobilization of NT-3mRNA/JetMESSENGER® lipoplexes to aligned electrospun fibers functionalized with pDOPA enables local, sustained NT-3mRNA delivery to Schwann cells, increasing Schwann cell secretion of NT-3 and enhancing DRG neurite outgrowth. This study displays the potential benefits of electrospun fiber-mediated mRNA delivery platforms for neural tissue engineering.