Department of Anesthesiology and Critical Care, Johns Hopkins School of Medicine, Baltimore, MD, USA.
Present address: Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI, USA.
J Neuroinflammation. 2020 Oct 25;17(1):319. doi: 10.1186/s12974-020-01984-1.
Neuroinflammation mediated by microglia plays a central role in the pathogenesis of perinatal/neonatal brain injury, including cerebral palsy (CP). Therapeutics mitigating neuroinflammation potentially provide an effective strategy to slow the disease progression and rescue normal brain development. Building on our prior results which showed that a generation-4 hydroxyl poly(amidoamine) (PAMAM) dendrimer could deliver drugs specifically to activated glia from systemic circulation, we evaluated the sustained efficacy of a generation-6 (G6) hydroxyl-terminated PAMAM dendrimer that showed a longer blood circulation time and increased brain accumulation. N-acetyl-L-cysteine (NAC), an antioxidant and anti-inflammatory agent that has high plasma protein binding properties and poor brain penetration, was conjugated to G6-PAMAM dendrimer-NAC (G6D-NAC). The efficacy of microglia-targeted G6D-NAC conjugate was evaluated in a clinically relevant rabbit model of CP, with a mild/moderate CP phenotype to provide a longer survival of untreated CP kits, enabling the assessment of sustained efficacy over 15 days of life.
G6D-NAC was conjugated and characterized. Cytotoxicity and anti-inflammatory assays were performed in BV-2 microglial cells. The efficacy of G6D-NAC was evaluated in a rabbit model of CP. CP kits were randomly divided into 5 groups on postnatal day 1 (PND1) and received an intravenous injection of a single dose of PBS, or G6D-NAC (2 or 5 mg/kg), or NAC (2 or 5 mg/kg). Neurobehavioral tests, microglia morphology, and neuroinflammation were evaluated at postnatal day 5 (PND5) and day 15 (PND15).
A single dose of systemic 'long circulating' G6D-NAC showed a significant penetration across the impaired blood-brain-barrier (BBB), delivered NAC specifically to activated microglia, and significantly reduced microglia-mediated neuroinflammation in both the cortex and cerebellum white matter areas. Moreover, G6D-NAC treatment significantly improved neonatal rabbit survival rate and rescued motor function to nearly healthy control levels at least up to 15 days after birth (PND15), while CP kits treated with free NAC died before PND9.
Targeted delivery of therapeutics to activated microglia in neonatal brain injury can ameliorate pro-inflammatory microglial responses to injury, promote survival rate, and improve neurological outcomes that can be sustained for a long period. Appropriate manipulation of activated microglia enabled by G6D-NAC can impact the injury significantly beyond inflammation.
小胶质细胞介导的神经炎症在围产期/新生儿脑损伤的发病机制中起着核心作用，包括脑瘫（CP）。减轻神经炎症的治疗方法可能提供一种有效的策略来减缓疾病进展并挽救正常的大脑发育。基于我们之前的研究结果，即第四代羟丙基聚酰胺胺（PAMAM）树枝状大分子可以从体循环中特异性地将药物递送至活化的神经胶质细胞，我们评估了具有更长血液循环时间和增加脑积累的第六代（G6）羟基封端 PAMAM 树枝状大分子的持续疗效。N-乙酰-L-半胱氨酸（NAC）是一种抗氧化剂和抗炎剂，具有高血浆蛋白结合特性和较差的脑穿透性，已被共轭到 G6-PAMAM 树枝状大分子-NAC（G6D-NAC）。在具有轻度/中度 CP 表型的临床相关 CP 兔模型中评估了靶向小胶质细胞的 G6D-NAC 缀合物的疗效，为未治疗的 CP 试剂盒提供了更长的存活时间，从而能够在 15 天的生命期内评估持续疗效。
对 G6D-NAC 进行共轭和表征。在 BV-2 小胶质细胞中进行细胞毒性和抗炎测定。在 CP 兔模型中评估了 G6D-NAC 的疗效。CP 试剂盒在出生后第 1 天（PND1）随机分为 5 组，接受单次静脉注射 PBS、G6D-NAC（2 或 5mg/kg）或 NAC（2 或 5mg/kg）。在出生后第 5 天（PND5）和第 15 天（PND15）评估神经行为测试、小胶质细胞形态和神经炎症。
单次全身“长循环”G6D-NAC 显示出显著穿透受损的血脑屏障（BBB）的能力，将 NAC 特异性递送至活化的小胶质细胞，并显著降低了皮质和小脑白质区域中小胶质细胞介导的神经炎症。此外，G6D-NAC 治疗至少在出生后 15 天（PND15）显著提高了新生兔的存活率，并将运动功能恢复到接近健康对照组的水平，而用游离 NAC 治疗的 CP 试剂盒在 PND9 之前死亡。
将治疗药物靶向递送至新生儿脑损伤的活化小胶质细胞，可以改善损伤后的促炎小胶质细胞反应，提高存活率，并改善可维持较长时间的神经发育结果。通过 G6D-NAC 进行适当的小胶质细胞活化操纵，可以显著影响炎症以外的损伤。
