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.

中文翻译：

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全身树枝状药物纳米药物长期治疗兔模型轻中度脑瘫


小胶质细胞介导的神经炎症在围产期/新生儿脑损伤（包括脑瘫（CP））的发病机制中发挥着核心作用。减轻神经炎症的治疗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。我们之前的结果表明，第 4 代羟基聚酰胺胺 (PAMAM) 树枝状聚合物可以将药物特异性地输送到体循环中的活化神经胶质细胞，在此基础上，我们评估了第 6 代 (G6) 羟基末端 PAMAM 树枝状聚合物的持续功效这表明血液循环时间更长，大脑积累增加。 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渗透性，与 G6-PAMAM 树枝状聚合物-NAC (G6D-NAC) 缀合。小胶质细胞靶向 G6D-NAC 缀合物的功效在临床相关的 CP 兔模型中进行了评估，该模型具有轻度/中度 CP 表型，可提供未经治疗的 CP 试剂盒的更长存活期，从而能够评估生命 15 天的持续功效。 G6D-NAC 被缀合并表征。在 BV-2 小胶质细胞中进行细胞毒性和抗炎测定。 G6D-NAC 的功效在兔 CP 模型中进行了评估。 CP 试剂盒在出生后第 1 天（PND1）随机分为 5 组，并接受单剂量 PBS 或 G6D-NAC（2 或 5 mg/kg）或 NAC（2 或 5 mg/kg）静脉注射。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。 单剂量的全身“长循环”G6D-NAC 可以显着穿透受损的血脑屏障 (BBB)，将 NAC 特异性递送至活化的小胶质细胞，并显着减少皮质和小脑白质中小胶质细胞介导的神经炎症地区。此外，G6D-NAC 治疗显着提高了新生兔的存活率，并在出生后至少 15 天（PND15）将运动功能恢复到接近健康的对照水平，而用游离 NAC 治疗的 CP 套件在 PND9 之前死亡。对新生儿脑损伤中活化的小胶质细胞进行靶向治疗可以改善促炎性小胶质细胞对损伤的反应，提高存活率，并改善可长期持续的神经系统结果。对 G6D-NAC 激活的小胶质细胞进行适当的操作可以显着影响炎症以外的损伤。