Department of Biochemistry and Cell Biology, Geisel School of Medicine, Dartmouth College, Hanover, NH, USA.
Department of Molecular and Systems Biology, Geisel School of Medicine, Dartmouth College, Hanover, NH, USA.
J Neurosci Methods. 2022 Feb 1;367:109437. doi: 10.1016/j.jneumeth.2021.109437. Epub 2021 Dec 7.
Acyl-CoA:cholesterol acyltransferase (ACAT) inhibitors have been considered as potential therapeutic agents to treat several diseases, including Alzheimer's disease, atherosclerosis, and cancer. While many ACAT inhibitors are readily available, methods to encapsulate them as nanoparticles have not been reported.
We report a simple method to encapsulate ACAT inhibitors, using the potent hydrophobic ACAT inhibitor F12511 as an example. By mixing DSPE-PEG egg phosphatidylcholine (PC), and F12511 in ethanol, followed by drying, resuspension and sonication in buffer, we show that F12511 can be encapsulated as stealth liposomes at high concentration.
We successfully incorporated F12511 into nanoparticles and found that increasing PC in the nanoparticles markedly increased the amount of F12511 incorporated in stealth liposomes. The nanoparticles containing F12511 (Nanoparticle F) exhibit average size of approximately 200 nm and are stable at 4 ºC for at least 6 months. Nanoparticle F is very effective at inhibiting ACAT in human and mouse neuronal and microglial cell lines. Toxicity tests using mouse primary neuronal cells show that F12511 alone or Nanoparticle F added at concentrations from 2 to 10 µM for 24-, 48-, and 72-hours produces minimal, if any, toxicity.
COMPARISON WITH EXISTING METHOD(S): Unlike existing methods, the current method is simple, cost effective, and can be expanded to produce tagged liposomes to increase specificity of delivery. This also offers opportunity to embrace water soluble agent(s) within the aqueous compartment of the nanoparticles for potential combinatorial therapy.
This method shows promise for delivery of hydrophobic ACAT inhibitors at high concentration in vivo.
酰基辅酶 A：胆固醇酰基转移酶（ACAT）抑制剂已被认为是治疗多种疾病的潜在治疗剂，包括阿尔茨海默病、动脉粥样硬化和癌症。虽然有许多 ACAT 抑制剂，但尚未报道将它们封装为纳米颗粒的方法。
我们报告了一种简单的方法来封装 ACAT 抑制剂，以强效疏水性 ACAT 抑制剂 F12511 为例。通过将 DSPE-PEG 卵磷酯（PC）和 F12511 混合在乙醇中，然后干燥、在缓冲液中重新悬浮和超声处理，我们表明 F12511 可以高浓度封装为隐形脂质体。
我们成功地将 F12511 掺入纳米颗粒中，并发现增加纳米颗粒中的 PC 会显著增加隐形脂质体中掺入的 F12511 的量。含有 F12511 的纳米颗粒（纳米颗粒 F）的平均大小约为 200nm，在 4°C 下至少稳定 6 个月。纳米颗粒 F 非常有效地抑制人源和鼠源神经元和小胶质细胞系中的 ACAT。使用小鼠原代神经元细胞进行的毒性测试表明，F12511 单独或浓度为 2 至 10µM 的纳米颗粒 F 添加 24、48 和 72 小时，产生的毒性最小，如果有，则几乎没有。
与现有方法不同，该方法简单、经济有效，并且可以扩展以产生标记的脂质体，以提高递送的特异性。这也为在纳米颗粒的水相部分中纳入水溶性剂（s）以实现潜在的联合治疗提供了机会。
该方法有望在体内以高浓度递送电荷性 ACAT 抑制剂。
