Nag Okhil K, Oh Eunkeu, Delehanty James B
Center for Bio/Molecular Science and Engineering Code 6900, US Naval Research Laboratory, Washington, DC 20375, USA.
Optical Sciences Division, Code 5600, US Naval Research Laboratory, Washington, DC 20375, USA.
Pharmaceuticals (Basel). 2024 Oct 10;17(10):1351. doi: 10.3390/ph17101351.
Maintaining intracellular adenosine triphosphate (ATP) levels is essential for numerous cellular functions, including energy metabolism, muscle contraction, and nerve impulse transmission. ATP is primarily synthesized in mitochondria through oxidative phosphorylation. It is also generated in the cytosol under anaerobic conditions using phosphocreatine (PCr) as a phosphate donor to adenosine diphosphate. However, the intracellular delivery of exogenous PCr is challenging as it does not readily cross the plasma membrane. This complicates the use of PCr as a therapeutic agent to maintain energy homeostasis or to treat conditions like cerebral creatine deficiency syndrome (CDS), which results from defective creatine transporters. This study employs the use of fusogenic liposomes to deliver PCr directly into the cytosol, bypassing membrane impermeability issues. We engineered various 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based fusogenic liposomes, incorporating phospholipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in combination with phospholipid-aromatic dye components to facilitate membrane fusion and to enhance the delivery of the PCr cargo. Liposomal formulations were co-loaded with membrane-impermeable chromophores and PCr and studied on live cells using confocal microscopy. We demonstrated the successful intracellular delivery of these agents and observed a 23% increase in intracellular ATP levels in cells treated with PCr-loaded liposomes. This increase was not observed with free PCr, confirming the effectiveness of the liposome-based delivery system. Additionally, cell viability assays showed minimal toxicity from the liposomes. Our results indicate that fusogenic liposomes are a promising method for the delivery of PCr (and potentially other cell-impermeable therapeutic agents) to the cellular cytosol. The approach demonstrated here could be advantageous for treating energy-related disorders and improving cellular energy homeostasis.
维持细胞内三磷酸腺苷（ATP）水平对于众多细胞功能至关重要，这些功能包括能量代谢、肌肉收缩和神经冲动传递。ATP主要通过氧化磷酸化在线粒体中合成。在无氧条件下，它也可利用磷酸肌酸（PCr）作为二磷酸腺苷的磷酸供体在细胞质中生成。然而，外源性PCr的细胞内递送具有挑战性，因为它不易穿过质膜。这使得将PCr用作维持能量稳态或治疗诸如脑肌酸缺乏综合征（CDS）等病症（该病症由肌酸转运体缺陷引起）的治疗剂变得复杂。本研究采用融合脂质体将PCr直接递送至细胞质中，绕过膜不透性问题。我们设计了各种基于1,2 - 二油酰基 - 3 - 三甲基铵丙烷（DOTAP）的融合脂质体，将磷脂如1,2 - 二油酰基 - sn - 甘油 - 3 - 磷酸乙醇胺（DOPE）与磷脂 - 芳香族染料成分结合，以促进膜融合并增强PCr货物的递送。脂质体制剂与膜不透性发色团和PCr共同装载，并使用共聚焦显微镜在活细胞上进行研究。我们证明了这些试剂成功地递送至细胞内，并观察到用装载PCr的脂质体处理的细胞中细胞内ATP水平增加了23％。游离PCr未观察到这种增加，证实了基于脂质体的递送系统的有效性。此外，细胞活力测定显示脂质体的毒性极小。我们的结果表明，融合脂质体是将PCr（以及潜在的其他细胞不透性治疗剂）递送至细胞溶质的一种有前途的方法。此处展示的方法对于治疗能量相关疾病和改善细胞能量稳态可能是有利的。
