Luna Arthur Cássio de Lima, Saraiva Greice Kelle Viegas, Filho Otaviano Mendonça Ribeiro, Chierice Gilberto Orivaldo, Neto Salvador Claro, Cuccovia Iolanda Midea, Maria Durvanei Augusto
Biochemistry and Biophysical Laboratory, Butantan Institute, University of Sao Paulo, Sao Paulo, Brazil; Department of Medical Sciences, Medical School, University of Sao Paulo, Sao Paulo, Brazil.
Department of Biochemistry, Institute of Chemistry, University of Sao Paulo, Sao Paulo, Brazil.
Int J Nanomedicine. 2016 Apr 18;11:1577-91. doi: 10.2147/IJN.S90850. eCollection 2016.
In recent studies, we showed that synthetic phosphoethanolamine (PHO-S) has a great potential for inducing cell death in several tumor cell lines without damage to normal cells. However, its cytotoxic effect and selectivity against tumor cells could increase with encapsulation in cationic liposomes, such as dioctadecyldimethylammonium chloride (DODAC), due to electrostatic interactions between these liposomes and tumor cell membranes. Our aim was to use cationic liposomes to deliver PHO-S and to furthermore maximize the therapeutic effect of this compound. DODAC liposomes containing PHO-S (DODAC/PHO-S), at concentrations of 0.3-2.0 mM, prepared by ultrasonication, were analyzed by scanning electron microscopy (SEM) and dynamic light scattering. The cytotoxic effect of DODAC/PHO-S on B16F10 cells, Hepa1c1c7 cells, and human umbilical vein endothelial cells (HUVECs) was assessed by MTT assay. Cell cycle phases of B16F10 cells were analyzed by flow cytometry and the morphological changes by SEM, after treatment. The liposomes were spherical and polydisperse in solution. The liposomes were stable, presenting an average of ∼ 50% of PHO-S encapsulation, with a small reduction after 40 days. DODAC demonstrated efficient PHO-S delivery, with the lowest values of IC50% (concentration that inhibits 50% of the growth of cells) for tumor cells, compared with PHO-S alone, with an IC50% value of 0.8 mM for B16F10 cells and 0.2 mM for Hepa1c1c7 cells, and without significant effects on endothelial cells. The Hepa1c1c7 cells showed greater sensitivity to the DODAC/PHO-S formulation when compared to B16F10 cells and HUVECs. The use of DODAC/PHO-S on B16F10 cells induced G2/M-phase cell cycle arrest, with the proportion significantly greater than that treated with PHO-S alone. The morphological analysis of B16F10 cells by SEM showed changes such as "bleb" formation, cell detachment, cytoplasmic retraction, and apoptotic bodies after DODAC/PHO-S treatment. Cationic liposomal formulation for PHO-S delivery promoted cytotoxicity more selectively and effectively against B16F10 and Hepa1c1c7 cells. Thus, the DODAC/PHO-S liposomal formulation presents great potential for preclinical studies.
在最近的研究中，我们发现合成磷酸乙醇胺（PHO-S）在几种肿瘤细胞系中具有诱导细胞死亡的巨大潜力，且不会对正常细胞造成损伤。然而，由于这些脂质体与肿瘤细胞膜之间的静电相互作用，其对肿瘤细胞的细胞毒性作用和选择性在包裹于阳离子脂质体（如二辛基二甲基氯化铵，DODAC）中时会增强。我们的目的是利用阳离子脂质体递送PHO-S，并进一步最大化该化合物的治疗效果。通过超声处理制备的浓度为0.3 - 2.0 mM的含PHO-S的DODAC脂质体（DODAC/PHO-S），通过扫描电子显微镜（SEM）和动态光散射进行分析。通过MTT法评估DODAC/PHO-S对B16F10细胞、Hepa1c1c7细胞和人脐静脉内皮细胞（HUVECs）的细胞毒性作用。处理后，通过流式细胞术分析B16F10细胞的细胞周期阶段，并通过SEM分析形态变化。脂质体在溶液中呈球形且多分散。脂质体稳定，PHO-S的平均包封率约为50%，40天后略有降低。与单独的PHO-S相比，DODAC显示出高效的PHO-S递送，对肿瘤细胞的IC50%（抑制细胞生长50%的浓度）值最低，B16F10细胞的IC50%值为0.8 mM，Hepa1c1c7细胞的IC50%值为0.2 mM，且对内皮细胞无显著影响。与B16F10细胞和HUVECs相比，Hepa1c1c7细胞对DODAC/PHO-S制剂表现出更高的敏感性。在B16F10细胞上使用DODAC/PHO-S诱导了G2/M期细胞周期阻滞，其比例显著高于单独用PHO-S处理的细胞。通过SEM对B16F10细胞进行形态分析，结果显示在DODAC/PHO-S处理后出现了如“泡状”形成、细胞脱离、细胞质收缩和凋亡小体等变化。用于递送PHO-S的阳离子脂质体制剂对B16F10和Hepa1c1c7细胞具有更具选择性和有效性的细胞毒性。因此，DODAC/PHO-S脂质体制剂在临床前研究中具有巨大潜力。
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