目的:含有阳离子脂质的脂质纳米粒越来越多地用于药物和基因递送,因为它们可以显示出改善的细胞摄取,阴离子货物(例如siRNA和mRNA)的负载增强或表现出额外的功能性(例如针对癌细胞的细胞毒性)。本研究检验了阳离子脂质的分子结构影响脂质纳米粒结构的假设,细胞摄取,以及由此产生的细胞毒性。
方法:三种潜在的细胞毒性阳离子脂质,随着疏水部分的系统变化,被设计和合成。合成的所有三种阳离子脂质都含有药效团,例如双环香豆素基团(CCA12),三环依托度酸部分(ETD12),或与含有C12双链的季铵阳离子脂质缀合的大型五环三萜类“熊果酸”基团(U12)。将阳离子脂质以0.1mol%至5mol%的浓度范围掺杂到单油酸甘油酯立方体中,并使用小角X射线散射(SAXS)的组合评估了脂质分子结构对立方体相行为的影响。动态光散射(DLS),ζ电位和低温透射电子显微镜(Cryo-TEM)。评估了这些颗粒对一系列癌细胞和非癌细胞系的细胞毒性。以及它们的细胞摄取。
结果:阳离子脂质的分子结构与所得阳离子立方体的内部纳米结构连接,通常观察到过渡到更弯曲的立方和六方相。发现在脂质浓度≥75µg/mL时,由阳离子脂质CCA12形成的立方体对胃癌细胞系(AGS)的细胞摄取和细胞毒性均高于阳离子脂质ETD12和U12。CCA12阳离子立方体在脂质浓度≥100µg/mL时也对前列腺癌PC-3细胞系表现出合理的细胞毒性。相比之下,2.5mol%ETD12和2.5mol%U12立方体在整个测试的浓度范围内通常对癌细胞系和非癌细胞系都是无毒的。发现阳离子脂质的分子结构会影响长方体相行为,细胞摄取和毒性,尽管需要进一步研究以确定一系列细胞系中结构与细胞摄取之间的确切关系。
OBJECTIVE: Lipid nanoparticles containing a cationic lipid are increasingly used in drug and gene delivery as they can display improved cellular uptake, enhanced loading for anionic cargo such as siRNA and mRNA or exhibit additional functionality such as cytotoxicity against cancer cells. This research study tests the hypothesis that the molecular structure of the cationic lipid influences the structure of the lipid nanoparticle, the cellular uptake, and the resultant cytotoxicity.
METHODS: Three potentially cytotoxic cationic lipids, with systematic variations to the hydrophobic moiety, were designed and synthesised. All the three cationic lipids synthesised contain pharmacophores such as the bicyclic coumarin group (CCA12), the tricyclic etodolac moiety (ETD12), or the large pentacyclic triterpenoid \"ursolic\" group (U12) conjugated to a quaternary ammonium cationic lipid containing twin C12 chains. The cationic lipids were doped into monoolein
cubosomes at a range of concentrations from 0.1 mol% to 5 mol% and the effect of the lipid molecular architecture on the cubosome phase behaviour was assessed using a combination of Small Angle X-Ray Scattering (SAXS), Dynamic Light Scattering (DLS), zeta-potential and cryo-Transmission Electron Microscopy (Cryo-TEM). The resulting cytotoxicity of these particles against a range of cancerous and non-cancerous cell-lines was assessed, along with their cellular uptake.
RESULTS: The molecular architecture of the cationic lipid was linked to the internal nanostructure of the resulting cationic
cubosomes with a transition to more curved cubic and hexagonal phases generally observed.
Cubosomes formed from the cationic lipid CCA12 were found to have improved cellular uptake and significantly higher cytotoxicity than the cationic lipids ETD12 and U12 against the gastric cancer cell-line (AGS) at lipid concentrations ≥ 75 µg/mL. CCA12 cationic
cubosomes also displayed reasonable cytotoxicity against the prostate cancer PC-3 cell-line at lipid concentrations ≥ 100 µg/mL. In contrast, 2.5 mol% ETD12 and 2.5 mol% U12
cubosomes were generally non-toxic against both cancerous and non-cancerous cell lines over the entire concentration range tested. The molecular architecture of the cationic lipid was found to influence the cubosome phase behaviour, the cellular uptake and the toxicity although further studies are necessary to determine the exact relationship between structure and cellular uptake across a range of cell lines.