DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)

DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine) is a neutral phospholipid containing two unsaturated oleic acid chains (C18:1). DOPE has a wide range of applications in biofilm research and drug delivery systems, especially as one of the components in liposomes or lipid nanoparticles (LNPs). It can work synergistically with other phospholipid molecules or cholesterol to modulate the physical and chemical properties of membranes, thereby optimizing the stability and delivery efficiency of drug or gene delivery systems^[1][2].

In vitro, DC-Chol/DOPE liposomes (molar ratios 1:1, 2:3, 1:2) were prepared at 4µmol/mL DC-Chol with 4, 6, or 8µmol/mL DOPE respectively, then diluated in 20µL HEPES buffer and incubated with 20µL of pDNA (1µg) at room temperature for 25min. Liposome/pDNA complexes containing 0.5µg pDNA were added to HepG2 cells for 8h. There was no significant difference in pDNA binding affinity between DC-Chol/DOPE liposomes containing various molar ratios of DC-Chol/DOPE. After transfection, cell viability was about 80% and the cytotoxicity of DC-Chol/DOPE liposomes containing a relatively high DOPE content was relatively low^[3]. HEK-293, mouse colon cancer (CT-26), and human tongue squamous cell carcinoma (CAL-27) cells were incubated with two mono cationic lipids Toc-Cim liposomes or Chol-Cim liposomes with lipid/DOPE molar ratio=1:1 at concentrations of 10, 20, 40, and 80μg/mL and lipid/pDNA ratios of lipoplexes ranging from 1:1 to 8:1 for 4h. All the liposomal concentrations and N/P ratios, the synthesized lipids exhibited limited cytotoxicity (more than 80% of cell viability) except at 80μg/mL liposomal concentration and 8:1 lipid/pDNA ratio. at 4:1 and 2:1 lipid/pDNA ratios, the Toc-Cim:pCMV-β-gal were produced the most efficient as closely to lipofectamine:pCMV-β-gal in the HEK-293 cell line compared to Chol-Cim:pCMV- β-gal. Toc-Cim:pCMV-β-gal at a 2:1 lipid/pDNA ratio is as effective against the CT-26 cell line as compared to the CAL-27 cell line^[4].

In vivo, DC-Chol (1.046mg), DOPE (1.451mg; 52.2mol%) and PEG2000-DSPE (0.281mg; 2.0 mol%) were mixed first to produce liposome. Next, p_shEg5@LS lipoplexes were prepared by mixing cationic liposomes with pAAV-shEg5 plasmids, and then become a final concentration of 0.89mg/mL at liposome/pDNA ratio = 1.65:1 in HBG buffer. A2780 tumor-bearing BALB/c nude mice intravenously administered with Cy5.5-p_shEg5@LS (1.5mg/kg) via tail vein showed a significant delay in tumor growth. The tumor tissues were significantly reduced in weight (58.0% reduction) at the completion of experiment (two weeks after injection)^[5]. The optimized formulation of 306-O12B LNP had 50% leading tail-branched bioreducible lipidoid (306-O12B), 38.5% cholesterol, 10% DOPC, and 1.5% DMG-PEG molar composition with a 7.5:1 weight ratio of 306-O12B/mRNA. C57BL/6 mice were intravenously administrated with 306-O12B LNPs containing Cas9 mRNA and Angptl3-targeting single-guide RNA (sgAngptl3) with a total RNA dose of 3.0mg/kg. At day 7 post-injection, 306-O12B LNP-mediated delivery resulted in a median editing rate of 38.5% in liver. The serum ANGPTL3 protein, LDL-C, and TG levels in 306-O12B LNP treatment group (65.2, 56.8, and 29.4% reductions, respectively)^[6].

References:
[1] MacDonald R C, Rakhmanova V A, Choi K L, Rosenzweig H S, Lahiri M K. O-ethylphosphatidylcholine: A metabolizable cationic phospholipid which is a serum-compatible DNA transfection agent. J Pharm Sci. 1999 Sep;88(9):896-904.
[2] Benedicto E A, Farbiak L, Ramírez M M, et al. Optimization of phospholipid chemistry for improved lipid nanoparticle (LNP) delivery of messenger RNA (mRNA). Biomater Sci. 2022 Jan 18;10(2):549-559.
[3] Chen Y, Sun J, Ying Y, et al. Complexes containing cationic and anionic pH-sensitive liposomes: comparative study of factors influencing plasmid DNA gene delivery to tumors. Int J Nanomedicine. 2013:8:1573-93.
[4] Manturthi S, Bhattacharya D, Sakhare K R, Narayan K P, Patri S V. Cimetidine-Based Cationic Amphiphiles for In Vitro Gene Delivery Targetable to Colon Cancer. ACS Omega. 2022 Aug 25;7(35):31388-31402.
[5] Seraj S, Lee J J, Ahn H J. Systemic delivery of Eg5 shRNA-expressing plasmids using PEGylated DC-Chol/DOPE cationic liposome: Long-term silencing and anticancer effects in vivo. Biochem Pharmacol. 2019 Aug:166:192-202.
[6] Qiu M, Glass Z, Chen J J. Lipid nanoparticle-mediated codelivery of Cas9 mRNA and single-guide RNA achieves liver-specific in vivo genome editing of Angptl3. Proc Natl Acad Sci U S A. 2021 Mar 9;118(10):e2020401118.

DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine)是一种中性磷脂，含两条不饱和油酸链(C18:1)。DOPE在生物膜研究及药物递送体系中具有广泛应用，尤其作为脂质体或脂质纳米颗粒(LNPs)的组分之一，可与其他磷脂分子或胆固醇协同作用，调节膜的理化性质，从而优化药物或基因递送体系的稳定性及递送效率^[1][2]。

体外实验中，以4µmol/mL DC-Chol分别与4、6或8µmol/mL DOPE制备摩尔比为1:1、2:3、1:2的DC-Chol/DOPE脂质体，取20µL与20µL HEPES缓冲液稀释，再加入20µL pDNA（1µg），室温孵育25min。将含0.5µg pDNA的脂质体/pDNA复合物加入HepG2细胞作用8h。不同摩尔比DC-Chol/DOPE脂质体对pDNA结合亲和力无显著差异；转染后细胞活力约80%，且含较高DOPE含量的DC-Chol/DOPE脂质体细胞毒性相对更低^[3]。人胚肾细胞系（HEK-293）、小鼠结肠癌细胞系（CT-26）和人舌鳞状细胞癌细胞系（CAL-27）细胞分别与两种单阳离子脂质Toc-Cim脂质体或Chol-Cim脂质体(脂质/DOPE摩尔比=1:1)在10、20、40、80μg/mL浓度及1:1–8:1的脂质/pDNA比例下孵育4h。除80μg/mL脂质浓度与8:1脂质/pDNA比例外，其余条件下合成脂质均显示有限细胞毒性（细胞活力>80%）。在4:1及2:1脂质/pDNA比例下，Toc-Cim:pCMV-β-gal在HEK-293细胞系中的转染效率最高，接近lipofectamine:pCMV-β-gal；Toc-Cim:pCMV-β-gal在2:1脂质/pDNA比例时对CT-26细胞系的效果与CAL-27细胞系相当^[4]。

体内实验：先将DC-Chol(1.046mg)、DOPE(1.451mg；52.2mol%)及PEG2000-DSPE(0.281mg；2.0mol%)混合制备脂质体；随后将阳离子脂质体与pAAV-shEg5质粒混合，在HBG缓冲液中制成脂质体/pDNA=1.65:1、终浓度0.89mg/mL的p_shEg5@LS脂质复合物。A2780荷瘤BALB/c裸鼠经尾静脉注射Cy5.5-p_shEg5@LS（1.5mg/kg），监测期内肿瘤生长显著延迟；实验结束（注射后两周）时，肿瘤重量显著下降（约58.0%）^[5]。经优化的306-O12B LNP配方为50%领先尾支化生物可还原类脂质306-O12B、38.5%胆固醇、10% DOPC和1.5% DMG-PEG（摩尔组成），306-O12B/mRNA重量比7.5:1。C57BL/6小鼠经尾静脉注射含Cas9 mRNA及靶向Angptl3单导向RNA(sgAngptl3)的306-O12B LNPs，总RNA剂量3.0mg/kg。注射后第7天，306-O12B LNP介导的肝脏中位编辑率达38.5%；306-O12B LNP治疗组血清ANGPTL3蛋白、LDL-C及TG水平分别下降65.2%、56.8%及29.4%^[6]。