CL4H6 is a pH-sensitive cationic lipid. CL4H6 is the main component of lipid nanoparticles (LNPs). CL4H6 can be used to target and deliver siRNA, which can induce effective gene silencing response[1-2].
LNPs-CL4H6 can transport siRNA to human conjunctival fibroblasts to achieve efficient silencing of MRTF-B gene expression [1]. CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. It can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS[3].
siRNA-loaded CL4H6-LNPs(i.v.) is highly selectively absorbed in TAMs, showing strong gene silencing activity and significant antitumor therapeutic effect such as a significant increase in the infiltration of macrophages (CD11b+ cells) into TME by 59% and an insignificant increase in the proportion of M1 Mφ (CD169+ cells) by 50%[2]. CL4H6 (Cl4H6-LNPs) showed potent gene silencing activity (ED50: 0.0025 mg/kg) in a mouse factor VII (FVII) model, and was biodegradable and tolerable. In vivo experiments showed that CL4H6-LNPs exhibited high endosomal escape, cytoplasmic release, and RNA-induced silencing efficiency in siRNA-supported complexes (RISCs)[4].
参考文献:
[1]. Sanghani A, Kafetzis KN, et,al. Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts. Pharmaceutics. 2021 Mar 13;13(3):382. doi: 10.3390/pharmaceutics13030382. PMID: 33805660; PMCID: PMC7998417.
[2]. Shobaki N, Sato Y, et,al. Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy. J Control Release. 2020 Sep 10;325:235-248. doi: 10.1016/j.jconrel.2020.07.001. Epub 2020 Jul 8. PMID: 32649972.
[3]. Luo J, Tan G, et,al. Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery. Pharmaceutics. 2022 Nov 16;14(11):2472. doi: 10.3390/pharmaceutics14112472. PMID: 36432663; PMCID: PMC9693853.
[4]. Sato Y, Hashiba K, et,al. Understanding structure-activity relationships of pH-sensitive cationic lipids facilitates the rational identification of promising lipid nanoparticles for delivering siRNAs in vivo. J Control Release. 2019 Feb 10;295:140-152. doi: 10.1016/j.jconrel.2019.01.001. Epub 2019 Jan 2. PMID: 30610950.
CL4H6 是一种 pH 敏感的阳离子脂质。 CL4H6 是脂质纳米粒 (LNPs) 的主要成分。 CL4H6可用于靶向和递送siRNA，可诱导有效的基因沉默反应[1-2]。
LNPs-CL4H6 可将 siRNA 转运至人结膜成纤维细胞，从而实现 MRTF-B 基因表达的高效沉默[1]。 CL4H6-LNP 能够安全有效地将 MRTF-B siRNA 递送到人类 TM 细胞中。它可以作为一种很有前途的非病毒基因疗法来预防 MIGS 纤维化[3]。
载有 siRNA 的 CL4H6-LNPs(i.v.) 被 TAMs 高度选择性地吸收，显示出强大的基因沉默活性和显着的抗肿瘤治疗作用，例如巨噬细胞（CD11b+ 细胞）向 TME 的浸润显着增加 59% 和M1 Mφ（CD169+ 细胞）的比例增加了 50%[2]。 CL4H6 (Cl4H6-LNPs) 在小鼠因子 VII (FVII) 模型中显示出有效的基因沉默活性（ED50:0.0025 mg/kg），并且可生物降解且耐受。体内实验表明，CL4H6-LNPs 在 siRNA 支持的复合物 (RISCs) 中表现出高内体逃逸、细胞质释放和 RNA 诱导的沉默效率[4]。
| Nanoparticle Formulations（ preparation of CL4H6-LNP） [1]: | |
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Preparation Method |
CL4H6, DOPE and PEG-DMG were prepared in 90% t-BuOH at a final volume of 400 μL, a molar ratio of 50:50:1 and a final total lipid concentration of 1.4 mg/mL. Then, 100 μL of a 0.4 mg/mL siRNA solution were mixed with the lipids to give an N/P ratio of 7.5. Next, the LNPs were prepared in 20 mM MES buffer (pH 6.0). A portion of the resulting LNPs were mixed with peptide cY at a weight ratio of 4:1 (peptide:siRNA) by rapid mixing to obtain the LNP+cY nanoparticles. The LNPs were stored at 4℃ until needed. |
| Cell experiment [1]: | |
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Cell lines |
Human conjunctival fibroblasts |
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Preparation Method |
Four types of LNPs(including CL4H6) were incubated with fibroblasts (40% density) at 37℃ for 48h according to the following instructions: LNP-MRTF-B siRNA, LNP+cY-MRTF-B siRNA, LNP-IRR siRNA, and LNP+cY-IRR siRNA. Each type of LNP was tested at two different siRNA concentrations (50 nM and 100 nM). |
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Reaction Conditions |
Cell with LNP(including CL4H6) and siRNA for 48 h at 37℃. |
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Applications |
LNPs(including CL4H6) Achieve Efficient Silencing of MRTF-B Gene Expression in Human Conjunctival Fibroblasts. |
| Animal experiment [2]: | |
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Animal models |
BALB/cAjcl-nu/nu mice |
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Preparation of siRNA-loaded LNPs |
The LNPs were prepared by alcohol dilution procedure. The lipids, which include 1800 nmol of original lipids, such as CL4H6, 1200 nmol of chol, and 30 nmol of PEG-lipid (DMG-PEG 2000 or DSG-PEG 2000), which represents the optimized molar ratio of 60/40/1 mol%, respectively- were first dissolved in 400 μL of 90% (v/v) tertiary butanol (t-BuOH). When a fluorescent lipophilic tracer (Dil or DiD) was incorporated into the LNPs, it was added at a concentration of 0.15-0.5 mol% of the total lipid of the lipid solution at this point. Then, 80 ug of siRNA suspended in a 200 μL portion of an aqueous solution was added gradually to the lipid solution under vigorous mixing, resulting in a siRNA/lipid ratio of 0.042 (wt/wt). The siRNAlipid mixture was then gradually added to 2 mL of 20 mM citrate buffer (pH 4.0), also under vigorous mixing to facilitate the formation of the LNPs through the electrostatic interaction between positively-charged lipids and negatively-charged siRNAs under this acidic condition. This led to a final t-BuOH concentration of 60% (v/v). Finally, a 2-round ultrafiltration was performed using amicon ultra centrifugal tubes (100K) to remove the solvent t-BuOH, the pH was adjusted by replacing the acidic buffer with phosphate-buffered saline (PBS) (pH 7.40), and the final LNP preparation was concentrated. The centrifuge conditions of ultrafiltration were (1000 g, 21-23 min, r.t.). |
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Preparation method |
BALB/cAjcl-nu/nu mice were injected subcutaneously (s.c.) in the back with OS-RC-2 cells. Once the tumor reached a volume of 50 to 100 mm3, mice were injected i.v. with siRNA-loaded CL4H6-LNPs, in which DiD was incorporated at 0.5 mol% of the total lipid. The injection volume was 250 μL (this volume was customized to obtain a dose of 1 mg of siCD45/kg in a 25 g weighted mouse) and was fixed to minimize variation between mice which had an average weight of 25 g. At 24 h after the injection, the mice were sacrificed and their body organs were collected. |
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Dosage form |
250 μL siRNA-loaded CL4H6-LNPs(i.v.) |
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Applications |
siRNA-loaded CL4H6-LNPs is highly selectively absorbed in tumor-associated macrophages (TAMs), showing strong gene silencing activity and significant antitumor therapeutic effect. |
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参考文献: [1]. Sanghani A, Kafetzis KN, et,al. Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts. Pharmaceutics. 2021 Mar 13;13(3):382. doi: 10.3390/pharmaceutics13030382. PMID: 33805660; PMCID: PMC7998417. [2]. Shobaki N, Sato Y, Suzuki Y, Okabe N, Harashima H. Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy. J Control Release. 2020 Sep 10;325:235-248. doi: 10.1016/j.jconrel.2020.07.001. Epub 2020 Jul 8. PMID: 32649972. |
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