CL4H6 |
| Catalog No.: GC62690 |
CL4H6 is a pH-sensitive cationic lipid.
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
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Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| 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℃. |
|
Applications |
LNPs(including CL4H6) Achieve Efficient Silencing of MRTF-B Gene Expression in Human Conjunctival Fibroblasts. |
| Animal experiment [2]: | |
|
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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References: [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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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].
References:
[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.
| Cas No. | 2256087-35-9 | SDF | |
| Formula | C59H113NO5 | M.Wt | 916.53 |
| Solubility | DMSO : 100 mg/mL (109.11 mM; Need ultrasonic) | Storage | Store at -20°C |
| General tips | For obtaining a higher solubility , please warm the tube at 37 ℃ and shake it in the ultrasonic bath for a while.Stock solution can be stored below -20℃ for several months. | ||
| Shipping Condition | Evaluation sample solution : ship with blue ice All other available size: ship with RT , or blue ice upon request |
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The nanomedicine rush: New strategies for unmet medical needs based on innovative nano DDS
The era of Nanomedicine has arrived with the approval of ONPATTRO? by the FDA in 2018. Lipid nanoparticle (LNP) technology has succeeded in delivering siRNA to the human liver in genetic diseases and has also been applied to mRNA vaccinations for COVID-19 using a similar LNP technology. In this review, we focus on the current status of new lipids for use in LNP formulations including our original lipids (CL4H6/CL4C6/CL4D6) as well as mechanisms of targeting without a ligand. Clinical applications of nano DDS are moving forward rapidly in the field of cancer immunology since the successful introduction of OPDIVO? in 2014. Antigen presentation and the maturation of immune cells can be controlled by nano DDS for cancer immunotherapy. YSK12-C4, a newly designed ionizable amino lipid can induce successful immune activation by silencing mRNA in DC and NK cells, which are expected to be evaluated for clinical use. Finally, new cancer therapy by targeting mitochondria involving the use of a MITO-Porter, a membrane fusion-type mitochondrial delivery system, has been introduced. The importance of delivering a photo sensitizer to mitochondria was clearly demonstrated in photodynamic cancer therapy. Clinical applications of MITO-Porters started in collaborative efforts with LUCA Science Co., Ltd. And was established in 2018. The future direction of Nanomedicine is discussed.
Non-Viral Gene Therapy in Trabecular Meshwork Cells to Prevent Fibrosis in Minimally Invasive Glaucoma Surgery
The primary cause of failure for minimally invasive glaucoma surgery (MIGS) is fibrosis in the trabecular meshwork (TM) that regulates the outflow of aqueous humour, and no anti-fibrotic drug is available for intraocular use in MIGS. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a promising anti-fibrotic target. This study aims to utilise a novel lipid nanoparticle (LNP) to deliver MRTF-B siRNA into human TM cells and to compare its effects with those observed in human conjunctival fibroblasts (FF). Two LNP formulations were prepared with and without the targeting peptide cΥ, and with an siRNA concentration of 50 nM. We examined the biophysical properties and encapsulation efficiencies of the LNPs, and evaluated the effects of MRTF-B silencing on cell viability, key fibrotic genes expression and cell contractility. Both LNP formulations efficiently silenced MRTF-B gene and were non-cytotoxic in TM and FF cells. The presence of cΥ made the LNPs smaller and more cationic, but had no significant effect on encapsulation efficiency. Both TM and FF cells also showed significantly reduced contractibility after transfection with MRTF-B siRNA LNPs. In TM cells, LNPs with cΥ achieved a greater decrease in contractility compared to LNPs without cΥ. In conclusion, we demonstrate that the novel CL4H6-LNPs are able to safely and effectively deliver MRTF-B siRNA into human TM cells. LNPs can serve as a promising non-viral gene therapy to prevent fibrosis in MIGS.
Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy
The tumor-microenvironment contains large numbers of tumor-associated macrophages (TAMs) which are largely M2 phenotypes and are involved in pro-tumorous functions. Targeting TAMs so as to manipulate them and to modify their functions could be a novel immunotherapy for the treatment of cancer. Such a strategy would involve targeting TAMs with short interfering RNA (siRNA) to modify their functions by silencing certain genes that are responsible for their M2 polarization. In this study, a lipid nanoparticle (LNP) formulation was used to target and deliver siRNA to TAMs. The LNP was mainly composed of a novel, pH-sensitive cationic lipid, referred to as the CL4H6 lipid, which had previously been optimized to target hepatocytes. The optimized siRNA-loaded CL4H6-LNPs were selectively and efficiently taken up and showed strong gene silencing activity in TAMs in a human tumor xenograft model in nude mice. Furthermore, an anti-tumor therapeutic response in the same tumor model was obtained by targeting TAMs using the optimized siRNA-loaded CL4H6-LNPs. The anti-tumor therapeutic response was obtained through the silencing of the signal transducer and activator of transcription 3 (STAT3) and hypoxia inducible factor 1 α (HIF-1α), which resulted in an increase in the level of infiltrated macrophage (CD11b+ cells) into the tumor-microenvironment (TME) as well as a tendency to increase the concentration of M1 macrophages (CD169+ cells). The treatment also resulted in reversing the pro-tumorous functions of TAMs -mainly angiogenesis and tumor cell activation-, as evidenced by a decrease in the related gene expression at the mRNA level. This research has promising clinical and pharmaceutical applications for achieving novel macrophage-based cancer immunotherapy.
Innovative nanotechnologies for enhancing nucleic acids/gene therapy: Controlling intracellular trafficking to targeted biodistribution
Nanomedicine promises to play an important role in next generation therapy, including Nucleic acid/Gene therapy. To accomplish this, innovative nanotechnologies will be needed to support nanomedicine by controlling not only the biodistribution but also the intracellular trafficking of macromolecules such as RNA/DNA. A multifunctional envelope-type nano device (MEND) was developed to meet this requirement. We herein provide an update regarding the functions of the MEND system focusing on the introduction of different functional biomaterials that enhance efficiency. The octaarginine (R8) peptide enhances cellular uptake and controls intracellular trafficking to induce synergism in transgene expression. The R8 was also used for developing a MITO-Porter system for mitochondrial targeting. The function of the MITO-Porter system was extended by developing a mitochondrial reporter gene for mitochondrial gene therapy. For efficient in vivo gene delivery, new pH-sensitive lipids have been introduced to achieve controlled biodistribution and to enhance endosomal escape. For example, the CL4H6 lipid exerts a more efficient in vivo gene silencing than that of ONPATTROTM, a preparation that has been approved by the US Food and Drug Administration. We further summarize new technologies that have been successfully applied to cancer immunotherapy leading to the introduction of a new strategy based on the concept of the Cancer-Immunity Cycle.
Novel PEGylated Lipid Nanoparticles Have a High Encapsulation Efficiency and Effectively Deliver MRTF-B siRNA in Conjunctival Fibroblasts
The master regulator of the fibrosis cascade is the myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway, making it a key target for anti-fibrotic therapeutics. In the past, inhibitors and small interfering RNAs (siRNAs) targeting the MRTF-B gene have been deployed to counter fibrosis in the eye, with the latter showing promising results. However, the biggest challenge in implementing siRNA therapeutics is the method of delivery. In this study, we utilised the novel, pH-sensitive, cationic lipid CL4H6, which has previously demonstrated potent targeting of hepatocytes and endosomal escape, to safely and efficiently deliver an MRTF-B siRNA into human conjunctival fibroblasts. We prepared two lipid nanoparticle (LNP) formulations, incorporating targeting cleavable peptide cY in one of them, and measured their physicochemical properties and silencing effect in human conjunctival fibroblasts. Both proved to be non-cytotoxic at a concentration of 50 nM and effectively silenced the MRTF-B gene in vitro, with the targeting cleavable peptide not affecting the silencing efficiency [LNP with cY: 62.1% and 81.5% versus LNP without cY: 77.7% and 80.2%, at siRNA concentrations of 50 nM (p = 0.06) and 100 nM (p = 0.09), respectively]. On the other hand, the addition of the targeting cleavable peptide significantly increased the encapsulation efficiency of the LNPs from 92.5% to 99.3% (p = 0.0005). In a 3D fibroblast-populated collagen matrix model, both LNP formulations significantly decreased fibroblast contraction after a single transfection. We conclude that the novel PEGylated CL4H6-MRTF-B siRNA-loaded LNPs represent a promising therapeutic approach to prevent conjunctival fibrosis after glaucoma filtration surgery.
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