CL4H6

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 lines

Human conjunctival fibroblasts

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).

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 models

BALB/cAjcl-nu/nu mice

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.).

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.

Dosage form

250 μL siRNA-loaded CL4H6-LNPs(i.v.)

Applications

siRNA-loaded CL4H6-LNPs is highly selectively absorbed in tumor-associated macrophages (TAMs), showing strong gene silencing activity and significant antitumor therapeutic effect.

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.