Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver

Abstract

Ionizable lipid nanoparticles (LNPs) have been widely used for in vivo delivery of RNA therapeutics into the liver. However, a main challenge remains to develop LNP formulations for selective delivery of RNA into certain types of liver cells, such as hepatocytes and liver sinusoidal endothelial cells (LSECs). Here, we report the engineered LNPs for the targeted delivery of RNA into hepatocytes and LSECs. The effects of particle size and polyethylene glycol-lipid content in the LNPs were evaluated for the hepatocyte-specific delivery of mRNA by ApoE-mediated cellular uptake through low-density lipoprotein receptors. Targeted delivery of RNA to LSECs was further investigated using active ligands. Incorporation of mannose allowed the selective delivery of RNA to LSECs, while minimizing the unwanted cellular uptake by hepatocytes. These results demonstrate that engineered LNPs have great potential for the cell type-specific delivery of RNA into the liver and other tissues.

Fig. 1. Preparation of LNPs derived from developed ionizable lipids.

(A) Six different piperidine- and piperazine-containing amines used in this study. (B) Ionizable lipids were synthesized by epoxide ring opening with amines and epoxide containing 10 carbon tails. (C) LNPs were prepared via a microfluidic system with ionizable lipids, helper lipid, cholesterol, and PEG-lipid. Prepared LNPs were examined with cryo–transmission electron microscopy for their size and structural analysis.

Fig. 2. In vitro evaluation of ionizable lipid candidates.

Ionizable lipid candidates were formulated into LNPs with mFLuc, and their efficacy was evaluated in vitro. After incubation with ApoE, LNPs were treated to HeLa cells (96 wells) at mRNA dose of 100 ng (n = 4). (A) Expression of mFLuc after transfection of HeLa cells in the presence of serum and (B) in the presence or absence of ApoE. (C) ApoE rescues the transfection efficiency of 246C10 in the absence of serum. (D) Pretreatment of anti-LDLR antibody (Ab) to HeLa cells decreased luciferase expression in 246C10 LNP–treated HeLa cells. P > 0.05, ***P < 0.001 as compared with antibody-pretreated cells. FBS, fetal bovine serum.

Fig. 3. In vivo evaluation of ionizable lipid candidates (mFLuc).

Ionizable lipid candidates were formulated with mFLuc. mFLuc-loaded LNPs were injected to C57BL/6 mice at mRNA dose of 0.1 mg/kg. Three hours after injection, bioluminescence was analyzed. 244C10- to 246C10-formulated LNPs resulted in potent luciferase expression. Ex vivo organ image showed that LNPs were mostly uptaken into liver. p, photons; PDI, polydispersity index.

Fig. 4. In vivo evaluation of ionizable lipid candidates (mCre).

Ionizable lipid candidates were formulated with mCre. mCre-loaded LNPs were injected to LSL-tdTomato at mRNA dose of 0.5 mg/kg. Two-days after injection, tdTomato fluorescence was analyzed. (A) Ex vivo organ image showed that LNPs were mostly uptaken into the liver. (B) Liver histology image showed significant tdTomato fluorescence in the hepatocyte. cv, central vein. (C) Transfection efficiency in hepatocytes, LSECs, and Kupffer cells was confirmed by histological analysis (n = 5). DAPI, 4,6-diamidino-2-phenylindole.

Fig. 5. In vivo delivery of mRNA-loaded LNPs to different types of liver cells by controlling their size and PEG-lipid content.

(A) Formulation ratio between lipid components. PEG-lipid was changed from 1.0 to 3.0% by expending that of cholesterol. (B) Resulting size and encapsulation efficiency of mRNA-loaded LNPs with different PEG-lipid density. (C) LSL-tdTomato mice were injected intravenously with mCre (0.5 mg/kg). After 2 days of injection, tdTomato fluorescence–positive liver cells were confirmed by histological analysis (n = 5).

Fig. 6. In vitro evaluation of receptor-mediated cellular uptake of mannose-LNPs.

(A) Structure of mannose-PEG lipid. (B) Formulation details. (C) HepG2 cells were incubated with LNPs at 25 ng of mFLuc (n = 4). Mannose-lipid–incorporated LNPs allowed enhanced cellular internalization compared to LNPs with PEG-lipid only. (D) Relative FLuc expression of mannose-incorporated LNPs with respect to that of LNPs with PEG-lipid only. RLU, relative luminescence units; RFU, relative fluorescence units. Pretreatment of anti-CD206 resulted in decreased mRNA expression in LNPs with mannose-PEG lipid–treated HepG2 cells (E) but not in LNPs with PEG-galactose–treated HepG2 cells (F). ns, not significant: P > 0.05; **P < 0.01 and ****P < 0.0001 as compared with LNPs formulated with PEG-lipid only.

Fig. 7. Targeted in vivo genome editing in LSECs by mannose-LNPs (mCre).

Ionizable lipid candidates were formulated with mCre. mCre-loaded LNPs were injected to LSL-tdTomato mice at mRNA dose of 0.5 mg/kg. After 2 days of injection, tdTomato fluorescence was analyzed. (A) Ex vivo organ image showed that LNPs with mannose were mostly uptaken into the liver. (B) Liver histology image showed significant tdTomato fluorescence along the liver vessel. α-SMA, α–smooth muscle actin. (C) Transfection efficiency of mannose-LNPs was evaluated among hepatocytes, LSECs, and Kupffer cells by histological analysis (n = 5).

Fig. 8. Selective in vivo gene silencing in LSECs using mannose-LNPs.

C57BL/6 mice were injected with siFVII (0.2 mg/kg) or siFVIII (0.5 mg/kg; n = 3 to 5). (A and C) Formulation details. (B) Incorporation of mannose to LNPs with 3.0% of PEG-lipid increased the delivery of siRNA to LSECs but incorporation of galactose did not. (D) Incorporation of mannose to LNPs with 1.5 and 3.0% PEG-lipid reduced the delivery of siRNA to hepatocytes, whereas incorporation of galactose to LNPs with 3.0% PEG-lipid did not. (****P < 0.0001 as compared between sugar moiety–incorporated LNPs and LNPs with PEG-lipid only; ns: P > 0.05).