Different kinetics for the hepatic uptake of lipid nanoparticles between the apolipoprotein E/low density lipoprotein receptor and the N-acetyl-d-galactosamine/asialoglycoprotein receptor pathway

Abstract

Lipid nanoparticles (LNPs) are one of the more promising technologies for efficiently delivering nucleic acids in vivo. Hepatocytes are the primary target cells of LNPs that are delivered via the apolipoprotein E (ApoE)-low density lipoprotein receptor (LDLR) pathway, an endogenous targeting pathway. This robust targeting mechanism results in the specific and efficient delivery of nucleic acids to hepatocytes. Trivalent N-acetyl-D-galactosamine (GalNAc) is known to be a high-affinity exogenous ligand against the asialoglycoprotein receptor (ASGPR), which is highly expressed on hepatocytes. In this study, we report that the kinetics of the hepatic uptake process between the two types of targeting pathways are different. Rapid blood clearance, accumulation to the space of Disse and a subsequent slow cellular uptake was observed in the case of the endogenous ApoE-LDLR pathway. On the other hand, both blood clearance and cellular uptake were more gradual in the case of the exogenous GalNAc-ASGPR pathway. Interactions between ApoE-bound LNPs and hepatic heparan sulfate proteoglycans (HSPGs) were involved in the rapid blood clearance and accumulation to the space of Disse in the case of the endogenous pathway. The findings presented here contribute to a more precise understanding of the mechanism of hepatic uptake and to the rational design of hepatocyte-targeting nanoparticles.

Introduction

The accumulation of nucleic acids in target tissues or cells is severely limited due to the following characteristics of these molecules: high molecular weight, negative charges and hydrophilicity [1,2]. Therefore, appropriate systems for delivering nucleic acids are essential for their efficient delivery and therapeutic applications. Lipid nanoparticles (LNPs) are one of the most promising technologies for the delivery of nucleic acids, and, as of this writing, a huge number of different LNPs have been designed [[3], [4], [5], [6], [7], [8], [9]]. Many efforts, including rational design or high throughput screening of potent cationic lipids, have dramatically improved the efficiency of nucleic acid delivery. This resulted in the approval of the first LNP-based short interfering RNA (siRNA) therapy for the treatment of hereditary ATTR amyloidosis in 2018 [10].

The liver is the primary target of LNPs containing nucleic acids because thousands of human diseases are estimated to be caused by genetic disorders originating in hepatocytes [11]. The following unique structural features in liver tissue enable size-controlled LNPs (diameters of 100 nm or less) to reach hepatocytes: 1) the presence of fenestrae which are pores with diameters of 100–150 nm in liver sinusoidal endothelial cells (LSECs), and, 2) a lack of basement membranes between LSECs and hepatocytes [[12], [13], [14]]. In addition, the presence of the apolipoprotein E (ApoE)-low density lipoprotein receptor (LDLR)-mediated endogenous uptake pathway in hepatocytes is a major contributor to the success of LNP-based therapy of liver-associated diseases, including fibrosis [15], hypercholesterolemia [16] and hepatitis [17,18]. It is known that LNPs interact with serum proteins, exchanging components and acquiring a protein-coated interface (i.e. protein corona) upon their intravenous injection [[19], [20], [21]]. It is particularly interesting that ApoE was found to be generally adsorbed on LNPs, leading to an enhanced uptake of such particles by hepatocytes [22,23]. ApoE is typically located on chylomicrons, very low density lipoproteins (VLDLs) and high density lipoproteins, and facilitates the clearance of VLDLs and chylomicron remnants in hepatocytes [24,25]. It is known that ApoE is an endogenous ligand for the LDLR family and heparan sulfate proteoglycans (HSPGs) [26]. The lipid-binding site in the NH₂-terminal domain of ApoE strongly interacts with LNPs through tryptophan residues [27]. The interaction results in a conformational change in ApoE. This results in a high affinity to its receptors, namely, the LDLR family and HSPGs, through an arginine-rich receptor-binding domain [28,29].

In another strategy for targeting hepatocytes, galactose or N-acetyl-D-galactosamine (GalNAc) are used as ligands for the asialoglycoprotein receptor (ASGPR) which is expressed on hepatocytes at high levels [30]. GalNAc is known to have an approximately 50-fold higher affinity for ASGPR compared to galactose [31]. Previous studies have demonstrated that the clustering of GalNAc greatly enhances its affinity (dissociation constant of up to single-digit nM) through the simultaneous occupation of several GalNAc-binding sites of ASGPR [32,33]. Because ASGPR is expressed at high levels (~500,000 ASGPRs per individual hepatocyte), is specific for hepatocytes and the recycling time of the ASGPR is rapid (approximately 15 min), clustered GalNAc has been utilized for delivering nucleic acids to hepatocytes as an exogenous ligand [22,34,35].

Although many previous reports have demonstrated that both targeting mechanisms can be useful, little is known regarding the kinetics of the hepatic uptake process between the two types of targeting pathways. In the present study, we prepared 4 types of LNPs with different surface modifications in an attempt to modify the hepatocyte-targeting pathway. Rapid blood clearance, accumulation in the space of Disse and a subsequent slow cellular uptake was observed in the case of the endogenous ApoE-LDLR pathway. On the other hand, both blood clearance and cellular uptake progressed gradually in the case of the exogenous GalNAc-ASGPR pathway. Interactions between ApoE-bound LNPs and hepatic HSPGs were found to be involved in the rapid blood clearance and accumulation to the space of Disse in the endogenous pathway. These findings contribute to a more precise understanding of the mechanism of the hepatic uptake process and to the rational design of hepatocyte-targeting nanoparticles.