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
Graphical abstract
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.
Section snippets
Materials
pH-sensitive cationic lipids, YSK05, and GalNAc3-PEG2k-DSG were synthesized as described previously [22,36,37]. Cholesterol (chol) was purchased from SIGMA Aldrich (St. Louis, MO). 1,2-Dimirystoyl-rac-glycero, methoxyethyleneglycol 2000 ether (mPEG2k -DMG) and 1,2-disrearoyl-rac-glycero, methoxyethyleneglycol 2000 ether (mPEG2k-DSG) were obtained from NOF Corporation (Tokyo, Japan). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) and Ribogreen were purchased from
LNPs that are taken up by the different cellular uptake pathways show different blood half-lives
For a model of LNP that accumulates to the liver via the ApoE/LDLR pathway, an LNP composed of a pH-sensitive cationic lipid, YSK05, cholesterol and mPEG2k-DMG with a molar ratio of 70/30/3 was used (Fig. 1a). Because the YSK05 has an apparent pKa of 6.4, these LNPs are electrostatically near neutral in the blood stream at pH 7.4 [17]. Cholesterol and mPEG2k-DMG was used for stability and size control, respectively. The mPEG2k-DMG, which contains two myristic acid (C14:0)-derived scaffolds, can
Discussion
In the present study, we discovered that the hepatic uptake process of LNPs between ApoE/LDLR and GalNAc/ASGPR pathway have different kinetics. ApoE-mediated binding to hepatic HSPG resulted in a rapid blood clearance followed by cellular uptake after residing in the space of Disse for 10 to 20 min in the former pathway (Fig. 6). This was clearly different from the gradual progress in both blood clearance and cellular uptake that was observed in the latter pathway (Fig. 6).
The blood clearance
Conclusions
In summary, the findings presented herein show that the hepatic uptake process for LNPs between the ApoE/LDLR and GalNAc/ASGPR pathways show different kinetics. ApoE-mediated binding to hepatic HSPG resulted in a rapid blood clearance followed by cellular uptake after the particles resided for 10 to 20 min in the space of Disse in the former pathway, which is clearly different from the gradual progress of both blood clearance and cellular uptake in the latter pathway. Interactions between the
Declaration of Competing Interest
The authors who have taken part in this study declare that they have nothing to disclose regarding funding or conflicts of interest with respect to this manuscript.
Acknowledgements
This work was supported in parts by Japan Society for the Promotion of Science (JSPS) KAKENHI, Japan Grant Numbers JP15K20831 and JP17H05052. The authors also wish to thank Dr. Milton S. Feather for his helpful advice in preparing the English manuscript.
References (62)
- et al.
Neutralization of negative charges of siRNA results in improved safety and efficient gene silencing activity of lipid nanoparticles loaded with high levels of siRNA
J. Control. Release
(2018) - 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) - et al.
Genetic heterogeneity in human disease
Cell
(2010) - et al.
The role of liver sinusoidal cells in hepatocyte-directed gene transfer
Am. J. Pathol.
(2010) - et al.
Novel pH-sensitive multifunctional envelope-type nanodevice for siRNA-based treatments for chronic HBV infection
J. Hepatol.
(2016) - et al.
Targeted delivery of RNAi therapeutics with endogenous and exogenous ligand-based mechanisms
Mol. Ther.
(2010) - et al.
The role of apolipoprotein E in the elimination of liposomes from blood by hepatocytes in the mouse
Biochem. Biophys. Res. Commun.
(2005) - et al.
Remnant lipoprotein metabolism: key pathways involving cell-surface heparan sulfate proteoglycans and apolipoprotein E
J. Lipid Res.
(1999) - et al.
New insights into the heparan sulfate proteoglycan-binding activity of apolipoprotein E
J. Biol. Chem.
(2001) - et al.
Lipid binding-induced conformational changes in the N-terminal domain of human apolipoprotein E
J. Lipid Res.
(1999)
The receptor-binding domain of human apolipoprotein E. Monoclonal antibody inhibition of binding
J. Biol. Chem.
Human apolipoprotein E. determination of the heparin binding sites of apolipoprotein E3
J. Biol. Chem.
Asialoglycoprotein receptor mediated hepatocyte targeting - strategies and applications
J. Control. Release
Human hepatic lectin. Physiochemical properties and specificity
J. Biol. Chem.
Binding and endocytosis of cluster glycosides by rabbit hepatocytes. Evidence for a short-circuit pathway that does not lead to degradation
J. Biol. Chem.
Hepatocyte-targeted RNAi therapeutics for the treatment of chronic hepatitis B virus infection
Mol. Ther.
A pH-sensitive cationic lipid facilitates the delivery of liposomal siRNA and gene silencing activity in vitro and in vivo
J. Control. Release
pH-labile PEGylation of siRNA-loaded lipid nanoparticle improves active targeting and gene silencing activity in hepatocytes
J. Control. Release
Influence of polyethylene glycol lipid desorption rates on pharmacokinetics and pharmacodynamics of siRNA lipid nanoparticles
Mol Ther Nucleic Acids
Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA
J. Control. Release
Elucidation of the physicochemical properties and potency of siRNA-loaded small-sized lipid nanoparticles for siRNA delivery
J. Control. Release
Plasma PCSK9 preferentially reduces liver LDL receptors in mice
J. Lipid Res.
In vivo clearance of ternary complexes of vitronectin-thrombin-antithrombin is mediated by hepatic heparan sulfate proteoglycans
J. Biol. Chem.
Two receptor systems are involved in the plasma clearance of tissue factor pathway inhibitor in vivo
J. Biol. Chem.
Intravenous heparinase inhibits remnant lipoprotein clearance from the plasma and uptake by the liver: in vivo role of heparan sulfate proteoglycans
J. Lipid Res.
Highly specific delivery of siRNA to hepatocytes circumvents endothelial cell-mediated lipid nanoparticle-associated toxicity leading to the safe and efficacious decrease in the hepatitis B virus
J. Control. Release
Binding of arginine-rich (E) apoprotein after recombination with phospholipid vesicles to the low density lipoprotein receptors of fibroblasts
J. Biol. Chem.
Hepatic heparan sulfate proteoglycans and endocytic clearance of triglyceride-rich lipoproteins
Prog. Mol. Biol. Transl. Sci.
Heparan sulfate proteoglycan expression in normal human liver
Hepatology
Next-generation lipids in RNA interference therapeutics
ACS Nano
Principles of nanoparticle design for overcoming biological barriers to drug delivery
Nat. Biotechnol.
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These authors contributed equally to this work