Peptide-mediated targeting of hepatocytes via low density lipoprotein receptor-related protein (LRP)

Abstract

It has previously been reported that a peptide sequence of T7 phage protein p17 mediates uptake of its cargo by liver parenchymal cells. The aim of this study was to identify the phage-binding receptor. The involvement of LRP was confirmed by the observations that phage binding to Hepa 1c1c7 cells was inhibited by the LRP-binding receptor-associated protein, LRP-deficient mouse embryonic fibroblasts bound phage with lower efficiency than their wild-type counterparts, and using mouse models with ablated LRP liver expression. The identification of LRP as a cognate receptor for this sequence offers a new ligand-receptor combination for hepatocyte delivery of therapeutic agents.

Keywords::

• Hepatocyte targeting
• LRP
• T7 Phage

Abbreviations:
ASGPr,	=	asialoglycoprotein receptor
GPr,	=	galactose particle receptor
HSPG,	=	heparan sulfate proteoglycans
Lf,	=	lactoferrin; LDL, low density lipoprotein
LDLr,	=	low density lipoprotein receptor
LRP,	=	LDLr-related protein
MEF,	=	mouse embryonic fibroblasts

Introduction

We have previously reported that a peptide sequence present in the T7 phage tail fiber protein (p17) efficiently targets phage particles and p17 derivatives to hepatocytes in vivo. Up to 95% of injected phage is taken up by liver, with ∼ up to 80% of the phage being associated with hepatocytes. The rest of the liver-sequestered phage is mostly associated with Kupffer cells (Sokoloff et al., 2003; Ludtke et al., 2007). The phage reaches hepatocytes within 5 min after injection. Preinjection of asialofetuin has no effect on phage uptake, suggesting that the uptake is not mediated by asialoglycoprotein receptor (ASGPr) (Sokoloff et al., 2003). Immunohistochemistry studies have shown that a large portion of hepatocyte-associated phage is internalized (Sokoloff et al., 2003; Ludtke et al., 2007). A synthetic analogue of the p17 sequence imparts hepatocyte targeting onto fusion proteins, polymers, siRNA, liposomes, and DNA particles (Wong et al., 2006). While these observations suggest that the p17 sequence can potentially be used as a hepatocyte targeting ligand in various delivery applications, the mechanism of the p17 interaction with hepatocytes has so far remained unknown. The study identifies LRP as a major hepatocyte receptor that mediates phage uptake.

Materials and methods

In vitro phage binding assay

Hepa 1c1c7 cells were obtained from ATCC (cat. # CRL-2026). Wild-type and LRP-deficient MEF were obtained from ATCC (cat. # CRL-2214 and CRL-2216, respectively). Approximately 400,000 cells were plated into  35 mm wells and grown overnight in 10% FCS in DMEM. Cells were washed once with PBS and then fixed with 2% formaldehyde (Sigma, St. Louis, MO) for 30 min at RT and washed three times with 2 ml portions of PBS. Cell treatments were conducted as described in the figure legends. 10⁹ pfu of phage was applied to the cells in 1 ml of OptiMEM (Invitrogen) and incubated at 37°C for 30 min. Unbound phage was removed by washing cells three times with 2 ml portions of PBS. Bound phage was eluted with 0.5 ml of 1% SDS in TBS for 10 min at RT. The amount of eluted phage was determined using a plaque assay (Novagen, 2000).

Tail vein injections

10⁹ or 10¹¹ pfu of phage was slowly injected via the tail vein of mice in 250–300 μl of PBS. After 30 min, the liver was perfused with 30–50 ml of PBS containing 1 U/ml heparin through a 24 gage catheter inserted into the inferior vena cava and placed into a 50 ml tube containing 20 ml of T7 lysis buffer (PBS/2% Triton X-100/1M NaCl) (Sokoloff et al., 2003). Livers were homogenized and infective phage content was determined using a plaque assay (Sokoloff et al., 2003).

LRP gene ablation experiments

Adenovirus expressing Cre from the CMV promoter (Ad5CMVCre) was purchased from the Gene Transfer Vector Core at the University of Iowa. Approximately 3 × 10¹¹ Ad5CMVCre particles (∼ 3 × 10⁹ pfu) was injected into the tail vein of mice following a protocol described by Rohlmann et al. (1996). Twenty days later, when the liver LRP expression level was expected to decrease by ∼ 100% (Rohlmann et al., 1998), 10¹¹ pfu of phage was injected. The amount of liver-sequestered phage was determined 30 min later as described above in the section Tail vein injections. Mice lacking LRP in their hepatocytes were produced by crossing LRP-Flox +/+ LDLr −/− mice (Rohlmann et al., 1998) with Cre +/+ mice (B6.Cg-Tg(Alb-cre)21Mgn/J) expressing Cre from the albumin promoter (Jackson Lab; Bar Harbor, ME). F2 progeny and backrosses between F2 and LRP-Flox +/+ LDLr −/− mice were used in all experiments. Genotyping was done following an established protocol (Rohlmann et al., 1998). Phage detection in liver by immunohistochemistry was described previously (Sokoloff et al., 2003). Briefly, frozen liver sections (5–7 μm) were fixed in 2% formaldehyde, blocked with 10% normal donkey serum, and incubated with rabbit anti-T7 antibody (IgG fraction depleted against Hela cells) followed by Cy3-conjugated donkey anti-rabbit antibody. Stained sections were counterstained with To-Pro-3 to visualize nuclei and with Alexa 488-phalloidin to visualize actin filaments near basolateral and bile canalicular membranes. Sections were analyzed under a confocal Zeiss LSM 510 microscope and images were constructed as a flattened projection of a stack of twelve 0.4 μm optical sections.

Statistics

Phage liver and blood contents were compared among groups using 1-way analysis of variance (ANOVA). Group comparisons were adjusted for the phage concentration using 2-way ANOVA. P-values were adjusted for multiple pairwise comparisons using Tukey’s Honest Significant Difference method. A two-sided 5%-level test result was regarded as statistically significant.

Results

Development of a phage binding assay

To facilitate the identification of the P17-binding hepatocyte receptor, an in vitro phage-binding assay was developed based on the observation that the T7 phage 20-6F, which targeted hepatocytes in vivo (Sokoloff et al., 2003), efficiently bound to immortalized mouse hepatocytes, Hepa 1c1c7 (Figure 1). Approximately 2–4% of input phage 20-6F were bound under these conditions. The phage binding was specific since its efficiency was dramatically reduced when HeLa cells were substituted for Hepa 1c1c7 cells or when phage 20-6F was replaced with mutant phage 20-6 that did not target hepatocytes in vivo (Sokoloff et al., 2003). The phage binding efficiency for these two sets of conditions was ∼ 0.1% and ∼ 0.01%, respectively. For direct quantitation of phage binding, Hepa 1c1c7 cells were fixed first with formaldehyde, which did not significantly affect the phage binding activity of these cells while blocking phage internalization. The suppression of phage internalization by incubating cells at 4°C could not be employed as it interfered, for undetermined reasons, with phage-cell interactions (data not shown).

Figure 1.  Phage binding to Hepa 1c1c7 cells and MEF. (A and B) Hepa 1c1c7 cells incubated with liver-targeting phage 20-6F in OptiMEM in the presence or absence of Lf or RAP. (C) Wild-type and LRP-deficient MEF (congenic cell line PEA-13) incubated with liver-targeting phage 20-6F or non-targeting phage 20-6 in OptiMEM. The amount of cell-bound phage was measured by a plaque assay. Duplicate samples were used. Data are shown as mean ± SE.

Phage binding to Hepa 1c1c7 cells and hepatocytes is inhibited by Lf

Phage binding to Hepa 1c1c7 cells was tested in the presence of potential binding competitors that reacted with major hepatocyte receptors (Shayakhmetov et al., 2005). Polymerized BSA, asialofetuin and low density lipoprotein (LDL), which react with the scavenger receptor SR-BI, ASGPr and LDL receptor (LDLr), respectively, had no significant effect on phage binding (data not shown). In contrast, bovine lactoferrin (Lf), which reacts with LRP and heparan sulfate proteoglycans (HSPG) (Herz & Strickland, 2001), considerably inhibited phage binding in a dose-dependent manner (Figure 1A). Bovine Lf reacts with LRP due to the presence of a positively charged cluster of four R residues at the Lf N-terminus, which relates it to the major LRP ligand, ApoE (Huettinger et al., 1988; 1992). Notably, the phage p17 targeting sequence also contains several positively charged residues that are critical for hepatocyte targeting (Sokoloff et al., 2003). Using our newly designed p17 display system, with the p17 targeting sequence replaced with a peptide display site, we have found that all p17-displayed random sequences selected for hepatocyte targeting are also positively charged (Ludtke et al., 2007).

Phage binding to Hepa 1c1c7 cells in vitro is mediated by LRP

To determine whether phage bound to LRP or HSPG or both, Hepa 1c1c7 cells were incubated with phage after being treated with heparinase I or heparinase III. These treatments had no effect, suggesting that HSPG did not contribute to phage binding. The involvement of LRP in the binding process was confirmed by the observations that the LRP-binding protein RAP (receptor-associated protein) strongly inhibited phage binding to Hepa 1c1c7 cells (Figure 1B), and that the LRP-deficient mouse embryonic fibroblasts (MEF; line PEA-13) bound phage far less efficiently than wild type MEF (Figure 1C). Overall, these observations provided strong evidence that LRP served as a phage-binding receptor in vitro. The preinjection of mice with  1 mg of Lf 5 min prior to injection of phage reduced the efficiency of phage uptake by liver from ∼ 72 to ∼ 40%, which suggested that LRP also reacted with phage in vivo. Asialofetuin was injected as a negative control (Sokoloff et al., 2003), and showed no effect on phage uptake (data not shown).

LRP mediates phage binding to hepatocytes in vivo

Phage 20-6F is taken up in vivo by both hepatocytes and Kupffer cells, with up to 80% of liver-sequestered phage being associated with the hepatocyte fraction (Sokoloff et al., 2003). Since LRP is expressed both by hepatocytes and macrophages (Moestrup, Gliemann, & Pallesen, 1992; Lupu et al., 1994), the phage uptake by hepatocytes and Kupffer cells could proceed through the same, LRP-mediated mechanism. The general LRP involvement in phage uptake by liver was demonstrated employing a ‘floxed’ LRP mouse model, in which the LRP gene was removed from liver cells by injection of Cre-expressing adenovirus (Rohlmann et al., 1998). Mice without liver LRP showed ∼ 3-fold reduction (p = 0.0002) in liver phage uptake as determined by a plaque assay (Figure 2A, closed bars). A combination of confocal microscopy and immunohistochemistry (Sokoloff et al., 2003) showed that just a small portion of the phage sequestered by liver in LRP-deficient mice was inside hepatocytes, with the bulk of phage being present in clusters that apparently corresponded to Kupffer cells (Figures 2B and C). The decrease in phage uptake was predictably accompanied by an increase (p = 0.0002) in the proportion of injected phage that remained in the blood (Figure 2A, open bars).

Figure 2.  Reduced phage uptake by LRP-deficient liver in Ad5/Cre-injected mice. (A) Phage contents in liver and blood were determined by a plaque assay. Data are shown as mean ± SD, n = 3. (B) Immunohistochemistry of normal liver after phage injection. (C) Immunohistochemistry of LRP-deficient liver after phage injection. Red, green, and blue colors correspond to phage, actin, and nuclei, respectively.

The role of LRP in phage uptake by hepatocytes was more specifically demonstrated in another ‘floxed’ mouse model lacking LRP, where Cre was specifically expressed in hepatocytes from the albumin promoter. The liver phage uptake in LRP−/LDLr+ mice, as compared to normal mice (LRP+/LDLr+), was reduced by ∼ 50% (Figure 3A; p = 0.002). The reduction was corroborated by immunohistochemistry data (Figures 3B and C). It appeared that LRP+/LDLr+ hepatocytes took up phage somewhat more efficiently than those lacking LDLr (LRP+/LDLr−; Figure 3A). However, the difference did not reach the level of statistical significance (p = 0.06). The role of LDLr was also assessed by comparing the phage uptake by liver in LRP−/LDLr+ and LRP−/LDLr− mice. No statistically significant difference was observed for this combination either (Figure 3A; p = 0.86). Moderate contribution of LDLr to the phage uptake by hepatocytes was detected in immunohistochemical experiments, where the residual phage uptake observed in LRP−/LDLr+ mice declined to nearly undetectable levels in LRP−/LDLr− mice (Figure 3C).

Figure 3.  Reduced phage uptake by LRP-deficient liver in ‘floxed’ LRP mice expressing Cre. (A) Liver phage uptake as determined by a plaque assay. Data are shown as mean ± SD, n = 4. (B) Confocal images of immunostained LRP+LDLr+ liver sections (Flox+/+, Cre−, LDLr+/+). (C) Confocal images of immunostained LRP−LDLr+ liver sections (Flox+/+, Cre+, LDLr+/+). (D) Confocal images of immunostained LRP−LDLr+ liver sections (Flox+/+, Cre+, LDLr−/−). Red, green, and blue image colors correspond to phage, actin, and nuclei, respectively.

Discussion

This study demonstrates that the T7 phage uptake by hepatocytes is largely mediated by an LDLr family member, LRP. LRP binds a broad variety of ligands, including lipoproteins, clotting factors, proteinases, proteinase-inhibitor complexes, intracellular proteins, extracellular matrix proteins, viruses, and bacterial toxins (Herz & Strickland, 2001). Complement-type, cysteine-rich repeats clustered in four of five structural units commonly found in the LDLr family members are largely responsible for the bulk of the LRP binding activity. The majority of ligand-binding sites are located in the LRP clusters II and IV, with most ligands binding equally well to both clusters. Most ligands bind to LRP with high affinity due to their interactions with multiple ligand-binding repeats (Willnow & Herz, 1994; Springer, 1998). The result from recent studies suggests that LRP mediates the liver uptake of adenoviral particles complexed to blood proteins from the coagulation and complement cascades, such as FIX, FX, and complement C4-binding protein (Shayakhmetov et al., 2005; Kalyuzhniy et al., 2008; Waddington et al., 2008).

Detectable amounts of LRP mRNA are observed in spleen, thymus, bone, heart, kidney, muscle, and placenta. The highest levels of LRP mRNA are observed in liver, brain, lung, and intestine (Herz et al., 1988; Moestrup et al., 1992). Detailed immunohistological analysis showed that LRP is expressed in a variety of cell types, including hepatocytes, Kupffer cells, lymph and spleen node macrophages, fibroblasts, smooth muscle cells, monocytes, syncitiotrophoblasts, astrocytes, and several types of epithelial cells (Moestrup et al., 1992; Lupu et al., 1994). However, the LRP-mediated endocytosis of apoE-enriched chylomicron remnants, beta-VLDL, and LRP antibodies occurs largely in liver, where both hepatocytes and Kupffer cells are accessible to small particles due to vasculature fenestration (Kita, Brown, et al., 1982; Kita, Goldstein, et al., 1982; Herz et al., 1990). The predominant occurrence of the LRP internalization activity in liver is consistent with our observations that up to 95% of T7 phage injected into mice is recovered from liver provided that the phage is rendered ‘invisible’ to natural IgM antibodies by removing its coat protein C-terminus (Sokoloff et al., 2000, 2003).

Importantly, LRP expression is not observed in normal endothelial cells despite the presence in these cells of some LRP mRNA (Moestrup et al., 1992; Lupu et al., 1994). Nor is LRP expressed in mature blood cells, dendritic cells, or lymphoid cells (Moestrup et al., 1992). The absence of LRP expression in these cells, which are readily accessible to blood-born LRP-targeting particles and proteins, is an important prerequisite for the therapeutic use of LRP-targeted bulky agents, as it essentially limits the targeting of blood-restricted agents to hepatocytes and macrophages. Macrophages can be differentially and reversibly suppressed, which would enhance the specificity of hepatocyte targeting (Sokoloff et al., 2003; Ludtke et al., 2007; Russell et al., 2007). Also, internalized phage is degraded by Kupffer cells much faster than by hepatocytes (Sokoloff et al., 2003), which suggests that the endosomal release and functional expression in Kupffer cells of genes, oligonuclotides, and siRNA is less likely than in hepatocytes. Due to the LRP expression in many tissues, specific LRP-mediated targeting of liver cells is restricted to particles and proteins that are large enough to avoid significant extravasation (Herz et al., 1990). The contribution of LDLr to the p17 targeting of hepatocytes is rather limited and is likely to be even smaller in normal animals as compared to LRP knock-outs, since the latter over-express LRP by at least 2-fold (Rohlmann et al., 1998). The limited role of LDLr in phage uptake is consistent with our previous observations that only the phage-displayed sequence derived from ApoE, which reacts with both LRP and LDLr, but not the sequence derived from ApoB, which mostly reacts with LDLr, mediates efficient phage internalization by hepatocytes (Ludtke et al., 2007).

Unlike adenoviruses that react with LRP via binding first to LRP-reactive plasma proteins, T7 phage evidently binds to liver cells via direct phage-LRP interactions. Using the same methodology that was successfully employed earlier to identify the blood factors required for adenovirus-LRP interactions (Shayakhmetov et al., 2005; Kalyuzhniy et al., 2008; Waddington et al., 2008), we found that phage which was passed through perfused liver (in the absence of blood) was taken up by liver cells as efficiently as if injected through the tail vein (data not shown). Direct phage-LRP interactions also were suggested by phage binding to cultured cells in OptiMEM (Figure 1).

The LRP delivery route is rather similar in terms of its cell specificity to the ASGPr route. In addition to the ASGPr expressed on hepatocytes, the ASGPr-reacting oligosaccharide ligands typically bind to the galactose particle receptor (GPr) expressed on Kupffer cells (Lehrman, Haltiwanger, & Hill, 1986; Lehrman & Hill, 1986; Sliedregt et al., 1999; Rensen et al., 2001). The uptake ratio between the ASGPr and GPr is defined in a complex manner by the size of a targeting agent and the surface distribution and exposure of Gal ligands. As with the LRP route, the specificity of the ASGPr route is enhanced by suppressing Kupffer cells (Lehrman, Pizzo, et al., 1986; Bijsterbosch & Van Berkel, 1990; Biessen et al., 1994; Kuiper et al., 1994; Sliedregt et al., 1999). The specificity of the ASGPr route also is increased by blocking the expression of ASGPr on activated T cells (Park, Kim, & Cho, 2006).

To summarize, this study demonstrates that the LRP targeting by the protein-incorporated or conjugated synthetic p17 sequence represents a new promising ligand-receptor combination for therapeutic delivery of particulate and proteinaceous agents to hepatocytes. This targeting route is suited particularly well for the hepatocyte delivery of proteinaceous agents that are readily amenable to genetic but not chemical modifications.

Acknowledgements

This study was partially supported by the NIH grant R01DK065090. The authors would like to thank Joachim Herz for the useful discussion of this study. Michael Evans’ help with conducting statistical analysis is greatly appreciated.

Declaration of interest: Dr Jon A. Wolff has a proprietary interest in Mirus Bio Corp. The authors alone are responsible for the content and writing of the paper.