Stabilin-1 is required for the endothelial clearance of small anionic nanoparticles

Clearance of nanoparticles (NPs) after intravenous injection – mainly by the liver – is a critical barrier for the clinical translation of nanomaterials. Physicochemical properties of NPs are known to influence their distribution through cell-specific interactions; however, the molecular mechanisms responsible for liver cellular NP uptake are poorly understood. Liver sinusoidal endothelial cells and Kupffer cells are critical participants in this clearance process. Here we use a zebrafish model for liver-NP interaction to identify the endothelial scavenger receptor Stabilin-1 as a non-redundant receptor for the clearance of small anionic NPs. Furthermore, we show that physiologically, Stabilin-1 is required for the removal of bacterial lipopolysaccharide (LPS/endotoxin) from circulation and that Stabilin-1 cooperates with its homolog Stabilin-2 in the clearance of larger (~100 nm) anionic NPs. Our findings allow optimization of anionic nanomedicine biodistribution and targeting therapies that use Stabilin-1 and -2 for liver endothelium-specific delivery.

sinusoids), recognize and internalize NPs and were long thought to be the only liver cell type responsible for NP clearance in vivo. However, recent studies on the cellular distribution of NPs within the liver have revealed important contributions of Blymphocytes, hepatocytes, hepatic stellate cells and especially liver sinusoidal endothelial cells (LSECs). 4 The relative contribution of KCs and LSECs to the clearance of circulating NPs depends mainly on size. Phagocytosis by KCs is responsible for clearance of particles with a size >500 nm. 5 , 6 These NPs are captured by KCs either directly through endocytic receptors on their surface (such as scavenger and mannose receptors), or indirectly, after binding of serum proteins to the NP surface (i.e. opsonization) and subsequent recognition of the bound proteins by Fc-and complement receptors. 7 Compared to KCs, clearance by the more numerous LSECs (present in an approximately 1:2 ratio) 4 is largely dependent on clathrinmediated endocytosis and is limited to particles with a size <500 nm. 8 Although for LSECs the molecular mechanisms leading to cellular NP uptake are less well understood, it is known that LSECs internalize biological colloidssuch as viral particles, 9 bacterial lipopolysaccharide (LPS), 10 , 11 oxidized lipoprotein particles 11 , 12 and immune complexes 13 at a similar rate to, or exceeding uptake by KCs.
Analysis and identification of the molecular mechanisms that mediate NP clearance will help in the challenging task of translation of nanomedicines. The analysis of LSEC function is complicated due to the rapid dedifferentiation of LSECs in vitro 14 and relies, consequently, on in vivo animal models. Recently, we have established an in vivo zebrafish model for NP clearance and have identified a cell type homologous to LSECs in the early zebrafish embryo. 15 These cellswhich we named scavenging endothelial cells (SECs), in analogy with previously identified non-mammalian LSEC homologs 16 are not located in the liver (as in mammals). Instead, they line the first embryonic veins and provide blood clearance before the liver vasculature becomes functional. Zebrafish is a convenient model owing to its ease of genetic manipulation and optical transparency in embryonic stages that provides the opportunity to combine genome-editing techniques and non-invasive microscopy imaging in real-time. Furthermore, zebrafish embryos are used to screen and optimize formulations prior to clinical studies. Remarkably, the clearance function of SECs and their gene expression signature in the zebrafish are comparable to those of the mammalian LSECs, as demonstrated by the presence of several endocytic receptors including several scavenger receptors. 17 LSEC-specific scavenger receptors are prominent candidates for mediating NP clearance in this cell type, since they have been reported to interact and endocytose a wide range of ligands including modified lipoprotein particles, 12 bacterial and viral pathogens, 18 and exosomes. 19 One of the LSEC-specific scavenger receptors -Stabilin-2has been found to bind and internalize apoptotic bodies 20 as well as exogenous ligands, such as antisense oligonucleotides 21 and NPs. 22 Indeed, by generating a zebrafish stab2 knockout line, we showed that Stabilin-2 is an important receptor for SEC-mediated clearance of anionic NPs. 15 However, we also observed that some negatively charged NPs were not only dependent on this receptor for clearance, demonstrated by the uptake of NPs in the SECs even in the absence of a functional Stabilin-2.
In this study, we identify a requirement for the stab2 homolog, Stabilin-1 (encoded by stab1 gene), in the removal of small (~6-30 nm) NPs from circulation. These two scavenger receptors have highly similar domain structure, consistent with the binding of Stabilin-1 to most (but not all) Stabilin-2 ligands in vitro, 23 suggestive of functional redundancy. 24 In addition, we generated stab1/stab2 double knockout zebrafish embryos to provide evidence that Stabilin receptors complement each other in NP clearance of larger NPs (~100 nm size), as well as in the removal of bacterial LPS from the circulation. Differential scavenging function between Stabilin-1 and Stabilin-2 suggests size as a determinant for receptor specificity.

NPs
Fluorescent Alexa488-LPS from Salmonella minnessota, PS NPs, and Qdots layered with an organic CdSeS/ZnS and a carboxylic acid as a reactive group (consisting of a monolayer of octylamine-modified poly acrylic acid and a monolayer of poly acrylic acid -PnOAm-co-PAA-copolymer cap) were purchased from Life Technologies (Eugene, US) by Thermo Fisher Scientific and Sigma-Aldrich (The Netherlands). SiNPs were purchased from HiQ-Nano SRL (Arnesano, Italy). FluoHA was prepared through conjugation of hyaluronic acid (100 kDa) with fluorescein isothiocyanate (Isomer I, Sigma-Aldrich The Netherlands) as previously described. 25 Formulation of SCNPs and synthesis and labeling are described in this work in the Supporting Information. PLGA NPs were formulated in house with a microfluidic system. DOPG-liposomes containing 1 mol % DOPE-lissamine rhodamine were formulated with an extrusion system as described previously. 15 Rhodamine-loaded polymersomes 26 on PIB/PEG block copolymers were a kind gift from S. Askes & S. Bonnet (Leiden University, The Netherlands). Atto-647 labeled CCMV-VLPs 27 were a kind gift from R. van der Hee & J. Cornelissen (University of Twente, The Netherlands).

Zebrafish handling and strains
Zebrafish (Danio rerio) were maintained and handled according to the guidelines from the Zebrafish Model Organism Database (http://zfin.org) and in compliance with the directives of the local animal welfare committee of Leiden University. Housing and husbandry recommendations were followed as recommended by Alestrom et al. 28 Fertilization was performed by natural spawning at the beginning of the light period and eggs were raised at 28.5°C in egg water (60 μg/ml Instant Ocean sea salts). The following established strains were used: Tg(mpeg1: mCherry), 29 Tg(flt1 enh :RFP) hu5333 , 30 Tg(flt4 BAC :YFP) hu7135 , 31 stab2 ibl2 , 15 stab1 ibl3 (described in this work).

CRISPR/Cas9 mutagenesis
Cloning-free sgRNA for CRISPR/Cas9 mutagenesis was designed and synthesized as described. 32 , 33 125 pg of sgRNA (Table S2) and 300 pg cas9 mRNA were co-injected into a single-cell wt embryo fish. Primers nucleotide sequences, sgRNA sequence and predicted stab1 and stab2 amino acid sequences in the stab1 ibl3 and stab2 ibl2 were used as reported. 15 Double stab1 ibl3 and stab2 ibl2 mutants were generated by crossing adult homozygous zebrafish stab2 ibl2 and stab ibl3 mutants.

In situ hybridization
Whole-mount ISH was performed as described previously. 34 The sequences for probes generation (stab1, stab2, mrc1 ) were used as reported. 15 Zebrafish i.v. microinjections and imaging NPs formulation were injected into 2-day old zebrafish embryos (52-56 hpf) using a modified microangraphy protocol. 35 One nanoliter volume of NP formulation was calibrated and injected into the duct of Cuvier after embryos were embedded in 0.4% agarose containing 0.01% tricaine as described. 15 We created a small injection space by penetrating the skin with the injection microneedle and gently pulling the needle back, thereby creating a small pyramidal space in which the NPs were injected. Representative embryos were randomly selected according to successful injections and imaged by confocal microscopy after one hour post injection. Confocal z-stacks were captured on a Leica TCS SPE or LEICA TCS SP8 confocal microscope, using a 10× air objective (HCX PL FLUOTAR), a 40× water-immersion objective (HCX APO L), or a 63× oil-immersion objective (HC PL APO CS). In order to compare images between strains, microscopy settings (laser intensity, gain and offset) were identical between stacks and sessions. Whole-embryo images were a compilation of 3-4 overlapping z-stacks. Fiji 36 distribution of ImageJ was used to process and quantify images. At least 6 images were used for quantification.

Imaging quantification
Quantification was performed in the caudal region of the zebrafish, known to contain Stabilin endothelium and that includes the dorsal aorta, the caudal vein, and the caudal hematopoietic tissue and could include macrophages associated with SECs. First, an average intravascular intensity, within the dorsal aorta, was measured within a rectangular area in a single confocal slice that captured the center of the dorsal aorta. This measurement was repeated three times per embryo in independent sites within the dorsal aorta. Next, the maximum intensity value obtained per image was used to adjust the threshold according to the max value measure in the aorta (in circulation), generating a binary image. The strong fluorescence signal observed by accumulated phagocytosed NPs could lead to a misinterpretation of a SEC signal. For that reason and since the aim of this quantification is to compare the contribution of stabilins in the clearance of NPs and not directly the phagocytosis of macrophages, we attempt to remove the signal potentially associated with macrophages by means of size filtering (0.25-20 um) and to use a qualitative approach to refer to macrophages uptake. From the resulting image, a value of 254 was subtracted in order to get values of 0 (no signal) or 1 (fluorescence). The image of interest was multiplied (max × mask) to obtain the mean intensity and the area (%) of the analyzed image. Having these values, the total area with signal (% area × total signal /100) and the total signal (mean signal × total area) were calculated. The median intensity value of the total signal obtained from the mutants was normalized against the wt counterpart. The angle of the dorsal aspect of the dorsal aorta (a straight line) was measured and then concatenated. Images were rotated to orient the DA horizontally within the image and were subsequently cropped.

Statistical analysis
For comparisons between multiple groups, we used Kruskal-Wallis tests followed by two-tailed Dunn's tests with Bonferroni correction using the PMCMR package in R or GraphPad Prism. No statistical methods were used to predetermine sample size, but group sizes were greater than 5 in order for the null distribution of the Kruskal-Wallis statistic to approximate the Χ 2 distribution (with k − 1 degrees of freedom). Graphs show all individual data points and the median. Confocal image stacks (raw data) are available from the corresponding author upon request.

Generation and characterization of stab1 and stab2 double knockout zebrafish
Liver endothelium is characterized by the presence of scavenger receptors strongly expressed on the membrane cell surface. Given that the clearance of some anionic NPs is not exclusively dependent on the scavenging function of Stabilin-2, 15 we hypothesized that one or more other scavenger receptor (s) expressed in LSECs might be involved in the removal of NPs. To identify additional clearance receptors, we first analyzed the RNA expression of all scavenger receptors in LSECs from mouse liver, based on published single-cell RNA sequencing datasets [37][38][39] (Table S1). Although this analysis revealed that Stab2 is the most abundant scavenger receptor expressed in LSECs, the expression of seven other scavenger receptors (Msr1, Scarb1, CD36, Scarf1, Mrc1, CXCl16 and Stab1) was consistently observed. Of these, the mannose receptor (Mrc1) and Stab1 are the most abundant scavenger receptors expressed by LSECs besides Stab2. We previously reported that the zebrafish orthologues of these genes (mrc1a and stab1) are also highly expressed on SECs. 15 Since Stabilin-1 binds similar ligands as Stabilin-2, 23 we further analyzed the role of this receptor in NP clearance.
To this end, we generated a zebrafish stab1 mutant line through CRISPR/Cas mutagenesis (guide RNA sequence in Table S2). In this strain (stab1 ibl3 ), a deletion of one nucleotide causes a frame-shift, leading to a premature stop codon after amino acid 85. The predicted gene product is a truncated protein lacking most conserved domains, including the fasciclin, EGFlike and LINK domains (Figure 1, A). This gene knockout approach allows us to study the biodistribution of NPs in zebrafish embryo and to compare the clearance by SECs in the presence and/or absence of functional Stabilin receptor(s). To study the combined contributions of Stabilin-1 and Stabilin-2, embryos with mutations in both stab1 and stab2 (stab DKO ) were also generated intercrossing stab1 ibl3 and stab2 ibl2 carriers.
Characterization of the generated mutants was performed by whole-mount in situ hybridization (ISH). Using antisense RNA probes we found a strong reduction of stab1 expression in stab1 ibl3 homozygous mutant embryos, consistent with nonsense-mediated decay (NMD) of the mutant RNA, whereas stab2 and mrc1a expression was unaffected, indicating normal SEC differentiation (Figure 1, B). In stab DKO embryos, expression of both stab1 and stab2 was also reduced through NMD. Of note, stab2 expression was found to be slightly increased in stab DKO compared to the signal in stab2 mutant embryos. Importantly, mrc1a expression in stab DKO embryos was maintained, indicating that SEC differentiation occurred even after the combined loss of stab1 and stab2.
Homozygous stab1 zebrafish mutants develop a normal blood and lymphatic vascular system (Figure 1, C) and we did not observe the previously described defects in lymphatic development induced by morpholino oligonucleotide-mediated after stab1 knockdown. 40 Stab1 mutant embryos develop without obvious morphological defects into viable and fertile adults (Figure 1, D) similar to stab2 mutant zebrafish, as well as adult Stab1 knockout mice. Strikingly, although Stab1/2 double knockout mice display reduced viability due to kidney failure, adult stab DKO zebrafish did not show increased mortality or pathology.

Identification of Stabilin-1 function in the clearance of anionic NPs
Previously, we found that two types of NPs were efficiently cleared by SECs even in the absence of stab2 expression. 15 Specifically, these were quantum dots (Qdots) with a negatively charged surface coating and Cowpea chlorotic mottle virus derived virus-like particles (CCMV-VLPs), which are nonenveloped protein capsids with a hexagonal closed packed structure. 27 As most viruses, these VLPs have a negative surface charge. Biodistribution of the Qdots was unchanged in stab2 mutants, while only a small reduction in the clearance of CCMV-VLPs was observed. The clearance of these two particle types, however, is apparently mediated through scavenger receptors, since it was completely inhibited by pre-injection with the general scavenger receptor inhibitor, dextran sulfate. Therefore, we injected fluorescently labeled Qdots and CCMV-VLP i.v. into the Duct of Cuvier of wild-type (wt), stab1, stab2 and stab DKO zebrafish embryos at 56 h post fertilization (hpf) and subsequently imaged their biodistribution with confocal microscopy. The resulting accumulation of NP fluorescence representing SEC-mediated clearance was quantified (see Methods for details) on a cellular level in the caudal region of the zebrafish tail ( Figure 2, A-B). Importantly, this region also contains plasma-exposed macrophages, commonly known to remove NPs from circulation and analogous to the mammalian Kupffer cells (exemplified in Figure S1).
Strikingly, for both Qdots and CCMV-VLPs, we observed a strong reduction in NP clearance in both stab1 mutants and stab DKO embryos indicating a dominant role for Stabilin-1 in the clearance of these NP types (Figure 2, C-F and Figure S2, A-B). NPs that were cleared mainly through Stabilin-1 differ in their composition (inorganic vs. viral capsids) and surface chemistry, and were also chemically distinct from the NPs cleared mainly through Stabilin-2 (which included lipid and polymeric particles). The difference in surface chemistry of all NPs studied suggests a more general mechanism where particle size might be an important factor for receptor specificity. To further strengthen this hypothesis, we next injected chemically distinct (polymeric) small NPs. To this end, fluorescently labeled anionic singlechain polymeric NPs (SCNPs) were synthesized and characterized (see Supporting Information for more details). SCNPs are a polymeric NP type which consists of biodegradable polymer chains that are covalently cross-linked and folded to form a small particle. 41 The resulting NP size is defined by the length of the polymer chain and can be used to obtain small, uniform polymer NPs of 10-20 nm. Injection of~10 nm SCNPs in the zebrafish embryo revealed that clearance of these particles occurred through SECs as expected. Importantly, SCNP clearance was strongly affected in stab1 mutants, similar to Qdots and CCMV-VLPs (Figure 2, G-H and Figure S2, C). These results indicate thatfor negatively charged NPssize, rather than chemical composition, is a predominant factor for receptor specificity.

Stabilin-1 and Stabilin-2 are complementary receptors in the clearance of anionic NPs by SECs
Previous studies on the biodistribution of polymer-and lipidbased NPs including polymersomes, 15 , 26 fibrillar supramolecu-lar polymers, 42 solid polystyrene (PS) NPs and liposomes 15 in zebrafish stab2 mutants revealed a residual uptake of NPs in SECs in the absence of stab2 expression. Common characteristics of these NPs were a negative surface, but these NPs differed in size, shape, composition and rigidity.
To investigate a complementary role of Stabilin-1 to Stabilin-2 in the clearance of NPs~100 nm, we injected negatively charged DOPG-liposomes and polymersomes in stab DKO embryos. Interestingly, the clearance of both NPs in stab DKO embryos was strongly affected, more than that observed in stab2 single mutants. The loss of a functional Stabilin-1 did not affect the biodistribution of these particles compared to wt embryos (Figure 3, A-D and Figure S3, A-B), in striking contrast to the biodistribution of the smaller NPs. This indicates a cooperative role of Stabilin-1 and Stabilin-2 in NP clearance, consistent with their highly similar ligand profile. 23 We next analyze the behavior of more rigid solid NPs. Therefore, we used anionic poly(D,L-lactide-co-glycolide) (PLGA) NPs and spherical silica NPs (siNPs) due to their relevant importance in drug delivery or biomedical imaging agent applications. 43 , 44 Similar to polymersomes and DOPGliposomes, anionic PLGA NPs and siNPs are cleared through SECs in wt zebrafish. Their biodistribution remained unchanged in stab1 mutants, but clearance was strongly affected in stab2 mutants, and even more in stab DKO mutants (Figure 3, E-H and Figure S3, C-D). Interestingly, for the PLGA NPs, the absence of SEC clearance resulted in increased macrophage uptake (indicated by white arrows in Figure 3, E, G and Figure S1). The absence of molecular interactions between NPs for most of the~100 nm NPs and SECs in the stab DKO embryos is an evidence of a combined contribution of Stabilin-1 and Stabilin-2, where Stabilin-2 is the predominant receptor involved in the clearance in this size range. However, this contribution seems to be a more complex process that depends not only on size but also on the particle composition. We observed the behavior for anionic NP type (polystyrene NPs) of different sizes (40-100 nm) ( Figure S4, A). In this case, we first qualitatively confirmed SEC-uptake of these particles through co-injection with fluorescently labeled hyaluronic acid (fluoHA), a Stabilin-2 ligand and a marker for SEC endocytosis ( Figure S4, B-C). SEC clearance is only partly affected in stab DKO mutants, indicating the presence of at least one additional clearance receptor besides Stabilin-1 and Stabilin-2 ( Figure S4, D-G).

Identification of an endogenous Stabilin ligand: bacterial LPS
Clearance of synthetic nanoparticles can only be reflective of physiological mechanisms that are required for the removal of naturally occurring particles in the 10 to 200 nm size range. We therefore sought to identify naturally occurring circulating particles of this dimension as a probable physiologic Stabilin ligand. One such ligand is LPS, which is the main component of the outer membrane of Gram-negative bacteria with the lipid A portion, an anchor in the bacterial cell wall, which provides toxicity and activates immune responses in mammals. 45  single and stab1 ibl3 stab2 ibl2 double mutants at 1-1.5 h post injection (hpi). White arrows indicate apparent NP uptake within plasma-exposed macrophages. Scale bar: 50 μm. Graphs represent intensity of fluorescent NPs in wt and stab mutants. Bar height represents median values, dots are individual data points, and brackets indicate significant values (*P 0.05, **P 0.01, ***P 0.001) based on Kruskal-Wallis tests followed by two-tailed Dunn's tests with Bonferroni correction.
We rationally hypothesized that bacterial endotoxin LPS would be one of this natural ligands for three reasons. First, LPS is highly toxic and must be rapidly eliminated from host circulation, which is performed mainly by LSECs scavenger receptors of unknown identity. 10 , 18 Second, due to its amphiphilic nature, LPS self-assembles into small anionic NPs with a diameter of~50 nm, resembling synthetic NPs. 46 , 47 Third, Stabilin-2 has been shown to bind to Gram-negative bacteriawhich contain LPSin vitro. 48 To investigate whether circulating LPS is indeed cleared by SECs through Stabilin receptors, fluorescently labeled LPS (Alexa488-LPS) from the Gram-negative bacterium Salmonella minnesota 49 was i.v. administered. LPS diluted into a salt suspension is proposed to aggregate into micelles at concentrations above the critical micelle concentration (CMC). 46 , 47 , 50 The LPS concentration injected (500 μg/ml) is above the CMC (~10 μg/ml) 50 even after dilution into the blood of a zebrafish embryo (estimated 30-fold dilution factor, excluding red blood cells, at 2 dpf). 51 Distribution of fluorescent LPS in Tg(mpeg1:mCherry) zebrafish shows no extensive co-localization with labeled macrophages ( Figure S5). This result indicates that phagocytosis by plasmaexposed macrophages does not represent the main clearance route of LPS in the zebrafish embryo, at 56 hpf. Instead, LPS was associated mainly with SECs located in the caudal vein region of wt zebrafish (Figure 4, A, E), confirming their functional homology to mammalian LSECs. 10 Next, we injected LPS in stab2, stab1 and double mutant zebrafish embryos (Figure 4, B-D, F-H). Association of LPS with SECs was maintained in stab2 mutants (Figure 4, B, G). In this case, although a slight decrease in the LPS clearance was observed compared to wt embryos, SEC-uptake was not significantly changed between these two groups (Figure 4, I). Importantly, LPS uptake by SECs was reduced in stab1 knockout and completely abrogated in stab DKO mutants, leading to a strong increased level of LPS in circulation (Figure 4, D, H). This result revealed a cooperative function of Stabilin-1 and Stabilin-2 in LPS uptake and clearance. The loss of LPS uptake in the double knockouts was very similar to that observed after pre-administration of a competitive inhibitor dextran sulfate ( Figure S6), indicating LPS is a common ligand for Stabilin-1 and Stabilin-2, and both receptors are redundantly required for the removal of LPS from the circulation.

Discussion
The understanding of NPs in vivo behavior of their molecular and cellular interactions after i.v. administrations is essential to improve efficacy and pharmacokinetics of nanomaterials. Here, we identify that Stabilin-1, a scavenger receptor expressed in LSECs in mammals, is involved in the clearance of small anionic NPs. Interestingly, while mice lacking both Stab1 and Stab2 revealed a glomerulofibrotic nephropathy secondary to impaired liver clearance of noxious blood factors 52 , 53 leading to strongly reduced viability, we observed that stab DKO adult zebrafish were obtained in mendelian ratios and were phenotypically indistinguishable from single mutant and wt zebrafish. Since the reduced viability observed in Stab1/Stab2 double knockout mice is due mainly to kidney failure, the viability of stab DKO fish potentially reflects the high regenerative capacity of the zebrafish kidney. 54 Through comparison of NP biodistribution in wt and single/ double Stabilin mutants we could quantify the relative contribution of Stabilin receptors to the clearance of specific NPs in zebrafish embryos. The analysis of physicochemical properties (Table S3) and in vivo biodistribution of NPs involved in differential uptake, for both Stabilin receptors, revealed a dependency on size of the particle. Particle size is a critical parameter affecting cellular uptake of nanotherapeutics. 55 Depending on the intended applicationi.e. drug targeting, vaccine delivery or nucleic acid deliveryan optimal size range is desired. 56,57 In addition, improved internalization associated with small nanoparticles (~25-50 nm) has been previously shown. 58,59 Gold nanoparticles of this size range coated with antibodies, for example, display improved endocytosis and regulation of cellular functions. 58 Similarly,~25-50 nm range has been suggested as an optimal size to reach the maximum cellular uptake. 59 For in vivo activity of small particles, NP clearance by the liver is an important factor influencing biodistribution, but so far has not been linked to a specific receptor. The preference between Stabilin-1 and Stabilin-2 could be attributed indirectly to biological factors or to differences in the structural domains. Biologically, although NP protein corona formation in vivo is known to affect the fate of NPs, the different chemistries of the various NPs involved in this study strongly suggest that charge and size are predominant requirements in the interactions between NPs and Stabilin receptors. Structurally, both receptors are initially expressed as a 310 kDa protein, have a very similar domain structure, and are known to share a very common ligand binding profile. 23 So far, it is unknown which structural differences could explain the differential requirement for both receptors to NP clearance.
Besides NPs, the identification of an endogenous ligand of Stabilins, LPS, provides important information in the mechanism of clearance of endotoxins. Mechanistically, LPS is known to be detected through toll-like receptor factor 4 and myeloid differentiation factor 2 (TLR4/Md-2) complex in mammals. 60 Inflammatory responses to LPS have been previously observed in the zebrafish, 5 but the signaling involved in LPS-sensing is not well understood. Since SECs in the zebrafish are functionally homologous to LSECs 15 and LPS is known to be recognized by LSECs in mammals, 10,18 we believe our results contribute to the mechanistic understanding of recognition and clearance of endotoxin LPSespecially at high concentrations above the CMCimportant not only in the identification and study of host-pathogen interactions but also in inflammation and immunity responses.
In conclusion, by using the zebrafish as model that allows genetic analysis and imaging of NP clearance in vivo, we demonstrate that Stabilin-1 is required independently of Stabilin-2 for endothelial clearance of small anionic Qdots, CCMV-VLP, and SCNPs (6-30 nm) from the circulation. Since NPs with very different chemistries are cleared by Stabilin-1, this strongly suggests negative surface charge and size as the predominant factors that determine a requirement for Stabilin-1 in NP clearance. We also show a combined contribution between Stabilin-1 and Stabilin-2 in the clearance of anionic liposomes, polymeric PS and siNPs (~100 nm) and in the removal of LPS. These results reveal a partial redundancy between stab1 and stab2, both important for NPs clearance, and suggest a differential uptake where size is one of the key parameters determining the selective uptake by each receptor. Given size is particularly important in vaccine development, biomedical imaging applications and delivery technologies, improved mechanistic insights into the interactions between size-selected NPs and the liver at the molecular level contribute to the optimization of small nanomaterials and avenues for receptor-specific targeting.

Associated Content
Supporting Information: Tables, figures, methods, characterization of all NPs used and synthesis and formulation of SCNPs.

Author Information
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