Immunomodulatory Therapy of Inflammatory Liver Disease Using

Aug 22, 2017 - inflammation depending on the underlying immunopathology. The nonsulfated random ... candidate for the treatment of inflammatory diseas...
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Immunomodulatory Therapy of Inflammatory Liver Disease Using Selectin-Binding Glycopolymers Matthias Bartneck,†,∥ Christopher Thorsten Schlößer,†,∥ Matthias Barz,‡ Rudolf Zentel,‡ Christian Trautwein,† Twan Lammers,§ and Frank Tacke*,† †

Department of Medicine III and §Department of Experimental Molecular Imaging, Helmholtz Institute for Biomedical Engineering, RWTH-University-Hospital Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany ‡ Institute for Organic Chemistry, Johannes Gutenberg-University Mainz, 55122 Mainz, Germany

ABSTRACT: Immunotherapies have the potential to significantly advance treatment of inflammatory disease and cancer, which are in large part driven by immune cells. Selectins control the first step in immune cell adhesion and extravasation, thereby guiding leukocyte trafficking to tissue lesions. We analyzed four different highly specific selectin-binding glycopolymers, based on linear poly(2-hydroxypropyl)-methacrylamide (PHPMA) polymers. These glycopolymers contain either the tetrasaccharide sialyl-LewisX (SLeX) or the individual carbohydrates fucose, galactose, and sialic acids mimicking the complex SLeX binding motive. The glycopolymers strongly bind to primary human macrophages, without activating them, and also to primary human blood leukocytes, but poorly to fibroblasts and endothelial cells in vitro. After intravenous injection in mice, all glycopolymers accumulated in the liver without causing hepatotoxicity. The glycosylated binder most potently targeted resident hepatic macrophages (Kupffer cells) and protected mice from acute toxic liver injury in the two different experimental models, carbon tetrachloride (CCl4) or Concanavalin A (ConA)-based hepatitis. Its sulfated counterpart, on the other hand, induced a decrease in infiltrating and resident macrophages, increased T helper cells, and aggravated immune-mediated liver injury. We demonstrate that, in the context of selectin-binding glycopolymers, minor modifications strongly impact leukocyte influx and macrophage activation, thereby ameliorating or aggravating liver inflammation depending on the underlying immunopathology. The nonsulfated random glycopolymer is a promising candidate for the treatment of inflammatory disease. The modulation of hepatic immune cells by selectin-binding glycopolymers might breach the immunosuppressive hepatic microenvironment and could improve efficacy of immunotherapies for inflammatory disease and cancer. KEYWORDS: macrophages, selectin-binding glycopolymers, acute liver injury, carbon tetrachloride, concanavalin A, immunotherapy

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selectins are among the initial adhesion molecules, which mediate the transendothelial migration of leukocytes. Selectinmediated adhesion of neutrophils, monocytes, or lymphocytes not only contributes to atherosclerosis1,3 but also promotes

electins are essential cell adhesion molecules mediating trafficking of leukocytes in immune surveillance and inflammation. They are transmembrane, calcium-dependent lectins that mediate leukocyte rolling on vascular surfaces, which is the first adhesive step during inflammation and immune surveillance.1 The naming of the selectins is based on the original discovery of their cellular expression, with L standing for leukocytes, E for endothelium, and P for platelets.2 During the course of interactions between the endothelium and leukocytes, © 2017 American Chemical Society

Received: July 3, 2017 Accepted: August 22, 2017 Published: August 22, 2017 9689

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Figure 1. Selectin-binding glycopolymers in liver homeostasis and inflammation. Selectins and their ligands are expressed by leukocytes and endothelial cells in the liver. Upon inflammation, selectins or their ligands rapidly flip from the intracellular compartment to the outer part of the cell membrane, a process, which is illustrated by opaque intracellular receptors. In the liver, inflammatory processes furthermore lead to an activation of hepatic stellate cells, which become myofibroblasts, and of macrophages, which acquire pro-inflammatory activation, leading to the release of inflammatory mediators such as the tumor necrosis factor (TNF). Four different selectin-binding glycopolymers (termed P1−P4) were analyzed in this study, and their selectin binding affinities are schematically illustrated (A). Chemical structures of the selectin binders (B).

selectin, and ligands for L-selectin, to the cellular surface, which promotes monocyte adhesion and allows for further chemokinedirected tissue invasion. Following their migration to inflamed sites, monocyte-derived macrophages can release pro-inflamma-

recruitment of inflammatory cells into injured parenchymal organs like the liver (Figure 1A).4 As an example, circulating monocytes constitutively express L-selectin and the ligands for Eand P-selectin.2 Upon activation, endothelial cells flip P- and E9690

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ACS Nano Table 1. Composition and Characterization of the Selectin-Binding Glycopolymersa PHPMA carbohydrate type and content [% of repeating units]

tyramine sulfate content (% of repeating units) DP and Mn (kg/mol) rh (nm, PBS buffer at c = 1g/L( ζ-potential (mV, PBS buffer at c = 0.25g/L)

P1

P2

−

− 100 14.6 4.0 −2 ± 3

SLeX10

SLeX10

− 100 24.1 5.1 −16 ± 5

10 100 25.4 5.4 −18 ± 4

P3

P4

Gal 10 Fuc 10 Neu 10 − 100 24.8 5.4 −29 ± 5

Gal 10 Fuc 10 Neu 10 10 100 26.1 6.0 −26 ± 5

a

Binders are multivalent SLeX (P1), or additionally sulfated (P2), and those decorated with randomly linked carbohydrates (P3), or with additional sulfation (P4).

Figure 2. Binding of the selectin-binding glycopolymers to different human primary cell types. The glycopolymers are multivalent SLeX (P1), additionally sulfated (P2), and those decorated with randomly linked carbohydrates (P3), with additional sulfation (P4). Incubation was performed for 60 min at 37 °C using RPMI1640 medium with 5% fetal calf serum (FCS). Binding was assessed by flow cytometry for the fluorescent binders signal, with untreated cells serving as controls. Data represent mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 (one-way ANOVA).

heart, and skin inflammation, atherosclerosis, and cancer.6 For instance, selectin-blocking agents are already in clinical trials to treat chronic obstructive pulmonary disease (COPD)7 and sickle cell disease.8 The therapeutic potential of polymer-based selectin binders for the treatment of liver diseases, however, has yet to be

tory factors such as interleukin 1β (IL1β) and IL6 or differentiate into restorative macrophages that promote resolution of inflammation (for example, by producing IL10) (Figure 1A).5 Targeting selectins has been proposed for a wide variety of inflammatory disorders including post-ischemic, brain, lung, 9691

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Figure 3. Changes in inflammatory gene expression induced by the selectin-binding glycopolymers in human primary macrophages. Polymerbased selectin binders based on multivalent SLex (P1), additionally sulfated (P2), and those decorated with randomly linked carbohydrates (P3), with additional sulfation (P4). Macrophages were generated by 7 days of culture of monocytes isolated from human blood in RPMI1640 medium with 5% autologous serum. Incubation with binders was done 1 h before treatment with lipopolysaccharides (LPS) for 24 h. Data represent mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed t tests).

evaluated. In experimental models of hepatic ischemiareperfusion and hemorrhagic shock models, selectin inhibition has demonstrated efficacy in improving liver pathology.9,10 It is known that the migration and activation of the different leukocyte subsets can aggravate or ameliorate toxic or autoimmune liver damage,4 but the exact therapeutic capabilities of selective selectin binders are unclear. Nanotechnology offers perspectives for combatting liver disease based on immunomodulation,11,12 and high concentrations of nanomedicines typically accumulate in the liver upon systemic application,12,13 making targeted treatment with immunomodulatory nanotherapeutics attractive. We recently reported the development of polymeric nanomedicines that bind to selectins (Figure 1B),14 based on an activated ester approach established by Barz and colleagues.15−17 These polymers prompted us to test their capacity to target monocytes/macrophages in the liver and their therapeutic potential in liver injury. Here, we assessed the binding capabilities to human primary blood cells, macrophages, fibroblasts, and endothelial cells and the potential pharmacological effects of four

selected poly(2-hydroxypropyl)methacrylamide (PHPMA)coupled selectin binders on macrophage activation in vitro.14 We further analyzed the in vivo biodistribution of the four selectin binders at the level of organs and cells. Finally, we studied the effects of these immunomodulatory nanomedicines in two mechanistically different models of liver injury and related the course of liver disease progression to immune cell activation.

RESULTS AND DISCUSSION Nanomedicines may allow therapeutic approaches for the treatment of liver diseases, including inflammation, fibrosis or cancer.5,18,19 While cell-specific targeting of drug delivery systems has shown remarkable progress in the field,20 pharmacologically active polymers or polymeric drugs binder with concomitant immunomodulatory properties could impact hepatic inflammation based on cell binding only. Given the relevance of selectins for inflammatory cell recruitment in liver injury,4 we comprehensively explored immunomodulatory effects of recently developed selectin-binding polymers14 in the context of healthy and injured liver. 9692

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Figure 4. Effects of selectin-binding glycopolymers on liver in homeostasis. The glycopolymers used were multivalent SLeX (P1), additionally sulfated (P2), and binders decorated with randomly linked carbohydrates (P3), with additional sulfation (P4). Immunomodulatory polymers were injected intravenously at a concentration of 7.5 mg/kg body weight into 8 weeks old C57BL/6J wild type mice. Animals were sacrificed 24 h after the injection of the binders. Liver histology (A), alanine aminotransferase activity (ALT, B), and serum levels of cytokines (C) were analyzed.

the PHPMA backbone, which leads to ζ-potentials of −29 ± 5 mV and 26 ± 5 mV, respectively. To delineate the specific binding capacities of the selectin binding polymers to different cell types, we incubated six types of primary cells with the nanosized binders and analyzed binding using flow cytometry. We observed a low binding to fibroblasts and endothelial cells and noted accelerated binding to lymphocytes and granulocytes. The strongest interactions were observed with monocytes and macrophages, as evidenced by strong peak shifts in flow cytometry, compared to a low binding to fibroblasts and endothelial cells (Figure 2A). Incubation at 4 °C had no impact on the strength of the fluorescent signal induced by the binders. These data indicate that the nanoparticles bind to the cellular surfaces, since an active uptake mediated by endocytosis is energy dependent and would have been strongly reduced by incubation at 4 °C (Figure 2B), which occurs for example with 50 nm sizing gold nanospheres.11 Synthetic glycopolymers were shown to regulate cellular differentiation of embryonic stem cells based on mimicking glycosaminoglycans (GAG) which bind to the fibroblast growth factor 2. These authors demonstrated that a lipid anchor leads to membrane insertion, whereas a GAG mimicking polymer was internalized by cells.20 Similarly, a terpolymer of sodium 4vinylbenzenesulfonate, 2-methacrylamido glucopyranose, and fluorescein o-methacrylate was bound to the cell membrane when coupled to the lipid 1,2-dipalmytol-sn-glycero-3-phosphoethanolamine, but the terpolymer alone was internalized by cells.21 To study possible effects of the selectin-binding polymers on immune cell activation mediated by binding to the selectin

Natural ligands of selectins within the inflammatory cascade are the glycoproteins E-selectin ligand-1 (ESL-1, E-selectin dissociation constant: 62 μM) and P-selectin glycoprotein ligand-1 (PSGL-1, P-selectin dissociation constant: 0.3 μM). In these glycoproteins, the Sialyl LewisX tetrasaccharide (SLeX) is the common structural binding motive.1,2 The binding affinity, however, is modulated by O-sulfation of tyrosine residues at the N-terminal binding site of PSGL-1. The tetrasacchride SLeX itself has rather low binding affinities to all members of the selectin family (IC50 = 0.6−1 mM). In this regard, we recently established selectin-binding PHPMA-based glycopolymers, which contain either SLeX or the individual carbohydrates fucopyranose, galactose, and neuramic acid, which are known to be the ones contributing to the interaction of SLex with selectins. The synthesis of glycopolymers P1 to P4 and the PHPMA control polymer PHPMA was carried out according to Moog et al.14 The characteristics of the polymers are outlined in Table 1. With a view toward systemic application, we also investigated the hydrodynamic radii (rh) and the zeta (ζ)-potential of all polymers in phosphate-buffered saline (PBS) at pH 7.4. The rh values were found to be 4.0 nm for PHPMA, 5.1 and 5.4 nm for the nonsulfonated polymers P1 and P2 as well as 5.4 and 6.0 nm for the sulfonated glycopolymers P3 and P4 (see Table 1). In line with these findings the ζ potential increases from −2 ± 3 mV for the PHPMA to −16 ± 5 mV and −18 ± 4 mV for P1 and P2, which can be attributed to the incorporation of 10% Nacetylneuraminic acid or SLeX, which both contain a carboxylic acid group with a pKa of around 2.5. The glycopolymers P3 and P4 additionally contain 10% tyramine-O-sulfate groups among 9693

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Figure 5. Organ distribution of the selectin-binding glycopolymers. Binders were injected intravenously into 8 week old C57BL/6J wild-type mice at the dose of 7.5 mg/kg body weight, and mice were sacrificed after 24 h. Organs were homogenized and digested using type IV collagenase. Flow cytometric analysis of organ distribution was done based on a quantification of the binders: multivalent SLeX (P1), additionally sulfated (P2), and binders decorated with randomly linked carbohydrates (P3), with additional sulfation (P4) in single cell suspensions of different organs. Representative dot plots and histograms of binders decorated with randomly linked carbohydrates (P3) compared to an untreated control (A), and a quantification of the mean fluorescent intensity of all binders P1−P4 (B). Data represent mean ± SD; *P < 0.05, ***P < 0.001 (one-way ANOVA).

HPMA copolymers with saccharide moieties have been used for a huge number of biomedical applications. For instance, labeling of a hepatic cancer cell line was shown to be increased by trivalent galactoside and lactoside-containing copolymers.22 Bojarova and colleagues discovered that chitooligosaccharide chains composed of one to five N-acetyl-D-glucosamine (GlcNAc) are highly efficient ligands for lectin.23 We have reported earlier that nanotherapeutics including polymers accumulate in the liver upon systemic administration.13,24,25 To study putative consequences of the binders on the liver in vivo, we analyzed liver histology, alanine aminotransferase (ALT), a serum indicator for liver injury, and inflammatory cytokines 24 h after i.v. injection of particles in wild-type mice. Liver histology was unaffected by all binders, and only the nonsulfated binder with random carbohydrates (P3) resulted in a slight (but nonsignificant) increase in serum ALT levels (Figure 4A,B). However, we observed divergent effects of the different selectin binders on inflammatory mediators. The two polymeric binders displaying multivalent SLeX (P1 and P2) induced systemic IL6 levels, sulfated random carbohydrates (P4) induced IL1β, and

receptors, we incubated human primary macrophages for 60 min with the binders at three different concentrations (0.03, 0.3, or 3 μM), with or without additional stimulation by lipopolysaccharides (LPS) for another 24 h. We observed that the binders had no effects on macrophage activation at the two lowest concentrations. However, at the highest concentration of 3 μM, multivalent SLeX (P1) induced low levels of IL1β expression, while all binders slightly increased TNF. Multivalent SLeX with O-sulfated tyramine (P2) and randomly linked carbohydrates (P3) increased TNF expression further, possibly based on synergistic effects evoked by the binders and LPS. The control polymer PHPMA and P1 led to IL10 up-regulation. The expression of CCL2 was significantly increased by multivalent SLeX with O-sulfated tyramine (P2) and LPS; CCL2 was also increased by both randomly linked carbohydrates, with and without O-sulfated tyramine (P3, P4) (Figure 3A−C). Thus, despite effective binding of the selectin-binding glycopolymers to macrophages, they only had modest activating effects on the cells, which was limited to the highest concentration. 9694

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Figure 6. Effects of selectin-binding glycopolymers on liver cells. Polymer-based selectin binders functionalized with multivalent SLeX (P1), additionally sulfated (P2), or decorated with randomly linked carbohydrates (P3), with additional sulfation (P4). Selectin binders were injected intravenously into 8 week old C57BL/6J wild-type mice at the dose of 7.5 mg/kg body weight, and mice were sacrificed after 24 h. Flow cytometric analysis of liver leukocytes was done based on combined analysis of antibodies and of the signal of the binders. Representative flow cytometric plots and statistical summary are given for the effects of binders on hepatic macrophages (A) as well as binders binding to these cell populations (B). Similar analyses are provided for hepatic CD4 T cells (C) and B cells (D). Abbreviations: KC, Kupffer cells; MoMF: monocytederived macrophages. Data represent mean ± SD; *P < 0.05, **P < 0.01 (two-tailed t tests).

fluorescent intensity (signal strength) confirmed the significant accumulation of P3 in the liver (Figure 5B). We next performed multicolor flow cytometry combined with the fluorescence of the binders to study their cellular localization in the liver. Notably, we found that the sulfated binder with random carbohydrate (P4) had modest immunomodulatory effects, because its administration slightly reduced the numbers of hepatic macrophages such as monocytic macrophages (MoMF) and Kupffer cells (KC) (Figure 6A). In addition to the effects on cell numbers, the

the random carbohydrate decoration of polymers (P3) evoked IL10 expression (Figure 4C). The distribution of the selectin-binding polymers in organs and their respective cellular compartments is a key factor to assess their immunomodulatory effects in vivo. Animals were studied 24 h after i.v. injection in wild-type mice using flow cytometry, and specifically the binders with random carbohydrates (P3) accumulated in liver, which becomes clear from dot plots and histrograms (Figure 5A). Quantifications of the mean 9695

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Figure 7. Effects of selectin-binding glycopolymers on liver injury. Binders are multivalent SLeX (P1) or additionally sulfated (P2), and those decorated with randomly linked carbohydrates (P3) or with additional sulfation (P4). Selectin-binding glycopolymers were administered at a concentration of 7.5 mg/kg body weight. After 40 h, either Con A was administered at 15 mg/kg body weight, and mice were sacrificed after another 8 h (A−C), or carbon tetrachloride (CCl4) was administered after 24 h, and animals were sacrificed after another 24 h (D−F). Liver histology, liver injury (ALT), and serum cytokine levels are given. Data represent mean ± SD; *P < 0.05, **P < 0.01 (two-tailed t tests).

binders possessed different binding capabilities to specific cell types. All binders except the sulfated polymer with random sugars (P4) efficiently bound to the MoMF, whereas all of the particles well targeted KC. Especially the glycopolymer without O-sulfate tyramine (P3) induced a strong binding-associated signal and bound to the vast majority of the liver resident KC, shown as a representative flow cytometric plot (Figure 6B). In addition to the interactions with the myeloid MoMF and KC, there were interactions with lymphocytes, which belong to the

adaptive immune system. Interestingly, the sulfated selectinbinding polymer based on randomly attached fucose, galactose, and sialic acids (P4) further led to an increase in CD4+ T helper cells, which function in coordinating adaptive immune responses (Figure 6C). The number of B cells was not affected by the same type of binder (P4), correlating with its low binding efficiency (Figure 6D). Thus, despite minor immune-activating effects in vitro, selectin binders exhibited distinct and differential immunomodulatory effects in vivo, with the sulfated selectin9696

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Despite their inertness in healthy liver, the selectin-binding polymers exhibited very specific effects in mouse models of liver injury, which can be attributed to selective actions on different immune cells. Multivalent SLeX presenting PHPMA (P1) is comparatively inert and has hardly shown any effects on the course of liver injury. The sulfated variants SLeX (P2) and random carbohydrates (P4) aggravated liver injury in two independent models. While the liver injury induced by P2 might originate from macrophage activation, the mechanism underlying P4 is more likely to be independent of macrophages, but instead due to the cytokine profile and the reduced macrophage numbers in livers of P4-injected animals. The P2-associated liver injury in both models might be aggravated by the larger number of T helper cells evoked by the compound. This increased liver damage by P2 and P4, which correlates with increased numbers of T helper cells, relates to data of other groups which have shown that T helper cells can induce liver injury upon their activation.29 In summary, the selectin-binding polymers (Figure 1, Table 1) exert their actions by differential binding to selectin-regulated receptors on different cell types, namely demonstrated low binding to non-immune cells such as fibroblasts and endothelial cells, with major effects on human primary immune cells in vitro (Figure 2). Direct effects on macrophage activation mediated by a potential activation of macrophages could be excluded, either as a single substance or in synergy with LPS since there was no induction of pro-inflammatory macrophage cytokines at clinically relevant doses of binders (Figure 3). In healthy liver, the selectin-binding glycopolymers did not trigger liver injury (Figure 4), which underlines the safety of these nanopharmaceuticals. Our data further demonstrate that chemical modifications allow the binders to accumulate in liver, as shown for the construct equipped with randomly linked carbohydrates (P3) (Figure 5). Interestingly, P3 strongly binds to resident liver macrophages in vivo, and its sulfated counterpart (P4) exhibits strikingly different effects through slight chemical modifications, leading to a reduction in hepatic macrophages (Figure 6). Liver disease models further support feasibility for using P3 as a therapeutics to combat liver disease, exclusively based on mimicking selectins (Figure 7). Corroborating our findings, other groups have used E-selectintargeted nanomimetics and have also discovered it as a useful target to reduce tissue injury.30 Studies of other groups have shown that targeting P selectin has very low pharmacological effects.7 Most interestingly, the nonsulfated random-glycobinder P3 significantly inhibited toxic liver injury and reduced the injury of Con A-induced immune-mediated hepatitis. Thus, it apparently represents the most interesting compound with respect to a potential candidate for treating liver diseases. The exact mechanism of protection is not known, but we hypothesize that it might be related to its strong binding to KC. This could potentially favor tissue-restoring activities of KC, which are related to phagocytosis or matrix remodeling.31

binding polymer (P4) having the strongest impact on the composition of immune cell populations in the liver (Figure 6). Clinical studies at phase II on pan-selectin inhibitors such as rivipansel (GMI-1070), which reduces the stickiness of blood cells, have generated promising data in patients suffering from vaso-occlusive crisis.26 We therefore investigated the complex interplay of selectin inhibition and leukocyte activation using selectin-binding glycopolymers. Considering anti-inflammatory treatment, the most interesting binding activities were identified for the polymer with randomly attached carbohydrates (P3), which binds to macrophages, but does not affect their migration. Interestingly, its sulfated variant (P4) bound to hepatic macrophages with a low intensity, but significantly reduced their numbers in the liver. The inhibition of hepatic macrophage numbers by P4 is in well agreement with earlier studies of our group, in which we demonstrated an inhibition of the migration of human primary macrophages by P4 in vitro.14 We hypothesized that the immunomodulatory potential of the selectin binders could be relevant for the regulation of inflammatory responses in the liver, which might block or accelerate liver disease progression. In order to address the functions of the binders in liver disease, we analyzed two different mouse models for acute liver injury. Both experimental models are based on single injections of toxins in mice: the immune cellmediated model of Concanavalin A (Con A) damage leads to both T cell and monocyte-dependent liver injury,12 and the model for acute toxic liver injury based on injection with CCl4 results in hepatocyte cell death and subsequent infiltration of immune cells.12 Both types of models are highly relevant, but reflect different (clinical) scenarios of hepatic inflammation. In case of Con A, immune cells become activated and attack hepatocytes (thereby modeling autoimmune hepatitis and immune-mediated forms of drug-induced liver injury),27 whereas the CCl4 model induces immune cell recruitment activation as an indirect consequence of liver injury (thereby modeling pathogen or alcohol-induced toxicity).28 These prototypic models reveal important insight into the hepatotoxicity of drug delivery systems or the efficacy of anti-inflammatory compounds.12,13 For instance, liposomal delivery of the anti-inflammatory drug dexamethasone is efficient to reduce liver injury in both models.13 Inhibiting immune cell activation by selectin binders would first be an attractive therapeutic alternative to ameliorate liver inflammation based on a binding site. Second, the binders allow to generate synergistic nanomedicines with additive therapeutic efficacy, when combined with encapsulated antiinflammatory drugs. In the immune-driven Concanavalin A model, the sulfated polymer presenting multivalent SLeX (P2) and the sulfated random sugar binder (P4) increased liver injury, based on liver histology and alanine aminotransferase (ALT; an indicator for liver injury) (Figure 7A,B), although with a relatively high variability between mice, which is typical for strong biological reactions. Accordingly, P2 led to increased IL1β levels, and P2 and P4 to increased IL6 levels in serum (Figure 7C); cytokines that are typically generated by activated macrophages. In the toxic model of CCl4 injury, liver damage is not driven by immune cells per se, but they become activated in response to hepatocyte cell death and modulate subsequent inflammation or recovery.17 Strikingly, P2 still increased liver injury, while P3 protected from CCl4-mediated hepatotoxicity (Figure 7D,E). The increased injury reflected by ALT mediated by P2 was confirmed using liver histology, whereas IL6 and IL10 were unaffected (Figure 7F).

CONCLUSIONS Altogether, our findings provide compelling evidence that carbohydrate-based selectin-binding polymers accumulate in the liver and, by simply binding to the surface of leukocytes, impact inflammatory response after injury. Nonetheless, even small changes determine the efficacy or toxicity of these constructs in vivo. It is tempting to speculate that the antiinflammatory potential of the most promising compound (P3) could be further enhanced by additional modifications, such as 9697

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isolated using Ficoll-based density gradient centrifugation as reported earlier.36 To isolate monocytes for macrophage culture, PBMC were incubated at 37 °C on bacterial grade Petri dishes at a density of 3 × 106 cells per mL in RPMI1640 (Sigma-Aldrich, St. Louis, MO, USA) containing 5% human autologous serum for 35 min in a humidified incubator with 5% CO2. During this period, monocytes become adherent, and lymphocytes were removed with the supernatant. To obtain macrophages, monocytes were cultured for 7 days in RPMI1640 medium supplemented with 5% autologous serum. To detach adherent macrophages from culture plates, the plates were put on ice for 20 min, and the cells were then detached using a rubber-based cell scraper. Serum was recovered from commercial collection tubes (Sarstedt). Normal human dermal fibroblasts and human umbilical vein endothelial cells (Huvec) were obtained from Lonza. Primary murine hepatocytes were isolated based on in situ perfusion with enzymes from the liver of C57BL6/J wild-type mice, as explained earlier in detail.34 Cell Binding Assays and Flow Cytometry. Analysis of binding of the binders to human and murine primary cells was done at 37 °C for 60 min in RPMI1640 cell culture medium supplemented with 5% fetal calf serum (FCS). Flow cytometric analysis of the cells, based on labeling with fluorescently labeled antibodies and fluorescent selectin binders after the in vivo injections, was done as described earlier in detail.12 Liver Enzymes, Histology, and Immunohistochemistry. Alanine aminotransferase (ALT) was determined at 37 °C in serum using the Modular Preanalytics System (Roche, Penzberg, Germany). Hematoxilin and eosin (H&E) section staining was conducted according to established protocols at the interdisciplinary center for clinical research (IZKF) Aachen. Isolation and Flow Cytometric Analysis of Blood and Intrahepatic Leukocytes. Blood was gained from the right ventricle. Cell lysis was done using Pharm Lyse (BD, Franklin Lakes, USA), and the remaining lysis buffer was removed by washing with Hank’s buffered salt solution containing 5 μM ethylenediaminetetraacetic acid and 0.5% bovine serum albumin. Hepatic leukocytes were isolated, and flow cytometry and intracellular flow cytometric analysis were done as described before.12 Serum Cytokine Measurements. Serum cytokines were measured in using ELISA assays (ebioscience), according to the instructions of the manufacturer. Statistical Analysis. Statistical analysis was performed using Graph Pad Prism 5.0 (LaJolla, California, USA). Unpaired t tests or one-way analysis of variance (ANOVA) were performed to test significance of differences between experimental groups. P = 0.05 was considered as statistically significant.

coupling of anti-inflammatory mediators. Since inflammation is regarded as the driving force in liver disease progression toward fibrosis and cirrhosis,4 this approach deserves further evaluation in chronic liver injury models. On the other hand, immunotherapy currently evolves as a treatment option in hepatocellular carcinoma, which often arises in chronically inflamed liver.4 Unfortunately, the objective response rate to treatment with the anti-PD-1 inhibitor nivolumab is as low as 20% of patients with advanced liver cancer.32 In this setting, modulation of hepatic immune cells by activating selectin binders might breach the immunosuppressive hepatic microenvironment and could improve efficacy of immunotherapy. The beneficial pharmacological properties of the simple glycobinders used in our study suggest that further tailoring of their targeting and activating/ suppressing effects on specific immune cells deserves further exploration, especially in the context of liver diseases.

METHODS Selectin-Binding Glycopolymers. The PHPMA-based selectinbinding glycopolymers were generated as described earlier in detail.14 Briefly, the selectin tetrasaccharide sialyl-LewisX (SLeX) is multivalently presented on a biocompatible PHPMA backbone either alone (P1) or in combination with O-sulfated tyramine side chains (P2). In order to compare, corresponding glycopolymers were prepared in which the crucial “single carbohydrate” substructures fucose, galactose, and sialic acid side chains were randomly linked to the PHPMA backbone (P3 or P4 (O-sulfated tyramine)). All polymers have an identical degree of polymerization, since they are derived from the same polymeric precursor, which is labeled with a fluorescent signal (Oregon Green, Thermo Fisher). Binding assays to selectins have shown that PHPMA with SLeX is an efficient binder to E-, L-, and P-selectins.14−17 The binding efficiency for the different binders (P1−P4) is schematically illustrated in Figure 1A, structures are given in Figure 1B, and a summary on the chemical composition is presented in Table 1. Glycopolymer Characterization. Dynamic light scattering (DLS) measurements were performed at 25 °C using a Malvern Zetasizer NanoZS with a 633 nm He/Ne laser at a fixed scattering angle of 173°. The polymer solutions (1 mg/mL in PBS buffer) were filtered for DLS measurements (Whatman Anotop 0.02 μm) and measured in triplicates. The ζ-potential measurements were done at a polymer concentration of 0.25 mg/mL in PBS buffer. Mice. C57BL6/J wild-type mice were housed in a specific pathogenfree environment. All experiments were done with male animals at 8 weeks of age under ethical conditions approved by the appropriate authorities according to German legal requirements. In an earlier in vitro study, we used these binders at a concentration of 3 μM in cell culture with human primary macrophages.14 This concentration resembled a polymer binder concentration of 7.46 mg/kg body weight, based on the assumption that one mouse has a total body blood volume (where polymers can accumulate) of 2.5 mL, a comparative study of human in vitro and mouse in vivo experiments that we have done earlier with gold nanorods11,12,33 and with liposomal dexamethasone.13,34 Liver Injury Models. To study the effects of the selectin-binding glycopolymers in liver injury models, these were adjusted to the desired dose in 0.9% sodium chloride (NaCl) and administered i.v. at a concentration of 7.46 mg/kg body weight into 8 week old C57BL6/J wild-type mice. In order to study biodistribution of the binders, mice were sacrificed after 24 h. To follow up on the effects of the binders in models of immune-driven liver injury, these were injected, followed by Concanavalin A (Con A) administration i.v. 15 mg/kg body weight 40 h later, and animals were sacrificed after another 8 h.35 In order to test the effects in a model for acute toxic liver injury, binders were administered, and carbon tetrachloride (CCl4) (Merck, Darmstadt, Germany) was injected intraperitoneally at a concentration of 0.6 mL/kg body weight (dissolved in corn oil) 24 h later, and animals were sacrificed after another 24 h. Control animals received NaCl instead of nanoparticles. Cell Isolation and Culture. Human primary peripheral blood mononuclear cells (PBMC) were obtained from healthy volunteers and

AUTHOR INFORMATION Corresponding Author

*E-mail: [email protected]. ORCID

Matthias Bartneck: 0000-0003-1516-9610 Rudolf Zentel: 0000-0001-9206-6047 Twan Lammers: 0000-0002-1090-6805 Author Contributions ∥

These authors contributed equally.

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by the German Research Foundation (DFG Ta434/3-1, Ta434/5-1, SFB/TRR57 to F.T., SFB/ TRR57, SFB1066 and ERC StG-309495 to T.L., and SFB1066 to M.Barz and R.Z.), by the Interdisciplinary Center for Clinical Research (IZKF), and by the Wilhelm Sander Stiftung (2015.124.1 to M.Bartneck). 9698

DOI: 10.1021/acsnano.7b04630 ACS Nano 2017, 11, 9689−9700

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ACS Nano

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