Dexamethasone Conjugation to Biodegradable Avidin-Nucleic-Acid

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Dexamethasone Conjugation to Biodegradable Avidin-Nucleic-Acid-NanoAssemblies Promotes Selective Liver Targeting and Improves Therapeutic Efficacy in an Autoimmune Hepatitis Murine Model Martina Bruna Violatto,† Elisabetta Casarin,‡ Laura Talamini,† Luca Russo,† Simone Baldan,§ Camilla Tondello,§ Marie Messmer,∥ Edith Hintermann,∥ Alessandro Rossi,† Alice Passoni,⊥ Renzo Bagnati,⊥ Stefania Biffi,# Chiara Toffanin,† Sara Gimondi,† Stefano Fumagalli,∇ Maria-Grazia De Simoni,∇ Donatella Barisani,¶ Mario Salmona,† Urs Christen,∥ Pietro Invernizzi,■ Paolo Bigini,*,†,● and Margherita Morpurgo*,§,● †

Department of Biochemistry and Molecular Pharmacology, ⊥Department of Environmental Health Sciences, and ∇Department of Neuroscience, Istituto di Ricerche Farmacologiche “Mario Negri” IRCCS, Milano, 20156, Italy ‡ ANANAS nanotech S.r.l., Padova, 35131, Italy § Department of Pharmaceutical and Pharmacological Sciences, University of Padova, Padova, 35131, Italy ∥ Pharmazentrum Frankfurt/ZAFES, Goethe University Hospital Frankfurt, Frankfurt am Main, 60488, Germany # Institute for Maternal and Child Health-IRCCS “Burlo Garofolo″, Trieste, 34137, Italy ¶ Department of Medicine and Surgery, and ■Division of Gastroenterology and Center for Autoimmune Liver Diseases, Department of Medicine and Surgery, University of Milano-Bicocca, Monza, 20900, Italy S Supporting Information *

ABSTRACT: Steroids are the standard therapy for autoimmune hepatitis (AIH) but the long-lasting administration is hampered by severe side effects. Methods to improve the tropism of the drug toward the liver are therefore required. Among them, conjugation to nanoparticles represents one possible strategy. In this study, we exploited the natural liver tropism of Avidin-Nucleic-AcidNano-Assemblies (ANANAS) to carry dexamethasone selectively to the liver in an AIH animal model. An acidlabile biotin-hydrazone linker was developed for reversible dexamethasone loading onto ANANAS. The biodistribution, pharmacokinetics and efficacy of free and ANANAS-linked dexamethasone (ANANAS−Hz−Dex) in healthy and AIH mice were investigated upon intraperitoneal administration. In ANANAS-treated animals, the free drug was detected only in the liver. Super-resolution microscopy showed that nanoparticles segregate inside lysosomes of liver immunocompetent cells, mainly involved in AIH progression. In agreement with these observational results, chronic low-dose treatment with ANANAS−Hz−Dex reduced the expression of liver inflammation markers and, in contrast to the free drug, also the levels of circulating AIH-specific autoantibodies. These data suggest that the ANANAS carrier attenuates AIH-related liver damage without drug accumulation in off-site tissues. The safety and biodegradability of the ANANAS carrier make this formulation a promising tool for the treatment of autoimmune liver disorders. KEYWORDS: avidin-nucleic-acid-nano-assemblies, autoimmune hepatitis, steroid treatment, targeted drug release, pH reversible linker

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utoimmune hepatitis (AIH) is a rare but severe autoimmune liver disease, characterized by progressive hepatic morphological alterations concurrent with © XXXX American Chemical Society

Received: December 21, 2018 Accepted: March 11, 2019

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DOI: 10.1021/acsnano.8b09655 ACS Nano XXXX, XXX, XXX−XXX

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Cite This: ACS Nano XXXX, XXX, XXX−XXX

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ACS Nano increased immunoglobulin G, aminotransferases, and circulating autoantibodies.1,2 Clinical treatments rely on chronic administration of high doses of prednisolone (sometimes associated with an immunosuppressant such as azathioprine). The wide distribution of steroids, which easily pass through biological barriers,3 leads AIH patients to withdraw from therapy because of severe steroid-related complications (osteopenia with vertebral collapse, brittle bones diabetes, hypertension, mood and cognitive disorders and cosmetic changes or obesity).4 Very often, these patients lack any reliable therapy and will require liver transplantation at a later stage of the disease.5−7 A possible solution could be to “concentrate” the pharmacological activity only at the liver, with the 2-fold scope of reducing the overall dose and avoiding off-site side effects. Nowadays, tissue-targeted therapies are pursued through several strategies, from the development/use of small molecules or monoclonal antibodies capable to interfere specifically with disease-related biological functions, to drug delivery systems (e.g., polymer- or antibody-drug conjugates or nanoparticle-(NP)-based systems) engineered to deliver or concentrate the drug at the desired body site. In particular, NPs can carry large drug payloads and, if necessary, their size allows concomitant loading of therapeutic, diagnostic, and targeting moieties. NP-based carriers appear to be highly suitable for liver targeting. Indeed, most NPs display natural tropism for this filter organ,8,9 or can be targeted to it by surface functionalization with liver targeting ligands.10 In particular, the liver resident macrophages (Kupffer Cells-KCs), which are closely involved in inflammatory disease onset and progression, have a strong ability to recognize, ingest, and degrade foreign materials, and can thus capture circulating NPs of a different nature, including hard nanomaterials,8 liposomes,11 and polymeric NPs,12 making these systems the ideal drug carriers for KC-related diseases. Controlled release (nano) formulations for corticosteroids have been described in the literature for different purposes.13−15 Liposomes and mannose-modified albumin specifically designed to target the liver have also been investigated for the treatment of liver inflammatory conditions, but the results were poor and controversial.16,17 Liposomes failed to achieve selective liver delivery and the mannose-modified albumin carrier, even if capable to carry the drug selectively to the liver KCs, failed to prevent inflammatory-related collagen deposition due to a combination of cell signaling events induced by the carrier interacting with the target mannose receptor. The limited effect observed in these studies is likely due to the evidence that steroid delivery into the liver is necessary but not sufficient to permit a successful therapeutic approach. The present study was aimed at achieving an effective therapy using a dexamethasone-carrying nanoformulation, based on Avidin-Nucleic-Acid-Nano-Assemblies (ANANAS), for patients affected by liver inflammation. An integrated platform was developed to evaluate its efficacy in a murine model of AIH. ANANAS are polyavidin-based NPs generated from the high-affinity interaction between the egg-white protein avidin and a nucleic acid filament (Figure 1).18,19 These NPs, which are made of soft biodegradable and biocompatible components, are poorly immunogenic,20 and can be considered “superavidins”, with diagnostic and therapeutic potential21−24 that expand those of the individual avidin protein, including targeted drug delivery.25,26 Interestingly ANANAS offer some features that make them particularly

Figure 1. Process of ANANAS−PEG−Hz−Dex assembly.

suitable as drug carriers to treat a broad range of liver disorders. It has been recently demonstrated that, besides systemic injection, ANANAS freely circulate in the bloodstream for more than 2 h, progressively accumulating in the liver parenchyma before being eliminated within 24−48 h.20,21 From a technical point of view, thanks to the high affinity (Kd = 10−15 M) between avidin and biotin, these NPs can be easily loaded with bioactive24,26 or contrast agents20,21 with controlled stoichiometry. All these features permitted a strict control of each physicochemical parameter during the synthesis, which is necessary for future translation from preclinical to clinical settings. In this work, we conjugated the steroid dexamethasone (Dex) to ANANAS. After a series of in vitro characterizations, we moved to in vivo studies to assess its kinetics and efficacy in mice. Dex was selected as the active agent because, among all corticosteroid drugs, it has the highest direct anti-inflammatory potencyabout 7.5 times higher than prednisoloneand the longest duration of action.27 Dex is indeed widely used (both orally and parenterally) in several inflammatory clinical applications including some immune-related disorders, thanks to its ability to inhibit the function of lymphocytes, macrophages, and other immune cells. The drug was tethered to ANANAS via a biotin−PEG linker using an acid-reversible hydrazone bond,14 which should guarantee stability at the neutral pH of plasma and permit drug release in the endosome/lysosome acidic environment (Figure 2E,F). The therapeutic potential of ANANAS−Hz−Dex on AIH was evaluated in the well-established cytochrome P450 2D6 (CYP2D6) mouse model that reflects many aspects of human AIH, including elevated serum aminotransferase and γ-globulin levels, generation of CYP2D6-specific antibodies and T cells, as well as liver infiltration and fibrosis.28−31 In this CYP2D6 model, the induction of AIH-like disease by injection of an adenovirus encoding the major liver autoantigen in human type 2 AIH, human CYP2D6, has the advantage that the immunopathogenic processes can be evaluated at precise times after induction. An initial in vitro study was performed to verify B


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Figure 2. Physicochemical properties of ANANAS−Hz−Dex. (A) Biotin binding site coverage (% over total available BBS) with biotin− PEG−Hz−Dex as a function of the amount of compound 2 in assembly solution. (B) Size of the assembly as a function of BBS coverage with compound 2. (C) UV−vis spectrum of ANANAS−Hz−Dex30. (D) Colloidal stability of ANANAS−Hz−Dex30 over storage time in solution at 2.7 mg/mL at 4 °C. (E) Scheme of pH-dependent release of Dex from ANANAS−Hz−Dex30. (F) In vitro drug release at (△) pH 7.4, (□) pH 5.0, and (●) pH 4.0.

with ANANAS. Each NP contains about 350 avidins, which are available for docking any kind of biotinylated moiety thanks to the high affinity between avidin and biotin (Kd = 10−15 M). When in close contact with cells, ANANAS were internalized through an endocytic pathway20,26 and this also occurs in the liver parenchyma following intravenous administration.20 Therefore, we decided to exploit the low pH reversible hydrazone bond to promote the release of the drug only upon cell internalization. To maximize the PEG layer necessary for surface protection concomitantly with Dex loading, we introduced a 5KDa poly(ethylene glycol) spacer between the

the different stability of the drug hydrazo linker at neutral and acidic pH. Then optical imaging, confocal and super-resolution microscopy were carried out to localize fluorescently labeled ANANAS in the whole body and in liver sections, and HPLC MS/MS to measure the level of free drug in plasma and in different organs. Lastly, the clinical signs of AIH mice were examined with biochemical and histological assays.

RESULTS AND DISCUSSION ANANAS−Hz−Dex Design and Characterization. Figure 1 summarizes the process of assembly of the Dex C


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Figure 3. In vivo nanoformulation biodistribution and pharmacokinetics. (A) The fluorescent signal associated with ANANAS, ANANAS− Hz−Dex, free alexa633 drops (2.5 mg NPs/mL, 4 μL/spot, same amount of dye). Water and background were used as controls. (B) Quantification of in vivo optical imaging signal related to the whole body of animals. (C) In vivo optical imaging of mice treated with alexa633, ANANAS, or ANANAS−Hz−Dex and scanned 30 min, 4 h, and 24 h after the treatment. (D) Ex vivo optical imaging of excised organs from animals sacrificed 4 and 24 h after vehicle, alexa633, ANANAS, and ANANAS−Hz−Dex administration. Li. = liver, Sp. = spleen, Ki. = kidneys, Lu. = lungs, Br. = brain. (E) Quantification of ex vivo optical imaging signal. Data are reported as mean ± SE. The data were analyzed by unpaired Student’s t test. * = ANANAS vs ANANAS−Hz−Dex at the same time point; # = 4 h vs 24 h after ANANAS treatment; ° = 4 h vs 24 h after ANANAS−Hz−Dex treatment. *p < 0.05, **p ≤ 0.005, ***p ≤ 0.0005.

biotin and the drug hydrazo moiety (biotin−PEG−Hz−Dex, compound 2, Scheme S1). Dex carrying NPs were prepared by mixing this reagent with core (empty) NPs previously obtained in freeze-dried form.24

These contain the smallest amount of a biotin−methoxy−PEG to ensure colloidal stability during preparation and freezedrying. This formulation can be functionalized in a one-pot reaction with biotin reagents without the need for additional D


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Figure 4. Localization of ANANAS and ANANAS−Hz−Dex in the liver of healthy and AIH mice. Confocal microscopy liver images (A and B) from healthy mice sacrificed 30 min and 24 h after ANANAS (A) and ANANAS−Hz−Dex (B). The blue signal refers to the nuclei (Hoechst 33258 staining), green corresponds to the lysosomal component of macrophages (CD 68 Antibody), red is associated with the alexa633 dye linked to the NPs. (C) Quantification of the area occupied by ANANAS overlapping the area occupied by CD 68 staining of liver sections from healthy mice. Data are reported as a scatter plot. Statistical analysis is performed by one-way ANOVA followed by Tukey’s post hoc test ***p < 0.0001. (D, E, and F) Super-resolution microscopy images of liver sections from healthy (D and E) or AIH (F) mice sacrificed 24 h after ANANAS−Hz−Dex treatment.

ANANAS and ANANAS−Hz−Dex Concentrate in the Liver Parenchyma of Healthy Mice. One of the main goals of NP dependent drug delivery is to increase the tropism toward the target organ. The biodistribution of both ANANAS and ANANAS−Hz−Dex after intraperitoneal (i.p.) administration was first investigated in healthy, immunocompetent, and specific pathogen-free mice to assess their behavior in physiological conditions. For this part of the analyses, the two formulations were fluorescently labeled with biotin-alexa633 covalently linked to ANANAS to permit longitudinal tracking for every single mouse by optical imaging (IVIS Lumina XRMS). Since minimal changes in NP composition, including the link to a steroid, may alter the interaction with host tissues, we carefully compared the two formulations. Whole-body longitudinal tracking (30 min, 4 and 24 h) of the fluorescent labeled NPs showed a similar profile for the two formulations regarding permanence and biodistribution in the body (Figure 3). The signal associated with the two formulations remains in the abdominal cavity for a long time (about 40% of the signal recorded 30 min after injection is still present at 24 h). To exclude that the signal was related to the free circulation of dye detached from ANANAS,32 we administered the same concentration of low molecular-weight biotin-alexa633 and we compared the fate of the dye to that of the two fluorescent nanoformulations. The signal related to the free fluorophore, despite its intrinsic higher fluorescence (Figure 3A), rapidly disappeared over time (Figure 3B,C).

purification steps unless the biotin binding sites (BBS) or the available surface area are saturated.22 The maximum NP loading capacity for the Dex derivative was therefore assessed in preliminary experiments. Mixtures of core NPs and biotin−PEG−Hz−Dex were generated in PBS buffer at biotin/BBS molar ratios between 0.25 and 0.8 and analyzed for their content of unbound PEG reagent by gel permeation chromatography. A maximum of 410 units of biotin−PEG−Hz−Dex/NP can be loaded, as also confirmed by HPLC titration of the releasable Dex from purified assemblies. This amount corresponds to a BBS coverage of about 30% (Figure 2A). On the basis of this information, ANANAS−Hz−Dex were later prepared by mixing biotin−PEG−Hz−Dex and the core NPs at 30% BBS (ANANAS−Hz−Dex30) coverage and used without purification. Upon addition of the Dex-carrying reagent, the size of the NPs increased from 125.8 ± 1.2 nm to 132.9 ± 2.9 nm, in agreement with the formation of a PEG layer around the core particle. Dynamic light scattering experiments also showed that the Dex-loaded formulations are colloidally stable for at least 48 h after preparation (Figure 2 and Figure S4). The ability of ANANAS−Hz−Dex30 to release free Dex selectively at acidic pH was demonstrated in vitro. At neutral pH, the drug remains stably tethered to the biotin linker, while at pH below 6.0, drug release occurs with a kinetics that increases in correlation with the decrease of pH (Figure 2E,F). E


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Figure 5. Pharmacokinetics study. Levels of free Dex measured in plasma (A), brain (B), and liver (C) from healthy mice and in liver (D and E) from AIH mice. Mice were sacrificed at different time points after intraperitoneal administration of Dex (blue bars) or ANANAS−Hz− Dex (gray bars) (0.4 mg/kg). Data are reported as mean ± SE. One-way ANOVA followed by Bonferroni’s post hoc test. Significant difference (*p < 0.05, **p < 0.005). LoQ = Limit of quantitation of the HPLC MS/MS method: liver, 0.45 ng/g; brain, 0.8 ng/g; plasma, 1.2 ng/mL.

Dexamethasone Loading Accelerates Tropism of ANANAS toward Liver Kupffer Cells. Liver tissue is made up of 80% hepatocytes (parenchymal cells) and 6.5% nonparenchymal cells (sinusoidal endothelial, Kupffer, and hepatic stellate cells). KCs are tissue macrophages with marked endocytic and phagocytic capacity. They play an essential role in the innate immune defense. They can secrete several mediators of the inflammatory response and thus control the early phase of liver inflammation. Indeed, aberrant activation of KCs can trigger inflammation contributing to both the start of disease and its progression. Their anti-inflammatory activity means macrophages can be considered as a therapeutic target in AIH.17,33 To see whether the ANANAS formulations selectively interact with liver cell types, we used confocal laser and super-resolution microscopy to analyze the liver tissue of NP treated mice and assessed the extent of NP and KC colocalization using a fluorescent marker for macrophages (anti-CD68).34 This experiment was carried out on both healthy and AIH mice models (Figure 4, Figures S10, S11), to evaluate if the NP fate changes when the liver is inflamed. To this end, we used the CYP2D6 mouse model.28 Wild-type C57BL/6 mice are infected with an adenovirus encoding for the major liver autoantigen in human type 2 AIH, cytochrome

The signal associated with ANANAS-treated mice, independently on the formulation, decayed more slowly, thus confirming the more lasting permanence of the NPs in the body compared to the dye, as already observed after intravenous injection.20 The colocalization experiments between alexa633 and an antibody directed against avidin (Figure S8) furthermore confirmed the reliability of our results and consequently the stability of the nanoassembly. Ex vivo analysis (Figure 3D,E) showed that, 4 h after administration, the fluorescent signal associated with ANANAS was well detectable in all selected organs, including the lungs and brain. However, 1 day after treatment, the pattern of fluorescence showed almost selective tropism toward the liver and spleen (see also Figure S5) and was much lower in other organs. These data suggest that, despite their size and hydrophilic nature, both NP formulations are capable of reaching the blood circulation from the pelvic area and then follow the same fate as after intravenous administration, when free circulation is followed by liver and spleen sequestration.20 However, the i.p. route led to slower but longer hepatic accumulation. This is extremely important in terms of pharmacokinetics because it enables ANANAS, and consequently Dex, to stay longer in the body of treated subjects. F


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The free drug penetrates the bloodstream rapidly and then follows the classic pharmacokinetic profile of Dex (t/2 = 1.8− 3.5 h in humans) (reference.medscape.com), reaching all major tissues, including liver and brain, and being fully cleared 24 h after administration. Notably, more than 10% of the level measured in plasma was found in the brain, the tissue in which steroid accumulation is linked to psychiatric/neurological side effects.36,37 However, when mice were treated with ANANAS−Hz−Dex, the free drug was found only in the liver. In both healthy and AIH models, the drug kinetics in this tissue is in line with what is expected from slow release from the carrier. Drug levels increased in the first hours after administration and then slowly decreased. Dex levels decreased faster than the NP ones (Figure 3 and Figure S5), suggesting that in vivo drug release occurs faster than NP degradation. It is also interesting that the kinetics in the liver of AIH mice was slower than in healthy animals, in both the accumulation and elimination. For example, 24 h after administration, the amounts of free drug were respectively 8.6% and 52.1% of that measured at 4 h in healthy and AIH animals. This is presumably due to an altered metabolism or different bioavailability linked to pathological changes occurring during the disease progression. Independently of the cause, more lasting permanence of the drug in the tissue could give a pharmacological advantage, since it may reduce the frequency of serial treatments in patients. Overall, these results are promising regarding both safety and the potential efficacy of the treatment in the liver. However, to gain a proof of this potential effect, a subchronic treatment in affected mice has been carried out using the same doses as for pharmacokinetics (Figure 7). Treatment with ANANAS−Hz−Dex Is More Effective than the Free Drug in Controlling AIH Disease in the Animal Model. Figure 6 schematizes the hypothetical kinetics of ANANAS−Hz−Dex (panel 1) after i.p. injection. Our pharmacokinetics analysis indicated that (at least for the 24 h experimental window) Dex remains attached to the nanoassembly both in the bloodstream (step 2) and in offtarget organs. ANANAS−Hz−Dex migrates to the liver within the first hours after administration (step 3) and selectively interacts with CD68-positive cells localized in the parenchyma (step 4). Once inside these cells, ANANAS−Hz−Dex does not penetrate the nucleus but efficiently enters the CD68-positive lysosomal vesicles (step 5). The lower pH inside these vesicles may induce the release of the drug from the acid-sensitive hydrazo linker (step 6). The lipophilic nature of Dex means it is potentially able to cross biological membranes (lysosomal, plasmatic, but also nuclear) and reach the target to exert its immunomodulatory activity. To evaluate the therapeutic potential of ANANAS−Hz−Dex on AIH, we scheduled a subchronic treatment in the AIH Ad2D6 mouse model.28 Briefly, Ad-2D6-infected mice received six i.p. Dex administrations either nanoformulated or as free drug (0.4 mg Dex/kg). Drug administration started (day −1) the day before virus infection (day 0) and was repeated on days 3, 7, 10, 14, and 17. The results (Figure 7) clearly show that ANANAS−Hz−Dex is effective and more efficient than the free drug in controlling the hepatic immune response in this AIH model. Indeed, despite the lower concentration of drug reaching the liver (Figure 5) in the case of the nanoformulation, the histopathological effects generated by the two treatments were equivalent. In both experimental groups, interlobular collagen I deposition (marker for fibrosis

P450 2D6 (CYP2D6). Infected mice show several features of human AIH, including interface hepatitis with infiltrating cells of the innate and adaptive immune system, generation of LKM-1-like antibodies, and CYP2D6-specific T cells, as well as fibrosis (Figure S9).29 The nanoassemblies were efficiently internalized by KCs; this selective tropism of ANANAS was not influenced by the pathology or by the loading of the steroid (Figure 4). Thirty minutes after administration, the red fluorescent signal associated with the drug-free ANANAS (Figure 4A, left) is mainly confined within a narrow region surrounding the vessels and does not overlap with the macrophage marker CD68 (green). On the contrast, the Dex-carrying formulation leads to faster tropism for KCs at the same time. Interestingly, the signal for ANANAS−Hz−Dex is homogeneously spread in the liver parenchyma, and almost exclusively overlaps the CD68 signal (Figure 4B, left). The difference between the two formulations at the two time-points was quantified by measuring the percentage of NP- and CD68-associated signal colocalization (Figure 4C), thus confirming the qualitative analysis. On the other hand, 24 h after treatment (Figure 4A,B, middle and right) the alexa633 signal related to both formulations was closely associated with that of KCs (CD68). Although we cannot exclude an early, transient interaction between the NPs and other cell populations (e.g., sinusoidal, endothelial, and B cells), these results do indicate that CD68-positive cells play a key role in the sequestration of ANANAS, independently of the nature of the NP cargo. It is hard to explain why the presence of Dex on the NP surface accelerates their uptake by KCs, but one possibility is that it facilitates the NP interaction with the cell membrane, either because of its hydrophobic nature or by “hijacking” an interaction with steroid receptors. The sublocalization of the NPs inside the cells was also investigated by super-resolution microscopy (Figure 4D−F). This high-resolution imaging tool is considered the most appropriate for analyzing soft materials since it avoids the generation of artifacts (agglomeration, deformation, collapse) which can otherwise occur during the drying process necessary for transmission electron microscopy (TEM).35 Considering the pH-dependent stability of the hydrazone bond adapted to tether Dex to ANANAS (Figure 2), reaching the mature acidic lysosomal compartment is fundamental for the drug to be released from ANANAS. NPs do appear clustered inside single lysosomes of hepatic macrophages of both healthy and AIH mice, holding promise that drug can indeed also be released in vivo. ANANAS−Hz−Dex Releases Free Dexamethasone Only in the Liver but Not in Other Tissues of Healthy and AIH Models. To assess the ability of the ANANAS−Hz− Dex to selectively release the steroid in the liver, we performed a pharmacokinetics study to monitor the levels of free drug in plasma and in the main target organs of both healthy and AIH mice. Although we had shown in vitro (Figure 2F) that the hydrazone linker was stable at neutral pH, the chemical milieu in the body is more complex than a saline buffer and a direct evidence of the level of Dex in organs is needed in order to rule out the risk of the drug being released before it reaches the liver. Figure 5 shows the results of the pharmacokinetics study carried out in mice treated i.p. with either ANANAS−Hz−Dex or the free drug (0.4 mg Dex/kg). G


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Hepatic fibrosis is a dynamic and highly regulated process. Inflammation stimulates the transition of perivascular hepatic stellate cells into myofibroblasts through the induction of molecules such as transforming growth factor β (TGF-β) and/ or platelet-derived growth factor (PDGF).38 Although not statistically significant, there is a drastic trend of reduction of both these pro-inflammatory factors in the liver of ANANAS− Hz−Dex treated mice (n = 3 see Figure S12A,B). It has already been reported that the decrease of myofibroblast activation may lead to a reduction of collagen expression, which is a typical pathological feature of AIH livers (see Figure 7B). According to the anti-inflammatory effect of ANANAS−Hz− Dex, a significant reduction of collagen (1α2 and 3α1) was seen in treated mice (Figure 8A,B). Marked activation of metalloproteinases naturally occurs in many organs, including the liver, to counteract fibrosis during chronic inflammation.39 The reduction of MMP13 is therefore to be expected in a tissue with lower and less extensive fibrosis (see Figure S12C). There was also a reduction of the expression of caveolin 1 (one of the important proteins found in caveolae) (Figure 8C). Though the role of this membrane protein has not been fully elucidated, it has been shown to play a role in tissue repair and fibrosis.40 The significant reduction of IL-1β gene-expression in the liver of ANANAS−Hz−Dex treated mice (Figure 8D) provides direct evidence of the anti-inflammatory effect of chronic treatment with the nanoformulations.41 In line with this immunomodulatory action played by the interaction of the steroid with hepatic macrophages, an important trend of decrease of gene expression level of three markers involved in chemotaxis and cell proliferation was found in the liver of treated AIH mice (see Figure S12DE,F).

CONCLUSIONS Overall, this evidence shows that the ANANAS−Hz−Dex nanoformulation is more effective than the free drug in controlling the disease in the animal model. Even though this is only a preliminary outcome, combined with the fact that the nanoformulation does not release the steroid in any body district other than the liver, it suggests that this is a promising carrier for controlling chronic liver inflammatory conditions. This result stems from the combination of several formulation features: (a) the target (KC) cell tropism, (b) the pathway of cell entry which fits, (c) the acid−labile hydrazone−mediated drug release mechanism, and (d) the slow degradation of the nanoassembly in the liver parenchyma. These combined features generate a relatively long-lasting availability of the free drug at the relevant site, where even a low Dex concentration is enough to control the disease hallmarks. It is worth noticing that, despite repeated administrations, the drug-free ANANAS does not lead to any measurable negative effect on liver functionality, such as liver fibrosis or AST and ALT levels. This suggests that the carrier itself is neither toxic, nor pro-inflammatory. The composition of the assembly is in fact such that its degradation leads only to welltolerated elements. Even if avidin is an exogenous protein and low-affinity antiavidin antibodies have been described in humans,42 tolerance to this protein has also been shown,43 quite likely as a consequence of egg products in the every day diet. Despite these encouraging elements, the formulation here described still needs further implementation before clinical translation. For example, its drug loading capability is quite low, and this would affect costs. Current efforts in our

Figure 6. Hypothetical scheme of ANANAS−Hz−Dex fate in the liver. To summarize the process, it is divided here into six steps. However, it is extremely important to underline that it complies with a hierarchical and not a strictly temporal pattern.

onset) was markedly reduced compared to control animals treated with either the drug-free ANANAS formulation (Figure 7B) or PBS (not shown). Histological examination of the lymphocyte infiltration in liver parenchyma (Figure 7C) showed no effects of the treatments on the amounts of CD4 T cells (quantification of several sections CD4 and collagen I). However, it is important to note that in contrast to mice of the FVB strain,28 C57BL/6 mice only show a lower infiltration, even in the presence of significant liver pathology. However, the ANANAS−Hz−Dex formulation was more effective than the free drug in controlling other liver inflammation parameters. For example, ALT and AST plasma levels, which also increase secondarily to the adenovirus infection, were reduced by Dex and ANANAS−Hz−Dex administration, but this effect was much stronger in the NPtreated group (Figure 7D,E). Besides, ANANAS−Hz−Dex was also more effective than the free drug in controlling the levels of AIH specific markers, namely the magnitude of circulating anti-CYP2D6 autoantibodies (Figure 7F). In fact, while free Dex did not affect this marker, in ANANAS−Hz−Dex treated animals this was 10 times lower. To further evaluate the beneficial effect of ANANAS−Hz− Dex, the expression of specific genes measured in the liver of treated and untreated AIH mice was compared (Figure 8). H


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Figure 7. In vivo treatment of the AIH mouse model. (A) The experimental procedure in AIH mice. (B,C) Collagen I and CD4 T cell staining, respectively, on liver sections from mice, sacrificed 20 days after virus infection. (D,E) Plasmatic levels of ALT and AST (U/L) measured 6 days after infection. The dashed lines represent the level of aminotransferases from noninfected mice. (F) Anti-CYP2D6 IgG levels analyzed in serum collected at euthanasia (day 20 postinfection). The measured values are no infection, n.d. (not detectable); vehicle, 12150 (8100−24300); free Dex, 24300 (8100−72900); ANANAS−Hz−Dex, 1620 (900−2700); ANANAS, 9000 (2700−24300).

laboratory involve the development of other biotin−Dex derivatives to improve both NP Dex loading and the linker hydrolytic stability to extend the drug release time frame. On the positive side, the fact that efficacy here was achieved even with a very low local drug concentration means that availability at the proper site is more important than the concentration. This also permits us to hypothesize that nanoformulation

dosages lower than the one used here may still be effective, reducing loadability requirements.

METHODS Reagents and Instrumentation. Biotin−PEG5KDa−succinimidylcarbonate (biotin−PEG5KDa−SC) α-biotin, ω-methoxy−PEG5KDa (MPEG−biotin) were from Laysan Bio (Arab, AL, USA); triethylamine (TEA, cat no. 15791) was from Acros Organics; absolute I


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Figure 8. mRNA levels of fibrosis-associated genes in hepatic tissue of chronically treated AIH mice. RNA expression was evaluated by realtime PCR in 3 vehicle- and in 3 ANANAS−Hz−Dex-treated mice. Data were normalized on beta glucoronidase and expressed as 2-(ΔΔCt) compared to an external referral sample. CAV1, caveolin 1; IL-1β, interleukin-1 β. ethanol (EtOH) was from Carlo Erba reagents, Italy. Ethyl acetate (EtOAc) AnalaR Normapur was supplied by WVR, Radnor, Pennsylvania, US. 2-(4-Hydroxyphenylazo)benzoic acid (HABA, cat. no. 54793) tert-butyl carbazate (BOC-Hz, cat no. B91005); 2,4,6trinitrobenzenesulfonic acid (TNBSA, cat no. 92823), dexamethasone (cat. no. 1756), trifluoroacetic acid (TFA, cat. no. T6508), dichloromethane (DCM), dimethyl sulfoxide (DMSO), and all other reagents were obtained from Sigma-Aldrich, Italy. All water was double-distilled (dd-H2O) grade. Gravity NAP-10 disposable columns prepacked with Sephadex G-25 DNA grade were from GEHealthcare. Spectroscopic analyses were conducted with a Varian Cary 50 UV−vis spectrophotometer. Fluorescence was determined with a Jasco FP-6200 spectrofluorimeter. Nuclear magnetic resonance analyses were done with Bruker AMX 300 and 400 MHz NMR spectrometers; NMR spectra were analyzed with Bruker’s TopSpin software. Gel permeation chromatography was done with an AKTA fast protein liquid chromatography (FPLC) purifier system (GEHealthcare) with a Waters 2414 RI (refractive index detector), integrated with a Superose 6 10/300 GL column (GE Healthcare). Reverse-phase high-performance liquid chromatography (RP-HPLC) was done with an Agilent chromatography system (USA), model 1220 Infinity LC equipped with diode array detector, using a Phenomenex Kinetex C18 5 μm 4.6 mm × 250 mm) column. NPs were measured by dynamic light scattering (DLS) using the Zetasizer Nano ZS (Malvern, Malvern UK). The mass of PEG compounds was measured

using the AB SCIEX 4800 MALDI TOF/TOF Analyzer (SCIEX, Toronto, Canada). For detecting the mass of low-molecular-weight compounds we employed the XEVO G2-S ESI-TOF mass spectrometer (Waters, Milford, MA, USA). Synthesis of Biotin Conjugates. Biotin−PEG5KDa−SC−Hydrazide. (Compound 1; Scheme S1). Biotin−PEG5 kDa−SC was mixed with 1.2 equiv of BOC-Hz in DCM/EtOAc (1:1) followed by 1 equiv of TEA. After 3 h, the product (biotin−PEG5kDa−SC−Hz−Boc, compound 1) was precipitated with cold Et2O, recovered by Gooch filtration and dried in vacuo. 1H NMR analysis (DMSO, 300 MHz) (δ = 3.504 ppm, m, 454 H-PEG main chain; δ = 1.396, m s, 9-H BOC) confirmed the product identity. Excess BOC-Hz was removed by several cycles of warm EtOAc and cold Et2O. The BOC group was removed by 1 h treatment in 95% TFA and the product was isolated as a solid by precipitation and extensive washing with cold dry Et2O. Product characterization was confirmed by 1H NMR, TNBSA, and HABA analysis. Biotin−PEG 5KDa −SC−Hz−Dexamethasone. (Compound 2; Scheme S1). Compound 1 was mixed with 10 equiv of Dex in DMF, and a catalytic amount of CH3COOH was added. After 100 h, full conversion of the hydrazine groups was verified by TNBSA assay (Snyder). The product was isolated by cold precipitation with Et2O and purified of excess Dex by repeated washes with cold Et2O, recovered by Gooch filtration, and dried in vacuo. The Dex/PEG molar ratio in the product was calculated by 1H NMR analysis and by J


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ACS Nano titrating an aqueous solution for PEG and releasable Dex contents using, respectively, the iodine assay44 and RP-HPLC analysis after 4 h hydrolysis at 50 °C in 0.1 HCl. The product was also analyzed by UV−vis spectrometry, matrix assisted laser desorption ionizationtime-of-flight mass spectroscopy (MALDI-TOF), and gel permeation chromatography. ANANAS−PEG−Hz−Dex Formulations. Core ANANAS containing the minimum amount of 5KD methoxy-PEG (12.5% BBS) to guarantee buffer solubility was prepared according to Pignatto19 and freeze-dried immediately after purification.26 NPs were reconstituted in PBS buffer and compound 2 was added to the desired biotin/BBS molar ratio. Dex-free formulations were generated using methoxyPEG-biotin instead of compound 2. When needed, biotin-alexa633 was also added at a biotin/BBS molar ratio of 0.2.20 Titration of Releasable Dexamethasone from ANANAS− PEG−Sc−Hz−Dex. ANANAS−PEG−SC−Hz−Dex samples were generated by mixing core ANANAS with a large excess of compound 2. The sample was purified by gel permeation chromatography and the purified NPs were treated in 0.1 M HCl at 37 °C for 4 h. The solution was then analyzed for free Dex content by RP-HPLC (eluent A, H2O + TFA 0.1%; eluent B, 5% A in ACN; gradient from 5% to 90% B in 40 min). Before HPLC analysis, the protein fraction was removed from the solution by cold CH3CN precipitation. Dexamethasone Release. Dex release from compound 2 both as a free molecule and when linked to the NPs was examined at three pH values (100 mM phosphate pH 7.4, 100 mM Na acetate, pH 5.0 and 100 mM Na acetate, pH 4.0. Samples (10 μg/mL in Dex) were incubated in the selected buffer at 37 °C, and at scheduled timepoints the concentration of free Dex in solution was measured by RPHPLC, as described above. Nanoparticle Titration on the Liver Tissue by Dot Blot. Liver tissues were mixed with hot 1% SDS in ddH2O at a 1:5 weight-tovolume ratio and disrupted using a Potter homogenizer, then sonicated at room temperature (RT) for 60 s and heated 10 min at 100 °C. The supernatant was isolated by centrifugation and mixed with 1/5 of its volume of 5X GSMT solution (glycerol/1.5 M TRIS/ SDS/β-mercaptoethanol = 630:330:150:22.2 weight/weight), then heated at 100 °C for 1 h. Samples were diluted 1:10 with PBS and spotted (2 μL/spot) on nitrocellulose paper. After blocking (3% BSA in PBST, 1 h, RT), dot quantification was carried out with incubation with rabbit antiavidin IgG (Abcam, P.N. no. ab6675, diluted 1:2500 with PBST+ 0.1% BSA, 1 h, RT), followed by HRP-conjugated goat antirabbit IgG (Millipore, AP307P, diluted 1:5000 in PBST/BSA 0.1%, 45 min, RT), and ECL development (GE Healthcare RPN2232). For ECL readout we used a Versadoc 4000MP (Biorad) luminescence reader, and dot intensity quantification was done with ImageJ Fiji. For quantification, samples were analyzed in parallel with calibrated solutions obtained by treating the same as liver samples of untreated animals spiked with known amounts of avidin (0, 3, 9, 26, 77 μg avidin/g of tissue). Animals. The “Mario Negri” Institute for Pharmacological Research IRCCS adheres to the principles set out in the following laws, regulations, and policies governing the care and use of laboratory animals: Italian Governing Law (D.lgs 26/2014; Authorisation n.19/ 2008-A issued March 6, 2008 by Ministry of Health); Mario Negri Institutional Regulations and Policies providing internal authorization for persons conducting animal experiments (Quality Management System Certificate, UNI EN ISO 9001:2015, Reg. No. 6121); the NIH Guide for the Care and Use of Laboratory Animals (2011 edition), and EU directives and guidelines (EEC Council Directive 2010/63/UE). The Statement of Compliance (Assurance) with the Public Health Service (PHS) Policy on Human Care and Use of Laboratory Animals was recently reviewed (9/9/2014) and will expire on September 30, 2019 (Animal Welfare Assurance no. A5023-01). This work was reviewed by the IRCCS-IRFMN Animal Care and Use Committee (IACUC) and then approved by the Italian “Istituto Superiore di Sanità” (code: 42/2016-PR). Eight-week-old male C57BL/6J mice were maintained under specific pathogen-free conditions in the Institute’s Animal Care

Facilities; they received food and water ad libitum and were regularly checked by a certified veterinarian who is responsible for animal welfare supervision and experimental protocol review. To induce AIH, C57BL/6J littermates were infected (i.p. and i.v.) with 3 × 108 pfu adenovirus encoding for the human CYP2D6 (Ad-2D6) as described for FVB mice.28 All experimental procedures with Ad-2D6-infected mice were approved by the local Animal Ethics Review Board, Darmstadt, Germany (V54−19c20/15-FU/1094). The experimental groups used for in vivo and ex vivo analyses are reported in Table 1.

Table 1. Animals, Treatment Schemes, and Type of Analysis mouse

no.

treatment

type of analysis

C57BL/6J

3

PBS

C57BL/6J C57BL/6J C57BL/6J

3 12 12

alexa633 ANANAS ANANAS−Hz−Dex

C57BL/6J Ad-2D6 infected C57BL/6J Ad-2D6 infected C57BL/6J Ad-2D6 infected C57BL/6J Ad-2D6 infected C57BL/6J C57BL/6J

12 3

Dex PBS

optical imaging, histology, HPLC MS/MS optical imaging optical imaging, histology optical imaging, histology, HPLC MS/MS HPLC MS/MS histology, HPLC MS/MS

12

ANANAS

histology

12

ANANAS−Hz−Dex

histology, HPLC MS/MS

12

Dex

HPLC MS/MS

3

PBS (multiple)

3

PBS (multiple)

6

ANANAS (multiple)

5

ANANAS−Hz−Dex (multiple) Dex (multiple)

histopathology, ALT and AST, Anti-CYP2D6 IgG histopathology, ALT and AST, Anti-CYP2D6 IgG histopathology, ALT and AST, Anti-CYP2D6 IgG histopathology, ALT and AST, Anti-CYP2D6 IgG histopathology, ALT and AST, Anti-CYP2D6 IgG

Ad-2D6 infected C57BL/6J Ad-2D6 infected C57BL/6J Ad-2D6 infected C57BL/6J Ad-2D6 infected C57BL/6J

5

In Vivo and ex Vivo Fluorescence Imaging. A total of 30 C57BL/6J animals were used for the biodistribution study. Twelve mice per group were injected intraperitoneally with 40 mg/kg of ANANAS, and ANANAS−Hz−Dex with the same amount of NPs loaded with 0.4 mg/kg of Dex. The remaining three were treated with PBS and were used as controls. Mice treated with alexa633 were enrolled to compare the signals of the free fluorophore and the fluorescent NPs. To reduce fluorescence background, mice were fed an AIN-76A alfalfa-free diet (Mucedola srl) for 2 weeks before analysis. In vivo optical imaging was done 30 min, 4 h, and 24 h after treatment. Fluorescence images were acquired with an IVIS Lumina III imaging system (PerkinElmer). The following acquisition parameters were used: excitation filter range 680 to 740 nm, emission filter (790 nm, exposure time 2 s), binning factor 4, and f/Stop 2. At the end of the study mice were euthanized 4 and 24 h after the treatment with an overdose of ketamine (150 mg/kg) and medetomidine (2 mg/kg). Liver, kidneys, spleen, lung and brain were removed and scanned for ex vivo imaging. Organs were collected without perfusing the animal so the signal also refers to their blood vessels. Spectral unmixing, image processing, and analysis were done using Living Image 4.3.1 software (PerkinElmer). Tissue Collection and Immunofluorescence. At the moment of sacrifice, livers from healthy and AIH mice treated with ANANAS and ANANAS−Hz−Dex were collected, postfixed in 4% paraformaldehyde for 24 h and transferred to a 20% sucrose solution until immunofluorescence staining. Cryostat sections were cut at 10 μm and mounted on glass slides. Slides were washed three times in phosphate-buffered saline (PBS) for 5 min and incubated for 1 h with a blocking solution (PBS-NGS 10%-Triton X-100 0.1%) then washed again with PBS. For subcellular localization, the antibody anti-CD68 (specific for lysosome and endosome membranes of macrophages) K


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ACS Nano was employed as follows: primary monoclonal rat antibody anti-CD68 (1:200, Serotec, Kidlington, UK) + Triton X-100 0.1% + NGS 3% in PBS O/N at 4 °C. After washing, secondary alexa488 conjugated antibody (Invitrogen) (1:500) was incubated for 1 h at RT in a 1% PBS-NGS solution. Then, the slides were washed and nuclei were stained with Hoechst 33258 (2 μg/mL in PBS, for 10 min). For avidin staining, livers from healthy mice treated with ANANAS were collected, frozen with liquid nitrogen and cryostat sections were cut at 10 μm and mounted on glass slides. Slides were fixed in 4% paraformaldehyde for 30 min, washed three times in PBS for 5 min and incubated with a blocking solution (15 min with 3% H2O2 to block peroxidase and 1 h with PBS-NGS 10%-Triton X-100 1% for nonspecific blocking). We used the antibody antiavidin (HRP) (1:100, Abcam) + 2% NGS + 0.5% Triton-X 100 in PBS O/N at 4 °C. After washing (TBS + 0.05% Tween-20), the slides were incubated for 8 min at RT with TSA-Fluorescein (1:50 in amplification diluent from the same kit, PerkinElmer, Inc.). At the end of incubation, slides were washed and nuclei were stained with Hoechst 33258 (2 μg/mL in PBS, for 10 min). Confocal and Super-resolution Microscopy. Specific lasers λexc 405 nm, λexc 488 nm, and λexc 640 nm, were employed to visualize the signals related to Hoechst 33258 (nuclei), alexa488/fluorescein (CD 68/avidin staining), and alexa633 (ANANAS), respectively. Samples were acquired using Nikon A1 Confocal and Nikon N-SIM microscopes and pseudocolored (blue for Hoechst 33258, green for alexa488/fluorescein, and red for alexa633). Confocal images were collected using 20× and 100× CFI SR HP Apochromat TIRF 100XC oil objectives. In 20× confocal acquisitions stitched fields were obtained automatically during acquisition with Nikon NIS-Element Software. In 100× confocal acquisition the stack thickness varies, from 0.12 to 0.96 μm between different samples. For N-SIM superresolution acquisitions a 100× CFI SR HP Apochromat TIRF 100XC oil objective was used. Images were acquired in 3D-SIM mode. Superresolution reconstruction and 3D rendering were obtained with Nikon NIS-Element Software. Images were quantified with Fiji (ImageJ) software, and 10 acquisitions were captured with the 20× objective for each group. The values indicate the percentage ANANAS signal within the CD68 positive area of liver sections from healthy and AIH mice. Data are reported as a scatter plot. Pharmacokinetics. Experimental Groups. To quantify the levels of Dex, plasma, brain, and liver from the group of healthy C57BL/6J mice treated with ANANAS−Hz−Dex were used. The other 12 animals were intraperitoneally injected with free Dex (0.4 mg/kg) and sacrificed at the same time points. For this analysis, Ad-2D6-infected mice were also examined 28 days after viral infection; 12 animals in both groups (ANANAS−Hz−Dex and Dex) were treated and sacrificed following the same procedure. Sample Preparation and Extraction. As a first step of the analytical methods, the internal standard, fludrocortisone (10 ng) was added to the sample from treated mice, and Dex (0−100 ng) and fludrocortisone (20 ng) were included for the calibration curve. Liver and brain slices were digested with methanol (1:2 w/v) and acetonitrile (1:1 w/v), stirred and sonicated for 20 min. Then water (1:10 w/v) was added and stirring and sonication were repeated. The resulting samples were centrifuged at 7000g for 15 min at 4 °C. The supernatant was further cleaned up with solid-phase extraction using Sep-Pak C18 1 cc Vac Cartridges, conditioned before use with 1 mL of methanol, followed by 1 mL of water. Samples were loaded on the SPE columns and passed through dropwise. Then cartridges were rinsed with 1 mL of water/acetone (80:20) and then with 1 mL of water before drying the columns under vacuum for 5 min. Samples were eluted with 1.8 mL of acetonitrile into glass receiving tubes. Plasma aliquots were directly eluted with acetonitrile (1:4v/v) and centrifuged at 7000g for 15 min at 4 °C. All the samples were evaporated to remove the organic phase. Just before analysis, they were suspended in 100 μL of 0.05% acetic acid/ acetonitrile (80:20) in autosampler vials. Liquid Chromatography (HPLC) and Tandem Mass Spectrometry (MS/MS). All experiments were carried out on an Agilent 1200 series HPLC system interfaced to an Agilent 6410 triple-quadrupole

mass spectrometer equipped with an electrospray ionization source (Agilent Corporation, MA, USA). All data were acquired and analyzed using Agilent MassHunter data processing software. Separation was performed with a Superspher 100 RP-18 (2.1 × 100 mm, Merck, Darmstadt, Germany) column, maintained at 30 °C. The elution solvents were 0.05% acetic acid in water (mobile phase A, MPA) and acetonitrile (mobile phase B, MP-B). The injection volume was 10 μL and the flow rate was 200 μL/min. The autosampler temperature was 6 °C. Elution started with 80% of MP-A and 20% MP-B for 2 min, followed by a 12 min linear gradient to 60% of MPB, and a 2 min linear gradient to 99% of MP-B, held for 4 min and a 1 min linear gradient to 80% of MP-A, which was maintained for 6 min to equilibrate the column. Dex and fludrocortisone, were analyzed using electrospray in negative ionization mode, with the spray voltage set at 2000 V. Nitrogen was used as nebulizer gas at a pressure of 35 psi. Desolvation gas (nitrogen) was heated to 250 °C and delivered at a flow rate of 7.5 L/min. Analysis data were acquired in the multiple reaction monitoring (MRM) mode. Table 2 shows the optimized MRM parameters for each component.

Table 2. Optimized MRM Parameters for Dexamethasone and Fludrocortisone (IS) component dexamethasone (quantifier) dexamethasone (qualifier) fludrocortisone (IS)

precursor ion (m/Z)

fragmentor energy (V)

collision energy (eV)

product ion (m/Z)

361.2

170

18

307.2

361.2

170

18

325.2

349.2

170

20

295.1

Chronic Treatments and Pathological Scores. We used Ad2D6 infected mice to evaluate the therapeutic efficacy of the nanoformulation. Animals received six serial intraperitoneal injections of PBS (n = 3), ANANAS (40 mg/kg, n = 6), Dex (0.4 mg/kg, n = 5), ANANAS−Hz−Dex (40 mg/kg of NPs and 0.4 mg/kg of drug, n = 5). Three remaining mice were not infected and served as the control. Injections were given on days −1, 3, 7, 10, 14, and 17 after Ad-2D6 infection. Blood was collected on days 6, 13, and 20 postinfection to determine serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, while anti-CYP2D6 IgG levels were only analyzed on day 20. At the moment of sacrifice, livers were harvested, fixed in formalin and embedded in paraffin. Serum Aminotransferase and anti-CYP2D6 Antibody Determination. Blood samples were collected with heparin-coated capillaries and serum was stored at −80 °C until further analysis. ALT and AST levels were measured with the Reflotron Plus blood analysis system (Roche Diagnostics, Mannheim, Germany). For the assessment of anti-CYP2D6 antibodies, 96-well microtiter plates were coated overnight at 4 °C with 100 μL of 0.25 μg/mL recombinant human CYP2D6 (Invitrogen) in 100 nM carbonate-buffer (pH 9.6) and plates were blocked with 2% FCS in PBS for 90 min at RT. Sera were added in PBS containing 2% FCS and were incubated for 90 min at 37 °C. The dilution series started at 1:100, followed by 1:3 dilution steps down to a dilution of 1/72 900. Alkaline-phosphatase-labeled goat antimouse IgG antibody (1:2000, Southern Biotech, Birmingham, USA) was added for 90 min and the reaction was developed by the addition of ECF substrate (GE Healthcare Bio-Sciences). Fluorescence intensity was determined using a Pharos FX molecular imager (Bio-Rad). Immunohistochemistry of Liver Sections. Mice were euthanized and livers were perfused with 5 mL of PBS in situ. Livers were removed, immersed in Tissue-Tek OCT (Bayer), and quickfrozen on dry ice. Using a cryomicrotome and sialin-coated Superfrost Plus slides (Fisher Scientific), 7 μm tissue sections were cut, then fixed with ethanol at −20 °C, washed in PBS, and an avidin/biotinblocking step was included (Vector Laboratories). Primary and biotinylated secondary antibodies (Vector Laboratories) were reacted with the sections for respectively 120 and 60 min, and color reaction L


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ACS Nano was obtained by sequential incubation with avidin-peroxidase conjugate (Vector Laboratories) and diaminobenzidine-hydrogen peroxide. Primary antibodies used were rat antimouse CD4 (BD Biosciences), rat antimouse CD11b (eBioscience), rat antimouse F4/ 80 (Serotec), and rabbit antimouse collagen I (Merck-Millipore). Biotinylated secondary antibodies were antirabbit and antirat IgG (both from Vector Laboratories). RNA Extraction, cDNA Synthesis, and Real-Time PCR. RNA was extracted from the liver of AIH mice undergoing chronic treatment, using the Quick RNA kit (Zymo Research, Irvine, CA) according to the manufacturer’s directions; the procedure included a DNase I treatment. RNA was quantified using Nanodrop (Thermo Fisher Scientific, Milan, Italy), and 1 μg was retrotranscribed using the High-Capacity cDNA reverse transcription kit (Thermo Fisher Scientific). Real-time PCR was done with the 96-well PrimePCR Pathway Plates (fibrosis pathway) (Biorad, Milan, Italy) with 15 ng of template for each well. Amplification and data acquisition were performed using a HT7900 Fast Real-Time PCR system (Thermo Fisher Scientific). Data were analyzed using the 2-(ΔΔCt) method, normalizing on beta glucoronidase expression. Statistics. All data were analyzed with the use of GraphPad Prism software (version 7). The differences in fluorescence intensity during ex vivo optical imaging were analyzed with Student’s unpaired t test (*p < 0.05, **p ≤ 0.005, ***p ≤ 0.0005). Data are reported as mean ± SE. For CD68 and NP colocalization we used one-way ANOVA followed by Tukey’s post hoc test (***p < 0.0001). Data are reported as a scatter plot. For pharmacokinetics, one-way ANOVA was followed by Bonferroni’s post hoc test (*p < 0.05, **p < 0.005). Data are reported as mean ± SE.

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ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b09655. Detailed information on the NP synthesis and characterization, HPLC chromatograms, immunofluorescence, and histopathology (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Martina Bruna Violatto: 0000-0002-9016-410X Laura Talamini: 0000-0001-6010-2947 Paolo Bigini: 0000-0002-0239-9532 Margherita Morpurgo: 0000-0002-8767-1995 Author Contributions ●

P.B. and M.M. equally contributed to the coordination of this study. Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENTS The authors gratefully acknowledge the financial support of the Italian Ministry of Health: Project Code RF-2013-02356221 and cc-2015-2365332 (Agreement No. 43/2017). REFERENCES (1) Lohse, A. W.; Mieli-Vergani, G. Autoimmune Hepatitis. J. Hepatol. 2011, 55, 171−182. (2) Heneghan, M. A.; Yeoman, A. D.; Verma, S.; Smith, A. D.; Longhi, M. S. Autoimmune Hepatitis. Lancet 2013, 382, 1433−1444. M


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