Highly Selective Targeting of Hepatic Stellate Cells for Liver Fibrosis

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Highly selective targeting of hepatic stellate cells for liver fibrosis treatment using a D-enantiomeric peptide ligand of Fn14 identified by mirror-image mRNA display Luying Huang, Jing Xie, Qiuyan Bi, Zhuoxuan Li, Sha Liu, Qing Shen, and Chong Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b01174 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on March 31, 2017

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Molecular Pharmaceutics

Highly selective targeting of hepatic stellate cells for liver fibrosis treatment using a D-enantiomeric peptide ligand of Fn14 identified by mirror-image mRNA display

Luying Huanga, Jing Xiea, Qiuyan Bia, Zhuoxuan Lia, Sha Liua, Qing Shenb,c,*and Chong Lia,*

a

College of Pharmaceutical Sciences, Southwest University, Chongqing 400715, China

b

School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of

Education, Fudan University, Shanghai 201203, China c

State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute,

Renji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai 200032, China

*

Author to whom correspondence should be addressed. Mailing address: College of Pharmaceutical Sciences, Southwest University, No. 2 Tiansheng Road, Beibei District, Chongqing 400715, China. Tel.: +86-23-68251225. Fax: +86-23-68251225. E-mail: [email protected]. School of Pharmacy, Key Laboratory of Smart Drug Delivery, Ministry of Education, Fudan University, Shanghai 201203, China. E-mail: [email protected]. 1

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Abstract: Although liver fibrosis is a major public health issue, there is still no effective drug therapy in the clinic. Fibroblast growth factor-inducible 14 (Fn14), a membrane receptor highly specifically expressed in activated hepatic stellate cells (HSCs), is the key driver of liver fibrosis, and thus it has a great potential as a novel target for the development of effective treatment. Here, we identified a D-enantiomeric peptide ligand of Fn14 through mirror-image mRNA display. This included the chemical synthesis of a D-enantiomer of the target protein (extracellular domain of Fn14), identification of an L-peptide ligand of D-Fn14 using a constructed mRNA peptide library, and identification of a D-enantiomer of the L-peptide, which is a ligand of the natural Fn14 for reasons of symmetry. The obtained D-peptide ligand showed strong binding to Fn14 while maintaining high proteolytic resistance. As a targeting moiety, this D-peptide successfully mediated high selectivity of activated HSCs for liposomal vehicles compared to that of other major cell types in the liver and significantly enhanced the accumulation of liposomes in the liver fibrosis region of a carbon tetrachloride-induced mouse model. Moreover, in combination with curcumin as an encapsulated load, a liposomal formulation conjugated with this D-peptide showed powerful inhibition of the proliferation of activated HSCs and reduced the liver fibrosis to a significant extent in vivo. This Fn14-targeting strategy may represent a promising approach to targeted drug delivery for liver fibrosis treatment. Meanwhile, the mirror-image mRNA display can provide a new arsenal for the development of D-peptide-based therapeutics against a variety of human diseases.

Keywords: mirror-image mRNA display; curcumin; liposomes; liver fibrosis

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Introduction

Liver fibrosis is the major pathological feature in most types of chronic liver diseases. Without intervention, it can deteriorate into liver cirrhosis, resulting in substantially high morbidity and mortality worldwide.1 There is overwhelming evidence that the activation and proliferation of hepatic stellate cells (HSCs) play a pivotal role in this progressive fibrogenesis, providing a promising cell target for therapy.2, 3 Unfortunately, because of the compromised functions of the fibrotic liver, the drug uptake by fibrogenic cells is usually low, while off-target effects can be high, which greatly limits the application of antifibrotic agents in the clinic.4 Therefore, the key factor in medicinal intervention is whether the drug is specifically accumulated in the responsible cells, HSCs. Accordingly, active drug-targeting strategies have been investigated to improve the drug efficacy and to reduce the side effects on non-target tissues. An obvious progress has been made in the design of HSC-targeting drug delivery systems (TDDS) such as carriers with surface-coupled mannose-6-phosphate (M6P) to target HSCs via the M6P receptor, retinol-modified liposomes targeting the retinol-binding

protein

receptor,

cyclic

arginylglycylaspartic

acid

(RGD)

peptide-conjugated particles recognizing integrin, etc.5-8 However, most of the systems still remain in the preclinical development stage, and some, such as RGD-modified

particles,

cause

concerns

that

they

would

affect

normal

intergrin-expressing hepatic cells.9 Therefore, it is necessary to identify other potential receptor–ligand systems and construct corresponding TDDS to achieve the precise drug delivery. Fibroblast growth factor-inducible 14 (Fn14) is a cell-surface receptor belonging to the tumor necrosis factor (TNF) receptor family. Importantly, Fn14 was found to be significantly upregulated in activated HSCs, while its levels were very low in quiescent HSCs and many other hepatic cells.10 Even more importantly, Fn14 itself participates in the progression of fibrotic liver disease,10 thus making it a very attractive potential target for the development of TDDS and therapy against liver fibrosis. Peptides, especially peptide ligands of certain receptors, have been widely used 3

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in targeted drug delivery as a trafficking moiety because of their small sizes, ease of synthesis and modification, and a low probability of undesirable immunogenicity. Since targeting ligands are usually exposed on the surface of drug carriers, stability of these peptides under physiological conditions may greatly influence the in vivo fate of TDDS. Therefore, proteolysis-resistant D-enantiomeric peptides have raised increasing interest as biostable targeting molecules in recent years.11-14 Thus, a peptide named angiopep-2 is a brain-targeting molecule, owing to its affinity to the low-density lipoprotein receptor-related protein 1 (LRP1) distributed in the blood–brain barrier (BBB). A D-peptide ligand of LRP1, although its binding to the receptor is weaker than that of angiopep-2, more efficiently mediates the transport of drug carriers across BBB, suggesting the superiority of the D-peptide to the natural L-peptide ligand.14 Considering that the liver is a key metabolic organ with a great variety of enzymes, it is possible that a D-peptide targeting ligand will better qualify for targeted drug delivery against liver diseases than its linear L-peptide counterpart. Nevertheless, there is currently no approach to directly obtain a D-peptide targeting moiety. Retro-inverso isomerization is currently the most explored method to generate a possible D-peptide ligand based on an existing L-peptide, i.e., to reverse the sequence of the parental peptide and replace all residues with D-amino acids (except glycine, which has no enantiomer). The postulate is that the side-chain topology of the produced retro-inverso isomer is similar to that of its parent peptide and can emulate the bioactivity.15, 16 However, retro-inverso isomerization is not suitable for all kinds of peptides; in particular, it works very poorly for molecular mimicry of helical peptides.17 Alternatively, mirror-image phage display is a promising method to directly screen for a D-peptide ligand of a target protein.18,

19

The idea of the

symmetry transition is the key to the whole process. It involves first replacing the original target protein by a chemically synthesized D-enantiomer as a target for screening. After obtaining an L-peptide that binds to the D-protein, the D-enantiomer of this L-peptide is the D-peptide ligand of the original protein for reasons of symmetry. More importantly, the above symmetry transition strategy may also be 4

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combined with other ligand-screening technologies, thus leading to more methods to obtain biostable ligand molecules.20 The extracellular part of Fn14 is mainly comprised of a cysteine-rich domain stabilized by three disulfide bonds as the recognition site.21 Through the mature solid-phase peptide chemistry and thiol/disulfide chemistry, it is quite possible to synthesize and correctly fold the Fn14 extracellular domain, creating a good starting point to screen for a biostable ligand of Fn14. Besides phage display, there are many other molecular display technologies such as bacterial display, yeast display, ribosome display, etc., among which mRNA display has its unique advantages. For example, the sizes of mRNA libraries are well beyond the limit of most other screening technologies, thus increasing the probability of selecting very rare sequences (a strong binder to the target).22 We report here the identification of a D-peptide ligand of Fn14 via mirror-image mRNA display and its systematic evaluation as a highly stable targeting moiety for liver fibrosis-targeting drug delivery and therapy.

Experimental Section Materials and Animals. Oligonucleotides and a random cassette were ordered from Sangon BioTech (Shanghai, China). EasyTaq DNA polymerase was purchased from TransGen BioTech (Beijing. China). The RNAsin ribonuclease inhibitor was supplied by Promega (Madison, WI, USA). Streptavidin (SA) magnetic beads and a six-tube magnetic separation rack were supplied by New England Biolabs (USA). Escherichia coli JM109 competent cells were obtained from Takara BioTech (Tokyo, Japan). Phosphatidylethanolamine distearoyl methoxy polyethylene glycol conjugate (mPEG2000–DSPE) was supplied by AVT (Shanghai, China), and maleimide -derivatized PEG–DSPE, MAL–PEG3400–DSPE (MPD), was purchased from Laysan Bio (New York, NY, USA). 1,1′-Dioctadecyl-3,3,3′,3′-tetramethylindotricarbo cyanine iodide (DiR), a near-infrared dye, was obtained from Invitrogen (Carlsbad, CA, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was purchased from Solarbio (Beijing, China). Coumarin-6 and curcumin (CUR) were provided by TCI (Tokyo, 5

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Japan). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Sephadex CL-4B were purchased from Sigma–Aldrich (St. Louis, MO, USA). All other reagents were of analytical grade. Male Kunming mice (4-6-week-old, 20 ± 2 g) were purchased from the Third Military Medical University’s experimental animal center (Chongqing, China) and maintained at 25 °C with free access to food and water in a special pathogen-free laboratory of the animal environmental facilities. To induce liver fibrosis, 2 mL/kg of 40% (v/v) CCl4 in olive oil was injected intraperitoneally two times per week for 8 weeks. Animal care and use were performed in compliance with the guidelines of the Ethics Committee of the College of Pharmaceutical Sciences, Southwest University. Cells. Activated HSCs (HSC-T6) and normal liver cell line BRL-3A were purchased from KeyGEN (Nanjing, China). HSC-T6 cells, primary murine HSCs, liver sinusoidal endothelial cells (LSECs), and Kupffer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, high glucose; Gibco) containing 10% newborn calf serum (NBCS; Gibco, Grand Island, NY, USA). BRL-3A cells were cultured in RPMI-1640 medium (Gibco) supplemented with 10% NBCS. All cells were maintained at 37 °C in a humidified atmosphere containing 5% CO2. Chemical Synthesis of Target Protein (D-Fn14). The extracellular domain of Fn14 (Fn14ECD, sequence: EQAPGTAPCSRGSSWSADLDKCMDCASCRARPHS DFCLGCAAA) was synthesized by Boc solid-phase peptide synthesis. After the chain assembly, the peptide was cleaved from the resin by HF containing 5% p-cresol for 1 h at 0 °C. The crude product was then precipitated by cold ether and purified by preparative reverse-phase high-performance liquid chromatography (HPLC) to homogeneity. Oxidative folding of the purified Fn14ECD was performed by dissolving the peptide at 0.5 mg/mL in 6 M guanidine-HCl containing 12 mM reduced glutathione and 1.2 mM oxidized glutathione, followed by rapid dilution with 0.25 M NaHCO3. After stirring gently overnight at room temperature, the final product was purified by HPLC. Mirror-Image mRNA Display. The mRNA display library (the constructal methods refer to supporting Information) was incubated with biotin–Fn14 in binding 6

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buffer [1 M NaCl, 5 mM ethylenediaminetetraacetic acid, and 10 mM 4-(2-hydroxy ethyl)-1-piperazineethanesulfonic acid (HEPES), pH 7.2] at room temperature for 30 min. Then, the binding reaction was transferred to 50 mL of preblocked SA magnetic beads for approximately 20 min, and the beads were washed five times with phosphate-buffered saline (PBS) with Tween 20 to remove excess antigen. Finally, for the elution of selected binding molecules, RNase A was added to degrade mRNA. The antigen-bound molecules were digested with proteinase K in elution buffer at 37 °C for 30 min, then a magnet was applied, and the supernatant was transferred to a clean tube. Elution was repeated three times as above, the eluted fractions were combined, and the selected cDNA molecules were amplified by PCR to conclude the first round of selection. The remaining rounds followed the same protocol, and the PCR products amplified after the eighth round of selection were cloned into the pMD19-T vector and sequenced. The selected peptide was named the D-Fn14-binding peptide (D-FNB peptide) (Scheme 1). Characterization of D-FNB Peptide. Binding affinity of the D-FNB peptide was evaluated by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare). Using the biotin–SA-coupling method, the biotinylated target protein was immobilized on SA sensor chips. Serial dilutions of the peptide were prepared and injected over the chip surface in HBS-P buffer (0.01 M HEPES, 0.15 M NaCl, and 0.005% surfactant P20, pH 7.4). A crystal structure of the Fn14 protein was downloaded from www.pdb.org (Protein Data Bank code: 2rpj). Missing atoms in the protein structure were added using the Protein Preparation package in the Schrödinger software. The structure model of the D-FNB peptide was built with the Schrödinger software and optimized in the Optimized Potential for Liquid Simulations 2005 force field. The peptide was docked to the Fn14 protein using the Glide package in the Schrödinger software with default parameters.23-26 After analyzing the resulting binding mode, the figure presented later was created using the PyMOL software. Synthesis of D-FNB–MPD. MPD and D-FNB (DCDHDPDRDEDVDDDVDEDLD YDSDTDVDFDGDH) were combined through the addition reaction between the sulfhydryl group in D-FNB and maleimide in MPD. Briefly, 5 mg of MPD and 2 mg 7

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of D-FNB were dissolved in dimethylformamide (DMF) and gently stirred for 48 h at room

temperature.

The

synthetic

process

was

monitored

by

thin-layer

chromatography. The reaction mixture was dialyzed against deionized water to remove the free MPD. The remaining liquid was lyophilized for 24 h and stored at −20 °C until required. The conjugate (D-FNB–MPD) of D-FNB and MPD was confirmed using a matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-TOF MS) (autoflex speed™, Bruker, Germany). Preparation of Liposomes. CUR-loaded liposomes were prepared by a thin-film hydration method.27, 28 In brief, hydrogenated soybean phosphatidylcholine (HSPC):cholesterol:PEG2000-DSPE:D-FNB-MPD

(molar

ratio:

76:20:2:2)

for

D-FNB-modified liposomes (D-FNB-LIPs) and HSPC:cholesterol:mPEG2000-DSPE (molar ratio: 76:20:4) for PEGylated liposomes (mPEG-LIPs) were dissolved in chloroform and mixed with CUR, which was hydrated in PBS (pH 7.4) at 60 °C for 1 h, at a ratio of CUR to the total lipids of 1:30 (w/w). The obtained liposomes (CUR-LIPs) were homogenized five times on ice using a probe sonicator (Ningbo Scientz BioTech, China) at 600 W for 10 s and purified through Sephadex CL-4B to remove the free CUR. Coumarin-6/DiR-loaded liposomes were prepared using the same procedure, except that CUR was replaced with the fluorescent reagents. Evaluation of In Vitro Uptake and Cellular Endocytosis Mechanism. Cells (3 × 104 cells per well) were seeded into 48-well plates, allowed to adhere overnight, and treated with coumarin-6-loaded formulations for 2 h. Then, the liquid was removed, and the cells were rinsed with PBS. Cellular uptake of the liposomes was visualized using a fluorescence microscope (Olympus, Japan) after staining with DAPI and washing three times with PBS. For quantitative analysis, the cells were detached with trypsin and analyzed by flow cytometry (ACEA NovoCyte™, China). In inhibition competition experiments, cells were pre-incubated with the free D-FNB peptide and TNF-related weak inducer of apoptosis (TWEAK) for 1 h and incubated with D-FNB-LIPs for 2 h at 37 °C afterwards. The cells were rinsed with PBS, then trypsinized, suspended in the culture medium, and collected for analyzing by flow 8

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cytometry. In order to study the mechanism of D-FNB-LIP endocytosis, HSC-T6 cells were pre-incubated with diverse inhibitors, including chlorpromazine (20 µg/mL), filipin (10 µg/mL), monensin (4 µg/mL), colchicine (250 µg/mL), brefeldin A (10 µg/mL), and methyl-β-cyclodextrin (20 mg/mL) for 30 min.29 Then, coumarin-6-loaded D-FNB-LIPs were added, and the cells were incubated for an additional 2 h. After that, the fluorescent intensity of the cells was determined using a flow cytometer. Evaluation of Pathology. Liver tissue was fixed in 4% paraformaldehyde overnight and embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) and observed under an Eclipse CI microscope (Nikon, Japan). For Sirius red staining, sections were dewaxed in warm xylene and hydrated in graded alcohol and water. Thereafter, nuclei were re-dyed with hematoxylin for 30 min, and the slides were rinsed three times with water, followed by Sirius red staining for 1 h. The sections were washed in two changes of acidified water, dehydrated in ethyl alcohol, cleared in xylene, and mounted with neutral balsam. In Vivo Imaging Assay. Normal mice and mice with CCl4-induced liver fibrosis were injected with D-FNB-LIP/DiR or mPEG-LIP/DiR via the tail vein at different times during the 8-week treatment. The mice were anesthetized, and the hair was removed from the mice using 8% sodium sulfide solution. The mice were imaged 2, 6, 8, 12, and 24 h later using an in vivo imaging system (FX-Pro, Bruker) at an excitation wavelength of 730 nm and an emission wavelength of 790 nm. The corresponding ex vivo imaging assays were also performed. Tissue Distribution. Mice with CCl4-induced liver fibrosis were injected with different CUR-loaded formulations (10 mg/kg) via the tail vein. The mice were sacrificed 2, 6, 8, and 12 h later, and tissues were collected and homogenized in saline. Tissue lysates were extracted with ethyl acetate, air-dried, redissolved in a mobile phase (acetonitrile:2% glacial acetic acid = 70:30), and analyzed by HPLC. The concentration of CUR in the samples was calculated using a standard curve. Co-localization of Formulations in Liver Tissues. To study the cellular distribution of formulations in a normal and fibrotic liver, the mice were sacrificed 6 h 9

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after they were intravenously injected with coumarin-6-loaded D-FNB-LIPs or mPEG-LIPs. Their liver tissues were collected and fixed in 4% paraformaldehyde overnight. The tissues were embedded in Tissue-Tek (Sakura, USA) and sectioned (10 µm thick) by using a cryostat microtome (Leica, Germany). Then, the sections were incubated with an α-smooth muscle actin (α-SMA) antibody (Proteintech, catalog no. 14395-1-AP), Fn14 antibody (Santa Cruz Biotechnology, catalog no. sc-27142) or F4/80 antibody (Bioss, catalog no. bs-11182R) for 12 h at 4 °C. Next, the sections were washed and incubated, respectively, with either a cyanine 3 dye-conjugated anti-rabbit α-SMA secondary antibody (Proteintech, catalog no. SA00003-8) or with a fluorescein isothiocyanate-conjugated anti-goat Fn14 secondary antibody (Proteintech, catalog no. SA00009-3) for 6 h at 4 °C. Nuclear staining was performed with DAPI and visualized using a fluorescence microscope. In Vivo Anti-Fibrosis Effect. Kunming mice intraperitoneally injected with CCl4 for 4 weeks were divided into three groups (n = 6) to evaluate the effect of different CUR-loaded preparations (10 mg/kg), including D-FNB-LIP, mPEG-LIP, and PBS groups. Each group was treated with the CUR-loaded formulations twice per week for 4 weeks via the tail vein, in parallel with the continued CCl4 administration. The efficacy of the treatments was assessed by determining biochemical factors, evaluating the pathology, and by immunofluorescence staining. For the biochemical assays, alanine transaminase (ALT) and aspartate transaminase (AST) were measured at 505 nm using commercial kits (Jiancheng BioTech, Nanjing, China). Statistical Analysis. All collected data are expressed as the mean ± standard deviation (SD) or standard error of the mean (SEM). Comparisons between groups were achieved by one-way analysis of variance tests. All statistical data were calculated using the SPSS 11.5 statistical software (Chicago, IL, USA), and p < 0.05 was considered statistically significant.

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Results and Discussion Screening and Characterization of a D-peptide Ligand of Fn14. As shown in Figure 1, Fn14ECD was successfully synthesized and folded. The determined MW of 4,433.60 kD was in good agreement with the theoretical MW of 4,433.94 kDa and the loss of six mass units confirming the formation of three disulfide bridges. The mRNA display screening was carried out for eight rounds, until a dominant sequence was identified and chemically synthesized with the following D-amino acids: DCDHDPDR D D

E VDDDVDEDLDYDSDTDVDFDGDH. Figure 2A shows the binding between Fn14 and

the D-FNB peptide, determined by SPR. After fitting with a 1:1 Langmuir model, the affinity of the D-FNB peptide to Fn14 was calculated to be 261 nM, suggesting its potential as an Fn14-targeting moiety. We also applied a molecular docking approach to further explore the interactions between the peptide ligand and the Fn14 receptor (Figure 2B). In a possible binding mode of the D-peptide ligand, the N-terminal amino groups of His1 and Arg3 formed an electrostatic interaction network with Asp45 and Asp62 in the Fn14ECD protein, and Glu4 formed an electrostatic interaction with Arg58 in Fn14ECD. At the tail of the peptide, Phe14 formed a positive ion–π interaction with Arg56 in Fn14. The Val5, Val7, Tyr10, and Val13 residues were bound to the protein via hydrophobic interactions. Preparation and Characterization of Liposomes. Since the D-FNB peptide contains a sulfhydryl group that can react with maleimide, the targeting compound was synthesized by the conjugation of D-FNB (MW 1,988.21 Da) with MPD (MW 4,200 Da) in DMF through the addition reaction. As shown in Figure 3A, the difference between the experimental MW of MPD and D-FNB–MPD, determined by MALDI-TOF MS, was about 2,000 Da, which was in accordance with the theoretical value and confirmed the identity of the synthetic product. The particle sizes of the liposomes modified with D-FNB–MPD or mPEG2000–DSPE were all less than 150 nm (Table S1). In particular, for the liposomes loaded with the model drug CUR, the encapsulation efficacy was approximately 90%. The spherical shape and lipid bilayer of the drug-loaded 11

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liposomes were observed under TEM, and no aggregation was detected (Figure 3B). In vitro drug release profiles of the CUR-loaded formulations are shown in Figure 3C, suggesting that 65% of the loaded CUR was released and that no significant differences existed between D-FNB-LIP/CUR and mPEG-LIP/CUR at various time points. Thus, the results indicated that the modification of liposomes with D-FNB–MPD showed no obvious effect on the particle size, EE%, and the release profile compared with those of mPEG-LIPs. D-Peptide Mediated Selective Targeting of Activated Hepatic Stellate Cells in Vitro. Various model cell lines were examined by immunofluorescent staining to detect the expression of biomarkers. Then, the cellular internalization of D-FNB-LIPs was

investigated

qualitatively

and

quantitatively

via

the

detection

of

coumarin-6-positive cells by fluorescence microscopy and flow cytometry, respectively (Figures 4A, 4B, S1, S2 and S3). D-FNB-LIPs/coumarin-6 were more efficiently integrated by HSC-T6 cells than mPEG-LIPs/coumarin-6 based on the finding that after the incubation for 2 h, the fluorescence intensity was approximately 2.54-fold higher in the HSC-T6 cells treated with the D-FNB-modified vesicles than in those treated with mPEG-LIPs (Figure 4B). The similar trend was also found in cellular uptake assay using activated primary murine HSCs (Figure S3C). However, there were no significant differences in the vesicle uptake among BRL-3A cells, LSECs, and Kupffer cells (Figures S1B and S2B). Preincubation of HSC-T6 cells with the free D-FNB peptide or TWEAK greatly inhibited the uptake of the targeting vesicles by the cells (Figure 4C). These data indicated that the D-FNB peptide specifically recognized Fn14-overexpressing cells, which supported the potential use of D-FNB-LIPs for drug delivery into activated HSCs in vivo. Our data indicated that cellular endocytosis of D-FNB-LIPs, unlike that of mPEG-LIPs, involved two main mechanisms, clathrin- and caveolin-dependent pathways, since chlorpromazine and filipin markedly reduced the cellular uptake of D-FNB-LIPs (by 49.6% and 26.0%, respectively) (Figure 5A, B).29 CUR, a well-known and widely used natural active compound isolated from turmeric, has been reported to exert an inhibitory effect against HSCs and has been 12

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considered in recent decades a promising anti-fibrotic agent.30 Therefore, it was selected as a potential therapeutic agent in this study. In vitro cytotoxicity of the peptide-modified CUR formulation for target cells was detected and quantified by the MTT assay. As expected, FNB-LIPs/CUR demonstrated significantly higher cytotoxicity against HSCs (for HSCs-T6, IC50 = 7.3 µg/mL, and for primary HSCs, IC50 = 9.1 µg/mL) than mPEG-LIPs/CUR (IC50 = 18.6 and 24.2µg/mL, respectively) (Figure 6A and S4) while normal hepatocyte cells, BRL-3A, showed no clear selectivity for the different treatments (Figure 6B). It has been reported that curcumin can inhibit HSC proliferation and promote apoptosis while induce HSC senescence through PPARγ signaling pathways.31, 32 Therefore, the in vitro anti-fibrosis effect of CUR formulations was further evaluated by the TUNEL and Western blot assay (Figure 7). Compared to the mPEG-LIP/CUR treated group, the D-FNB-LIP/CUR treatment could induce more target cell apoptosis and also significantly alleviate the expression of α-SMA, suggesting the inhibition of HSCs activation.33, 34 D-Peptide Mediated Active Targeting of Diseased Tissues in Vivo. The levels of hepatic fibrosis were evaluated in the model mice at designated time points (0, 2, 4, 6, and 8 weeks) using several assessment methods, including H&E and Sirius red staining and biochemical assays (Figure S5). After 4 weeks of continuous treatment with CCl4, central necrosis of the hepatic lobule and inflammation with central vein bridging necrosis were obvious, and Sirius red+ collagen appeared in fibrous septa and portal areas in the model mice.35 The serum levels of ALT and AST, which are the markers of hepatic damage, were significantly higher in the model mice, especially in the groups with long-term CCl4 induction (4, 6, or 8 weeks), than in the normal mice (Figure S5B). In addition, the livers of the normal mice had a smooth surface with homogeneous and flexible textures, while those of the model mice (4 weeks of CCl4 treatment) were covered with fibrous capsules, and their textures became sclerosal (Figure S5C). These results demonstrated that the liver fibrosis model was successfully established and 4 weeks could be considered the key time point. Accordingly, in vivo imaging assays were performed as shown in Figure 8 in the normal and CCl4-treated model mice treated with different formulations. It was 13

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observed that the FNB-modified liposomes significantly improved the distribution of encapsulated fluorescence in the fibrotic liver compared to the non-modified vesicles at the same time points. Moreover, as the model mice deteriorated from 2 to 8 weeks of CCl4 induction, the fluorescence intensity of FNB-LIP/DiR-treated livers became increasingly stronger, indicating a positive response of the Fn14 expression levels in the fibrotic livers to the FNB-embellished formulation. It is also worth noting that the FNB-LIP/DiR treatment produced relatively long-lasting fluorescence signals in the livers in all groups, unlike the quick decrease by mPEG-LIP treatment, suggesting that, to some extent, the FNB-LIPs can avoid the uptake by mononuclear phagocyte system (MPS) in the livers via targeting the HSCs. A similar trend was also detected by the quantitative analysis of in vivo drug distribution in the normal and model mice after a single dose of 10 mg/kg of the CUR formulations. Based on Figure 9A and Table S2, FNB-LIPs/CUR were more prominently accumulated in liver tissues than mPEG-LIPs/CUR. Furthermore, the fluorescence signals of coumarin-6-loaded FNB-LIPs were co-localized strongly with the α-SMA- and Fn14-positive areas while weakly with the F4/80-positive areas in fibrotic liver tissue, indicated that the modification of liposomes with FNB remarkably enhanced the liver-targeting efficiency of the drug delivery systems, especially their ability to precisely hit the diseased sites (Figure 10 and Figure S6). Besides, the fluorescent immunostaining against F4/80 also revealed evidence of markedly increased number of macrophages in fibrotic liver tissues (Figure S6), mainly due to recruitment of circulating monocyte subsets into the injured liver and subsequent differentiation in the inflamed hepatic environment.36 It can therefore provide a possible explanation for our observation that the mPEG-LIPs were equally taken up in fibrotic livers versus normal livers while the abnormal neovascular structure and function altered during angiogenesis in hepatic fibrosis may also influence the uptake of liposomes (Figure 8 and Figure 9).37 In Vivo Anti-Fibrosis Effect. The model mice treated with CCl4 for 4 weeks were continuously induced by CCl4 administration for the following 4 weeks while simultaneously receiving CUR (10 mg/kg) in different formulations twice per week. 14

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The therapeutic effects were then assessed by the evaluation of pathology, immunofluorescence staining, and biochemical factors. H&E and Sirius red staining showed that the treatment with CUR-loaded FNB-LIPs obviously alleviated hepatic fibrosis, unlike treatments with CUR-loaded mPEG-LIPs and the free drug (Figure 11A, S7). We also detected, by examining the expression of Fn14 and the fibroblast marker α-SMA in liver tissues after the therapy with the different formulations of CUR, that FNB-LIPs/CUR remarkably decreased the infiltration of Fn14+ and α-SMA+ myofibroblasts in the fibrotic livers of the treated mice (Figure 11B). Furthermore, the serum levels of ALT and AST were significantly lower in the mice treated with the targeting formulation compared with the mPEG-LIP/CUR- and free CUR-treated groups (Figure 11C), once again showing a notable curative effect of the liposomal CUR coupled with the Fn14-targeting ligand. Liposomes have a natural homing activity to the liver, mainly owing to the uptake into MPS, which can be regarded as passive targeting to whole liver tissues. However, active-targeted drug delivery to a certain region in the liver has been reported relatively rarely, especially since HSCs are present in a relatively small amount and lack specific biomarkers to differentiate them from neighboring parenchymal cells. Therefore, Fn14, which is significantly upregulated in fibrotic tissues, provides a very attractive target to mediate drug targeting. Moreover, anti-Fn14 antibodies have already been tested in clinical trials for some other diseases related to Fn14,38 which, in turn, may greatly support further development of the Fn14-targeting strategy and corresponding formulations to overcome the current situation of no effective drugs available for liver fibrosis therapy. Compared with small chemicals and macromolecules, peptides have unique properties, which make them promising targeting ligands; however, their metabolic stability is the major bottleneck. Since proteolysis is a stereoselective process, i.e., proteases specifically recognize natural peptides/proteins with L-amino acids, the use of D-amino acids may provide a fundamental solution under certain circumstances. Upon a parallel evaluation of this D-FNB peptide and the L-FNB peptide identified by conventional mRNA display, the D-peptide ligand showed superiority to its natural 15

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counterpart (Figures S8 and S9). A new drug, etelcalcetide, very recently approved in the European Union, has a D-amino acid backbone, and more D-peptide drug candidates are currently in clinical trials, showing a bright future for D-peptide-based therapeutics.

Conclusion We successfully designed an innovative approach, the mirror-image mRNA display, to screen for a biostable peptide ligand of the Fn14 receptor for targeted delivery of drugs for liver fibrosis. Our study may offer an efficient strategy for the development of D-peptide targeting moieties against a variety of human diseases.

Supporting Information. Supporting Information is available free of charge on the ACS Publications website at DOI: ***. More experimental details: the construction of DNA libraries for mRNA display, isolation of primary HSCs, LSECs and Kupffer cells, characterization of liposomes and the cell viability assays (PDF). Acknowledgment. This work was supported by the National Natural Science Foundation of China (Grant Nos. 21272187 and 81673376), Fundamental Research Funds for the Central Universities (Grant Nos. XDJK2015A012, XDJK2016E126, and XDJK2016E127), and National Natural Science Foundation of Chongqing (Grant No. cstc2015jcyjBX0100).

Conflict of Interest. The authors declare no competing financial interests.

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Scheme 1. Screening for a D-peptide ligand of Fn14 by mirror-image mRNA display.

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Figure 1. Evaluation of the synthesized Fn14ECD by MS and HPLC. (A, B) Mass spectra of the folded (A) and unfolded (B) Fn14 protein, illustrating its molecular mass by the mass-to-charge ratio (m/z). (C) HPLC chromatogram of the folded Fn14 protein.

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Figure 2. Interaction between the D-FNB peptide and Fn14ECD. (A) Surface plasmon resonance assay of the binding of the D-FNB peptide and Fn14ECD. Data are presented as the means ± SD (n = 3). (B) Details of the binding mode of the D-peptide (blue color). Fn14ECD (gray color) is shown as a ribbon. Residues involved in the binding are represented by sticks, and hydrogen-bond networks are denoted by black dashed lines.

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Figure 3. Characterization of D-FNB-modified liposomes. (A, B) MALDI-TOF MS analysis of D-FNB–MPD (A) and MAL–PEG3400–DSPE (MPD) (B). The arrow indicates the peak corresponding to the targeting product. The molecular weight of D-FNB–MPD was determined to be approximately 6,200 Da. (C) Transmission electron microscopy images of D-FNB-LIPs (scale bar = 100 nm). (D) In vitro CUR release from D-FNB-LIPs/CUR and mPEG-LIPs/CUR in PBS (pH 7.4) with 2% SDS at 37 °C. Data are presented as the means ± SD (n = 3).

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Figure 4. Evaluation of in vitro cellular uptake of formulations. (A) Expression of α-SMA and Fn14 in BRL-3A and HSC-T6 cells. Representative fluorescence images show the association between the expression of Fn14 (red) and the fibroblast marker α-SMA (green) in cells (40×). Flow cytometric analysis of (B, C) the interaction of HSC-T6 and BRL-3A cells with different formulations containing coumarin-6 and (D) the competitive inhibition by D-FNB and TWEAK of the D-FNB-LIP uptake by HSC-T6 cells.

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Figure 5. A study of the uptake mechanism of (A) D-FNB-modified liposomes and (B) mPEG-LIPs by HSC-T6 cells (mean ± SD, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001.

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Figure 6. In vitro cytotoxicity of CUR in different formulations to (A) HSC-T6 cells and (B) BRL-3A cells. Cells were treated with CUR at 0.5–50 µg/mL in different formulations such as free CUR, D-FNB-LIP/CUR, and mPEG-LIP/CUR for 48 h (n = 3). The data are the mean ± SEM.

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Figure 7. TUNEL staining of cells with (A) mock treatment (negative control), (B) mPEG-LIP/CUR (20 µM) treatment, (C) D-FNB-LIP/CUR (20 µM) treatment, (D) DNase I (1500 U/ml) treatment (positive control), (E) no TdT added (negative control). Scale bar = 200 µm. (F) Levels of α-SMA on HSC-T6 were quantified by western blot. HSC-T6 were treated with mPEG-LIP/CUR (20 µM) or D-FNB-LIP/CUR (20 µM) for 24 h.

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Figure 8. Evaluation of in vivo targeting by formulations in normal and model mice. (A) DiR-loaded formulations were administered via the tail vein, and images of the normal and model mice treated with mPEG-LIP/DiR (a) or D-FNB-LIP/DiR (b) were taken after 2, 6, 8, 12, and 24 h. (B) Ex vivo fluorescent images of collected tissues of the normal and model mice treated with mPEG-LIP/DiR (a) or D-FNB-LIP/DiR (b).

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Figure 9. Biodistribution of D-FNB-MPD-LIP/CUR and mPEG-LIP/CUR in livers (A), blood (B), lungs (C), kidneys (D), and spleens (E) following the tail vein injection of a dose of 10 mg/kg. All values are expressed as the mean ± SEM.

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Figure 10. Co-localization of different coumarin-6 formulations in liver tissues. Fluorescence images of the liver uptake of coumarin-6 in different formulations (free coumarin-6, coumarin-6-loaded mPEG-LIPs, and coumarin-6-loaded D-FNB-LIPs) and the expression of (A) Fn14 (20×) and (B) α-SMA (20×).

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Figure 11. CUR-loaded D-FNB-LIPs decreased the liver fibrosis in a CCl4-induced fibrosis model. (A) H&E (200×) and Sirius red (40×) staining of liver tissue from the mice treated with CCl4 for 8 weeks and co-treated with different CUR formulations. (B) Expression of α-SMA and Fn14 in liver tissues of the mice treated with CUR-loaded formulations. Representative fluorescent images show the association between the expression of Fn14 (green) and the fibroblast marker α-SMA (red) in tissue samples from the mice with hepatofibrosis (20×). (C) AST and (D) ALT levels in the serum of the model mice after the therapy with CUR-loaded formulations. Values are the mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001.

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