Extracellular matrix-penetrating nanodrill micelles for liver fibrosis therapy

Journal Pre-proof Extracellular matrix-penetrating nanodrill micelles for liver fibrosis therapy Qian-Qian Fan, Cheng-Lu Zhang, Jian-Bin Qiao, Peng-Fei Cui, Lei Xing, Yu-Kyoung Oh, Hu-Lin Jiang PII:

S0142-9612(19)30715-X

DOI:

https://doi.org/10.1016/j.biomaterials.2019.119616

Reference:

JBMT 119616

To appear in:

Biomaterials

Received Date: 28 September 2019 Accepted Date: 9 November 2019

Please cite this article as: Fan Q-Q, Zhang C-L, Qiao J-B, Cui P-F, Xing L, Oh Y-K, Jiang H-L, Extracellular matrix-penetrating nanodrill micelles for liver fibrosis therapy, Biomaterials (2020), doi: https://doi.org/10.1016/j.biomaterials.2019.119616. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Ltd.

Extracellular matrix-penetrating nanodrill micelles for liver fibrosis therapy Qian-Qian Fan,a, 1 Cheng-Lu Zhang,a, 1 Jian-Bin Qiao,a Peng-Fei Cui,a Lei Xing,a, b, c Yu-Kyoung Oh,*, d Hu-Lin Jiang,*, a, b, c

a

State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China

Pharmaceutical University, Nanjing 210009, China b

Jiangsu Key Laboratory of Druggability of Biopharmaceuticals, China Pharmaceutical

University, Nanjing 210009, China c

Jiangsu Key Laboratory of Drug Screening, China Pharmaceutical University, Nanjing 210009,

China d

College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National

University, Seoul 08826, Korea *Corresponding Author Prof. Hu-Lin Jiang, State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China. E-mail: [email protected] (H.L. Jiang). Or Prof. Yu-Kyoung Oh, College of Pharmacy and Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul 08826, Republic of Korea. E-mail: [email protected] (Y.K. Oh).

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These authors contributed equally.

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ABSTRACT As hepatic stellate cells (HSCs) are essential for hepatic fibrogenesis, HSCs targeted nano-drug delivery system is a research hotspot in liver fibrosis therapy. However, the excessive deposition of fibrosis collagen (mainly collagen I) in the space of Disse associated with hepatic fibrogenesis would significantly hinder nano-formulation delivery to HSCs. Here, we have prepared a collagenase I and retinol co-decorated polymeric micelle that possess nanodrill-like and HSCstarget function based on poly-(lactic-co-glycolic)-b-poly (ethylene glycol)-maleimide (PLGAPEG-Mal) (named CRM) for liver fibrosis therapy. Upon encountering collagen I barrier, CRM exerted a nanodrill-like function, efficiently degrading pericellular collagen I and showing greater uptake by human HSCs than other micelle formulations. Besides, CRM could realize excellent accumulation in the fibrotic liver and accurate targeting to activated HSCs in mouse hepatic fibrosis model. Moreover, CRM loaded with nilotinib (CRM/NIL), a second-generation tyrosine kinase inhibitor used in the treatment of liver fibrosis, showed optimal antifibrotic activity. This work suggests that CRM with dual function is an efficient carrier for liver fibrosis drug delivery and collagenase I decorating could be a new strategy for building more efficient HSCs targeted nano-drug delivery system. Keywords: Liver fibrosis therapy; Hepatic stellate cell; Extracellular matrix; Polymeric micelle; Collagenase I; Retinol

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1. Introduction Liver fibrosis is a critical pathological feature that accompanies all forms of chronic hepatic diseases [1,2]. Progressive fibrosis often results in cirrhosis, which is associated with high morbidity and mortality rates worldwide [3]. Fortunately, statistics from laboratory research and clinical trials have shown that hepatic fibrosis, unlike cirrhosis, is reversible and can be remedied by drugs [4,5]. Accordingly, a number of antifibrotic drugs have been developed and researched for use in liver fibrosis therapy in recent decades [6,7]. Most of these chemical agents have shown potential antifibrotic activity in vitro or in vivo. However, efficacy and applications are limited owing to their low water solubility, poor oral absorption, and/or lack of targeting ability [8-10]. As an example, nilotinib (NIL), a second-generation tyrosine kinase inhibitor, has been researched extensively for potential efficacy in the treatment of liver fibrosis [11-13]. However, the poor water solubility and low lipid solubility of NIL have seriously limited its application in humans [14]. Following liver injury, hepatic stellate cells (HSCs), a type of liver-resident nonparenchymal cell present in the space of Disse [15], are activated from their resting state by reactive oxygen intermediates or cytokines and are transformed to proliferative, fibrogenic, and contractile myofibroblasts. If the insult is sustained, activated HSCs secrete excessive fibrosis collagen, which subsequently becomes deposited in excess in the space of Disse and ultimately promotes liver fibrosis [5,16]. Activated HSCs are the major fibrogenic effecter in the pathogenesis of liver fibrosis and thus have become the main target cells of antifibrotic therapy [17]. Therefore, HSC-targeted nanodrug-delivery systems have become a focus in recent research on liver fibrosis therapy [18,19]. It has previously been reported that HSCs in the resting state are a major type of retinol-storing cell in the body, taking up and storing ~80% of

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retinol from the circulation [20]. Interestingly, it has been found that decorating nanocarriers with retinol helps address such carriers to activated HSCs in vivo [21-25]. To date, however, the fact that excessive fibrosis collagen (mainly collagen I) deposited in the space of Disse upon hepatic fibrogenesis would hinder the delivery of nano-formulations to HSCs has been largely ignored [26,27]. Notably, it has been demonstrated that endowing nanocarriers with a proteolytic surface could facilitate their permeation in the tumor extracellular matrix (ECM) [28-31]. Therefore, removal and/or proteolysis of excessive collagen I may facilitate nanocarrier permeation in hepatic fibrosis tissue. Monomeric type I collagen consists of two extended α1 chains and one α2 chain, twisted into a triple helix, which also makes it resistant to most proteases except collagenase [32]. It has been reported that collagenase firstly partially decomposes the triple helix structure of collagen, which makes collagen more susceptible to proteolytic enzymes [33]. Inspired by these previous findings, we developed a polymeric micelle based on poly-(lactic-coglycolic)-b-poly(ethylene glycol)-maleimide (PLGA-PEG-Mal) that is co-decorated with collagenase I and retinol (termed CRM). The PLGA component of CRM improves the solubility of hydrophobic antifibrotic drugs, and the presence of a PEG chain confers a long circulation time in the body [34]. In the fibrotic liver, the collagenase I decorating CRM facilitates permeation of fibrotic ECM; thereafter, the retinol decorating the surface enables CRM to efficiently recognize and target HSCs. The synergistic effects of modified collagenase I and retinol in CRM create a “nanodrill”-like action that allows precise delivery of cargos to HSCs. For comparison, we also prepared undecorated PLGA-PEG polymeric micelles (M), retinoldecorated PLGA-PEG polymeric micelles (RM), and collagenase I-decorated PLGA-PEG polymeric micelles (CM) as controls (Figure 1). The physicochemical properties of these four

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types of polymeric micelles were investigated and the efficiency of their uptake by human HSCs (LX-2 cells) in the presence of an excessive collagen I barrier were compared. The in vivo biodistribution of these polymeric micelles was also studied at the level of organs and cells in a nonlethal hepatic fibrosis model. In addition, the antifibrotic activity and toxicity profile of polymeric micelles loaded with the antifibrotic drug NIL were analyzed.

Figure 1. (A) Schematic illustration of the preparation of four different polymeric micelles. Polymeric micelles were fabricated by a dialysis method. Collagenase I and retinol (vitamin A, VA) were linked to the polymeric micelles surface via a maleimide-thiol coupling reaction. (B) Schematic illustration of the proposed destiny of the four different polymeric micelles in vivo. The nanodrill-like CRM/NIL is able to penetrate the collagen barrier and target activated HSCs. Internalization of CRM/NIL allows the release of NIL, which reduces expression of the

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metallopeptidase inhibitor, TIMP-1, which in turn enhances collagen I degradation, thereby exerting therapeutic action against liver fibrosis. 2. Materials and methods 2.1 Materials PLGA20k-PEG5k-maleimide (PLGA: lactic acid:glycolic acid, 50:50), Mal-PEG2k-COOH SH-PEG2k-COOH were purchased from Daigang (Jinan, China). Vitamin A was obtained from Energy Chemical (Shanghai, China). Dulbecco’s Modified Eagle Medium (DMEM) and 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from KeyGEN Biotech (Nanjing, China). Fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Waltham, Massachusetts, USA). Trypsin-EDTA solution (0.25%) was obtained from Gibco (Burlington, Canada). 4-Dimethylaminopyridine (DMAP) and N-hydroxysuccinimide (NHS) was purchased from Aladdin Industries Inc. (Nashville, TN, USA). Collagenase I was purchased from Invitrogen (Carlsbad, California, USA). Traut’s reagent and nilotinib was obtained from J&K Chemicals (Beijing, China). All other chemicals and reagents of the highest purity available were obtained from commercial sources. 2.2 Preparation of polymeric micelles NIL was loaded into PLGA20k-PEG5k-maleimide (PLGA20k-PEG5k-Mal) using a dialysis method [35]. Briefly, PLGA20k-PEG5k-Mal and NIL (4:1 w/w ratio) were dissolved in dimethyl sulfoxide (DMSO). The polymer and drug solution was added drop-wise into deionized water (1:3 v/v). Then, the solution was dialyzed against deionized water (14 kD MWCO) for 24 h and centrifuged at 800 × g. The supernatant was filtered and stored at 4 °C. Collagenase I was first thiolated via Traut’s reaction by adding 5x Traut’s reagent to 5 mg/mL of collagenase I solution

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in PBS and allowing the reaction to proceed for 1 h at room temperature with stirring. After the reaction, the thiolated collagenase I (SH-Collagenase I) was purified on a desalting column (Thermo Fisher Scientific, Waltham, Massachusetts, USA) and used immediately. Mal-PEG2kVA and SH-PEG2k-VA were synthesized by mixing Mal-PEG2k-COOH or SH-PEG2k-COOH (0.05 mmol) and DMAP (0.01 mmol), NHS (0.1 mmol) and vitamin A (VA) (0.25 mmol) in a 20-mL DMSO solution with stirring in the dark at room temperature for 24 h. The solutions were then dialyzed (0.5 kD MWCO) for 48 h against DMSO to remove unreacted vitamin A, and then against deionized water for 48 h at 4 °C to remove DMSO. After dialysis, the solutions were stored at 4 °C. Four types of polymeric micelles were prepared via step-by-step grafting. M/NIL and RM/NIL were prepared by reacting NIL-loaded PLGA20k-PEG5k-Mal with SH-PEG2kCOOH or SH-PEG2k-VA at a 25:2 w/w ratio for 4 h at room temperature. The solution was then centrifuged at 9500 × g for 30 min, and the supernatant was removed. Precipitated polymeric micelles were resuspended in PBS and stored at 4 °C. CM/NIL and CRM/NIL were prepared by first reacting NIL-loaded PLGA20k-PEG5k-Mal with SH-Collagenase I for 12 h at room temperature. Mal-PEG2k-COOH was then added and reacted for 4 h at room temperature. SHPEG2k-COOH or SH-PEG2k-VA was subsequently added to the solution and reacted for 4 h at room temperature. The final ratio (w/w) of NIL-loaded PLGA20k-PEG5k-Mal to SH-Collagenase I to Mal-PEG2k-COOH to SH-PEG2k-COOH or SH-PEG2k-VA was 25:10:1:2. Finally, the solution was centrifuged at 9500 × g for 30 min. The supernatant was removed, and precipitated polymeric micelles were resuspended in PBS and stored at 4 °C. Other cargo-loaded polymeric micelles used in this study were prepared in the same manner. 2.3 Characterization of polymeric micelles

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The morphology of polymeric micelles was assessed using an H-7650 TEM system (Hitachi, Tokyo, Japan). Samples were diluted with distilled water and then loaded onto Formvar-coated copper grids, dried at room temperature, and stained with a 2% (w/v) aqueous solution of phosphomolybdic acid. Intensity-average diameters, polydispersity index (PDI) and zeta-potential values of polymeric micelles were measured at a concentration of 1 mg/mL using a ZetaPlus particle size and zeta potential analyzer (Brookhaven Instruments, Holtsville, NY, USA). 2.4 The quantification of collagenase I and vitamin A Collagenase I was quantified by the fluorescence spectroscopy (Spectra Max M5, Molecular Devices, USA). Firstly, 20 µL of FITC-collagen I (1 mg/mL in PBS) was incubated with 200 µL of four types of polymeric micelles or collagenase I (0 to 1 mg/mL in PBS) at 37 °C for 2 hours. Then the mixed solution was centrifuged at 6000 × g for 15 min. The fluorescence intensity of FITC in the supernatant was monitored at its excitation wavelength (450 nm) and an emission wavelength of 538 nm by fluorescence spectroscopy. The amount of vitamin A attached to the polymeric micelle was determined by ultraviolet-visible (UV-vis) spectroscopy at 328 nm. The concentration of polymeric micelles was measured after lyophilized. The graft ratio was calculated by dividing the weight of collagenase I or vitamin A in polymeric micelle by the combined weight of polymeric micelle. Four types of polymeric micelles were measured three times parallelly. 2.5 Assay of NIL loading content NIL loading content was analyzed by HPLC using a Shimadzu LC-20A system with an Ultimate XB-C18 (5 µm, 4.6 × 250 nm) column and a photodiode array (PDA) detector. The

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column

was

eluted

with

2%

acetic

acid

(pH

adjusted

to

6.8

with

a

25:75

triethylamine:acetonitrile solution) at a flow rate of 1 mL/min, and UV-absorption was monitored at 258 nm.

2.6 Cell culture The L02 human hepatocyte and HSC-T6 rat liver stellate cell line were purchased from the Chinese Academy of Sciences Shanghai Institute of Cell Bank (Shanghai, China). The LX-2 human hepatic stellate cell line was purchased from Xiangya Central Experiment Laboratory, Central South University (Hunan, China). L02, LX-2 and HSC-T6 cells were cultured in DMEM supplemented with 10% (v/v) FBS, 100 µg/mL streptomycin and 100 U/mL penicillin at 37 °C in a humidified 5% CO2 atmosphere. 2.7 Cell survival assay Briefly, L02 cells and LX-2 cells were seeded in 96-well plates at an initial density of 6 × 103 cells/well in 200 µL of growth medium and incubated for 12 h. The medium was then replaced with serum-free medium containing different concentrations of polymeric micelles. After further incubation for 24 or 48 h, 20 µL of MTT (5 mg/mL in PBS) was added to the cells. After incubating for 4 h at 37 °C in the dark, the medium was carefully removed and the formazan crystals formed by metabolically active cells were dissolved with 150 µL of DMSO. The absorbance of the solution in each well at 570 nm was recorded using a Multiskan Go ELISA plate reader (Thermo Fisher Scientific, Bremen, Germany). Untreated cells were used as a control. All experiments were conducted in quadruplicate. 2.8 Assay of cellular uptake

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L02 cells and LX-2 cells were seeded in 24-well plates at an initial density of 1 × 104 cells/well in 1 mL of growth medium and incubated for 24 h. The medium in each well was then carefully removed, and 500 µL of FBS-free medium containing free Cor6, M/Cor6, RM/Cor6, CM/Cor6 or CRM/Cor6 was added (Cor6 concentration, 0.025 µg/well). After incubating for an additional 6 h at 37 °C in the dark, cells were washed three times with PBS and harvested by trypsinization. The mean fluorescence intensity was measured by flow cytometry using a BD Accuri C6 system (BD Biosciences, New Jersey, USA). LX2 cells were seeded in 6-well plates at a density of 3.5 × 105 cells/well and incubated for 24 h. The medium was then replaced with serum-free medium containing M/NIL, RM/NIL, CM/NIL or CRM/NIL (NIL concentration, 30 µg/mL). After 2 h, cells were washed three times with PBS and collected, and the number of cells in each well was counted. Next, cells were lysed with cell lysis buffer and NIL was dissolved in methanol. After centrifuging at 13680 × g for 10 min, the supernatant was collected and lyophilized. The lyophilized powder was dissolved in mobile phase solution, and the NIL in each sample was analyzed by HPLC. 2.9 Cellular uptake of polymeric micelles in the presence of a collagen I barrier Polycarbonate membranes of Transwell chambers (8 µm pore size) were uniformly coated with 50 µL of rat tail type I collagen (3 mg/mL; Thermo Fisher Scientific) according to the manufacturer’s instructions to form a collagen I layer. LX-2 cells were seeded in 24-well plates at an initial density of 1 × 104 cells/well in 1 mL of growth medium and incubated for 24 h. The growth medium was then replaced with 500 µL of FBS-free medium, after which 100 µL of FBS-free medium containing free Cor6, M/Cor6, RM/Cor6, CM/Cor6 or CRM/Cor6 (Cor6 concentration, 0.025 µg/well) was added into the Transwell chambers. Cells were incubated for 1 or 6 h at 37 °C in the dark, then washed three times with PBS and harvested by trypsinization.

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The mean fluorescence intensity was measured by flow cytometry using a BD Accuri C6 system (BD Biosciences, USA). In addition, LX-2 cells were seeded in thin, glass-bottomed, 35-mm petri dishes at a density of 1 × 104 cells/dish in 1 mL of growth medium and incubated for 12 h. The medium was then replaced with 1 mL serum-free medium containing 150 µg FITC-conjugated collagen type I (1 mg/mL). After 5 h, 1 mL of FBS-free medium containing M/Dox, RM/Dox, CM/Dox or CRM/Dox (Dox concentration, 60 µg/well) was added and incubated with cells at 37 °C in the dark for 2 h. Media were then removed and cells were washed three times with PBS, after which cells were fixed with 4% paraformaldehyde and counterstained with Hoechst 33342 as per the manufacturer’s instructions. Finally, cells were imaged under an FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). 2.10 Immunofluorescence staining of collagen I in vitro LX-2 cells or L02 cells were washed and then fixed by incubating with 4% paraformaldehyde in Hank’s balanced salt solution (HBSS) solution for 20 min. Thereafter, cells were washed three times with HBSS and incubated with blocking buffer (5% donkey serum in HBSS) for 30 min at room temperature. After carefully removing blocking buffer, cells were incubated for 12 h at 4 °C with a rabbit anti-human collagen I primary antibody (Abcam, Cambridge, UK) diluted in blocking buffer. Cells were then washed three times with HBSS and incubated for 1 h at 4 °C with Cy3-conjugated donkey anti-rabbit secondary antibody (ZSGBBio, Beijing, China), diluted in blocking buffer. Thereafter, cells were washed three times, and nuclei were counterstained by incubating with Hoechst 33342 dye. Finally, cell fluorescence was analyzed using an FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). The intensity of Cy3-collagen I fluorescence was per cell was quantified using Image J.

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2.11 Intracellular distribution LX-2 cells were seeded in thin, glass-bottomed, 35-mm petri dishes at a density of 1 × 104 cells/dish in 1 mL of growth medium and incubated for 12 h. The medium was then replaced with serum-free medium containing CRM/Cor6 (Cor6 concentration, 0.05 µg/dish). After 2 or 4 h, media were removed, cells were washed three times with PBS, and LysoTracker Red DND99 (100 nM; Molecular Probes, OR, USA) was added and incubated with cells for 1 h. Cells were then stained with Hoechst 33342 as per the manufacturer’s instructions, and then fixed with 4% paraformaldehyde and visualized under an FV1000 laser-scanning confocal microscope (Olympus, Tokyo, Japan). 2.12 Assay of hemolytic activity Hemolytic activity was evaluated as previously reported [36,37]. Briefly, 200 µL of freshly diluted blood from 8-wk-old healthy male C57BL/6 mice was added to 800 µL of PBS (pH 7.4) containing different amounts (0.01, 0.05, 0.1, 0.5, 1 mg) of drug-free M or CRM polymeric micelles, or PEI 25K polymer. After incubating at 37 °C for 4 h, sample solutions were centrifuged at 800 × g for 5 min and 200 µL of each supernatant was placed in individual wells of 96-well plates. The released hemoglobin in each sample was measured at 540 nm using an ELISA plate reader. Blood mixed with PBS or PBS containing Triton X-100 served as negative and positive controls, respectively. The relative rate of hemolysis (%) was calculated as [(As – An)/(Ap – An)] × 100%, where As, An and Ap represent the absorbance of samples, negative controls and positive controls, respectively. All experiments were conducted in triplicate. 2.13 Animal treatments

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Eight-week-old male C57BL/6 mice (20 ± 2 g) in the same background were obtained from the Animal Centre of Yangzhou University (Yangzhou, China). All animal experiments were conducted under protocols approved by the Ministry of Health of the People’s Republic of China and followed Guidelines for the Care and Use of Laboratory Animals of China Pharmaceutical University (acceptance number: 2019-07-001). All mice were housed at 25 °C and 40-60% humidity with a 12 h light/dark cycle and free access to water and laboratory chow. Mice used for the CCl4-induced liver fibrosis model were randomly divided into groups and provided free access to water and laboratory chow. Fibrosis model mice received intraperitoneal injections of CCl4 in olive oil (1:4, 2.5 µL/g body weight) twice weekly for 4 or 8 wk, whereas mice in the control group received olive oil intraperitoneally [38]. 2.14 Acute toxicity study The acute toxicity of CRM was assessed by intravenously injecting normal C57BL/6 mice with drug-free CRM at doses of 25, 50, 125 and 250 mg/kg in a total volume of 0.1 mL once a day for three consecutive days. Control mice were injected intravenously with the same volume of sterile PBS. Twenty-four hours after the last injection, mice were sacrificed, and hearts, livers, spleens, lungs and kidneys were dissected and stored in 10% paraformaldehyde for subsequent histological analyses. 2.15 Tissue distribution study The tissue distribution of polymeric micelles was determined using DiR-loaded micelles instead of NIL-loaded micelles. Mice were anesthetized and the hair on their chest and abdomen was removed. Free DiR, M/DiR, RM/DiR, CM/DiR or CRM/DiR (DiR:body weight = 1:1 mg/kg), suspended in 100 mL of sterile PBS, was intravenously injected into fibrotic mice and nonfibrotic controls. At 5 and 10 h, and 1, 2, 3, 5, 7, and 9 d after injection, mice were

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anesthetized using isoflurane and subjected to in vivo fluorescence imaging with an in vivo imaging system (FX-Pro; Bruker) using excitation and emission wavelengths of 745 and 800 nm, respectively. On day 9, mice were sacrificed by cervical dislocation, and livers, lungs, spleens, and kidneys were removed for ex vivo imaging using the same system and settings as above. 2.16 Staining of Nanodrill micelles in liver tissues The cellular localization of polymeric micelles in liver was studied using DiI-loaded instead of NIL-loaded polymeric micelles. Free DiI, M/DiI, RM/DiI, CM/DiI or CRM/DiI (DiI:body weight = 1:1 mg/kg), suspended in 100 mL of sterile PBS, was intravenously injected into fibrotic mice once a day for three consecutive days. Twenty-four hours after the last injection, mice were sacrificed, and liver tissue was harvested and paraffin-embedded. HSCs were then labeled by incubating sections first with a rabbit anti-mouse α-SMA antibody (Abcam) at 4 °C overnight and then with an AlexaFluor 488-labeled goat anti-rabbit secondary antibody at 37 °C for 45 min. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI; SigmaAldrich) to label nuclei and frozen until analysis. Frozen sections were imaged with a Panoramic MIDI Slide scanner (3D HISTECH, Budapest, Hungary). 2.17 Assay of anti-fibrosis efficacy in vivo The antifibrotic activity of polymeric micelles in vivo was tested in fibrotic model mice. Mice in the control group were only received olive oil intraperitoneally. Free NIL concentration of 100 µg/mL was dissolved in a solution consisting of 4% DMSO, 30% PEG 300, and 5% Tween 80. Then, free NIL, M/NIL, RM/NIL, CM/NIL or CRM/NIL, suspended in 100 mL of sterile PBS, were intravenously injected into fibrotic mice (NIL:body weight = 1:2 mg/kg) twice a week for 3 weeks, respectively. CCl4 was also injected as above during the same 3weeks period. Three days after the last injection, blood was collected for immediate measurements of

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serum cytokines. Mice were then sacrificed, and their hearts, livers, spleens, lungs and kidneys were dissected and stored in 10% paraformaldehyde for subsequent histological analysis. Part of the liver was stored in liquid nitrogen for subsequent Western blot analysis. 2.18 Serum cytokine and hydroxyproline measurements Serum cytokines were measured using commercial ELISA kits (Jiancheng BioTech, Nanjing, China) according to the manufacturer’s instructions. Hydroxyproline (Hyp) contents were measured using hydroxyproline assay kit (Jiancheng Biotech) according to the manufacturer’s instructions. 2.19 Histological analysis and Fibrosis Quantification Paraffin-embedded liver tissues were sectioned with 5 µm. After deparaffinization and hydration, sections were stained using an H&E staining Kit (Jiancheng Biotech) or 0.1% (w/v) Sirius Red (Direct Red 80; Sigma-Aldrich) in a saturated aqueous solution of picric acid for 1 h. Slides were then rinsed twice in 0.01N HCl for 15 min each to remove unbound dye. After dehydration, slides were mounted and photographed. Sections were also stained using a Masson’s trichrome staining Kit (Jiancheng Biotech) according to the manufacturer’s instructions. The METAVIR scoring system was applied to quantify hepatic fibrosis by Wuhan Servicebio Technology CO., LTD. (Wuhan, China). 2.20 Western blot analysis For Western blot analysis, 60 µg protein was resolved by sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF (polyvinylidene difluoride) membranes (Merck Millipore, Billerica, Massachusetts, USA) by electroblotting. Membranes

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were blocked by incubating for 12 h at 4 °C in Tris-buffered saline/0.1% Tween-20 (TBST) containing 5% skim milk, and then incubated first with the appropriate primary antibodies in 5% skim milk at 4 °C overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 2 h. After washing, protein bands were analyzed using a CCD image system (Tanon 4200, Shanghai, China) and densitometrically quantified using Image J. Protein expression levels were normalized against β-actin. 2.21 Statistical analysis All experiments were repeated at least three times. All data are presented as means ± standard deviation (SD), and were analyzed using one-way analysis of variance ANOVA and Student-Newman-Keuls post hoc test. Statistical analyses were performed using SPSS 19.0. P-values < 0.05 were considered statistically significant; individual P-values are indicated in figure legends.

3. Results and discussion 3.1 Characterization of polymeric micelles In this study, four types of polymeric micelles—M/NIL, RM/NIL, CM/NIL and CRM/NIL—were prepared using step-by-step grafting procedure (Figure 1A). The average size of M/NIL, RM/NIL, CM/NIL and CRM/NIL, measured by dynamic light scattering (DLS), were determined to be 178.8 ± 12.8, 187.8 ± 8.9, 179.4 ± 12.9 and 217.0 ± 8.1 nm, respectively (Figure 2A). All types of polymeric micelles had a polydispersity index (PDI) less than 0.2, and transmission electron microscopy (TEM) images showed that all types of polymeric micelles displayed a uniform spherical morphology with an apparent core-shell structure (Figure 2B).

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Average diameters of M/NIL, RM/NIL, CM/NIL and CRM/NIL by TEM were 78.7 ± 9.6, 101.7 ± 4.7, 89.1 ± 8.6, and 87.6 ± 8.3 nm, respectively, which are smaller values than those yielded by DLS analysis. The difference is mainly attributable to the fact that polymeric micelles are in a hydrated state for DLS analysis and are in a dried state for TEM observations. M/NIL, RM/NIL, CM/NIL, and CRM/NIL exhibited zeta potential values of -5.01 ± 0.76, -7.33 ± 1.42, 13.01 ± 1.18, and -16.55 ± 2.92 mV, respectively (Figure 2C). The clear decrease in zeta potential values of CM/NIL and CRM/NIL is explained by the surface grafting of collagenase I. All types of polymeric micelles exhibited good size stability over 3 days in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) at 37 °C, likely owing to the presence of a PEG shell (Figure 2D). We further used fluorescein isothiocyanate (FITC)-labeled collagenase I to prepare CRM/NIL, which was then imaged using a Cyto Viva hyperspectral microscopy system. The green fluorescence emitted by FITC completely overlapped with CRM/NIL, demonstrating successful grafting of type I collagenase (Figure 2E). To measure the grafted collagenase I, we preincubated FITC-labeled collagen I with polymeric micelles at 37 °C for 2 h, and then measured FITC fluorescence in the supernatant. The amount of collagenase I attached to the CM and CRM was 0.25 ± 0.08 and 0.23 ± 0.06 wt%, respectively (Figure 2F). These results demonstrate that grafted collagenase I has similar enzymatic activity in CM/NIL and CRM/NIL. Besides, the amount of vitamin A attached to the RM and CRM was 0.32 ± 0.03 and 0.37 ± 0.02 wt%, respectively.

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Figure 2. (A) Average size distribution of different polymeric micelles, as determined by DLS. (B) Representative TEM images of different polymeric micelles. Scale bar: 100 nm. (C) Zeta potential of different polymeric micelles. (D) Size change of different polymeric micelles in DMEM containing 10% FBS at 37 °C. (E) Cyto Viva hyperspectral microscopy system images of CRM/NIL containing FITC-labeled collagenase I. (F) Collagenase I quantification. Standard

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curve of FITC in the supernatant at 538 nm. Values are expressed as means ± SD (error bars; *P < 0.05, **P < 0.01, ***P < 0.001; NS: no significant difference). 3.2 Cell viability and intracellular distribution of polymeric micelles in vitro The cell viability of drug-free polymeric micelles in the human hepatocyte line L02, human hepatic stellate cell line LX-2, and rat liver stellate cell line HSC-T6 were investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Figure S1). After a 24- or 48-h incubation, the viability of all cell types was higher than 80% at all polymeric micelle concentrations below 100 µg mL-1, demonstrating good biocompatibility of these drugfree polymeric micelles at these concentrations. Next, the pericellular collagen I matrix of LX-2 and L02 cells cultured on plastic plates was confirmed by immunofluorescence staining. Confocal laser-scanning microscopy (CLSM) images clearly showed excessive red fluorescence of collagen I surrounding LX-2 cells compared with that surrounding L02 cells (Figure 3A). A quantitative analysis confirmed this, showing that the average fluorescence intensity on the surface of LX-2 cells was 4.9-fold higher than that on L02 cells (Figure 3B). We further preincubated LX-2 cells with different polymeric micelles loaded with the fluorescent dye coumarin 6 (Cor6) for 6 h, and then analyzed the collagen I surrounding LX-2 cells by immunofluorescence staining. The collagen I surrounding LX-2 cells was clearly degraded following treatment with CM/Cor6 or CRM/Cor6 (Figure 3C). We then investigated the uptake of Cor6-loaded polymeric micelles by LX-2 cells. A flow cytometric examination revealed higher intracellular accumulation of RM/Cor6 in LX-2 cells compared with M/Cor6 after a 1- or 6-h incubation (Figure 3D). It is known that HSCs, which express high levels of surface receptors involved in retinol uptake, are the main cell type responsible for storing retinol in the body [20-22]. Consistent with this, several studies have

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reported that modification of nano-formulations with the ligand, retinol, promotes cellular uptake by LX-2 cells [23-25]. Notably, we found that intracellular accumulation of CRM/Cor6 and CM/Cor6, containing collagenase I, was significantly greater than that of the corresponding polymeric micelles RM/Cor6 and M/Cor6 lacking collagenase I, demonstrating that the presence of excessive collagen I around LX-2 cells impedes the absorption of nanoparticles and that CM/Cor6 and CRM/Cor6 achieved higher internalization through proteolysis of collagen I. Thus, among these four types of polymeric micelles, CRM/Cor6 achieved the highest cellular uptake by LX-2 cells through both the proteolysis function of collagenase I and enhanced-uptake effect of the retinol ligand (Figure 3D). We also tested the efficiency of NIL-loaded polymeric micelle uptake by LX-2 cells using high-performance liquid chromatography (HPLC), and found that CRM/NIL, like CRM/Cor6, showed the highest uptake among these four types of polymeric micelles (Figure S2). To assess the intracellular distribution of CRM/Cor6 in LX-2 cells, we investigated colocalization of Cor6 fluorescence with LysoTracker Red, which labels lysosomes/late endosomes, by CLSM after treatment with nanoparticles (Figure S3). These experiments revealed strong colocalization of the green fluorescence of Cor6 with the red fluorescence of LysoTracker Red at 30 min, indicating the CRM/Cor6 was internalized through the endocytic pathway and was eventually trapped inside lysosomal vesicles. The green fluorescence of Cor6 subsequently appeared in the cytoplasm at 2 h, and became diffusely distributed throughout the cytoplasm after 4 h. These observations indicate that drugs carried by CRM could achieve good intracellular release.

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Figure 3. Cellular uptake of polymeric micelles in vitro. (A) CLSM images of collagen I immunostaining (red) in L02 and LX-2 cells, and (B) Cy3-collagen I (Cy3-COL I) fluorescence intensity in L02 and LX-2 cells, quantified by Image J (n = 5). (C) CLSM images of collagen I immunostaining (red) in LX-2 cells preincubated for 6 h with different Cor6-loaded polymeric micelles. (D) Mean fluorescence intensity in LX-2 cells treated with free Cor6 or different Cor6loaded polymeric micelles for 1 or 6 h, measured by flow cytometry (n = 3). In (A) and (C), blue corresponds to nuclei counterstained with DAPI. Scale bar: 10 µm. Values in (B) and (D) are presented as means ± SD (error bars; *P < 0.05, **P < 0.01, ***P < 0.001; NS, no significant difference).

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3.3 Anti-collagen I activity in vitro Collagen I is the most abundant ECM protein and forms the scar tissue in liver fibrosis. In the next experiment, collagen I was pre-layered on polycarbonate membranes of Transwell chambers, after which free Cor6 or different Cor6-loaded polymeric micelles were added to the Transwell chambers, and uptake of polymeric micelles in LX-2 cell was measured by flow cytometry at 1 and 6 h (Figure 4A). This analysis revealed no significant differences in intracellular accumulation among Cor6, M/Cor6 and RM/Cor6 in LX-2 cells at 1 and 6 h (Figure 4B), indicating that excessive collagen I is truly a barrier against access of polymeric micelles to LX-2 cells and further that this obstacle is positively correlated with the quantity of collagen I. By comparison, CM/Cor6 and CRM/Cor6 showed higher intracellular accumulation, indicating that the presence of collagenase I on the surface of CM and CRM facilitated penetration of the collagen I barrier. In addition, CRM/Cor6 accumulated to a greater extent than CM/Cor6, indicating that decoration of the surface with retinol further promoted CRM/Cor6 targeting to HSCs, and enhanced uptake by LX-2 cells compared with CM/Cor6 (Figure 4B). Doxorubicin (Dox) is hydrophobic as same as nilotinib. Besides, Dox could emits a distinct red light under the laser of 475-485 nm, which is clearly distinguishes from the green fluorescence of FITC-Collagen I and could easily captured by confocal laser scanning microscopy. Considering these excellent properties of Dox, we chosen Dox as a model drug as a replacement of nilotinib to study the “nanodrill-like” performance of our designed polymeric carrier. Firstly, we formed a collagen barrier layer close to cells by adding FITC-labeled collagen I directly to a plate on where LX-2 cells were grown. We then added different Dox-loaded polymeric micelles to the plate, and analyzed cellular uptake of polymeric micelles by LX-2 cells and degradation of FITC-labeled collagen I after 2 h using CLSM (Figure S4A) and Image J

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(Figure S4B,C). As expected, collagen I barriers in CM/Dox and CRM/Dox groups were clearly degraded, demonstrating that decoration with collagenase I endowed polymeric micelles with a nanodrill-like function that allows better penetration under conditions of excessive collagen fibrosis. Besides, it had been proved that our polymeric nano-carriers, especially the CRM, could exhibit a good uptake behavior in LX-2 cells (Figure 3D) and achieve good intracellular release within 2 h (Figure S3), so we believe that our CRM/NIL could function as a “nanodrill” to penetrate collagen and then achieve good intracellular uptake and release in short time.

Figure 4. Cellular uptake of polymeric micelles through anti-collagen I barrier activity in vitro. (A) Illustration of the in vitro collagen I barrier model. (B) Mean fluorescence intensity in LX-2 cells measured by flow cytometry 1 and 6 h after adding free Cor6 or different Cor6-loaded polymeric micelles to Transwell chambers (n = 3). Values are expressed as means ± SD (error bars; *P < 0.05, **P < 0.01, ***P < 0.001; NS, no significant difference). 3.4 Distribution of DiR-loaded polymeric micelles in normal and fibrotic mice The compatibility of polymeric carriers with blood is very important for their persistence in the systemic circulation. Before testing the distribution of polymeric micelles in vivo, we

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evaluated the hemocompatibility of M- and CRM-type micelles using an in vitro hemolytic activity assay (Figure S5A,B) [34,35]. The hemolytic action of the cationic polymer PEI 25K caused complete lysis at concentrations greater than 0.5 mg/mL; by contrast, M- and CRM-type micelles caused no hemolysis, even at concentration as high as 1 mg/mL, implying negligible erythrocyte membrane disturbance. The greater hemolysis produced by PEI 25K compared with M and CRM may be a consequence of the high charge density of PEI 25K. In comparison, the low charge surface of M and CRM promote their high blood compatibility. The safety profile of CRM was further evaluated by assessing its acute toxicity (Figure S5C). To this end, healthy mice were intravenously injected with different concentrations of CRM once a day for three consecutive days. A histological analysis of dissected, hematoxylin and eosin (H&E)-stained tissues revealed no obvious tissue damage at concentrations up to 250 mg/kg, further supporting the biocompatibility of CRM. To establish a model with different stages of liver fibrosis, we intraperitoneally injected C57BL/6 mice with carbon tetrachloride (CCl4; diluted in olive oil) twice weekly for 4 or 8 wk (Figure 5A). As shown in Figure 5B, both Sirius Red and Masson’s trichrome staining of dissected liver tissues revealed continuously increased collagen fibrosis accumulation with increased CCl4 treatment duration. A semi-quantitative analysis of the area of Sirius Red staining by Image J indicated that hepatic collagen deposition in mice treated with CCl4 for 8 wk was 3fold higher than that in mice treated with CCl4 for 4 wk and 6-fold higher than that in normal mice (Figure 5C). These differences in pathological collagen deposition at different stages of liver fibrosis led us to speculate that the distribution of polymeric micelles would depend on pathological status. To test this, we injected normal mice or mice treated with CCl4 for 4 or 8 wk with different DiR-loaded polymeric micelles via the tail vein and measured near infrared

24

fluorescence emission by in vivo imaging. Consistent with our speculations, the distribution of these polymeric micelles differed according to pathological status. Differences among normal mice, mice treated with CCl4 for 4 wk and 8 wk are not only in fibrotic level, but also the hepatic metabolic capacity. As the fibrotic level increased, (i) fibrotic livers deteriorated gradually and were unable to clear micelles efficiently, (ii) HSCs activation and proliferation increased the binding efficiency of micelles, and (iii) collagen content increasing led to poor micellar permeability. In normal group, polymeric micelles reached a peak in livers at 10 h and then declined (Figure 5D,E), which may due to the normal liver metabolic capacity resulting the fast declining. In contrast, the fluorescence intensity of all types of polymeric micelles in the liver area of mice treated with CCl4 for 4 or 8 wk was maintained at a higher level from 5 h to 9 days (Figure 5D,E), which may result from the dysfunction of livers and the accumulated collagen in the space of Disse. Besides, different collagen content levels between mice in CCl4 for 4 wk and CCl4 for 8 wk further hindered the permeability of micelles and caused different maximum accumulation and peak time. However, CRM/DiR all exhibited notably excellent accumulation in normal and liver fibrosis model mice treated with CCl4 for 4 or 8 wk. Generally speaking, a believable reason for the difference of maximum accumulation and peak time of micelles among normal mice, mice treated with CCl4 for 4 wk and CCl4 for 8 wk is the exist of a balance between the fibrotic level, hepatic metabolic capacity and the property of polymeric micelles. At day 9, the liver, spleen, kidneys, lung and heart were removed and fluorescence emission was measured by ex vivo imaging (Figure S6). Fluorescence in the livers of normal and fibrotic mice was much higher than that in other tissues. In addition, CRM/DiR and CM/DiR yielded stronger signals in the liver of both normal and fibrotic mice than RM/DiR and M/DiR.

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Figure 5. Distribution of DiR-loaded polymeric micelles in nonfibrotic and fibrotic mice. (A) Schematic summary of animal studies. (B) Representative Sirius Red and Masson staining of liver tissue sections from normal mice, and mice treated with CCl4 for 4 or 8 wk. Scale bar: 100 µm. (C) Semi-quantitative analysis of the area of Sirius Red staining in the liver by Image J. Results are from five randomly selected fields for each specimen. (D) Representative in vivo

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near-infrared fluorescent images of normal mice, and mice treated with CCl4 for 4 or 8 wk, acquired 5, 10 h and 1, 2, 3, 5, 7, and 9 d after intravenous injection of different formulations. (E) Fluorescence intensity in livers of normal mice, and mice treated with CCl4 for 4 or 8 wk, expressed as average radiant efficiency units. (F) Colocalization of DiI and DiI-labeled polymeric micelles with activated HSCs in the livers of fibrotic mice treated with CCl4 for 8 wk. Liver tissue was sectioned and immunostained with an anti-α-SMA (smooth muscle actin) antibody (green) for activated HSCs and counterstained with the nuclear dye, DAPI (blue). Fluorescent DiI is shown in red. Colocalization was determined as yellow signals corresponding to perfect merges of green and red fluorescence. Values are expressed as means ± SD (error bars; *P < 0.05, **P < 0.01, ***P < 0.001; n = 3). The optimal polymeric carrier is expected to ultimately target activated HSCs in the liver. To test the targeting ability, we loaded different polymeric micelles with the hydrophobic fluorochrome,

1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine

iodide

(DiI),

then

intravenously administered these DiI-labeled polymeric micelles once a day for three consecutive days into mice that had been treated with CCl4 for 8 wk. Twenty-four hours after the last intravenous injection, mice were sacrificed and liver sections were immunostained for αSMA, a marker of activated myofibroblasts (a subset of HSCs) and counterstained with the nuclear dye DAPI. As shown in Figure 5F, in mice injected with CRM/DiI, red DiI fluorescence was found to have largely merged with the green fluorescence of HSCs to generate strong yellow signals compared with other groups, indicating that most DiI was located in activated HSCs in CRM/DiI treated group. Taken together, these results clearly demonstrate that during circulation in the bloodstream, collagenase I decoration helps polymeric micelles permeate ECM deposited in the fibrotic liver, whereas decoration with retinol helps polymeric micelles target HSCs in

27

vivo. Therefore, collagenase I and retinol co-decorated CRM carriers exhibit better delivery efficiency, as evidenced by their high liver accumulation and good HSC-targeting in vivo. 3.5 Antifibrotic activity of NIL-loaded polymeric micelles in vivo Serial intraperitoneal treatment of mice with CCl4 twice a week for 8 wk has been shown to cause nonlethal liver fibrosis as above. Adapting this additional fibrosis model to mice, we further assessed the antifibrotic potential of polymeric micelles loaded with the antifibrotic drug NIL. To this end, we administered different polymeric micelle formulations to mice twice weekly for 3 weeks, and evaluated therapeutic efficacy 3 d after the last treatment using biochemical assays and immunohistological analyses (Figure 6A). Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were clearly decreased to a greater extent in fibrotic mice injected with NIL, M/NIL, RM/NIL, CM/NIL or CRM/NIL than in those injected with PBS. Notably, in the CRM/NIL treated group, AST and ALT levels approached those of normal mice level (Figure 6B). These results demonstrate that CRM/NIL functions efficiently in liver fibrosis therapy. In addition to biochemical assays, we also evaluated therapeutic efficacy by investigating liver collagen fiber content using semi-quantitative immunohistochemical assays together with Sirius Red staining of fibrous tissues. In normal liver tissues, only perivascular collagen fibers stained red (Figure 6C). In contrast, in PBS-treated fibrotic mice, red-stained tissue areas were observed throughout the liver, indicating widespread fibrosis (Figure 6C) and the fibrotic areas comprised 8.2 ± 3.0% of the total area (Figure 6D). Treatment with NIL, M/NIL, RM/NIL, CM/NIL and CRM/NIL decreased liver fibrotic areas to 5.9 ± 2.1%, 5.2 ± 2.9%, 5.2 ± 1.0%, 4.3 ± 0.7%, and 3.3 ± 1.7%, respectively (Figure 6D). CRM/NIL treatment restored fibrotic areas to normal levels, demonstrating that CRM/NIL has a greater ability to repair fibrous tissues than other formulations. Further, semi-quantitative assays

28

of Masson’s trichrome staining of dissected liver tissues also showed that CRM/NIL treatment resulted in significantly reduction of collagen area (Figure S7A,B). As shown in Figure S7C, hydroxyproline contents in livers of CRM/NIL-treated fibrotic mice showed significantly decrease compared with those of NIL-, M/NIL-, RM/NIL- and CM/NIL-treatment. Besides, the Masson’s trichrome staining of dissected liver tissues were used to quantify hepatic fibrosis by METAVIR scoring. As shown in Figure S8A, there exists a scoring decline in CM/NIL and CRM/NIL groups. As shown in Figure 6C, H&E staining of liver tissues revealed clear inflammatory infiltration around hepatic sinusoids and tissue necrosis in the PBS group. This inflammatory response was mitigated by treatment with NIL, M/NIL, RM/NIL, CM/NIL or CRM/NIL. Therapeutic effects on fibrosis were also assessed by immunohistochemical staining of α-SMA in dissected liver tissues of all groups, and a distinct decline of α-SMA was observed in CRM/NIL treated group (Figure S8B). In addition, Western blot analysis (Figure 6E) showed that treatment with CM/NIL or CRM/NIL significantly decreased the content of tissue inhibitor of metalloproteinase-1 (TIMP-1) in fibrotic liver tissue, restoring it to normal levels, as determined by densitometric quantification of Western blots (Figure 6F and Figure S9). Moreover, few abnormalities were found in histological sections of heart, spleen, lung or kidney tissue after treatment in each group, further confirming the safety of these polymeric micelles (Figure S10). In our research, we have evaluated hepatic fibrosis by various methods including immunohistochemical staining, Western blots, hydroxyproline content measurements and quantification of hepatic fibrosis by METAVIR scoring. We also evaluated liver functions by checking serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT). These results show that CRM/NIL could restored hepatic function greatly, repaired most fibrotic

29

tissues, reduced the number of activated-HSCs, clearly improved hepatic necrosis and reduced the content of TIMP-1 to a normal level, demonstrating that the optimal treatment efficiency of CRM/NIL reflects its best delivery efficiency compared with other polymeric micelle formulations.

30

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Figure 6. Antifibrotic activity of NIL-loaded polymeric micelles in vivo. (A) Schematic diagram showing the induction of hepatic fibrosis by CCl4 in mice, and administration of NIL and NILloaded polymeric micelles. (B) Serum levels of AST and ALT, as measured by biochemical assays. (C) Representative Sirius Red- and H&E-stained liver tissue sections. Red areas in Sirius Red-stained sections indicate collagen deposition in fibrotic liver tissues. Scale bars: 100 µm (200x). (D) Semi-quantitative analysis of the area of Sirius Red-stained sections of all animals in each group. Results are from five randomly selected fields in each specimen (n = 5). (E) Detection of TIMP-1 protein by Western blotting in the livers of animals following different treatments. (F) Densitometric quantification of Western blotting results of three randomly selected animals in each group. Values are means ± SD (error bars; *P < 0.05, **P < 0.01, ***P < 0.001; NS: no significant differences). 4. Conclusions HSCs targeted nano-drug delivery system was significantly hindered by the excessive deposition of fibrosis collagen in the space of Disse associated with hepatic fibrogenesis. In the current study, we designed a nanodrill-like polymeric micelle (CRM) and demonstrated that it could penetrate the excessive collagen barrier characteristic of the fibrotic liver and ultimately achieve highly efficient HSC targeting. In parallel, we also prepared three other undecorated polymeric micelles (M, RM and CM) for comparison. These four different polymeric micelles were found to have similar physicochemical properties. In the presence of an excessive collagen I barrier, the nanodrill-like CRM realized the highest cellular uptake among these four types of polymeric micelles, mainly owing to the proteolysis function of collagenase I and enhanceduptake effect of the ligand retinol decorating CRM. In addition, CLSM revealed that the CRM carrier effectively released intracellular cargo in LX-2 cells. We also established a two-stage,

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nonlethal hepatic fibrosis model by serial intraperitoneal treatment with CCl4 for 4 or 8 wk, and used it to assess the stage-dependent accumulation of these polymeric micelles in the liver. These experiments showed that CRM exhibited the best accumulation in the liver in mice treated with CCl4 for 8 wk, whereas CRM and CM showed similarly high accumulation in the liver in mice treated with CCl4 for 4 wk. Furthermore, immunofluorescence staining demonstrated that CRM delivered cargos to activated HSCs more efficiently than CM. In addition, CRM loaded with the antifibrotic drug NIL produced optimal antifibrotic activity in the nonlethal hepatic fibrosis model produced by serial intraperitoneal treatment with CCl4 for 8 wk, demonstrating high liver accumulation and good HSC targeting. Importantly, we also demonstrated that CRM possesses excellent cell compatibility and hemocompatibility in vitro, and exhibits no acute or chronic toxicity in vivo. On the basis of these findings, we propose that CRM is an ideal HSC-targeting nanodrug delivery system for liver fibrosis therapy. Moreover, we are the first to show that decorating a nanocarrier with collagenase I facilitates penetration of the fibrotic liver and promotes good accumulation. This demonstrates that modification with collagenase I could be a new strategy for the design of more efficient vehicles for targeted liver fibrosis therapy. Conflicts of interest The authors declare no competing financial interest. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. 1 These authors contributed equally. Abbreviations

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HSCs, hepatic stellate cells; CCl4, carbon tetrachloride; NIL, nilotinib; ECM, extracellular matrix; PDI, polydispersity index; TEM, transmission electron microscopy; DLS, dynamic light scattering; FBS, fetal bovine serum; L02 cells, human hepatocyte line; LX-2 cells, human hepatic stellate cell line; HSC-T6 cells, rat liver stellate cell line; MTT, 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide; Cor6, coumarin 6; COL I, collagen I; Dox, doxorubicin; DiR,

1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine

iodide;

DiI,

1,1-dioctadecyl-

3,3,3,3-tetramethylindocarbocyanine iodide; i.p., intraperitoneal injections; H&E, hematoxylin and eosin staining; AST, aspartate aminotransferase; ALT, alanine aminotransferase; TIMP-1, tissue inhibitor of metalloproteinases-1. Acknowledgements This work was financially supported by the National Science and Technology Major Project (2017YFA0205400) and the National Natural Science Foundation of China (81773667, 81573369), and NSFC Projects of International Cooperation and Exchanges (81811540416). This work was also supported by the First-class Project (CPU2018GY06) and the “111” Project from the Ministry of Education of China and the State Administration of Foreign Experts Affairs of China (B16046). We thank the Cellular and Molecular Biology Center of China Pharmaceutical University for assistance with confocal microscopy work. Data Availability Appendix A. Supplementary data The following files are available free of charge in pdf format: cytotoxicity study, intracellular colocalization, hemolytic activity assay, acute toxicity test, organizational distribution, test of

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