Route to Rheumatoid Arthritis by Macrophage-Derived Microvesicle

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Expressway to Rheumatoid Arthritis by Macrophagederived Microvesicle-Coated Nanoparticles Ruixiang Li, Yuwei He, Ying Zhu, Lixian Jiang, Shuya Zhang, Jing Qin, Qian Wu, Wentao Dai, Shun Shen, Zhiqing Pang, and Jianxin Wang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.8b03439 • Publication Date (Web): 06 Dec 2018 Downloaded from http://pubs.acs.org on December 6, 2018

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Expressway to Rheumatoid Arthritis by Macrophage-derived MicrovesicleCoated Nanoparticles

Ruixiang Li 1,2*, Yuwei He 1*, Ying Zhu 3, Lixian Jiang 2, Shuya Zhang 1, Jing Qin 1, Qian Wu4, Wentao Dai4, Shun Shen1, Zhiqing Pang 1,#, and Jianxin Wang 1,5# 1 Department

of Pharmaceutics, School of Pharmacy, Fudan University & Key Laboratory of

Smart Drug Delivery, Ministry of Education, Shanghai 201203, China. 2

Innovation Research Institute of Traditional Chinese Medicine, Shanghai University of

Traditional Chinese Medicine, Shanghai 201203, China. 3 Institute

of Tropical Medicine, Guangzhou University of Chinese Medicine, Guangzhou 510405,

Guangdong Province, China. 4

Shanghai Center for Bioinformation Technology, Shanghai Industrial Technology Institute,

Shanghai 201203, China 5 Institute

of Materia Medica, The Academy of Integrative Medicine of Fudan University,

Shanghai 201203, China.

* These authors contributed equally to this work. # Corresponding Authors: Zhiqing Pang Tel.: +86-21-51980069; fax: +86-21-51980069; E-mail address: [email protected] Jianxin Wang Tel.: +86-21-51980088; fax: +86-21-51980088; E-mail address: [email protected] 1

ACS Paragon Plus Environment

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Abstract The targeted delivery of therapeutics to sites of rheumatoid arthritis (RA) has been a long-standing challenge. Inspired by the intrinsic inflammation-targeting capacity of macrophages, macrophage-derived microvesicle (MMV)-coated nanoparticle (MNP) was developed for targeting RA. MMV was efficiently produced through a novel method. Cytochalasin B (CB) was applied to relax the interaction between the cytoskeleton and membrane of macrophages, thus stimulating MMV secretion. The proteomic profile of the MMV was analyzed by iTRAQ (isobaric tags for relative and absolute quantitation). The MMV membrane proteins were similar to those of macrophages, indicating that the MMV could exhibit bioactivity similar to that of RAtargeting macrophages. Poly (lactic-co-glycolic acid) (PLGA) nanoparticle was subsequently coated with MMV, and the inflammation-mediated targeting capacity of the MNP was evaluated both in vitro and in vivo. The in vitro binding of MNP to inflamed HUVECs was significantly stronger than that of red blood cell membrane coated-nanoparticle (RNP). Compared with bare NP and RNP, MNP showed a significantly enhanced targeting effect in vivo in a collagen-induced arthritis (CIA) mouse model. The targeting mechanism was subsequently revealed according to the proteomic analysis, indicating that Mac-1 and CD44 contributed to the outstanding targeting effect of the MNP. A model drug, tacrolimus, was encapsulated in MNP (TRNP) and significantly suppressed the progression of RA in mice. The present study demonstrates MMV as a promising and rich material with which to mimic macrophages and that MNP is an efficient biomimetic vehicle for RA targeting and treatment. 2


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Keywords: rheumatoid arthritis, biomimetic nanoparticles, macrophage-derived microvesicle, proteomics, tacrolimus

Introduction Rheumatoid arthritis (RA) is one of the main causes of disability1 and has long been a threat to global health. As numerous studies have characterized, RA is an autoimmune disease associated with chronic joint inflammation and cartilage destruction2, 3. The common treatment for RA is a combination of anti-inflammatory drugs and immunosuppressants4, 5. However, due to the nonspecific targeting of these drugs, there is always the risk of severe side effects, such as osteoporosis, muscular atrophy and impaired immune function, thus greatly limiting their therapeutic efficacy. Recently, nanoparticle (NP)-based drug delivery systems (DDSs) have been developed for the targeting and treatment of RA with relatively reduced doses and side effects6, 7. Among them, biomimetic DDSs have attracted considerable attention. The combination of synthetic NPs with natural cell membranes can camouflage the drug carriers as endogenous cells, significantly reducing the elimination and prolonging the circulation of NPs8. Moreover, cell membranes can enable the fabrication of DDSs with specific functions mimicking those of the membrane’s cell of origin9, 10. Numerous inflammatory cells can infiltrate the synovium with the progression of RA. A large proportion of infiltrating cells are macrophages, which play a pivotal role in RA11. Macrophages are recruited by inflammatory chemokines, roll on the inflamed endothelium, and extravasate into the synovium. Evidence has shown that macrophages 3


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adhere to the synovium or the pannus of inflamed vascular tissue via specific ligands and become “resident”12,

13.

These properties raise the possibility of developing

macrophage-mimetic NPs, which could actively reach sites of RA and be trapped in the inflamed synovium, thus maintaining a high concentration of drugs in the target sites. In recent years, macrophage membranes have been successfully applied to develop biomimetic DDSs for targeting cancer or inflammation14-16. However, the capacity of macrophage-mimetic NPs for targeting RA has rarely been studied, and the underlying mechanism remains to be discovered. Based on membrane-coating technology, in preparing macrophage-mimetic NPs, the most important element is the outer cell membrane. Ensuring the efficacy and bioactivity of the macrophage membrane is essential for biomimetic NP preparation and RA targeting. However, the traditional method for macrophage membrane extraction is complicated and inefficient and involves several steps, including cell disruption, density gradient centrifugation and ultracentrifugation17, 18. To address these problems, macrophage-derived microvesicles (MMVs), mainly composed of the plasma membrane, may be used as a reliable substitute. Researchers have successfully stimulated the secretion of microvesicles from fibroblasts and embryonic kidney cells using cytochalasin B (CB)19, 20. As previously reported, one cell could produce more than 50 microvesicles. CB can reversibly affect the interaction between the cytoskeleton and the cell membrane, which strongly promotes microvesicle production21. Inspired by this discovery, in this study, CB was first applied to stimulate macrophages to produce many MMVs for NP cloaking and macrophage imitation. 4


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According to previous studies, membrane proteins play a dominant role in the biofunctions22, 23. Thus, performing a comprehensive analysis of the protein composition of MMV is essential to guarantee that the MMV will function well and even act similar to macrophages. Proteomics is an integrated approach for mapping proteins in biological systems. Recently, an increasing number of studies have revealed that proteomics is promising for studying the protein corona or protein fingerprint of NPs24. Accordingly, proteomic analysis may also be a viable approach for identifying the protein composition of MMV. In the present study, MMVs were prepared from macrophages by CB treatment (Fig. 1A). To systematically explore the protein composition of the MMV, the membrane proteins were determined by iTRAQ (isobaric tags for relative and absolute quantitation) and compared with those of the macrophage membrane. MMV-coated NP (MNP) was developed, and the capacity of the MNP for targeting sites of RA was highlighted in comparison with that of bare NP and red blood cell membrane coatednanoparticle (RNP). Moreover, the targeting mechanism was determined based on the proteomic analysis. Finally, the therapeutic efficacy of tacrolimus-loaded MNP (TMNP) was evaluated in an RA mouse model. The immunogenicity and safety of the MNP were also preliminarily examined.

Methods Preparation of MMV RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, 5


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USA) and cultured in Dulbecco’s minimum essential medium (DMEM, Corning, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% penicillin (100 IU/mL, Corning, USA), and streptomycin (100 µg/mL, Corning, USA). MMV was prepared using a previously reported process with some modifications19. Briefly, the cells were washed twice with phosphate buffer saline (PBS) and then incubated in 5 mL of serum-free DMEM containing 10 μg/mL CB (Abcam, UK) for 1 h at 37 °C. To observe

the

formation

of

MMV,

the

cells

were

stained

with

3,3′-

dioctadecyloxacarbocyanine perchlorate (DIO, Fanbo Biochemical, China) and subjected to confocal laser scanning microscopy. The cells without CB treatment were applied as control. To isolate MMVs, the cells and MMVs were detached from the culture dish by rinsing with 5 mL of DMEM, transferred into a 15-mL tube and vortexed for 5 min. Five milliliters of FBS was added to the tube to obtain a final FBS concentration of 50%. The suspension was subjected to centrifugation at 1000 rpm for 10 min to remove the cells and large MMV aggregates. Then, the supernatant was centrifuged at 4000 rpm for 15 min to collect the MMVs. The collected MMVs were then washed twice with deionized water (containing 0.25% EDTA) to remove the cytoplasm content and prepare the purified MMVs for later use. The protein quantity of the purified MMV samples was determined by bicinchoninic acid protein (BCA, Beyotime, China) assay. Analysis of the MMV protein composition The MMV protein composition was determined by an iTRAQ process25. Briefly, after the MMVs were collected by centrifugation, lysis buffer was added and the samples 6


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were sonicated for 60 s on ice. The MMV lysate was then subjected to centrifugation at 13000 rpm for 20 min at 4 °C. Then, the supernatant was collected into a 1.5-mL tube, and acetone was added to a 6:1 ratio (acetone to supernatant). The mixture was incubated at -20 °C overnight, followed by centrifugation to collect the protein pellet. After washing, the pellet was redissolved in buffer (300 mM triethylamine borane, TEAB, 6 M guanidine hydrochloride), followed by vortexing for 15 min. Then, the protein was quantitated by BCA assay and diluted with 100 mM TEAB at 1 mg/mL. The protein was subsequently digested by trypsin at 37 °C overnight. The resulting peptide was labeled using the iTRAQ kit according to the manufacturer’s protocol (iTRAQ Reagent-Multiplex Buffer Kit, ABSCIEX, Darmstadt, Germany). Then, the labeled peptide was subjected to a series of analyses; details can be found in the supplementary materials. An equivalent amount (by protein) of macrophage membrane served as the control in the same analysis. The macrophage membrane (Mmembrane) was isolated via a density gradient centrifugation method, as previously17. Preparation and characterization of MNP MMV membrane-coated NP (MNP) was prepared according to a previously described method26, 27. First, 0.67 dL/g carboxy-terminated 50:50 poly (lactic-co-glycolic acid) (PLGA) (LACTEL Absorbable Polymers, USA) was applied to form PLGA NP cores via a nanoprecipitation method. After PLGA was dissolved in acetone at a concentration of 10 mg/mL, 1 mL of the PLGA solution was added rapidly to 2 mL of deionized water and placed under vacuum to remove the acetone. The resulting NP solution was mixed with the MMVs at a protein ratio of 1:10 (w/w, protein to PLGA) 7


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and sonicated for 3 min using a water bath sonicator (GuTel, China) at a power of 100 W. Red blood cell membrane-coated nanoparticle (RNP) was prepared as previously described26. The RNP or MNP were labeled with 1,1’-dioctadecyl-3,3,3’,3’– tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt (DID) and 1,1’dioctadecyl-3,3,3’,3’-tetramethylindotricarbocyanineiodide (DIR, Fanbo Biochemical, China) using the same methods except that DID or DIR was incorporated into the polymer solution at a 0.1% (w/w) concentration. Tacrolimus-loaded NP (T-NP), RNP (T-RNP) or MNP (T-MNP) were prepared by adding 10% (w/w) tacrolimus (MedChemexpress, USA) during the PLGA core preparation. The morphology of the MNP was observed using a transmission electron microscope (Tecnai G2 20, FEI, USA) after staining with 1% uranyl acetate. The size distribution and zeta potential of the MMV, PLGA NP, RNP and MNP were measured using a Malvern Zetasizer (Nano ZS, Malvern, UK). To determine the encapsulation eﬃciency (EE) and drug loading capacity (DLC) of tacrolimus, T-MNP was collected through centrifugation at 15000 g for 30 min and dissolved in dimethyl sulfoxide. The amount of tacrolimus in the TMNP was measured by ELISA (Abnova, China)28. The EE and DLC were calculated using the following formulas: EE（%） =

Tacrolimus encapsulated in T - MNP Total tacrolimus

Tacrolimus encapsulated in T - MNP Weight of T - MNP

× 100%

DLC（%） =

× 100%

To evaluate the stability of the MNP, the change in MNP size was observed over one week at 4 ℃. To further determine the stability in blood, MNP was incubated with 50% FBS (Gibco), and changes in the turbidity over time were observed by determining the 8


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absorbance at 560 nm using a microplate reader. Bare NP served as a control. To determine the rate of tacrolimus release from the T-MNP, 2 mg of T-MNP was suspended in 1 mL of 0.5% (v/v) Tween 80 solution (in 1×PBS) in a tube, and the supernatant (1 mL) was sampled by centrifugation after 1, 2, 6, 12, 24, 48, 72, 96, 144 and 192 h (3 tubes for each time point). Bare T-NP was applied as control. Finally, the amount of tacrolimus was measured by ELISA according to the manufacturer’s protocol, and the release profile of tacrolimus was plotted. Protein composition of MNP The protein composition of the MNP was investigated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE). Briefly, 2 mg of MNP was collected by centrifugation at 15000 g for 30 min, mixed in SDS sample buffer (Invitrogen, USA) and heated at 90 °C for 5 min. Then, 20 µL of each sample was run on a 10% SDSpolyacrylamide gel (Bio-Rad, SDS-PAGE Gel Preparation Kit, USA) at 120 V for 1 h, followed by Coomassie blue staining and imaging. The expression of CD44 and Mac1 on the MNP was measured by western blotting. Briefly, NuPAGE® 1 × Laemmli sample buffer (LDS) sample buffer (Invitrogen, USA) was used to lyse the MNP and extract the total protein. Then, equivalent amounts of protein were separated by SDSPAGE and transferred to nitrocellulose membranes (PALL, USA). After being blocked with 5% nonfat milk in Tris-buffered saline (TBS, Amresco, USA) containing 0.1% Tween-20, the membranes were incubated with primary antibodies overnight at 4 °C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:200 dilution); the proteins were then detected using Immobilon™ 9


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Western Chemiluminescent HRP Substrate (Millipore, USA). Band intensities were quantified using densitometry with ImageJ software. The MMV was analyzed as a control. In vitro binding of MNP To determine whether the MMV coating could facilitate the active targeting of MNP, an in vitro binding experiment was performed. Briefly, Human Umbilical Vein Endothelial Cells (HUVECs, ATCC, USA) were seeded on cover slips in 24-well plates at a density of 1 × 105 cells/well in 1 mL of culture medium. After the cells reached 7080% confluence, the proinflammatory cytokine TNF-α (50 ng/mL, PeproTech, USA) was added to the HUVECs culture and incubated for 6 h to establish a model of inflamed vasculature. DID-labeled MNP was then added to the plate, cultured for 4 h at 4 °C, and then washed 3 times with PBS. The DID-labeled RNP was applied as a control. To further investigate the binding mechanism, the HUVECs were stained with Pselectin or ICAM-1. Briefly, after incubation with MNP, the cells were fixed with 4% paraformaldehyde and washed 3 times with PBS. Then, the cells were blocked with 10% BSA in PBS with 0.5% Tween 20 for 30 min. Primary antibody against P-selectin (1:100, 3633R, Biovision, USA) or ICAM-1 (1:100, ab171123, Abcam, UK) was subsequently added and incubated with the cells overnight at 4 ℃. After the cells were washed three times, fluorescein isothiocyanate (FITC)-labeled secondary antibody and 4’,6-diamidino-2-phenylindole (DAPI, Beyotime, China) were added and incubated with the cells for 30 min at room temperature. The colocalization of MNP with P10


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selectin or ICAM-1 was observed by confocal microscopy. Distribution of MNP in mice with RA The RA mouse model was established using collagen according to the manufacturer's instructions (Chondrex, USA). Bovine type II collagen (CII, Chondrex, USA) was dissolved in 0.1 M acetic acid at 4℃ (2 mg/mL) and then emulsified with an equal volume of complete Freund's adjuvant (CFA, Chondrex, USA) containing 2 mg/mL mycobacterium tuberculosis. Then, 100 μL of the emulsion was injected subcutaneously into 7-week-old DBA/1 mice at the base of the tail. Twenty-one days after the primary injection, the DBA mice received a booster injection of CII emulsified in incomplete Freund's adjuvant (IFA, Chondrex, USA). The in vivo circulation time of the MNP was determined as previously reported26. DID dye was applied to label the MNP, which was then injected into male ICR mice (20–22 g) through the tail vein (20 mg/kg). Then, 50 μL of blood was collected from the submaxillary vein after 1, 5, and 30 min and 1, 3, 8, and 24 h. After the plasma was obtained by centrifugation, fluorescence measurements were obtained using a microplate reader (excitation/emission = 644/665 nm). The same dose of DID-labeled NP was used as a control, and the relative signal-time curve was plotted. After the RA model was established, the biodistribution of the MNP in RA mice was observed by in vivo imaging. DIR dye was encapsulated in MNP, which was then intravenously injected into the mice (20 mg/kg) with RA (with one hindlimb morbid and the other hindlimb normal). Fluorescence images were obtained after 1, 2, 6, 12, and 24 h using an in vivo imaging system (IVIS, Caliper, USA). The same amount of 11


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DIR-loaded NP, RNP or free DIR solution were injected as controls. Twenty-four hours after the injection, the mice were sacrificed for ex vivo tissue distribution analysis. The fluorescence intensity of organs in the different groups was calculated after background subtraction with PBS group using the IVIS system. To explore the targeting mechanism, DID-loaded MNP was injected into mice (20 mg/kg) with or without RA. The mice were sacrificed 2 h after the injection. The synovial tissue was dissected and dehydrated in 30% sucrose solution overnight and then cryosectioned at 10 μm. The sections were then blocked with 10% BSA at room temperature for 30 min and incubated with P-selectin or ICAM-1 primary antibody at 4 ℃ for 12 h. After the samples were washed twice, Alexa Fluor 555-labeled second antibody (goat-anti rabbit antibody, Invitrogen, USA) was added to the sections and incubated for 30 min; then, the samples were stained with DAPI and washed twice. Fluorescence images were obtained using a confocal microscope (Carl Zeiss, German). The in vivo drug release of T-MNP was studied by intravenous injection of T-MNP (1 mg/kg tacrolimus) in the RA mice (n=15). The mice were sacrificed after 1, 2, 6, 12 and 24 h (n=3). The arthritic paws were obtained and weighted, which was then homogenized with 1 mL of 0.5% (v/v) Tween 80 solution (in 1×PBS). The homogenate was subjected to centrifugation at 15000 g for 30 min. The supernatant was collected and measured by ELISA. Free tacrolimus, T-NP and T-RNP were administrated at the same dose as controls. Treatment of RA mice with T-MNP The state of RA could be assessed visually by grading the paw swelling as previously 12


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described29: 0, no swelling; 1, one toe inflamed and swollen; 2, more than one toe but not entire paw inflamed and swollen; 3, moderate swelling of entire paw; 4, most severe erythema and swelling of the entire paw and ankle. The total score of the four paws was recorded for each mouse as an arthritis index. After the RA model was established, the mice with a score of 3 were divided into six groups randomly to receive different treatments: saline; free tacrolimus; T-NP; T-RNP; MNP and T-MNP (n=5). For each group, 1 mg/kg tacrolimus was administered every two days. The arthritis index of each group was recorded over time. After 14 days of treatment, the mice were sacrificed, and the limbs were collected for micro-computed tomography (micro-CT) analysis and histological examination. For the micro-CT analysis, the limbs of mice from the different groups were fixed for 24 h with 4% paraformaldehyde and then examined using a micro-CT imaging system (aluminum filter (0.5 mm), 50 kV, 497 μA, 18 μm resolution, SkyScan 1176, Beckman Coulter, USA). The images were further analyzed using CTvox version 2.7 software (SkyScan) to calculate the bone volume. All animal experiments were performed according to the Guiding Principles for the Care and Use of Experimental Animals in Fudan University (Shanghai, China). The protocols of the study were evaluated and approved by the Ethical Committee of Fudan University. Histological study The collected limbs were decalcified with PBS containing 15% EDTA for 1 month, embedded in paraffin and sectioned at a thickness of 4 µm. The specimens were then 13


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deparaffinized, and the antigens were retrieved by microwave. The sections were blocked using 3% BSA for 30 min, followed by incubation with primary antibodies (TNF-α, IL-Iβ, or IL-6) overnight at 4 °C. After washing, the sections were incubated with biotinylated secondary antibody for 50 min and subsequently incubated with streptavidin solution. Then, 10 μL of 3,3-diaminobenzidine tetrahydrochloride (DAB) was added as a chromogen, followed by counterstaining with hematoxylin. After staining, the sections were dehydrated through increasing concentrations of ethanol and xylene. The sections were then observed using an ECLIPSE TI-SR microscope (NIKON, Japan), and the staining intensity in each of 6 randomly selected fields per sample was calculated using ImageJ software (n=3). Moreover, the sections were stained using hematoxylin and eosin (H&E) to determine the level of cartilage erosion. Safety examination To determine the immunogenicity of the MNP, 0.5 mg of MNP was injected into ICR mice every two days (n=3). The blood was sampled after 7 days and subsequently analyzed by hematology and blood chemistry tests. The safety of the MNP was further evaluated by examining the main organs via histological sectioning and H&E staining. Samples from untreated mice were used as controls. Statistical analysis The data were analyzed using GraphPad Prism 5 (GraphPad Software, Inc., La Jolla, CA, USA). Significant differences were evaluated using an unpaired Student's t-test for the comparison of two groups and one-way ANOVA for multiple-group comparisons. 14


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The data are expressed as the mean ± standard deviation (SD), and a p value

Route to Rheumatoid Arthritis by Macrophage-Derived Microvesicle

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