Modulation of macrophage phenotype by biodegradable polyurethane

Subscriber access provided by University of Winnipeg Library

Biological and Medical Applications of Materials and Interfaces

Modulation of macrophage phenotype by biodegradable polyurethane nanoparticles: the possible relation between macrophage polarization and immune response of nanoparticles Yen-Jang Huang, Kun-Che Hung, Huey-Shan Hung, and Shan-hui Hsu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 18 May 2018 Downloaded from http://pubs.acs.org on May 18, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Modulation of macrophage phenotype by biodegradable polyurethane nanoparticles: the possible relation between macrophage polarization and immune response of nanoparticles Yen-Jang Huang†, Kun-Che Hung†, Huey-Shan Hung, Shan-hui Hsu, ,* §

Institute of Polymer Science and Engineering, National Taiwan University, Taipei,

Taiwan, R.O.C.

Translational Medicine Research, China Medical University Hospital, Taichung,

Taiwan, R.O.C. §

Institute of Cellular and System Medicine, National Health Research Institutes,

Miaoli County, Taiwan, R.O.C.

＊

Shan-hui Hsu (Corresponding author)

Institute of Polymer Science and Engineering, National Taiwan University, No. 1, Sec. 4 Roosevelt Road, Taipei 10617, Taiwan, R.O.C. Phone: (886) 2-33665313 Fax: (886) 2-33665237 E-mail: [email protected]

1

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 55

ABSTRACT Nanomaterials with surface functionalized by different chemical groups can either provoke or attenuate the immune responses of the nanomaterials, which is critical to their biomedical efficacies. In this study we demonstrate that synthetic waterborne polyurethane nanoparticles (PU NPs) can inhibit the macrophage polarization towards the M1 phenotype, but not M2 phenotype. The surface-functionalized PU NPs decrease the secretion levels of proinflammatory cytokines (TNF-α and IL-1β) for M1 macrophages. Specifically, PU NPs with carboxyl groups on the surface exhibit a greater extent of inhibition on M1 polarization than those with amine groups. These water-suspended PU NPs reduce the nuclear factor-κB (NF-κB) activation and suppress the subsequent NLR family pyrin domain containing 3 (NLRP3) inflammasome signals. Furthermore, the dried PU films assembled from PU NPs have a similar effect on macrophage polarization and present a smaller shifting foreign body reaction (FBR) in vivo than the conventional poly(L-lactic acid) (PLA). Taken together, the biodegradable waterborne PU NPs demonstrate surface-dependent immunosuppressive properties and macrophage polarization effects. The findings suggest potential therapeutic applications of PU NPs in anti-inflammation and macrophage related disorders, and propose a mechanism for the low FBR observed for biodegradable PU materials. 2


Page 3 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


KEYWORDS: polyurethane nanoparticles, macrophage polarization, autophagy, NLRP3 inflammasome, foreign body reaction.

3



Page 4 of 55

1. INTRODUTION Nanoparticles (NPs) present a novel approach for biomedical applications, and have been increasingly used in medical diagnosis, drug delivery, and tissue engineering.1 The NP characteristics such as size, form, and surface modification may influence the efficiencies of the NPs in translational medicine.2 Meanwhile, the oxidative stress and inflammation initiated by NPs may reduce the therapeutic efficacy and cause harmful effects on biological systems.3 The link between NP characteristics and inflammation has been an important issue that needs to be investigated and understood before the biomedical applications of NPs can be fully achieved. Macrophages are the first line to perform phagocytic clearance of foreign pathogens and materials to maintain local homeostasis in innate immunity and contribute antigen processing in adaptive immunity. Nevertheless, the unwanted interactions between nanomaterials and macrophages may lead to acute and chronic inflammatory diseases.4 Two polarized forms of macrophages have been suggested to play different roles in macrophage-mediated immune responses, the classically activated (M1) macrophage phenotype and the alternatively activated (M2) macrophage phenotype. The M1 macrophages are induced via interaction with proinflammatory signals such as interferon-γ (IFN-γ) and microbial products such as lipopolysaccharide (LPS). M1 macrophages produce proinflammatory cytokines (such 4


Page 5 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


as TNF-α and IL-1β) in response to prevent pathogen expansion. In the case of biomaterial implants, the prolonged inflammation of M1 macrophages leads to a severe foreign body reaction (FBR), granuloma, and fibrous encapsulation resulting in the failure of biomaterial integration.5 M2 macrophages consistently express the scavenger and mannose receptors (CD206) and anti-inflammatory cytokines such as IL-10. Within the M2 subsets, the M2a and M2b subsets exert immune regulatory functions by anti-inflammatory cytokines. The M2c subset plays an important role in tissue remodeling and suppression of inflammatory immune reactions.6 Hence, M2 macrophages can reduce the fibrous tissue formation and improve the intended function of biomaterials.5 Meanwhile, M1 macrophages promotes the initiation and propagation in acute and chronic inflammatory processes through IL-1β.7 In the latter, the inactive precursor pro-IL-1β is processed into active IL-1β by the enzyme caspase-1 in the inflammasome complex NLRP3 (NLR family, pyrin domain containing 3), leading to cell pyroptosis.8 Certain NPs have been reported to activate the inflammasome complex of macrophages and provoke the inflammatory responses.9-11 Although macrophages can be activated by NPs, the surface modification of the NPs can significantly modify the immune responses.12 The polymeric poly(L-lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) NPs could stimulate the 5



proinflammatory

cytokine

production,

which

is

Page 6 of 55

important

for

vaccine

development.13-14 Biodegradable polyurethane (PU) is also gaining attention in biomedical related fields because of the excellent elastic properties and biocompatibility. PU has another major advantages in the tunable physico-chemical properties.15 With these benefits, the NPs made of various PUs are used as drug nanovectors and can self-assemble into hydrogel or other biomaterials.16-18 The interaction of PU NPs and macrophages is thus an unexplored but essential issue for their further applications. PU elastomers are composed of hard and soft segments with a linear structure. and have been applied in medical devices for many decades. The hydrolysis-prone polyester diols, including PCL diol, PLA diol, and their copolymer, can be used as the soft segments to synthesize biodegradable PU, and the degradation products through hydrolysis had no obvious cytotoxicity.19-21 The surface characteristics and polymer chemistry are important in mediating the inflammatory responses and the secretion of cytokines to stimulate a cascade of events leading to the encapsulation of implants. PU films with the net surface negative charge may influence the early phase acute inflammatory response to an implant by reducing neutrophil invasion and macrophage activation.22

6


Page 7 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In this study, we evaluated the effect of PU NPs on the phenotype of THP-1 human macrophages. The THP-1 cell models have been widely used to study macrophage polarization, because the transcriptional responses of THP-1 cells were very similar to those of human peripheral blood mononuclear cell (PBMC) derived macrophages.23 We demonstrated that, unlike most NPs with immune stimulating properties, the PU NPs with different functional groups could not only suppress the immune response of macrophages but also reduce their polarization into M1 phenotypes. The preferential M2 macrophage population was preserved for the films cast from the PU NP dispersion as well. Therefore, the PU NPs containing specific surface functional group may be used to realize and design bioactive functional polymer NPs with good biocompatibility.

2. MATERIALS AND METHODS 2.1. Synthesis and physico-chemical analyses of waterborne PU NPs. PU NPs were made from a waterborne method. The reaction was conducted under nitrogen atmosphere and was verified by dibutylamine back titration method. PCL (poly ε-caprolactone) diol (Mn ~ 2000 Da, Sigma) was selected to be the oligodiol soft segment and reacted with IPDI (isophorone diisocyanate, Evonik Degussa GmbH) in a vessel purged nitrogen at 75°C for ~3 h with 0.03% stannous octoate (Sn(Oct)2) as 7



Page 8 of 55

the catalyst. After the prepolymerization, the ionic chain extender DMPA (2,2-bis(hydroxymethyl)propionic acid, Sigma) and a limited amount of methyl ethyl ketone (MEK, J.T. Baker) were further added to react for another hour. When the vessel was cooled down to 45°C, triethylamine (TEA, RDH) was applied to neutralize the carboxylic group of DMPA. After half an hour, the neutralized prepolymer was dispersed by deionized water and stirred vigorously. In the end, ethylenediamine (EDA, Tedia) was employed for chain extension in another half an hour. The remaining MEK and TEA were eliminated through vacuum distillation. The product of PUwas in the form of NPs stably suspended in deionized water with a solid content of approximately 30 wt% PU. The ionic contents of DMPA ratios in PU NPs were 4.0 or 4.4 wt% in the whole PU molecule. The abbreviations of such PU NPs were PU-C′ (4.0 wt% DMPA) and PU-C (4.4 wt% DMPA). The stoichiometric ratios of IPDI/PCL diol/DMPA/EDA were 3.47/1/0.95/1.52 (PU-C′) and 3.57/1/1.05/1.52 (PU-C). For the preparation of amine-rich PU NPs abbreviated as PU-N, N-methyldiethanolamine (N-MDEA, Acros) with positively charged amine group was substitute for DMPA that contains COO− group. EDA was not used as chain extender in the synthesis of PU-N. The cationic charge was provided from the secondary amines of urethane group. The hydrodynamic diameter of PU NPs was examined by dynamic light scattering and the zeta potential was determined by electrophoretic light 8


Page 9 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


scattering, using a submicron particle analyzer (Delsa Nano, Beckman Coulter, USA). We made PU films by casting PU NP dispersion on 15 mm glass cover slips at 25οC. The spectra of PU surface were collected by the attenuated total reflection-Fourier transform infrared spectroscopy (ATR-IR, Spectrum 100, Perkin-Elmer, USA) on PU films.

2.2. Cell culture. Macrophages were obtained after differentiation of the human monocytic THP-1 cells. The THP-1 cells were grown in continuous culture in RPMI-1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco), which was previously heated for 30 minutes at 56oC to inactivate complement, and 1% penicillin–streptomycin–amphotericin (PSA) (Gibco). Cells were maintained at 37oC in a humidified 5% CO2 incubator, and subcultured for two times each week. For experiments, cells were seeded into a six-well culture plate (Greiner CELLSTAR®, Sigma) and treated with phorbol myristate acetate (PMA, 200 ng/ml) for 48 h to generate THP-1 macrophages macrophages (M0). To generate M1-polarized THP-1 macrophages, THP-1 cells were treated with 200 ng/ml PMA for 48 h and then cultured with PMA plus 100 ng/ml LPS and 20 ng/ml IFN-γ for 24 h. To generate M2-polarized THP-1 macrophages, THP-1 cells were treated with 200 ng/ml PMA for 48 h, and then cultured with PMA plus 20 ng/ml IL-4 and 20 ng/ml IL-13 for 9



Page 10 of 55

another 24 h. The morphology was observed under an inverted microscope (Leica, DMIRB). The polarized macrophages were further analyzed the expressions of CD surface markers with anti-CD86 antibody (ab53004, Abcam) for M1 macrophages and anti-CD206 antibody (ab64693, Abcam) for M2 macrophages, which were normalized to the macrophage marker CD68 (ab955, Abcam) by the BD FACSCalibur™ flow cytometer. The mean fluorescence intensity (MFI) was used for the quantitative analysis. The PBMCs were isolated from human fresh blood buffy coats by the density gradient centrifugation (Taichung Veterans General Hospital, Taichung, Taiwan, IRB-TCVGH approval number CG16100A) and were incubated with GM-CSF (50 ng/ml) or M-CSF (20 ng/ml) for 6 days to acquire the precursor cells of M1 and M2 macrophages, respectively. The precursor cells of M1 macrophages were treated with LPS (200 ng/ml) and IFN-γ (20 ng/ml) for 24 h, while the precursors of M2 macrophages were treated with IL-4 (20 ng/ml).21 The polarized macrophages were further characterized by cytokine secretion.

2.3. Enzyme-linked immunosorbent assay (ELISA) for secretion cytokines. To evaluate the secretion cytokines after macrophage differentiation and PU NP treatment, cells were treated with PMA (200 ng/ml) for 48 h in the complete medium at 37oC. After being incubated with PU NPs (solid content 100 µg/ml) for 30 min, 10


Page 11 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


cells were treated with LPS/IFN-γ or IL-4/IL-13 for 24 h respectively. The released cytokines in the cell media were measured according to manufacturers’ instructions by ELISA kits (R&D Systems, USA).

2.4. Quantification of nuclear:cytoplasmic ratios of NF-κB staining. Cells were fixed with 4% paraformaldehyde on glass coverslips and washed with phosphate-buffered saline (PBS). Cells were incubated sequentially with anti-NF-κB p65 (phospho Ser529) antibody (GTX50254, GeneTex) and anti-rabbit IgG (Alexa Fluor 594) (ab150080, Abcam). The nuclei were stained with DAPI. The images were obtained using a fluorescence microscope and were analyzed according to the protocol established previously by ImageJ.24

2.5. Cell viability assay. To evaluate the cell viability after macrophage differentiation and PU NP treatment, cells were treated with PMA (200 ng/ml) for 48 h in the complete medium at 37oC. After being incubated with PU NPs (solid content 100 µg/ml) for 30 min, cells were treated with LPS/IFN-γ or IL-4/IL-13 for 24 h. Cell viability was measured by the acridine orange (AO)/propidium iodide (PI) staining and the WST-8 assay (Sigma). For AO/PI staining, cells were stained with AO/PI (Chemometec) for 5 min and then the cellular fluorescence was analyzed and 11



Page 12 of 55

quantified by the NucleoCounter® NC-3000™. For WST-8 assay, the absorbance was determined at the wavelength of 450 nm by a microplate reader (SpectraMax® M5, USA).

2.6. Quantitative real time polymerase chain reaction (qRT-PCR) analysis. The mRNA expression for M1 markers and M2 markers was examined by qRT-PCR. Total RNA was isolated using the Trizol® reagent (Invitrogen) by the manufacturer's instructions (Invitrogen, Carlsbad, CA). The extracted RNA were incubated with DNase I (Thermo Scientific, USA) to remove genomic DNA contamination. The RNA (1 µg) was reverse-transcribed into cDNA by the RevertAid First Strand cDNA Synthesis Kit (MBI Fermentas, St. Leon-Rot, Germany). QRT-PCR was performed in a Chromo 4 PTC200 Thermal Cycler (MJ Research, USA) by the DyNAmo Flash SYBR Green qPCR Kit (Finnzymes Oy, Espoo, Finland). QRT-PCRs were detected using primers for autophagy related 5 (ATG5), beclin 1, mechanistic target of rapamycin (Mtor), IL-1β, IL-6, NLR family pyrin domain containing 3 (NLRP3), CD36, CD68, nitric oxide synthase (NOS2), arginase 1 (ARG-1), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH was the control gene for normalization and the values were shown as the relative ratio compared to that in the mock group. The sequences of primers are listed in Table S1. 12


Page 13 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2.7. Western blot analysis. To evaluate the signal transduction of inflammasome activation, THP-1 cells were seeded in a 6-well culture plates at a density of 2.5×105 cells/well and cultured with PMA (200 ng/ml) for 48 h. Cells were incubated in the presence of PU NPs for 30 min and subsequently treated with LPS/IFN-γ or IL-4/IL-13, respectively. After treatment for 24 h, cells were collected and lysed in RIPA lysis buffer. Proteins were separated by mass via sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto polyvinylidene fluoride (PVDF) membranes, and bound to polyclonal anti-LC3 antibody (GTX127375,

Genetex),

monoclonal

anti-p-NF-κB

(GTX38622,

Genetex),

monoclonal anti-IL-1β antibody (#12242, Cell Signaling), monoclonal anti-caspase 1 antibody (GTX62815, Genetex), and monoclonal anti-GAPDH (GTX100118, Genetex). The band intensity was detected by Labwork software (UVP). The quantified protein expression was analyzed using the software ImageJ.

2.8. Preparation of PU Films. The PU films for macrophage polarization in vitro were made by casting PU dispersion (300 µl; 10 wt% solid contents) in 24-culture wells at 25 °C. For rat subcutaneous implantation, the PU films were

13



Page 14 of 55

assembled by casting PU dispersion on teflon dishes and were further trimmed into a square shape (10 mm × 10 mm, 0.2 mm thickness).

2.9. Rat subcutaneous implantation. The Adult Sprague-Dawley rats (300–350 g, n=6 for each groups) were created with a dorsal incision of ~10 mm × 10 mm under anesthesia. The PLA films and PU films were inserted into the subcutaneous sites. After implantation for 4 weeks, the specimens were removed and wash with PBS. The specimens with surrounding tissue were fixed in formaldehyde solution. After H&E staining, the thickness of the fibrous capsule was analyzed by software from the six average sites of the capsule. For the macrophage detection, the slices of specimens

were

incubated

with

mouse

monoclonal

anti-CD86

antibody

(MCA2874GA, AbD Serotec) for M1 macrophages and mouse monoclonal anti-CD163 antibody (MCA342GA, AbD Serotec) for M2 macrophages. The second antibodies were anti-mouse IgG (Alexa Fluor 594) (Biolegend) for CD86 and anti-mouse IgG (Alexa Fluor 488) (Biolegend) for CD163. Macrophages were analyzed using a fluorescence microscope. The number of macrophages labeled positively for each marker was counted for each image. The mean of the numbers for three high power images was then calculated for each sample. The M2/M1 ratio was defined as the number of M2 macrophages divided by the number of M1 14


Page 15 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


macrophages of each group, and was further normalized to the poly(D,L-lactide) (PLA) polymer (Mw 121.4 kDa, low crystalline, Ingeo 2002D, NatureWorks) that served as the conventional biodegradable control group. Results are expressed as mean ± SD (N = 6). Statistical differences among the groups were examined by one way ANOVA. All procedures followed the ethical guidelines and were approved by the Animal Care and Use Committee (NTU105-EL-00012).

2.10. Statistical analyses. Data were collected from a multiple number of samples and were expressed as the mean ± standard deviation. The representative data were shown in this study. Statistical differences among the experimental groups were examined by one-way ANOVA. Differences were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001 among the indicated groups.

3. RESULTS AND DISCUSSION 3.1. Synthesis and physico-chemical analyses of waterborne PU NPs. The two types of PU NPs prepared in this study are illustrated in Figure 1A. The ionic component N-MDEA containing positive amine groups was employed for the synthesis of PU-N and DMPA with negative carboxyl groups was used for PU-C. The surface chemistry of PU NPs was defined by the attenuated total reflectance-Fourier 15



Page 16 of 55

transform infrared spectroscopy (ATR-FTIR), as shown in Figure 1B. The IR absorption peak at 1200 cm-1 indicated the amine group of PU-N and the peak at 1660 cm-1 was assigned to the COO- group in the PU-C structure. We further measured the hydrodynamic size and surface (zeta) potential of the NPs by dynamic light scattering apparatus. The NPs of PU-N possessed a hydrodynamic diameter of 62.8±4.5 nm and a positive zeta potential of 58.1±0.2 mV. The NPs of PU-C owned a smaller hydrodynamic diameter (35.2±1.2 nm) and a negative zeta potential (-70.3±1.2 mV) (Table 1). Based on the ATR-IR spectra and the values of zeta potential, we had prepared two kinds of PU NPs with different surface functional groups.

3.2. Polarization of macrophages upon treatment of PU NPs. The influence of PU NPs on the macrophage polarization of human THP-1 monocytes was evaluated after the cells were incubated with different surface-functionalized PU NPs. The suspended THP-1 monocytes become adherent when they are differentiated into macrophages (M0) with increased gene expression of macrophage markers (CD36 and CD68) by incubation with PMA (Figure S1A,B). The normal morphology of M0 macrophages is in round shape. Following the treatment of different PU NPs, the morphology of M0 macrophages was comparatively normal. We further assessed the effects of PU NPs on macrophage polarization into M1 and M2 macrophages in the 16


Page 17 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


presence of PU NPs. Cells were treated with LPS/IFN-γ for M1 polarization, and at this time the mock macrophages showed a spindle-shaped M1 morphology. In contrast, the macrophages still kept relatively round morphology in either of the PU NP-treated groups. Meanwhile, cells were treated with IL-4/IL-13 for M2 polarization. The morphology of M2 macrophages was round and it remained unchanged with either PU NP treatment (Figure 2A). We analyzed the cell shape by the ImageJ software. The direction in which the cells had the greatest length was defined as the long axis, and the length across the nucleus in the direction perpendicular to the long axis was the short axis. The elongation factor was defined as the ratio of the two axes.25 M1 macrophages treated with PU NPs showed a significantly lower degree of elongation compared with the untreated M1 macrophages (Figure S2). M1 macrophages which were stimulated by LPS/IFN-γ showed an upregulation of the M1 proinflammatory cytokines, TNF-α and IL-1β, compared to M0 macrophages. It was observed that the treatment of PU NPs significantly reduced these proinflammatory cytokines in protein levels, and in particular, PU-C exhibited the inhibitory capacities and rescued the cell viability of M1 macrophages in a dose-dependent manner (Figure S3) and showed greater inhibitory capacities than PU-N (Figure 2B). M2 macrophages which were stimulated by IL4/IL13 showed the upregulated secretion levels of the M2 immunosuppressive cytokines, TGF-β and IL-10. However, the 17



Page 18 of 55

treatment of PU NPs did not obviously affect the protein expressions of M2 immunosuppressive cytokines, and only PU-C slightly decreased the TGF-β secretions (Figure 2B). The PU NPs affected neither the proinflammatory cytokines in M2 macrophages nor the immunosuppressive cytokines in M1 macrophages (Figure S4). Furthermore, PU NPs inhibited the expression of M1 surface marker, CD86, but not the expression of M2 surface marker, CD163 (Figure S5). The gene expression of NOS2 decreased after the treatment of PU-N or PU-C, but the gene expression of ARG-1 did not (Figure S6). Taken together, PU NPs showed an inhibitive effect on M1 polarization, but not M2 polarization. Especially, PU NPs with surface carboxyl groups revealed a larger extent of inhibition on M1 polarization than those with amine groups.

3.3. Activation of the NF-κB in M1 macrophages by PU NPs. Since NF-κB is well established as a transcriptional regulator of M1 related genes during macrophage polarization, we examined whether PU NPs would affect the NF-κB activation. The localization of NF-κB was examined by fluorescence microscopy. It was found that NF-κB in M1 macrophages was in clear nuclear localization, but the trend was less obvious after PU-NP treatment (Figure 3A). Furthermore, Western blots showed that the p-NF-κB activation was reduced in M1 macrophages with the PU NP treatment, 18


Page 19 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and in particular, PU-C had a greater inhibitive effect on NF-kB activation than PU-N (Figure 3B). We also evaluated the level of p-NF-κB activation in M2 macrophages. Conversely, the p-NF-κB activation in M2 macrophages was not significantly changed compared to that in M0 macrophages, and neither was it altered by the treatment of PU NPs (Figure S7). These data suggested that PU NPs specifically influenced the NF-κB activation on M1 macrophages, but not on M2 macrophages.

3.4. Enhancement of autophagy in M1 macrophages by PU NPs. Since autophagy is mainly involved in NF-κB p65 degradation,26-27 we examined if the level of autophagy of M1 macrophages was enhanced by the PU NP treatment. For this intent, the endogenous expression of LC3-II, a representative autophagosomal marker, was detected by Western blots. It was observed that the level of LC3-II protein expression increased slightly in M0 cells (1.4-fold) after treatment with either type of PU NPs. Meanwhile, the level of LC3-II protein expression in M1 macrophages was also enhanced by 4.2 folds. After the treatment with either type of PU NPs, the LC3-II expression in M1 macrophages was significantly upregulated by an extent of 7 folds, but there was no obvious difference between the treatments of PU-N and PU-C (Figure 4A). The autophagy-related genes, ATG5 and BECN1, as well as the autophagy inhibition gene MTOR, was further analyzed. Results revealed that ATG5 19



Page 20 of 55

and BECN1 genes in M1 macrophages were upregulated for all groups compared to those in M0 untreated macrophages. Moreover, the increased level of these two genes in the PU-C treated group was significantly higher than that in the PU-N treated group. However, the MTOR gene expression in M0 and M1 macrophages showed no difference among the groups (Figure 4B).

3.5. Inhibition of NLRP3 inflammasome by PU NPs. We then investigated whether PU NP treatment led to the possible inhibition of NLRP3 inflammasome in M1 macrophages. As a matter of fact, the mRNA expression of NLRP3 was reduced by PU NP incubation (Figure 5A). Formation of NLRP3 inflammasome is known to subsequently trigger the cleavage and activation of caspase-1 and IL-1β. We also observed that both PU-N and PU-C prevented the activation of caspase-1 and IL-1β. Moreover, PU-C exerted more blocking effect on the activities of these two proteins than PU-N (Figure 5B,C). Since the cell death triggered by NLRP3 inflamasome is a feature for pyroptosis, we further examined the change in cell viability of polarized macrophages with the PU NP treatment. As shown in Figure 6, the cell viability of M0 and M2 macrophages remained unchanged after exposure to either type of PU NPs. We used the AO/PI staining to assess live and dead cells. For polarized M1 macrophages without the PU NP treatment, the PI positive cells increased at 24 h. 20


Page 21 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Meanwhile, the treatment of PU NPs partially rescued the cell viability of M1 macrophages, and in particular, PU-C showed a greater recovering effect than PU-N (Figure 6A,B). The WST-8 cell viability assay also confirmed that the viability of M1 macrophages was higher in PU NP treated groups (Figure 6C). These findings suggested that PU NPs may decrease the macrophage M1 polarization via the mechanism associated with the NLRP3 inflamasome inhibition.

3.6. The differentiation of M1 macrophages cultured on different PU films. Since PU NPs could assemble to form dense films, we further investigated that whether PU films, when used in cell culture, could inhibit the differentiation of M1 macrophages as that had already observed for PU NPs. Macrophages were cultured on the films or on the tissue culture polystyrene (TCPS). The M0 macrophages were in round shape in all groups, but they were less adherent on the PU-N and PU-C films compared to the TCPS and the commercial non-biodegradable PU (Pellethane) films. The M1 macrophages cultured on Pellethane films showed the same spindle-shaped morphology as those on TCPS. In contrast, the M1 macrophages kept relatively round and slightly aggregated morphology on both PU-C and PU-N (Figure 7A). Also, culturing cells on PU-N and PU-C films, but not Pellethane films, significantly reduced the TNF-α and IL-1β secretion levels of M1 macrophages (Figure 7B). The 21



Page 22 of 55

TGF-β and IL-10 secretion levels of M2 macrophages did not change obviously on Pellethane, PU-N and PU-C films. Only the IL-10 level of M2 macrophages on PU-C films was slightly reduced (Figure 7B). Hence, the biodegradable PU inhibited the activities of M1 macrophages in both the NP and self-assembled (film) forms.

3.7. Biocompatibility in the rat subcutaneous implantation. FBR of an implant are characteristic of the fibrous encapsulation and macrophage accumulation at the periphery of the implanted material.28 Both PU films, which formed from the assembly of PU NPs, showed a lower thickness of fibrous capsule than PLA films after implantation for 4 weeks (Figure 8A,B). We further confirmed that PU films recruited less M1 macrophages and more M2 macrophages compared to PLA films (Figure 8C,D). Meanwhile, PU-C films exhibited less FBR than PU-N films. Taken together, these data suggested that the waterborne PU was superior to the conventional PLA in biocompatibility and the surface groups could influence on the immune responses of PU in vivo. NP surface can be functionalized with different chemical groups to provoke or prevent immune responses. The undesired immune activities may reduce the efficiency of NPs for biomedical applications. Studies showed that the amino-functionalized polystyrene NPs, but not carboxyl- or nonfunctionalized 22


Page 23 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


polystyrene, could trigger the NLRP3 inflammasome activation and subsequent release of IL-1β by the lysosomal destabilization in macrophages.29 In the present study, we observed that PU NPs could specifically attenuate M1 macrophage activities, including cell morphology, cytokine expressions, and cell viability, but not those of M0 or M2 macrophages. Exposure of macrophages to bacterial products such as lipopolysaccharide (LPS) results in NF-κB activation and proinflammatory cytokine secretions. In this study, M0 macrophages did not activate NF-κB or increase TNF-α and IL-1β secretions with PU NP treatment. Furthermore, the NF-κB activation and proinflammatory cytokine secretions decreased after PU NP treatment. Hence, we excluded the possibility of bacterial contamination of PU NPs in this study. Both amine and carboxylic functionalized PU NPs, i.e. PU-N and PU-C, could reduce the M1 macrophage activities, though PU-C exhibited a greater inhibitive effect than PU-N. The chemical structure of waterborne PU NPs consists of PCL diol as the soft segments and ionic chain extender as partial hard segments. PU NPs had microphase-separated structure, and the major distribution of ionic chains was on the NP surface. Hence, PU NPs could be evenly dispersed in water. Without the ionic chain extenders, PU NPs cannot be synthesized from the water-based process. The zeta potential of PU-C′ NPs was -47.2±1.6 mV, which was less negative than PU-C 23



Page 24 of 55

NPs (-70.3±1.2 mV). PU NPs had microphase-separated structure and the ionic chains (offering negative charge) were mostly located on the PU NP surface. Therefore, the lower COOH content (of 0.4% difference) could result in significant decreases of surface negative charge and the subsequent ability to inhibit M1 macrophages. Because PU-C′ had the similar hydrodynamic sizes as PU-N but had different surface charge from PU-N, we compared the immunosuppression activities of PU-N and PU-C′. The effect of PU-C′ to inhibit TNF-α and IL-1β secretions of M1 macrophages was greater than that of PU-N and less than that of PU-C, suggesting that negative charge may be critical for immunosuppression (Figure S9). We further polarized the macrophages to M1 or M2 phenotype and subsequently treated the cells with PU NPs for 24 h. We observed the same tendency that PU NPs inhibited

the

proinflammatory

cytokines

in

M1

macrophages,

but

not

immunosuppression cytokines in M2 macrophages (Figure S10). These data indicated that surface functionalization of PU NPs may affect the immunosuppression properties in macrophages and is an important factor in immune regulation of NPs. To validate the work with primary human monocytes, we further performed key experiments

with

PBMCs.

We

demonstrated

that

PU

NPs

exhibited

immunosuppressive activity on PBMC derived M1 macrophages by reducing the secretion of TNF-α and IL-1β, as observed in THP-1 (Figure S11). Meanwhile, PU 24


Page 25 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NPs increased the production of IL-10 and TGF-β in M2 macrophages derived from PBMCs, which was different from the non-significant effect of PU NPs on M2 macrophages derived from THP-1. The difference may indicate promotion of the M2 phenotype and deserves further studies. The surface charge of nanoparticles (NPs) can affect the phagocytotic activity of polarized macrophages.30 Thus, the uptake amounts of PU NPs with different surface charges may be different in polarized macrophages and influence the subsequent autophagy pathway. In this study, we showed that PU-C activated more autophagy response than PU-N in M1 macrophages. Studies indicated that enhanced autophagy can reduce the inflammation by the attenuation of NF-κB pathway.31-33 We also observed that PU-C induced the same amounts of LC3-II activation as PU-N, and more gene expressions of Beclin 1 and ATG5 than PU-N. We suggested that PU-C may induce more autophagy activity than PU-N and subsequently reduce NF-kB activity. Hence, PU NPs had the suitable biocompatibility by regulation of NF-κB and autophagy may be treated as drug carrier with lower side effects of immune responses. Since PU NPs may promote the autophagy-lysosome pathway function without the disruption of intracellular pH, the possible relationship between immunosuppressive properties and lysosome functions is worthy of further investigations. Since NF-κB is a key transcription factor related to proinflammatory cytokine 25



Page 26 of 55

production and NLRP3 expression in M1 macrophage,34 the enhanced autophagy could reduce NF-κB activities in M1 macrophages. In contrast, the predominant activation of M2 macrophages was through the STAT6 pathway by IL-4/13, not NF-κB. Hence, the expression of p-NF-κB was the same in M0 and M2 macrophages. After PU NP treatment, the expression of p-NF-κB remained unchanged. The carboxyl groups on the surface of PU NPs transported Ca2+ from the extracellular milieu into macrophages to activate autophagy.35 Meanwhile, PU-N induced the same level of LC3-II as PU-C, but less autophagy-related factor expressions in M1 macrophages than PU-C in this study. This may indicate that factors other than surface functionalization may play potential roles in immunosuppression of PU NPs. Calcium (Ca2+) has been implicated in autophagic signaling pathways.36 Autophagy inhibition contributes to the NLRP3 inflammasome activation and the secretion of proinflammatory cytokines in macrophages.37 Therefore, PU NPs may affect the Ca2+ and subsequent autophagic signals. The possible relationship between PU NPs and Ca2+ signals is worthy of further exploration. The NLRP3 inflammasome has been implicated in a wide variety of organ diseases and metabolic disorders.38-39 The NLRP3 inflammasome activation leads to the processing and secretion of the proinflammatory cytokine IL-1β by caspase 1. Several studies have indicated that NLRP3 inflammasome plays an important role in 26


Page 27 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NP induced cytotoxicity through the autophagic-lysosomal disruption.40-41 Here, we demonstrated that PU NPs inhibited the gene expressions of NLRP3 and IL-1β via autophagy activation. After that, the protein processing of caspase-1 and IL-1β was further reduced. Moreover, the pyroptosis was attenuated after treatment with PU NPs in M1 macrophages. The mechanism by which the PU NPs modulate the macrophage polarization is portrayed in Figure 9. Previous studies also indicated that the NF-κB and NLRP3 degradation could be promoted by enhancing autophagy, thereby suppressing downstream immune responses.33-34 The observed downregulated NLRP3 activation pathway supported that PU NP exposure led to immunosuppression in macrophages and may have potential therapeutic implications for NLRP3 inflammasome related disorders. Since PU has unique features, including microphase-separated structure, good mechanical properties, and blood compatibility, it gains increasing attention in many biomedical fields. The degradation time of PU can be tuned by employing various biodegradable oligodiols in synthesis.15, 35 Due to the self-assembly characteristics, PU NPs become the film form after water removal. We observed that PU films exhibited a similar immunosuppressive effect as PU NPs on M1 macrophages. The M1 macrophages cultured on PU films inhibited proinflammtory cytokine expressions while those cultured on conventional PU (Pellethane) films showed upregulated 27



Page 28 of 55

proinflammtory cytokine expressions. M2 macrophages on PU films showed less expression of IL-10 than those on TCPS and Pellethane films. We hypothesized that the STAT3 pathway which regulates the IL-10 expression may have been influenced by PU films. Thus, our waterborne PU had higher immunosuppressive properties compared to the conventional PU, probably because of the difference in surface functional groups. PU with the net surface negative charge may influence the early phase acute inflammatory response to an implanted material by reducing neutrophil invasion and macrophage activation.42 The biodegradable polyurethane grafts also revealed the switch in macrophage polarization from M1 proinflammatory toward M2 anti-inflammatory phenotype for constructive remodeling.43,44 The biodegradable elastomeric grafts implanted into rat abdominal aorta yield neoarteries with less FBR and a large number of infiltrating M2 macrophage which are generally considered as anti-inflammatory and facilitate constructive remodeling.45 The FBR of implanted materials is of critical importance for the success of a medical device. Although the initial inflammatory response would promote the wound healing, the implant materials often lead to excessive inflammatory responses resulting in the formation of FBR and hinder the medical applications. The macrophage polarization plays a critical role in the FBR of implanted materials and can be affected by the material 28


Page 29 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


form, size, and porous structure.46 In this study, PU films induced lower thickness of fibrous capsule and less M1 macrophage recruitment than PLA films after the subcutaneous implantation. Moreover, the PU-C films showed less FBR than PU-N films. These data suggest that the good biocompatibility of biodegradable waterborne PU may be regulated by the surface chemistry through macrophage polarization. Mechanical biocompatibility gains increasing importance in biomedical engineering and impacts the development of medical devices. The mechanical properties including tensile strength, modulus, density, and porosity may facilitate the clinical acceptance of implant materials. The more flexible implant was associated with the smaller foreign body response.42 PU with excellent flexibility may reduce the FBR. After cellular uptake, most nanomaterials enter the lysosomal compartment, which is the most common intracellular site of sequestration and degradation. While the autophagy and lysosomal pathways play potential roles in the disposition of nanomaterials, autophagy and lysosomal dysfunction are the emerging mechanisms of nanomaterial toxicity.47 The disruption of lysosomal pH is considered as the factor causing lysosomal dysfunction.29, 48 The surface functional groups also affected the phagocytotic

activity of

polarized

macrophages.

The cationic polystyrene

nanoparticles decreased the phagocytosis in M1 and M2 macrophages.30 Here, we demonstrate that the waterborne PU NPs are immunosuppressive through the 29



Page 30 of 55

activation of autophagy but without causing toxicity on M1 macrophages. PU NPs may promote the autophagy-lysosome pathway function without the disruption of intracellular pH. The unique characteristics of PU NPs, their influence on the intracellular pH, and the possible relationship between immunosuppressive properties and lysosome functions deserve further investigation.

4. CONCLUSIONS The immunological impact of waterborne PU NPs on the phenotype of macrophages was investigated in this study. Results revealed that PU NPs could reduce NLRP3 inflammasome activation and subsequent proinflammatory cytokine expression in M1 macrophages. The inflammasome inhibition was mediated by PU NP-induced autophagy and NF-κB inactivation. These processes led to attenuation of M1 macrophage activities and shift of the population towards M2 macrophages. The surface chemistry of PU NPs had influence on immunoregulation and may account for the low inflammatory and foreign body response of biodegradable PU as implanted materials. The findings also suggested potential therapeutic applications of PU NPs in anti-inflammation and macrophage related disorders.

ASSOCIATED CONTENT 30


Page 31 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Supporting Information Information for each primer used for the real-time RT-PCR, the marker expressions of THP-1 monocyte differentiation induced by PMA, the cell shape of polarized macrophages, the secretion cytokines of M1 macrophages exposed to different amounts of PU-C NPs, the surface markers of polarized macrophages, the gene expression of polarized macrophages, the NF-κB phosphorylation of macrophage, the differentiation of M1 macrophages exposed to PU NPs containing different amount of surface carboxyl groups, and the effect of PU NPs with different surface functional groups on the differentiation of human monocytes and macrophages derived from PBMCs.

AUTHOR INFORMATION Corresponding Author *Shan-hui Hsu Institute of Polymer Science and Engineering, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei 10617, Taiwan, R.O.C. Phone: (886) 2-33665313. Fax: (886) 2-33665237. E-mail: [email protected]. Notes 31



Page 32 of 55

Any additional relevant notes should be placed here.

ACKNOWLEDGMENTS This research was supported by the Aim for the Top University Plan (104R4000) from the Ministry of Education and grants from the Ministry of Science and Technology (MOST106-2221-E-002-079-MY2; 106-3114-E-002-019), Taiwan. We are grateful to Dr. Shiu-Huey Chou for her assistance with the culture of THP-1 monocytes.

REFERENCES (1) Subbiah, R.; Veerapandian, M.; Yun, K. S. Nanoparticles: Functionalization and Multifunctional Applications in Biomedical Sciences. Curr. Med. Chem. 2010, 17, 4559-4577. (2) Wang, E. C.; Wang, A. Z. Nanoparticles and Their Applications in Cell and Molecular Biology. Integr. Biol. (Camb) 2014, 6, 9-26. (3) Buzea, C.; Pacheco, II; Robbie, K. Nanomaterials and Nanoparticles: Sources and Toxicity. Biointerphases 2007, 2, MR17-71. (4) Lameijer, M. A.; Tang, J.; Nahrendorf, M.; Beelen, R. H.; Mulder, W. J. Monocytes and Macrophages as Nanomedicinal Targets for Improved Diagnosis and Treatment of Disease. Expert. Rev. Mol. Diagn. 2013, 13, 567-580. 32


Page 33 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(5) Sridharan, R.; Cameron, A. R.; Kelly, D. J.; Kearney, C. J.; O’Brien, F. J. Biomaterial Based Modulation of Macrophage Polarization: a Review and Suggested Design Principles. Materials Today 2015, 18, 313-325. (6) Gordon, S.; Martinez, F. O. Alternative Activation of Macrophages: Mechanism and Functions. Immunity 2010, 32, 593-604. (7) Martinon, F.; Mayor, A.; Tschopp, J. The Inflammasomes: Guardians of the Body. Annu. Rev. Immunol. 2009, 27, 229-265. (8) Bergsbaken, T.; Fink, S. L.; Cookson, B. T. Pyroptosis: Host Cell Death and Inflammation. Nat. Rev. Microbiol. 2009, 7, 99-109. (9) Martinon, F.; Petrilli, V.; Mayor, A.; Tardivel, A.; Tschopp, J. Gout-Associated Uric Acid Crystals Activate the NALP3 Inflammasome. Nature 2006, 440, 237-241. (10) Morishige, T.; Yoshioka, Y.; Inakura, H.; Tanabe, A.; Yao, X.; Narimatsu, S.; Monobe, Y.; Imazawa, T.; Tsunoda, S.; Tsutsumi, Y.; Mukai, Y.; Okada, N.; Nakagawa, S. The Effect of Surface Modification of Amorphous Silica Particles on NLRP3 Inflammasome Mediated IL-1beta Production, ROS Production and Endosomal Rupture. Biomaterials 2010, 31, 6833-6842. (11) Reisetter, A. C.; Stebounova, L. V.; Baltrusaitis, J.; Powers, L.; Gupta, A.; Grassian, V. H.; Monick, M. M. Induction of Inflammasome-Dependent Pyroptosis by Carbon Black Nanoparticles. J. Biol. Chem. 2011, 286, 21844-21852. 33



Page 34 of 55

(12) Dobrovolskaia, M. A.; McNeil, S. E. Immunological Properties of Engineered Nanomaterials. Nat. Nanotechnol. 2007, 2, 469-478. (13) Nicolete, R.; dos Santos, D. F.; Faccioli, L. H. The Uptake of PLGA Micro or Nanoparticles by Macrophages Provokes Distinct in Vitro Inflammatory Response. Int. Immunopharmacol. 2011, 11, 1557-1563. (14) Thomas, C.; Rawat, A.; Hope-Weeks, L.; Ahsan, F. Aerosolized PLA and PLGA Nanoparticles Enhance Humoral, Mucosal and Cytokine Responses to Hepatitis B Vaccine. Mol. Pharm. 2011, 8, 405-415. (15) Hung, K. C.; Tseng, C. S.; Hsu, S. H. Synthesis and 3D Printing of Biodegradable Polyurethane Elastomer by a Water-Based Process for Cartilage Tissue Engineering Applications. Adv. Healthc. Mater. 2014, 3, 1578-1587. (16) Kuo, Y. C.; Hung, S. C.; Hsu, S. H. The Effect of Elastic Biodegradable Polyurethane Electrospun Nanofibers on the Differentiation of Mesenchymal Stem Cells. Colloids Surf. B Biointerfaces 2014, 122, 414-422. (17) Tsai, M. C.; Hung, K. C.; Hung, S. C.; Hsu, S. H. Evaluation of Biodegradable Elastic Scaffolds Made of Anionic Polyurethane for Cartilage Tissue Engineering. Colloids Surf. B Biointerfaces 2015, 125, 34-44. (18) Hsieh, F. Y.; Lin, H. H.; Hsu, S. H. 3D Bioprinting of Neural Stem Cell-Aaden Thermoresponsive Biodegradable Polyurethane Hydrogel and Potential in Central 34


Page 35 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Nervous System Repair. Biomaterials 2015, 71, 48-57. (19) Hsu, S. H.; Hung, K. C.; Lin, Y. Y.; Su, C. H.; Yeh, H. Y.; Jeng, U. S.; Lu, C. Y.; Dai, S. A.; Fu, W. E.; Lin, J. C. Water-Based Synthesis and Processing of Novel Biodegradable Elastomers for Medical Applications. J. Mater. Chem. B 2014, 2, 5083-5092. (20) Guo, Q.; Lu, Z.; Zhang, Y.; Li, S.; Yang, J. In Vivo Study on the Histocompatibility and Degradation Behavior of Biodegradable Poly(Trimethylene Carbonate-Co-D,L-Lactide). Acta. Biochim. Biophys. Sin. (Shanghai) 2011, 43, 433-440. (21) Tatai, L.; Moore, T. G.; Adhikari, R.; Malherbe, F.; Jayasekara, R.; Griffiths, I.; Gunatillake, P. A. Thermoplastic Biodegradable Polyurethanes: the Effect of Chain Extender Structure on Properties and in-Vitro Degradation. Biomaterials 2007, 28, 5407-5417. (22) Hunt, J. A.; Flanagan, B. F.; McLaughlin, P. J.; Strickland, I.; Williams, D. F. Effect of Biomaterial Surface Charge on the Inflammatory Response: Evaluation of Cellular Infiltration and TNF Alpha Production. J. Biomed. Mater. Res. 1996, 31, 139-144 (23) Sharif, O.; Bolshakov, V. N.; Raines, S.; Newham, P.; Perkins, N. D. Transcriptional Profiling of the LPS Induced NF-kappaB Response in Macrophages. 35



Page 36 of 55

BMC Immunol. 2007, 8, 1. (24) Noursadeghi, M.; Tsang, J.; Haustein, T.; Miller, R. F.; Chain, B. M.; Katz, D. R. Quantitative Imaging Assay for NF-kappaB Nuclear Translocation in Primary Human Macrophages. J. Immunol. Methods 2008, 329, 194-200. (25) McWhorter, F. Y.; Wang, T.; Nguyen, P.; Chung, T.; Liu, W. F. Modulation of Mcrophage Phenotype by Cell Shape. Proc. Natl. Acad. Sci. U S A 2013, 110, 17253-172588. (26) Pei, F.; Wang, H. S.; Chen, Z.; Zhang, L. Autophagy Regulates Odontoblast Differentiation by Suppressing NF-KappaB Activation in an Inflammatory Environment. Cell Death Dis. 2016, 7, e2122. (27) Chang, C. P.; Su, Y. C.; Lee, P. H.; Lei, H. Y. Targeting NFKB by Autophagy to Polarize Hepatoma-Associated Macrophage Differentiation. Autophagy 2013, 9, 619-621. (28) Brown, B. N.; Londono, R.; Tottey, S.; Zhang, L.; Kukla, K. A.; Wolf, M. T.; Daly, K. A.; Reing, J. E.; Badylak, S. F. Macrophage Phenotype as a Predictor of Constructive Remodeling following the Implantation of Biologically Derived Surgical Mesh Materials. Acta Biomater. 2012, 8, 978-987. (29) Lunov, O.; Syrovets, T.; Loos, C.; Nienhaus, G. U.; Mailander, V.; Landfester, K.; Rouis, M.; Simmet, T. Amino-Functionalized Polystyrene Nanoparticles Activate the 36


Page 37 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NLRP3 Inflammasome in Human Macrophages. ACS Nano 2011, 5, 9648-9657. (30) Fuchs, A. K.; Syrovets, T.; Haas, K. A.; Loos, C.; Musyanovych, A.; Mailander, V.; Landfester, K.; Simmet, T. Carboxyl- and Amino-Functionalized Polystyrene Nanoparticles Differentially Affect the Polarization Profile of M1 and M2 Macrophage Subsets. Biomaterials 2016, 85, 78-87. (31) Xi, C.; Zhou, J.; Du, S.; Peng, S. Autophagy Upregulation Promotes Macrophages

to

Escape

Mesoporous

Silica

Nanoparticle

(MSN)-Induced

NF-KappaB-Dependent Inflammation. Inflamm. Res. 2016, 65, 325-341. (32) Chuang, S. Y.; Yang, C. H.; Chou, C. C.; Chiang, Y. P.; Chuang, T. H.; Hsu, L. C. TLR-Induced PAI-2 Expression Suppresses IL-1beta Processing via Increasing Autophagy and NLRP3 Degradation. Proc. Natl. Acad. Sci. U S A 2013, 110, 16079-16084. (33) Chang, C. P.; Su, Y. C.; Hu, C. W.; Lei, H. Y. TLR2-Dependent Selective Autophagy Regulates NF-KappaB Lysosomal Degradation in Hepatoma-Derived M2 Macrophage Differentiation. Cell Death Differ. 2013, 20, 515-523. (34) Bauernfeind, F. G.; Horvath, G.; Stutz, A.; Alnemri, E. S.; MacDonald, K.; Speert, D.; Fernandes-Alnemri, T.; Wu, J.; Monks, B. G.; Fitzgerald, K. A.; Hornung, V.; Latz, E. Cutting edge: NF-KappaB Activating Pattern Recognition and Cytokine Receptors License NLRP3 Inflammasome Activation by Regulating NLRP3 Expression. J. 37



Page 38 of 55

Immunol. 2009, 183, 787-791. (35) Huang, Y. J.; Hung, K. C.; Hsieh, F. Y.; Hsu, S. H. Carboxyl-Functionalized Polyurethane Nanoparticles with Immunosuppressive Properties as a New Type of Anti-Inflammatory Platform. Nanoscale 2015, 7, 20352-20364. (36) Bootman, M. D.; Chehab, T.; Bultynck, G.; Parys, J. B.; Rietdorf, K. The Regulation of Autophagy by Calcium Signals: Do We Have a Consensus? Cell Calcium 2018, 70, 32-46. (37) Dai, J.; Zhang, X.; Li, L.; Chen, H.; Chai, Y. Autophagy Inhibition Contributes to ROS-Producing

NLRP3-Dependent

Inflammasome

Activation

and

Cytokine

Secretion in high Glucose-Induced Macrophages. Cell Physiol. Biochem. 2017, 43, 247-256. (38) De Nardo, D.; De Nardo, C. M.; Latz, E. New Insights into Mechanisms Controlling the NLRP3 Inflammasome and its Role in Lung Disease. Am. J. Pathol. 2014, 184, 42-54. (39) Benetti, E.; Chiazza, F.; Patel, N. S.; Collino, M. The NLRP3 Inflammasome as a Novel Player of the Intercellular Crosstalk in Metabolic Disorders. Mediators Inflamm. 2013, 2013, 678627. (40) Mishra, A. R.; Zheng, J.; Tang, X.; Goering, P. L. Silver Nanoparticle-Induced Autophagic-Lysosomal Disruption and NLRP3-Inflammasome Activation in HepG2 38


Page 39 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Cells is Size-Dependent. Toxicol. Sci. 2016, 150, 473-487. (41) Chen, Z.; Liu, Y.; Sun, B.; Li, H.; Dong, J.; Zhang, L.; Wang, L.; Wang, P.; Zhao, Y.; Chen, C. Polyhydroxylated Metallofullerenols Stimulate IL-1beta Secretion of Macrophage

through

TLRs/MyD88/NF-KappaB

Pathway

and

NLRP(3)

Inflammasome Activation. Small 2014, 10, 2362-2372. (42) Sohal, H. S.; Clowry, G. J.; Jackson, A.; O'Neill, A.; Baker, S. N. Mechanical Flexibility Reduces the Foreign Body Response to Long-Term Implanted Microelectrodes in Rabbit Cortex. PLoS One 2016, 11, e0165606. (43) McBane, J. E.; Matheson, L. A.; Sharifpoor, S.; Santerre, J. P.; Labow, R. S. Effect of Polyurethane Chemistry and Protein Coating on Monocyte Differentiation towards a Wound Healing Phenotype Macrophage. Biomaterials 2009, 30, 5497-5504. (44) Enayati, M.; Eilenberg, M.; Grasl, C.; Riedl, P.; Kaun, C.; Messner, B.; Walter, I.; Liska, R.; Schima, H.; Wojta, J.; Podesser, B. K.; Bergmeister, H. Biocompatibility Assessment of a new Biodegradable Vascular Graft via in Vitro Co-Culture Approaches and in Vivo Model. Ann. Biomed. Eng. 2016, 44 , 3319-3334. (45) Wu, W.; Allen, R. A.; Wang, Y. Fast-Degrading Elastomer Enables Rapid Remodeling of a Cell-Free Synthetic Graft into a Neoartery. Nat. Med. 2012, 18, 1148-1153. (46) Sussman, E. M.; Halpin, M. C.; Muster, J.; Moon, R. T.; Ratner, B. D. Porous 39



Page 40 of 55

Implants Modulate Healing and Induce Shifts in Local Macrophage Polarization in the Foreign Body Reaction. Ann. Biomed. Eng. 2014, 42, 1508-1516. (47) Stern, S. T.; Adiseshaiah, P. P.; Crist, R. M. Autophagy and Lysosomal Dysfunction as Emerging Mechanisms of Nanomaterial Toxicity. Part. Fibre. Toxicol. 2012, 9, 20. (48) Ma, X.; Wu, Y.; Jin, S.; Tian, Y.; Zhang, X.; Zhao, Y.; Yu, L.; Liang, X. J. Gold Nanoparticles Induce Autophagosome Accumulation Through Size-Dependent Nanoparticle Uptake and Lysosome Impairment. ACS Nano 2011, 5, 8629-8639.

40


Page 41 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


FIGURE CAPTIONS Figure 1. Preparation of PU NPs with different surface functional groups. (A) The chemical structure and synthetic process of PU NPs with N-MDEA (PU-N) or DMPA (PU-C) incorporated in the chemical compositions. (B) The IR spectra of PU NPs showing the amine groups and carboxyl groups on PU-N and PU-C.

Figure 2. The differentiation of macrophages exposed to PU NPs with different surface functional groups. (A) The morphologies of macrophages (6 × 105/per well in the 6-well plate) after incubation with PU NPs (100 µg/ml) for 30 min, and then treated with LPS/IFN-γ or IL-4/IL-13 for 24 h. The scale bar represents 50 µm. (B) The secretion levels of cytokines for M1 macrophages or M2 macrophages after incubation with PU NPs (100 µg/ml) for 30 min and then treated with LPS/IFN-γ or IL-4/IL-13 for 24 h. Data are representative of three independent experiments. Results are expressed as mean ± SD, N = 3. *p < 0.05, **p < 0.01, ***p < 0.001, among the indicated groups. ns, no significant difference.

Figure 3. The inhibition of NF-κB activation in M1 macrophages by the treatment of PU NPs as described. (A) Fluorescent images and the nuclear/cytoplasmic ratios of pNF-κB p65 for M1 macrophages exposed to PU NPs (100 µg/ml) and then LPS/IFN-γ for 4 h. The scale bar represents 10 µm. Data from analysis of five 41



Page 42 of 55

separate field images for each group. (B) The p-NF-κB protein expression for M1 macrophages treated with PU NPs (100 µg/ml) and then LPS/IFN-γ for 24 h. GAPDH was used as the internal control. Band intensities were quantified and normalized to GAPDH. Data are representative of three independent experiments. Results are expressed as mean ± SD, N = 3. **p < 0.01, ***p < 0.001 among the indicated groups. ns, no significant difference.

Figure 4. Upregulation of autophagy in M1 macrophages by the combined treatment of PU NPs as described. (A) Western blot analysis for related LC3-II protein expression for M1 macrophages treated with PU NPs (100 µg/ml) and then LPS/IFN-γ for 24 h. GAPDH was used as the internal control. The band intensities of LC3-II were quantified and normalized to GAPDH. (B) The mRNA expressions of autophagy-related genes for M1 macrophages after incubation with PU NPs (100 µg/ml) and then LPS/IFN-γ for 24 h. Data are representative of three independent experiments. Results are expressed as mean ± SD, N = 3. *p < 0.05, **p < 0.01, among the indicated groups. ns, no significant difference.

Figure 5. The inhibition of NLRP3 inflammasome activation in M1 macrophages by the treatment of PU NPs as described. (A) The mRNA expressions of NLRP3 gene of 42


Page 43 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


M1 macrophages after incubation with PU NPs (100 µg/ml) and then LPS/IFN-γ for 24 h. (B) (C) The caspase 1 and IL-1β protein expression of M1 macrophages treated with PU NPs (100 µg/ml) and then LPS/IFN-γ for 24 h. GAPDH was used as the internal control. Band intensities were quantified and normalized to GAPDH. Data are representative of three independent experiments. Results are expressed as mean ± SD, N = 3. *p < 0.05, **p < 0.01, among the indicated groups. ns, no significant difference.

Figure 6. Cell viability in different macrophage groups (M0, M1, and M2) after treatment with PU NPs. (A) The fluorescence histogram of live/dead stain after treatment with PU NPs (100 µg/ml) for 30 min and then treated with LPS/IFN-γ or IL-4/IL-13 for 24 h. (B) The PI positive cells after treatment with PU NPs. (C) Cell viability determined by the WST-8 assay after treatment with PU NPs. Results are expressed as mean ± SD, N = 3. *p < 0.05, **p < 0.01, among the indicated groups. ns, no significant difference.

Figure 7. The differentiation of M1 macrophages cultured on different PU films. (A) The morphologies of macrophages cultured on different PU films treated with LPS/IFN-γ for 24 h. The scale bar represents 50 µm. (B) The extent of differentiation 43



Page 44 of 55

of M1 or M2 macrophages cultured on different PU films treated with LPS/IFN-γ or IL-4/IL-13 for 24 h, expressed by the secretion levels of cytokines. Data are representative of three independent experiments. Results are expressed as mean ± SD, N = 3. *p < 0.05, ***p < 0.01, among the indicated groups. ns, no significant difference.

Figure 8. The FBR of PU films after subcutaneous implantation. (A) The histology of hematoxylin and eosin stained sections after implantation for 28 days. (B) The extent of FBR was represented by the capsule thickness (arrows) based on the histology. The scale bar represents 100 µm. (C) The immunofluorescent images (marker protein expression) of macrophages in response to the implants. CD86 (M1) = red, CD163 (M2) = green. The scale bar represents 10 µm. (D) Quantification of M1 and M2 populations. Data are representative of two independent experiments.

Results are

expressed as mean ± SD, N = 3. *p < 0.05, **p < 0.01, and ***p < 0.001, among the indicated groups.

Figure 9. The proposed mechanism by which the M1 macrophage polarization was significantly inhibited after exposure to PU NPs.

44


Page 45 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Table 1. The hydrodynamic diameter and zeta potential of main PU NPs prepared in the study. Sample

Hydrodynamic

Zeta potential (mV)

diameter (nm)

Polydispersity index (PDI)

PU-N

62.8±4.5

58.1±0.2

0.163

PU-C

35.2±1.2

-70.3±1.2

0.179

45



Page 46 of 55

Figure 1

46


Page 47 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 2

47



Page 48 of 55

Figure 3

48


Page 49 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 4

49



Page 50 of 55

Figure 5

50


Page 51 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6

51



Page 52 of 55

Figure 7

52


Page 53 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 8

53



Page 54 of 55

Figure 9

54


Page 55 of 55 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Graphical Table of Contents

55


Modulation of macrophage phenotype by biodegradable polyurethane

Recommend Documents