Nitric Oxide Nanosensors for Predicting the Development of

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Nitric oxide nanosensors for predicting the development of osteoarthritis in rat model Pan Jin, Christian Wiraja, Jinmin Zhao, Jinlu Zhang, Li Zheng, and Chenjie Xu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b06404 • Publication Date (Web): 10 Jul 2017 Downloaded from http://pubs.acs.org on July 11, 2017

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ACS Applied Materials & Interfaces

Nitric oxide nanosensors for predicting the development of osteoarthritis in rat model Pan Jin,a,b,c‡ Christian Wiraja,d‡ Jinmin Zhao,a,b,c‡ Jinlu Zhang,a,b,c Li Zheng,a,b,c* and Chenjie Xud,e* a

Guangxi Engineering Center in Biomedical Materials for Tissue and Organ Regeneration, The

First Affiliated Hospital of Guangxi Medical University, Nanning, People’s Republic of China b

Guangxi Collaborative Innovation Center for Biomedicine, The First Affiliated Hospital of

Guangxi Medical University, Nanning, People’s Republic of China c

Guangxi Key Laboratory of Regenerative Medicine, The First Affiliated Hospital of Guangxi

Medical University, Nanning, People’s Republic of China d

School of Chemical and Biomedical Engineering, Nanyang Technological University, 70

Nanyang Drive, Singapore 637457 e

NTU-Northwestern Institute for Nanomedicine, Nanyang Technological University, 50

Nanyang Avenue, Singapore 639798 ‡Pan Jin, Christian Wiraja and Jinmin Zhao contributed equally.

KEYWORDS: nanosensor, nitric oxide sensing, osteoarthritis, DAF-FM, PLGA

ACS Paragon Plus Environment

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ABSTRACT: Osteoarthritis (OA) is a chronic arthritic disease with the overproduction of inflammatory factors such as nitric oxide (NO). This study develops a NO nanosensor to predict the OA development. The nanosensor is synthesized by encapsulating NO sensing molecules (i.e. 4-amino-5-methylamino-2',7'-difluorofluorescein Diaminofluorescein-FM, DAF-FM) within biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles. In vitro, the nanosensor allows the monitoring of NO release in IL-1β-stimulated chondrocytes and the alleviative effect of NG-Monomethyl-L-arginine (L-NMMA, a NO inhibitor) and Andrographolide (an antiinflammatory agent). In the rat OA model, it permits the quantification of NO level in joint fluid. The proposed NO nanosensor may facilitate non-invasive and real time evaluation of the OA development.


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1. INTRODUCTION Osteoarthritis (OA) is a chronic condition with significant disease burden, affecting 15% of the population worldwide.1, 2A key molecule in the inflammatory and degradative cascade of arthritis, nitric oxide (NO) is noted as a potential biomarker of inflammation for OA.3 Its overproduction is the result of the overexpression of inducible nitric oxide synthase (iNOS or NOS2) caused by the stimuli of proinflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukine-1 beta (IL-1β).4, 5 Studies showed that NO increases the expression of active matrix metalloproteinases (MMPs), inhibits the matrix protein synthesis, modulates PGE2 synthesis, and under some conditions may initiate chondrocyte apoptosis.3,6 NO is present in plasma of OA patients and more prominently in the synovial fluid.7, 8 The in vitro NO secretion is often measured via fluorescence microscopy using diaminofluorescein-based probes (e.g. 4-Amino-5-methylamino-2′,7′-difluorescein Diaminofluorescein-FM, DAF-FM).9 The aromatic vicinal diamines of diaminofluorescein can react with NO in the presence of O2 to form the corresponding triazole compounds, which results in a turn-on emission because the quenching extent of triazole groups is smaller than electro-rich amino groups.10 These fluorescent sensors are quite sensitive to NO with a detection limit in the nanomolar range. Their cell permeability can be improved by converting the probe (e.g. DAFFM) to a diacetateform (e.g. DAF-FM diacetate). Despite their sensitivity to NO and the convenience in usage, these molecular sensors also come with several disadvantages.11 First, they react with other biological molecules such as dehydroascorbic acid and ascorbic acid, which contributes to false signals. Secondly, pH condition may also affect the fluorescence intensity.12 Finally, as small molecules, these probes are poorly retained within cells or tissue and are


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quickly excreted or secreted. Thus, there hasn’t been any progress to track NO production in OA animal model. While efforts are being spent on developing new molecular sensors or setting independent verification using spectroscopic or electrochemical methods,13 an alternative solution is needed to allow continual usage of these sensitive molecular sensors to track NO in OA model. Previously, we explored the utilization of nanoparticle encapsulation to extend cellular retention of molecular beacons or viability sensing molecules in stem cell tracking.14, 15 Inspired by those works, we hypothesize that nanoparticle encapsulation could address the abovementioned challenges of diaminofluorescein-based molecular probes. As a proof-of-concept, we synthesize a NO nanosensor by encapsulating the DAF-FM within poly(lactic-co-glycolic acid) (PLGA) nanoparticles (Figure 1) for predicting OA progression. 2. MATERIALS AND METHODS 2.1 Synthesis and characterization of NO nanosensor NO nanosensor (DAF-FM-containing PLGA nanoparticle) was synthesized using the single emulsion method. Briefly, 500 µg DAF-FM (D1821, Sigma-Aldrich, Saint Louis, Missouri, USA) was mixed with 50 mg PLGA (50:50, MW of 10 kDa) in 2 ml chloroform at 4 °C. Subsequently, the mixture was added dropwise to 3% polyvinyl alcohol (PVA) solution and homogenized (Tissue Master 125, Omni International) for 60 s at 24,000 rpm. Then, the emulsion was placed in chemical hood for 4 hrs to evaporate chloroform. Finally, the nanosensors were centrifuged at 10,000 rpm and rinsed thrice with distilled (DI) water prior to freeze-drying. Hydrodynamic diameter and zeta potential of nanosensors were quantified using


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Zetasizernano Z (Malvern Instruments Ltd., UK). Their morphology was examined with JSM6700F field-emission scanning electron microscope (FESEM, JEOL USA, Inc.). The release profile of DAF-FM was studied in cell culture medium at 37 °C. Absorbance of the supernatants at 495 nm was obtained at designated time points (i.e. 0, 6, 12, 24, 48, 72, 96 and 144 hr) using a UV-2450 UV-Visible spectrophotometer (Shimadzu corporation, Kyoto, Japan). The DAF-FM in the supernatants was quantified by fitting the concentration-absorbance standard curve of DAF-FM and then normalizing it against the original loaded quantity. Stability of DAF-FM probe inside nanosensor was similarly evaluated, by measuring fluorescence intensity of nanosensor release supernatant (495/515nm) against DAF-FM solution incubated for the same period at 37 °C. 2.2 Articular chondrocytes isolation and culture Primary articular chondrocytes were isolated from the knee joints of neonatal Sprague-Dawley (SD) rats (3-7 days old, Animal Resources Centre of Guangxi Medical University, Nanning, Guangxi, China) by collagenase digestion as described previously.16 Briefly, cartilage was separated from joints, sectioned into pieces, dissociated with 2 mg/mL collagenase type II (Gibco), and cultured in alpha-modified eagle’s medium (α-MEM, Gibco) containing 10% (v/v) fetal bovine serum (FBS, Gibco) and 1% (v/v) penicillin/streptomycin (Solarbio). Chondrocytes at passage 2 were used for further studies. 2.3 Cytotoxicity assay Cytotoxicity was examined with cell counting Kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan). Briefly, chondrocytes were treated with IL-1β, Andrographolide (Abbreviation: Andro,)


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and L-NMMA, and then added with CCK-8 reagent at 37°C for 4 hours. Absorbance at 450 nm was measured using a microplate reader (Thermo Scientific Multiskan GO Microplate Spectrophotometer) and cell numbers were calculated with calibration curve. 2.4 IL-1 β-induced chondrocytes and treatment Cells were divided into four groups: normal control (untreated chondrocytes), IL-1β group (chondrocytes treated with 10 ng/mL IL-1β), IL-1β+L-NMMA group (chondrocytes cultured with 10 ng/mL IL-1β along with L-NMMA (an iNOS inhibitor) of recommended concentration), and IL-1β+Andro group (chondrocytes incubated with 10 ng/mL IL-1β and Andro (an antiinflammatory drug) of optimal dose). Cells were cultured for 2, 4 and 6 days respectively. Interleukin-1β (IL-1β) was acquired from PeproTech (New Jersey, USA).L-NMMA (NGMonomethyl-L-arginine, Monoacetate Salt) was purchased from Beyotime Institute of Biotechnology, Jiangsu, China.Andro (Chengdu Must Bio-technology Co. LTD., Sichuan, China) was prepared in dimethyl sulphoxide (DMSO) with the final concentration of 100 mM. The final concentration of DMSO was less than 0.1%. 2.5 Fluorescence intensity analysis with NO nanosensors After 2, 4 and 6 days of culture, 1 mg/mL NO nanosensors were added in the culture medium in all of the groups. After 6 hours incubation at 37°C, the supernatants were collected and the fluorescence intensity was recorded with fluorescence microplate reader (Synergy™ H1, BioTek Instruments, Inc.) at 495/515 nm. 2.6 NO quantification in the cell supernatant


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At day 2, 4 and 6, cell supernatants in all of the groups were collected for detection of NO levels using nitric oxide detection kit (Nanjing Jiancheng Bioengineering Research Institute, China) according to the manufacture’s protocol. 2.7 Cell labelling and imaging with NO nanosensors For imaging purposes, cells were labeled with nanosensor and DAF fluorescence were captured. Firstly, cells were treated with 1 mg/mL nanosensor or DAF-FM. Subsequently, cells were washed with PBS, incubated in a dark environment with 70 nM rhodamine-phalloidin (Cytoskeleton, Inc.) for 30 min, and then stained with 1µg/mL Hoechst 33258 (Sigma) for 5 minutes. Images were obtained by a laser scanning confocal microscope (Nikon A1). 2.8 Gene analysis with quantitative real-time polymerase chain reaction (RT-PCR) RNA was extracted from cells using an RNA extraction kit (Tiangen Biotech Co.,Ltd., China). The RNA was reverse transcribed using a reverse transcription kit (FermentasCompany, USA). The RT-PCR reaction was carried out using a quantitative PCR detection system (Realplex 4, Eppendorf Corporation) with FastStart Universal SYBR Green Master (Roche). The PCR primers for OA related genes are presented in Table 1. Gene expression was analyzed using the 2-∆∆CT method with β-actin as the control. Each sample was repeated in triplicate to reduce operation bar errors. 2.9 OA induction in rats and the treatment A total of 100 male Sprague-Dawley (SD) rats weighing 210-240 g were used in the experiment. All of the animal experiments were performed in accordance with ethical approval by the Institutional Ethics Committee of Guangxi Medical University (2015-03-21). After being


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anesthetized with 2% pentobarbital sodium solution via intraperitoneal injection, anterior cruciate ligament transection (ACLT) was performed on the right hind limb of the rats to induce arthritis.17 The same incision was created on the left hind limb as the sham group. The animals were randomly divided into two groups: OA group and treatment group. In OA group, the animals received an intra-articular injection of 0.3 mL normal saline post ACLT operation once per week for 4, 8 and 12 weeks. The treatment group was divided into 2 subgroups: one received intra-articularly injection of 1000 µM L-NMMA and the other with 50µM Andro once per week for 8 weeks after 4 weeks post ACLT operation. 2.10 In vivo imaging of NO nanosensorlabeled joints Animals were immobilized using 2% anesthetic isoflurane (2% in O2). For in vivo imaging, the hair of the rat hind limb was removed to reduce autofluorescence. Each rat was intra-articularly injected with 0.3 mL 40 mg/mL NO nanosensors or fluorescent dye solution. Throughout the whole experiment, the body temperature of the rat was kept constant with heating pads and rectal measurement of their body temperature. Fluorescence imaging was obtained at hour 0.5, 1, 2 and 3 after the injection of the probe using the fluorescence imaging system (In-Vivo FX PRO, Bruker Corporation, Baden-Württemberg, Germany) at 495 nm excitation and 515 nm emission. Regions of interest were placed identically over both knees, within which the total fluorescence intensity was measured. 2.11NO quantification in joint synovial fluid The rats were sacrificed post the fluorescence imaging. Knee joint fluid was obtained through joint extract and stored at -20°C before analysis. NO concentration in the joint fluid was assessed by nitric oxide detection kit (Nanjing Jiancheng Bioengineering Research Institute, China).


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2.12 Macroscopic observation of the cartilage damage in the joints The joints of all of the experimental animals were harvested for macroscopic observation and histological evaluation. Macroscopic evaluation was performed by two independent observers who were blinded to the treatment group. The depth of lesions of the articular cartilage was performed on a scale of 0-4 as described by Pelletier et al.18 2.13 Histology analysis of the cartilage damage in the joints After macroscopic observation, knee joints were immediately fixed in 4 % paraformaldehyde and then decalcified with buffered ethylenediaminetetraacetate (EDTA). Samples were embedded in paraffin and serially sectioned from the medial compartment of the joints in the mid-sagittal plane. After de-waxing, 5-µm thick sections were stained with hematoxylin and eosin (H&E) staining and Safranin-O-Fast green staining respectively. Cartilage destruction was scored by two blinded observers using the histological grading system.19 2.14 Immunohistochemical analysis Immunohistochemical analysis was performed on articular cartilage to investigate the protein expressions of collagen type II alpha 1 chain (COL2A1). Briefly, sections were incubated with primary antibody of COL2A1 (BA0533, Boster Biological Technology, Ltd., Wuhan, China, 1:100). After incubation with the second antibody and biotin-labeled horse radish peroxidase, the specimens were processed with a 3, -3’-diaminobenzidine tetrahydrochloride (DAB) kit (Boster) and counter-stained with hematoxylin. Images were captured using an inverted phase contrast microscope (Olympus, Tokyo, Japan). 2.15 Western blot analysis Cartilage tissues were scraped with a blade and pulverized in liquid nitrogen. 60 µg protein was extracted, loaded on a 10% (v/v) polyacrylamide gel, and transferred to PVDF membrane


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(Millipore, USA) using electro-blotting. After blocked with 5% non-fat dry milk, the specimens were incubated with primary antibody of matrix metallopeptidase 13 (MMP13, ab75606, Abcam, 1:200), PTGS2 (cst12282, Cell Signaling Technology, Inc., 1:500), NOS2 (ab15323, Abcam, 1:40) and β-actin (ACTB, A2228, 1:1000), probed with an Alexa infrared dye-conjugated secondary antibodies (Invitrogen), and visualized using the Odyssey Infrared Imaging System (LI-COR Biotechnology, USA). Data were normalized against β-actin for each protein. 2.16 Correlation analysis Associations between NO concentration and fluorescence intensity were tested by calculating Spearman correlation coefficients (r2, very weak: 0 - 0.20, weak: 0.20 - 0.39, moderate: 0.40 0.59, strong: 0.60 - 0.79, very strong: 0.80 -1).20 2.17 Statistical Analysis Student’s t test and ANOVA with post hoc tests were used for pairwise comparisons and multi comparisons, respectively, after confirming a normal distribution using the Shapiro-Wilk test. SPSS software (version 16.0, SPSS Inc., Chicago, Illinois, USA) was used and p < 0.05 considered significant difference. 3. RESULTS 3.1 NO nanosensor synthesis and characterization NO nanosensors were synthesized by encapsulating DAF-FM into biodegradable PLGA nanoparticles by the single emulsion method. The nanosensors were about 1 µm in size (Figure 2A&B). They carried almost neutral charges (-0.05 mV), except when being coated with Poly-Llysine (PLL) (+16.8 mV) to facilitate the cellular internalization (Figure 2C). Upon dispersion in cell culture medium, the PLGA shells degraded over time due to the hydrolysis. In consequence,


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the encapsulated DAF-FM molecules were continuously released over a period of ~144 hours, with an initial burst release of ~50% by the first 12 hours, followed by a slower release of the remaining 50% until 144 hours (Figure 2D). Interestingly, DAF-FM stability to hydrolysis was significantly improved after nanoparticle encapsulation (Figure 2E). As compared to when an equivalent amount of DAF-FM was dissolved in culture medium which showed complete degradation to DAF by day 5 (120 hours), fluorescence intensity of nanosensor-containing supernatant rose significantly slower (p

Nitric Oxide Nanosensors for Predicting the Development of

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