- Email: [email protected]
Acid-activatable polymeric curcumin nanoparticles as therapeutic agents for osteoarthritis
Journal Pre-proof Acid-activatable polymeric curcumin nanoparticles as therapeutic agents for osteoarthritis
Changsun Kang, Eunkyeong Jung, Hyejin Hyeon, Semee Seon, Dongwon Lee PII:
S1549-9634(19)30188-1
DOI:
https://doi.org/10.1016/j.nano.2019.102104
Reference:
NANO 102104
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Revised date:
7 September 2019
Please cite this article as: C. Kang, E. Jung, H. Hyeon, et al., Acid-activatable polymeric curcumin nanoparticles as therapeutic agents for osteoarthritis, Nanomedicine: Nanotechnology, Biology, and Medicine(2019), https://doi.org/10.1016/ j.nano.2019.102104
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© 2019 Published by Elsevier.
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Acid-activatable polymeric curcumin nanoparticles as therapeutic agents for osteoarthritis
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Changsun Kang, PhD a,b, Eunkyeong Jung, BS a, Hyejin Hyeon, BS a, Semee Seon, BS a,
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Dongwon Lee, PhD a,c,* a
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Department of BIN Convergence Technology, Chonbuk National University, Jeonju, Chonbuk 54896, Republic of Korea b
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Department of Pharmaceutical Sciences, Texas A&M University, College Station, TX 77843, United States c
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Department of PolymerNano Science and Technology, Chonbuk National University, Jeonju, Chonbuk 54896, Republic of Korea
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* Corresponding author: Dongwon Lee, Email: [email protected]. A word count in Abstract : 148 A word count in text : 4428
A number of references : 51 A number of figures : 7 Supporting information included Conflict of interest: The authors declare no competing financial interest. Funding: This work was supported by the National Research Foundation of Korea grant by the Ministry of Science, ICT and Future Planning (2017R1A4A1015681). 1
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Abstract
Curcumin, a primary active element of turmeric, has potent antioxidant and anti-inflammatory activity, but its low bioavailability is a major hurdle in its pharmaceutical applications. To enhance the therapeutic efficacy of curcumin, we exploited polymeric prodrug strategy. Here, we report rationally designed acid-activatable curcumin polymer (ACP), as a therapeutic prodrug of
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curcumin, in which curcumin was covalently incorporated in the backbone of amphiphilic
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polymer. ACP could self-assemble to form micelles that rapidly release curcumin under the
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acidic condition. The potential of ACP micelles as therapeutics for osteoarthritis was evaluated
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using a mouse model of monoidoacetic acid (MIA)-induced knee osteoarthritis. ACP micelles drastically protected the articular structures from arthritis through the suppression of tumor
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necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). Given their pathological stimulus-
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responsiveness and potent antioxidant and anti-inflammatory activities, ACP micelles hold
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inflammatory diseases.
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remarkable potential as a therapeutic agent for not only osteoarthritis but also various
Keywords: osteoarthritis; inflammation; curcumin; polymeric prodrug; drug delivery 2
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Background Reactive oxygen species (ROS) are engendered as unavoidable byproducts of aerobic metabolism and include hydrogen peroxide (H2O2) and radicals such as singlet oxygen and hydroxyl radicals.1,2 ROS play a crucial role as a signal messenger for cell communication and a
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regulator for gene expression in normal physiological events.3 However, excessive and
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upregulated ROS induce oxidative stress and are considered as a major culprit of acute and/or
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chronic inflammatory diseases which are related with various pathological processes of
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Alzheimer’s disease, asthma, atherosclerosis, dermatitis and arthritis.1,4,5 Osteoarthritis is a common chronic degenerative disease of cartilage and is considered as a
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major cause of functional disability in daily living of the elderly populations.6 During
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osteoarthritis progression, the articular cartilage is most affected through the fibrosis formation and matrix degradation which provoke osteoarthritis development with inflammatory
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mediators.7,8 Chondrocytes, the only cells in cartilage, are sensitive to redox balance under
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hypoxic condition. However, when chondrocytes are mechanically and chemically stressed, they generate ROS and induce redox imbalance which stimulates matrix metalloproteinase (MMPs) with up-regulated inflammatory cytokines, and affect cellular structures associated with extracellular matrix, consequently leading to cell death and cartilage degradation.9,10 Moreover, oxygen free radicals lead to the instability of telomere to progress the replicative senescence for cartilage aging and dysfunction of chondrocytes.11 Conservative treatments for osteoarthritis include nonpharmacologic, pharmacologic, and alternative therapy.12 Pharmacologic therapy involves the use of nonsteroidal anti-inflammatory drugs (NSAIDs). However, these applications 3
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not only alleviate progression of degeneration but also have side effects to circulatory and digestive systems.12 In this regard, development of substances which can regulate ROS level without side effects would be a logical strategy for treatment of osteoarthritis. Curcumin, a main component of curcuma longa, has been extensively utilized as a traditional supplement and regarded as a safe compound.13 Curcumin was determined to be an effective antioxidant through radical scavenging and metal chelating and exert remarkable anti-
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inflammatory activity in musculoskeletal pathologies.14 However, despite its great therapeutic
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potential, curcumin has not been used in therapeutic applications because of several drawbacks
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such as low water solubility, poor absorption, rapid elimination and rapid metabolism.15 In order
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to overcome these hurdles of conventional therapeutics such as curcumin, drug delivery using
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nanoscale drug carriers have been extensively exploited.14,16,17 In particular, curcuminencapsulated micellar systems have drawn great attractions in the treatments for various diseases
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such as cancer and Alzheimer’s diseases.18 However, manipulation between release and
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retention, low drug loading content, dose dependent toxicity and immunogenicity remain a common challenge of traditional micellar drug carriers. A new promising logical strategy for
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targeted and controlled drug delivery involves polymeric prodrugs, in which active drugs are incorporated in the environment-sensitive backbone of biodegradable polymers.2 Unlike conventional polymeric micelles, polymeric prodrugs could retain a high percentage (60%) of drugs that are available as the polymer degrades.19 However, to our best understanding, there are no studies on the polymeric prodrug strategy of curcumin for osteoarthritis. Poly(-amino ester) (PAE), synthesized from the conjugate addition polymerization of diacrylate and amine molecules holds great potential as a platform of polymeric prodrugs 4
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because of its excellent structural tunability, positive charges and biocompatibility.20,21 Among several features of PAE, acid-triggered hydrophobic/hydrophilic transition of tertiary amine groups is a key property to achieve controlled-release of drug payloads under acidic condition.22 In addition, the positively charged PAE would provide benefits in the treatment of osteoarthritis because it could have electrostatic interactions with glycosaminoglycans (GAGs) in cartilage and
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therefore achieves targeted drug delivery.23,24 We therefore reasoned that acid-sensitive and cartilage-targeted PAE would provide a promising strategy in the development of drug carriers
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for osteoarthritis.
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The primary objective of this work is the development of polymeric prodrugs of curcumin to
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explore their potential as a therapeutic agent for osteoarthritis. By taking advantages of
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pathological characteristics of osteoarthritic joint such as low pH (6.6-7.1) and high level of ROS25, we developed anti-inflammatory polymeric prodrug of curcumin (ACP) exploiting PAE
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chemistry. ACP was designed to incorporate curcumin in its hydrophobic backbone of PAE and
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possess a hydrophilic block. The amphiphilic ACP could be fabricated into micelles under aqueous conditions through self-assembly and dissociate under acidic conditions (Figure 1).
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Based on the physicochemical properties of ACP micelles and pathological relationship between low pH and osteoarthritis, we hypothesized that ACP micelles could undergo acid-accelerated hydrolysis to release curcumin in the inflammatory joint. In this study, we examined the potential of ACP micelles as a targeted therapeutic agent for inflammatory diseases using a mouse model of monoidoacetic acid (MIA)-induced osteoarthritis.
Methods 5
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Materials Curcumin was obtained from Alfa Aesar (Haverhill, MA, USA). Acryloyl chloride, poly(ethylene glycol) (PEG)-acrylate (MW 2000 Da), 4,4’-trimethylene dipiperidine, dimethyl sulfoxide-d6 (DMSO-d6), deuterium oxide (D2O), pyrene and MIA were obtained from SigmaAldrich (St. Louis, MO, USA). Tetrahydrofuran (THF), dichloromethane (DCM) and hexane
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were obtained from Showa (Japan).
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Synthesis and Characterization of ACP
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Curcumin (5.42 mmol), acryloyl chloride (10.84 mmol) and trimethylamine (TEA) (10.84 mmol)
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were dissolved in 100 mL of dry THF on ice. The reaction was kept at 4°C for 12 h in dark and
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compound 1 (retardation factor value = 0.7 in 3:7 (v/v) of ethyl acetate-hexane) was obtained from the silica gel column chromatography. ACP was synthesized from the polymerization
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reaction of 1, 4,4’-trimethylene dipiperidine (2.2 mmol) and PEG acrylate (0.22 mmol) in dry
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DCM at 50°C. After 48 h of reaction, ACP was obtained from DCM/water extraction, followed by cold hexane precipitation. The chemical structure of 1 and ACP was analyzed using 1H NMR
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(400 MHz, DMSO-d6) (JEOL, Japan). The molecular weight of ACP was verified by gel permeation chromatography (GPC, Waters, Milford, MA, USA) equipped with a refractive index detector. THF was used as an eluent.
Preparation and Characterization of ACP Micelles Briefly, ACP (1 mg) was dissolved in 1 mL of THF and the solution was mixed with 10 mL of phosphate buffer saline (PBS, pH 7.4). The mixture was gently mixed and THF was completely 6
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evaporated using a rotary evaporator to give ACP micelles. To determine critical micelle concentration (CMC) of ACP, various concentrations of ACP micelles were prepared in PBS, which encapsulate pyrene (5 µM). Excited at 334 nm, fluorospectrometer (FP-6500, JASCO Corp., Japan) was used to measure their fluorescence emission intensity. UV-vis absorbance and fluorescence emission spectra of ACP were obtained using
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spectrometer (S-3100, Scinco, Korea) and fluorospectrometer. For the determination of curcumin content in ACP, ACP micelles were treated by esterase to release curcumin. ACP micelles were
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incubated in the presence of esterase at 37°C for 3 days and then lyophilized. Esterase-incubated
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ACP was dissolved in ethanol and the content of curcumin in ACP was verified with HPLC
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(Agilent Technologies, Willington, DE, USA) by measuring its absorbance at 420 nm.
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A particle size analyzer (Brookhaven Instrument Corp., Holtsville, NY, USA) was used to determine the hydrodynamic diameter of ACP micelles. ACP micelles were stained with
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phosphotungstic acid solution and then observed under a transmission electron microscope at
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100 kV (TEM, Hitachi Corp., Japan).
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Release Kinetics of Curcumin from ACP Micelles ACP micelles (1 mg/mL) were added in a dialysis tube (molecular weight cut off 2,000, Membrane Filtration Product, Inc., Seguin, TX, USA). The tube was immersed in 15 mL of buffer with 1% tween 80 solution (pH 7.4, 6.6, 6.0) and kept with mechanical stirring at 37°C for 10 days. At appropriate time intervals, 100 µL of solution was collected and replaced with fresh solution. The content of curcumin released from ACP micelles was verified using HPLC.
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H2O2 Scavenging ability of ACP Curcumin or ACP micelles were added to 1 mL of H2O2 solution (2 µM). The Amplex Red assay (Invitrogen, Carlsbad, CA, USA) was employed to verify the concentration of H2O2 according to the manufacturer’s protocol.
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Cytotoxicity of ACP Micelles The cytotoxicity of ACP micelles was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium bromide (MTT) assay. When reaching ~80% confluency, RAW 264.7 cells
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and chondrocytes were treated with ACP micelles for 24 h and then given 100 µL of MTT
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solution. After 4 h of incubation, 200 µL of dimethyl sulfoxide was added to each well to
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dissolve the resulting formazan crystals. A microplate reader was used to evaluate the cell
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viability by measuring the absorbance at 570 nm.
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Evaluation of Protective Activity of ACP Micelles To evaluate anti-inflammatory effects of ACP micelles, RAW264.7 cells and chondrocytes were
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stimulated with H2O2 (200 µM) or lipopolysaccharide (LPS, 1 µg/mL). The stimulated cells were treated with ACP micelles or curcumin for 24 h. The cell viability was measured as afore addressed.
Evaluation of Antioxidant Effects of ACP micelles The antioxidant activity of ACP micelles was verified by measuring the level of ROS in RAW264.7 cells stimulated with 1 µg/mL of LPS. Cells were incubated with ACP micelles and 8
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curcumin for 24 h. The culture medium was carefully collected and centrifuged (8000g for 10 min).
The
level
of
ROS
production
was
determined
using
DCFH-DA
(2’,7’-
Dichlorohydrofluorescein diacetate) purchased form Sigma-Aldrich.
Anti-inflammatory Activities of ACP Micelles in vitro
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Curcumin dispersion or ACP micelles were added to LPS-stimulated RAW 264.7 cells. After 24 h of incubation, the culture medium was collected. The level of cytokines was measured with a
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mouse TNF-α ELISA kit (eBioscience, San Diego, CA, USA) and a mouse IL-1β ELISA kit
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(eBioscience).
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A Mouse Model of MIA-Induced Osteoarthritis
Monoidoacetic acid (MIA) (Sigma-Aldrich) was dissolved in PBS at 10 mg/mL. Mice
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anaesthetized with an intraperitoneal injection of mixture of ketamine and xylazine (8:1 ratio)
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were given a single intra-articular injection of 10 µL of MIA solution through patellar tendon, perpendicular to the tibia for the induction of osteoarthritis.26 Five days after MIA injection, the
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mice were given intramuscular injection close to the osteoarthritic joint with one of the following: saline, curcumin (0.7 mg/kg), curcumin (1.4 mg/kg), ACP micelles (2.5 mg/kg) and ACP micelles (5 mg/kg). Each group was intramuscularly injected every 3 days for 28 days. To investigate the fluorescence recovery of ACP at MIA-induced osteoarthritis, 10 µL of MIA (10 mg/mL) was intra-articularly injected to synovial cavity of knee. After 3 h, 50 µL of ACP micelles (1 mg/mL) were given to the knee joint. Fluorescence images of the inflammatory osteoarthritic knees were obtained using IVIS (Lumina2, PerkinElmer, Waltham, MA, USA) 9
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with excitation at 500 to 600 nm 1 h after the injection of ACP micelles. The animal experiments were approved by the Institutional Animal Care and Use Committee of Chonbuk National University (CBNU2015-043) and carried out under the guidelines of the Institutional Animal Ethical Committee. Evaluation of Therapeutic Activity of ACP Micelles
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Knee tissues were excised at 28 days and homogenized in cold PBS. The tissue lysates were centrifuged (10,000×g) at 4°C for 10 min. The level of cytokines in the supernatants were
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measured with a mouse TNF-α ELISA kit (eBioscience) and a mouse IL-1β kit (eBioscience).
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Histological Examination
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Knee joint tissues were fixed in 4% paraformaldehyde. The tissues were decalcified for 6 h using Decalcifying Solution-Lite (Sigma-Aldrich). Tissues were embedded in paraffin and sectioned.
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Tissue sections were stained with H&E (hematoxylin and eosin), Masson’s Trichrome, safranin
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Toxicity studies
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O and aggrecan (sc-33695, Santa Cruz, Dallas, TX, USA).
Mice were intravenously injected ACP micelles (10 or 20 mg/kg) through a tail vein. On day 7, blood serum was collected and a microplate reader was used with an ALT assay kit (Asan Pharma, Korea) to determine the level of ALT (alanine transaminase) in serum. Organs were excised and were fixed in 4% paraformaldehyde. The tissues were stained with H&E for histological examination.
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Statistical Analysis Statistical analysis was conducted employing the one-way ANOVA (analysis of variance). P values of <0.1 were considered statistically significant. Data are expressed as mean ± standard deviation (S.D.).
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Results Synthesis of ACP
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ACP was designed as an acid-sensitive polymeric prodrug of curcumin. As shown in Figure
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S1, diacrylate-containing curcumin (1) was synthesized from the reaction of curcumin with
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acryloyl chloride. ACP was synthesized from the polymerization reaction of 1, trimethylene
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dipiperidine and PEG acrylate at a molar ratio of 0.95:1.0:0.1. 1H NMR analysis confirmed the successful synthesis of ACP (Figure S2). From the end group analysis in NMR spectrum, we
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found that ACP contains ~9 curcumin molecules and ~10 trimethylene dipiperidine molecules in
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its backbone. GPC analysis revealed that APC has molecular weight of ~8000 Da.
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Characterization of ACP micelles
Amphiphilic ACP could self-assemble to form micelles with curcumin-based hydrophobic core and hydrophilic PEG corona. Above the concentrations of ~7 µg/mL, ACP formed hydrodynamically stable micelles through self-assembly in PBS containing 10% serum (Figure 2A). ACP micelles displayed a spherical shape with a mean diameter of ~170 nm, demonstrated by size analysis and TEM (Figure 2B-C). Micelle formulation was further confirmed by 1H NMR (Figure 2D). As a result of self-assembly of ACP in D2O, the protons of curcumin-based 11
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hydrophobic segments were not detected, but one strong peak corresponding to hydrophilic PEG corona was observed, indicating that ACP forms micelles with curcumin-based hydrophobic segments in their core and PEG on their surface. In design, ACP has pH-responsiveness due to the presence of protonable tertiary amine groups in its backbone. In order to investigate acid-accelerated demicellization of ACP micelles,
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their diameter was observed at different pH values for 48 h. As shown in Figure S3, ACP micelles showed increasing proportions in the large diameter under acidic condition with time. In
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addition, ACP micelles displayed the increment of size at acidic pH 6.6 (Figure 2E). It can be
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explained by the rationale that ACP loosely packed because of the decreased hydrophobicity
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resulting from acid-triggered hydrophobic/hydrophilic transition in the hydrophobic core.
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On the basis of the instability of ACP micelles in acidic environments, pH-dependent curcumin release profile from ACP micelles was also examined. At acidic pH, ACP micelles
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continuously released more than 95 % of curcumin within 7 days because of acid-catalyzed
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hydrolysis of ester linkages. On the other hand, ACP micelles displayed a remarkably slow
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release profile at neutral pH (Figure 2F). In addition, ACP micelles could significantly scavenge H2O2 because of electron transferring and H atom donating activities of curcumin.27,28 As shown in Figure S4, 40% of H2O2 was scavenged by 100 µg/mL of ACP micelles.
Photophysical property of ACP micelles The photochemical property of ACP micelles formulated in water was studied by UVspectroscopy and fluorescence spectroscopy. Curcumin dispersed in water exhibited two distinct photoabsorption peaks at 270 nm and 420 nm. However, orange colored ACP showed a distinct 12
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absorption peak at 405 nm in concentration-dependent manners without a curcumin’s characteristic absorption peak ranging from 230 nm to 300 nm (Figure 3A). In ethanol which is a good solvent for both ACP and curcumin, ACP showed a strong absorption at a shorter wavelength than curcumin (Figure S5). The blue-shifted absorption of ACP is a result of acrylation of curcumin compared with unmodified curcumin.29 However, esterase-treated ACP
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showed an absorption peak identical to those of curcumin because esterase-induced cleavage of -amino ester linkages leads to the release of curcumin from ACP. HPLC analysis revealed that
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ACP incorporates ~28 wt% of curcumin in its backbone.
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To explore the potential of ACP micelles as environment-sensitive fluorophore, the
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fluorescence spectra of ACP were recorded in water and THF. ACP exhibited fluorescence
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emission at 570 nm, with a higher intensity at a higher concentration (Figure 3B). ACP micelles formed in water showed fluorescence quenching because of the close proximity of fluorescent
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curcumin in the hydrophobic core. In contrast, the same concentration of ACP exhibited a
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remarkably higher fluorescence intensity in THF than water (Figure 3C-D). In order to evaluate the pH-dependent changes in fluorescence properties, fluorescence spectra of ACP micelles were
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recorded over a wide range of pH values. The peak fluorescence at 560 nm underwent a monotonic increase by acidity (Figure 3E-F). ACP micelles showed 5 times higher fluorescence intensity at pH 2 than at pH 8. These observations demonstrate that ACP micelles are stable at neutral pH, but readily disrupt at acidic pH to recover fluorescence.
Biological activity of ACP micelles
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To verify the cellular uptake of ACP micelles, RAW264.7 cells were observed under a confocal laser scanning microscope. After 30 min of incubation with ACP micelles, the fluorescence was observed in the periphery of cells (Figure S6). Fluorescence became spread to cytosol and its intensity increased with time. The observation suggests that ACP micelles are readily internalized by probably endocytosis. The enhanced fluorescence intensity results from
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the dissociation of micelles and subsequent fluorescence recovery. Cytotoxicity of ACP micelles was evaluated using RAW264.7 cells and chondrocytes. ACP
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micelles induced no or negligible cytotoxicity against both cells at concentrations less than 100
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µg/mL (Figure 4A & Figure S7A). We next evaluated the beneficial effects of ACP micelles (10
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or 25 µg/mL) compared with their equivalent free curcumin (2.8 or 7 µg/mL). After stimulation
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with H2O2 or LPS, a large amount of ROS was generated in cells, evidenced by the strong fluorescence of DCFH-DA (Figure 4B and Figure S7B). Curcumin reduced the level of
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intracellular ROS concentration dependently. However, ACP micelles suppressed the ROS
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production more effectively compared to equivalent curcumin. Cells stimulated with H2O2 or LPS showed remarkably decreased cell viability. ACP micelles significantly inhibited the cell
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death from H2O2- or LPS-induced toxicity (Figure 4C-D and Figure S7C). Anti-inflammatory effects of ACP micelles were also evaluated in LPS-stimulated cells. LPS-stimulated cells showed drastically elevated level of anti-inflammatory TNF-α and IL-1β. The level of TNF-α and IL-1β was substantially diminished by the treatment of ACP micelles, concentration-dependently. In contrast, equivalent curcumin showed moderate anti-inflammatory effects (Figure 4E-F). These results strongly demonstrate that ACP micelles exert higher
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therapeutic activities than free curcumin and also have great potential as an antioxidant and antiinflammatory agent.
Inflammation-responsive fluorescence switch As illustrated in Figure 5A, ACP micelles were injected into a MIA-induced osteoarthritic
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joint and fluorescence images were made to observe their inflammation-responsiveness. ACP
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micelles injected into a normal joint showed no discernable fluorescence signals, but markedly enhanced fluorescence was observed at the site of MIA-induced inflammation in joint (Figure
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5B-C). It can be reasoned that ACP maintains the stable micellar structure to undergo
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inflammatory site to recover fluorescence.
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fluorescence quenching under normal physiological conditions, but readily disrupt at the acidic
Therapeutic activity of ACP micelles
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The antioxidant activity of ACP micelles was determined by measuring the level of H2O2.
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MIA-induced osteoarthritic joint displayed the significantly higher level of H2O2 than nonosteoarthritic joint. ACP micelles suppressed the generation of H2O2 to a level similar to the noninjured group, but equivalent curcumin dispersion exhibited moderate antioxidant effects (Figure 6A). We also measured the level of TNF-α and IL-1β to examine the therapeutic efficacy of ACP micelles on MIA-induced osteoarthritis (Figure 6B-C). MIA significantly upregulated the level of TNF-α. While curcumin dispersion exhibited marginal effects on level of TNF-α, ACP micelles significantly suppressed TNF-α expression. MIA also significantly upregulated
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expression of IL-1β. The treatment of ACP micelles significantly suppressed the MIA-induced upregulation of IL-1β, with stronger effects than equivalent free curcumin.
Histological analysis of osteoarthritic joints Histological analysis was carried out to further examine the therapeutic effects of ACP
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micelles (Figure 7). Non-injured of articular cartilage exhibited regular and smooth surface with evenly distributed aggrecan, collagen and proteoglycan. MIA injection resulted in severe damage
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of cartilage and loss of ECM components. However, after treatment with ACP micelles MIA-
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induced osteoarthritic joints showed smooth surface with structural integrity of cartilage along
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with strong expression of proteoglycan, aggrecan and collagen. In contrast, equivalent curcumin
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exerted marginal effects on the expression of collagen, aggrecan and proteoglycan. This is probably because free curcumin is prone to aggregate in physiological environments and rapidly
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cleared while ACP micelles have prolonged retention time in osteoarthritic joint and release
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curcumin in inflammation-triggered manners.30 These results indicate that ACP micelles have
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highly potent anti-inflammatory and anti-arthritic activity.
Toxicity of ACP micelles
The level of serum ALT was measured to evaluate the potential toxicity of ACP micelles. At a dose less than 20 mg/kg, ACP micelles have no significant difference compared to the control group (Figure S8A). In addition, there was no apparent structural change of main organs in the histological examination (Figure S8B). These results indicate that ACP micelles have excellent safety profile. 16
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Discussion Conventional drug delivery systems such as liposomes, micelles and solid nanoparticles aim to maximize therapeutic efficacy of drugs, but the content of drugs loaded in these carriers is limited.31,32 It has been well accepted that in general, the drug loading content of polymeric nanoparticles is less than ~20 wt%.33 The drug loading content of recently developed delivery
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systems for curcumin is not exceeding 20 wt%.32,34 If drugs have poor therapeutic potency and
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drug loading efficiency is low, a large quantity of drug carriers must be used to achieve sufficient
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therapeutic efficacy, which could lead to toxicity resulting from poor metabolism of excessively
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used carriers. Therefore, there is great need for the development of vehicles which could deliver
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a large amount of drugs. In this regard, as shown in Figure 1, ACP was developed as a polymeric prodrug of curcumin, in which curcumin is covalently incorporated in the backbone of PAEs that
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possesses the tertiary amine groups (pKb = 6.5) to be protonated in acidic inflammatory
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environments. This molecularly engineered polymeric prodrug could be a logical strategy to dramatically enhance therapeutic efficacy of curcumin because of a large quantity of curcumin
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incorporated and pathological stimulus-triggered therapeutic actions. As designed, ACP incorporates ~28% of curcumin, which is a higher ratio compared to conventional drug delivery systems because curcumin serves as one of comonomers for the synthesis of polymers. Acidic pH could be a potential indicator of various pathological conditions and therefore has been extensively exploited as a therapeutic and diagnostic biomarker in the development of drug delivery systems and imaging agents for inflammatory diseases.35 ACP micelles could realize acid-triggered curcumin release and subsequent therapeutic actions. As presented in Figure 3E, 17
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ACP micelles also showed pH-dependent fluorescence quenching-recovery phenomena. At neutral pH, stable ACP micelles displayed fluorescence quenching because of the close proximity of curcumin in the hydrophobic core. In contrast, ACP micelles quickly dissociated at acidic pH because of rapid hydrophobic/hydrophilic transition of protonable amine groups and recovered fluorescence. Acid-dependent fluorescence recovery of ACP micelles was
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substantiated by optical imaging of osteoarthritic knee induced by MIA which was directly injected in the synovial cavity. ACP micelles were injected 3 h after the induction of MIA-
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induced osteoarthritis because intra-articular injection of MIA significantly increases rolling and
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adherent of leucocytes for 3 h, which is a typical response of inflammation.36,37 As shown in
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Figure 5, ACP micelles displayed markedly increased fluorescence signal in the acidic
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inflammatory and osteoarthritic knee associated with accumulation of macrophages and neutrophils38, compared to non-osteoarthritic knee. The fluorescence signal was detected in the
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small area of the joint because inflammation occurs only in the tiny synovial cavity.39
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Fluorescence of curcumin with low intensity at 540 nm could not provide significance in diagnostics of osteoarthritis. However, the recovered fluorescence indicates that ACP micelles
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are activated to exert therapeutic actions under acidic inflammatory conditions. The antioxidant and anti-inflammatory activity of ACP micelles were evaluated using LPSstimulated cells. After LPS stimulation, cells generated a large amount of ROS, which activates inflammatory transcription factors. 40 ACP micelles suppressed ROS generation and expression of inflammatory factor, TNF- and IL-1, more effectively than equivalent free curcumin (Figure 4D-F). The inferior antioxidant and anti-inflammatory activity of curcumin can be
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explained by its low solubility and poor cellular uptake because curcumin was dispersed in the culture medium to avoid the cytotoxicity of organic solvents as a vehicle.41,42 In the progression of osteoarthritis, ROS are known to play a critical role in articular cartilage degeneration.43 ACP micelles significantly reduced the level ROS in MIA-induced osteoarthritic joints. ACP micelles also exerted highly potent anti-inflammatory effects by
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suppressing TNF- and IL-1. One of the major indications of osteoarthritis is cartilage degradation which is characterized by loss of extracellular matrix (ECM), including
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proteoglycan, collagen and aggrecan.44 The anti-osteoarthritic activity was further evidenced by
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histological analysis. As shown in Figure 7, ACP micelles markedly suppressed the damage in
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ECM and helped maintain the structural integrity of articular cartilages. The therapeutic effects
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of ACP micelles are significantly higher than equivalent curcumin dispersion. The excellent antiosteoarthritic activity of ACP micelles could be explained by the controlled and sustained release
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of anti-inflammatory curcumin.45
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Natural compounds have been contributing to the development of new drugs.46 Over 50% of all new-approved drugs were inspired from nature, for examples: biological macromolecules,
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unaltered natural compounds and natural product derivatives. Natural product-derived drugs account for 45% of best-selling drugs.47 Among numerous natural compounds, curcumin has been extensively utilized as traditional medicine and studied in a field of new drug discovery due to its strong anti-inflammatory activity. In the discovery of new drugs, natural drug sources have a number of advantages such as chemical diversity, but many of natural compounds are not highly soluble in blood and therefore have poor bioavailability.48,49 About 30% of natural compounds and 40% of drugs in market are poorly water soluble 50 and therefore these drugs 19
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should be dissolved in water-miscible solvents which could result in toxicity.51 However, as polymeric prodrug nanoparticles can be evenly dispersed in water with high stability, polymeric prodrug strategy eliminates the use of toxic organic solvents because polymeric prodrugs can be formulated into nanoparticles well-dispersible in PBS. The avoidance of organic solvents is an additional advantage of polymeric prodrugs of curcumin.
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Although the findings of the present study demonstrate the remarkable therapeutic potential of ACP micelles, the limitation of the present study is that their therapeutic efficacy was
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evaluated for only acute MIA-induced acute inflammation. Further studies are needed on various
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animal models of osteoarthritis such as genetically modified-, surgically induced- and naturally
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occurred-osteoarthritis. In addition, preclinical studies using rigorous models are warranted to
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examine the pharmacokinetics, therapeutic efficacy, optimal dosing and side effects of ACP micelles.
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In summary, ACP was developed as a polymeric prodrug of curcumin and was formulated
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into hydrodynamically stable micelles by the self-assembly in aqueous solutions. ACP micelles exhibited pH-dependent fluorescence and exerted highly potent antioxidant and anti-
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inflammatory effects in LPS- and H2O2-stimulated cells. In the mouse model of MIA-induced osteoarthritis, ACP micelles emitted the clearly noticeable fluorescence in the tiny osteoarthritic knee joint and suppressed the expression of pro-inflammatory TNF- and IL-1. ACP micelles also remarkably inhibited the cartilage degradation, evidenced by the maintained structural integrity of articular cartilages and extracellular matrix. Given their excellent biocompatibility, high drug content, pathological stimulus-triggered therapeutic action and highly potent
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therapeutic efficacy, ACP micelles have remarkable translational potential for management of various inflammatory diseases such osteoarthritis.
Supporting Information. The Supporting Information is available free of charge. Characterization of compounds and polymers, Characterization of micelles, Histological
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examination of tissues.
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ACKNOWLEDGMENTS
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The authors thank the Center for University-Wide Research Facility (CURF) at Chonbuk
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National University for TEM, HPLC and flow cytometric analysis.
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References
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1. Kwon J, J Kim, S Park, G Khang, PM Kang, D Lee. Inflammation-Responsive Antioxidant Nanoparticles Based on a Polymeric Prodrug of Vanillin, Biomacromolecules 2013; 14: 1618-26. 2. Singh SVB, E Jung, J Noh, D Yoo, C Kang, H Hyeon, et al. Hydrogen peroxide-activatable polymeric prodrug of curcumin for ultrasound imaging and therapy of acute liver failure, Nanomedicine 2019; 16: 45-55. 3. J Jackson M, S Papa, J Bolaños, K Bruckdorfer, H Carlsen, M Ruan, et al. Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function, Mol Aspects Med 2002; 23: 209-85. 4. Nowak WN, J Deng, XZ Ruan, Q Xu. Reactive Oxygen Species Generation and Atherosclerosis, Arterioscler Thromb Vasc Biol 2017; 37: e41-e52. 5. Sivaranjani N, SV Rao, G Rajeev. Role of reactive oxygen species and antioxidants in atopic dermatitis, J Clin Diagn Res 2013; 7: 2683-5. 6. Agas D, F Laus, G Lacava, A Marchegiani, SY Deng, F Magnoni, et al. Thermosensitive hybrid hyaluronan/p(HPMAm-lac)-PEG hydrogels enhance cartilage regeneration in a mouse model of osteoarthritis, J Cell Physiol 2019; 234: 20013-27. 7. Rahmati M, A Mobasheri, M Mozafari. Inflammatory mediators in osteoarthritis: A critical review of the state-of-the-art, current prospects, and future challenges, Bone 2016; 85: 81-90.
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Figure legends
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Figure 1. Schematic showing pH-responsive polymeric prodrug of curcumin as a therapeutic
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system for osteoarthritis.
Figure 2. Characterization of ACP micelles. (A) Determination of CMC of ACP micelles. The
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intensity ratio (I384/I375) from pyrene emission spectra was plotted as a function of ACP concentrations in PBS (pH 7.4). (B) Size distribution of ACP micelles in PBS. (C) TEM image
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of ACP micelles. (D) 1H NMR spectrum of ACP micelles formulated in D2O. (E) TEM image of ACP micelles at pH 6.6. (F) Release kinetics of curcumin from ACP micelles at pH 7.4 and 6.0.
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Values are mean SD (n=4).
Figure. 3. Photophysical properties of ACP micelles. (A) UV-vis absorbance of ACP and curcumin in water. (B) Fluorescence emission spectra of ACP and curcumin in water. (C) Fluorescence spectra of ACP micelles in water and THF. (D) Representative fluorescence image of ACP in water and THF. (E) Fluorescence spectra of ACP micelles at various pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.4, 8.0). (F) Plot of fluorescence intensity (FI) of ACP micelles at 560 nm against pH values. Figure 4. Therapeutic activities of ACP micelles. (A) Cytotoxicity of ACP micelles against RAW 264.7 cells. (B) The inhibitory effect of ACP micelles on the intracellular ROS generation 25
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in LPS-stimulated cells. (C) Protective effect of ACP micelles from H2O2-induced toxicity. (D) Protective effect of ACP micelles from LPS-induced toxicity. Anti-inflammatory effect of ACP micelles on the expression of IL-1β (E) and TNF-α (F) in LPS-stimulated cells. Values are mean ± SD (n=4). **P<0.05, ***P<0.01, ****P<0.001. Figure 5. Fluorescence recovery of ACP micelles at the site of inflammation. (A) Experimental scheme of MIA-induced knee joint osteoarthritis. (B) Representative fluorescence image of dissected joint injected with ACP micelles. (C) Quantitative analysis of fluorescence signal in
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joints. Values are mean ± SD (n=4). ****P<0.001.
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Figure 6. Therapeutic anti-inflammatory activity of ACP micelles in inflamed joint. (A) The level of H2O2 in inflamed joints after treatment with curcumin or ACP micelles. Inhibitory
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effects of ACP micelles on the expression of IL-1β (B) and TNF-α (C) in the inflamed joint.
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Values are mean ± SD (n=4). *P<0.1, **P<0.05, ***P<0.01, ****P<0.001.
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Figure 7. Histological examination of MIA-induced knee osteoarthritic joints. The cartilage
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sections were stained with H&E, Safranin-O and aggrecan.
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Graphical Abstract Acid-responsive anti-inflammatory curcumin polymer, termed ACP is rationally designed to incorporate curcumin in the backbone of acid-sensitive amphiphilic poly(β-amino ester). ACP could form micelles through self-assembly that rapidly release curcumin and recover fluorescence signal in the acidic inflammatory site. ACP micelles enhance fluorescence signals
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and drastically protect the articular structures from arthritis through the suppression of tumor
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necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). Given their pathological stimulusresponsiveness and potent antioxidant and anti-inflammatory activities, ACP micelles hold
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remarkable potential in the capacity of a therapeutic agent for osteoarthritis.
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Figure 1
Figure 2
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Changsun Kang, Eunkyeong Jung, Hyejin Hyeon, Semee Seon, Dongwon Lee PII:
S1549-9634(19)30188-1
DOI:
https://doi.org/10.1016/j.nano.2019.102104
Reference:
NANO 102104
To appear in:
Nanomedicine: Nanotechnology, Biology, and Medicine
Revised date:
7 September 2019
Please cite this article as: C. Kang, E. Jung, H. Hyeon, et al., Acid-activatable polymeric curcumin nanoparticles as therapeutic agents for osteoarthritis, Nanomedicine: Nanotechnology, Biology, and Medicine(2019), https://doi.org/10.1016/ j.nano.2019.102104
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© 2019 Published by Elsevier.
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Acid-activatable polymeric curcumin nanoparticles as therapeutic agents for osteoarthritis
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Changsun Kang, PhD a,b, Eunkyeong Jung, BS a, Hyejin Hyeon, BS a, Semee Seon, BS a,
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Dongwon Lee, PhD a,c,* a
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Department of BIN Convergence Technology, Chonbuk National University, Jeonju, Chonbuk 54896, Republic of Korea b
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Department of Pharmaceutical Sciences, Texas A&M University, College Station, TX 77843, United States c
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Department of PolymerNano Science and Technology, Chonbuk National University, Jeonju, Chonbuk 54896, Republic of Korea
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* Corresponding author: Dongwon Lee, Email: [email protected]. A word count in Abstract : 148 A word count in text : 4428
A number of references : 51 A number of figures : 7 Supporting information included Conflict of interest: The authors declare no competing financial interest. Funding: This work was supported by the National Research Foundation of Korea grant by the Ministry of Science, ICT and Future Planning (2017R1A4A1015681). 1
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Abstract
Curcumin, a primary active element of turmeric, has potent antioxidant and anti-inflammatory activity, but its low bioavailability is a major hurdle in its pharmaceutical applications. To enhance the therapeutic efficacy of curcumin, we exploited polymeric prodrug strategy. Here, we report rationally designed acid-activatable curcumin polymer (ACP), as a therapeutic prodrug of
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curcumin, in which curcumin was covalently incorporated in the backbone of amphiphilic
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polymer. ACP could self-assemble to form micelles that rapidly release curcumin under the
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acidic condition. The potential of ACP micelles as therapeutics for osteoarthritis was evaluated
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using a mouse model of monoidoacetic acid (MIA)-induced knee osteoarthritis. ACP micelles drastically protected the articular structures from arthritis through the suppression of tumor
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necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). Given their pathological stimulus-
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responsiveness and potent antioxidant and anti-inflammatory activities, ACP micelles hold
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inflammatory diseases.
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remarkable potential as a therapeutic agent for not only osteoarthritis but also various
Keywords: osteoarthritis; inflammation; curcumin; polymeric prodrug; drug delivery 2
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Background Reactive oxygen species (ROS) are engendered as unavoidable byproducts of aerobic metabolism and include hydrogen peroxide (H2O2) and radicals such as singlet oxygen and hydroxyl radicals.1,2 ROS play a crucial role as a signal messenger for cell communication and a
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regulator for gene expression in normal physiological events.3 However, excessive and
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upregulated ROS induce oxidative stress and are considered as a major culprit of acute and/or
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chronic inflammatory diseases which are related with various pathological processes of
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Alzheimer’s disease, asthma, atherosclerosis, dermatitis and arthritis.1,4,5 Osteoarthritis is a common chronic degenerative disease of cartilage and is considered as a
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major cause of functional disability in daily living of the elderly populations.6 During
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osteoarthritis progression, the articular cartilage is most affected through the fibrosis formation and matrix degradation which provoke osteoarthritis development with inflammatory
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mediators.7,8 Chondrocytes, the only cells in cartilage, are sensitive to redox balance under
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hypoxic condition. However, when chondrocytes are mechanically and chemically stressed, they generate ROS and induce redox imbalance which stimulates matrix metalloproteinase (MMPs) with up-regulated inflammatory cytokines, and affect cellular structures associated with extracellular matrix, consequently leading to cell death and cartilage degradation.9,10 Moreover, oxygen free radicals lead to the instability of telomere to progress the replicative senescence for cartilage aging and dysfunction of chondrocytes.11 Conservative treatments for osteoarthritis include nonpharmacologic, pharmacologic, and alternative therapy.12 Pharmacologic therapy involves the use of nonsteroidal anti-inflammatory drugs (NSAIDs). However, these applications 3
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not only alleviate progression of degeneration but also have side effects to circulatory and digestive systems.12 In this regard, development of substances which can regulate ROS level without side effects would be a logical strategy for treatment of osteoarthritis. Curcumin, a main component of curcuma longa, has been extensively utilized as a traditional supplement and regarded as a safe compound.13 Curcumin was determined to be an effective antioxidant through radical scavenging and metal chelating and exert remarkable anti-
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inflammatory activity in musculoskeletal pathologies.14 However, despite its great therapeutic
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potential, curcumin has not been used in therapeutic applications because of several drawbacks
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such as low water solubility, poor absorption, rapid elimination and rapid metabolism.15 In order
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to overcome these hurdles of conventional therapeutics such as curcumin, drug delivery using
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nanoscale drug carriers have been extensively exploited.14,16,17 In particular, curcuminencapsulated micellar systems have drawn great attractions in the treatments for various diseases
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such as cancer and Alzheimer’s diseases.18 However, manipulation between release and
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retention, low drug loading content, dose dependent toxicity and immunogenicity remain a common challenge of traditional micellar drug carriers. A new promising logical strategy for
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targeted and controlled drug delivery involves polymeric prodrugs, in which active drugs are incorporated in the environment-sensitive backbone of biodegradable polymers.2 Unlike conventional polymeric micelles, polymeric prodrugs could retain a high percentage (60%) of drugs that are available as the polymer degrades.19 However, to our best understanding, there are no studies on the polymeric prodrug strategy of curcumin for osteoarthritis. Poly(-amino ester) (PAE), synthesized from the conjugate addition polymerization of diacrylate and amine molecules holds great potential as a platform of polymeric prodrugs 4
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because of its excellent structural tunability, positive charges and biocompatibility.20,21 Among several features of PAE, acid-triggered hydrophobic/hydrophilic transition of tertiary amine groups is a key property to achieve controlled-release of drug payloads under acidic condition.22 In addition, the positively charged PAE would provide benefits in the treatment of osteoarthritis because it could have electrostatic interactions with glycosaminoglycans (GAGs) in cartilage and
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therefore achieves targeted drug delivery.23,24 We therefore reasoned that acid-sensitive and cartilage-targeted PAE would provide a promising strategy in the development of drug carriers
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for osteoarthritis.
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The primary objective of this work is the development of polymeric prodrugs of curcumin to
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explore their potential as a therapeutic agent for osteoarthritis. By taking advantages of
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pathological characteristics of osteoarthritic joint such as low pH (6.6-7.1) and high level of ROS25, we developed anti-inflammatory polymeric prodrug of curcumin (ACP) exploiting PAE
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chemistry. ACP was designed to incorporate curcumin in its hydrophobic backbone of PAE and
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possess a hydrophilic block. The amphiphilic ACP could be fabricated into micelles under aqueous conditions through self-assembly and dissociate under acidic conditions (Figure 1).
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Based on the physicochemical properties of ACP micelles and pathological relationship between low pH and osteoarthritis, we hypothesized that ACP micelles could undergo acid-accelerated hydrolysis to release curcumin in the inflammatory joint. In this study, we examined the potential of ACP micelles as a targeted therapeutic agent for inflammatory diseases using a mouse model of monoidoacetic acid (MIA)-induced osteoarthritis.
Methods 5
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Materials Curcumin was obtained from Alfa Aesar (Haverhill, MA, USA). Acryloyl chloride, poly(ethylene glycol) (PEG)-acrylate (MW 2000 Da), 4,4’-trimethylene dipiperidine, dimethyl sulfoxide-d6 (DMSO-d6), deuterium oxide (D2O), pyrene and MIA were obtained from SigmaAldrich (St. Louis, MO, USA). Tetrahydrofuran (THF), dichloromethane (DCM) and hexane
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were obtained from Showa (Japan).
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Synthesis and Characterization of ACP
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Curcumin (5.42 mmol), acryloyl chloride (10.84 mmol) and trimethylamine (TEA) (10.84 mmol)
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were dissolved in 100 mL of dry THF on ice. The reaction was kept at 4°C for 12 h in dark and
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compound 1 (retardation factor value = 0.7 in 3:7 (v/v) of ethyl acetate-hexane) was obtained from the silica gel column chromatography. ACP was synthesized from the polymerization
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reaction of 1, 4,4’-trimethylene dipiperidine (2.2 mmol) and PEG acrylate (0.22 mmol) in dry
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DCM at 50°C. After 48 h of reaction, ACP was obtained from DCM/water extraction, followed by cold hexane precipitation. The chemical structure of 1 and ACP was analyzed using 1H NMR
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(400 MHz, DMSO-d6) (JEOL, Japan). The molecular weight of ACP was verified by gel permeation chromatography (GPC, Waters, Milford, MA, USA) equipped with a refractive index detector. THF was used as an eluent.
Preparation and Characterization of ACP Micelles Briefly, ACP (1 mg) was dissolved in 1 mL of THF and the solution was mixed with 10 mL of phosphate buffer saline (PBS, pH 7.4). The mixture was gently mixed and THF was completely 6
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evaporated using a rotary evaporator to give ACP micelles. To determine critical micelle concentration (CMC) of ACP, various concentrations of ACP micelles were prepared in PBS, which encapsulate pyrene (5 µM). Excited at 334 nm, fluorospectrometer (FP-6500, JASCO Corp., Japan) was used to measure their fluorescence emission intensity. UV-vis absorbance and fluorescence emission spectra of ACP were obtained using
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spectrometer (S-3100, Scinco, Korea) and fluorospectrometer. For the determination of curcumin content in ACP, ACP micelles were treated by esterase to release curcumin. ACP micelles were
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incubated in the presence of esterase at 37°C for 3 days and then lyophilized. Esterase-incubated
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ACP was dissolved in ethanol and the content of curcumin in ACP was verified with HPLC
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(Agilent Technologies, Willington, DE, USA) by measuring its absorbance at 420 nm.
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A particle size analyzer (Brookhaven Instrument Corp., Holtsville, NY, USA) was used to determine the hydrodynamic diameter of ACP micelles. ACP micelles were stained with
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phosphotungstic acid solution and then observed under a transmission electron microscope at
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100 kV (TEM, Hitachi Corp., Japan).
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Release Kinetics of Curcumin from ACP Micelles ACP micelles (1 mg/mL) were added in a dialysis tube (molecular weight cut off 2,000, Membrane Filtration Product, Inc., Seguin, TX, USA). The tube was immersed in 15 mL of buffer with 1% tween 80 solution (pH 7.4, 6.6, 6.0) and kept with mechanical stirring at 37°C for 10 days. At appropriate time intervals, 100 µL of solution was collected and replaced with fresh solution. The content of curcumin released from ACP micelles was verified using HPLC.
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H2O2 Scavenging ability of ACP Curcumin or ACP micelles were added to 1 mL of H2O2 solution (2 µM). The Amplex Red assay (Invitrogen, Carlsbad, CA, USA) was employed to verify the concentration of H2O2 according to the manufacturer’s protocol.
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Cytotoxicity of ACP Micelles The cytotoxicity of ACP micelles was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-
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diphenyltetrazolium bromide (MTT) assay. When reaching ~80% confluency, RAW 264.7 cells
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and chondrocytes were treated with ACP micelles for 24 h and then given 100 µL of MTT
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solution. After 4 h of incubation, 200 µL of dimethyl sulfoxide was added to each well to
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dissolve the resulting formazan crystals. A microplate reader was used to evaluate the cell
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viability by measuring the absorbance at 570 nm.
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Evaluation of Protective Activity of ACP Micelles To evaluate anti-inflammatory effects of ACP micelles, RAW264.7 cells and chondrocytes were
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stimulated with H2O2 (200 µM) or lipopolysaccharide (LPS, 1 µg/mL). The stimulated cells were treated with ACP micelles or curcumin for 24 h. The cell viability was measured as afore addressed.
Evaluation of Antioxidant Effects of ACP micelles The antioxidant activity of ACP micelles was verified by measuring the level of ROS in RAW264.7 cells stimulated with 1 µg/mL of LPS. Cells were incubated with ACP micelles and 8
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curcumin for 24 h. The culture medium was carefully collected and centrifuged (8000g for 10 min).
The
level
of
ROS
production
was
determined
using
DCFH-DA
(2’,7’-
Dichlorohydrofluorescein diacetate) purchased form Sigma-Aldrich.
Anti-inflammatory Activities of ACP Micelles in vitro
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Curcumin dispersion or ACP micelles were added to LPS-stimulated RAW 264.7 cells. After 24 h of incubation, the culture medium was collected. The level of cytokines was measured with a
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mouse TNF-α ELISA kit (eBioscience, San Diego, CA, USA) and a mouse IL-1β ELISA kit
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(eBioscience).
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A Mouse Model of MIA-Induced Osteoarthritis
Monoidoacetic acid (MIA) (Sigma-Aldrich) was dissolved in PBS at 10 mg/mL. Mice
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anaesthetized with an intraperitoneal injection of mixture of ketamine and xylazine (8:1 ratio)
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were given a single intra-articular injection of 10 µL of MIA solution through patellar tendon, perpendicular to the tibia for the induction of osteoarthritis.26 Five days after MIA injection, the
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mice were given intramuscular injection close to the osteoarthritic joint with one of the following: saline, curcumin (0.7 mg/kg), curcumin (1.4 mg/kg), ACP micelles (2.5 mg/kg) and ACP micelles (5 mg/kg). Each group was intramuscularly injected every 3 days for 28 days. To investigate the fluorescence recovery of ACP at MIA-induced osteoarthritis, 10 µL of MIA (10 mg/mL) was intra-articularly injected to synovial cavity of knee. After 3 h, 50 µL of ACP micelles (1 mg/mL) were given to the knee joint. Fluorescence images of the inflammatory osteoarthritic knees were obtained using IVIS (Lumina2, PerkinElmer, Waltham, MA, USA) 9
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with excitation at 500 to 600 nm 1 h after the injection of ACP micelles. The animal experiments were approved by the Institutional Animal Care and Use Committee of Chonbuk National University (CBNU2015-043) and carried out under the guidelines of the Institutional Animal Ethical Committee. Evaluation of Therapeutic Activity of ACP Micelles
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Knee tissues were excised at 28 days and homogenized in cold PBS. The tissue lysates were centrifuged (10,000×g) at 4°C for 10 min. The level of cytokines in the supernatants were
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measured with a mouse TNF-α ELISA kit (eBioscience) and a mouse IL-1β kit (eBioscience).
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Histological Examination
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Knee joint tissues were fixed in 4% paraformaldehyde. The tissues were decalcified for 6 h using Decalcifying Solution-Lite (Sigma-Aldrich). Tissues were embedded in paraffin and sectioned.
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Tissue sections were stained with H&E (hematoxylin and eosin), Masson’s Trichrome, safranin
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Toxicity studies
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O and aggrecan (sc-33695, Santa Cruz, Dallas, TX, USA).
Mice were intravenously injected ACP micelles (10 or 20 mg/kg) through a tail vein. On day 7, blood serum was collected and a microplate reader was used with an ALT assay kit (Asan Pharma, Korea) to determine the level of ALT (alanine transaminase) in serum. Organs were excised and were fixed in 4% paraformaldehyde. The tissues were stained with H&E for histological examination.
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Statistical Analysis Statistical analysis was conducted employing the one-way ANOVA (analysis of variance). P values of <0.1 were considered statistically significant. Data are expressed as mean ± standard deviation (S.D.).
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Results Synthesis of ACP
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ACP was designed as an acid-sensitive polymeric prodrug of curcumin. As shown in Figure
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S1, diacrylate-containing curcumin (1) was synthesized from the reaction of curcumin with
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acryloyl chloride. ACP was synthesized from the polymerization reaction of 1, trimethylene
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dipiperidine and PEG acrylate at a molar ratio of 0.95:1.0:0.1. 1H NMR analysis confirmed the successful synthesis of ACP (Figure S2). From the end group analysis in NMR spectrum, we
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found that ACP contains ~9 curcumin molecules and ~10 trimethylene dipiperidine molecules in
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its backbone. GPC analysis revealed that APC has molecular weight of ~8000 Da.
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Characterization of ACP micelles
Amphiphilic ACP could self-assemble to form micelles with curcumin-based hydrophobic core and hydrophilic PEG corona. Above the concentrations of ~7 µg/mL, ACP formed hydrodynamically stable micelles through self-assembly in PBS containing 10% serum (Figure 2A). ACP micelles displayed a spherical shape with a mean diameter of ~170 nm, demonstrated by size analysis and TEM (Figure 2B-C). Micelle formulation was further confirmed by 1H NMR (Figure 2D). As a result of self-assembly of ACP in D2O, the protons of curcumin-based 11
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hydrophobic segments were not detected, but one strong peak corresponding to hydrophilic PEG corona was observed, indicating that ACP forms micelles with curcumin-based hydrophobic segments in their core and PEG on their surface. In design, ACP has pH-responsiveness due to the presence of protonable tertiary amine groups in its backbone. In order to investigate acid-accelerated demicellization of ACP micelles,
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their diameter was observed at different pH values for 48 h. As shown in Figure S3, ACP micelles showed increasing proportions in the large diameter under acidic condition with time. In
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addition, ACP micelles displayed the increment of size at acidic pH 6.6 (Figure 2E). It can be
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explained by the rationale that ACP loosely packed because of the decreased hydrophobicity
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resulting from acid-triggered hydrophobic/hydrophilic transition in the hydrophobic core.
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On the basis of the instability of ACP micelles in acidic environments, pH-dependent curcumin release profile from ACP micelles was also examined. At acidic pH, ACP micelles
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continuously released more than 95 % of curcumin within 7 days because of acid-catalyzed
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hydrolysis of ester linkages. On the other hand, ACP micelles displayed a remarkably slow
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release profile at neutral pH (Figure 2F). In addition, ACP micelles could significantly scavenge H2O2 because of electron transferring and H atom donating activities of curcumin.27,28 As shown in Figure S4, 40% of H2O2 was scavenged by 100 µg/mL of ACP micelles.
Photophysical property of ACP micelles The photochemical property of ACP micelles formulated in water was studied by UVspectroscopy and fluorescence spectroscopy. Curcumin dispersed in water exhibited two distinct photoabsorption peaks at 270 nm and 420 nm. However, orange colored ACP showed a distinct 12
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absorption peak at 405 nm in concentration-dependent manners without a curcumin’s characteristic absorption peak ranging from 230 nm to 300 nm (Figure 3A). In ethanol which is a good solvent for both ACP and curcumin, ACP showed a strong absorption at a shorter wavelength than curcumin (Figure S5). The blue-shifted absorption of ACP is a result of acrylation of curcumin compared with unmodified curcumin.29 However, esterase-treated ACP
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showed an absorption peak identical to those of curcumin because esterase-induced cleavage of -amino ester linkages leads to the release of curcumin from ACP. HPLC analysis revealed that
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ACP incorporates ~28 wt% of curcumin in its backbone.
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To explore the potential of ACP micelles as environment-sensitive fluorophore, the
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fluorescence spectra of ACP were recorded in water and THF. ACP exhibited fluorescence
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emission at 570 nm, with a higher intensity at a higher concentration (Figure 3B). ACP micelles formed in water showed fluorescence quenching because of the close proximity of fluorescent
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curcumin in the hydrophobic core. In contrast, the same concentration of ACP exhibited a
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remarkably higher fluorescence intensity in THF than water (Figure 3C-D). In order to evaluate the pH-dependent changes in fluorescence properties, fluorescence spectra of ACP micelles were
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recorded over a wide range of pH values. The peak fluorescence at 560 nm underwent a monotonic increase by acidity (Figure 3E-F). ACP micelles showed 5 times higher fluorescence intensity at pH 2 than at pH 8. These observations demonstrate that ACP micelles are stable at neutral pH, but readily disrupt at acidic pH to recover fluorescence.
Biological activity of ACP micelles
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To verify the cellular uptake of ACP micelles, RAW264.7 cells were observed under a confocal laser scanning microscope. After 30 min of incubation with ACP micelles, the fluorescence was observed in the periphery of cells (Figure S6). Fluorescence became spread to cytosol and its intensity increased with time. The observation suggests that ACP micelles are readily internalized by probably endocytosis. The enhanced fluorescence intensity results from
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the dissociation of micelles and subsequent fluorescence recovery. Cytotoxicity of ACP micelles was evaluated using RAW264.7 cells and chondrocytes. ACP
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micelles induced no or negligible cytotoxicity against both cells at concentrations less than 100
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µg/mL (Figure 4A & Figure S7A). We next evaluated the beneficial effects of ACP micelles (10
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or 25 µg/mL) compared with their equivalent free curcumin (2.8 or 7 µg/mL). After stimulation
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with H2O2 or LPS, a large amount of ROS was generated in cells, evidenced by the strong fluorescence of DCFH-DA (Figure 4B and Figure S7B). Curcumin reduced the level of
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intracellular ROS concentration dependently. However, ACP micelles suppressed the ROS
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production more effectively compared to equivalent curcumin. Cells stimulated with H2O2 or LPS showed remarkably decreased cell viability. ACP micelles significantly inhibited the cell
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death from H2O2- or LPS-induced toxicity (Figure 4C-D and Figure S7C). Anti-inflammatory effects of ACP micelles were also evaluated in LPS-stimulated cells. LPS-stimulated cells showed drastically elevated level of anti-inflammatory TNF-α and IL-1β. The level of TNF-α and IL-1β was substantially diminished by the treatment of ACP micelles, concentration-dependently. In contrast, equivalent curcumin showed moderate anti-inflammatory effects (Figure 4E-F). These results strongly demonstrate that ACP micelles exert higher
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therapeutic activities than free curcumin and also have great potential as an antioxidant and antiinflammatory agent.
Inflammation-responsive fluorescence switch As illustrated in Figure 5A, ACP micelles were injected into a MIA-induced osteoarthritic
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joint and fluorescence images were made to observe their inflammation-responsiveness. ACP
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micelles injected into a normal joint showed no discernable fluorescence signals, but markedly enhanced fluorescence was observed at the site of MIA-induced inflammation in joint (Figure
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5B-C). It can be reasoned that ACP maintains the stable micellar structure to undergo
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inflammatory site to recover fluorescence.
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fluorescence quenching under normal physiological conditions, but readily disrupt at the acidic
Therapeutic activity of ACP micelles
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The antioxidant activity of ACP micelles was determined by measuring the level of H2O2.
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MIA-induced osteoarthritic joint displayed the significantly higher level of H2O2 than nonosteoarthritic joint. ACP micelles suppressed the generation of H2O2 to a level similar to the noninjured group, but equivalent curcumin dispersion exhibited moderate antioxidant effects (Figure 6A). We also measured the level of TNF-α and IL-1β to examine the therapeutic efficacy of ACP micelles on MIA-induced osteoarthritis (Figure 6B-C). MIA significantly upregulated the level of TNF-α. While curcumin dispersion exhibited marginal effects on level of TNF-α, ACP micelles significantly suppressed TNF-α expression. MIA also significantly upregulated
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expression of IL-1β. The treatment of ACP micelles significantly suppressed the MIA-induced upregulation of IL-1β, with stronger effects than equivalent free curcumin.
Histological analysis of osteoarthritic joints Histological analysis was carried out to further examine the therapeutic effects of ACP
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micelles (Figure 7). Non-injured of articular cartilage exhibited regular and smooth surface with evenly distributed aggrecan, collagen and proteoglycan. MIA injection resulted in severe damage
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of cartilage and loss of ECM components. However, after treatment with ACP micelles MIA-
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induced osteoarthritic joints showed smooth surface with structural integrity of cartilage along
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with strong expression of proteoglycan, aggrecan and collagen. In contrast, equivalent curcumin
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exerted marginal effects on the expression of collagen, aggrecan and proteoglycan. This is probably because free curcumin is prone to aggregate in physiological environments and rapidly
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cleared while ACP micelles have prolonged retention time in osteoarthritic joint and release
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curcumin in inflammation-triggered manners.30 These results indicate that ACP micelles have
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highly potent anti-inflammatory and anti-arthritic activity.
Toxicity of ACP micelles
The level of serum ALT was measured to evaluate the potential toxicity of ACP micelles. At a dose less than 20 mg/kg, ACP micelles have no significant difference compared to the control group (Figure S8A). In addition, there was no apparent structural change of main organs in the histological examination (Figure S8B). These results indicate that ACP micelles have excellent safety profile. 16
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Discussion Conventional drug delivery systems such as liposomes, micelles and solid nanoparticles aim to maximize therapeutic efficacy of drugs, but the content of drugs loaded in these carriers is limited.31,32 It has been well accepted that in general, the drug loading content of polymeric nanoparticles is less than ~20 wt%.33 The drug loading content of recently developed delivery
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systems for curcumin is not exceeding 20 wt%.32,34 If drugs have poor therapeutic potency and
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drug loading efficiency is low, a large quantity of drug carriers must be used to achieve sufficient
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therapeutic efficacy, which could lead to toxicity resulting from poor metabolism of excessively
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used carriers. Therefore, there is great need for the development of vehicles which could deliver
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a large amount of drugs. In this regard, as shown in Figure 1, ACP was developed as a polymeric prodrug of curcumin, in which curcumin is covalently incorporated in the backbone of PAEs that
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possesses the tertiary amine groups (pKb = 6.5) to be protonated in acidic inflammatory
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environments. This molecularly engineered polymeric prodrug could be a logical strategy to dramatically enhance therapeutic efficacy of curcumin because of a large quantity of curcumin
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incorporated and pathological stimulus-triggered therapeutic actions. As designed, ACP incorporates ~28% of curcumin, which is a higher ratio compared to conventional drug delivery systems because curcumin serves as one of comonomers for the synthesis of polymers. Acidic pH could be a potential indicator of various pathological conditions and therefore has been extensively exploited as a therapeutic and diagnostic biomarker in the development of drug delivery systems and imaging agents for inflammatory diseases.35 ACP micelles could realize acid-triggered curcumin release and subsequent therapeutic actions. As presented in Figure 3E, 17
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ACP micelles also showed pH-dependent fluorescence quenching-recovery phenomena. At neutral pH, stable ACP micelles displayed fluorescence quenching because of the close proximity of curcumin in the hydrophobic core. In contrast, ACP micelles quickly dissociated at acidic pH because of rapid hydrophobic/hydrophilic transition of protonable amine groups and recovered fluorescence. Acid-dependent fluorescence recovery of ACP micelles was
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substantiated by optical imaging of osteoarthritic knee induced by MIA which was directly injected in the synovial cavity. ACP micelles were injected 3 h after the induction of MIA-
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induced osteoarthritis because intra-articular injection of MIA significantly increases rolling and
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adherent of leucocytes for 3 h, which is a typical response of inflammation.36,37 As shown in
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Figure 5, ACP micelles displayed markedly increased fluorescence signal in the acidic
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inflammatory and osteoarthritic knee associated with accumulation of macrophages and neutrophils38, compared to non-osteoarthritic knee. The fluorescence signal was detected in the
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small area of the joint because inflammation occurs only in the tiny synovial cavity.39
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Fluorescence of curcumin with low intensity at 540 nm could not provide significance in diagnostics of osteoarthritis. However, the recovered fluorescence indicates that ACP micelles
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are activated to exert therapeutic actions under acidic inflammatory conditions. The antioxidant and anti-inflammatory activity of ACP micelles were evaluated using LPSstimulated cells. After LPS stimulation, cells generated a large amount of ROS, which activates inflammatory transcription factors. 40 ACP micelles suppressed ROS generation and expression of inflammatory factor, TNF- and IL-1, more effectively than equivalent free curcumin (Figure 4D-F). The inferior antioxidant and anti-inflammatory activity of curcumin can be
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explained by its low solubility and poor cellular uptake because curcumin was dispersed in the culture medium to avoid the cytotoxicity of organic solvents as a vehicle.41,42 In the progression of osteoarthritis, ROS are known to play a critical role in articular cartilage degeneration.43 ACP micelles significantly reduced the level ROS in MIA-induced osteoarthritic joints. ACP micelles also exerted highly potent anti-inflammatory effects by
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suppressing TNF- and IL-1. One of the major indications of osteoarthritis is cartilage degradation which is characterized by loss of extracellular matrix (ECM), including
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proteoglycan, collagen and aggrecan.44 The anti-osteoarthritic activity was further evidenced by
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histological analysis. As shown in Figure 7, ACP micelles markedly suppressed the damage in
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ECM and helped maintain the structural integrity of articular cartilages. The therapeutic effects
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of ACP micelles are significantly higher than equivalent curcumin dispersion. The excellent antiosteoarthritic activity of ACP micelles could be explained by the controlled and sustained release
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of anti-inflammatory curcumin.45
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Natural compounds have been contributing to the development of new drugs.46 Over 50% of all new-approved drugs were inspired from nature, for examples: biological macromolecules,
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unaltered natural compounds and natural product derivatives. Natural product-derived drugs account for 45% of best-selling drugs.47 Among numerous natural compounds, curcumin has been extensively utilized as traditional medicine and studied in a field of new drug discovery due to its strong anti-inflammatory activity. In the discovery of new drugs, natural drug sources have a number of advantages such as chemical diversity, but many of natural compounds are not highly soluble in blood and therefore have poor bioavailability.48,49 About 30% of natural compounds and 40% of drugs in market are poorly water soluble 50 and therefore these drugs 19
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should be dissolved in water-miscible solvents which could result in toxicity.51 However, as polymeric prodrug nanoparticles can be evenly dispersed in water with high stability, polymeric prodrug strategy eliminates the use of toxic organic solvents because polymeric prodrugs can be formulated into nanoparticles well-dispersible in PBS. The avoidance of organic solvents is an additional advantage of polymeric prodrugs of curcumin.
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Although the findings of the present study demonstrate the remarkable therapeutic potential of ACP micelles, the limitation of the present study is that their therapeutic efficacy was
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evaluated for only acute MIA-induced acute inflammation. Further studies are needed on various
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animal models of osteoarthritis such as genetically modified-, surgically induced- and naturally
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occurred-osteoarthritis. In addition, preclinical studies using rigorous models are warranted to
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examine the pharmacokinetics, therapeutic efficacy, optimal dosing and side effects of ACP micelles.
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In summary, ACP was developed as a polymeric prodrug of curcumin and was formulated
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into hydrodynamically stable micelles by the self-assembly in aqueous solutions. ACP micelles exhibited pH-dependent fluorescence and exerted highly potent antioxidant and anti-
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inflammatory effects in LPS- and H2O2-stimulated cells. In the mouse model of MIA-induced osteoarthritis, ACP micelles emitted the clearly noticeable fluorescence in the tiny osteoarthritic knee joint and suppressed the expression of pro-inflammatory TNF- and IL-1. ACP micelles also remarkably inhibited the cartilage degradation, evidenced by the maintained structural integrity of articular cartilages and extracellular matrix. Given their excellent biocompatibility, high drug content, pathological stimulus-triggered therapeutic action and highly potent
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therapeutic efficacy, ACP micelles have remarkable translational potential for management of various inflammatory diseases such osteoarthritis.
Supporting Information. The Supporting Information is available free of charge. Characterization of compounds and polymers, Characterization of micelles, Histological
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examination of tissues.
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ACKNOWLEDGMENTS
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The authors thank the Center for University-Wide Research Facility (CURF) at Chonbuk
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National University for TEM, HPLC and flow cytometric analysis.
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Figure legends
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Figure 1. Schematic showing pH-responsive polymeric prodrug of curcumin as a therapeutic
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system for osteoarthritis.
Figure 2. Characterization of ACP micelles. (A) Determination of CMC of ACP micelles. The
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intensity ratio (I384/I375) from pyrene emission spectra was plotted as a function of ACP concentrations in PBS (pH 7.4). (B) Size distribution of ACP micelles in PBS. (C) TEM image
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of ACP micelles. (D) 1H NMR spectrum of ACP micelles formulated in D2O. (E) TEM image of ACP micelles at pH 6.6. (F) Release kinetics of curcumin from ACP micelles at pH 7.4 and 6.0.
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Values are mean SD (n=4).
Figure. 3. Photophysical properties of ACP micelles. (A) UV-vis absorbance of ACP and curcumin in water. (B) Fluorescence emission spectra of ACP and curcumin in water. (C) Fluorescence spectra of ACP micelles in water and THF. (D) Representative fluorescence image of ACP in water and THF. (E) Fluorescence spectra of ACP micelles at various pH values (2.0, 3.0, 4.0, 5.0, 6.0, 7.4, 8.0). (F) Plot of fluorescence intensity (FI) of ACP micelles at 560 nm against pH values. Figure 4. Therapeutic activities of ACP micelles. (A) Cytotoxicity of ACP micelles against RAW 264.7 cells. (B) The inhibitory effect of ACP micelles on the intracellular ROS generation 25
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in LPS-stimulated cells. (C) Protective effect of ACP micelles from H2O2-induced toxicity. (D) Protective effect of ACP micelles from LPS-induced toxicity. Anti-inflammatory effect of ACP micelles on the expression of IL-1β (E) and TNF-α (F) in LPS-stimulated cells. Values are mean ± SD (n=4). **P<0.05, ***P<0.01, ****P<0.001. Figure 5. Fluorescence recovery of ACP micelles at the site of inflammation. (A) Experimental scheme of MIA-induced knee joint osteoarthritis. (B) Representative fluorescence image of dissected joint injected with ACP micelles. (C) Quantitative analysis of fluorescence signal in
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joints. Values are mean ± SD (n=4). ****P<0.001.
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Figure 6. Therapeutic anti-inflammatory activity of ACP micelles in inflamed joint. (A) The level of H2O2 in inflamed joints after treatment with curcumin or ACP micelles. Inhibitory
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effects of ACP micelles on the expression of IL-1β (B) and TNF-α (C) in the inflamed joint.
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Values are mean ± SD (n=4). *P<0.1, **P<0.05, ***P<0.01, ****P<0.001.
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Figure 7. Histological examination of MIA-induced knee osteoarthritic joints. The cartilage
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sections were stained with H&E, Safranin-O and aggrecan.
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Graphical Abstract Acid-responsive anti-inflammatory curcumin polymer, termed ACP is rationally designed to incorporate curcumin in the backbone of acid-sensitive amphiphilic poly(β-amino ester). ACP could form micelles through self-assembly that rapidly release curcumin and recover fluorescence signal in the acidic inflammatory site. ACP micelles enhance fluorescence signals
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and drastically protect the articular structures from arthritis through the suppression of tumor
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necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). Given their pathological stimulusresponsiveness and potent antioxidant and anti-inflammatory activities, ACP micelles hold
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remarkable potential in the capacity of a therapeutic agent for osteoarthritis.
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Figure 1
Figure 2
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