Polymeric Nanomedicine with âLegoâ Surface Allowing Modular

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Biological and Medical Applications of Materials and Interfaces

Polymeric Nanomedicine with “Lego” Surface Allowing Modular Functionalization and Drug Encapsulation Chen Sun, Haipeng Zhang, Shengke Li, Xiangjun Zhang, Qian Cheng, Yuanfu Ding, Lianhui Wang, and Ruibing Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06598 • Publication Date (Web): 11 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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ACS Applied Materials & Interfaces

Polymeric Nanomedicine with “Lego” Surface Allowing Modular Functionalization and Drug Encapsulation Chen Sun,a Haipeng Zhang,b Shengke Li,a Xiangjun Zhang,a Qian Cheng,a Yuanfu Ding,ac LianHui Wang,c Ruibing Wang *a a

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical

Sciences, University of Macau, Avenida da Universidade, Taipa, Macau, China. b

Department of Gynecology, The First Hospital of Jilin University, Changchun, China.

c

Key Laboratory for Organic Electronics and Information Displays, Jiangsu Key Laboratory of

Biosensors, Institute of Advanced Materials, Nanjing University of Posts and Telecommunications, 9 Wenyuan Road, Nanjing 210023, China KEYWORDS: cucurbituril; surface functionalization; nanomedicine; PLA/PLGA; drug delivery

ABSTRACT: Surface functionalization of nanoparticles (NPs) is of pivotal importance in nanomedicine. However, current strategies often require covalent conjugation that involves laborious design and synthesis. Herein, cucurbit[7]uril (CB[7])-decorated PLA/PLGA NPs are developed and exploited for the first time as a novel, biocompatible and versatile drug delivery platform with a noncovalently tailorable surface. CB[7] on the surface of NPs, acting as a

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“Lego” base block, allowed facile, modular surface modification with a variety of functional moieties or tags that are linked with amantadine (a complementary “Lego” piece to the base block), including amantadine conjugated folate, polyethylene glycol and fluorescein isothiocyanate. In addition, surface CB[7] also provided an opportunity for encapsulation of a secondary drug, such as oxaliplatin, into the cavity of the base block CB[7], in addition to a primary drug (e.g. paclitaxel) loaded into PLA/PLGA NPs, for a possible synergistic chemotherapy. This proof of concept not only provides the first versatile PLA/PLGA nanomedicine platform with “Lego” surface for modular functionalization and improved drug delivery, but also offers new insights to the design and development of novel nanomedicine with a modular surface.

INTRODUCTION Nanomedicine often exhibits unique medical effects including controlled payload release, targeted

delivery,

lower

immunogenicity,

fluorescence-traceability,

and

synergistic

chemotherapy.1-3 These functionalities are usually realized via nanomedicine’s surface modification,4-6 for instance, active targeting is achieved by functionalization of nanoparticles (NPs) with targeting ligands;7-8 PEGylation of nanocarriers would reduce immunogenicity and extend systemic circulation time;9 tagging a fluorescent probe onto the surface of nanomedicine would allow traceable delivery.10 However, conjugation of these functional moieties and tags to nanomaterials often involves laborious chemical design and synthesis, and nanomedicine serving different purposes would have to be functionalized via different chemical methods, as there is no “magic” tool box available for facile “build-and-serve” conjugation, particularly for nanomedicine based on established biomaterials.11-14 In addition, covalent functionalization may


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also affect the integrity of the nanomedicine.12, 15 On the other hand, highly selective, strong yet dynamic host-guest interactions have been employed in the design and construction of functional nanostructures for potential biomedical applications.16-20 Among several families of macrocyclic host molecules, cucurbit[n]urils (CB[n], n = 5-8, and 10) have recently emerged as important building blocks for novel nanostructures with biomedical potential.18, 21-24 For instance, by virtue of the strong interactions between CB[6] and spermine, Kim et al. developed perfunctionalizedCB[6] based nanocapsules and nanoparticles as a versatile platform for multimodal cancertargeted bioimaging and drug delivery,25-28 where a variety of functional moieties were noncovalently attached to the nanocapsules via CB[6]-spermine-functional moiety host-guest interactions. Although fundamentally interesting, none of these nanomaterials are made of established biomaterials geared for real-world biomedical applications,29 and the use of CB[6] on surface, due to its small cavity size, doesn’t allow encapsulation of a secondary drug molecule for synergistic drug delivery.25-28 More importantly, CB[6]-spermine conjugation could be competitively broken apart during targeted cancer therapy, as spermine is an over-expressed biomarker of several types of tumors such as breast, lung and colorectal tumors.30-32 Attributed to the strong binding affinities between CB[7] and amantadine (ADA) and its derivatives,33 CB[7]ADA binding pair, literally without a guest competitor in Nature, would be a superior noncovalent strategy for nanomedicine surface functionalization. To the best of our knowledge, CB[7] has never been employed in decoration of nanomedicine surface, partly due to the significant difficulty in functionalization of CB[7].34-36 Herein, we report the first development of CB[7]-decorated PLA/PLGA NPs as a novel drug delivery platform with a “Lego” surface allowing modular functionalization (Scheme 1). Due to the amphiphilicity of CB[7]-conjugated PLA (CB[7]-PLA), nanoprecipitation of the


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mixture of CB[7]-PLA and PLGA afforded CB[7]-PLA/PLGA NPs with quantitative CB[7] decorated on the surface of the NPs. CB[7] on the surface of NPs allowed noncovalent surface modulation based on host-guest complexations between CB[7] and adamantane (ADA) derivatives (Ka ≈ 108 M-1),37 yielding a variety of surface functionalized NPs, including but not limited to: (1) folate-amantadine (FA-ADA) modified NPs that exhibited targeted delivery to folate-receptor overexpressed cell line; (2) fluorescein isothiocyanate-amantadine (FITC-ADA) modified NPs for fluorescence trackable delivery in cells; (3) PEG-amantadine (PEG-ADA) modified NPs for lowering phagocytosis by macrophage; and (4) a secondary drug encapsulated inside CB[7] on the surface of NPs for synergistic therapy (together with a primary drug loaded into the NPs) against cancer cells.

Scheme 1. a) Synthesis of CB[7]-PLA. b) Schematic diagram exhibiting CB[7]-PLA/PLGA NPs with noncovalently tailorable surface.


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EXPERIMENTAL SECTION Materials and Methods. AO1-CB[7] was prepared according to a literature report.38 DL-Lactide (DL-LA, 98%), 6,6'-disulfanediylbis(hexan-1-ol) (SS-OH, 98%), stannous octoate (Sn(Oct)2, 98%), folic acid (FA, 98%), 1-amantadine amine hydrochloride (ADA, 99%), tris(2carboxyethyl)phosphine

(TCEP,

98%),

triethylamine

(TEA,

99%),

1-ethyl-3-(3-

dimethylaminopropyl) carbodiimide hydrochloride (EDC·HCl, 97%), N-hydroxysuccinimide (NHS, 98%) and Fluorescein isothiocyanate isomer I (FITC, 90%) were purchased from SigmaAldrich (China). Poly(D, L-lactic-co-glycolic acid) [50:50] (PLGA) with intrinsic viscosity of 0.50-0.65 was purchased from Polysciences, Inc. mPEG2k-NHS (95%) was purchased from Xi’an ruixi Biological Technology Co., Ltd (Xi’an, China). Paclitaxel (PTX, 97%) was purchased from Xi’an haoxuan Biological Co., Ltd (Xi’an, China). Oxaliplatin (OX, Reference Standard) was purchased from Shanghai Machlin Biochemical Co., Ltd (Shanghai, China). Penicillin and streptomycin were provided by HyClone (Waltham, MA, USA). Dulbecco's Modified Eagle Medium (DMEM) medium and fetal bovine serum (FBS) were obtained from Gibco (USA). Cyanine Dyes (Cy5, 95%) and 4’, 6-Diamidino-2-phenylindole (DAPI, 95%) were purchased from Invitrogen (USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT, 98%) was supplied by Amresco. All the reagents and solvents employed were commercially available and used as supplied without further purification. Dialysis was performed using Slide-A-Lyzer® dialysis cassette (MWCO, 2 kD). Milli-Q water was purified using a Milli-Q Integral system from Merck Millipore.


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The

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H NMR spectra were acquired on a Bruker Ultra Shield 600 PLUS NMR

spectrometer. Fourier transform infrared assay (FT-IR) was performed on Thermo Nicolet iS10 FT-IR Spectrometer. The number-average molecular weight (MW) and molecular weight distribution (MW/Mn) of thiol-PLA and CB[7]-PLA were determined by advanced polymer chromatography (APC). The thermal stability (weight loss) of the polymers were assessed via thermal gravimetric analysis (Perkin-Elmer TGA-7) and the glass transition temperatures (Tg) were obtained on differential scanning calorimetry (Perkin-Elmer DSC-7), with a typical heating rate of 10 °C/min in nitrogen. The fluorescence spectroscopy was performed using a Lumina™ fluorescence spectrophotometer, Thermo Scientific, with a 1.0 cm path length quartz cell. The size and zeta potential of micelles were determined by doing dynamic light scattering (DLS) at 25°C with a Zetasizer (Malvern. Co., UK). Transmission electron microscopy (TEM) analysis was performed using a Tecnai G20 TEM (FEI, Co., USA) at operation voltage of 200KV. The amount of platinum in OX loaded CB[7]-PLA/PLGA NPs was examined by inductively coupled plasma mass spectrometry (ICP-MS). The drug concentration was detected using HPLC, the chromatographic conditions were as follows: the column used was an XDB C18 (4.6×250 mm, 5 mm), and the mobile phase consisted of acetonitrile and water (60/40, v/v). Cell uptake and cell apoptosis was analyzed immediately by a FACS flow cytometer (Beckman coulter). A confocal laser scanning microscopy (CLSM, Zeiss LSM710) was used to directly visualize the intracellular location of micelles. Cell viability was measured by a multi-mode microplate reader (FlexStation 3). The L-02 and RAW 264.7 cell lines were obtained from the Committee of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). The MCF-7 cell lines were purchased from American Type Culture Collection (ATCC, Shanghai, China).


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Synthesis of thiol-PLA

Scheme 2. Synthesis of thiol-PLA. Thiol-PLA was prepared by ring opening polymerization of DL-LA initiated by SS-OH (Scheme 2). Certain amounts of DL-LA, SS-OH (molar ratio = 70 : 1; 70 : 5; 70 : 10) and Sn(Oct)2 (Catalyst/monomer ratio = 5/10000) were weighted into a flask. The mixture was heated to 80 °C under vacuum to remove oxygen and water. The polymerization was carried out under vacuum at 140 °C for 48 h. The resulting thiol-PLA was dissolved in chloroform and precipitated in ethyl ether. The collected thiol-PLA was a pale yellow powder, which was further dried under vacuum. The product was purified by dissolving precipitated method (yield: 74%). According to NMR results, molar ratio=70 : 5 shown the highest molecular weight, so used it do the following experiment. 1H NMR (600 MHz, DMSO) δH (ppm) 5.21 (q, 1H), 4.23 (q, 1H), 4.11 (t, 2H), 2.69 (t, 2H), 1.58 (m, 2H), 1.46 (d, 3H). Synthesis of CB[7]-PLA AO1-CB[7] was synthesized following Kim’s method.38 CB[7]-PLA was synthesized by the addition of AO1-CB[7] (925 mg, 0.75 mmol) to thiol-PLA (750 mg, 0.4 mmol), catalyzed by


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TCEP (1025 mg, 4 mmol.), through thiol-ene click reaction upon irradiation of UV light (λmax = 256 nm) in 35 mL DMSO in a quartz tube for 12 h. The product was deposited by acetone and washed with deionized water, and dried under the reduced pressure to give the target compound (1 g, 60%). 1H NMR (600 MHz, DMSO) δH (ppm) 5.65 (m, 1H), 5.41 (m, 1H), 5.21 (q, 1H), 4.21 (m, 1H), 4.11 (t, 2H), 3.62 (t, 2H), 2.68 (t, 2H), 1.74 (m, 2H), 1,59 (m, 2H), 1.46 (d, 3H). RESULTS AND DISCUSSION Preparation and characterizations of CB[7]-PLA/PLGA NPs

CB[7]-PLA was synthesized by a thiol-ene click reaction between AO1-CB[7] ((allyloxy)1CB[7]) to thiol-PLA oligomer upon UV irradiation (SI for experimental details), and 1H-NMR, FT-IR, APC analysis (MW), TGA and DSC supported the successful conjugation and formation of CB[7]-PLA (Figure S1, S2, S3, S4,

S5 and Table S1). CB[7]-PLA/PLGA NPs were

subsequently prepared via oil/water precipitation/centrifugation method as described by Jeon et al (SI for experimental details).39 In fact, initially the use of 100% CB[7]-PLA (without any PLGA) in this preparation process generated large particles with diameters of micron-scale (~ 1920 ± 350 nm), similar in scale to the previously reported large particles self-assembled from short-chain allyl and long-chain alkyl derivatized CB[7].40-41 This result suggested that CB[7]PLA alone was unable to form suitable-sized NPs for biomedical applications with the precipitation method, likely attributed to its amphiphilicity and a relatively low molecular weight of the PLA moiety. We subsequently blended commercial PLGA with CB[7]-PLA for preparing NPs with the same precipitation/centrifugation method, where we expected CB[7]-PLA to act, at least in part, in a role of surfactant. With an increasing weight percentage of CB[7]-PLA (0%, 20%, 40%, 60%, 80%) in the CB[7]-PLA and PLGA mixture, NPs with suitable sizes (200 ~ 322


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nm range) were achieved (Figure S6). DLS analysis (Figure 1a) of the NPs prepared with 60% CB[7]-PLA and 40% PLGA revealed that the NPs were around 220 (± 100) nm in diameter in aqueous solution, consistent with the size derived from TEM analysis showing solid nanospheres of 150 ± 30 nm in solid phase (Figure 1b). The relatively larger particle size derived from DLS, in comparison with that from TEM, is likely attributed to the solvation effects of the NPs in aqueous solutions. CB[7]-PLA/PLGA NPs were surface-functionalized by simple mixing the NPs with modular “blocks", such as FA-ADA, FITC-ADA, PEG-ADA and OX, and subsequent centrifugation to remove excess ligands. FTIR analysis (Figure S7) of the functionalized NPs revealed that the modular units were conjugated on the surface of the NPs. Their size-stability and zeta potentials were further evaluated by Zetasizer for 11 days under ambient conditions. As shown in Figure S8, most of these NPs were relatively stable in size, except for FA-and Oxfunctionalized NPs that tended to aggregate over time. On the other hand, PEG-functionalized NPs were extremely stable in size and surface properties. During clinical applications, CB[7]PLA/PLGA NPs would be freshly functionalized shortly before administration, with this novel modular approach by clinicians based on the clinical needs of their patients. Surfacefunctionalized NPs wouldn’t involve extended storage.


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Figure 1. a) DLS and b) TEM image of CB[7]-PLA/PLGA NPs containing 60% CB[7]-PLA and 40% PLGA (w/w). c) Quantity of CB[7] on CB[7]-PLA/PLGA NPs containing 0%, 20%, 40%, 60%, 80% and 100% CB[7]-PLA. d) Drug (paclitaxel) release profile of CB[7]-PLA/PLGA NPs at pH 6.2 and 7.4 at 37 oC, respectively. Data are expressed as the mean values (n = 3) ± standard deviation. Quantification of CB[7] on the surface of CB[7]-PLA/PLGA NPs

CB[7] on the surface of CB[7]-PLA/PLGA NPs was quantified according to a method reported by A. Hennig et al. (SI for experimental details, Figure S9).42 With the increasing CB[7]-PLA w% (from 0, 20, 40, 60, to 80%), the number of CB[7] on CB[7]-PLA/PLGA NPs generally increased in a more or less linear manner until CB[7]-PLA content reached about 60% (w/w) with the rest being PLGA, and it became stable when CB[7]-PLA content was about 6080% (Figure 1c), suggesting a probable surface-CB[7] saturation. Interestingly, NPs made of 100% CB[7]-PLA exhibited an even lower quantity of CB[7] on their surface, likely attributed to the smaller surface to volume ratio of larger particles.43 Considering the particle size/size


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distribution as well as surface CB[7] quantity, CB[7]-PLA/PLGA NPs made of 60% (w/w) CB[7]-PLA were selected for subsequent studies. Biocompatibility of CB[7]-PLA/PLGA NPs

As a biocompatibility study in vitro, the cytotoxicity of CB[7]-PLA/PLGA NPs (up to 750 µg/mL) was evaluated first via MTT assays on RAW 264.7 and L-02 cell lines upon incubation for up to 48 h, with PLGA NPs as the “Gold Standard“ for its superior biocompatibility. To our pleasant surprise, the results on both cell lines suggested that the biocompatibility of CB[7]PLA/PLGA NPs was comparable with, and even moderately better than, that of the Gold Standard (Figure S10). Drug loading and release profile

To test the drug loading and release profile of the NPs, a natural anticancer drug, paclitaxel (PTX), was used as a hydrophobic model drug loaded into CB[7]-PLA/PLGA NPs during the precipitation process, the encapsulation efficiency (EE) and drug loading content (DL) of PTX in CB[7]-PLA/PLGA NPs were determined by HPLC (Table S2, Supporting Information) and optimized by varying PTX feeding ratio (details described in SI). When monitored for drug release at pH 7.4 and 6.2 (simulative of tumor tissue pH) respectively, PTX loaded CB[7]PLA/PLGA NPs (Figure 1d) exhibited similar release kinetics, with moderately higher cumulative amount of drug released, in comparison with those of PTX loaded PLGA NPs (Figure S11). Cellular uptake of FA-ADA/CB[7]-PLA/PLGA NPs


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Encouraged by these results, we exploited the potential application of CB[7]-PLA/PLGA NPs as a versatile drug delivery platform with a surface that can be noncovalently tailored for facile functionalization. Thanks to the strong complexations between CB[7] and ADA derivatives, we firstly prepared ADA-conjugated FA (FA-ADA, details of synthesis and characterization in the SI) for surface modification on CB[7]-PLA/PLGA NPs by simple mixing and centrifugation to remove free FA-ADA, aiming to have FA-functionalized PLA/PLGA NPs for targeted drug delivery. MCF-7 cell line, known for folate receptors overexpression, was employed to study a possible targeted delivery of the NPs. Flow cytometry analysis was performed to evaluate the cellular uptake of FA-ADA/CB[7]-PLA/PLGA NPs loaded with Cy5 as a fluorescent cargo. Cy5 loaded CB[7]-PLA/PLGA NPs without FA-ADA were prepared as a control. Upon incubation with these two groups of NPs at 50 µg/mL concentration for different time lengths, or with various concentrations for 8 h, the relative fluorescence intensity of MCF-7 cells, as a result of cellular uptake of Cy5-loaded NPs, exhibited time-dependent and NPs-dose dependent manners, respectively. And FA-ADA/CB[7]-PLA/PLGA NPs exhibited significantly enhanced cellular uptake by MCF-7 cells, in comparison with CB[7]-PLA/PLGA NPs (Figure 2a,b). In addition, different quantities of FA-ADA were noncovalently attached to the surface of the NPs for studying the influence of folate quantity on the cellular uptake behaviors of the NPs. As shown in Figure 2c, the fluorescence intensity of MCF-7 cells increased until the surface CB[7] of CB[7]-PLA/PLGA NPs was saturated with FA-ADA due to the formation of 1:1 ADACB[7] host-guest complexes (around 0.8 µg/mL of FA-ADA), suggesting that noncovalent surface functionalization with FA significantly improved the receptor-mediated endocytosis of the NPs in a dose-dependent manner (until surface saturation). On the other hand, the fluorescence intensity of MCF-7 cells decreased, when the cells were co-treated with Cy5-loaded


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FA-ADA/CB[7]-PLA/PLGA NPs and excess FA that may competitively occupy FA-receptor on the cell membrane (Figure 2d), suggesting that the uptake of FA-ADA/CB[7]-PLA/PLGA NPs was indeed via FA-receptor mediated endocytosis. To further demonstrate that the surface functionalization was via noncovalent interactions between surface CB[7] and FA-ADA, FAADA/CB[7]-PLA/PLGA NPs, containing 20% CB[7]-PLA and 60% CB[7]-PLA, respectively, were loaded with Cy5 and incubated with MCF-7 cells for endocytosis studies. As expected, in comparison with those incubated with Cy5-loaded PLGA NPs and Cy5-loaded FA-ADA/CB[7]PLA/PLGA NPs with 20% CB[7]-PLA, the fluorescence intensity of MCF-7 cells incubated with FA-ADA/CB[7]-PLA/PLGA NPs containing 60% CB[7]-PLA was significantly higher (Figure 2e), suggesting that higher quantity of CB[7] on the surface of NPs would lead to higher degree of surface functionalization through non-covalent modulation. Meanwhile, Cy5-loaded PLGA NPs were employed as the control NPs without any CB[7] on the surface. Figure 2f shows that the fluorescence intensities of MCF-7 cells treated with Cy5-loaded PLGA NPs mixed with or without FA-ADA, or co-treatment with FA, exhibited no differences. Confocal Laser Scanning Microscopy (CLSM) was employed to further examine the cellular uptake and distribution of Cy5 loaded CB[7]-PLA/PLGA NPs and FA-ADA/CB[7]-PLA/PLGA NPs within MCF-7 cells. As shown in Figure 2g, 2h and S12, after incubation of MCF-7 cells with the NPs for 4-24 h, strong intracellular red fluorescence was observed in the cytoplasm of MCF-7 cells incubated with Cy5 loaded FA-ADA/CB[7]-PLA-PLGA NPs, in contrast to that of the NPs without FA-ADA functionalization. Taken together, all these results demonstrated that the surface of CB[7]-PLA/PLGA NPs can be noncovalently functionalized with a targeting molecule for targeted payload delivery and the extent of surface functionalization are controllable. The mechanism of transcellular delivery of CB[7]-PLA/PLGA NPs is likely similar to that of PLGA


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NPs due to the chemical and nanosized nature of these NPs, which has been extensively studied previously.44-45

Figure 2. Cellular uptake of Cy5-loaded CB[7]-PLA/PLGA NPs, FA-ADA/CB[7]-PLA/PLGA NPs, and PLGA NPs by MCF-7 cells determined by flow cytometry. MCF-7 cells were treated with a) 50 µg/mL NPs for different time lengths, or b) different concentrations of NPs for 8 h, or c) 50 µg/mL CB[7]-PLA/PLGA NPs prepared with different quantities of FA-ADA for 8 h, or d) 50 µg/mL FA-ADA/CB[7]-PLA/PLGA NPs for 8 h, co-treatment with different concentrations of FA, or e) PLGA NPs, CB[7]-PLA-PLGA NPs with 20% and 60% of CB[7]-PLA, respectively pre-treated with the same quantities of FA-ADA, or f) 50 µg/mL PLGA NPs mixed with or without FA-ADA, or co-treatment with FA, respectively, for 8 h. Data are expressed as the mean values (n = 3) ± standard deviation. Unpaired t-test analysis was used for statistical analysis


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