F68 Micelles of Baicalein Could Interfere with

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Pluronic P85/F68 Micelles of Baicalein Could Interfere with Mitochondria to Overcome MRP2-Mediated Efflux and Offer Improved Anti-Parkinsonian Activity Tongkai Chen, Ye Li, Chuwen Li, Xiang Yi, Ruibing Wang, Simon Ming Yuen Lee, and Ying Zheng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00374 • Publication Date (Web): 14 Aug 2017 Downloaded from http://pubs.acs.org on August 15, 2017

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Molecular Pharmaceutics

Pluronic P85/F68 Micelles of Baicalein Could Interfere with Mitochondria to Overcome MRP2-Mediated Efflux and Offer Improved Anti-Parkinsonian Activity

Tongkai Chen1, 2, Ye Li1, Chuwen Li1, Xiang Yi3, Ruibing Wang1, Simon Ming-Yuen Lee1, Ying Zheng1* 1

State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macau, China 2

Institute of Clinical Pharmacology, Guangzhou University of Chinese Medicine, Guangzhou, China 3

Division of Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, North Carolina, USA

*To whom correspondence should be addressed: Ying Zheng, Ph.D. Institute of Chinese Medical Sciences, University of Macau Tel: (+853) 88224687; Fax: (+853) 28841358 E-mail: [email protected]

ACS Paragon Plus Environment


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Abstract

Overexpression of the drug efflux transporter multidrug resistance-associated protein 2 (MRP2) in the gastrointestinal tract and blood-brain barrier compromises the oral delivery of drugs to the circulation system and brain in the treatment of Parkinson’s disease (PD).

In this study, we aim to develop small-sized Pluronic

P85/F68 micelles loaded with baicalein (B-MCs) to overcome MRP2-mediated efflux and to investigate related mechanism, as well as the anti-Parkinsonian efficacy. Spherical and sustained-release B-MCs have a mean particle size of 40.61 nm, a low critical micelle concentration (CMC) of 5.01 × 10-3 mg/mL with an encapsulation efficiency of 95.47% and a drug loading of 7.07%.

In comparison with the free

baicalein, the cellular uptake and apparent permeability coefficient (Papp) of B-MCs were significantly enhanced (p 99%) was purchased from Chengdu Preferred

Biological Technology Co., Ltd. (Chengdu, China). from Invitrogen (Carlsbad, CA, USA).

DiO and DiI were purchased

Dulbecco’s modified Eagle medium (DMEM)

and fetal bovine serum (FBS) were obtained from Gibco (Carlsbad, CA, USA). MK-571 [5-(3-(2-(7-chloroquinolin-2-yl)ethenyl)phenyl)-8-dimethyl-carbamyl-4,6-dithiaoc-ta noic

acid

sodium

salt

hydrate,

C26H26CIN2NaO3S2·xH2O],


MTT


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[3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

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bromide],

MPTP,

1-methyl-4-phenylpyridinium ion (MPP+), and L-deprenyl (L-dep, Selegiline) were purchased from Sigma-Aldrich (St. Louis, MO, USA).

SH-SY5Y cells were

obtained from the American Type Culture Collection (Manassas, VA, USA).

Permeability Study of Baicalein in MDCK-MRP2 Cells

An MTT assay was also carried out to select the concentration for permeability study (Supporting Information, section S1).

For the transport study, the cells were

seeded on Transwell polycarbonate membranes in 12-well plates and allowed 4 days to reach confluence.

The integrity of cell monolayers was examined by Lucifer

yellow combined with trans-epithelial electrical resistance (TEER).

Cell monolayers

were considered to be intact and suitable for transport experiments when TEER values were above 250 Ωcm2 and Lucifer yellow permeability less than 1 × 10-6 cm/s.

The

transport study of baicalein across MDCK-MRP2 cell monolayers was performed to determine the apparent permeability coefficient (Papp) in two directions.

Papp(A→B)

and Papp(B→A) represented the permeability values from the apical (A) to basolateral (B) side and from the B to A side, respectively.

A-to-B transport was conducted by

adding 0.5 mL of medium containing of baicalein to the A chamber and 1.5 ml of Hank’s buffered salt solution (HBSS, pH7.4) to the B chamber.

B-to-A transport

was evaluated by adding 1.5 mL of medium containing of baicalein to the B chamber and 0.5 mL of HBSS to the A chamber.

After incubation for 2 h at 37 °C, samples

(0.2 mL) were collected from the B chamber at prescheduled time intervals (0.25, 0.5,


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1, 1.5, and 2 h) to obtain the Papp(A→B), and vice versa.

The concentration of

baicalein was detected by high performance liquid chromatography (HPLC) using a ZORBAX Eclipse Plus C18 (4.6×250 mm, 5 µm) analytical column in Agilent 1200 series HPLC system (Santa Clara, USA).

A 10 µL sample was subjected to the

column, eluted by methanol/0.05% (v/v) phosphoric acid solution (70: 30, v/v) at a flow rate of 1 mL/min for 7 min and detected by UV 276 nm.

The Papp (cm/s) was

calculated using the eq 1:21

Papp =

dQ / dt ×1 A× C0

1

where dQ/dt is the transport rate (ng/s), C0 is the initial drug concentration on the apical chamber (ng/ml), and A is the surface area of the membrane filter (cm2).

The

efflux ratio was obtained according to the following eq 2:22

Efflux ratio =

Papp ( B → A) Papp (A → B )

2

Experiments involving inhibition of baicalein efflux were carried out by incubating baicalein (50 µM) together with each of the 0.01 mg/mL excipients on the A side of the monolayer.

Mixed excipients (0.005 mg/mL + 0.005 mg/mL) were also

evaluated for their combined inhibition effects following similar procedures. MK571 (20 µM, a specific MRP2 inhibitor) was served as positive control in this study.

B-MCs Preparation and Characterization

B-MCs were prepared by a thin-film hydration method.23, 24


Briefly, 2 mg of


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baicalein and 100 mg of Pluronic mixture containing P85 and F68 (1:1, w/w) were dissolved in 10 mL of acetonitrile.

The solution was subsequently evaporated under

vacuum rotation at 60 °C to yield a thin film of drug/polymer, and the film was hydrated in 10 mL of water at 60 °C for 1 h.

Finally, the solution was filtered

through a 0.22 µm membrane to remove unincorporated drug aggregates.

The empty

micelles (MCs) were prepared according to the same procedure without baicalein. To optimize the B-MCs formulation, B-MCs were also prepared under multiple conditions by varying the solvent composition, the amount of baicalein, the weight ratio of Pluronic P85 and Pluronic F68, the volume of water, the hydration temperature, and the hydration time.

The obtained B-MCs were then characterized

in terms of their physicochemical properties including particle size, polydispersity index (PDI), zeta potential, critical micelle concentration (CMC), morphology, encapsulation efficiency (EE), and drug loading (DL).

A detailed description of the

B-MCs characterization is provided in the Supporting Information, section S2.

Physical Stability of B-MCs

The physical stability of B-MCs was performed in simulated gastric fluid with pepsin (SGF, pH 1.2), simulated intestinal fluid with trypsin (SIF, pH 6.8), and phosphate-buffered saline (PBS) for physiological pH of 7.4. prepared according to the direction in US Pharmacopeia.

These media were

1 mL of B-MCs were

dispersed in 10 mL of simulated fluid and shaken in a thermostatic shaker at 37 °C. Samples were then taken at prescheduled time intervals (0, 1, 2, 4, and 6 h). Mean


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particle size, PDI, zeta potential, EE, and DL were measured before and after incubation.

In Vitro Drug Release Study

The in vitro release of baicalein from the B-MCs was evaluated under SGF, SIF and PBS buffer pH 7.4 conditions (37 °C, 100 r/min).

Briefly, 0.4 mg/mL of B-MCs

in 1 mL was dialyzed through a dialysis bag with the molecular weight cut off 3500 (Biotopped, USA) into 20 mL of release medium.

To maintain the sink condition,

release medium was replaced with fresh medium at prescheduled time points.

The

concentration of baicalein was detected by HPLC as described in Permeability Study of Baicalein in MDCK-MRP2 Cells.

MRP2 inhibition evaluation

MDCK-MRP2 cells (200,000 cells/mL) were seeded in a 12-well plate and allowed to attach for 48 h.

Subsequently, the cell culture media were replaced with

fresh media containing 20 µM baicalein, B-MCs or physical mixture of baicalein with Pluronic P85 and F68 (B-PM).

After incubation for 2 h, the cells were washed with

cold PBS (4 °C) and lysed with lysis buffer. as the positive control.

Besides, MK571 (20 µM) was selected

To evaluate the levels of cellular uptake of baicalein,

acetonitrile was added to the samples to precipitate the protein by vortexing (2 min) and subsequent centrifugation (15, 000 × g, 20 min) at room temperature.

The

obtained supernatants were processed to determine the baicalein contents by HPLC as



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described in Permeability Study of Baicalein in MDCK-MRP2 Cells.

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All data

were expressed as the amount (in micrograms) of baicalein per milligram of the total cellular protein.

The A-to-B transport of baicalein, B-MCs or B-PM was also

investigated as described in Permeability Study of Baicalein in MDCK-MRP2 Cells.

Intracellular Integrity Monitoring by FRET

To evaluate the physical state of micelles in cells, cells were treated with DiO/DiI-MCs at the concentration of 5 µg/mL (equal to total fluorophore concentration of 100 ng/mL) at 37 °C for 15, 30 and 60 min, respectively. After treatment, the incubation medium was removed, and the cells were rinsed three times with ice-cold PBS and subsequently fixed in 4% paraformaldehyde and examined using a confocal laser scanning microscopy (CLSM) (Leica TCS SP8, Solms, Germany) 20.

The release of the fluorophores from DiO/DiI-MCs was monitored by

the FRET ratio IDiI/(IDiO + IDiI),25 where IDiO and IDiI were the fluorescence intensities of DiO at 505 and DiI at 565 nm, respectively using excitation at 420 nm.

To

identify whether DiO/DiI-MCs could transport across the cell monolayers, we measured the fluorescence spectra of samples withdrawn from the basolateral chamber after 2 h of exposure of the apical chamber of the cell monolayers to DiO/DiI-MCs.


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Measurement of Mitochondrial Membrane Potential

Mitochondrial membrane potential was assessed using JC-1 mitochondrial membrane potential assay kit (Beyotime, Nantong, China) as reported previously.26 Briefly, MDCK-MRP2 cells were treated with FBS-free DMEM (negative control), MCs (20, 50, 100 µg/mL) or NaN3 (10 µM, positive control) at 37 °C for 1, 2 or 4 h, respectively.

After incubation, the cells were washed and stained with a

pre-prepared JC-1 working solution based on the manufacture’s protocol.27

Finally,

all samples were analyzed by an Incell Analyzer 2000 (GE Healthcare Life Sciences, USA) system to detect red fluorescence at excitation/emission wavelengths of 525/590 nm and green fluorescence at excitation/emission wavelengths of 490/530 nm.

Determination of Intracellular ATP Level

Intracellular ATP levels of MDCK-MRP2 cells were quantified using an ATP Assay Kit (Beyotime, Nantong, China).28

MDCK-MRP2 cells were treated with

FBS-free DMEM (negative control), MCs (20, 50, 100 µg/mL), and NaN3 (10 µM, positive control) at 37 °C for 1, 2 or 4 h, respectively.

After treatment, the cells were

washed and lysed with 200 µL of lysis buffer based on the manufacture’s protocol. The ATP concentration was determined using a standard curve (a known amount from 1 nM to 10 µM), and ATP levels were normalized to protein concentrations using a BCA Protein Assay Kit (Beyotime, Nantong, China).



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Analysis of Respiration Rate

The mitochondrial respiration was measured with an XF24 Cell Mito Stress Test Kit (Seahorse Bioscience, North Billerica, MA, USA).29

The cells were cultured in

an XF24 cell culture microplate (Seahorse Bioscience, North Billerica, MA, USA) at a seeding density of 2500 cells/well, then allowed to attach and grow for 24 h.

In the

concentration-dependent study, the cells were incubated with various concentrations of MCs (20, 50, 100 µg/mL) for 2 h.

In the time-dependent study, the cells were

incubated with MCs (50 µg/mL) for 1 and 4 h, respectively.

FBS-free DMEM was

used as a negative control, and NaN3 (10 µM) was selected as a positive control. The oxygen consumption rate (OCR, indicative of mitochondrial respiration) was evaluated using a Seahorse XF24 Extracellular Flux Analyzer (Seahorse Bioscience, North Billerica, MA, USA) after subsequent injections of the following metabolic toxins: oligomycin A (1 µM), carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) (1 µM), and a mixture of antimycin A (1 µM) and rotenone (1 µM).30

Neuroprotective Effects on Zebrafish

The neuroprotective effects of B-MCs were evaluated using MPTP treated zebrafish as previously reported.31

The immunohistochemistry study was carried out

using zebrafish that had been exposed to MPTP (200 µM) in an embryo medium at 1 day post fertilization (dpf).

The whole-mount anti-TH immunostaining was

performed using a previously developed method.32


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Statistical Analysis

All data reported are expressed as means ± standard deviation.

Statistical

significance of the results was analyzed using a two-tailed independent samples t-test where p

F68 Micelles of Baicalein Could Interfere with

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