Ionic liquid form of donepezil: Preparation, characterization and formulation development

Journal Pre-proof Ionic liquid form of donepezil: Preparation, characterization and formulation development

Hao Wu, Fang Fang, Lulu Zheng, Weijie Ji, Minghui Qi, Minghuang Hong, Guobin Ren PII:

S0167-7322(19)34142-X

DOI:

https://doi.org/10.1016/j.molliq.2019.112308

Reference:

MOLLIQ 112308

To appear in:

Journal of Molecular Liquids

Received date:

25 July 2019

Revised date:

4 December 2019

Accepted date:

12 December 2019

Please cite this article as: H. Wu, F. Fang, L. Zheng, et al., Ionic liquid form of donepezil: Preparation, characterization and formulation development, Journal of Molecular Liquids(2019), https://doi.org/10.1016/j.molliq.2019.112308

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© 2019 Published by Elsevier.

Journal Pre-proof

Ionic liquid form of donepezil: Preparation,

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characterization and formulation development

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Hao Wua, Fang Fanga, Lulu Zhengb, Weijie Jia, Minghui Qia, Minghuang Honga* and Guobin

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Rena*

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a State Key Laboratory of Bioreactor Engineering; Engineering Research Centre of

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Pharmaceutical Process Chemistry, Ministry of Education; Laboratory of Pharmaceutical Crystal

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Engineering & Technology, School of Pharmacy, East China University of Science and

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Technology, No. 130 Meilong Road, Shanghai 200237, China

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b Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of

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Science and Technology, 200237, P. R. China

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ABSTRACT

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The aim of this study was to transform donepezil into ionic liquids (ILs) to promote its skin

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permeability for developing a new delivery mode of donepezil. Donepezil ILs were formed by

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pairing with docusate, ibuprofen and four unsaturated fatty acids. The synthesized ionic liquids

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were fully characterized by thermal analysis, fourier transform infrared spectroscopy, and

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nuclear magnetic resonance spectrometer analysis. Physicochemical properties, such as solubility

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and partition coefficients, were measured at 25°C. The potential toxicity of ILs was evaluated

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against

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acetylcholinesterase inhibitory activity and docking study were also conducted to evaluate the

human

neuroblastoma

SH-SY5Y

cells.

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In

vitro

Electrophorus

electricus

Journal Pre-proof potential effect on inhibition of acetylcholinesterase activity after the formation of ILs. Blood-

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Brain Barrier (BBB) Parallel Artificial Membrane Permeability Assay and Skin Parallel

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Artificial Membrane Permeability Assay were carried out to evaluate the BBB permeability and

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skin permeability, respectively. Furthermore, drug-in-adhesive transdermal patches were

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prepared to explore the feasibility of developing donepezil ILs for transdermal delivery. Ionic

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liquids with α-linolenic and docosahexaenoic acid were more permeable through artificial skin

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membrane than the free base of donepezil, indicated by 1.9- and 1.55-fold increase in the

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permeability coefficients, respectively. In addition, donepezil ILs loaded adhesive patches had

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advantage in skin permeation compared to the corresponding free base patch. The formation of

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ionic liquid provided a new versatile platform to facilitate transdermal delivery.

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Keywords: Ionic liquids; Alzheimer’s disease; Donepezil; Drug-in-adhesive patch; Skin

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permeability

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Alzheimer’s disease (AD), a progressive multifarious neurodegenerative disorder, affects up to

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47 million elderly people, by causing memory impairment, aphasia, misuse, loss of recognition,

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visual spatial impairment, executive dysfunction and losing ability to learn[1]. As one of three

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FDA-approved acetylcholinesterase inhibitors for the treatment of AD, donepezil is superior to

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others because of its high potency and selectivity for the acetylcholinesterase (AChE) in the

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central nervous system[2, 3]. However, the oral form of donepezil is associated with a high

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incidence of gastrointestinal side effects, such as nausea, diarrhea, and vomiting, and these

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adverse effects affect 72% subjects[4]. Therefore, it is necessary to develop novel delivery

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systems to overcome these drawbacks.

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Journal Pre-proof Transdermal drug delivery system (TDDS) could overcome gastrointestinal side effects and

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prolong drug action to improve patient compliance, which further simplifies drug therapy for

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caregivers[5]. While the transdermal route of administration is promising, stratum corneum

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serves as a primary rate-limiting barrier for drug absorption through skin[6]. Different

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approaches were adopted to overcome the stratum corneum barrier, such as percutaneous

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enhancers and microneedles delivery system. For example, fatty acids were used as skin

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permeation enhancer to improve the transdermal permeation of donepezil[7]. Donepezil was also

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encapsulated in the tips of dissolving microneedles for rapid transdermal delivery[8].

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Ionic liquids (ILs) are currently defined as organic salts composed of ionized species with

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melting point (mp) or glass transition temperature (Tg) below 100°C, and the term IL is loosely

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defined such that hydrogen bonded complex with a significant proportion of neutral species also

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fulfill the definition of ILs[9-12]. In this study, hydrogen bonded complex was referred to as ILs.

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A number of studies have proven that ionic liquids are promising in the field of transdermal

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administration as active pharmaceutical ingredients (APIs), and lower ionicity may favor higher

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permeability through biological membranes[13-16]. Ionic liquids may stay as ion pairs in the

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biological system with increasing lipophilicity, and cross biological membranes more

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rapidly[17].

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Although there are some methods to enhance the transdermal permeation of donepezil[7, 8,

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18], the report of applying donepezil ILs in drug delivery system has not been found. In the

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present study, donepezil was converted into ionic liquid with six anionic counterions. The

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physicochemical and biochemical properties of these ionic liquids were characterized, and

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Blood-Brain Barrier Parallel Artificial Membrane Permeability Assay (BBB PAMPA) and Skin

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Parallel Artificial Membrane Permeability Assays (Skin PAMPA) were employed to evaluate the

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Journal Pre-proof permeability. Furthermore, a controlled release drug-in-adhesive (DIA) transdermal patch of

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ionic liquids was designed and prepared. Thermal analysis and fourier transform infrared

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spectroscopy (FT-IR) study were conducted to reveal the interaction among ionic liquids and

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pressure sensitive adhesive (PSA). The effect of ionic liquids formation on donepezil skin

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permeation was evaluated by in vitro permeation assay.

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2. Materials and methods

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2.1. Materials

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Donepezil hydrochloride and Ibuprofen sodium were purchased from Adamas-beta

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(Shanghai, China). Docusate sodium, oleate sodium, linoleate sodium and α-linolenic acid were

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purchased from Shanghai Macklin Biochemical Co., Ltd. (Shanghai, China). DHA was bought

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from Acros (Geel, Belgium). Ultrapure water (resistivity = 18.2 MΩ·cm) was prepared using a

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Merck Millipore Ming Che D24 UV system (Germany). Acetylthiocholine iodide, 5,5′-dithiobis

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(2-nitrobenzoic acid) (DTNB; Ellman’s reagent) and acetylcholinesterase from Electrophorus

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electricus acetylcholinesterase (EeAChE) were purchased from Sigma-Aldrich (Missouri, USA).

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DURO-TAK®87-4098 adhesive was purchased from Henkel AG&Co. (Holthausen, Germany).

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All the rest chemicals and reagents were at least of analytical grade.

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2.2. Preparation of donepezil ionic liquids

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The ionic liquids described herein were synthesized by metathesis reaction. Docusate

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sodium, oleate sodium, linoleate sodium and ibuprofen sodium were commercially available

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compounds. The α-linolenic sodium and docosahexaenoic sodium could be prepared by reaction

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of their acid with NaOH. In brief, the solution of NaOH (1M in water, 1.0 mL, 1.0 mmol) was

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slowly added to the suspension of α-linolenic or DHA (1.0 mmol in 10 mL ultrapure water), then

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the mixture was stirred at room temperature for 2 hours. Donepezil hydrochloride (2.0 mmol) and anionic sodium (2.0 mmol) were dissolved in 40

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mL ultrapure water with stirring at room temperature for 4 hours. The products were extracted

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with dichloromethane, then the dichloromethane phase was washed with ultrapure water to

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remove the formed inorganic salt. Silver nitrate test was used to confirm that the chloride ions

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was completely removed. Dichloromethane was removed on a rotary evaporator at 50°C for 2

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hours, then dried at 50°C under high vacuum for 24 hours. The final product was obtained. The

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yield of donepezil-docusate, donepezil-ibuprofen, donepezil-oleate acid, donepezil-linoleate

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acid, donepezil-α linolenic acid and donepezil-docosahexaenoic acid were 96.54%, 91.35%,

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92.13%, 90.71%, 83.71% and 81.69%, respectively.

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2.3. Formulation preparation

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Drug-in-adhesive (DIA) patch was prepared using solvent evaporation method[19, 20]. The

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specific composition and donepezil content in the PSA layer were listed in Table S1, Supporting

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Information. Donepezil and donepezil ILs were dissolved in ethyl acetate, mixed with DURO-

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TAK®87-4098 (DT4098) adhesive, and stirred for 3 h until it became homogeneous. The mixture

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was poured on a release liner 3M Scotchpak™ 9741 (Minnesota Mining and Manufacturing

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Company, Minnesota, USA) using a laboratory coating unit. Then, the PSA layer was dried at

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70°C for 3 h. Finally, the layer was covered with a backing film of Scotchpak™ 9723

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(Minnesota Mining and Manufacturing Company, Minnesota, USA).

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2.4. Modulated differential scanning calorimetry (M-DSC) analysis

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M-DSC experiments were performed on a TA Instruments Q2000 (TA instruments,

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Delaware, USA). Donepezil ILs (ca. 1 to 3 mg) were added into nonrecyclable aluminum

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hermetic pans and analyzed under nitrogen atmosphere by heating from -80°C to 100°C at the

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rate of 1°C/min. The modulation period was 60 s. Donepezil and donepezil ILs DIA patch dissolved in ethyl acetate was added into aluminum

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hermetic pans and then stored at room temperature for 12 h. Finally, sample was dried at 60°C

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under high vacuum for 12 h. The glass transition temperatures (Tg) were calculated using TA

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Universal Analysis software (TA instruments, Delaware, USA).

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2.5. Thermal gravimetric analysis (TGA)

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The thermal stabilities and decomposition temperatures of the obtained products were

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determined using a thermal gravimetric analyzer (TA Instrument Model TGA Q500) (TA

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instruments, Delaware, USA). Donepezil and donepezil ILs were placed inside aluminum pans

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and heated up to 400°C at a heating of 10°C/min and under a nitrogen atmosphere of 50 mL/min.

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TA Universal Analysis software (TA instruments, Delaware, USA), was used to determine the

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onset (T5%onset) temperatures, as the temperatures at which 5% of weight loss was observed.

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2.6. Fourier transform infrared spectrophotometer (FTIR) analysis

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Infrared spectra were recorded on an Agilent Cary 630 FTIR spectrometer (Agilent

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Technologies, California, USA) and evaluated using Microlab software (Agilent Technologies,

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California, USA). Each spectrum was scanned in the range of 400−4000 cm −1 with a resolution

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of 8 cm−1, and a minimum of 32 scans were collected and averaged to obtain good quality

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spectra. The spectra were normalized and background corrected.

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2.7. Nuclear magnetic resonance (NMR) analysis The NMR experiments were performed on a Bruker Avance III 400 MHz spectrometer 1

(Bruker GmbH, Germany). The

H NMR spectra of all obtained products at various

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concentrations in deuterated dimethyl sulfoxide (DMSO) were recorded at 298 K and using

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tetramethylsilane (TMS) as an internal standard.

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2.8. High performance liquid chromatography (HPLC) analysis

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An HPLC method was established to determine the concentration of donepezil using a

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reversed phase Agilent 1260 system (Agilent Technologies, California, USA). Donepezil was

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separated by a CAPCELL PAK C18 column (250 mm × 4.6 mm, 5 μm) and detected at 268 nm.

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The mobile phase was 0.01 M potassium dihydrogen phosphate buffer/acetonitrile (65/35, v/v) at

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the flow rate of 1.0 mL/min. The column temperature was set at 25°C. The injection volume was

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20 μL.

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2.9. Equilibrium solubility determination

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An excess amount of donepezil and donepezil ILs were added to 5 mL ultrapure water in 10

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mL glass vials. Then, the vials were placed in a shaker and kept under shaking at 200 rpm and

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25°C (± 0.3°C), and the samples were withdrawn after 24 hours. The remaining solid was

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removed by filtration (syringe filter membranes, 0.2 μm pore size), and donepezil concentration

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in the resultant filtrate was determined by HPLC.

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2.10. Partition coefficients determination.

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Journal Pre-proof The octanol-water partition coefficients of ILs and donepezil were measured by shake-flask

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method, according to Organization for Economic Co-operation and Development (OECD)

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guidelines[21]. Firstly, two bottles charged with sufficient ultrapure water in n-octanol and

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sufficient n-octanol in ultrapure water were placed in a thermostat shaker for 24 hours, and

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through standing enough time to separate obtained mutually saturated two phases. Then,

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donepezil ILs and donepezil dissolute in octanol that is saturated with ultrapure water, and the

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same volume of ultrapure water saturated with octanol was added. Next, the vials were placed in

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shaker, kept under stirring at 200 rpm and controlled the temperature at 25°C (± 0.3°C) for 24 h

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to reach the equilibrium. Subsequently, both samples were centrifuged to ensure the complete

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phase separation. Finally, the concentration of donepezil in the water phase and octanol phase

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were determined by HPLC.

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The aqueous solution was replaced by 0.01 M phosphate buffer saline (PBS 7.4), which was

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carried out to determine the distribution coefficients at pH values of 7.4 (LogD7.4). The

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remaining steps are consistent with the steps for determining LogP.

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2.11. Skin Parallel Artificial Membrane Permeability Assay (Skin PAMPA)

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The skin-permeation tests were performed using Skin PAMPA that was purchased from

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Pion Inc. (Massachusetts, USA). Standard Britton-Robinson buffers were made, and 0.5 M

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sodium hydroxide was applied to adjust the pH to 7.4 to serve the acceptor solution and donor

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solution. Donepezil and donepezil ILs were dissolved in the donor solution, and the

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concentration of each sample is 0.263 mM. The Skin PAMPA sandwiches were used after

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overnight hydration. Two hundred microliters of sample solution was added to each well of the

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donor (bottom) plate. The top plate was filled with 200 μL fresh acceptor solution, and this

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system was incubated at 32°C. The acceptor solution was sampled (150 μL) after 2, 4, 6, 8, 10,

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and 12 hours of incubation, and the same volume of fresh donor solution was added. The

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concentration of each sample was determined by HPLC method as described above. Four parallel

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measurements were conducted for each drug.

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The percentage of the applied dose transported across the membrane was calculated using the following equation;

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j=1 Permeation(% applied dose) = moles API donor × 100% (1) phase

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C; the concentration of the API in the receiver phase (μg/mL)

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i and j; samples numbers

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Ci ×0.2+∑i−1 Cj ×0.15

0.2; the receiver chamber volume (mL)

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0.15; the volume of the collected sample (mL)

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The cumulative amount of donepezil per unit area versus time was plotted to determine the

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penetration parameters. The transdermal flux (J, μg/cm2/h) was calculated as the slope, and the

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permeability coefficient (P, μm/h) was calculated as the transdermal flux divided by the initial

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drug concentration (μg/cm3).

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2.12. Blood-Brain Barrier Parallel Artificial Membrane Permeability Assay (BBB PAMPA)

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The BBB-permeation tests were performed using BBB PAMPA. Porcine brain lipid (PBL)

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was obtained from Avanti Polar Lipids, Inc. (Alabama, USA). The donor microplate

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(MATRNPS50) and the acceptor microplate (MAIPN4550) were purchased from Merck

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Journal Pre-proof Millipore (Massachusetts, USA). The 96-well UV plate was purchased from Corning Inc. (New

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York, USA). 300 μL 0.01 M PBS 7.4 buffer/alcohol (8:2) mixture was added to the acceptor

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microplate, and the filter membrane of donor microplate was impregnated with 4 μL of PBL in

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dodecane (20 mg/mL). Donepezil and donepezil ILs were dissolved in DMSO at a concentration

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of 5 mg/mL, then diluted 50-fold at a concentration of 100 μg/mL. 200 μL of donepezil solution

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was added to the donor microplate within 10 minutes after the filter membrane was impregnated.

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A sandwich was formed and incubated for 12 h at 25°C. After incubation, the donor plate was

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carefully removed, and the concentrations of the compound in the acceptor microplate were

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determined using a microplate reader (Biotek, Vermont, USA).

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2.13. Inhibition of Electrophorus electricus AChE

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Inhibitory capacity of compounds on Electrophorus electricus AChE (EeAChE) biological

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activity was evaluated by using Ellman’s method[22]. EeAChE was dissolved in 20 mM HEPES

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buffer pH 8.0 to produce concentrations of 2.5 units/mL enzyme activity. The other assay

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solutions were dissolved in 0.2 M PBS pH 7.4. In the procedure, 50 μL tested compound solution

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and 50 μL enzyme solution were added into the 96-well plate, then 100 μL 0.3 mM DTNB was

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added. After preincubation at 37°C for 10 min, the reaction was initiated by adding 50 μL 10

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mM acetylthiocholine iodide solution. The hydrolysis of acetylthiocholine was monitored by the

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formation of yellow 5-thio-2nitrobenzoate anion, which was measured at a wavelength of 412

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nm every minute for 10 min using a microplate reader (Biotek, Vermont, USA). IC50 values were

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determined graphically from six-point inhibition curves using the IBM SPSS 22.0 (IBM, New

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York, USA).

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2.14. Docking Study of donepezil and anionic counterions with AChE

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Journal Pre-proof All the computational studies were performed on Maestro 10.5 embedded in Schrödinger

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software package (2016-1) (Schrödinger, LLC, Oregon, USA). Firstly, LigPrep and Protein

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Preparation wizard were utilized to prepare small molecules and recombinant human

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acetylcholinesterase (rhAChE), respectively. Water molecules within 10 Å around active site of

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AChE were retained for the reason that waters play an important role in the binding of donepezil.

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Secondly, Glide extra precision (XP) model was used to dock donepezil, protonated donepezil

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and their anionic counterions to the active site of AChE. Finally, Prime Molecular Mechanics-

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Generalized Born Surface Area (MM-GBSA) was applied to calculate binding energies between

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small molecules’ most superior conformation and AChE. 3D interaction figures were generated

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by Pymol 1.5 (DeLano Scientific LLC, California, USA).

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2.15. In vitro cytotoxicity test

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Human neuroblastoma SH-SY5Y cells were purchased from the Cell Bank of the Shanghai

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Institute of Cell Biology, Chinese Academy of Science. SH-SY5Y cells were cultured in

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Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal calf serum and

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incubated at 37°C in a humidified atmosphere of 95% air and 5% CO2. SH-SY5Y cells were

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seeded into 96-well plates at a density of 4 × 103 cells/well and incubated for 12 h. Then, the cell

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medium was removed and replaced with the solutions of donepezil ILs whose concentration in

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fresh culture medium ranged from 1 nM to 1 mM. After 24 h incubation, 20 μL CCK8 (MCE

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Kits, New Jersey, USA) was added to the cells for another 2 h incubation. Finally, the plate was

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gently shaken for 10 seconds, and absorbance was measured at 450 nm with Microplate reader

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(Biotek, Vermont, USA). Data were means ± SD of at least three independent experiments.

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2.16. In vitro skin permeation study

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Journal Pre-proof A vertical Franz diffusion cell (Kaikai, Shanghai, China) with a chamber volume of 7.0 mL

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and an effective diffusion area of 1.77 cm2 was maintained at 32.0 ± 0.3°C and champed with

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Start-M® membrane (Merck Millipore, Massachusetts, USA) for in vitro permeation study. In

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this study, the patches with similar weight (30 mg PSA layer, 1.77 cm2) and similar donepezil

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content (2.4 mg, 8% w/w of PSA layer) were selected for this experiment. The receiver size was

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filled with 7.0 mL 0.01 M PBS pH 7.4 and was continuously stirred at 300 rpm. Two hundred

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microliter samples were withdrawn at 2 h, 4 h, 6 h, 8 h, 10 h, 12 h, 24 h, 36 h, 48 h, 60 h, 72 h

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for HPLC analysis and replaced with the same volume of fresh buffer. The cumulative

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permeation amount (Q, μg/cm2) was calculated by the following equation: Ci ×7+∑i−1 j=1 Cj ×0.2

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Q=

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C; the concentration of the API in the receiver phase (μg/mL)

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i and j; samples numbers

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7; the receiver chamber volume (mL)

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0.2; the volume of the collected sample (mL)

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A; effective diffusion area (cm2)

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2.17. Statistical analysis

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The results were expressed as the mean ± stand deviation. ANOVA was carried out to

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determine the significant difference between groups data, and a difference was statistically

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significant when the P < 0.05.

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3. Results and discussion

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3.1. Characterization of ILs. A total of six donepezil ionic liquids were synthesized, of which donepezil-docusate

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([DON][DOC]) and donepezil-ibuprofen ([DON][IBU]) were colloidal semisolid, while

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donepezil-oleate acid ([DON][OA]), donepezil-linoleate acid ([DON][LA]), donepezil-α

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linolenic acid ([DON][ALA]) and donepezil-docosahexaenoic acid ([DON][DHA]) were viscous

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liquid. Their structure and appearance were summarized in Fig. 1.

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Fig. 1. Chemical structure and appearance of synthesized ionic liquids.

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Journal Pre-proof The stoichiometry ratio between the anion and donepezil was calculated by 1H NMR. The

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NMR spectra of six donepezil ILs were shown in Supporting Information (Figure S1 ~ Figure

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S6). A molar ratio of 1 : 1 of donepezil to anion were found for all the six ionic liquids. In

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addition, the peaks that were not belong to donepezil and anions were hardly found in NMR

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spectrum. The purity of synthesized ILs were determined by HPLC, and the results were listed in

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Table 1. The purities of the obtained donepezil ILs were in the range of 95% to 98%.

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H NMR could also be used to assess the degree of proton transfer between donepezil and

the cation. The chemical shifts of hydrogen (-NCH2Ph) from donepezil, donepezil hydrochloride

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and donepezil ILs were listed in Table 1. As shown in the 1H NMR of ILs, the signals of

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hydrogen attached to the carbon between piperidine ring and benzene ring (-NCH2Ph) in the

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donepezil structure shifted downfield when donepezil ILs were formed, indicating the possible

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formation of hydrogen bond or ionic bond. Meanwhile, the ionicity of donepezil ILs could be

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assessed by 1H NMR, and the degree of ionicity is related to the states of hydrogen bonded

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complex (Fig. S8, Supporting Information). The percentage of ionized donepezil in

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synthesized ILs was calculated by comparing the interpolation of chemical shifts (Equation 1,

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Supporting Information), according to Ruth’s report[12]. The major intermolecular interactions

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were hydrogen bonds, when the data of %Salt was less than 10[11, 12]. Donepezil hydrochloride

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was considered as fully ionized, so the data of %Salt was 100. The chemical shifts of -NCH2Ph

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belonging to [DON][HCL] and [DON][DOC] were δ 4.260 ppm and δ 4.290 ppm, respectively.

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Compared with donepezil hydrochloride, the chemical shifts of [DON][DOC] revealed no

21

obvious differences. The major intermolecular interaction of [DON][DOC] was SO3-···NH+

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ionic bond. The salt% values of other donepezil ILs were less than 5%, which suggested the

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other synthesized ILs were mostly unionized proton transfer compound. The proton transfer

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process could be improved though the use of stronger acids[23]. The acidity of ibuprofen and

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fatty acids were significant weaker than docusate, which may lead to the low degree of ionicity. Fig. 2 displayed the FT-IR spectra of donepezil, donepezil hydrochloride and donepezil ILs.

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The characteristic stretching vibrations of donepezil hydrochloride’s ammonium NH+ was

5

located in the wavenumber from 2600 to 2300 cm-1, indicating the formation of salt (Fig. 2A).

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According to Fig. 2B, a wide vibration peak of [DON][DOC] was also observed in the

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wavenumber from 2700 to 2500 cm-1, which was not found in other five donepezil ILs. Thus, the

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major intermolecular interactions of [DON][DOC] may be SO3-···NH+ ionic bond. The other

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five donepezil ILs are largely unionized, and the major intermolecular interactions may be O-

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H···N hydrogen bonds rather than COO- ···NH+ ionic bonds.

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Based on the above experimental data, the major intermolecular interactions of

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[DON][DOC] was ionic bond, and the major intermolecular interactions of [DON][IBU],

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[DON][OA], [DON][LA], [DON][ALA] and [DON][DHA] were hydrogen bond. All the 6

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synthesized donepezil-based products will be referred to as donepezil ILs.

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Fig. 2. FT-IR spectra of donepezil, donepezil hydrochloride and donepezil ILs.

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(A - all data, B – expanded view of 70%-100% transmittance)

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of donepezil and ILs.

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-NCH2Ph δ (ppm)

% Salt

Purity (%)

Tg (°C)

T5%onset (°C)a

[DON]

3.434

0.00

> 98.00

-

273.52

[DON][HCL]

4.260

100.00

> 98.00

-

235.81

[DON][DOC]

4.290

103.63

97.22

6.96

251.68

[DON][IBU]

3.450

1.94

9.49

154.46

[DON][OA]

3.440

0.84

96.81

-29.50

212.35

[DON][LA]

3.443

1.09

97.11

-40.93

199.51

[DON][ALA]

3.459

3.02

96.22

-42.41

197.67

[DON][DHA]

3.450

1.94

95.34

-40.12

229.52

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95.23

a: T5%onset is defined as the temperatures at which loss 5% weight.

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3

Compound

of

Table 1. 1H NMR Data, Percentage of Ionized Complex, Purities and thermochemical properties

ro

1

3.2. Thermochemical properties

6

Glass transition temperature (Tg) was detected by Modulated differential scanning

7

calorimetry (M-DSC, Table 1). For colloidal semisolid, the Tg values of [DON][DOC] and

8

[DON][IBU] were 6.96°C and 9.49°C, respectively. For viscous liquid, the Tg values of

9

[DON][OA], [DON][LA], [DON][ALA] and [DON][DHA] were -29.50°C, -40.93°C, -42.41°C

17

Journal Pre-proof and -40.12°C, respectively. The above results were in agreement with previous report that

2

ILs with lower Tg showed better fluidity[17]. The weaker viscosity of IL was directly correlated

3

with the weaker the cohesive energy which was indicated by the lower Tg value [11, 23]. Tg

4

could be decreased through modification of the anionic component[24]. The anions of [OA],

5

[LA], [ALA] and [DHA] were all long chain unsaturated fatty acids, which were less prone to

6

form intermolecular interaction than [DOC] and [IBU]. Therefore, the cohesive energy of these

7

four ILs might be lower than that of [DON][DOC] and [DON][IBU], which was reflected by

8

lower Tg and lower viscosity. In addition, the more asymmetrical chemical structure of these

9

unsaturated fatty acids than [DOC] and [IBU] might also contributed to the lower Tg. Indeed, the

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similar phenomenon was also observed in the literature[25].

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The thermal decomposition values of donepezil ILs were reported here in terms of losing

12

5% weight, which could reflect the thermal stability[26-28]. According to Table 1, TGA results

13

suggested one-step decomposition with thermal stability up to 150°C for all ILs, indicating

14

reasonably good thermal stability, even though the T5%onset values of formed ILs were reduced

15

compared to donepezil.

16

3.3. Equilibrium solubility and partition coefficients

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17

The solubility of donepezil and donepezil ILs in ultrapure water and 0.01 M phosphate

18

buffer saline (PBS) pH 7.4 buffer were listed in Table 2. It is possible to observe that the

19

solubility of the donepezil ILs spans over a wide range of values (from 42.61 μg/mL to 839.22

20

μg/mL in ultrapure water, from 19.58 μg/mL to 720.06 μg/mL in PBS 7.4), either lower or much

21

higher than the solubility measured for donepezil. Donepezil revealed poor water solubility

22

(44.70 μg/mL), and the solubility increased to 108.78 μg/mL when placed in PBS 7.4 buffer

18

Journal Pre-proof condition. The formation of these donepezil ILs significantly affected the solubility of donepezil.

2

The solubility of [DON][DOC] were 42.61 μg/mL in water and 19.58 μg/mL in PBS 7.4, which

3

were slightly lower than that of donepezil. Significantly higher concentrations for [DON][IBU]

4

was observed, indicated by almost 20-fold and 7-fold increase of solubility values in ultrapure

5

water and PBS 7.4, respectively. [DON][OA], [DON][LA], [DON][ALA] and [DON][DHA]

6

showed slightly higher level of solubility than donepezil, as indicated by from 2.59- to 8.12-fold

7

increase in aqueous solubility values and from 1.41- to 2.73-fold increase in PBS 7.4 solubility

8

values.

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of

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The octanol-water partition coefficients (LogP) and distribution coefficients at pH value of

10

7.4 (LogD7.4) were listed in Table 2. Because of its hydrophobic nature, donepezil displayed a

11

positive LogP (1.97) and LogD7.4 (2.64). Apart from [DON][DOC], the other donepezil ILs

12

revealed similar LogP values, with the difference of less than 0.2 unit. Based on the above

13

characterization, [DON][DOC] was the salt form but not unionized proton transfer compound,

14

which may reduce the lipophilicity of donepezil. The distribution coefficient increased to 2.49

15

when placed in PBS 7.4 buffer, which could be explained by the decreased ammonium ionization

16

degree. The majority of these donepezil ILs showed decreased LogP and LogD7.4 values

17

comparing with donepezil, except for [DON][DHA] that was leading the values both in LogP

18

(1.99) and LogD7.4 (2.78). Another interesting point to be noticed is that [DON][DHA] also

19

improved the solubility of donepezil, which means IL technology allowed the simultaneous

20

enhancement of hydrophilicity and lipophilicity.

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21 22

19

Journal Pre-proof 1

Solubility in PBS7.4

4

LogD7.4

（μg/mL）

（μg/mL）

[DON]

44.70 ± 5.18

108.78 ± 9.25

1.97 ± 0.08

2.64 ± 0.05

[DON][DOC]

42.61 ± 1.36

19.58 ± 0.77

1.65 ± 0.07

2.49 ± 0.06

[DON][IBU]

839.22 ± 26.67

720.06 ± 12.59

1.90 ± 0.05

2.49 ± 0.05

[DON][OA]

115.78 ± 6.51

1.79 ± 0.10

2.57 ± 0.08

[DON][LA]

140.23 ± 15.12

277.36 ± 11.49

1.81 ± 0.05

2.54 ± 0.04

[DON][ALA]

169.30 ± 22.37

336.31 ± 15.79

1.85 ± 0.06

2.60 ± 0.10

[DON][DHA]

363.04 ± 34.28

297.14 ± 24.04

1.99 ± 0.14

2.78 ± 0.01

-p

ro

na

lP

238.65 ± 22.60

Jo

3

LogP

of

Solubility in water

Compound

re

Table 2. Equilibrium solubility (25°C，n = 3，± SD), LogP and LogD7.4 of donepezil ILs

ur

2

3.4. Skin Parallel Artificial Membrane Permeability Assays (Skin PAMPA)

5

In vitro permeation tests, Skin PAMPA were carried out to compare the skin permeability

6

of donepezil and donepezil ILs. Skin PAMPA is a rapid and cost-efficient system for the

7

prediction of skin penetration[29]. The recent studies illustrated that the new artificial PAMPA

8

membrane was highly consistent with an ex vivo human skin membrane[30].

20

Journal Pre-proof The permeation percentage of the applied dose calculated from eqn (1) for donepezil and

2

donepezil ILs was presented in Fig. 3A. The permeation percentage of donepezil ILs had an

3

advantage over that of donepezil during the first 6 h. Specifically, the permeation percentage

4

values of [DON][DHA] and [DON][ALA] were 1.53 and 1.80 times higher than that of

5

donepezil, respectively. However, the results of 12 h in vitro permeation tests suggested that the

6

gap between donepezil ILs and donepezil had been narrowed. The 12 h permeation percentage of

7

drug permeated was in the following order: [DON][DHA] (47.01%) > [DON][ALA] (43.80%) >

8

[DON][LA] (38.40%) > [DON][DOC] (38.14%) > [DON][IBU] (37.66%) > [DON][OA]

9

(36.98%) > [DON] (36.51%). The permeation percentage of [DON][DHA] and [DON][ALA]

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were only 1.27 and 1.20 times higher than that of donepezil, respectively.

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1

The phenomenon discussed in the previous paragraph can be more clearly reflected by the

12

steady-state flux (J) and permeability coefficient (P). The J and P of 6 h and 12 h were all listed

13

in Table 3. The parameter J was conducted to evaluate the steady-state diffusion rate of the drug

14

through the membrane. As seen in Table 3, the 6 h steady-state flux of all donepezil ILs was

15

slightly higher than the 12 h steady-state flux, but there was little difference between the steady-

16

state flux of donepezil for 6 h and that for 12 h (1.409 ± 0.203 μg/cm2/h vs. 1.440 ± 0.169

17

μg/cm2/h). By eliminating the influence of initial concentration, the permeability coefficient (P)

18

that better reflects drug permeability was found. The 12 h permeability coefficient of donepezil

19

and donepezil ILs were about 0.021 cm/h, but donepezil ILs held an advantage over donepezil in

20

the 6 h permeability coefficients. Specifically, the 6 h permeability coefficients of [DON][ALA]

21

and [DON][DHA] were markedly increased (1.9- and 1.55-fold increase respectively).

Jo

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11

22

21

Journal Pre-proof 1 2 3

Table 3. Permeation parameters of donepezil through the Skin PAMPA model (32°C, n = 4, ±

4

SD) J12h (μg/cm2/h)

P6h (cm/h)

P12h (cm/h)

[DON]

1.409 ± 0.203

1.440 ± 0.169

0.020 ± 0.003

0.021 ± 0.002

[DON][DOC]

1.792 ± 0.215*

1.393 ± 0.136

[DON][IBU]

1.665 ± 0.054

[DON][OA]

1.858 ± 0.192*

1.381 ± 0.207

[DON][LA]

1.703 ± 0.138

[DON][ALA] [DON][DHA]

0.019 ± 0.002

1.590 ± 0.005

0.022 ± 0.001

0.021 ± 0.001

re

0.025 ± 0.003

0.019 ± 0.003

1.620 ± 0.078

0.023 ± 0.002

0.022 ± 0.001

2.465 ± 0.268*

1.391 ± 0.221

0.038 ± 0.004*

0.021 ± 0.003

2.051 ± 0.253*

1.647 ± 0.061

0.031 ± 0.004*

0.025 ± 0.001*

na

lP

-p

ro

0.025 ± 0.003

ur

*P < 0.05, vs. [DON]

of

J6h (μg/cm2/h)

Jo

5

Compound

6

Donepezil ILs were more permeable than the free base of donepezil in terms of both the

7

steady-state flux and permeability coefficient during the first 6 h, which indicated that the speed

8

of donepezil ILs crossing artificial skin membrane were faster than donepezil at similar

9

concentration gradients. LogP, molecular weight and melting point are the three factors to

10

influence drug skin permeation[6]. In the study, lipophilicity of donepezil was slightly affected

11

after the formation of donepezil ILs, but the glass transition temperature of donepezil ILs was

22

Journal Pre-proof significantly reduced. Overall, the thermodynamic properties might dominate the skin

2

permeation of donepezil, thus the ILs showed improved transdermal delivery property.

ur

3

na

lP

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-p

ro

of

1

Fig. 3. A. Permeation as a percentage of applied dose in mol% versus time from Skin PAMPA.

5

B. Pe values for evaluating BBB permeability of donepezil and donepezil ILs (*P < 0.05, vs.

6

[DON]). C. Inhibition of Electrophorus electricus AChE by donepezil and donepezil ILs (*P <

7

0.05, vs. [DON]). D. The cytotoxicity of donepezil and donepezil ILs (**P < 0.01, vs. [DON]).

8

3.5. Blood-Brain Barrier Parallel Artificial Membrane Permeability Assay (BBB PAMPA)

Jo

4

9

BBB PAMPA was used to evaluate the effect of IL formation on brain permeation of

10

donepezil. BBB PAMPA established by Di et al. predicted passive BBB permeability ability

11

with high success, high throughput, low cost and reproducibility[31]. The method described in

23

Journal Pre-proof this study was modified based on Di’s method and widely applied to predict the BBB permeation

2

potential of compounds[32, 33]. By comparing the experimental permeability with the reported

3

values of 7 commercial drugs, a good lineal correlation, Pe(exp) = 1.028Pe(bibl) - 0.81 (R2 =

4

0.978) (Fig. S7, Supporting Information), was observed. Based on the limit established by Di

5

et al. for BBB permeation and the above self-established equation, compounds with

6

permeabilities above 3.302 × 10-6 cm·s-1 was considered to have the ability to cross the blood-

7

brain barrier (Table S3, Supporting Information). Pe values of donepezil and donepezil ILs

8

were presented in Fig. 3B. The formation of donepezil ILs slightly affected the brain permeation

9

of donepezil. All test donepezil ILs showed permeability values above the 3.302 × 10-6 cm·s-1,

-p

ro

of

1

which pointed out that donepezil ILs could cross the blood-brain barrier.

11

3.6. In Vitro Inhibition Studies of Electrophorus electricus AChE

lP

re

10

Recently, a series of studies on IL-induced inhibition or activation of enzyme activity have

13

been reported[34-36]. For example, chitin deacetylase was activated by [C4MIM][Br] at low

14

concentrations[37], and trypsin was inhibited by imidazolium- and ammonium-based ILs[38].

ur

na

12

To evaluate the potential effect of IL formation on acetylcholinesterase activity, in vitro

16

EeAChE inhibitory activity of donepezil ILs were determined. Donepezil was used as a reference

17

standard, and the results were provided in Fig. 3C. The IC50 value of donepezil published by

18

Benjamin et al was 0.096 μM[2], which was similar to our data (0.128 μM). Among the ILs,

19

[DON][DHA] was the most potent EeAChE inhibitor with an IC50 value of 0.41 μM (*P < 0.05,

20

vs. [DON]). Although IC50 values of the other ILs were higher than that of donepezil, there was

21

no significant difference between these ILs and donepezil. In this study, the formation of ILs had

Jo

15

24

Journal Pre-proof 1

little effect on the inhibition of acetylcholinesterase activity, and all donepezil ILs showed

2

significant inhibition of EeAChE.

3

3.7. Docking Study of donepezil and anionic counterions with AChE A human acetylcholinesterase (AChE) in Complex with donepezil (PDB ID: 4EY7) was

5

used to detect the anionic counterions’ potential impact on the binding affinity of donepezil and

6

protonated donepezil. As shown in Table 4, donepezil was allocated about 1.5 and 3 times more

7

than its anionic counterions in XP GScore and DG bind, respectively. Compared with donepezil,

8

the protonated donepezil displayed a slightly higher values in XP GScore and DG bind. The

9

interaction pattern of donepezil and anionic counterions were presented in Fig. S9, Supporting

10

Information. Given that donepezil and protonated donepezil have much stronger binding

11

capacities than their anionic counterions, it is certain that the anionic counterions couldn’t affect

12

the binding capacities of donepezil and protonated donepezil to the active site of AChE, which

13

were in consisted with in vitro EeAChE inhibitory activity experiments.

14

Table 4. The XP GScore and DG bind of donepezil and anionic counterions

ur

na

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4

XP GScore

Donepezil

-14.648

-60.4858

Protonated donepezil

-16.566

-88.5922

Docusate

-6.908

-22.5595

Ibuprofen

-9.212

-20.8348

Oleic acid

-8.003

-22.7702

Linoleic acid

-8.053

-22.1469

α-linolenic acid

-7.935

-17.1304

Jo

Compound

25

DG bind (kcal/mol)

Journal Pre-proof DHA 1 2 3

-9.735

-25.4178

XP GScore of molecules’ most superior conformation and their binding energies. The binding energy is calculated according to the equation: DG bind = E_complex(minimized) - E_ligand(minimized) E_receptor(minimized).

4 Notably, it was found that protonated donepezil had a slightly higher binding activity

6

compared with donepezil, which could be interpreted by their different binding patterns to

7

AChE. As presented in Fig. 4, nitrogen atom in donepezil formed hydrogen bonds with TYR-337

8

and TYR-341 mediated by a water molecule, while nitrogen cation in protonated donepezil

9

straightforwardly formed electrostatic interactions with TYR-337, TRP-86 and PHE-338. Direct

ro

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electrostatic interactions may be more stable than indirect hydrogen bonds aided by water.

lP

10

of

5

Jo

ur

na

11

12 13 14

Fig. 4. AChE active site with bound donepezil (left) and protonated donepezil (right), respectively. Waters, key residues and small molecules are drawn as sticks. Carbons are colored pink in small molecules and rainbows in residues of the complex. All oxygen, nitrogen and

26

Journal Pre-proof 1 2

hydrogen atoms are colored red, blue and white, respectively. Hydrogen bonds are represented by purple dashes.

3 4

3.8. Cytotoxicity Toxicity of ILs was a long-lasting topic during the application, which is complicated and

6

unavoidable[39, 40]. To evaluate the relative cytotoxicity of these donepezil ILs, the widely

7

applied model system, neuroblastoma cell lines (SH-SY5Y), was chosen[2, 41].

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of

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The cytotoxicity of donepezil ILs were expressed as IC50 values and plotted in Fig. 3D. The

9

calculated IC50 value of donepezil was 0.104 μM, and a significant inhibition effect was

10

observed. [DON][IBU], [DON][ALA] and [DON][DHA] revealed similar toxicity or safety

11

profile with donepezil, yet [DON][DOC], [DON][OA] and [DON][LA] displayed attenuated

12

toxicity as compared with donepezil. Specifically, the formation of [DON][DOC] showed

13

significant lower toxicity than donepezil, as evidenced by IC50 values (9.813 μM) (**P < 0.01,

14

vs. [DON]). These reduced cytotoxicity after the formation of ILs could be observed in other

15

literature reports[42]. Overall, the formation of donepezil ILs did not cause more severe

16

cytotoxicity but reduced the toxicity of donepezil in some cases.

17

3.9. Characterization of the donepezil ILs PSA Patch

Jo

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8

18

A single layer or multilayer DIA patch is one of the three main types of transdermal drug

19

delivery systems and is mainly composed of PSA which is simple and stable[19, 20]. PSA could

20

not only help patch adhesion to the skin surface but also control drug release[43-45]. DT4098

21

(without functional group) was selected as model acrylic adhesive to minimize the interaction of

22

the drug with PSA for further investigation. [DON][ALA], [DON][LA] and [DON][IBU] were 27

Journal Pre-proof 1

selected as model ionic liquids after comprehensively considering physicochemical properties,

2

biochemical properties and permeability factors. These ILs enhanced donepezil’s aqueous

3

solubility and maintained its partition coefficient, and they also showed superior skin

4

permeability and similar BBB permeability. Glass transition temperature (Tg) of DT4098 and drug-loaded DT4098 patch were

6

determined by M-DSC, and the results were summarized in Fig. 5A. Tg was a simple indicator

7

of molecular mobility of PSA, and the high Tg value indicates the poor molecular flexibility and

8

the small chance of forming free volume[43, 45]. Tg values of black DT4098 patch,

9

[DON][ALA]-loaded DT4098 patch, [DON][LA]-loaded DT4098 patch, [DON][IBU]-loaded

10

DT4098 patch and [DON]-loaded DT4098 patch were -20.16°C, -29.10°C, -26.10°C, -18.18°C

11

and -19.19°C, respectively. Tg values of [DON]-loaded DT4098 patch and [DON][IBU]-loaded

12

DT4098 patch increased slightly compared with DT4098. Meanwhile, Tg values of

13

[DON][ALA]-loaded DT4098 patch and [DON][LA]-loaded DT4098 patch decreased about 9°C

14

and 6°C compared with DT4098, respectively. The decreased Tg values indicated that PSA

15

mobility of two patches could increase significantly.

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lP

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Jo

16

of

5

FT-IR spectra were recoreded to identify the molecular interactions between DT4098 and

17

drugs. As shown in Fig. 5B, the FT-IR spectra of donepezil was covered by blank PSA (DT4098)

18

because of low drug content. Only four stretching vibration peaks belonging to donepezil could

19

be found from 1000 cm-1 to 2000 cm-1 in donepezil-loaded DT4098 patch. Stretching vibrations

20

C=O (C=O) of donepezil changed significantly after donepezil was loaded into DT4098, and it

21

shifted from 1682 cm-1 to 1703 cm-1. This result indicated possible intermolecular interaction

22

between donepezil and DT4098.

28

Journal Pre-proof FT-IR spectra of [DON][ALA]-loaded DT4098 patch, [DON][LA]-loaded DT4098 patch

2

and [DON][IBU]-loaded DT4098 patch were displayed in Fig. 5C, Fig. 5D and Fig. 5E,

3

respectively. The spectra of ionic liquids-loaded DT4098 patch were almost completely

4

coincident with the spectra of donepezil-loaded DT4098 patch, which means that the

5

intermolecular interaction between donepezil and DT4098 did not change after the formation of

6

ionic liquids.

Jo

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1

7 8

Fig. 5. A. Thermal analysis of DT4098 patch and drug-loaded DT4098 patch. B. FT-IR spectra

9

of donepezil, DT4098 patch and donepezil-loaded DT4098 patch. C. FT-IR spectra of donepezil-

10

loaded DT4098 patch and [DON][ALA]-loaded DT4098 patch. D. FT-IR spectra of donepezil-

11

loaded DT4098 patch and [DON][LA]-loaded DT4098 patch. E. FT-IR spectra of donepezil-

12

loaded DT4098 patch and [DON][IBU]-loaded DT4098 patch.

29

Journal Pre-proof 1 2 3.10. In vitro skin permeation study

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4

Fig. 6. A. In vitro skin permeation profiles of donepezil-loaded DT4098 patch and donepezil ILs-

6

loaded DT4098 patch. B. In vitro skin permeation profiles of [DON][ALA]-loaded DT4098

7

patch and [DON][ALA].

na

ur Jo

8

lP

5

9

The results of in vitro skin permeation of DIA patches were shown in Fig. 6A. Obviously,

10

donepezil ILs patches showed advantage over donepezil patch in terms of cumulative permeation

11

amount. At the end of 72 h, the amount of donepezil permeated from donepezil-loaded DT4098

12

patch was 123.31 ± 30.28 μg/cm2. The corresponding amount of donepezil permeated from

13

[DON][ALA]-loaded DT4098 patch, [DON][LA]-loaded DT4098 patch and [DON][IBU]-

14

loaded DT4098 patch were 275.35 ± 39.29 μg/cm2, 224.29 ± 41.89 μg/cm2 and 197.44 ± 25.35

15

μg/cm2, respectively. In addition, ionic liquids could be directly applied to the skin without the

30

Journal Pre-proof aid of organic solvents. [DON][ALA] was chosen as a model ionic liquid to investigate the

2

difference between the directly applied IL and IL-loaded DT4098 patch, because [DON][ALA]

3

revealed good skin permeability both in SKIN PAMPA and in vitro skin permeation study. As

4

presented in Fig. 6B, [DON][ALA] had far superior forces in cumulative permeation amount

5

when [DON][ALA] was directly applied to the membrane. The cumulative permeation amount

6

of [DON][ALA] was 755.51 ± 101.56 μg/cm2, which was 2.75 times more than the cumulative

7

permeation amount of [DON][ALA]-loaded DT4098 patch.

8

Table 5. in vitro skin permeation parameters of DIA patch and directly applied [DON][ALA].

[DON]

ro

19.772 ± 0.623

0.788 ± 0.013

2.952 ± 0.382

13.920 ± 2.741

0.926 ± 0.038*

3.523 ± 0.821*

16.038 ± 1.748

0.932 ± 0.041*

10.207 ± 5.333*

0.933 ± 0.036*

1.685 ± 0.582*

0.983 ± 0.008*

lP

[DON][IBU]

na

[DON][LA]

4.166 ± 0.655*

ur

Jo

[DON][ALA]

(directly applied) 9

R2

re

1.783 ± 0.483

[DON][ALA]

Lag time (h)

-p

J (μg/cm2/h)

DIA patch

of

1

21.204 ± 2.239*

*P < 0.05, vs. [DON]

10

As mentioned above, the penetration parameters could be determined by plotting the

11

cumulative amount of donepezil per unit area versus time. The transdermal steady-state flux (J,

12

μg/cm2/h) was calculated as the slope. Lag time indicated the time that drug saturated the

13

membrane and reached the receiving phase, which could be calculated from the X-axis intercept

14

values of the regression lines. R2 was the coefficient of determination of the regression lines.

31

Journal Pre-proof 1

Donepezil rarely permeated through the skin membrane in the initial 10 h, and DIA patches

2

reached the pseudo steady state at 12 h. In contrast, the first 24 h was the pseudo steady state of

3

[DON][ALA], when it was directly applied to the skin membrane. Therefore, the parameters of

4

the DIA patches were calculated based on the cumulative permeation amount from 12 h to 72 h,

5

while the parameters of [DON][ALA] was based on the first 24 h. In vitro skin permeation parameters were listed in Table 5. The transdermal steady-state

7

flux of donepezil from the patch was 1.783 ± 0.483 μg/cm2/h, whereas from the donepezil ILs

8

DIA patches, they readily increased to 2.952 ± 0.382 μg/cm2/h ([DON][IBU]-loaded DT4098

9

patch), 3.523 ± 0.821 μg/cm2/h ([DON][LA] -loaded DT4098 patch) and 4.166 ± 0.655 μg/cm2/h

10

([DON][ALA]-loaded DT4098 patch), respectively. The latter two patches showed significantly

11

higher steady-state flux (P < 0.05, vs. [DON]-loaded DT4098 patch). In addition, PSA played

12

the major role in the controlled release of the DIA patch, and the abilities of controlling drug

13

release may be assessed by the coefficient of determination (R2) of the regression lines. The

14

better stability of steady-state flux could be reflected by a closer value of R2 to 1. According to

15

Table 5, it could be concluded that the stability of steady-state flux of donepezil ILs-loaded

16

DT4098 patches were significantly higher than that of donepezil-loaded DT4098 patch, as

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indicated by a significantly greater values of R2 (P < 0.05, vs. [DON]-loaded DT4098 patch).

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Furthermore, more time was required for donepezil-loaded DT4098 patch to saturate the

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membrane and reach the receiving phase, indicated by 1.420-, 1.233- and 1.937-fold increase of

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lag time value for [DON][IBU]-loaded DT4098 patch, [DON][LA]-loaded DT4098 patch and

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[DON][ALA]-loaded DT4098 patch, respectively. This results also echoed the previous SKIN

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PAMPA conclusion that donepezil ILs through artificial skin membrane were faster than

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donepezil at similar concentration gradients. Finally, when [DON][ALA] was directly applied to

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Journal Pre-proof the skin membrane, a significant permeation advantage was observed. The transdermal steady-

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state flux of [DON][ALA] was 21.204 ± 2.239 μg/cm2/h, which was 11.892 times than that of

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donepezil-loaded DT4098 patch. Lag time of [DON][ALA] was 1.685 ± 0.582 h, which was

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dramatically shortened by 18 h compared to donepezil-loaded DT4098 patch (19.772 ± 0.623 h).

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Drug release from patch and permeation crossing skin are the two main steps for DIA patch

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transdermal drug delivery system[45]. Directly application of ionic liquid to the skin could omit

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the step of drug release, which could be the reason why [DON][ALA] permeated mostly quickly.

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Drug-PSA interaction and molecular mobility of drug in DIA patch were two important factors

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that affected drug release from the patch. The more efficient transdermal delivery donepezil by

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IL DIA patches than donepezil DIA patch could be explained from the following aspects. First,

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the similar molecular interaction between donepezil and DT4098 might have little effect on drug

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release from DIA patches. Secondly, IL DIA patches showed higher molecular mobility than

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donepezil DIA patch, as indicated by their lower Tg, which would further accelerate drug release

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from the patch. That was also the reason why the stability of steady-state flux of [DON][LA]-

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loaded DT4098 patch was higher than that of [DON][IBU]-loaded DT4098 patch. Tg values of

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donepezil ILs-loaded DT4098 patches were ranked in the following order: [DON][IBU]-loaded

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DT4098 patch > [DON][LA]-loaded DT4098 patch > [DON][ALA]-loaded DT4098 patch,

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which was consistent with the order of corresponding donepezil ILs’ Tg values. The Tg value of

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donepezil ILs patch may be decreased by the low Tg value of donepezil ILs. Finally, the release

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donepezil IL might cross the skin more efficiently than donepezil itself, as indicated by the

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permeability determined by skin PAMAP assay (Fig. 3A). According to our previous work, the

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skin permeation of the drug could be improved by forming ILs with lipophilic ligands, and the

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LogP values of ILs were correlated with their permeability coefficients[17]. In this study, the

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Journal Pre-proof LogP values of the formed donepezil ILs could not be improved for the excellent lipophilicity of

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donepezil itself, but the Tg values of the formed donepezil ILs were decreased. As mentioned in

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skin PAMAPA (section 3.4), the thermodynamic properties of donepezil ILs might dominate the

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skin permeation of donepezil. Thus, the advantage of donepezil ILs’ permeation and the effect of

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donepezil ILs on DIA patches resulted in the higher permeation through the skin, and the ILs

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with low Tg values could be selected to improve the skin permeation in the case of similar

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lipophilicity.

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Donepezil ILs DIA patches and [DON][ALA] applied directly to the skin were used to

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explore the application of ILs formulation. Although these two formulations facilitated

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transdermal delivery of donepezil, we have to admit that they have non-negligible defects. When

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[DON][ALA] was applied directly to the skin, it was difficult to control the amount of

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administration and consistent attachment to the skin. These defects reduced the potential of

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donepezil ILs practical application in TDDS. It is necessary to explore a dosage form that can

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exploit the advantages of ionic liquids, such as DIA patch. On the other hand, lag time is the

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biggest problem for donepezil ILs DIA patches, because almost no donepezil could permeate the

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skin-mimic artificial membrane in the initial 10 h. Since lag time is a common characteristic for

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DIA patch, it may be shortened by pressure sensitive adhesive screening and adding

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percutaneous enhancers[20, 43, 44]. Further study will be performed to optimize the formulation,

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then the pharmacokinetic studies of prepared formulations will be carried out.

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4. Conclusion

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Donepezil ILs were successfully prepared, which was confirmed by thermal analysis, FT-IR

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analysis and NMR analysis. Except for [DON][DOC], the formed donepezil ILs enhanced the

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Journal Pre-proof solubility and maintained the partition coefficients, and they had little effect on the inhibition of

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acetylcholinesterase activity and toxicity. According to in vitro SKIN PAMPA test, these

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donepezil ILs had the advantage over the donepezil base in terms of skin permeability. Drug-in-

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adhesive patch was chosen as model type of transdermal drug delivery system to develop the

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formulation of donepezil ILs. DIA patches of donepezil ILs-loaded DT4098 were successfully

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prepared and more permeable than donepezil-loaded DT4098. Specifically, the advantage of

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[DON][ALA] permeability was more pronounced when it was applied directly to the skin

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membrane. The formed donepezil ILs facilitated transdermal delivery of donepezil, which may

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contribute to develop innovative therapeutic alternatives for enhanced donepezil administration for AD treatment.

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Acknowledgments

This work was supported by National Natural Science Foundation of China (No. 21576080)

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and the Fundamental Research Funds for the Central Universities (No. 222201714036).

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Journal Pre-proof Author Statement Hao Wu：Methodology, Investigation, Formal analysis, Writing - Original Draft

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Fang Fang: Investigation, Formal analysis

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Lulu Zheng: Investigation, Software

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Weijie Ji: Validation, Software

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Minghui Qi: Supervision

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Minghuang Hong*：Conceptualization, Methodology, Writing - Review & Editing

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Guobin Ren*：Conceptualization, Resources, Project administration

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Declaration of interests

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The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

5 ☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Journal Pre-proof 1

Graphical abstract

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Journal Pre-proof 1 2

Highlights

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Donepezil was transformed into six ionic liquids for the first time.

4 5

The formed donepezil ILs enhanced the solubility and maintained the partition coefficients.

6 7

Donepezil ILs had the advantage over the donepezil base in terms of skin permeability.

Donepezil ILs-loaded DT4098 patches were successfully prepared and more permeable than donepezil-loaded DT4098 patch.

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