Pharmaceutical Development and Technology

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Preparation and in vitro / in vivo evaluation of 6-

Gingerol TPGS/PEG-PCL polymeric micelles

Lijun Zhen, Qiuyu Wei, Qilong Wang, Huiyun Zhang, Michael Adu-Frimpong,
Caleb Kesse Firempong, Ximing Xu & Jiangnan Yu

To cite this article: Lijun Zhen, Qiuyu Wei, Qilong Wang, Huiyun Zhang, Michael Adu-Frimpong,
Caleb Kesse Firempong, Ximing Xu & Jiangnan Yu (2018): Preparation and in vitro / in vivo
evaluation of 6-Gingerol TPGS/PEG-PCL polymeric micelles, Pharmaceutical Development and
Technology, DOI: 10.1080/10837450.2018.1558239

To link to this article: https://doi.org/10.1080/10837450.2018.1558239

Accepted author version posted online: 17

Dec 2018.

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Preparation and in vitro / in vivo evaluation of 6-Gingerol
TPGS/PEG-PCL polymeric micelles

PHARMACEUTICAL DEVELOPMENT AND TECHNOLOGY

Lijun Zhen a, Qiuyu Wei a, Qilong Wang a, Huiyun Zhang a, Michael

Adu-Frimpong a, Caleb Kesse Firempong a,b, Ximing Xu a,*, Jiangnan Yu
a,
*

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a Department of Pharmaceutics, School of Pharmacy, Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu

University, Zhenjiang 212013, People’s Republic of China

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b Department of Biochemistry and Biotechnology, College of Science, KwameNkrumah University of Science and Technology,

Kumasi-Ghana
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* Corresponding authors.
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Ximing Xu; Jiangnan Yu

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Department of Pharmaceutics, School of Pharmacy, Center for Nano Drug/Gene

Delivery and Tissue Engineering, Jiangsu University, Zhenjiang 212013, China.

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Fax: +86 511 85038451.

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E-mail addresses: xmxu@ujs.edu.cn (X. Xu); yjn@ujs.edu.cn (J. Yu).

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Preparation and in vitro / in vivo evaluation of 6-Gingerol

TPGS/PEG-PCL polymeric micelles

Abstract: 6-Gingerol, an active herbal ingredient of ginger, has various

bioactivities such as anti-neurodegenerative disease, anti-inflammatory and so

on. The aim of the present study was to enhance the oral bioavailability and
1
 brain distribution of 6-Gingerol via polymeric micelles. A polymeric micelles

drug delivery system of 6-Gingerol consisting of D-α-Tocopheryl polyethylene

glycol 1000 succinate (TPGS) and Poly (ethylene glycol)-poly (ε-caprolactone)

(PEG-PCL) was prepared via solvent injection method. The developed

6-Gingerol-loaded TPGS/PEG-PCL micelles (6-GTPMs) were characterized

based on particle size, polydispersity index (PDI), Zeta potential, encapsulation

efficiency (EE), drug loading (DL) and in vitro release profile. The

pharmacokinetics and tissue distribution studies were also evaluated. The

nanoformulation produced a particle size of 73.24±2.84 nm with acceptable

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PDI (0.129±0.03), zeta potential (-2.74 ± 0.92 mV), DL (4.64%) and EE

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(79.68%). The in vitro release profile showed that the 6-GTPMs enhanced the

solubility of 6-Gingerol, while the pharmaceutical analysis in rats indicated

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that 6-GTPMs significantly improved the oral bioavailability of 6-Gingerol
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(about 3 folds) in circulation. The 6-GTPMs exhibited remarkable brain

targetability in the tissue distribution analysis. Collectively, a 6-Gingerol

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polymeric micelle with enhanced oral bioavailability coupled with excellent

brain distribution was successfully developed.

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Keywords: 6-Gingerol; TPGS; PEG-PCL; Binary mixed micelles;

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Bioavailability; Tissue distribution

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Introduction:
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6-Gingerol (1, [4′-hydroxy-3′- methyoxyphenyl-5-hydroxy]-3-decanone) (Lee et al.

2008) (Fig.1 A), one of the major pungent compounds isolated from ginger, is a

hydrophobic ingredient with scarce water-solubility. Historically, 6-Gingerol has been

widely reported for its beneficial effects such as antioxidant (Masuda et al. 2004),

anti-inflammatory (Young et al. 2005; Dugasani et al. 2010), antithrombotic (Liao et

2
al. 2012), inhibition of adipogenesis (Tzeng et al. 2014), anti-diabetic (Chakraborty et

al. 2012), anti-arthritic, anti-migraine (Gauthier et al. 2013) and anti-nausea (Pertz et

al. 2011), as well as alleviating the malaise caused by rheumatic disorders (Srivastava

and Mustafa 1989, 1992). 6-Gingerol can also inhibit cancer development by

suppressing metastasis of cancer cells (Lee et al. 2008) coupled with

anti-angiogenesis of cancerous tumors (Kim EC et al. 2005). It was also reported that

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6-Gingerol-rich fraction from Zingiber officinale have significantly antioxidative,

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anti-inflammatory and antiapoptotic activities in the brain (Abolaji et al. 2017)

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Moreover, 6-Gingerol has potent protective effect on the brain’s nervous system and

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studies has shown that the drug could attenuate amnestic deficits of Alzheimer’s
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disease by inhibiting different biochemical factors in the brain (Joshi and Parle 2006;
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Halawany et al. 2017).

However, the clinical use of 6-Gingerol, especially its utilization as tumor

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inhibitor and brain-active ingredient, has been limited by some setbacks such as the
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low oral bioavailability caused by poor water solubility of the drug (Jaapar et al.
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2014; Syed Jaapar et al. 2015; Matsumoto et al. 2016) as well as the fact that the
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P-glycoprotein mediated drug efflux effect could reduce the absorption of 6-Gingerol,

thereby decreasing its bioavailability (Yu et al. 2017). To improve the solubility of

6-Gingerol, microbial glucosylation synthesis reaction was used to modified the

structure of 6-Gingerol and its solubility was increased more than three hundred-times

(Matsumoto et al. 2016). Besides, hot compressed water method and co-solvent

3
method were used to improve the solubility of 6-gingerol (Jaapar et al. 2014; Syed

Jaapar et al. 2015). But above methods were limited to the field of chemical and less

of convenience on clinical utilized. Due to these challenges, there is the need for

innovative development of drug delivery systems to improve the pharmaceutical

applications of 6-Gingerol.

Several formulation have developed to correct the deficiencies of drugs, such

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as low solubility and unsatisfactory bioavailability. For example, the graft polymeric

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micelles of paclitaxel (Liu J et al. 2011), novel nanoparticles of capsaicin (Peng et al.

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2015), liposome of chlorogenic acid (Feng et al. 2016) and binary mixed micelles of

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icariside (Hou, Wang, et al. 2016) to increase the bioactivity of drugs, porous silica
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nanoparticles (Cao et al. 2013) and micelle-templated porous calcium phosphate
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microparticles of silybin(Zhu Y. et al. 2016) for ideal release position, as long as the

new self-codelivery system from redox-sensitive camptothecin-cytarabine conjugate

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assembly (He et al. 2017). However, in the case of 6-Gingerol, only limited
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preparations, such as novel transdermal delivery system (Chen et al. 2014),

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self-microemulsifying drug delivery system (Xu et al. 2016), proliposome (Wang Q et

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al. 2018) have been developed. Thus, novel formulations strategy with the potential to

improve the oral bioavailability of 6-Gingerol and increase the clinical application of

the drug are immediately needed. Micelles, as a novel drug delivery system, have

unique advantages including long retention in the body plus high solubility coupled

with enhanced oral bioavailability (Liu J et al. 2011; Wang L et al. 2014). The

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polymeric micelle system is one of the most effective nanodrug carriers that aggregate

of amphiphilic polymeric molecules which could spontaneously form micelles

(Abdullah et al. 2011; Hu X et al. 2018), with appropriate particle size and protective

effect for aqueous insoluble drug (Duan Y. et al. 2016; Shi et al. 2018).

D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) (Fig. 1 B),

consisting of natural Vitamin E succinate with polyethylene glycol (PEG) 1000, was

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selected as the carrier biomaterial to form the micelle structure (Yang et al. 2016). As

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a non-ionic surfactant, it has an amphiphilic structure with solubilization ability.

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Furthermore, TPGS could also work as a kind of P-glycoprotein (P-gp) inhibitor by

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suppressing P-gp ATPase activity, and overcoming the multidrug resistance (MDR),
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hence improving the absorption and oral bioavailability of the drugs (Zhu H et al.
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2014; Cheng et al. 2016). Due to the excellent properties on P-gp inhibiting, the

TPGS loaded liposomes could enhanced the cellular uptake and cytotoxicity of
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docetaxel in brain cancer cells (Muthu MS et al. 2011). What’s more, the TPGS
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mixed micelles could delivery protein as well as small-molecule drugs to the brain
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and resulting in a significantly increased brain targeting effect (Meng Xin et al. 2017).
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Above studies revealed that TPGS could utilized as efficient and cost-effective

pharmaceutic adjuvant on clinical application for the treatment of cerebral disease. As

an adjuvant utilized in novel drug delivery system, the TPGS usually mixed with

other materials, such as polyethylene glycol-polyethylene (PEG-PE) and polyethylene

glycol-polylactic acid (PEG-PLA) and so on, to obtain more stable and solubilized

5
pharmaceutical preparation (Guo et al. 2013). Poly(ethylene

glycol)-poly(ε-caprolactone) (PEG-PCL) (Fig. 1 C), a novel copolymer material, has

also been approved by FDA as pharmaceutically safe excipients, and was frequently

used in drug research and development due to its biodegradability, biocompatibility

along with other advantages (Gong et al. 2009; Cuong et al. 2010; Yang et al. 2017).

The use of both TPGS and PEG-PCL in binary mixed micelles could overcome the

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low hydrophobicity of the TPGS, meanwhile have no influence on the P-gp inhibited

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activity of TPGS (Yang et al. 2017). Therefore, it could be hypothesized that the

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synergy of TPGS and PEG-PCL in the preparation of mixed polymeric micelles may

provide a more ideal formulation.

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In the present study, the TPGS and PEG-PCL were used to prepare
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TPGS/PEG-PCL polymeric micelles to enhance the oral bioavailability and brain

distribution of 6-Gingerol. Herein, the optimum preparation of 6-Gingerol loaded

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polymeric micelles was successfully developed. The formulation was characterized

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using the particle diameter, zeta potential, DL and EE, as well as morphological
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characterization via transmission electron microscope (TEM). The in vitro release

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profile, in vivo pharmacokinetic analysis and tissue distribution were also evaluated in

this research.

6
Materials and methods

2.1 Materials

6-Gingerol (98% purity) was purchased from Aladdin Industrial Corporation

(Shanghai, China). D-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) was

obtained from Aladdin Industrial Co., Ltd (Shanghai, China). Poly (ethylene

glycol)-poly (ε-caprolactone) (PEG-PCL/5,000:20,000) was bought from Jinan

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Daigang Biomaterial Co., Ltd (Jinan, China). The syringe filter membrane was

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supplied by Jiangsu Mingjie Scientific Instruments Co., Ltd (Nanjing, China), while

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pure chromatographic grade methanol was obtained from Hanbon Sci. & Tech

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(Jiangsu, China). The other chemicals and reagents were of analytical grade and
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obtained commercially. Water was produced in-house by a Millipore water
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purification system (Millipore Corporation, Bedford, MA, USA).

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2.2 Animals

Male Sprague Dawley (SD) rats (200 ± 20 g) and Kunming mice (20 ± 2 g) were
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obtained from the Laboratory Animal Center of Jiangsu University, Zhenjiang, China.
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The animals were housed for 72 h under 12 h light/dark cycles throughout the
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experiment at a controlled temperature of 22 ± 2 ℃. They were fed with standard food

and water ad libitum under standard laboratory conditions. All the animals were

fasted 12 h prior to each experiment but allowed free access to water. The animal

experiments involving the use of animals were strictly followed based on the

guidelines of the Ethic Committee of Jiangsu University.

7
2.3 Preparation of 6-Gingerol-TPGS/PEG-PCL Micelles

The drug-loading micelles were prepared using the solvent injection method as

described previously (Cheng et al. 2016) with slight modifications. The varieties and

ratio of excipients were determined by formulation screening pre-experiment which

was not shown in this paper. Briefly, PEG-PCL, TPGS and 6-Gingerol were dissolved

in moderate volume of acetonitrile at a ratio of 5:5:2, respectively. The mixture was

injected into water at 45 ℃, stirred for 30 min by a Heating Magnetic Stirrer, to form

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the 6-GTPMs and the organic solvent was then removed from the product by long

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time volatilization. The ratio of acetonitrile and water was 1:10 (v/v). The formulation

was stored at 4 ℃ for pending further investigations.

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2.4 Establishment of HPLC Analysis of 6-Gingerol
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The detection of 6-Gingerol was performed by HPLC methods as described

previously(Xu et al. 2016) with suitable modifications. The quantitative of 6-Gingerol

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was detected using a Shimadzu High Performance Liquid Chromatography (HPLC)

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UV detector (LC-20AD, SHIMADZU, JAPAN) with RP-HPLC Symmetry C18

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column (4.6×150 mm, 5μm) (Waters, Ireland). The HPLC condition was as follows:
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UV wavelength of 230 nm; column temperature of 37 °C; methanol/water (65/35, v/v)

as mobile phase and flow rate at 1.0 mL/min.

To calculate the content of 6-Gingerol in vitro and in vivo, a series of

calibration curves were established by HPLC in this study.

8
2.5 Characteristics of 6-Gingerol-Loaded TPGS/PEG-PCL Micelles

2.5.1 Particle size and Zeta Potential Analysis

After filtering the formulated drug through 0.22 µm syringe filter, the particle size and

zeta potential of the nano-sized particle were determined using dynamic light

scattering technique at 25 ℃ and a scattering angle of 90° on a Brookhaven 90 Plus

PALS instrument (Brookhaven Instruments Crops., Holtsville, NY, USA). The

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concentration of 6-Gingerol in the samples was 2 mg/mL.

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2.5.2 Morphological Analysis

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The morphology characterization of 6-GTPMs was examined using TEM (JEM-2100,

JEOL, Japan). A fraction of 6-GTPMs (200 µL) was put on a copper grid, stained
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with phosphotungstic acid (pH 4.47). The copper grid was then dried at room
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temperature to observe the morphological properties (Hou, Wang, et al. 2016).

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2.5.3 Determination of Encapsulation Efficiency and Drug-Loading Capacity

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The 6-Gingerol encapsulated within the micelles was determined using the method
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described in other report (Zhu Yuan et al. 2015) with appropriate modifications. In

brief, fresh micelles (2 mg/mL) were prepared and 1 mL of the micellar solution was
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poured onto a 0.22 µm organic membranes to remove the unencapsulated 6-Gingerol.

The filtrate was then disrupted with absolute methanol at a ratio of 1:100 (v/v),

respectively. The content of 6-Gingerol was measured by aforementioned HPLC

9
methods. After that the drug loading capacity (DL) plus encapsulation efficiency (EE)

were calculated according the following equations:

weight of 6-Gingerol in micelles

DL= ×100% (1)
total weight of micelles

weight of 6-Gingerol in micelles

EE= weight of 6-Gingerol intially added ×100% (2)

2.5.5 In vitro Drug Release

The solubility of 6-gingerol in three different release media were measured in a

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pre-experiment and the solubility values were 252.31 μg/mL (DSW (pH 7.0), 226.24

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μg/mL (HCl (pH 1.2) and 258.87 μg/mL (PBS (pH 7.4), respectively. In vitro release

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of free 6-Gingerol and 6-GTPMs were conducted based on dialysis method (Zhu J et

al. 2018) with slight modifications. In brief, a triplicate assay was carried out in three
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different media viz., HCl solution (HCl (pH 1.2)), phosphate buffer solution (PBS (pH
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7.4)) and double-distilled water (DSW (pH 7.0)), performed on a constant temperature
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water-bath vibrator at 37 °C with a rotating speed of 100 rpm. The free 6-Gingerol

suspension (2 mg/mL, 1mL) and 6-GTPMs (equivalent 6-Gingerol content) were

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transferred into dialysis bags (3,500 Da, previously soaked in three media,
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respectively) then immersed in 100 mL of three different dissolution media,

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respectively. At predetermined intervals, 1 mL each of the different media was taken

and subsequently an equal volume of fresh media (preheated to 37 ℃) were added to

maintain the sink condition. All the samples were centrifuged at 10,000 rpm for 10

min. The supernatant (20 µL) was then injected into the HPLC for the sample

analysis.
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2.6 Pharmacokinetics

Before the experiments, six SD rats (200 ± 20 g) were randomly divided into two

equal groups and treated with free 6-Gingerol and 6-GTPMs respectively. Drugs

equivalent to a dose of 250 mg/kg 6-Gingerol were given to the rats via intragastric

administration. Blood samples (0.5 mL of each) were collected from infraorbital

venous plexus under ether anesthesia at different time intervals (5 min, 10 min, 15

min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min). The samples were

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centrifuged at 3700 rpm for 10 min to separate the plasma. Then 50 µL of curcumin

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(internal standard) and 500 µL of ethyl acetate were immediately added to 200 µL of

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plasma immediately. Afterwards, the samples were vortexed for 30 s plus
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centrifugation for 10 min at 10,000 rpm, the supernatant was carefully removed and

dried using nitrogen with gentle heat at 37 ℃ on a water bath. The residue was
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re-dissolved in 400 µL of the mobile phase and centrifuged at 10,000 rpm for 10 min.
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The supernatant (20 µL) was then injected into the HPLC for the sample analysis.
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2.7 In vivo Tissue Distribution

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Thirty male Kunming mice (20 ± 2 g) were randomly divided into two equal groups
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and were treated with free 6-Gingerol and 6-GTPMs. Drugs equivalent to 50 mg/kg

6-Gingerol were given via oral administration. After the drug administration, the

tissue samples including heart, liver, spleen, lung, kidney and brain were collected at

different time interval (5 min, 30 min, 60 min) from 5 animals and treated according

to earlier described method (Omari-Siaw, Wang, et al. 2016) with appropriate

11
modifications. In briefly, the tissues were homogenized with normal saline at a ratio

of 0.1 g/1 mL using an electromotive homogenizer to obtain a uniform mixture. An

aliquot (50 µL) of curcumin solution (internal standard) and 500 µL of ethyl acetate

were immediately added to 200 µL of each tissue sample, vortexed and centrifugated.

The supernatant was collected and nitrogen-dried with gentle heat at 37 ℃ on a water

bath. The residue was re-dissolved in 400 µL of the mobile phase (65 %

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methanol-water system), vortexed plus centrifuged at 10,000 rpm for 10 min. The

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supernatant (20 μL) was injected into the HPLC for the sample analysis.

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2.8 Statistical Analysis

Experimental data were expressed as mean ± SD. Origin 9.0 was used to construct the
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graphs. SPSS 19.0 software was used for the statistical analysis. The statistical
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differences between two different groups were analyzed using the Student’s t-test.
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Pharmacokinetic parameters were also calculated using BAPP 2.3 pharmacokinetic

software (Munyendo et al. 2013).

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3. Result and discussion

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3.1 Assay validation and Calibration curves of 6-Gingerol

The calibration curve established using the HPLC based on the different concentration

of 6-Gingerol standard solutions was used to calculate the in vitro content of

6-Gingerol. The curve produced an equation of A = 10571C - 2326.6 (n=5, R² =

0.9999) with a linear range of 1-200 µg/mL, where C represented the concentrations

12
of 6-Gingerol and A represented the peak area observed in the HPLC. The average

recovery and relative standard deviation (RSD) were 99.64% and 3.99%, respectively

(n=3). The limit of detection (LOD) was 0.5 μg/mL, while the intra- plus inter-day

variations of RSD were less than 5%. In vivo standard curves were shown in Table 1,

where C represented the concentrations of 6-Gingerol and A represented the peak area

observed in the HPLC.

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3.2 Characteristics of 6-Gingerol-Loaded TPGS/PEG-PCL Micelles

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The zeta potential of 6-GTPMs was -2.74 ± 0.92 mV while the average particle size

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distribution of 6-GTPMs was 73.24±2.84 nm, with polydispersity index (PDI) of

0.129±0.03 (Fig. 2. (A)). The results indicated that 6-GTPMs were nanosized with a
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typical lognormal distribution and homogeneous micelle system because of the low
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PDI (<0.3) (Chu et al. 2011). In addition, previous report(Gao and Jiang 2006) has
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established that particle size below 100 nm was beneficial to pass through the blood

brain barrier (BBB), which could increase the drug level in the brain. The TEM image
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of 6-GTPMs showed in Fig. 2. (B) also revealed that the polymeric micelles were
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homogeneous with spherical morphology, as well as well dispersed nanoparticles with

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no obvious sign of particle aggregation. The scale of micelles showed in Fig. 2. (B)

was about 40 nm, significantly smaller than Fig. 2. (A), hydrodynamic particle size

detected using DLS experiment. The difference between TEM and DLS data might be

due to the water absorbency of polymer materials. Other studies also stated that the

dehydration process in TEM experiment could result in smaller particle size of the
13
polymeric-micelles (Tao et al. 2013). Thus, the variation in TEM and DLS results

may be caused by the tendency for the polymeric-micelles to shrink in the dry state

(Tao et al. 2015). Altogether, the micelle system consisted of TPGS and PEG-PCL,

with appropriate particle size and zeta potential, has been successfully developed in

this research.

The EE of 6-GTPMs was estimated as 79.68%, while the DL was 4.64%. This

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finding indicated that the majority of 6-Gingerol was entrapped in the

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TPGS/PEG-PCL micelles, which contributed to better solubility and bioavailability.

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In vitro release experiments were carried out in sink condition and as shown in

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Fig. 3, 6-Gingerol release from the micelles was characterized with an initial burst
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followed by a slow-release phase. The initial burst release of 6-GTPMs might
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attributed to 6-Gingerol molecules located within the corona or at the interface

between the micelle core and corona (Liu SQ et al. 2005). Besides, the release of
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6-Gingerol from 6-GTPMs was faster than that from free 6-Gingerol, which was
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similar to previous reported preparations of 6-Gingerol (Xu et al. 2016; Wang Q et al.
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2018). The maximum release of 6-GTPMs was 96.42%, 88.95% and 96.84% in the
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three different medias (DSW (pH 7.0), HCl (pH 1.2), alongside PBS (pH 7.4)),

respectively. However, the maximum release of free 6-Gingerol in pH 7.0 DSW

(85.95%), pH 1.2 HCl (76.89%) and pH 7.4 PBS (80.71%) was lower than that of

6-GTPMs, respectively. Furthermore, the cumulative release profile of 6-GTPMs was

higher than previous reported self-microemulsifying drug delivery system of

14
6-Gingerol (Xu et al. 2016). These findings may attribute to the smaller particle size

with broad surface area plus increased permeability provided by 6-GTPMs

(Omari-Siaw, Zhu, et al. 2016). Above all, the polymeric micelles could significantly

promote the in vitro release of 6-Gingerol with increased desired solubility.

3.3 Pharmacokinetics

The plasma concentration of 6-Gingerol in 6-GTPMs groups showed sharp increase

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and later reached the maximum plasma concentration (C max) at 15 min after

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administration (Fig. 4). The C max of 6-Gingerol in 6-GTPMs groups was about

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4-folds higher than that of the free 6-Gingerol cohort. The plasma concentration was

also remarkably higher compared with the free 6-Gingerol group at each time point.
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The significant increase in plasma concentration indicated a desired
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bioavailability-enhancing effect via micellar formulation. The area under the

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concentration-time curve (AUC0-t) value of 6-GTPMs was about 5-folds of free

6-Gingerol, whereas that of C max value was about 4-folds of free 6-Gingerol (Table
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2). Similarly, the results confirmed the increased bioavailability effect of 6-GTPMs.
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The enhanced plasma concentration of 6-Gingerol in 6-GTPMs possibly

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through the three under listed reasons: (1) the polymeric micelles. As a novel

formulation strategy, the polymeric micelles have been widely used for the

solubilization of drugs with poor water solubility, thus improved the bioavailability.

(2) the utilization of TPGS and PEG-PCL. It has been reported that TPGS could

enhance the solubility of hydrophobic compound, overcome P-gp mediated drug

15
efflux (Sun et al. 2017) , and improve its oral bioavailability(Mei et al. 2013; Duan

Yuwei et al. 2016; Hu M et al. 2016). Additionally, the excipient PEG-PCL, consisted

of biocompatible hydrophilic groups and biodegradable lipophilic groups, could

spontaneously form micelles with TPGS, thereby encapsulate the drug as well as

solubility improving (Hou et al. 2017). The combined effect of TPGS and PEG-PCL

played important role on the bioavailability-enhancing effect of 6-GTPMs. (3) the

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narrow range of size distribution and desired smaller particle size could extend the

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retention time of micelles by reducing nonselective reticuloendothelial system

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scavenging, and also contributing to the prolonged retention time of 6-Gingerol in

plasma (Hou, Sun, et al. 2016; Yang et al. 2017).

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3.4 In vivo Tissue Distribution
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The concentration of 6-Gingerol in the six different organs obtained from 6-GTPMs
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group were higher compared with the free 6-Gingerol cohort (Fig. 5). Five minutes

after oral administration, the concentration of 6-Gingerol in liver was the highest in
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selected organs. Besides, the increasing range of 6-Gingerol concentration among six
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organs at 5 min between the free 6-Gingerol groups and 6-GTPMs groups was
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different. Briefly, the highest increase range among free 6-Gingerol and 6-GTPMs

groups at 5min was almost 26-folds in the brain, while the incremental trend in other

tissues is as follows: lung (10-folds) >kidney (9-folds) >spleen (3.8-folds) >liver

(3.5-folds) >heart (1.2-folds). Remarkably, the concentration of 6-Gingerol was

higher in the brain which suggested that 6-GTPMs could overcome the obstacle of
16
brain-blood barrier (BBB) and become distributed in brain. Previous studies on

6-Gingerol preparations (Singh et al. 2012; Xu et al. 2016; Wang Q et al. 2018) did

not report its brain targeting activity, thus the present study has revealed the unique

characteristic of 6-GTPMs. However, other reports have verified the utilization of

TPGS as a powerful biological response modifier (BRM) for the brain targeting drug

delivery (Muthu MS et al. 2011; Meng X. et al. 2017; Yu et al. 2017). The TPGS

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could also inhibit P-gp regulated drug efflux from cells and enhance drug transport

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across BBB (Omari-Siaw, Wang, et al. 2016). Furthermore, combined with the former

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reported TPGS/PEG-PCL mixed micelles of ginsenoside compound K for lung cancer

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treatment(Yang et al. 2017), the current article reported the brain targetability of
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TPGS/PEG-PCL mixed micelles, proved that TPGS/PEG-PCL mixed micelles was a
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meaningful preparation. Besides, the concentration of 6-Gingerol at 30 min and 60

min was similar high and combined with the rapidly in vitro release from micelles,
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indicated that the contribution of micelles to brain distribution may mostly due to the
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blockage of P-gp from BBB. Thus the utilized of TPGS could be the main reason of
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the brain targetable activity(Muthu M et al. 2012; Sonali et al. 2015; Meng Xin et al.
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2017) while further research seems to be needed to classify whether the dosage form

made from TPGS/PEG-PCL could influence the brain targetability. Several researches

showed that 6-Gingerol have potential neuroprotective effect, such as protecting

neuronal cells from prion peptide-induced damage (Jeong et al. 2013), alleviating

brain neuropathic pain (Gauthier et al. 2013) and attenuating learning and memory

17
impairments (Kim CY et al. 2018). The binary polymeric micelles made up with

TPGS and PEG-PCL might promote the development of intracerebral treatment.

Nevertheless, further research is required to confirm this preparation as nanocarrier

for brain targetability to treat brain neurodegenerative disease.

4. Conclusion

In the present study, the 6-Gingerol loaded polymeric micelles (6-GTPMs) were

t
ip
successfully developed using TPGS and PEG-PCL, which were homogeneous and

cr
spherical in morphology with narrow particle size. The in vitro and in vivo tests

us
proved that 6-GTPMs could increase solubility and significantly enhance the

bioavailability of 6-Gingerol. Furthermore, 6-GTPMs could increase the content of

an
6-Gingerol in each selected tissues of mice, with much improvement in the brain
M

leading to brain targetability. Therefore, the 6-GTPMs could effectively cross the
ed

BBB; however, the mechanism of its significant higher brain distribution needs

further investigation in future studies.

pt
ce

Conflict of interest

The authors declare that they have no conflict of interest.

Ac

Acknowledgements

This work was supported by the [National Natural Science Foundation of China]

under Grant [81371171]; [the Doctoral Fund of Ministry of Education of China] under

18
Grant [20113227110012]; [Special Funds for 333 and 331 projects] under Grant

[BRA2013198].

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Figure Captions

Figure 1. The chemical structure of (A) 6-Gingerol; (B) TPGS and (C) PEG-PCL.

Figure 2. (A) Particle size of 6-GTPMs; (B) Transmission electron morphology of

6-GTPMs

Figure 3. In vitro accumulative release profile of the 6-GTPMs and free 6-Gingerol in

100 mL of three different media: (A) double-distilled water; (B) HCl solution at pH

t
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1.2; (C) PBS solution at pH 7.4. Each data point represents mean ± SD (n=3). (* P<

0.05 compared to 6-Gingerol group; ** P < 0.01 compared to 6-Gingerol group.)

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Figure 4. The plasma concentration-time curve of 6-Gingerol after oral administration

us
of free 6-Gingerol and 6-GTPMs (250 mg/kg of 6-Gingerol). Each data point
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represents mean ± SD (n=3). (* P < 0.05 compared to 6-Gingerol group; ** P < 0.01

compared to 6-Gingerol group.)

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Figure 5. Tissue distribution of 6-Gingerol following oral administration of free

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6-Gingerol and 6-GTPMs to mice for 5 min (A); 30 min (B) and 60 min(C) (n=5). (*

P < 0.05 compared to 6-Gingerol group; ** P < 0.01 compared to 6-Gingerol group.)
pt
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27
Figure 1

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Figure 2
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28
Figure 3

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Figure 4

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Figure 5
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29
Table 1. Calibration curves and linearity of 6-Gingerol in various tissues respectively.
Liner Range
Sample Regression Equation R2
(µg/mL)
Plasma y = 0.3346x + 0.0521 0.9995 0.125-25
Heart y = 0.4043x - 0.1257 0.9986 0.125-25
Liver y = 0.3202x - 0.003 0.9997 0.125-25
Spleen y = 0.3835x - 0.0322 0.9966 0.125-25
Lung y = 0.4034x - 0.0431 0.9999 0.125-25
Kidney y = 0.396x - 0.0315 0.9987 0.125-25
Brain y = 0.362x - 0.0228 0.9993 0.125-25

Table 2. Pharmacokinetic parameters of 6-Gingrol after oral administration of free

t
6-Gingerol and 6-GTPMs (250 mg/kg of 6-Gingerol)

ip
AUC 0-t MRT 0-t T max C max

cr
(min*µg/mL) (min) (min) (µg/mL)
6-Gingerol 89.88±22.84 45.67±20.11 5 2.66±0.19
6-GTPMs 497.36±48.11* 54.28±4.14

us 15 9.55±1.12*
*P<0.05, compared with free 6-Gingerol. 6-GTPMs: 6-Gingerol-loaded TPGS/PEG-PCL micelles
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30