Ropivacaine loaded microemulsion and microemulsion-based gel for transdermal delivery: Preparation, optimization, and evaluation

Accepted Manuscript Title: Ropivacaine loaded microemulsion and microemulsion-based gel for transdermal delivery: Preparation, optimization and evaluation Author: Lili Zhao Yi Wang Yingjie Zhai Zimin Wang Jiyong Liu Guangxi Zhai PII: DOI: Reference:

S0378-5173(14)00728-5 http://dx.doi.org/doi:10.1016/j.ijpharm.2014.10.005 IJP 14373

To appear in:

International Journal of Pharmaceutics

Received date: Revised date: Accepted date:

11-7-2014 30-9-2014 3-10-2014

Please cite this article as: Zhao, Lili, Wang, Yi, Zhai, Yingjie, Wang, Zimin, Liu, Jiyong, Zhai, Guangxi, Ropivacaine loaded microemulsion and microemulsion-based gel for transdermal delivery: Preparation, optimization and evaluation.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2014.10.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Ropivacaine loaded microemulsion and microemulsion-based gel for transdermal delivery: preparation, optimization and evaluation Lili Zhao 1, Yi Wang 2, Yingjie Zhai1, Zimin Wang2, Jiyong Liu3*, Guangxi Zhai1*

1. Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan

250012, China

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2. Department of Orthopedics, Changhai Hospital, Second Military Medical University, Shanghai

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200433, China

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3. Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai

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200433, China

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* Corresponding author:

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Jiyong Liu, Ph.D.

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Department of Pharmacy, Changhai Hospital, Second Military Medical University, Shanghai

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200433, China, Tel.: (86) 21-31162308, E-mail: [email protected], [email protected]

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Guangxi Zhai, Ph D

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Department of Pharmaceutics, School of Pharmaceutical Sciences, Shandong University, Jinan

250012, China, Tel.: (86) 531-88382015. E-mail: [email protected]

Graphical abstract Abstract The objective of the present study was to prepare and evaluate a ropivacaine-loaded

microemulsion (ME) formulation and microemulsion-based carbopol gel (ME-gel) for transdermal

delivery. Pseudo-ternary phase diagrams and a simplex lattice experiment design were utilized to

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screen and optimize the ME formulation. In the process, drug solubility and particle size were

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inspected as dependent variables whilst Capryol® 90 (X1), Smix (X2, Labrasol®: absolute ethanol

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= 1:2 w/w), water (X3) as independent variables. Following the optimization, the optimal ME

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formulation was comprised of 15 % Capryol® 90, 53 % Smix and 32 % water, respectively.

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Ropivacaine loaded ME appeared to be spherical under transmission electron microscope, and the

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average particle size was 58.79 nm. The results of ex vivo permeation study showed that

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ropivacaine had a significant higher cumulative amount from ME than that from ME-gel.

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Histopathology study elucidated that the microstructure of skin surface was significantly changed

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by the treatment of ME formulation. Skin irritation study indicated that neither ME nor ME-gel

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caused any irritation responses. Both ME and ME-gel presented a remarkable analgesic activity on

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acetic acid-induced writhing in mice. In conclusion, ME could be a promising formulation for

ropivacaine transdermally administration.

Keywords ropivacaine; microemulsion; carbopol; transdermal delivery; analgesic activity

1. Introduction

Ropivacaine (1-propyl-2’, 6’-pipecoloxylidide, chemical structure) is a new-developed long-

acting local anesthetic drug, which is marketed in injection dosage form under the trade name of Naropin® (Ropivacaine hydrochloride) (Leisure and DiFazio, 1996; Zhai et al., 2014). By analogy

to the other amide-based local anesthetics such as lidocaine and bupivacaine, ropivacaine is

widely used in labor analgesia and post-operative pain relief (Gutton et al., 2013; Kau et al.,

anesthetic effects seen with bupivacaine, ropivacaine also

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2001). With the desirable local

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possesses more advantages, like lower cardiovascular toxicity (Scott et al., 1989), providing more

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differential block between sensory and motor (McClure, 1996). However, the pursuit of better

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patient compliance and a longer-acting of ropivacaine required more explorations on novel routes

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of administration, for instance, transdermal delivery. Furthermore, its low molecule weight

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(MW=274.41) and an appropriate log P value (2.9) (McClure, 1996) make ropivacaine a candidate

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for transdermal delivery.

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Transdermal drug delivery system (TDDS) has attracted multitudes of researches since the

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1970s when transdermal patches were popular in systemic drug delivery (Roy et al., 1996).

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Ropivacaine designed to be delivered through TDDS possesses numerous advantages, particularly

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with a long-lasting and uniform drug plasma level, thereby reducing the frequency of

administration and the side effects derived from plasma concentration fluctuation like central

nervous system and cardiovascular system toxicity (Chaudhary et al., 2013). Thus, ropivacaine

could be safer and provoded better patient compliance. Furthermore, circumvention of harsh

gastrointestinal environment and hepatic first pass metabolism (Liu et al., 2012), better patient

compliance, made TDDS a potential proposal for ropivacaine delivery (Kim et al., 2013).

However, ascribed to the ‘bricks and mortar’ structure composed of quasi-columnar, proteinrich

corneocytes of the stratum corneum, TDDS encounters a formidable obstacle (Cevc, 2004; Elias

and Menon, 1991; Prow et al., 2011). Hence, multiple measures, both active strategies and

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positive ones, were conducted to attenuate the physical barrier of skin, for instance, microneedles

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(Liu et al., 2012; Vitorino et al., 2014) and sonophoresis (Polat et al., 2012) were utilized to

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generate temporary holes on skin allowing drugs penetrated through, while chemical permeation

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enhancers (Jung et al., 2013; Lee et al., 2013; Vitorino et al., 2013) and nano-scale systems

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(Chaudhary et al., 2013; Jana et al., 2014; Khurana et al., 2013) were applied to enhance drug

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permeation through altering the surface property of skin.

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Microemulsion (ME) is a monophasic, transparent, thermodynamically stable mixture

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composed of oil, water and stabilized with surfactant and co-surfactant. Owing to its nano-scale

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particle size and certain ingredients acting as permeation enhancers, ME, working as a potential

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transdermal drug delivery carrier, has attracted a number of researches. Among these studies, an

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intense field was focused on the delivery of substances such as naproxen with analgesic activities

(Ustundag Okur et al., 2011). Ustundag Okur et al prepared a naproxen loaded microemulsion

formulation with the average droplet size of 45.83 nm, comprised of isopropyl myristate, Labrafil

M, Cremophor EL, isopropyl alcohol and 0.5 M NaOH solution, for transdermal delivery. The ex

vivo permeation rate of naproxen from the developed microemulsion was 8.44-folds that of the

commercial gel, which indicated that microemulsion could be a potential drug transdermal

transporter.

In this present work, a ME formulation was designed, optimized and evaluated for

ropivacaine. Ternary phase diagrams and the simplex lattice experiment design handled by Design

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expert® software were employed in the process of formulation design and optimization. Ex vivo

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and in vivo studies, including physicochemical properties, ex vivo permeation behavior, skin

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irritation and pharmacodynamics, were conducted to estimate the feasibility of ME for

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2.1. Materials

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2. Materials and methods

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matrix at 0.5% was also studied as control.

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transdermal drug delivery. In addition, the ME-based gel with the concentration of carbopol

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Ropivacaine was purchased from Dexinjia Pharmaceutical Co., Ltd. (Jinan, China). Capryol®

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90, Labrasol®, Plurol® Oleique CC 497 and Transcutol®HP were purchased from Gattefosse

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(Saint-Priest, France). Cremophor®EL and Solutol HS 15 (polyethylene glycol monostearate)

were obtained from BASF (ludwigshafen, Germany). Emulsifier OP, propylene glycol and Tween

80 were purchased from Guangcheng chemical agent Co., Ltd. (Tianjin, China). Ethyl oleate and

polyethylene glycol 400 (PEG 400) were from Shanghai chemical agent Co., Ltd. (Shanghai,

China). Castor oil was supplied by Sinopharm chemical reagent Co., Ltd. (Shanghai, China). Oleic

acid was purchased from Kemiou chemical reagent Co., Ltd. (Tianjin, China). Carbopol 940 was

acquired from Shenxing Pharmaceutical Manufactory (Shanghai, China). Methanol, acetonitrile,

triethylamine were of chromatographic grade. All of the other chemicals and reagents used were

of analytical grade.

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2.2. Solubility study of ropivacaine in oils, surfactants and co-surfactants

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Solubility studies were conducted to find out appropriate components of ME formulation with

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high drug-loading capacity. In the process, an excess amount of ropivacaine was separately added

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into 1g of oils such as ethyl oleate, isopropyl myristate®, castor oil, Capryol® 90, oleic acid,

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surfactants like Cremophor®EL, Tween® 80, Labrasol®, Emulsifier OP, Solutol HS 15 (all of the

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above vehicles were in liquid phase at 37℃), and various co-surfactants namely Plurol® Oleique

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CC497, Transcutol®HP, absolute ethanol, propylene glycol, PEG400. Then the samples were

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vortexed for 2 min for thoroughly mixing and followed by constantly shaken in a water bath

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shaker at 37℃ for 72h to achieve a dissolution equilibrium state. Afterward, the samples were

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centrifuged at 10000 rpm for 10 min and the concentrations of ropivacaine in the supernatant were

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quantified by HPLC analysis. All experiments were performed in triplicate.

2.3. Construction of pseudo-ternary phase diagrams

For the purpose of locating the ME region, especially obtaining the concentration ranges of

components such as oil, surfactant, co-surfactant and water for the existing ME region, the

pseudo-ternary phase diagrams were constructed.

The experiment was carried out with water titration method (Acharya et al., 2013). First of all,

a series of self-emulsifying systems consisting of oil/surfactant/co-surfactant were prepared with

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the weight ratio of oil to surfactant/co-surfactant (Smix) ranging in 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7,

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2:8 and 1:9, while the weight ratio of surfactant to co-surfactant (Km) was varied as 1:2, 1:1, 2:1.

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Then at each specific Km, the freshly prepared self-emulsifying systems were titrated with

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distilled water under magnetic stirring, respectively. The systems were examined visually and

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carefully after each addition of distilled water, until it changed from transparent to cloudy or

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opaque. At the end point, the addition quantity of distilled water was recorded and the percentage

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origin® 8.0 software.

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of each component was calculated out. ME region was clearly marked in phase diagrams with

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2.4. Simplex lattice experiment design

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In this study, an augmented simplex lattice experiment design was introduced to optimize the

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composition of ME formulation (Zhu et al., 2008). The simplex lattice experiment design for a

three-component system was represented by an equilateral triangle in two-dimensional space as

shown in Fig. 1. In this design, the total concentration of oil, Smix and water was kept constantly

100 % while the ratio of the three mixture factors was altered within the specified realm resulted

from pseudo-ternary phase diagrams. Fourteen batches of ropivacaine loaded ME were prepared

as shown in Table1. The distribution of them in the simplex lattice experiment model was marked

in Fig. 1: three vertexes (X1, X2, X3), each of them represented a formulation comprised of the maximum amount of one component, with other two components at minimum, three midpoints

between vertices (X1-2, X1-3, X2-3) which showed formulations containing the average of the

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minimum and maximum amounts of the two ingredients represented by two vertices, and the

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center point which represented a formulation containing one-third of each ingredient, while the

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points named CentEdge represent formulations containing two-thirds of one component and one-

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sixth of the other two, separately.

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Fig. 1

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In this study, the concentrations of oil (X1), Smix (X2) and water (X3) were opted as

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independent variables while equilibrium solubility of ropivacaine (Y1) and particle size (Y2) as

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dependent variables. The relationship between independent variables and dependent variable was

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analyzed with Design-Expert® software as well as the experiment arrangements displayed in Table

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1. In consideration of smaller particle size and higher solubilizing capacity, optimal formula

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would be recommended.

2.5. Preparation of ropivacaine loaded ME and ME-gel

According to the established formula, the corresponding amount of each component e.g. oil,

surfactant, and co-surfactant, were weighted out exactly and blended together adequately. Then

measured amount of ropivacaine was dissolved into the oily mixtures. Ultimately, weighted

quantity of distilled water was added into the system drop by drop under mild magnetic stirring for

5 min. The whole process was performed at ambient temperature.

Gel was prepared to enhance the adhesion of the optimized ME formulation, hence prolong its

retention time when applied to the skin. Firstly, plain Carbopol solution (1.5 %) was prepared by

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dissolving 1.5 g Carbopol 940 into 100 ml distilled water at 37 ℃ under magnetic stirring

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overnight. Ropivacaine loaded ME was mixed sufficiently with plain Carbopol solution in the

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ratio of 2:1(v/v). Then quantified triethanolamine was added into the mixture to neutralize with

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the acid residues on carbopol molecules, which led to gelatination.

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2.6. Particle size and zeta potential

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Both particle size and zeta potential are important indicators of the physical stability of a ME

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system. A small particle size indicates non-flocculation in the system while an appropriate zeta

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potential decreases flocculation phenomenon. The particle size and zeta potential of ropivacaine

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loaded ME formulation were measured by dynamic light scattering at 25°C using Malvern

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Zetasizer Nano-ZS (Malvern Instruments Ltd., Worcestershire, England). The particle size was

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expressed as average particle size of droplets in the system and polydispersity index (PI) which

indicated the width of the size distribution. All experiments were performed in triplicate.

2.7. Transmission electron microscopy (TEM)

The shape and surface morphology of ropivacaine loaded ME were observed under

transmission electron microscope (JEM-100CXII, Japan). Prior to the observation, the ME sample

was adsorbed to a copper grid with films and then stained with 2% (w/v) phosphotungstic acid for

30 s followed by drying at ambient temperature.

2.8. Ex vivo permeation study

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2.8.1 Preparation of skin

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Skin permeation study was reviewed and approved by the Institutional Animal Ethics

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Committee. Male Kunming mice weighing 22 ± 2 g were purchased from Experimental Animal

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Center of Shandong University (Shandong, China). The mice were housed in cages and supported

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with standard laboratory diet and water. Before the experiment, the mice were on fasting

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overnight. The abdominal skin was excised surgically after mice were sacrificed by cervical

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dislocation and the hair on the skin was shaved with electrical shaver. Then the subcutaneous

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tissues of skin were removed. The obtained skin was washed with normal saline and inspected for

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integrity before further use (Fouad et al., 2013; Ustundag Okur et al., 2011).

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2.8.2. Permeation experiment

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In order to evaluate the permeability of the prepared formulation, the ex vivo permeation study

was conducted with Franz diffusion cell apparatus and excised mice skin as permeable model

(Fouad et al., 2013; Yuan et al., 2006). The skin samples, possessing an effective diffusion area of 3.14 cm2, were tightly mounted to the donor compartment with stratum corneum side up. The

receptor compartment was filled with 15 ml of normal saline containing 30 % PEG 400 to

maintain sink condition. The whole system was kept at 37±1 ℃ and magnetic stirring at 600 rpm throughout the entire process.

Briefly, 1 g of ropivacaine loaded MEs to be tested or the optimized drug-loaded ME-gel (1g

of ropivacaine loaded ME mixed with 1.5 % carbopol solution at the volume ratio of 2:1), all

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containing an equal amount of drug e.g. 6 mg, was given to the stratum corneum side of the skin,

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respectively. Afterwards, the donor compartment was sealed with paraffin film to prevent water

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evaporation from the system. For each experiment, 0.5 ml of the sample was taken out from the

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receptor compartment at pre-determined time and replenished instantly with an equal volume of

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fresh receiver medium. All samples were treated with 1 ml of methanol, vortexed for 2 min and

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then filtered through 0.22 µm membrane filters before HPLC analysis. The cumulative amount of

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ropivacaine that permeated through the excised skin (Qn) was calculated based on the reported

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method (Vitorino et al., 2014; Zhu et al., 2009; Zhu et al., 2008).

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2.9. Histopathology study

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Histological examination of skin samples, with or without formulations treatment, was

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conducted to evaluate the mechanism of drug permeation through skin (Chaudhary et al., 2013).

Mice abdominal skin samples were treated with NS (as control group), the optimized ME and ME-

gel for 12h separately on the Franz diffusion cells under the same conditions as ex vivo permeation

study. Afterwards, skin samples were washed with NS and inspected for the integrity. After a

sufficient fixation in formalin solution, skin samples were dehydrated with ethanol and immersed

in dimethyl benzene for transparentizing. And then the skin samples were embedded in paraffin

and cut vertically into slices. Finally, the samples were stained with hematoxylin and eosin (H&E)

and then observed under light microscope.

2.10. Skin irritation study

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The skin irritation study was performed using three Wistar rats weighing 290~300g. The

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abdominal side of the rats was shaved clearly with electrical shaver and was divided into four

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regions prior to the test (Azeem et al., 2009). 50µl of the optimized ME (Ⅲ) and ME-gel (Ⅳ)

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were separately applied to an approximately area of 1 cm2 in order to test whether they show any

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irritation to rat skin or not. An equal volume of xylene (Ⅰ) was selected to show a standard

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irritant (Chatterjee et al., 2005) while normal saline (NS, Ⅱ) was selected as negative control. The

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development of signs of irritation, e.g. erythema was monitored for 24 h. Then all samples applied

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to the four regions were removed and mildly washed with N.S. The application sites were

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examined visually for erythema, which was repeated for 2 consecutive days.

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Chatterjee et al (2005) reported that the application of xylene on rat skin could induce a slight

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erythema within 8 h, subsequently, steadily increase and reach the maximum score in 104 h. It

was presumed that xylene could penetrate rapidly through the epidermis and thereby induce

cytokine/chemokine synthesis which plays an important role in skin irritation responses, for

example, erythema (Kano and Sugibayashi, 2006). Herein, xylene was selected as the positive

control substance in this study.

2.11. Assessment of analgesic activity

Acetic acid-induced writhing test

was conducted to assess the analgesic activity of the

optimized ME and ME-gel (Goyal et al., 2013; Koster et al., 1959).15 male Kunming mice

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weighing 22 ± 2 g were randomly divided into three groups (n=5 for each group). ME formulation

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and ME-gel both containing 6 mg ropivacaine were applied to an area of approximately 3.14 cm2

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on the dorsal skin of mice. After 6 h of drug treatment, mice were intraperitoneally injected with

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0.6 % acetic acid solution (0.1ml/10g) and observed for the following 20 min, respectively, in

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order to count the number of writhes. The analgesic activity was evaluated with the inhibition rate

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of writhes which could be calculated with the following equation (Goyal et al., 2013).

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Inhibiton rate=(C-T)/C x 100%

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In the above equation, C represents the number of writhes of the control groups and T

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represents the number of writhes of treated groups.

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2.12. High performance liquid chromatography (HPLC) analysis

Ropivacaine was analyzed by reversed phase HPLC using Agilent 1200 series. The HPLC

system applied in this study included a reversed phase Hypersil BDS C18 column (5µm, 4.6mm*250mm) and a UV/visible dual wavelength detector. The mobile phase was a mixture of

acetonitrile and phosphate buffered solution (60:40, v/v) with 0.05% (v/v) triethylamine for

modifying peak shape, constantly flowing at 1 ml/min. Samples with an injection volume of 20 µl

were injected into the column and monitored at 225 nm. All operations were carried out at

ambient temperature. The analytical method of ropivacaine was self-developed.

2.13. Statistical data analysis

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All skin permeation experiments and skin irritation tests were repeated three times. Data were

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expressed as the mean value ± S.D. The statistical analysis of the data was carried out using one-

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way analysis of variance (ANOVA) with P < 0.05 as the minimal level of significance.

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3. Results and discussion

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3.1. Solubility study

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Equilibrium solubilities of ropivacaine in various vehicles were presented in Fig. 2. Labrasol®

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presented the highest solubility of ropivacaine (17.18mg/g) in the screening realm and was opted

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as the surfactant component of ME formulation. Capryol® 90 (92.11mg/g) and oleic acid

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(43.16mg/g) were selected for further investigations due to the highest solubilizing capacity of

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Capryol® 90 compared with other candidates and the intrinsic permeation enhancing effect of

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oleic acid with the property of enhancing fluidity of lipid composition of the stratum corneum (Zhao et al., 2006), while Transcutol® HP (36.29mg/g) and absolute ethanol (34.37mg/g) were

chosen as co-surfactants.

Fig.2

3.2. Construction of pseudo-ternary phase diagrams

On the basis of preliminary screening result of solubility study, 14 batches of self-emulsifying

system under diverse Km were prepared for emulsification test. It was found out that the system comprised of Capryol® 90 as oil phase, Labrasol® as surfactant and absolute ethanol as co-

surfactant showed the bigger area of ME domains (shown in Fig. 3), which indicated better

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miscible ability with water, compared with other systems examined. Further, the system under

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Km=1:2 (shown in Fig. 3C) was chosen as the best combination.

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In the ME system, surfactants and co-surfactants were added to lower the oil/water surface

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intension through their surface adsorption property and balance the whole system. Acting as co-

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surfactants, medium chain alcohols were commonly added to further reduce the surface intension

Fig. 3

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(Tenjarla, 1999) and in this study ethanol was opted as co-surfactant.

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3.3. Optimization of ME formulation

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According to Fig. 3C, the ranges of three independent variables were selected as follows: 10%

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- 35% (w/w) for oil (X1), 50% - 75% (w/w) for Smix (Km=1:2, X2), 15% - 40% (w/w) for water

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(X3). A simplex lattice experiment design containing 14 runs generated by Design-Expert® software and corresponding responses were presented in Table 1. Afterwards, analysis and

optimization of the obtained data were conducted with the aid of the software (Fouad et al., 2013).

In the process of Fit Summary, the regression calculations were carried out to fit all of the

polynomial models such as linear, quadratic, special cubic and full cubic polynomials to the

selected responses, which referred to solubility and particle size in the present study. It produced

statistics like P-values, lack of fit, and R-squared values for comparing the models so as to select

statistically significant models for both responses. After ANOVA analysis of the suggested models obtained from aforesaid process, the models that possessed low Std Dev, high R2 and

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Logit (Solubility) = Ln [(Solubility - 6.76) / (74.00 - Solubility)] =

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smallest PRESS values were built for solubility and particle size shown below, respectively.

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-297.59324 * Oil - 31.77298 * Smix + 27.80678 * Water + 639.25932 * Oil * Smix

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Size = -39.26474 * Oil + 221.2152 * Smix - 162.56074 * Water

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Table 1

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Based on the two equations, contour diagrams (Fig. 4A&B) were drawn to visually depict the

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correlation between responses and different ratios of oil, Smix and water. Taking both responses

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into consideration, ME formulations marked with dark gray domain in Fig. 4C presented a relative

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high solubility for ropivacaine and a smaller particle size. A high solubility warrants the drug

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loading capacity of the optimized formulation and a smaller particle size was supposed to promote

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drug release and transport through membranes (Acharya et al., 2013; Zhao et al., 2013). Thereby,

three ME compositions within the dark gray zone in Fig. 4C were recommended as follows: ME1

composed of X1, X2 and X3 values of 15%, 53%, and 32% respectively, ME2 of 18%, 54.5%, and 27.5%, and ME3 of 10%, 50%, and 40%. Moreover, the bias in Table 2 showed that the predicted

values of mean particle size and equilibrium solubility of MEs calculated by equations were close

to those obtained from the experiment, indicating the simplex lattice method can accurately

predict the experiment results.

Fig. 4

For further screening of optimal ME formulation, the recommended formulations were

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investigated for the purpose of revealing the key factors affecting drug penetration. Among the

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three formulations, ME2 possessed the highest drug solubility and biggest particle size, while the

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lowest drug solubility and smallest particle size for ME3, and ME1 with medium values on both

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(details shown in Table 2). Ex vivo permeation studies were conducted to compare the skin

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permeation ability among formulations of ME1, ME2 and ME3. Cumulative amount of

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ropivavaine (Qn) permeated percutaneously were depicted in Fig. 5. ME1 displayed the highest

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Qn after 12 h (Q12) among the three recommended formulations in the light of preliminary

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optimization by the simplex lattice experiment, while there was no statistical significant difference

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between ME2 and ME3 (P > 0.05).

Table 2

Fig. 5

Particle size, components of ME formulation and drug solubility are key factors influencing

drug penetration. The smaller the particle size is, the larger the total surface area of the ME system

contacting with skin surface is. Herein, the small particle size provides more opportunities for

drug transferring into the skin (Khurana et al., 2013). Moreover, absolute ethanol in ME system

plays the role of permeation enhancers (El Maghraby, 2008; Zhang and Michniak-Kohn, 2011;

Zhao et al., 2006) as well as co-surfactant. The mechanism of ethanol working as a permeation

enhancer lies in extracting large amounts of lipid composition of stratum corneum and thus

temporarily enhancing the permeability of skin, which was verified through Differential Scanning

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Calorimetry (DSC) studies and Fourier Transform Infrared Spectroscopy (FTIRS) (Azeem et al., ®

®

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2012; Fang et al., 2008; Vaddi et al., 2002). It was reported that both Labrasol and Capryol

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90 had penetration enhancing effect on the skin (Hathout and El-Shafeey, 2012; Ogiso

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et al., 1996). Particularly, capryol® 90 can be incorporated into the stratum corneum

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increasing its fluidity and permeability and thus facilitating the passage of liquid

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disperse systems, like microemulsions (Ogiso et al., 1996). On the other hand, due to

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the high percentage components of surfactant and co-surfactant, microemulsions

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could significantly decrease the skin barrier function and consequently its electrical

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resistance, hence result in the high permeation fluxes (Hathout and Nasr, 2013).

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Additionally, it was reported that increasing the drug solubility of ME was also an effective

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method to enhance the skin permeation rate (Hathout et al., 2010; Zhao et al., 2006). Higher drug

solubility in ME guaranteed a higher drug loading dose, which could lead to a higher

concentration grade as power to enhance the permeation potential. The permeation ability of ME

might be balanced by the combined effects of particle size, drug solubility and components which

played the role of permeation enhancers of ME. Therefore, ME1 comprised of 15% Capryol® 90, 53% Smix (Labrasol® mixed with absolute ethanol, Km=1:2), and 32% water, was selected as the

optimal formulation for further evaluation.

3.4. Physicochemical evaluation of drug loaded ME

The solubility of ropivacaine in the optimal ME formulation was 20.38 mg/g, which

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represented a good drug loading capacity. The particle size was 58.79 ± 0.185 nm, and zeta

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potential -19.63 mV.

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The morphology of the optimal ME was observed using transmission electron microscope. ME

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appeared to be uniformly spherical. The approximate mean particle size of ME droplets obtained

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from TEM was around 50 nm to 70 nm, which was in accordance with result of the dynamic light

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scattering measurement.

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3.5. Effect of gel

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The optimal ME formulation was incorporated into carbopol gel and their percutaneous

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permeation behavior was investigated, depicted in Fig.5. Relative to ropivacaine loaded ME, a

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significant decrease (P < 0.05) in Q12 was observed after ME was incorporated into carbopol gel.

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More in-depth, the lag time (tlag) for prepared ropivacaine loaded ME-gel was found to be 3.83 ± 0.24 h, which was significantly higher (P < 0.05) than that of ME (1.77 ± 0.55 h). In a word, ME-

gel showed a sustained permeation of ropivacaine, compared to ME. The result is in accordance

with these of Jana S et al (2014) and Das B et al (2013). This phenomenon could be elucidated

with the release retarding effect of the polymeric matrix, mainly due to the increased viscosity

stemming from carbopol gelation (Chen et al., 2007; Zhu et al., 2009).

3.6. Histopathology study

The effect of formulations, including ME and ME-gel, on mice skin was examined with light

microscope. Microphotographs of H&E stained skin samples were shown in Fig. 6. In the control

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group (Fig. 6A), stratum corneum (SC), the outmost layer of skin, appeared to be an integral

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structure with tight connections between cells. Some skin flakes could be observed around the SC

SC

surface, which might be ascribed to skin hydration during NS treatment for 12 h (Chaudhary et al.,

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2013). Compared with the control group, SC of mice skin in both tested groups (Fig. 6B, C)

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became markedly thinner. Moreover, SC layers shown in Fig. 6B&C were loose, anomalistic and

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showed large cell gaps between each other. It was mainly owed to the ME compositions,

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especially ethanol (Ustundag Okur et al., 2011; Vitorino et al., 2014), with powerful function of

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extracting large amounts of lipids from SC, which also explained the skin permeation mechanism.

Fig. 6

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Overall, both ropivacaine loaded ME and ME-gel are effective for transdermal delivery.

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3.7. Skin irritation study

Rat skin irritation study was performed to estimate the potential irritant effect of the optimized

ME and ME-gel, while normal saline served as negative control and xylene as positive control.

After 24 h of the application, an obvious visible erythema was observed in region Ⅱ treated with

xylene and no signs for skin irritation were detected in other regions (Fig. 7). This study

demonstrated that neither the optimized ME nor ME-gel caused any irritation to rat skin, which

was in an agreement with the conclusion obtained from histopathology study.

Fig. 7

3.8. Assessment of analgesic activity

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Acetic acid-induced writhing test raised by Koster et al (1959) was widely employed by a lot

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of researchers to evaluate the analgesic activity of potential analgesics, especially for measuring

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peripheral analgesic activity (Alvarenga et al., 2013; Goyal et al., 2013; Matera et al., 2014;

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Shankarananth et al., 2007).

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Results of acetic acid-induced writhing experiment were summarized in Table 3. The average

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number of writhes of ME and ME-gel groups were 3.4 and 14.8, with the corresponding inhibition

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rate of 94.33 % and 75.33 %, respectively. Both ME and ME-gel presented a remarkable

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inhibition of writhing response induced by injection of acetic acid (P < 0.01), which indicated an

EP

effective analgesic activity. Furthermore, compared to ME-gel, ME showed a statistically

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significant better analgesic effect (P < 0.05). It was mainly ascribed to the delayed drug release

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from gel, which was in accordance with conclusions obtained in permeation study.

Table 3

In the histopathology study, it was confirmed that ropivacaine loaded ME could decrease the

barrier of SC so that drug molecules could permeate across the skin by passing through the

numerous cavities presented on the surface of SC and afterwards into the systemic circulation to

result in this remarkable analgesic effect. Furthermore, it was concluded that transdermal

administration of drugs loaded ME had a sustained and enhanced systemic absorption (Al Abood

et al., 2013; Gannu et al., 2010; Zhao et al., 2006).

4. Conclusion

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In the present study, ropivacaine loaded ME and ME-gel formulations were successfully

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developed and evaluated for transdermal delivery. Pseudo-ternary phase diagrams were explored

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to depict the ME regions for screening of the ME ingredients. And the ME formulation was

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optimized with a simplex lattice experiment design. Then the physiochemical properties were

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characterized to show the intact spherical morphology of the optimal ME with the average particle

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size of 58.79 nm. In ex vivo permeation study, ME showed a significant higher cumulative

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amount of ropivacaine permeated after 12 h application than ME-gel. The skin histopathology

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study showed that the microstructure of skin surface was significantly changed after the treatment

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of ME formulation, which might be the reason for the enhanced drug penetration. In addition, skin

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irritation study demonstrated that ME and ME-gel could not cause any irritation responses. And

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both ME and ME-gel presented a remarkable analgesic activity. It can be concluded from the

results that ME is a potential carrier for transdermal delivery of ropivacaine.

Acknowledgements

This work is supported by grants from Shanghai Municipality Science and Technology

Commission (12nm0500700, 11DZ1971400) and the National Nature Science Foundation

(No.81171766, No.81373896) and the Natural Science Foundation of Shandong Province, China

(No.ZR2011HM026).

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X3: water 0.192 0.192 0.275 0.233 0.275 0.150 0.150 0.317 0.400 0.150 0.150 0.400 0.233 0.150

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X2: Smix 0.667 0.542 0.500 0.583 0.625 0.500 0.750 0.542 0.500 0.625 0.500 0.500 0.583 0.750

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X1: oil 0.142 0.267 0.225 0.183 0.100 0.350 0.100 0.142 0.100 0.225 0.350 0.100 0.183 0.100

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Formulations 1 2 3 4 5 6 7 8 9 10 11 12 13 14

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Table 1 The formulation designed based on simplex lattice model and the response results Y1: solubility/mg·g-1 58.07 63.32 6.76 42.30 18.26 7.56 21.64 35.10 12.78 73.99 7.66 13.41 41.27 21.64

Y2: size /nm 168.8 115.1 84.7 63.1 63.8 65.3 65.3 29.6 51.3 76.4 65.3 51.3 63.1 138.5

Table 2 Three ME formulations recommended by Design Expert® software and validation

Formulation

Experimental value 19.54 33.26 11.67

Bias % -16.45 -4.57 -6.77

Predicted particle size /nm 59.74 69.00 41.80

Experimental value 58.79 61.66 46.30

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ME1 ME2 ME3

Predicted Solubility /mg·g-1 16.33 31.74 10.88

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Bias (%) = (Predicted value − Experimental value/Experimental value)

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×

Bias % 1.62 11.90 9.72

Table 3 Analgesic activity of optimized ME formulation and ME-based carbopol gel on acetic acid-induced writhing. Groups

Number of writhes (mean ± SEM) 60.0 ± 13.6 3.4 ± 2.7 * 14.8 ± 2.6 *,**

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Control ME ME-gel

Inhibition rate (%) 94.33 75.33

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1**P < 0.05, ME vs ME-gel group. 00

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* P < 0.01 compared with control group.

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Data were expressed as mean ± SEM.

Figure caption: Fig. 1. Equilateral triangle representing simplex lattice design. Fig. 2. The solubility of ropivacaine in oils, surfactants and cosurfactants. (n=3) Fig. 3. Pseudo-ternary phase diagrams of ME containing Capryol® 90 as

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oil phase, Labrasol® as surfactant, absolute ethanol as co-surfactant (A, B,

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C), or Transcutol HP as co-surfactant (D, E, F) and water.

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Fig. 4. Contour plot of the effect of independent variables on the

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(A) solubility of drug in ME formulations, (B) droplet size of ME and

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(C) the domain of the optimal ME formulations located.

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Fig. 5. In vitro permeation profiles of ropivacaine from three

a

ME1 > ME2 (P < 0.05), b ME1 > ME3 (P < 0.05), c P = 0.085, no

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Note:

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recommended ME formulations and the optimized ME-gel.

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significant difference between ME2 and ME3, d P <0.05. (n=3)

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Fig. 6. Photomicrographs of sections of mice skin treated with (A) N.S.,

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(B) optimized ME formulation and (C) ME-based carbopol gel (400×). Fig. 7. Photograph of the abdomen of Wistar rat showing signs for skin irritation. Various region was applied with different formulations as

following : (Ⅰ) Normal saline, (Ⅱ) Xylene, (Ⅲ) ME, (Ⅳ) ME-based

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gel.

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(B) Km=1:1

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(D) Km=1:2

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(C) Km=1:2

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(A) Km=2:1

(E) Km=1:1

(F) Km=2:1 Fig. 3

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