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Drug Development and Industrial Pharmacy

ISSN: 0363-9045 (Print) 1520-5762 (Online) Journal homepage: http://www.tandfonline.com/loi/iddi20

Formulation and Rheological Evaluation ofEthosome-loaded Carbopol Hydrogel forTransdermal Application

Shashank Jain, Niketkumar Patel, Parshotam Madan & Senshang Lin

To cite this article: Shashank Jain, Niketkumar Patel, Parshotam Madan & SenshangLin (2016): Formulation and Rheological Evaluation of Ethosome-loaded CarbopolHydrogel for Transdermal Application, Drug Development and Industrial Pharmacy, DOI:10.3109/03639045.2015.1132227

To link to this article: http://dx.doi.org/10.3109/03639045.2015.1132227

Accepted author version posted online: 04Jan 2016.

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Just Accepted by Drug Development and Industrial Pharmacy

Formulation and Rheological Evaluation of Ethosome-loaded Carbopol Hydrogel for Transdermal Application

Shashank Jain, Niketkumar Patel, Parshotam Madan, and Senshang Lin

doi: 10.3109/03639045.2015.1132227

Abstract

Objective: To select a suitable ethosome-loaded Carbopol hydrogel formulation, specifically tailored for transdermal application that exhibits (i) plastic flow with yield stress of approximately 50-80 Pa at low polymer concentration, (ii) relatively frequency independent elastic (G’) and viscous (G”) properties, and (iii) thermal stability.

Method: Carbopol (C71, C934, C941, C971 or C974) hydrogels were prepared by dispersing Carbopol in distilled water followed neutralization by sodium hydroxide. The effects of Carbopol grade, Carbopol concentration, ethosome addition and temperature on flow (yield stress and viscosity) and viscoelastic (G’ and G”) properties of Carbopol hydrogel were evaluated. Based on the aforementioned rheological properties evaluated, suitable ethosome-loaded Carbopol hydrogel was selected. In-

vitro permeation studies of diclofenac using rat skin were further conducted on ethosome-loaded Carbopol hydrogel along with diclofenac-loaded ethosomal formulation as control.

Results: Based on preliminary screening, C934, C971 and C974 grades were selected and further evaluated for flow and viscoelastic properties. It was observed that ethosome-loaded C974 hydrogel at concentration of 0.50% and 0.75% w/w, respectively, demonstrated acceptable plastic flow with distinct yield stress and a frequency independent G’ and G”. Furthermore, the flow and viscoelastic properties were maintained at the 4°C, 25°C and 32°C. The results from in-vitro skin permeation studies indicate that ethosome-loaded C974 hydrogel at 0.5% w/w polymer concentration exhibited similar skin permeation as that of ethosomal formulation.

Conclusion: The results indicate that suitable rheological properties of C974 could facilitate in achieving desired skin permeation of diclofenac while acting as an efficient carrier system for ethosomal vesicles.

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Formulation and Rheological Evaluation of Ethosome-loaded Carbopol Hydrogel for

Transdermal Application

Running Head: Rheological properties of ethosome-loaded Carbopol hydrogel

Shashank Jain, Niketkumar Patel, Parshotam Madan, and Senshang Lin*

College of Pharmacy and Health Sciences

St. John’s University, Queens, NY, USA

*Corresponding Author

Senshang Lin, Ph.D.

8000 Utopia Parkway, Queens, NY 11439, USA

Tel: (001) (718) 990 5344

Fax: (001) (718) 990 1877

E-mail: linse@stjohns.edu

Keywords: Yield stress, viscosity, viscoelastic modulus, elastic modulus, lipid based vesicles, skin

permeation

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Abstract

Objective: To select a suitable ethosome-loaded Carbopol hydrogel formulation, specifically tailored

for transdermal application that exhibits (i) plastic flow with yield stress of approximately 50-80 Pa at

low polymer concentration, (ii) relatively frequency independent elastic (G’) and viscous (G”)

properties, and (iii) thermal stability.

Method: Carbopol (C71, C934, C941, C971 or C974) hydrogels were prepared by dispersing

Carbopol in distilled water followed neutralization by sodium hydroxide. The effects of Carbopol

grade, Carbopol concentration, ethosome addition and temperature on flow (yield stress and viscosity)

and viscoelastic (G’ and G”) properties of Carbopol hydrogel were evaluated. Based on the

aforementioned rheological properties evaluated, suitable ethosome-loaded Carbopol hydrogel was

selected. In-vitro permeation studies of diclofenac using rat skin were further conducted on ethosome-

loaded Carbopol hydrogel along with diclofenac-loaded ethosomal formulation as control.

Results: Based on preliminary screening, C934, C971 and C974 grades were selected and further

evaluated for flow and viscoelastic properties. It was observed that ethosome-loaded C974 hydrogel at

concentration of 0.50% and 0.75% w/w, respectively, demonstrated acceptable plastic flow with

distinct yield stress and a frequency independent G’ and G”. Furthermore, the flow and viscoelastic

properties were maintained at the 4°C, 25°C and 32°C. The results from in-vitro skin permeation

studies indicate that ethosome-loaded C974 hydrogel at 0.5% w/w polymer concentration exhibited

similar skin permeation as that of ethosomal formulation.

Conclusion: The results indicate that suitable rheological properties of C974 could facilitate in

achieving desired skin permeation of diclofenac while acting as an efficient carrier system for

ethosomal vesicles. JUST A

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Introduction

Colloidal lipid-based vesicles, such as liposome, transferosome, and nanosome, have shown

promising results as carrier systems in delivering drugs through transdermal route. Moreover,

ethosome, an elastic lipid vesicle, has recently emerged as a new delivery system that could deliver

drugs more effectively and efficiently to the deeper skin tissues as compared to other colloidal lipid-

based vesicles.

Similar to liposome, ethosome is composed of distinct lipid bilayer that encapsulates aqueous

phase. However, unlike liposome that contains water as aqueous phase, ethosome contains hydro-

ethanolic solution (10-40%). The inclusion of ethanol enhances the drug delivery to deeper skin layer

by promoting reversible partial fluidization of lipophilic structure of the skin. Also, ethanol improves

the elasticity and reduces the size of vesicles that can consequently contribute to the skin permeation

enhancement of ethosome1. Furthermore, ethanol could act as a co-solvent to improve the entrapment

efficiency of lipophilic drugs in the ethosome1,2

.

However, colloidal lipid-based vesicles, like ethosome, are mainly prepared as a dispersion

form and are therefore typically incorporated in a carrier (e.g. iontophoretic system, transdermal

patch, ointment base, etc.) to prolong their skin retention time during the application3-6

. In this

regards, hydrogels have been utilized successfully as carrier for colloidal dispersions during

transdermal application7-9

. Because of their cross-linked gel network, hydrogels can effectively

prolong skin retention of the colloidal dispersions7. Furthermore, hydrogels are highly porous which

facilitate the loading of lipid vesicles into the gel matrix and their subsequent release from the gel10

.

They are also easy to apply on the surface of the skin resulting in high patient compliance.

Hydrogels are typically classified as non-Newtonian system. A non-Newtonian system

exhibits non-linear relationship between stress and shear (or deformation) rate. The flow behavior of

non-Newtonian system can either be plastic, Bingham or pseudoplastic flow. However, for

transdermal application, plastic flow is most desirable for hydrogels11

. A hydrogel with plastic flow is

characterized by its yield stress and viscosity. The yield stress of hydrogel represents a resistance that

needs to be overcome for a rested material to flow. Therefore, the hydrogel would not drip from

fingers (rest position) until applied on the affected area, with a pressure exceeding the yield stress,

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where upon it could then easily flow and spread on the skin surface12

. Ideally, hydrogel having yield

stress value of approximately 50-80 Pa is considered suitable for transdermal application12

.

Furthermore, viscosity of the hydrogel is more closely related to the diffusion of drug due to its

relationship with diffusion coefficient of drug13

. Therefore, yield stress and viscosity are important

rheological parameters to understand the flow behavior of the hydrogel to be applied in transdermal

route.

Although yield stress and viscosity provide useful information about the flow behavior of the

carrier system, both parameters are particularly not comprehensible tools to understand the

mechanical or structural properties of non-Newtonian system like hydrogels14

. The parameters that

define the structure of hydrogels include elastic modulus (storage, G’) and viscous modulus (loss,

G”). In rheological terms, G’ represents the energy stored and recovered per cycle of deformation. In

other words, it is a driving force for reformation of structure after the deformation of a material has

occurred under applied stress. On the other hand, G” is a measure of the energy lost per cycle and

reflects the liquid-like component12

. For enhancing skin retention during transdermal application, the

hydrogel should be viscoelastic (i.e. the magnitude of G’ should be greater than that of G” over wide

frequency range)12

. In addition, G’ and G” should be relatively independent of the applied

frequency12,15

.

Typically, hydrogels can be prepared using synthetic (e.g. carbomer and hydroxypropyl

methylcellulose) and natural (e.g. xanthan gum and guar gum) polymers. However, synthetic

polymers are more commonly used than natural polymers. Among various synthetic polymers,

carbomer (Carbopol) is the most commonly used because of its superior physical and rheological

properties16

. In addition, Carbopol has no reported skin irritation and stability issue with lipid

vesicles8,10,17

. Many studies have successfully shown the effectiveness of Carbopol as a carrier for

lipid vesicles for transdermal application8,9,18

. Furthermore, electron paramagnetic resonance study has

elicited effective transport of liposome from the Carbopol to the deeper skin19

.

As aforementioned, rheological properties of the Carbopol hydrogels are ideal for the

effective delivery of ethosome via transdermal route. However, despite the importance of

understanding the rheological properties of Carbopol hydrogels, literature provides limited

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information on the effect of various formulation variables of colloidal lipid-based vesicles on

rheological properties of Carbopol hydrogels, especially on viscoelastic properties (G’ and G”). Most

importantly, available studies in the literature are not specifically focused on transdermal route, which

requires special considerations during tailoring and optimization of rheological properties of Carbopol

hydrogels. Furthermore, though few research studies are available in the literature on liposome-loaded

Carbopol hydrogel, there is no comprehensive rheological information available on ethosome-loaded

Carbopol hydrogel8,18

. Since ethosome contains ethanol, it could play crucial role in modulating the

rheological properties of Carbopol hydrogels by affecting the polymer crosslinking. Also, the

inclusion of ethanol in ethosome decreases the transition temperature of lipid, rendering it temperature

sensitive1. Therefore, in the present investigation, effects of Carbopol grade, Carbopol concentration,

ethosome addition, and temperature on the rheological properties of Carbopol hydrogels were

evaluated. Based on these evaluations, the objective was to select a suitable transdermal ethosome-

loaded Carbopol hydrogel formulation that (i) exhibits plastic flow with yield stress of approximately

50-80 Pa at low polymer concentration, (ii) is relatively frequency independent G’ and G” (especially

G’) with G’ exceeding G” over wide frequency range, and (iii) possesses thermal stability12

.

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Materials and Methods

Materials

Carbopol® (C71, C934, C941, C971 or C974), diclofenac sodium, cholesterol, ethanol and

triethanolamine were purchased from Sigma Chemicals (St. Louis, MO). Soya phosphatidylcholine

(99% pure) was purchased from Avanti Polar Lipids (Alabaster, Al). All chemicals were analytical

grade and used as received.

Carbopol hydrogel preparation

Carbopol hydrogel (without ethosome) formulations were prepared in the manner described in

the literature12

. Briefly, the required quantity of Carbopol was weighed and dispersed in distilled

water. The dispersed mixture was then stirred at 800 rpm for 30 min to form a homogeneous

hydrogel. The formed un-neutralized hydrogel was then neutralized by drop wise addition of

triethanolamine till the pH reaching 7.4 ± 0.5. The entrapped air bubbles were removed by keeping

the formed hydrogel in a vacuum oven for 2 h at room temperature.

Evaluation of rheological properties of Carbopol hydrogel

Effect of Carbopol grade

The effect of Carbopol grade (C71, C934, C941, C971, and C974) on the rheological

properties Carbopol hydrogel was studied at two polymer concentration levels (0.25% and 1% w/w)

which represent lowest and highest polymer concentration utilized for hydrogel preparation. Carbopol

grade that exhibited relatively more satisfactory flow and the viscoelastic properties (i.e. posses

plastic behavior with frequency independent G’ and G” with G’ > G” over entire frequency range)

was selected for further investigation.

Effect of Carbopol concentration

The degree of polymer crosslinking and consequently the rheological properties of hydrogel

are strongly dependent on the polymer concentration used in the investigation20

. Typically, Carbopol

concentration used for hydrogel preparation ranges from 0.25% to 2% w/w21

. However, specifically

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for transdermal application of ethosome vesicles, higher polymer concentration is undesirable. At

higher concentration, there will be more polymer crosslinking that can hinder the movement of

ethosome vesicles from the hydrogel to the skin surface. Therefore, for transdermal application of

ethosome vesicles, appropriate polymer concentration range is from 0.25% to 1% w/w20,22

. To identify

the suitable Carbopol concentration, the rheological properties of selected Carbopol grade (screened

from previous study) were studied at 0.25%, 0.5%, 0.75% and 1% w/w concentrations, respectively.

Based on this study, the lowest Carbopol concentration at which the Carbopol hydrogel formulations

exhibit plastic behavior and have sufficient mechanical strength (frequency independent G’ and G”

with G’ > G”) were selected for the inclusion of ethosome formulation.

Effect of addition of ethosomal formulation

Based on the results from previous study, selected Carbopol grade and its concentration were

used to incorporate diclofenac-loaded ethosomal formulation. To prepare ethosome-loaded Carbopol

hydrogel, ethosomal formulation EO (i.e. soy phosphatidylcholine:cholesterol of 88.4:11.6 w/w,

ethanol concentration of 22.9% w/v, and diclofenac concentration of 1% w/v; an optimized

formulation from previous reported studies) was added to the neutralized Carbopol hydrogel obtained

earlier by mixing it at 200 rpm for 5 min1. The rheological properties of Carbopol hydrogel before and

after addition of ethosome dispersion were evaluated and compared.

Effect of temperature on ethosome-loaded Carbopol hydrogel

Since the ethosomes are composed of temperature sensitive soy phosphatidylcholine and

ethanol, the rheological properties of ethosome-loaded Carbopol hydrogels could be drastically

affected by change in temperature. Therefore, effect of temperature on ethosome-loaded hydrogels

was studied from 4°C to 45°C to cover the storage temperature (~ 4°C), room temperature (~ 25°C),

and skin temperature (~ 32°C) conditions to which these ethosome-loaded hydrogels might be

exposed23,24

. Furthermore, flow and viscoelastic properties of ethosome-loaded hydrogels were

studied at individual temperature of 4°C, 25°C and 32°C, respectively.

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Rheological properties of Carbopol hydrogel

Dynamic-Hybrid Rheometer (TA Instruments, New Castle, DE) along with cone plate fixture

(40 mm cone diameter and cone angle of 1.99°) was used to perform rheological examination of

Carbopol hydrogels. Temperature was controlled by peltier heating system. The TRIOS software

(version 2.6.1, TA Instruments, New Castle, DE) of Dynamic-Hybrid Rheometer was used for data

analysis.

Prior to the rheological experiment, Carbopol hydrogel formulation was placed on the peltier

plate just sufficient enough to cover the cone at the top of the plate. The Carbopol hydrogel on the

peltier plate was then equilibrated at a controlled temperature (depending upon the experimental

condition) for 3 min both before and after lowering the cone over the peltier plate. After lowering the

cone, the gap between cone and plate was trimmed with “trim gap” option in the rheometer to provide

thin uniform film of the Carbopol hydrogel under the cone. The excess Carbopol hydrogel on the

sides of the cone was removed carefully without disturbing the film between plate and cone.

Following tests were then performed for the hydrogel formulations.

Preliminary study: strain sweep test

Strain sweep test was performed as a preliminary study to determine the linear viscoelastic

region (LVR) and critical strain (γc, the endpoint of LVR). These parameters are important since

ideally rheological studies of the hydrogel formulations must be performed in linear viscoelastic

region. In non-linear viscoelastic region (i.e. region above γc), the mechanical structure of the

hydrogel is damaged due to excessive oscillatory strain resulting in the inability to obtain the reliable

information about inter-molecular and inter-particle forces in the material. Therefore, all rheological

studies (flow ramp, frequency sweep, and temperature ramp) of the hydrogel formulations must be

performed at an oscillatory strain lower than γc (i.e. in the linear viscoelastic region) to assure that the

structure of the hydrogel is not damaged. In order to determine LVR and γc, G’ was measured as a

function of oscillatory strain (0.03% - 50%) at a fixed frequency of 1 Hz. All experiments were

performed in triplicates. The LVR was defined as the region where G’ was independent of oscillatory

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strain in accordance to previous studies25

. The γc value was considered as a strain value at which the

elastic modulus drops by 10% of the G’ value of the most linear portion of LVR26

.

Continuous flow ramp test

The flow properties (plastic behavior, yield stress, and viscosity) were determined by

continuous flow ramp experiment. The hydrogels were subjected to stepwise increase in shear rate

from 1-200 s-1

and the corresponding stress was measured at 4°C, 25°C and 32°C, respectively. All

experiments were performed in triplicates. Flow curves obtained were further analyzed by Herchel-

bulkley model to determine flow behavior, yield stress, and viscosity (equation 1).

- 0 = cs (1)

where, τ is shear stress, τ0 is yield stress, γ is shear rate and η0 and s are viscosity and plasticity

descriptors, respectively.

Frequency sweep test

Examination of viscoelastic properties (elastic modulus and viscous modulus) was performed

by frequency sweep experiment at 4°C, 25°C and 32°C, respectively. The elastic (G’) and viscous

(G”) modulus were measured with a stepwise increase in angular frequency from 1 to 200 rad/s in the

linear viscoelastic region. All experiments were performed in triplicates. The obtained mechanical

spectra of log G’ and log G” versus log frequency was investigate to determine the frequency

dependence of these moduli.

Temperature ramp test

In temperature ramp test, elastic modulus and viscosity of the hydrogels were measured as a

function of temperature increasing from 4°C to 45°C at a rate of 5 ◦C/min at constant frequency (1

Hz) and oscillatory strain (1%).

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Evaluation of in-vitro skin permeation behavior of ethosome-loaded Carbopol hydrogel

Franz permeation cells (Crown Glass Co., Inc., Somerville, NJ) having receptor compartment

volume of 4.8 ml with effective surface area of 0.63 cm2, and synchronous driving assembly, were

used to evaluate the in-vitro skin permeation of diclofenac from ethosome-loaded Carbopol hydrogel

formulation. Male Sprague Dawley (SD) rats (200-250 g) were obtained from Charles River

Laboratories Inc., (Wilmington, MA). The protocol for performing animal studies was approved by

the Animal Care Committee at St. John’s University (Queens, NY). Briefly, SD rats were sacrificed

by carbon dioxide asphyxiation. A section of full-thickness abdominal skin was excised from the fresh

carcasses of animal, after removing the abdominal hairs. The skin was then thoroughly washed with

pH 7.4 phosphate buffer solution (PBS) and subcutaneous fat was carefully removed. The receptor

compartment of Franz permeation cells was then filled with degassed pH 7.4 PBS, equilibrated to 32

± 1°C (skin temperature) by surrounding water jacket and continuously stirred with a magnetic bar.

The skin sections obtained were mounted between donor and receptor compartments of the Franz

permeation cells with the stratum corneum side facing towards the donor compartment.

Rheological properties of the Carbopol hydrogel can affect the behavior of skin permeation of

ethosomal vesicles. Therefore, the selected ethosome-loaded Carbopol hydrogel along with the

ethosomal formulation EO in dispersion form (as a control) were studied. Ethosomal dispersion

(formulation EO) along with its corresponding Carbopol hydrogel (formulations EN50EO and

EN75EO) with the total drug concentration of 1% w/v (500 l), were then applied onto the donor

compartments. For Carbopol hydrogel formulations, the hydrogel was gently rubbed in circular

motion ten times by index finger on the skin surface. At pre-determined time (1, 2, 3, 4, 5, 6, 8 and 24

h), samples (300 l each) were taken from the receptor compartment and diclofenac concentration

was analyzed by the HPLC method. The withdrawn volume from the receptor compartment was

replaced with fresh PBS (maintained at 32 ± 1°C) to maintain constant volume for sink condition. the

drug concentrations measured were corrected for the dilution factor. Care was taken to avoid

introduction of air bubbles beneath the dermis during the entire course of the experiments. All

experiments were performed in triplicates.

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Analytical methodology of diclofenac

The diclofenac assay was performed based on the reported literature with slight modification

to avoid any interference by solvents27

. Briefly, the method employed a reverse-phase HPLC (HP1100

series, Agilent Technologies, Wilmington, DE) with a 4.6 mm × 250 mm C-18 column (Macherey-

Nagel, Bethelehem, PA). The mobile phase consisted of 60% v/v acetonitrile and 40% v/v of 0.5%

v/v acetic acid. A flow rate of 2 ml/min was set and diclofenac content was detected at UV

wavelength of 280 nm. The retention time of diclofenac was 4.5 min. The area under the peak was

used to calculate the concentration of diclofenac and linearity over the concentrations ranging

between 1 µg/ml and 500 µg/ml was evaluated. The peak area was observed to increase linearly with

respect to the increase in diclofenac concentrations with correlation coefficient (r2) of 0.9987.

Statistical analysis

The PRISM software (GraphPad, version 5, San Diego, CA) was utilized for student’s t test

or one-way ANOVA to evaluate statistical significance (P < 0.05) of the obtained data.

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Results and discussion

Carbopol hydrogel preparation

The prepared Carbopol hydrogel formulations, irrespective of Carbopol grade and

concentration, exhibited good spreadability and were free from agglomerates. The rheological

properties of these hydrogel formulations were then screened to select suitable Carbopol grade and

concentration that could be utilized for incorporating ethosomal dispersion for transdermal

application. As described above for transdermal application, a suitable ethosome-loaded hydrogel

carrier must exhibit (i) plastic flow with yield stress of approximately 50-80 Pa at low polymer

concentration, (ii) relatively frequency independent G’ and G” (especially G’) with G’ exceeding G”

over wide frequency range, and (iii) thermal stability. Once these criteria were met, in-vitro skin

permeation study of diclofenac from ethosome-loaded Carbopol hydrogel was performed and

compared with ethosomal dispersion (as control), to evaluate the feasibility of Carbopol hydrogel as

carrier system on the skin permeation of diclofenac-loaded ethosome.

Rheological properties of Carbopol hydrogel

Preliminary study: strain sweep test

As mentioned earlier, above critical strain (c), the structure of Carbopol hydrogel could be

disrupted under oscillatory strain resulting in unreliable rheological information. The strain sweep test

was performed as a preliminary study to determine the c. It is important to acknowledge that since

the c is strongly related to mechanical structure of the hydrogel, this study could also provide useful

information in identifying the factors that could affect the rheological properties of Carbopol

hydrogels.

As outlined in Table 1, the length of LVR indicated by γc values was predominantly affected

by neutralization process, Carbopol grade, Carbopol concentration, and addition of ethosome. It was

observed that at each respective concentration, neutralized Carbopol hydrogel exhibited higher critical

strain value as compared to the un-neutralized Carbopol hydrogel, indicating that neutralization of the

hydrogels could be an essential for a hydrogel to achieve maximum mechanical strength. This

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observation could be attributed to the fact that maximum polymer crosslinking for Carbopol hydrogel

occurred at pH 7.4. Therefore, all Carbopol hydrogel formulations were neutralized during their

preparation for further evaluations. However, at each respective concentration, both neutralized and

un-neutralized C71 as well as C941 hydrogel formulations showed a lower γc value as compared to

other Carbopol hydrogel formulations (Table 1). The results indicate that C71 and C941 hydrogels

were unable to maintain their hydrogel structure even at a very low oscillatory strain (< 1% in most

cases), reflecting the poor cross-linked gel network of these polymer grades. Therefore, C71 and C941

hydrogel formulations were excluded from the further rheological studies. Furthermore, since length

of LVR for C934, C971 and C974 hydrogel formulations was significantly affected by grade of

Carbopol and its concentration, comprehensive rheological studies are required to evaluate the

suitability of these Carbopol hydrogel formulations. In this regards, since the LVR was maintained at

oscillatory strain of 1% for neutralized C934, C971 and C974 hydrogel formulations, irrespective of

Carbopol concentration, all further rheological studies were performed keeping a constant oscillatory

strain of 1%. In order to further evaluate suitable Carbopol hydrogel for transdermal application,

several variables on flow and viscoelastic properties, systematic and comprehensive rheological

studies were performed on C934, C971 and C974 hydrogel formulations and described below.

Effect of Carbopol grade

As indicated in the Figure 1, all hydrogel formulations, irrespective of Carbopol grade,

exhibited non-Newtonian plastic flow. This suggests that the hydrogel network demonstrated

resistance to an external force before it started flowing. However, despite the similarity in their flow

behavior, the magnitude of yield stress and viscosity were significantly affected by the change in

Carbopol grade (Table 2). At respective concentration levels (0.25% or 1% w/w), the yield stress and

the viscosity increased in order of C971 < C934 C974. This might be due to the fact that C971 has

low polymer crosslinking as compared to C934 and C974. Also, C934 and C974 have similar cross-

linked structure among the polymers studied25

. Furthermore, as illustrated in Figure 2, though the

viscoelastic parameters (G’ and G”) at respective concentrations followed a similar trend (i.e. C971 <

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C934 C974) as yield stress and viscosity, both C971 (formulations DN25 and DN100) and C934

(formulations BN25 and BN100) hydrogels showed a relatively more frequency dependent

mechanical spectra over entire frequency range as compared to C974 hydrogel (formulations EN25

and EN100), indicated by uneven linear region with respect to angular frequency. As aforementioned,

a frequency independent mechanical spectrum over entire frequency range could be an essential

criterion for suitability of the Carbopol hydrogel for transdermal application. Therefore, based on the

results of this study, C934 and C971 polymers were discontinued from the current investigation. The

C974 polymer, on the other hand, was selected for further studies as it exhibited plastic flow with

distinct yield stress and a relatively frequency independent G’ and G” with G’ exceeding G” over

wide frequency range.

Effect of Carbopol concentration

After the selection of Carbopol grade (i.e. C974), it is important to understand the effect of

Carbopol concentration on rheological properties. At higher concentration level, there will be more

crosslinking of the polymer chain that can hinder the movement of ethosome vesicles from the

hydrogel to the skin surface. Therefore, for incorporation of ethosome in C974 hydrogel, the goal was

to select the lowest possible concentration level of C974 that shows plastic flow and has sufficient

mechanical strength (frequency independent G’ and G” with G’ > G”). Several C974 hydrogel

formulations were studied at 0.25% (formulation EN25), 0.50% (formulation EN50), 0.75%

(formulation EN75) and 1% w/w (formulation EN100) polymer concentration. The flow curves of

C974 hydrogels at various concentration levels are shown in Figure 1. It was observed that all

formulations, irrespective of concentration, exhibited non-Newtonian plastic flow with a distinct yield

stress. For C974 hydrogel formulations, both yield stress and viscosity at concentration 0.50%, 0.75%

and 1% w/w, respectively, were observed to be statistically insignificant (Table 2). Similarly, though

all the formulations exhibited viscoelastic behavior (G’ > G”) with the magnitude of G’ and G”

increasing with increase in C974 concentration, the mechanical spectra of formulations EN50, EN75

and EN100 hydrogels were relatively similar in comparison to formulation EN25 (Figure 3). Based on

the results, it could be inferred that the flow (yield stress and viscosity) and viscoelastic (G’ and G”)

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properties of C974 hydrogel at concentration of 0.50% (formulation EN50), 0.75% (formulation

EN75), and 1% w/w (formulation EN100) were similar. Nonetheless, as the goal of the investigation

was to select the lowest Carbopol concentration that exhibits plastic flow with sufficient mechanical

strength, formulations EN50 and EN75 were selected for incorporating ethosome dispersion and

subjected to further evaluation.

Effect of addition of ethosome formulation

Based on the previous studies, C974 hydrogel formulations with Carbopol concentrations of

0.50% and 0.75% w/w were selected to incorporate the ethosomal formulation EO. However, addition

of ethosome dispersion in Carbopol hydrogel could affect the flow and viscoelastic behavior of

Carbopol hydrogels. Therefore, flow ramp and frequency sweep tests of formulations EN50EO (0.50%

w/w) and EN75EO (0.75% w/w) were performed, to understand the effect of ethosome incorporation

on the hydrogel properties at room temperature.

The results of flow ramp test indicate that both Carbopol hydrogel formulations maintained

non-Newtonian plastic flow even after addition of ethosome (Figure 4). Furthermore, at respective

concentration, addition of ethosome formulation reduced the yield stress and viscosity values (Table

3). Similarly, at respective concentration, the magnitude of G’ and G” decreased after addition of

ethosome formulation (Figure 5), without affecting the viscoelastic nature of the hydrogel (G’ > G”

with G’ exhibiting relatively frequency independent mechanical spectra). The decrease in the

magnitude of flow (yield stress and viscosity) and viscoelastic (G’ and G”) parameters after addition

of ethosome (formulation EO) could be attributed to the presence of ethanol in ethosome formulation,

which increases the polymer chain mobility resulting in decrease of flow and viscoelastic

parameters28

. In particular, the decrease in the yield stress is desirable in this investigation because

formulations EN50 and EN75 (C974 hydrogel without ethosome) showed a yield stress of

approximately 120-170 Pa, which is not suitable for transdermal application. Dilution effect due to

ethosome addition facilitates the decrease in yield stress to the desirable limit (around 50 to 80 Pa). It

is also important to acknowledge that even after addition of ethosome, C974 at 0.50 % and 0.75%

w/w, respectively, was able to maintain both plastic flow and viscoelastic behavior with sufficient

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mechanical strength. This reiterates that C974 polymer at concentration of 0.50 % and 0.75% w/w,

respectively, could be suitable for incorporating ethosomal formulation.

Effect of temperature on ethosome-loaded Carbopol hydrogel

The results of temperature ramp test indicate that viscosity of formulations EN50EO and

EN75EO decreased with increase in temperature from 4°C to 45°C, which was expected due to loosing

of polymer entanglement at higher temperatures (Figure 6). However, irrespective of concentration,

both moduli (G’ and G”) were relatively frequency independent with G’ >G” (Figure 7), indicating

that decrease in viscosity (or loss of polymer entanglement) was not significant enough to affect the

overall mechanical structure of Carbopol hydrogel. Furthermore, the flow ramp and frequency sweep

tests were performed at refrigerated (~ 4°C), controlled room (~ 25°C) and skin (~ 32°C) temperature

conditions. It was observed that the plastic flow of formulations EN50 and EN75 was maintained

even after addition of ethosome formulation (formulations EN50EO and EN75EO) at all temperatures

studied (Figure 8). The results indicate that the flow properties (i.e. yield stress and viscosity) were

unaffected by change in temperature (Table 4). Moreover, the yield stress of formulations EN50EO

and EN75EO, irrespective of temperature, was close to the desired yield stress range (50 to 80 Pa) for

transdermal application (Table 4). Similarly, irrespective of temperature, the G’ and G” exhibited a

frequency independent mechanical spectra with G’ exceeding G” moduli (Figure 8). The results of

these studies (temperature ramp, flow ramp and frequency sweep) conclude that formulations

EN50EO and EN75EO are thermally stables.

Based on the results obtained in this investigation, formulations EN50EO and EN75EO

achieved the desired goal for transdermal application to (i) exhibit plastic flow with yield stress of

approximately 50-80 Pa at low polymer concentration (Figure 4 and Table 2), (ii) be frequency

independent G’ and G” (especially G’) with G’ exceeding G” over wide frequency range (Figure 5),

and (iii) have good temperature stability at storage, room and skin temperature (Figures 6, 7, 8 and

Table 4). As a result, both formulations were carried forward for in-vitro skin permeation study.

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Evaluation of in-vitro skin permeation behavior of ethosome-loaded Carbopol hydrogel

In-vitro skin permeation of diclofenac from formulations EN50EO and EN75EO was studied

and compared with formulation EO dispersion. It was observed that formulations EN50EO and EN75EO

showed a slightly lower skin permeation behavior as compared to formulation EO (Figure 9). This

could be attributed to cross-linked nature of Carbopol hydrogel, which might affect the skin

permeation of ethosome. Although the permeation profile of EN50EO and EN75EO formulation was

not significantly different, formulations EN50EO was observed slightly higher skin permeation, which

could be due to the lower level of Carbopol concentration and consequently the lower cross-linked

polymer network.

Conclusion

In the present study, the rheological properties of Carbopol hydrogel was studied for its

feasibility to incorporate ethosomal formulation and then specifically tailored for transdermal

application. Effects of Carbopol grade, Carbopol concentration, ethosome addition, and temperature

on the rheological properties of Carbopol hydrogel were evaluated. Based on the results of these

studies, Carbopol grade 974 at a concentration of 0.50% w/w was able to exhibit suitable rheological

properties and in-vitro skin permeation of diclofenac as a carrier system of ethosome for transdermal

application.

Acknowledgments

The authors acknowledge St. John’s University for providing financial assistance and

research facilities to carry out this research.

Declaration of interest

The authors declare no conflict of interest (monetary or otherwise) in conducting this

research. The authors alone are responsible for the content and writing of the paper.

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Table 1. Effect of Carbopol grade, neutralization, Carbopol concentration and ethosome addition of

Carbopol hydrogel formulations on critical strain (c) obtained from strain sweep test performed

at 25°C (n = 3).

Carbopol grade Neutralizationa

Carbopol

concentration

(%)

Ethosome

additionb

Critical strain

(%)

C71

U 0.25 - 0.3

1 - 1.3

N 0.25 - 1.1

1 - 1.6

C934

U 0.25 - 1.5

1 - 1.9

N 0.25 - 6.2

1 - 8.8

C941

U

0.25 - 0.2

1 - 0.6

N 0.25 - 1.5

1 - 1.8

C971

U

0.25 - 0.4

1 - 1.6

N 0.25 - 4.8

1 - 8.3

C974 U 0.25 - 1.5

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1 - 1.8

N

0.25 - 7.1

1% 2.2

1

- 8.5

1% 2.1

a. N: neutralized, U: un-neutralized

b Formulation EO was used.

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Table 2. Effect of Carbopol grade and its concentration of Carbopol hydrogel formulations on yield

stress and viscosity obtained from flow ramp test performed at 25°C (n = 3).

Formulation codea

Yield stress

(Pa ± S.D)

Viscosity

(Pa.s ± S.D.)

C934

BN25 174.2 ± 25.9 188.3 ± 10.0

BN100 243.9 ± 17.7 255.4 ± 14.9

C971

DN25 11.5 ± 1.5 37.3 ± 0.6

DN100 19.5 ± 2.8 58.3 ± 0.8

C974

EN25 153.0 ± 27.8 176.9 ± 13.2

EN50 176.9 ± 43.9 192.5 ± 13.1

EN75 124.5 ± 34.6 219.7 ± 46.2

EN100 234.8 ± 48.8 262.6 ± 5.8

a. N: neutralized, B: C934, C: C941, D: C971, E: C974, last two-digit: Carbopol

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Table 3. Effect of ethosome addition to C974 hydrogel formulations having polymer concentration of

0.50 and 0.75% w/w, respectively, on yield stress and viscosity obtained from flow ramp test

performed at 25°C (n = 3).

Carbopol

concentration

(w/w)

Yield stress (Pa ± S.D.) Viscosity (Pa.s ± S.D.)

Before addition

After addition

Before addition

After addition

0.5% 176.9 ± 43.9 93.1 ± 6.2 192.5 ± 13.1 95.0 ± 13.2

0.75% 124.5 ± 34.6 85.1 ± 1.8 219.7 ± 46.2 97.3 ± 0.7

Table 4. Effect of temperature on yield stress and viscosity of ethosome-loaded C974 formulations

having polymer concentration of 0.50 and 0.75% w/w, respectively, following flow ramp test

performed at 4°C, 25°C and 32°C (n = 3).

Formulation

codea

Storage

(~ 4°C)

Controlled room

(~ 25°C)

Skin

(~32°C)

Yield

stress (Pa

± S.D.)

Viscosity

(Pa.s ± S.D.)

Yield

stress (Pa ±

S.D.)

Viscosity

(Pa.s ± S.D.)

Yield

stress (Pa

± S.D.)

Viscosity (Pa.s

± S.D.)

EN50EO

105.3 ±

20.6 119.5 ± 2.8 93.1 ± 6.2 95.0 ± 13.2 72.2 ±1.8 77.1 ± 0.8

EN75EO

93.7 ±

3.3 130.0 ± 9.9 85.1 ± 1.8 97.3 ± 0.7

80.6 ±

6.0 100.8 ± 14.3

a. N: neutralized, E: C974 and EO: Ethosome formulation JU

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Figures Legend

Figure 1. Effect of Carbopol grade and its concentration of C934 (green), C971 (red) and C974 (blue)

hydrogel formulations on the flow behavior obtained from flow ramp test performed at

25°C (n = 3).

Figure 2. Effect of Carbopol grade of (a) C934 (green), (b) C971 (red) and (c) C974 (blue) hydrogel

formulations at respective polymer concentration of 0.50 and 1% w/w, respectively, on

elastic (G’, solid line) and viscous (G”, dotted line) modulus obtained from frequency

sweep test performed at 25°C (n = 3).

Figure 3. Effect of Carbopol concentration of C974 hydrogel formulations on elastic (G’, solid line)

and viscous (G”, dotted line) modulus obtained from frequency sweep test performed at

25°C (n = 3).

Figure 4. Effect of addition of ethosome to C974 hydrogel at polymer concentration of 0.50 % and

0.75% w/w, respectively, on the flow behavior following flow ramp test performed at

25°C (n = 3).

Figure 5. Effect of addition of ethosome to Carbopol hydrogel at a polymer concentration of 0.50%

and 0.75% w/w, respectively, on elastic (G’, solid line) and viscous (G”, dotted line)

following frequency sweep and frequency sweep tests performed at 25°C (n = 3).

Figure 6. Effect of temperature on the viscosity of formulations EN50EO and EN75EO at polymer

concentration of 0.50 and 0.75% w/w, respectively, following temperature ramp test

performed over 4°C to 45°C (n = 3).

Figure 7. Effect of temperature on the elastic and viscous modulus of formulations EN50EO and

EN75EO at polymer concentration of 0.50 and 0.75% w/w, respectively, following ramp

test performed over 4°C to 45°C, (n = 3).

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Figure 8. Effect of temperature on the flow behavior of (a) formulation EN50EO and (b) formulation

EN75EO following flow ramp test as well as on the elastic and viscous modulus of (c)

formulation EN50EO and (d) formulation EN75EO following frequency sweep test

performed at 4°C, 25°C and 32°C, respectively (n = 3).

Figure 9. In-vitro skin permeation profile of diclofenac from ethosome-loaded Carbopol hydrogel

(formulations EN50EO and EN75EO) along with formulation EO as control through SD rat

skin (n = 3).

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Fig. 1

0 50 100 150 2000

300

600

900

1200

1500

1800

2100

Shear rate (s-1)

Str

es

s (P

a ±

S.D

.)

EN25

EN100

DN25

DN100

BN25

BN100EN50EN75

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Fig. 2 (a)

(b)

(c)

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Fig. 3

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Fig. 4

Shear rate (s-1)

Str

es

s (P

a ±

S.D

.)

0 50 100 150 2000

500

1000

1500

2000

EN50

EN50EO

EN75

EN75EO

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Fig. 5

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Fig. 6

5 10 15 20 25 30 35 40 45

20

40

60

80

100

Temperature (°C)

Vis

co

sity

(Pa

.s ±

S.D

.)

EN50EOEN75EO

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Fig. 7

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Fig. 8

Shear rate (s-1)

Str

es

s (P

a ±

S.D

.)

0 50 100 150 2000

500

1000

1500

EN75EO (4°C)

EN75EO (25°C)

EN75EO (32°C)

Shear rate (s-1)

Str

es

s (P

a ±

S.D

.)

0 50 100 150 2000

500

1000

1500

EN50EO (4°C)

EN50EO (25°C)

EN50EO (37 C)

(a)

(d)(c)

(b)

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Fig. 9

0 4 8 12 16 20 240

50

100

150

200

250

300

350

400 Formulation Eo

Formulation EN75Eo

Formulation EN50Eo

Time (h)

Cum

ula

tive

drug

per

mea

ted

(µg/

cm2

)

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