Journal of Experimental Nanoscience

ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/tjen20

Formulation and evaluation of niosomes-based

chlorpheniramine gel for the treatment of mild to
moderate skin allergy

Urooj Afreen, Khairi Mustafa Fahelelbom, Syed Nisar Hussain Shah, Akram
Ashames, Uzma Almas, Shujaat Ali Khan, Muhammad Arfat Yameen, Naveed
Nisar, Muhammad Hassham Hassan Bin Asad & Ghulam Murtaza

To cite this article: Urooj Afreen, Khairi Mustafa Fahelelbom, Syed Nisar Hussain Shah,
Akram Ashames, Uzma Almas, Shujaat Ali Khan, Muhammad Arfat Yameen, Naveed Nisar,
Muhammad Hassham Hassan Bin Asad & Ghulam Murtaza (2022) Formulation and evaluation of
niosomes-based chlorpheniramine gel for the treatment of mild to moderate skin allergy, Journal of
Experimental Nanoscience, 17:1, 467-495, DOI: 10.1080/17458080.2022.2094915

To link to this article: https://doi.org/10.1080/17458080.2022.2094915

© 2022 The Author(s). Published by Informa

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Group.

Published online: 19 Jul 2022.

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JOURNAL OF EXPERIMENTAL NANOSCIENCE
2022, VOL. 17, NO. 1, 467–495
https://doi.org/10.1080/17458080.2022.2094915

Formulation and evaluation of niosomes-based

chlorpheniramine gel for the treatment of mild
to moderate skin allergy
Urooj Afreena, Khairi Mustafa Fahelelbomb, Syed Nisar Hussain Shaha,
Akram Ashamesc,d, Uzma Almase, Shujaat Ali Khanf, Muhammad Arfat Yameenf,
Naveed Nisara, Muhammad Hassham Hassan Bin Asadf and Ghulam Murtazag
a
Department of Pharmaceutics, BahauddinZakariya University, Multan, Pakistan; bDepartment of
Pharmaceutical Sciences, College of Pharmacy, Al Ain University, Al Ain, United Arab Emirates;
c
Medical and Bio-allied Health Sciences Research Centre, Ajman University, Ajman, UAE;
d
Department of Pharmaceutical Sciences, Ajman University, Ajman, UAE; eDepartment of
Dermatology, Bahawal Victoria Hospital, Bahawalpur, Pakistan; fDepartment of Pharmacy, COMSATS
University Islamabad, Abbottabad Campus, Pakistan; gDepartment of Pharmacy, COMSATS University
Islamabad, Lahore Campus, Lahore, Pakistan

ABSTRACT ARTICLE HISTORY

Purpose of present study was to develop eight formulations of Received 24 March 2022
chlorpheniramine (CPM) niosomes according to 23 factorial Accepted 21 June 2022
design, characterise on the basis of various evaluation tests, i.e.
KEYWORDS
in vitro drug release, SEM, FTIR, TGA and release kinetics, optimise
Factorial design; niosomal
the eight formulation on the basis in vitro drug release data, for- gel; In vitro drug release
mulate gel of optimised dispersion, and to perform in vivo and study; pharmacokinetic
histopathological study using gel of optimised dispersion on rab- study; histopatho-
bits. Here, N3 having low level of cholesterol and span-80 but logical study
high level of span-60(0.1:0.2:0.05) was selected as optimised dis-
persion of niosomes that showed highest drug release i.e. 88.25%
at pH 6 over 24 h of study and followed Korsmeyers-Peppas
release kinetics with Fickian diffusion mechanism. After applica-
tion of statistic by Analysis of variance (ANOVA) with 3D surface
plots construction, gel of optimised dispersion of CPM niosomes
was formulated, and evaluated by tests for i.e. viscosity,
Spreadability, Extrudibility, drug content, drug entrapment, stabil-
ity, SEM, FTIR, TGA, in vitro drug release, in vivo drug release fol-
lowing first order kinetics and histopathological study. Niosomal
gel of CPM ensured successful development using suitable com-
bination of non-ionic surfactants, and effective loading of drug for
targeted delivery of drug.

CONTACT Khairi Mustafa Fahelelbom [email protected] Department of Pharmaceutical Sciences,

College of Pharmacy, Al Ain University, Al Ain, United Arab Emirates; Ghulam Murtaza [email protected]
Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia.
ß 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/
licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
468 U. AFREEN ET AL.

1. Introduction
Transdermal delivery system (TDDS) ensures delivery of drug through transdermal route
of application that presents a unique non-invasive route of application, avoid gastrointes-
tinal tract passage, produce controlled effect maintaining sustained concentration of drug
in plasma for longer period of time, and hence produce high patient compliance [1,2]. It
has also been observed that transdermal route of application diminishes requirement for
intravenous route of application, due to lower levels of Cmax (peak plasma concentration
of drug) leading to lower dose associated side effects, i.e. both systemic hepatic first pass
metabolism, and pre-systemic side effects [3]. Other advantages of transdermal route are
to deliver not only hydrophilic drugs, but also lipophilic drugs directly into blood circula-
tion through skin, control amount of drug applied, control the area of application, release
kinetics and extended time of application [4]. Transdermal route of application also have
some disadvantages regarding skin irritation [5], lag time of drug absorption, onset of
action of drug, metabolism of drug in skin, and limited application dose [6]. Other disad-
vantage faced by transdermal route is low rate of turnover from transdermal products due
to barrier skin layer of dermis, i.e. the stratum corneum [7].
To avoid these disadvantages, to perform distinctive functions various dosage forms
have been developed, and used but gel of niosomes (semisolid formulations) was selected
as best dosage form, to encapsulate drug due to many reasons, i.e. gels melt at body tem-
perature being dried right after application on skin and gels are formulations that are
alcoholic based semisolid, viscous, non-greasy, easily speardable, easily extrudable, emolli-
ent, water soluble and highly compatible with excipients [8]. Niosomes based gel entrap
drug completely from surroundings and use for allergic diseases safely.
Basic component of gel is carbomer that is polymer of acrylic acid and forms hydrogel
due to hydration of carboxylic group present in structure when come in contact with
aqueous or alkaline solution [9]. Carbomers have widely been used during numerous pre-
vious research works [10] due to their unique characteristics, i.e. optimum viscosity even
at low concentration, good stability at heating, no effect on ageing, non-irritating and
non-supportive for growth of bacteria or fungi [11]. One of the most common polymer
used for topical drug delivery system is Carbopol that is synthetic,anionic polymer deriva-
tive of carbomer, and have ability to crosslink its chains to self-assemble into microgel
structure with permeation enhancement along with organic compound in gel formula, i.e.
polyethylene glycol (PEG) and propylene glycol (PG) [8].
Hypersensitivity developed due to allergen contact can proceed to systemic symptoms,
if not treated promptly. Inflammatory reaction initiated due to histamine in response to
antigen–antibody reaction after interaction and attachment of foreign antigen with anti-
bodies, produced from activated immune cells produced localised symptoms of itching,
erythema, pain, inflammation and blisters at site of contact but if not treated lead to gen-
eralised symptoms of fever, chills, sleep disturbances, allergic rhinitis, disturbed gastro-
intestinal motility and sleep disturbances [12].
Chlorpheniramine maleate (CPM) belonging to alkyl amine class has been widely used
to develop various dosage forms of topical drug delivery system due to its pharmacoki-
netic, physicochemical and pharmacodynamics characteristics suitable for transdermal
drug delivery [13]. CPM has been used for various mild to moderate acute condition of
inflammation and allergy, i.e. runny nose, sunburn, urticaria, pruritus, angioedema, cough
and insect bites symptoms [14]. Various dosage forms have been developed using CPM
i.e. syrups, tablets, capsules but their oral formulation produced side effects, i.e. dizziness,
muscular weakness, gastrointestinal disturbances and mild to moderate sedation to deep
sleep. CPM-based organogels based on span60/tween20 were developed as an alternative
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 469

dosage form avoiding oral route associated side effects where organogel having 21% span-
60, 3% tween-20, 5% menthol, 2% CPM and sunflower oil (to make 100 g) showed best
key results of physical properties along with good drug release and higher anti-inflamma-
tory effects [15]. Recently, topical skin therapy has obtained greater attention to treat der-
matological diseases due to targeted delivery of drug, reduced loss by systemic uptake
[16] with high efficacy, safety than systemic delivery, enhanced bioavailability and trans-
porting the drug to deeper and lower layers of skin, i.e. stratum corneum through various
carriers [17] where most feasible drug carriers were niosomes and liposomes [18].
Niosmes were preferred due to increased stability, decreased cost, ability to encapsulate
drugs with variety of physico-chemical properties [19], enhanced drug permeation rate,
release of drug in more sustained or controlled way, and ability to create drug depot.
Niosomes can be modified by controlling, ratio of hydrophilic and/or the hydrophobic
moiety and hydrated to form gel (hydrogel) using carbopol [20]. Niosomal gels are more
useful and advantageous than traditional semisolid dosage forms due to ability to have
long residence time, maintain sustained, higher concentration in skin and retain its rheo-
logical behaviour [19].
The present work was designed to develop and formulate CPM niosomes, optimise the
prepared niosomes and convert the optimised niosomes into gel based on low and high lev-
els of three variables, i.e. cholesterol, span 60 and span 80. Niosomal gel of CPM was formu-
lated to estimate feasibility of niosomal gel for topical delivery of CPM in rabbit model.

2. Materials and methods

Chlorpheniramine maleate (CPM) 99.99% purity (gifted by Pfizer Pvt. Ltd, Multan),
Cholesterol (Merck, Germany), Propylene glycol (PG), Polyethylene glycol (PEG-1000)
(Fluka, Germany), Methanol (HPLC grade), Carbopol-940 (polymer, China), dichlorome-
thane (Merck), PBS (pH 7.4), Double Distilled Water (Distillation Plant in Pharmacy
Department, BahauddinZakariya University, Multan). All reagents and chemicals of ana-
lytical grade were purchased and used during this research work.

2.1. Formulation of chlorpheniramine maleate loaded gel of niosomes

Design of experiment for preparation of CPM niosomes was 23 factorial design (Table 1).
A 23 full-factorial design was used to optimise the CPM noisomal gel by assessing the

Table 1. Experimental plan for niosomal dispersion formulation: (a) Three factorial design 23, and (b) Coded level
translation in actual units.
Coded factor levels Composition of factors (g) (Independent Variables)
(a) Formulation Codes A B C A (cholesterol) B (Span-60) C(Span-80)
N1 – – – 0.10 0.10 0.05
N2 þ – – 0.30 0.10 0.05
N3 – þ – 0.10 0.20 0.05
N4 þ þ – 0.30 0.20 0.05
N5 – – þ 0.10 0.10 0.10
N6 þ – þ 0.30 0.10 0.10
N7 – þ þ 0.10 0.20 0.10
N8 þ þ þ 0.30 0.20 0.10
(b) Coded levels – þ
A(Cholesterol) 0.10 0.30
B(Span-60) 0.10 0.20
C(Span-80) 0.05 0.10
470 U. AFREEN ET AL.

Table 2. Composition of CPM loaded niosomes displaying amount (g) of all factors used in formula to make 100 g
niosomal dispersion formulation.
Ingredients N1 N2 N3 N4 N5 N6 N7 N8
Cholesterol 0.10 0.30 0.10 0.30 0.10 0.30 0.10 0.30
Span-60 0.10 0.10 0.20 0.20 0.10 0.10 0.20 0.20
Span-80 0.05 0.05 0.05 0.05 0.10 0.10 0.10 0.10
DCM 10.0 10.0 10.0 10.0 10.0 10.0 10.0 10.0
CPM 2.0 2.0 2.0 2.0 2.0 2.0 2.0 2.0
PBS (pH 7.4) Q.S Q.S Q.S Q.S Q.S Q.S Q.S Q.S
DCM ¼ Dicholoromethane, CPM ¼ Chlorpheniramine maleate, PBS ¼ Phosphate buffer saline, Q.S. ¼Quantity sufficient
to make 100 g.

effect of independent variables (concentration of cholesterol, concentration of span-60

and span-80) at two levels of low and high on dependent variables (percentage yield, drug
content, drug loading, entrapment efficiency, size of vesicles, thermal stability of gel,
in vitro drug release and anti-inflammatory effect). The physical characteristics of the pre-
pared CPM loaded niosomal gel formulations were carried out by determining percentage
yield, drug content, drug loading, entrapment efficiency, size of vesicles, stability of gel,
in vitro drug release, release kinetics and anti-inflammatory effect [21].
Niosomal gel loaded with CPM was formulated using optimised dispersion of CPM-
loaded niosomes.
Eight formulations of niosomes were prepared by modified method of ether injection
introduced by Deamer and Bhangham in 1976.
Proper quantities (translated according to 23 three factorial design of experiment as in
Table 1) of cholesterol, span-60 and span-80 as mentioned in Table 2 were dissolved in
dichloromethane that resulted in organic phase.
This organic phase was injected slowly at rate of 0.25 ml/mint through 14-gauge needle
into PBS having CPM swirling at 500 rpm with thermostat temperature 55–65  C and pH
7.4 resulting in formation of niosomes loaded with CPM.
Niosomes were formulated due to difference in temperature between organic and phos-
phate buffer saline phase [22].
After formulation of dispersion and optimisation of dispersion of niosomes, gel of
optimised niosomal dispersion was prepared. First of all, 2% Carbopol-934 [23] 2.5 g was
dissolved in PG 25 g to form viscous solution and PEG 2 g was dissolved in methanol
(15 ml almost) to make thin solution with addition of N3 (optimised niosomal dispersion
of CPM) 50 g (give 10 mg/g of gel) in it. Thin solution was added to viscous solution at
once with continuous stirring at 55  C to form CPM loaded niosomal gel of pH 5.5–6
adjusted by dropwise Triethanolamine (TEA) under continuous stirring at temperature
55  C to get homogenous gel and finally stored in collapsible tubes for further study [24].
Concentration of drug CPM used in this niosomal gel formulation is 2 g (2000 mg) per
100 g of gel, larger as compared to total components in order to deliver 15–20 mg of CPM
from single application of CPM loaded niosomal gel, i.e. 0.1 g delivers 20 mg of CPM that is
standard dose of drug for therapeutic effect i.e. anti-inflammatory effect on allergic skin.

2.2. Physicochemical properties of CPM

The values of physicochemical properties of CPM are stated here: partition coefficient for
CPM 7.1, Log P 0.85, pH 4–5 and pKa 9.2, while BCS class is I [25].
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 471

2.3. Physical examination

The optimised niosomalgel of CPM was examined physically to determine its physical
state, uniformity, consistency, phase separation, colour, texture, presence of lumps and
homogeneity by pressing small quantity of gel between index finger and thumb.

2.4. Homogeneity
Optimised niosomal gel was examined for homogeneity by placing in glass container to
determine aggregate or lump [25].

2.5. pH determination
Average pH (n ¼ 3) of formulated dispersions of niosomes of CPM and niosomal gel of
Optimised CPM niosomal Dispersion at room temperature 25 ± 0.5  C by pH metre
(Digital, WTW, pH 526 Germany) with probe calibration each time before use and pH
adjustment up to 5.5–6 pH with Triethanolamine (TEA) [26].

2.6. Viscosity
Viscosity of optimised niosomal gel of CPM was determined by rotational digital viscom-
eter using N4 spindle at 12 rpm and room temperature [26].

2.7. Spreadability
Spreadability explains the coming out behaviour of gel from collapsible tube. Spreadability
of niosomal gel of CPM was determined using a wooden block apparatus (introduced by
Multimer et al.) consisting of fixed glass slide of 7.5 cm length on one end and movable
on other end that was tied to weight pan that was rolling on pulley set horizontally with
fixed glass slide. To perform test about 1 g of niosomal gel was placed between glass
slides, 1 kg weight was applied on slide for five minutes, then 60 g weight was added to
pan and two glass slides were separated (time in seconds was noted). Spreadability was
determined by formula:
M:L
S¼
T
where S¼spreadability in g cm/s, m¼ weight tied upper slide, t¼ time in seconds l¼
length of glass slide (7.5 cm) and w¼ weight tied to upper slide was (60 g). The process
was repeated three times to get average value of Spreadability (n ¼ 3) [27].

2.8. Extrudibility
Extrudibility test is simple test that determines flow ability and measures force required to
extrude the gel from aluminium tube using hardness tester. Extrudibility test was per-
formed by filling 5 g niosomal gel in lacquered aluminium collapsible tube, adjusting
plunger to hold tube with application of pressure of 1 kg/cm2 for 30 s on tube that would
extrude ribbon of study gel and calculating amount of extruded gel by calculating pressure
in grams. Procedure was repeated three times to get average value with standard deviation
(n ¼ 3) [26].
472 U. AFREEN ET AL.

Wt
Eb ¼
D
where Eb¼extrudability, Wt ¼ applied weight to extrude gel from tube (in g), D¼area
(in cm2).
Formula used to calculate extrudability was as stated below [28]:
Wg
Eb ¼
D
where Eb ¼ extrudability, W¼ Weight applied on tube to extrude gel from tube and D¼
Area (cm2).

2.9. Partition coefficient (KO/PB system) studies

Pinch of CPM was added in 5 ml of n-octanol and phosphate buffer and mixedby vigor-
ous shaking in separating funnel that was allowed to stand in vertical position in stand
for 24 h after which solvent was centrifuged for half hour or less at 2000 rpm and ana-
lysed at 265 nm by UV spectrophotometer [29]. Following formula was used (n ¼ 3) to
calculate KO/PB:
Partition coefficient of the drug (KP) ¼
Concentration of drug in organic phase
Partition coefficient of the drug ðKPÞ ¼
Concentration of drug in aqueous phase

3. Chemical examination
3.1. Determination of percentage yield
Percentage yield of all niosomal dispersions and optimised gel of CPM was calculated by
dividing measured weight of formulations with formula weight and multiplying with 100
[26] by following formula:
Total weight of CPM liposomes
Percentage practical yield ¼  100
Total Theoretical weight

3.2. Determination of percentage drug content and percentage drug loading

100 mg or 0.1 g of CPM niosomes or optimised CPM niosomal gel was sealed in cello-
phane membrane soaked in conical flask of water for 24 h with subsequent withdrawal of
1 ml sample to check absorbance then concentration in mg/ml using UV spectrophotom-
eter at 265 nm and formulas below [26]:
Drug conc: ðmg=100 mlÞ
Drug Content ¼
Wt: of CPM niosomal gel=dispersion taken
 Theoretical mass of formulation
Actual amount of drug content
Drug Content ð%Þ ¼  100
Theoretical amount of drug content
Formula stated below determines capacity of niosomal gel/dispersion to load CPM
[30].
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 473

Amount of drug in formulation

Drug Loading ð%Þ ¼  100
Amount of niosomal formulation

3.3. Drug content uniformity

Content uniformity was determined by repeating process three times to take three sam-
ples (n ¼ 3) from top, middle and bottom points of glass container of niosomal gel [31].

3.4. Drug entrapment efficiency of CPM niosomal gel

Free drug is always present in niosomal formulation of CPM as no dispersion/gel has
entrapped total drug. To separate free drug 0.1 g of niosomal gel of CPM was hydrated
with 10 ml PBS and sonicated in bath sonicator for 10 min. Dispersion of CPM niosomes
was centrifuged at 10,000 rpm at 25 ± 0.1  C for half hour and produced supernatant solu-
tion that was filtered to separate free drug. Supernatant solution was analysed at 265 nm
by UV spectrophotometer [21]. Results were used to estimate EE (%) by using formula:

Ct Cf
EE ð%Þ ¼  100
Ct

where Ct ¼ concentration of total drug and Cf ¼ Concentration of free drug.

Entrapped drug
Entrapment efficiency ð%Þ ¼  100
Total drug added

3.5. Fourier Transform infrared spectroscopy (FTIR)

FTIR spectrophotometer (Perkin Elmer-spectrum RX-I, Lamba USA), based on KBr disc
method under hydraulic press by applying 600 kg/cm2 pressure was used to obtain spectra
of formulated CPM niosomes, CPM niosomal gel and components of niosomes, i.e. chol-
esterol, span-60, span-80 and CPM individually. FTIR was used to investigate drug-ingre-
dients interaction, compatibility and structural features of samples [32].

3.6. Thermal gravimetric analysis

TGA was performed to estimate physical properties, compatibility between drug and
ingredients of formula of optimised niosomal dispersion, and gel by placing sample of gel
6.285 mg in aluminium pan under pressure of Nitrogen gas at rate 20.0 ml/min with heat-
ing at 40–400  C Perkin-Elmer thermal analyser. Heating under Nitrogen atmosphere pro-
duced sharp thermogram peaks due to initiation of reaction in pan [26].

3.7. Scanning electron microscopy (SEM)

Surface morphology, size, texture of vesicles of optimised niosomal dispersion of CPM,
and optimised niosomal gel of CPM were determined by Scanning electron microscopy
(SEM Hitachi S-3600 N Japan), using platinum sputter technique. SEM photographs of
niosomal dispersion (N3) were obtained [21].
474 U. AFREEN ET AL.

3.8. Stability studies and drug leakage study

Stability study was conducted to evaluate drug leakage, drug content, encapsulation effi-
ciency and other physical behaviour parameters of optimised niosomal gel of CPM. After
removal of free CPM niosomal dispersion was sealed in 10 ml sealed glass vial and nioso-
mal gel was sealed in 10 collapsible aluminium tubes (n ¼ 3), and kept at three different
temperatures, i.e. refrigeration temperature (2–8  C ± 2  C), ambient temperature
(20  C ± 2  C), elevated temperature (40  C ± 2  C) and relative humidity 75 ± 5% for
period of 3 months in stability chamber. Sample of 0.5 g from each was taken at preset
time interval, i.e. 1st day, 7th day, 2nd weeks, 1st month, 2nd month and 3rd month and
analysed for free drug content. Drug leakage was determined by comparing drug content
of niosomal formulations before and after storage. Procedure was followed by centrifuging
sample at 20,000 rpm for half hour to separate leaked CPM and get supernatant (having
free drug) evaluated spectrophotometrically at 265 nm by using formula:
a
Percentage drug leakage ¼  100
b
where a ¼ the amount of CPM measured in the supernatant (g); b ¼ the initial amount
of CPM entrapped in niosomes (g).
Samples of formulations were inspected visually to determine physical stability parame-
ters and evaluated for amount of free and entrapped drug to determine chemical stabil-
ity [20].

3.9. In vitro drug release study of niosomal gel of CPM

Dissolution profile of all niosomal dispersions of CPM (N1-N8) and optimised niosomal
gel of CPM was determined using USP apparatus Type II (paddle procedure) model no.
D15/68 (Digital instrument) having PBS pH 6 at 37.0 ± 0.5  C with speed 59 rpm [33].
Drug release from CPM niosomal dispersion/optimised niosomal gel of CPM was
determined by mounting Dialysis membrane (width 29.31 mm and average diameter
17.5 mm) in the USP apparatus type II (paddle). Dialysis membrane was soaked in freshly
prepared pure boiling distilled water for 12–24 h. Temperature of dissolution medium
outside glass dissolution bowl was maintained at 37 ± 1  C and speed of paddle rotation
was set at 100 rpm to rotate in dissolution medium of glass dissolution bowl (500 ml at
pH 6). Niosomal dispersions and Optimised niosomal gel of CPM were sealed in dialysis
membrane bag and attached to paddles. After starting apparatus 5 ml samples were taken
at preset interval by replacement with 5 ml PBS (temperature maintained at 37 ± 1  C) to
maintain constant volume of dissolution medium and analysed after dilution at 265 nm
by UV spectrophotometer by repeating process in triplicate n ¼ 3 [28]. Drug release (%)
was calculated by using following calibration curve equation for CPM, i.e.
Y ¼ 0:0217x þ 0:0414
where y¼absorbance, m¼slope, x¼concentration and b¼intercept. Whereas, the value of
R2 (Regression coefficient) was 0.9964 (values as per linearity curve of CPM).

3.10. Drug release kinetics study

Release kinetics were determined by using MS Excel 2010 with added DD Solver program
and applying release models, i.e. First order, Zero order, Hixon-Crowell, Higuchi and
Korsmeyer-Peppas models on drug release % data of CPM niosomal dispersions. All
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 475

models were applied individually to determine best fit model followed by niosomes of

CPM. Value of R2 (Regression analysis coefficient) of five models was used to determine
accuracy of best fit model and its value should be near to 1.00 8]. Value of Akaike
Information Criterion (A/C) of five models was used to determine validation of best fit
model [34]. Value of “n” was used to determine mechanism of drug release, i.e. if
n ¼ 0.45 mechanism is Fickian Diffusion, n lower than 0.45 but greater than 0.89 mechan-
ism is Non-Fickian Diffusion: Anomalous, and if n greater than 0.89 mechanism is
Erosion [28].

3.11. Optimisation of formulation

Design expert 7.0.0 was used to apply factorial design on in vitro drug release data to
optimise niosomal formulation of CPM that was further used for development and evalu-
ation of optimised niosomal gel of CPM [22].

4. In vivo study
4.1. Skin irritation study
Sample of about 1.00 g niosomal gel of CPM was applied in triplicate on back of hand
and forearm skin (3in2) of five human volunteers and graded for any type of erythema
and edoema for 3 h using Van-Abbe Dermal Scoring Criteria who stated that values
between 0 and 9 indicate that formulation applied is not an irritant to skin [26].

4.2. In vivo anti-inflammatory study

After approval from the Ethical Committee of Faculty of Pharmacy, B.Z. University
Multan for using rabbits for ex vivo, in vivo and histopathological studies. Six healthy rab-
bits of 2.5 ± 0.19 kg weight were selected by assuring that selected rabbits have not been
used for any laboratory experimental purpose for last 15 days, and not taken any medica-
tion for last six hours before starting study. Straight, broad and clearly seen skin area
(10 cm2) of dorsal side of rabbit above belly was selected, shaved and rubbed gently with
green chilli (Capsicum annum) for 1–2 min to induce inflammatory response. Optimised
niosomal gel of CPM was applied with custom made applicator on shaved area for 2 h,
i.e. 0.05 g of Niosomal gel (having CPM dose according to human dose, i.e. 10 mg) on
1 cm2 dorsal skin area. Inhibition of capsicum induced inflammatory response was used
as measure of in-vivo anti-inflammatory activity of CPM in form of its niosomal gel.

4.3. Pharmacokinetic (bioavailability) study

In vivo studies of optimised niosomal gel for pharmacokinetic study were performed to
determined drug uptake, clearance and release using rabbit model [35]. Blood samples
(5 cc) from marginal ear vein were withdrawn at preset time points, i.e. 0, 0.25, 0.5, 0.75,
1.0, 1.5, 2, 2.5, 3 and 4 h after application of gel on rabbit dorsal allergic skin (triplicate
n ¼ 3), and centrifuged for 25 min at 4000 rpm to obtain serum containing CPM (protein
bound) which was stored at 20  C until analysed. Serum was treated with de-proteinizer,
i.e. methanol (volume equal to serum volume), centrifuged for 25 min at 6000 rpm to sep-
arate protein, and was analysed by UV-spectrophotometer at 265 nm to determine con-
centration of drug in serum at different time intervals [21].
476 U. AFREEN ET AL.

Pharmacokinetic model by PK solver (Version 2) was applied to determine bioavail-

ability by calculating various pharmacokinetic parameters of niosomal gel of CPM using
Microsoft Excel, Windows Professional XP Version 2013 along with calculation of mean,
and standard deviation, i.e. time to reach peak serum concentration (tmax), Peak serum
concentration (Cmax), half-life (tmax), Volume of distribution (Vd), Clearance(Cl) and Area
under the plasma concentration time curve (AUC0-t and AUC0-a) [36].

4.4. Histopathological study

Histopathological method using histological techniques and optical microscope was used
to analyse numerous capsicum induced histopathological alterations in dorsal side pre,
and post (treatment) skin samples of rabbit. Procedure was performed sequentially, i.e.
rabbit skin tissues from normal, capsicum treated and gel treated areas were selected,
excised and used to prepare slides stained with H&E dye (haematoxylin and Eosin) to
locate, identify and demonstrate histopathological changes clearly. These slides were
viewed under high powers of optical microscope to obtain photo-microscopic images of
all slides to visualise whole skin tissue, i.e. Epidermis, Lamina propria, loose connective
tissues, all type cells, all glands, secretory vesicles, vascular tissues, basal layers, and to
confirm all histopathological alterations for detailed comparison of slides of various skin
tissues. Procedure was found to be similar to procedure used by Bozzatto (2013), and also
in accordance with histopathological method used by Javed (2018) to see histopathological
alterations in goat nasal membrane [26].
To perform histopathological studies 1 day after application of optimised CPM nioso-
mal gel rabbits were biopsied, biopsies of normal rabbit skin tissue, capsicum treated rab-
bit skin tissue, and niosomal gel treated rabbit skin tissue were collected for
histopathological studies [37]. Images were taken to capture, demonstrate all incidents
regarding capsicum induced inflammatory, vascular, and histological alterations from their
first appearance to treatment of symptoms. Green chilli (Capsicum annum) was used as
main allergen that damaged rabbit skin tissue mechanically with induction of inflamma-
tory responses on site of application.

4.5. Statistical analysis

To develop, niosomes and niosomes based gel of CPM, Microsoft Excel 2013 was used to
estimate values of mean and standard deviation with application of Analysis of variance
(ANOVA) (p < 0.05, Regression analysis to evaluate difference, and impact of various lev-
els of cholesterol, span-60 and span-80 (independent variables) on drug release (depend-
ent variable) [38].

5. Results and discussion

It was observed from current study that the independent variables (concentration of chol-
esterol, concentration of span-60 and span-80) greatly influenced the dependent variables
(percentage yield, drug content, drug loading, entrapment efficiency, size of vesicles, ther-
mal stability of gel, in vitro drug release and anti-inflammatory effect). A significant dif-
ference in all dependent variables was noticed using prepared CPM loaded niosomal gels,
which in turn influenced the CPM release from gel.
Niosomal dispersions of CPM were very light pale white (milky) in colour, clear with
no particular odour, and uniform in appearance. The percentage yield of CPM niosomes
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 477

Figure 1. (a) Optimised Niosomes (N3) 3D Surface plot of % Drug release response at y ¼ pH 6. (b) Optimised
Niosomes N3 interaction diagram.

was in a range of 95.04–98.51% while EE % results ranged between 87 and 96%

(N1 ¼ 94, N2 ¼ 87, N3 ¼ 96, N4 ¼ 90, N5 ¼ 95, N6 ¼ 89, N7 ¼ 96 and N8 ¼ 91) that indi-
cated its direct relation with amount of cholesterol and surfactant. In vitro drug release
study of 24 h was performed for CPM Niosomal formulation (N1-8). All the formula-
tions showed continuous and sustained release behaviour while selected formulation
(N3) showed more controlled but slowly enhancing release of CPM  40–50% at 4th
hour of study at pH 6 and at 24th hour study design with 12% amount of
drug remaining in niosomes for further hours after 24 h of application. As shown in
Figure 1a, b, it was clear from 3D surface graph that by decreasing amount of choles-
terol drug release increased in all formulations of Niosomes and by increasing amount
of cholesterol drug release was decreased. In case of span-60, by decreasing amount of
span-60, drug release was decreased but when amount of span-60 was increased drug
release was also increased. While in case of span-80, when amount of span-80 was
decreased drug release was decreased, and when amount of span-80 was increased drug
release was also increased.
478 U. AFREEN ET AL.

Figure 2. calibration (Linear regression) Curve at 265 nm (Phosphate buffer pH 6). In the stated equation
(Y ¼ 0.0217x þ 0.0414), Y is UV absorbance at 265 kmax, m is slope (0.0217), x is concentration (mg/ml) of CPM, b is
the intercept (0.0414), and R2 is correlation coefficient (0.9964).

5.1. Physical examination of niosomal dispersion of CPM

The visual inspection revealed that CPM niosomal dispersions were clear, transparent,
and showed good homogeneity with absence of lumps, aggregates or precipitates. The pH
of CPM niosomes was in range of 5.1 ± 0.1 to 5.8 ± 0.1 that in range of skin pH probably
producing, no skin irritation, and suitable for dermatological use. The particle size ana-
lysis ranged between 0.3 and 5.0 micrometer.

5.2. Calibration curve of CPM

CPM Linear regression curve displayed in Figure 2 was produced by plotting absorbance
at 265 nm against concentrations (mg/ml). The acquired linear regression equation was
y ¼ 0.0217x þ 0.0414, where R2 (regression coefficient) was 0.9964.

5.3. Solubility studies

The solubility of CPM was 681.51 ± 0.01 mg/ml in water, 731.51 ± 0.01 mg/ml in PBS and
542.15 ± 0.01 mg/ml in methanol, declaring that CPM was freely soluble in these solvents in an
order of PBS > Water > Methanol. These values were in accordance with the previous study
values [29], who studied microsponge based CPM gel where CPM solubility was as given here:
PBS (810.52 ± 3.8 mg/mL) > water (687.58 ± 2.9 mg/mL) > methanol (547.98 ± 2.3 mg/mL).

5.4. Partition coefficient (K O/PB) studies

The partition coefficient of CPM was 6.99 with logarithmic value (log P) equals 0.850
demonstrating good hydrophobicity of CPM, to formulate its topical dosage form. The
values were in accordance with the previous where CPM partition coefficient was 0.851
(log p ¼ 7.1) during the study of CPM transdermal patch [39].
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 479

Figure 3. FTIR peaks of a ¼ drug (CPM), b ¼ cholesterol, c ¼ span-60, d ¼ span-80, e ¼ PEG-1000, f ¼ PG, g ¼ carbopol-
940, and h ¼ the optimised niosomal gel of CPM (N3).

5.5. Fourier transform infrared spectroscopy (FTIR)

FTIR spectra of niosomal gel of CPM and its main components (cholesterol, span-60,
span-80 and carbopol-934) were recorded and shown in Figure 3. CPM spectra showed a
characteristic peak at 1431.09 and 1089.55/cm due to C–H group and phenyl group for
maleate salts, respectively. Cholesterol spectra showed characteristics and broad peaks at
2901.78, 2961.51, 3299.92, 2846.23 and 1790.24/cm due to strong aromatic stretching of
CH ¼ CH, acetyl groups, hydroxyl group, symmetric –CH3 and vinyl group, respectively.
In addition, span-60 spectra showed sharp, and broad peaks at 2916.22, 1735.58 and
2849.13/cm due to strong aromatic –CH3 group, strong C ¼ O ester bond and hydroxyl
OH group, respectively and small peaks in range of 1000–1100/cm were due to aliphatic
structure. Moreover, span-80 spectra showed characteristic peaks at 2922.48, 1738.97 and
1457.64/cm were due to –OH group, 5 membered ring and –CH3, respectively [40].
Furthermore, carbopol-934 spectra showed broad peaks at 2916.47 (in range of
3000–3800/cm) due to amine group.
FTIR of CPM niosomal gel showed sharp peaks at 1040.18/cm due to phenyl group
that were sharper in FTIR of niosomal gel of CPM than in FTIR of Carbopol 934 alone
due to coordination of linkages in niosomal gel, while the broad peaks at 921.73/cm were
480 U. AFREEN ET AL.

due to –OH group, and were more sharp in spectrum of niosomal gel of CPM.
Furthermore, FTIR of niosomal gel of CPM showed very slight shifting of peaks and
smoothening of peaks indicating strong physical interaction between CPM, and its com-
ponents responsible for formation of niosomal gel. No new peaks were obtained that indi-
cated no interaction between CPM and other components. No major shifting of peak was
seen that ensured no chemical interaction and good compatibility between formula com-
ponents leading to conclusion that CPM niosomes, can be successfully incorporated into
gel having span-60, span-80, cholesterol, PG, PEG and Carbopol-934. Hence FTIR study
has shown that pure CPM, cholesterol, PEG, PG, Carbopol 934, Span-60, and span-80
showed no significant difference among peaks alone or in combination ensuring their
good activity in final formulation without any chemical interaction [41].

5.6. Analysis of 23 factorial design

Analysis of 23 Factorial Design Based on 23 full-factorial design studies, it was observed
that the concentration of cholesterol, san-60 and span-80 have significant effect on per-
centage yield, drug content, drug loading, entrapment efficiency, size of vesicles, thermal
stability of gel, in vitro drug release, and anti-inflammatory effect. These results support
the selection of independent variables, in the current CPM loaded niosomal gel study,
and explained under concerned variable.

5.7. Effect of independent variables on percentage yield

The percentage yield of the optimised CPM niosomal gel was 98.88 ± 0.01 (n ¼ 3) as
found by using formula (N3 at A , B þ and C  levels), indicating that percentage yield
of niosomal gel was high, when low concentration of Cholesterol was used with high con-
centration of non-ionic surfactant span-60, and low concentration of span-80 keeping
concentration of Carbopol-934 constant.

5.8. Effect of independent variables on percentage drug content and percentage

drug loading
CPM content ranged from 96.88 ± 0.02 to 98.78 ± 0.02 after 24 h and 94.96 ± 0.02 to
96.77 ± 0.02 after 90 days (within required range for CPM, i.e. 93–107%), with percentage
loading of CPM niosomes was 0.94 ± 0.01 to 0.97 ± 0.01 after 24 h, and 0.969 ± 0.01 to
0.987 ± 0.01 after 90 days. These values demonstrated high drug content and loading val-
ues of CPM niosomal gel were due to, optimum formula having high amount of span-60,
and low amount of cholesterol and remained stable at 2–8  C, and ambient temperature
up to 90 days showing little changes at high temperature. These readings were found
almost similar to that of study of [21], i.e. 97% of acyclovir content was remained at end
of 3 months. According to previous reports, it was stated that nature of drug entrapped
in niosomal vesicles affect its percentage drug loading and tendency of drug to interact
with lipid bilayer by various forces for example, non-polar, polar and/or electrostatic
forces decide its loading in aqueous, or lipid bilayer portion of niosomes [42]. During this
study work it was suggested that CPM was incorporated into aqueous compartment of
niosomes, and showed maximum entrapment with span-60 (96.55%±0.02) [26].
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 481

5.9. Effect of independent variables on drug entrapment efficiency of niosomal gel

of CPM
The entrapment efficiency % of optimised niosomal gel was 97.62 ± 0.37%, and the reason
for this high value of entrapment efficiency was optimum concentration of cholesterol:
span-60: span-80 (in ratios 0.1:0.2:0.05), because various studies reported direct relation-
ship of Concentration of cholesterol, and non-ionic surfactant with entrapment efficiency
of niosomal vesicles. During this study, it was observed that on increase in concentration
of cholesterol from 0.5 to 1% entrapment efficiency was increased, but by increasing con-
centration from 1% to 1.5 or 2% entrapment, efficiency was decreased due to reason that
increase in cholesterol increased hydrophobicity, rigidity, and stability of lipid bilayer of
vesicles membrane, leading to low permeability, and entrapment that were similar previous
findings [43]. The entrapment efficiency of CPM niosomes of gel was also found in accord-
ance with that of aceclofenac niosomes by Mishra in 2014, who reported that when concen-
tration of cholesterol was low (upto optimum level) with constant level of Non-ionic
surfactant, the entrapment efficiency was high. Reason of this high entrapment efficiency
with low amount of cholesterol could be the low characteristic effects produced by choles-
terol, i.e. less rigidity, less cementing effect, decreased micro viscosity of lipid bilayer lead-
ing to high leaking spaces in lipid bilayer of niosomes, and increased chances of uptake of
CPM in vesicles of niosomes of gel. This phenomenon was observed at low concentration
of cholesterol upto particular level beyond which, membrane loses its integrity leading to
disruption, and breakage of vesicles. Another study, confirming values of this study was,
reports from Korchowiec (2006) stating that optimum concentration of cholesterol pro-
vided sufficient rigidity to stabilise niosomes with reduced leaky effect, leading to reduced
efflux with increased entrapment effect [44]. One other reason for high concentration of
cholesterol leading to low entrapment efficiency might be due to, high concentration of
cholesterol formed cluster affecting integrity of vesicles, causing non uniform distribution
of drug along lipid bilayer being reported by Finean [45]. Various contrasting conclusion
studies have been reported to study effect of cholesterol concentration on entrapment effi-
ciency, i.e. some studies showed that cholesterol has no effect on entrapment efficiency
[46], some studies showed that inverse relation was present between cholesterol concentra-
tion and entrapment efficiency [47], and some studies reported that direct proportional
relationship was present between cholesterol concentration, and entrapment efficiency [48].
Entrapment efficiency was also found to be affected by amount of non-ionic surfactants
(span-60 and span-80), i.e. in this case Entrapment efficiency of Optimised niosomal gel
was due to, high level of Span-60 might be attributed to high gel to liquid phase transition
temperature of Span-60, and longer saturated alkyl chain length of Span-60 leading to sta-
ble niosomes of its gel and these findings were found to be in accordance with that of
nimesulide-span-60 niosomes [49]. Other reason for high entrapment, when concentration
of non-ionic surfactant was increased, was due to reason of decreased drug leakage from
vesicles [50]. Optimised formula of niosomal gel of CPM of present study (cholesterol:-
span-60, 1:2) was also in accordance with formula optimised by Jigar (2011) during prepar-
ation of Erythromycin niosomal gel where concentration ratio of cholesterol:Noionic
surfactant was 1:2 High, amount of span-60 than span-80 produced, high entrapment than
with high amount of span-60 with high amount of span-80 also because span-80 alone pro-
duces high entrapment, but with span-60 its entrapment efficiency decreases, and same
effect was observed ring this study of Erythromycin niosmal gel where span-80 alone pro-
duced high entrapment efficiency (82.26%), while when combine with span-20 (49.51%) or
2pan-60 (72.62%) hence, confirming optimised formula of this study [47].
482 U. AFREEN ET AL.

Figure 4. SEM of optimised nisomal gel of CPM (N3). Where,a ¼ SEM of niosomal gel at 4000 magnification, b ¼ SEM
of niosomal gel at 10,000 magnification, and c&d ¼ SEM of niosomal gel at 15,000 magnification.

5.10. Effect of independent variables on vesicle size (scanning electron

microscopy SEM)
Study of SEM of optimised niosomal gel of CPM showed (SEM images) that niosomal gel
sample produced spherical shape and morphology of vesicles of size range 0.1–5mm on
different magnification power of 4000, 10,000 and 15,000 as shown in Figure 4. Optimum
size of niosomal vesicles of its gel was produced, due to reason of having optimum con-
centration of span-60, and cholesterol with span-80 in formula. Critical packing parameter
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 483

Figure 5. TG/DTA of optimised niosomal Gel of CPM (N3).

(CPP) of Span-60 was 0.5–1 that was considered to be responsible for formation of nioso-
mal vesicles in its gel formulation [51]. Span-60 and span-80 produced proper shaped and
spherical vesicles than tween in formula because Span are water insoluble, and have high
lipophilic nature leading to proper visualisation in aqueous medium, while tween is water
soluble and high hydrophilic property no vesicles are formed in aqueous medium [47].
High amount of Span-60 used in optimised formula have low free surface energy that
reduce vesicle size, because of high hydrophobicity and low HLB 4.3 of span-60 [27].
Vesicle size of niosomal vesicles of gel was in accordance with Diacerein niosomal vesicle
size produced by Khan, i.e. 1 lm, Diclofenac niosomes by Jaiswal (2016), i.e. 1–6 lm, ace-
clofenac niosomes by Mishra (1997), i.e. 4.2–4.8 lm, and spherical shape and morphology
of vesicles was similar to shape of naltrexone niosomal vesicles produced by Khan [41].

5.11. Effect of independent variables on thermal stability of CPM loaded niosomal

gel (thermal gravimetric analysis)
Thermal analysis of optimised CPM niosomes (N3) shows stability of niosomal gel of
CPM on different temperatures and melting point of niosomal gel (Figure 5). There is a
sharp single exothermic peak between 120 and 350  C with loading temperature of 30  C
that went to 50  C, and loading weight of niosomal gel of CPM was 6.285 mg that
decreased to 0.2 mg at 410–600  C crossing melting point of CPM (CAMEO chemicals
reports). Hence it declared the niosomal gel of CPM to be more thermostable than dis-
persion at molecular level in accordance with reported melting point [52].

6. Physical examination
6.1. Homogeneity
On physical examination it was found that CPM niosomal gel was translucent milky white
to off-white in colour, without any colour intensity difference, clear without any aggregate
484 U. AFREEN ET AL.

or phase separation, equally opaque with good tendency, odourless, without any grittiness,
and homogenous in appearance being similar to niosomal gel of Goyal [31].

6.2. pH determination
pH of Optimised CPM niosomal gel was almost 6.06 ± 0.15 (n ¼ 3) lying in range of pH
making it suitable for topical delivery of CPM, without causing any skin irritation, and
readings of pH were consistent with that of niosomal gel of Benzoyl peroxide by Goyal
(2015) where pH of niosomal gel, was 6.2 ± 0.162, Meloxicam niosomal gel by Usama
(2016) where pH was 6.2 ± 0.4 and readings of Kamboj (2013), where pH of mefenamic
acid niosmal gel was 6.4–6.7 ± 0.12 [53].

6.3. Viscosity
Viscosity of optimised niosomal gel of CPM calculated by Brookfield viscometer (Model
RVTDV 11, Brookfield Engineering Laboratories, lnc, Stoughton, MA using spindle 06,
and 2.5 rpm at 25 ± 1  C was 3768 ± 0.57102cP (n ¼ 3) was determined. Viscosity of for-
mulated niosomal gel of CPM was lying in desirable range providing sufficient consist-
ency, Spreadability, and Extrudability of gel leading to its easy application in thin film
with low resistance to flow (Non-Newtonian flow system) by applying small stress force,
easy removal with good feeling sensation, and enhanced permeability across skin [54]

6.4. Spreadability
Spreadability and Extrudability of Mefenemic acid niosomal gel by Kamboj [55] also
showed, good outcomes, due to optimum concentration of formula producing viscosity
smaller than CPM niosomal gel of this study, hence, higher spread ability values were
obtained by Kamboj and workers (2013). Values of Spread ability of optimised CPM nio-
somal gel obtained in triplicate was 3.91 ± 0.1 g cm/s, and showed inverse relation with
viscosity as confirmed by that of Goyal [31]. Optimum Spreadability with small force
application was considered to be because of optimum concentration of various formula
major components, and major reason was considered to be loose gel matrix of gel, due to
presence of vesicles of niosomes [38]. Readings were found to be similar to that of
Etodolac niosomal gel [50]. In fact spread ability value of niosomal gel of CPM was
affected by various factors, i.e. amount of cholesterol, span-60, PEG-1000, and span-80
and showed that gel was easily spread with small shear force due to PEG-1000 in formula
being in concordance with those reported by Boushra and Gosyal [56]. It was already
reported by Garg (2002) that PEG-1000 and PG (permeation enhancers) in formula of
niosomal gel enhanced spreadability, and consistency [57].

6.5. Extrudibility
Niosomal gel of CPM had optimum viscosity that allowed it to flow out from plastic col-
lapsible tubes easily, quickly and with application of small force on tube maintaining its
good consistency hence showed good Extrudibility determined as average of three values
(n ¼ 3), i.e. 0.950.03 g/cm. Values were found in accordance with values of mefenemic
acid niosomal gel [55].
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 485

All physical examination tests suggested good homogeneity, optimum viscosity, desir-
able pH, sufficient Spreadability [58], and Extrudibility of formulated optimised CPM nio-
somal gel being safe for topical delivery of CPM

6.6. Stability studies and drug leakage study

Stability studies of optimised CPM niosomal gel performedfor three months byusing
Stability chamber set at refrigerated temperature (2–8  C), ambient temperature (20  C),
and elevated temperature (40  C) showed no particular change in physical appearance, i.e.
colour, consistency, viscosity, homogeneity, phase, pH, drug entrapment, and drug con-
tent (drug leakage) at refrigerated temperature (2–8  C), very slight changes at ambient
temperature (20  C), and slight change at elevated temperature (40  C).
The findings of stability study of CPM niosomal gel were summarised that suggested
that gel was more stable at refrigerated conditions, less stable at ambient temperature after
2nd month, while unstable at elevated temperature after 3rd month, because its CPM con-
tent and CPM entrapment were non-significantly (p > 0.05) decreased to 95.01 and 96.56,
respectively.
Increased stability of niosomal gel of CPM at refrigerated and room temperature than
elevated temperature might be due to elevated temperature induced fluidity of niosomal
gel lipid bilayer, leading to higher CPM leakage at elevated temperature, and stability
were found parallel to the stability measured by Bhaskaran and Jousma [59], during their
research, where room or higher temperature enhanced drug leakage. Stability of CPM nio-
somal gel was higher that stability of niosomes might be, because of prevention of fusion
of niosomal vesicles after incorporation of them into Carbopol 934 P based gel, and this
value was found to be in accordance with values of [47].
Values of this stability study were almost similar to one month stability study of
Diclofenac niosomal gel by Jaiswal (2016), where initial entrapment efficiency was
89.55 ± 3.90 at 2–8  C, 89.47 ± 3.59 at 20  C and 89.38 ± 2.89 at 40  C, and after 1 month
entrapment efficiency was 89.38 ± 0.34 at 2–8  C, 89.23 ± 1.04 at 20  C and 88.98 ± 0.88 at
40  C. These observations were in concordance with three month stability study of nioso-
mal gel of Erythromycin by Jigar et al. [47], where amount of drug retained on skin and
drug release was reduced at end of 12th week at higher temperatures than at refrigerated
temperature.
Stability study of optimised CPM niosomal gel (at 2–8  C, 75 ± 5% RH) showed no
change in physical appearance (milky white), pH (6.2), viscosity (3768 cP102),
Spreadability (3.81–3.91 g cm/min), homogeneity (clear), drug entrapment efficiency
(97.22–97.62%), and drug content (98.12–98.78) during three months of stability study.
Stability study of optimised CPM niosomal gel (at 20  C, 75 ± 5% RH) showed mild
changes in parameters, i.e. same physical appearance (milky white), mild decrease in pH
(6.2–5.1), decrease in viscosity (3768 to 3710 cP102), increase in spreadability (up to
3.99 g cm/min), homogeneity (clear till end of 1st month, and lumps during 2nd and 3rd
month), decrease in drug entrapment efficiency to 97.11%, and decrease in drug content
to 97.09 with the passage of time. But the Stability study of optimised CPM niosomal gel
(at 40  C, 75 ± 5% RH), showed prominent changes in parameters, i.e. light yellow appear-
ance, decrease in pH to 4.1, decrease in viscosity to 3508 cP102, increase in spreadability
to 4.87 g cm/min, larger yellow lumps, decrease in drug entrapment efficiency to 97.11%,
and decrease in drug content to 97.09 on 3rd month of stability study [58].
According to this CPM niosomal gel was stable at 2–8  C showing little less stability at

40 C stating that best storage temperature for niosomal gel of CPM should be
486 U. AFREEN ET AL.

refrigerated temperature for maintained efficacy with reduced leakage, and decreased sta-
bility problems being in concordance with that of niosomal gel of erythromycin and
diclofenac [27].
In one study conducted by Nagalakshmi(2016), and his workers in 2016 to formulate
herbal niosomal gel stability study showed drug content was lower, i.e. 66.52% at high
temperature (25  C ± 2  C), than 82.28% at 4  C ± 2  C confirming findings of this study.
The main reason for lower retention of drug (drug content) on skin at higher tempera-
ture, was because high temperature cause phase transition of surfactant leading to degen-
eration of polymer, lipid vesicles leakage, and poor drug retention [59].

6.7. Effect of independent variables on in vitro drug release study of niosomal gel
of CPM
CPM release from Optimised niosomal gel studied for 24 h, using linear regression equa-
tion Y ¼ mx þ b, and R2 (regression coefficient) at pH 6, i.e. values for CPM
Y ¼ 0.0217x þ 0.0414 and R2 ¼ 0.9964 showed that maximum drug release was, 48.81% at
4th hour, 66.69% at 12th hour, 69.08% at 18th hour, and 69.64% at 24th hour of study as
shown in Figure 6. Study demonstrated that drug release was rapid during first 12 h, and
slow during last 24 h indicating sustained release of CPM from niosomal gel. Effect of this
slow and excellent CPM release study were in concordance with effects of Herbal nioso-
mal gel formulated, and evaluated by Nagalakshmi (2016) and his workers in 2016 who
stated that release of drug from niosomal gel was 52% at 12th hours, and 85% at 24th
hour of study (24 h study) [59].
Drug release from Optimised niosomal gel of CPM N3 was high (i.e. 69.64%) at ratios
of cholesterol:span-60:span-80 as 0.1:0.2:0.05, i.e. lower concentration of cholesterol with
high amount of span-60 and low amount of span-80 because as amount of span-80 was
also increased (from 0.05 to 0.1%) with increased amount of span-60 (0.2%) produced a
huge amount of surfactant in aggregate (0.3%) that might act as depot and prevent drug
leakage hence drug release from niosomal vesicles of gel leading to low drug release hence
authenticating the optimised formula that was further validated by Salih et al. (2013) [60]
formulated etodolac niosomal gel drug release. In case of etodolac niosomal gel higher,

Figure 6. In vitro release (%) of CPM from Niosomal gel at pH 6.

 JOURNAL OF EXPERIMENTAL NANOSCIENCE 487

Table 3. Drug release kinetics of optimised CPM niosomal gel (N3) at pH 6.

Zero order First order Higuchi Korsmeyer-Peppas Hixon Crowell
Form.
CPM-NG R 2
K0 A/C R
2
K1 A/C R
2
KH A/C R
2
Kkp n A/C R 2
KHC A/C
0.40 0.07 132.87 0.85 0.002 110.2 0.81 2.27 114.83 0.83 2.59 0.47 116.74 0.79 0.001 116.8
CPM-NG ¼ Niosomal Gel of CPM.

i.e. 94.91% drug release was obtained at end of 1 day at 1:1 ratio of cholesterol and surfac-
tant than drug release at 1:1.5 ratio of cholesterol and surfactant [50].
Values of drug release from CPM niosomal gel (69.64%) at 24th hour of study was
lower as compared to CPM simple organogel (80.3%) at even 8th hour of study [15],
because network of niosomal gel vesicles entrapped CPM making unavailable to sur-
rounding gel, and CPM entrapped in niosomal vesicles leaked out from vesicles into sur-
rounding gel slowly, and gradually than plain or organo gel of CPM being in
concordance with values of Korchowiec [44]. Another reason for this slow release from
niosomes, was presence of cholesterol in niosomal gel formula, because cholesterol present
in niosomal vesicle lipid layer limit drug mobility cause spontaneous mixing between two
membrane lipids, increase stability of lipid layer, and perform membrane stabilising
role [53].

6.8. Drug release kinetics study

Values of five models of release kinetics applied on in vitro drug release data from nioso-
mal gel of CPM using DD solver and M S Excel calculated, on the basis of coefficient of
regression (R2) [59], and Akaike Information Criterion (A/C) [26] showed that R2 Value
of First order kinetics was highest than that of others, i.e. 0.85 near to one suggesting the
First order kinetics was the best fit release model followed by niosomal gel (in vitro),
whose goodness was further validate by its low A/C 110.2 than that of others [34]. Values
of all release kinetics parameters, i.e. value of K (rate constants), R2, nd A/C of five release
models were displayed in Table 3. The outcome of this study confirmed that optimised
niosomal gel of CPM followed, the First order release kinetic model being similar to CPM
microsponge based gel [29].

6.9. In vivo study

In vivo studies of skin irritation study, anti-inflammatory study, pharmacokinetic, bio-
availability study, and histopathological study of CPM loaded optimised niosomal gel
were narrated individually. Only optimised gel is selected for study rather than eight nio-
somal gels of niosomal formulations (N1–8), as previous sections of this study published
earlier, as niosomal dispersion of CPM stated that N5 produced better result than all
others. Hence, to avoid extra length, and irrelevant features of study only optimised gel
was used. Although, the in vivo findings are not in agreement with that of in vitro results,
it could be due to narrow pore size of dialysis membrane. Furthermore, it was also
observed from results of current study that niosomal gel of CPM is far better than emul-
gel, or simple gel because it has enhanced targeting, enhanced loading due to its lipid
structure similar to skin, longer residence time sustained effect from lipid-based vesicles
serving as best drug depot system, and finally automatic absorption of lipid-based drug
delivery system due to its biodegradable nature.
488 U. AFREEN ET AL.

Figure 7. a ¼ Capsicum induced allergic skin area of Rabbit and b ¼ CPM niosomal gel treated skin area of rabbit.

6.10. Skin irritation study

Skin irritation study of formulated niosomal gel of CPM showed, mean skin irritation score
of 0.02 ± 0.01 confirming non irritancy, and safety of gel for topical use on human skin
without producing any sign of irritation, i.e. redness, lesion, abrasion or erythema [61]. The
effects were in accordance with those of Benzoyl peroxide niosomal gel skin irritation test
having score of 1.13 ± 0.21 being safe for skin use [31].

6.11. Effect of independent variables on anti-inflammatory studies

In vivo studies of CPM niosomal gel was conducted by applying gel sample on dorsal side
skin area (10 cm2) of six rabbits (2.5 ± 0.19 kg) (n ¼ 6) to observe inflammatory effect of
capsicum, and anti-inflammatory effect of CPM. After washing, drying, shaving and clean-
ing rabbit skin area was labelled as Normal skin area, and capsicum treated skin area where
normal skin area was area not having capsicum induced allergy or niosomal gel treatment
but, used to study histology of normal rabbit skin and to compare it with inflamed skin
area for observation of histopathological changes, and Capsicum treated area was the area
treated with allergen (green chilli: Capsicum annum) to study, histopathological changes
induced by capsicum treated allergy. Procedure was followed by rubbing half cut portion of
fresh green chilli (Capsicum annum), gently on selected area for 2 min that induced topical
allergic or inflammatory reaction by producing red small round spots or patches along with
itching, shivering, rashes, redness, and inflammation etc. After induction of symptoms half
of capsicum treated area, was kept untreated with gel, and used as control to observe capsi-
cum induced histopathological changes while remaining half capsicum treated area was
treated with 0.05 g of niosomal gel of CPM (10 mg dose of drug CPM) to observe effect of
treatment rapidly after 61 min of application of niosomal gel of CPM as shown in Figure 7.

6.12. Pharmacokinetic (bioavailability) study

The pharmacokinetic parameters of CPM niosomal gel by using rabbit as experimental
model are shown in Table 4.
Values were in concordance with pharmacokinetic of CPM muco-adhesive buccal
patch Sekhar (2008), i.e. for oral and buccal routes Cmax (ng/ml) 5.73 ± 1.08, and
6.16 ± 0.99, Tmax (h) 2.17 ± 0.41, and 3.33 ± 0.82 and AUC0  n (ng h/ml) 57.85 ± 15.50
and 84.99 ± 17.96, respectively demonstrating that Cmax of niosomal gel was higher with
lower Tmax. These readings suggested that niosomal gel was more fast and efficient drug
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 489

Table 4. Pharmacokinetic parameters of CPM (10.00 mg) (niosomal gel) applied topically on allergenic rabbits.
Parameters Unit Values (mean ± SD)
Cmax lg/ml 43.76 ± 0.200
Tmax h 0.75 ± 0.00
t1/2 h 7.338 ± 1.92
AUC0-t lg/ml  h 57.46 ± 0.29
AUC0-a lg/ml  h 149.86 ± 27.43
Cl/F (mg)/(lg/ml)/h 0.0687 ± 0.013
Vz/F (mg)/(lg/ml) 0.75 ± 0.072
Note: Cmax is Peak serum concentration, Tmax is Time to reach peak serum concentration, t1/2 is Half-life, AUC0-t is
Area under the plasma concentration time curve when time range from 0-t (where t ¼ 4h), AUC0-a, is Area under the
plasma concentration time curve when 0-infinity, Cl/F is Clearance/bioavailability, Vz/F is Volume of distribution/
Bioavailability.

delivery system than buccal or oral patch of CPM, and topical route of CPM niosomal gel
application was best route for enhanced absorption, and bioavailability due to high bio-
availability of CPM niosomal gel than both buccal, and oral CPM patch where low bio-
availability might be due to route of application and complications associated with patch
dosage form (buccal/oral). Another reason for high pharmacokinetic parameters of CPM
niosomal gel, might be suggested that CPM enclosed in lipid directly was directly applied
to skin, where these vesicles having lipid structure, and composition similar to that of
human bi-layered lipid membrane diffuse and permeate easily through membrane without
any hindrance hence showing enhanced bioavailability than buccal CPM patch, and oral
CPM tablets where CPM was embedded in matrix polymer causing delayed CPM release,
and low bioavailability and CPM was granulated in oral tablet facing first pass effects of
oral route, and reducing its bioavailability to only 25–45%, respectively [36].
In vivo CPM release study showed that cumulative drug released during 1st hour of
application being highest at 0.75th hour (43.76 ± 0.21), and decreased slowly and continu-
ously during late hours showing sustained drug release pattern. During in vivo study of
four hours, duration it was noticed that maximum drug was released during first hour,
and decreased during later being in concordance with previous study reports of CPM sus-
tained release tablet by Masumoto and his colleagues (1974) where, maximum drug
amount released was, i.e. Cmax (ng/ml) 10.71 ± 0.89. It was also noticed that mean peak
plasma concentration from in vivo CPM niosomal gel was higher than its in vitro values,
and mean time to reach peak plasma concentration of in vivo was lower than in vitro
being in agreement with great difference lying in mean peak plasma concentration, and
time obtained from in vitro than from in vivo with area under curve (AUC) value being
same ensuring no decrease in bioavailability from sustained release dosage forms [62].

6.13. Histopathological studies

Histological examination of capsicum treated skin tissue revealed major changes, and phe-
nomenon of histological examination of capsicum treated rabbit skin tissue demonstrated
that green chilli induced inflammatory process causing, histopathological alterations in
skin hence, acted as a strong irritant due to its major chemical ingredient, i.e. capsaicin
that caused dose dependent hypersensitivity in rabbit skin, because higher the amount of
capsaicin greater the histopathological, and morphophysiological damage produced in
skin. These changes were found in accordance with those of Bozzatto [63]. Capsicum
treated skin also showed some vascular changes along with stated histological changes, i.e.
some blood vessel haemorrhages in few portion of skin tissue. These all changes were also
observed by Germuth (1953), during study of hypersensitivity induced responses resulting in
490 U. AFREEN ET AL.

epitheloid, and giant cell reactions in skin tissue [64]. Observations of histological examination
of CPM niosomal gel treated area of rabbit skin tissue demonstrated, reduced inflammatory
process along with reduced necrosis, reduced tissue disorganisation, reduced haemorrhages,
and increased cell infiltration, improved epithelialization, and better tissue organisation, and
enhanced tissue strength being similar to observations of Oryan and Zakar [37].

6.14. Histopathological study of normal rabbit skin

Normal rabbit skin after selection of appropriate portion, staining with haematoxylin, and
eosin (H&E), and visualising under low, and high magnification powers (i.e. 10 and

Figure 8. (a) Normal rabbit skin tissue histology; (b–e) excised capsicum treated rabbit skin tissues; (f, g) 40 power,
enlarged view, (h) 10 power, (i) excised CPM niosomal gel treated rabbit skin tissues stained with haematoxylin and
eosin (high magnification).
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 491

40) of optical microscope displayed prominent histopathological alterations (Figure 8a)

showing regular and symmetrical arrangement of all layers and cells. Histopathological
study of normal skin showed, symmetrical and smooth surfaced lamina propria with sym-
metrical collagen fibres, and glands along with their normal secretions of eosinophilic leu-
kocytes, lymphocytes, macrophages, mast cells, plasma cells, and fibroblasts. Fibroblasts
and collagen fibres were found to make a regular network of loose connective tissue cov-
ering lamina propria, and performing function by mechanism of diffusion through capil-
laries to supply nutrients, and oxygen to the cells and to remove carbon dioxide, and
water from cell being in accordance with findings of normal skin histology by Javed [26].

6.15. Histopathological study of treated rabbit skin tissue

Two portions of excised rabbit skin, mainly capsicum treated skin tissue and CPM nioso-
mal gel treated skin, were selected, stained with H&E dye, and visualised using high mag-
nification power (40) of optical microscope.
Photo-microscopy of capsicum treated skin after induction of allergy and initiation
of inflammatory reaction showed, major histopathological changes as displayed in
Figure 8b–e, i.e. lamina propria showed clear surface changes, cells, and glands became
asymmetrically arranged, blood vessels became wide spaced, fibroblasts increased to huge
number, inflammatory cells increased in numbers, and made clusters called Inflamosomes,
collagen fibres became roughly connected to Inflamosomes, and loosely packed connective
tissue un evenly covered Inflamosomes. Photo-microscopic images also showed, various
other major visible changes beside these alterations, i.e. disruption and dissolution of whole
or part of epithelium called de-epithelialization, lamina propria composition modulation,
appearance of hypersensitivity cells mainly the neutrophils and lymphocytes in lamina prop-
ria, rupturing of blood capillaries leading to accumulation of blood, and clear haemor-
rhages, and rupturing, dissolution and accumulation of fibroblasts called disruption of
fibroblasts. These major alterations produced after induction of allergy with capsicum con-
firmed the capsicum (mainly its capsaicin) to be very strong irritant, and lethal allergen
because it dissolved epithelium, ruptured fibroblast, and even blood vessels.
Photo-microscopy of CPM niosomal gel treated skin slides after application of gel, and
initiation of anti-inflammatory reaction showed, major histological alterations as displayed
in Figure 8f–i, i.e. regeneration of epithelium where only 1-2 layers were regenerated
while, remaining layers were missing, somewhere total disruption of epithelium, and lam-
ina propria with reduced number of lymphocytes in lamina propria, and moderate fibro-
blasts indicating treatment of allergenic symptoms by applying anti-allergic gel
application. Vascular changes were much reduced, i.e. no haemorrhages were seen that
showed good response of anti-allergy treatment. Hence application of niosomal gel of
CPM reduced allergic reaction and declared CPM niosomal gel, as therapeutically active
anti-histaminic treatment. Figure 8f displayed gel induced alterations in skin, i.e. lamina
propria disappearance or asymmetry, clustering of inflammatory cells, extensive fibro-
blasts, and prominent blood vessels while, Figure 8g showed fibroblasts, vessels, and con-
nective tissue while Figure 8h showed lymphocytes, and neutrophils, and Figure 8i
showed, disruption of epithelium with 1–2 layers along with regeneration of epithelium
clearly. Niosomal vesicles being lipophilic were used to entrap CPM due to hydrophilic
nature CPM needed to be loaded in some lipophilic carriers to cross skin epithelium
more efficiently by trans-cellular passive diffusion mechanism. Gel of these niosomal
vesicles was used to obtain enhanced, facilitated permeation, and absorption of CPM
492 U. AFREEN ET AL.

through epidermis by concentration, dependent mechanism due to, presence of additional

permeation enhancers like Propylene glycol and Polyethylene glycol in gel [65].
After observing all slides form Figure 8f–i, it was clear that niosomal gel proved to be
a good medicine, because initiated healing response by activating defense mechanism,
attaching many inflammatory cells towards induced allergic site on skin, and production
of various Inflamosomes, i.e. lymphocytes, neutrophils, and fibroblast more than in nor-
mal skin. Enlarged view at 40 produced enlargement of histopathological changes for
prominent view. Underlying mechanism of action of CPM in its niosomal gel was, its
anti-histaminic activity (compete with Histamine for binding to H1 receptors) because,
various inflammatory agents mediate inflammatory response by involving Histamine H1
receptors [66].
In short, it was suggested that good alteration in lamina propria caused enhanced
CPM permeability, increased penetration of drug across skin for longer time, i.e. 1 day,
increased production of collagen by chronic fibroblasts, increased healing of wounds,
increased mucin the a-glycoprotein secretion overlapping the cell leading to, increased
anti-inflammatory reaction, and enhanced drug transport. Underlying mechanism for
mucin-action was described in few steps, i.e. increased mucin secretions, increased over-
lapping of all cells, increased tight attachment of niosomal lipophilic vesicles of CPM to
hydrophilic end of mucin, increased production of wide spaces in loose network of con-
nective tissue that allowed increased drug passage in membrane easily, hence, facilitated
transport of drug inside membrane [67].
During this study, capsicum was used as strong inflammatory agent to study induced
inflammatory response by infiltration of inflammatory cells, i.e. lymphocytes and neutrophil
into capsicum treated rabbit’s dorsal skin site. The major reason for selection of capsicum
induced inflammation, as a model inflammation induction method, was the smaller number
of reports or less available data regarding this method than frequently used histamine
induction method used to study rat paw edoema by many reporter [68].
Anti-histaminic and anti-inflammatory activity of CPM observed during this study,
was in concordance with effects of previous studies reports that measured anti-
inflammatory activity of CPM (administered by various routes) in terms of percent
inhibition of paw edoema, i.e. CPM administration by intravenous injection in rat’s
paws, inhibited histamine-induced paw edoema [69], CPM administered by subcuta-
neous injection in rat’s paw inhibited Substance p-induced (s/c) paw edoema, CPM
given by oral route in rat inhibited histamine induced paw edoema, and CPM admin-
istered by intradermal injection in rat’s paw also inhibited histamine induced
edoema [66].
Hence, topical application of niosomal gel of CPM proved, to be successful, and stable
dosage form providing sufficiently facilitated, and potentiated anti-inflammatory effect of
CPM by avoiding oral routes side effects like, poor bioavailability due to first pass metab-
olism causing only 25–45% of CPM reaching blood circulation [70], by the presence of
permeation enhancers like Propylene glycol, Polyethylene glycol etc., and by the presence
of penetration enhancers like Span-60 and Span-80 in formulation that enhanced parti-
tioning of CPM into skin, being in parallel to phenomenon of span-60 based topical CPM
organogel formulated by Balata and co-workers (2017)[15s].

7. Conclusion
It can be concluded that CPM-based niosomal gel was successfully developed by using
suitable combination of non-ionic surfactants for effective drug loading and good
 JOURNAL OF EXPERIMENTAL NANOSCIENCE 493

bioavailability. This gel could be an efficient carrier for sustained drug release across the
skin and an improved anti-allergic effect.

Data availability statement

All the data has been incorporated into this article.

Disclosure statement
No potential conflict of interest was reported by the authors.

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