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Original Article
CILNIDIPINE-LOADED TRANSDERMAL NANOEMULSION-BASED GEL: SYNTHESIS,
OPTIMISATION, CHARACTERISATION, AND PHARMACOKINETIC EVALUATION
MAHESH T. GAIKWAD1* , RAJENDRA P. MARATHE2
1*Govt College of Pharmacy, Opp. Govt. Polytechnic, Osmanpura, Chhatrapati Sambhajinagar-431005, Maharashtra, India. 2Department of
Pharmaceutical Chemistry, Govt College of Pharmacy, Opp. Govt. Polytechnic, Osmanpura, Chhatrapati Sambhajinagar-431005,
Maharashtra, India
*Corresponding author: Raman Rajeshkumar; *Email: maheshgaik7@gmail.com
Received: 16 Sep 2024, Revised and Accepted: 02 Nov 2024
ABSTRACT
Objective: The aim of the study was to enhance transdermal flux and bioavailability, thereby reforming the effectiveness of drug delivery by
synthesising and characterising cilnidipine-loaded nanoemulsion-based gel.
Methods: The research was conducted with meticulous planning and execution. After preformulation studies, cilnidipine-loaded nanoemulsions
were synthesised using probe sonication and optimised by a 2-factor central composite design. The optimised nanoemulsions were loaded in
Carbopol 940 and HPMC K4M gelling system. The optimised nanoemulsions were characterised for droplet size, zeta potential, viscosity, refractive
index, pH and TEM, and cilnidipine-loaded nanoemulsion gels were characterised for clarity, homogeneity, consistency, spreadability, extrudability,
pH, viscosity, in vitro diffusion study, dermal toxicity, and pharmacokinetic profiling. The process was accurately planned and accomplished at each
step to ensure the precision and reliability of the results.
Results: The findings of this research are not just significant; they are groundbreaking. The steady-state flux values observed ranged from
35.71±1.27 µg/cm²/h to 107.7±2.04 µg/cm²/h for DOE_CiL_1 to 9 and 40.88±1.44 µg/cm²/h to 80.64±1.38 µg/cm²/h for NEn_CiL_GeL_1 to 4. These
results underscore the diverse efficacy of different formulations in facilitating drug delivery through the skin. The pharmacokinetics profile of
cilnidipine also showed remarkable changes. The Cmax for the cilnidipine tablet was 332.3±14.2 ng/ml, whereas it significantly increased (p<0.05) to
593.00±24.8 ng/ml in the nanoemulsion gel, demonstrating a substantial enhancement in drug concentration. Additionally, the AUC0-12 showed a
significant (p<0.05) increase from 1279±34.1 ng/ml. h with the tablet to 1922.50±162.8 ng/ml. h with the nanoemulsion gel. The AUC0-∞ also
increased from 1395.5±156.7 ng/ml·h for the tablet to 1962.30±174.9 ng/ml. h for the nanoemulsion gel, further confirming the improved
bioavailability of cilnidipine with the nanoemulsion gel. These significant bioavailability improvements cause excitement about the potential impact
of this research, which could revolutionise transdermal drug delivery systems in the pharmaceutical business, leading to more effective and efficient
drug delivery methods.
Conclusion: The results of this novel study are not only promising but also hold the potential to be transformative. The significant improvement in
transdermal flux from the cilnidipine-loaded nanoemulsion gel reveals a substantial increase in the drug's bioavailability. This breakthrough could
eliminate several drawbacks of cilnidipine, like first-pass fate and poor solubility, and provide a safer, more convenient delivery method for
managing hypertension.
Keywords: Cilnidipine, Pseudo-ternary phase diagram, Transdermal flux, Bioavailability, Pharmacokinetics, Dermal toxicity
© 2025 The Authors. Published by Innovare Academic Sciences Pvt Ltd. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/)
DOI: https://dx.doi.org/10.22159/ijap.2025v17i1.52689 Journal homepage: https://innovareacademics.in/journals/index.php/ijap
INTRODUCTION
High blood pressure is a significant cause of cardiovascular disorders,
renal failure, and death if not detected and treated appropriately,
affecting more than nearly 213 million by the year 2025. In this
context, Cilnidipine, a novel class of anti-hypertensive medication that
is different from other L-type Ca2+channel blockers or even other
antihypertensives that work against both l and N types and is a
recently identified calcium channel blocker, was chosen as the drug of
interest for this research. Because N-type calcium is distributed
throughout the nerve and brain, it is expected to affect nerve action.
This data will aid in selecting antihypertensive medications [1, 2].
Cilnidipine (CiL) is a poorly water-soluble drug with a
Biopharmaceutical Classification System (BCS) Class II. It has a slow
dissolution rate, hence poor bioavailability (<13%) in humans, and
reported a pKa value of 11.39. It is highly hydrophobic, and its
partition coefficient value is 4.7; thus, it has good permeability [3–8].
Despite its poor oral efficacy, cilnidipine is considered safer and
more effective than other calcium antagonists in the treatment of
hypertension [3]. However, as indicated, its high solubility and
prolonged first-pass metabolism led to a very low oral
bioavailability [9]. Previous research showed various methods for
improving solubility and bioavailability and eradicating the
drawbacks of cilnidipine. Strategies like self-micro emulsifying drug
delivery systems [10], transferosomes [11], polymeric nanoparticles
[12], fast-dissolving tablets [13], nanosuspensions [14, 15], oral
films [16], Microemulsions were tried [17]. Transdermal drug
delivery may find benefit with patients suffering from long-lasting
hypertensive situations. The stratum corneum restricts the drug's
transport across the skin. Hence, many researchers reported nano
vesicles like nanoemulsion, transferosomes, and some other nano or
microcarriers for transdermal delivery of the drug [18].
Nanoemulsions are an excellent choice as a delivery system for
transdermal drug delivery. They are a subcategory of emulsions
with twenty to 500 nm globule sizes, sometimes called mini-
emulsions. Nanoemulsions can deliver lipophilic drugs, which may
show enhanced pharmacological action. Their robust stability
extends their expiry, making them a unique and practical choice for
drug delivery [19–21]. Non-magnetized views of stable
nanoemulsions reveal them to be transparent or slightly
transparent. Moreover, they do not cream or settle. They improve
absorption and bioavailability and remove variability in absorption,
it has been found [22]. Nanoemulsions are kinetically stable and are
extensively used because of their low rheological properties and
good optical characteristics [23]. Several theories have explained the
benefits of nanoemulsions for transdermal drug delivery. A greater
degree of drug solubilisation in the nanoemulsion system may
amplify the thermodynamic activity directed toward the skin.
Substances in nanoemulsions may improve drug absorption via the
skin through enhanced permeation. Last, the drug's penetration rate
from the nanoemulsion may be increased because it is easy to
change a drug's affinity for the internal phase to favour partition
International Journal of Applied Pharmaceutics
ISSN- 0975-7058 Vol 17, Issue 1, 2025
M. T. Gaikwad & R. P. Marathe
Int J App Pharm, Vol 17, Issue 1, 2025, 255-274
256
into the skin. These unique benefits make nanoemulsions a highly
effective choice for transdermal drug delivery [24].
This research is at the forefront of innovation, aiming to design and
synthesise a nanoemulsion and nanoemulsion gel for the
transdermal delivery of cilnidipine. We are breaking new ground in
drug delivery by enhancing solubility and transdermal flux.
Transdermal delivery can avoid the first-pass effect, facilitating a
more controlled and steady drug release into the bloodstream. This
approach aims to maximize the therapeutic potential of cilnidipine
by improving its systemic availability and seeking to reduce
potential side effects.
Fig. 1: Graphical abstract showing synthesis and optimisation of cilnidipine nanoemulsions and cilnidipine-loaded nanoemulsion gel
MATERIALS AND METHODS
Materials
Cilnidipine (CiL) was purchased from Yarrow Chem Products,
Mumbai. IMCD India Private Ltd., Bandra (East), Mumbai provided
gift samples of Polyoxyl 40 Hydrogenated Castor Oil
(Kolliphor®RH40), Oleyl alcohol (Kollicream®OA), Macrogolglycerol
Ricinoleate (Kolliphor®EL), and Macrogol (Kollisolv®PEG400).
Glyceryl mono-oleate (Paceol), Oleoyl polyoxyl-6 glycerides
(Labrafil®M 1944), Diethylene glycol monoethyl ether
(Transcutol®P), Propylene glycol monolaurate (Lauroglycol™ FCC)
were gifted by Gattefossé India, Vikhroli (East), Mumbai. Captex 355
was purchased from Abitee Corporation, USA. Glycerin,
Triethanolamine, oleic acid, Ethanediol, Tween 20, Ethanol,
Isopropyl alcohol, Isopropyl Myristate, PEG, PEG 200, and PEG 400
were procured from Thermo Fisher Scientific India Pvt. Ltd.,
Mumbai. Propylene glycol, Propanediol, and Ethyl oleate were
procured from Loba Chemie Pvt Ltd., Mumbai. Hydroxy propyl
methyl cellulose K4M and Carbo pol 940 were obtained from Sigma-
Aldrich Chemicals Pvt. Ltd, Bangalore. The solvents and remaining
chemical compounds were all analytical grade. All of the excipients
and chemicals were used precisely as obtained. A 0.45 µm
membrane filter (Deccan Plastics, Pvt. Ltd., Chh. Sambhajinagar,
India) was used to filter recently produced distilled water and when
needed.
Animals
Healthy Sprague Dawley and Wistar rats (Male and female) were
procured from LACSMI Biofarms Pvt Ltd, Pune, and used. Before
commencing the experiments, the Institutional Animal Ethical
Committee (IAEC) approved the experimental animal studies
(PCP/IAEC/2023/3-43 and PCP/IAEC/2023/3-44).
Methods
Preformulation assessment
Solubility study of cilnidipine
The solubility of Cilnidipine was assessed using the shake flask
system and measured in various oils, emulsifiers, and co-emulsifiers.
As per the methodology, these solvents were mixed with the right
amount of cilnidipine in 5 ml rubber-stoppered vials and 2 ml of
ethyl alcohol. The combination was then incubated for 48 h at 37 °C
in an orbital-shaker incubator (Remi Instruments, India). After ten
minutes of centrifugation at 5000 rpm, the supernatants were
filtered across a 0.45 μm membrane filter (Deccan Plastics, Pvt. L td.,
Chh. Sambhajinagar, India.) to remove any remaining particles. The
solubility of cilnidipine was determined by back dilution and
quantified using a UV spectrophotometer set at 243 nm. Each
experiment was carried out thrice to ensure consistent and
dependable results, reinforcing the reliability of our findings. Fig. 3
shows various components which solubilise the maximum amount
of the drug chosen for the study [25, 26].
Drug's chemical compatibility: excipients
100 mg of cilnidipine and 500 mg of each selected excipient were
carefully weighed and combined into five-milliliter glass vials with
rubber stoppers. Blends were maintained for 14 d in sealed vials in
humidity-controlled ovens at 60 °C and 40 °C/75% RH. A pure
standard cilnidipine sample was stored under the same conditions.
Two sets of drug-excipient combinations were analysed using FTIR
and DSC approaches after 14 d, as shown in fig. 4 [26–29].
Surfactant and co-surfactant testing: evaluation of the
emulsification efficiency
The emulsification properties were evaluated through visual
inspection. A specified amount of oil and emulsifier were measured
and blended in a 1:3 ratio to create a uniform mixture. The mixture
was thoroughly heated at 40-50 °C to promise uniformity.
Subsequently, 500 mg of the oil-surfactant mix was added to a 10 ml
beaker and gradually blended with a magnetic mixture until
dissolved. The degree of self-emulsification was optically assessed
based on the last appearance, dispersibility, and comfort of
emulsification, as indicated in table 1, and up to 10 ml of water was
increasingly added. Different co-surfactants were evaluated by
mixing specific emulsifiers in a 2:1 (w/w) mass ratio with co-
surfactants. To ensure a homogenous blend, the oily component was
added to the mixture at 1:3 (w/w), thoroughly vortexed, and heated
softly. This practical line evaluated the co-surfactants' capability to
emulsify [26].
M. T. Gaikwad & R. P. Marathe
Int J App Pharm, Vol 17, Issue 1, 2025, 255-274
257
Table 1: Evaluation of the emulsification efficiency
Dispersibility and occurrence
Self-emulsification time (min)
Score
Reference
Make a clear, transparent nanoemulsion by rapidly dispersing in water
<1
+++(very good)
[26]
The blend disperses into droplets that create a turbid emulsion in the water
3–5
+(good)
The mixture forms clusters of oil droplets that stay together in the water
Not emulsified
-(Poor)
A B
C D
E F
M. T. Gaikwad & R. P. Marathe
Int J App Pharm, Vol 17, Issue 1, 2025, 255-274
258
G H
I J
Fig. 2: Pseudo-ternary phase diagrams, A) Ethyl Oleate with Smix (Kollisolve®PEG400: Transcutol®P) 1:1, B) Ethyl Oleate with Smix
(Kollisolve®PEG400: Transcutol®P) 1:2, C) Ethyl Oleate with Smix (Kollisolve®PEG400: Transcutol®P) 1:3, D) Ethyl Oleate with Smix
(Kollisolve®PEG400: Transcutol®P) 3:1, E) Ethyl Oleate with Smix (Kollisolve®PEG400: Transcutol®P) 3:2, F) Kollicream®OA with Smix
(Kolliphor®RH40: Transcutol®P) 1:1, G) Kollicream®OA with Smix (Kolliphor®RH40: Transcutol®P) 1:2, H) Kollicream®OA with Smix
(Kolliphor®RH40: Transcutol®P) 1:3, I) Kollicream®OA with Smi x (Kolliphor®RH40: Transcutol®P) 3:1, J) Kollicream®OA with Smix
(Kolliphor®RH40: Transcutol®P) 3:2
Synthesis and characterisation of cilnidipine nanoemulsion
Creation of pseudo-ternary phase diagrams
Created pseudo-ternary phase diagrams are shown in fig. 2. This
study used triple combinations with diverse oil r atios, emulsifiers,
co-surfactants, and Chemix application ver. 10 was used to generate
them. Kollisolve®PEG 400 and Kolliphor®RH40's self-emulsification
ability played a role in the surfactant selection. The oil phase
consisted of Kollicream®OA and ethylene oleate, with Transcutol®P
as a co-surfactant. Less emulsification area led to the rejection of
ethyl oleate. Pseudo-ternary phase diagrams were created using
distilled water, Smix, and oil. The water titration method was used to
make it. The increasing surfactant concentration relative to co -
surfactant and vice-versa resulted in the identification of different
mass ratios between them (1:1, 1:2, 1:3, 3:1, and 3 is to 2). The oil
was blended in a discrete 10 ml borosilicate glass beaker with a
weight ratio of 1:9 to 9:1 using a specified mixing ratio. Phase
borders in each phase diagram were precisely created using 45
diverse ratios of oil to Smix (9:1, 8:2, 7:3 up to 1:9). When the various
o/w Nanoemulsions were clear or somewhat bluish, the titration
was ended, and water was calculated. Translucency was experienced
as the oil and Smix co mbinations were diluted in the water phase. We
discarded the remaining nanoemulsions. The mass percent of water,
oil, and Smix were recorded at these terminations because the sum
was 100 %. Phase diagrams showing the physical state of
nanoemulsions revealed the gathering of the Smix phase, oil phase,
and aqueous phase in a single region. Each diagram described the
nanoemulsion area, with a more extensive region indicating more
efficient emulsification [30, 31].
Formulation of cilnidipine-loaded nanoemulsions
Cilnidipine is carefully added to the nanoemulsion system after
investigating pseudo-ternary phase diagrams showing a more
extensive nano-emulsification region. Kollicream®OA, surfactant-
co-emulsifier mixture 1:3; (Ko lliphor®RH40/Transcutol®P) were
used. A weighed amount of cilnidipine is added to the oil in a 10 ml
beaker, heated at 40 to 50 °C in a bath sonicator (Enertech Pvt.
Ltd., Mumbai, India) with vor texing it, and then slowly mixed with
calculated Smix. The resultant mixture was titrated against water,
and this assembly was placed over a magnetic stirrer (1 MLH
Magnetic Stirrer, Remi Instruments, Mumbai, India) at 500 rpm to
produce a raw emulsion. Droplet size was reduced using probe
sonication from 0.33 min to<6 min, giving fine nanoemulsions
[32–34].
M. T. Gaikwad & R. P. Marathe
Int J App Pharm, Vol 17, Issue 1, 2025, 255-274
259
Table 2: Trial batches of cilnidipine nanoemulsions with ethyl oleate
Smix ratio
Formulation code
% w/w components in formulation
Oil (%)
Water (%)
Smix (S+CoS %)
NEn-A
Smix ratio 3:1
NEn_CiL_A1
7.5
36
56.5
NEn_CiL_A2
12.5
30
57.5
NEn_CiL_A3
17.5
26.7
55.8
NEn_CiL_A4
22.5
24
53.5
Table 3: Trial batches of cilnidipine nanoemulsions with kollicream®OA
Smix ratio
Formulation code
% w/w components in formulation
Oil (%)
Water (%)
Smix (S+CoS %)
NEn-B
Smix ratio 1:1
NEn_CiL_B1
5.2
75.2
19.6
NEn_CiL_B2
10.2
58.7
31.1
NEn_CiL_B3
15.2
40.7
44.1
NEn_CiL_B4
20.2
33.2
46.6
NEn-C
Smix ratio 1:3
NEn_CiL_C1
3
83.6
13.4
NEn_CiL_C2
8
63
29
NEn_CiL_C3
13
51.1
35.9
NEn_CiL_C4
18
40.6
41.4
NEn-D
Smix ratio 1: 2
NEn_CiL_D1
5.2
77.8
17
NEn_CiL_D2
10.2
60.5
29.3
NEn_CiL_D3
15.2
45
39.8
Optimisation of cilnidipine-loaded nanoemulsion by 2-factor
central composite design
Utilising response surface methodology (RSM), the analysis of
independent variables was conducted, like a mixture of surfactant
and co-surfactant [Smix] (X1) and ultrasonic irradiation time (X2),
affected response variables, such as drug content (Y2), particle size
(Y1), and PDI (Y3) in nanoemulsions. In table 4, the central
composite design and coded levels are displayed. A quadratic model
and a central composite design (five levels) were used in the
experiment's design. Table 9 lists the thirteen experimental runs,
coded as Design of Experiment (DOE_CiL_1 to 9), and included five
centre points (repeated, so only one is measured), four axial points,
and four factorial points, so a total of nine runs were conducted
randomly. Design-Expert®Software (Stat-Ease Inc., Trial version 13,
Minnesa, USA) was employed in designing the experiment to create
optimised cilnidipine-loaded nanoemulsions. Trail batch NEn_CiL_C2
that underwent this optimisation [35–37].
Table 4: Variables used in 2-factor central composite design
Independent variables
Levels
-α
-1
0
+1
+α
X1: Smix (%)
22.92
25
30
35
37.07
X2: Ultrasonic irradiation time (min)
0.1715
1
3
5
5.8284
Dependent variables
Y1: Particle size (nm)
Minimise
Y2: Drug content (%)
Maximise
Y3: PDI
Minimise
Thermodynamic stability studies
Multiple thermodynamic stability tests were carried out to assess
the physical stability of the formulations [38].
Cycle of heating-cooling
To evaluate the stability of the formulations, they were kept at 4° C
in the refrigerator and 45° C for a minimum of 48 h, changing the
temperature six times in succession [38].
Test for centrifugation
An investigation into phase separation and drug settling was conducted
by centrifuging nanoemulsions for 30 min at 3000 rpm [38].
Freeze-thaw cycle
Three freeze-thaw series were employed throughout 48 h from
temperatures-21 °C to 25 °C. [38].
Characterisation of cilnidipine nanoemulsion
Cilnidipine nanoemulsion globule size measurement
We used the Malvern particle size analyser (Zetasizer ver. 6.20,
Model: MAL1051945, Malvern Instruments, Ltd., UK). The globule
size of the nanoemulsions was assessed, with each size value stated
as the mean±SD of three samples. The polydispersity index was
computed to evaluate the uniformity of particle diameters [35, 38].
Zeta Potential estimation of cilnidipine nanoemulsion globules
Electrophoretic light scattering with a Malvern Zetasizer (Zetasizer
ver. 6.20, Model: MAL1051945, Malvern Instruments, Ltd., UK) was
used to estimate the zeta potential of the cilnidipine nanoemulsion.
The experiment was conducted with a constant electrical field of 1
volt using a dynamic light scattering particle size analyser set to 633
nm. The nanoemulsion was diluted at a 1:100 ratio using pre-
filtered, double-distilled water. The information was presented as
mean±SD based on three separate assessments [35, 38].
Viscosity
A Brookfield R ST rheometer (Brookfield Engineering Laboratories,
Mumbai) with a MS: CCT-14 was used to measure the viscosity of the
cilnidipine nanoemulsions at 25 °C. Three measurements were made
[35, 38].
Refractive index
The nanoemulsion's refractive index was determined by applying
one drop in triplicate to the slide at 25 °C using a refractometer
(Cyber-Lab, Cyber AB, Hyderabad) [35, 38].
M. T. Gaikwad & R. P. Marathe
Int J App Pharm, Vol 17, Issue 1, 2025, 255-274
260
pH determination
The apparent pH of the cilnidipine nanoemulsion was determined
using a pH meter in triplicate at 25 °C (Systronics, model 802, India)
[35, 38].
Transmission electron microscopy (TEM)
The morphology of the cilnidipine nanoemulsion was examined. To
ascertain its dimensions and form, a point-to-point-separable Gatan 626
cryo specimen holder electron microscope (TECNAI 12, Fei Company,
The Netherlands, Software: Tecnai Imaging and Analysis, Source:
Tungsten Filament) operating at 20–120 kV was utilised. Bright-field
imaging techniques and diffraction modes were applied [35, 38].
In vitro permeation study for optimised cilnidipine-loaded
nanoemulsions
In vitro skin permeation experiments were carried out using Franz
diffusion cells. To simulate the skin, the donor and r eceptor
compartments were separated by the Dialysis Membrane-60, with a
molecular size of 12000 Da. (Hi-Media Laboratories Pvt. Limited,
Thane). Additionally, the dialysis membrane received the calculated
dosage of optimised nanoemulsion, equivalent to 10 mg of
medication. A 3 x 12 mm magnetic bead (Orchid Scientific and
Innovative India Pvt. Ltd., Nashik, India) was utilised to ensure the
drug was distributed uniformly and at the proper temperature. The
medium used in this experiment consisted of 75% phosphate buffer
7.4 and 25% isopropyl alcohol. Approximately 1 ml of samples was
taken from the receptor compartment at 0.5, 1, 2, 4, 6, 8, 12, and 24
h. These samples were then replaced with equivalent medium
volumes to maintain sink condition. The dialysis membrane's total
drug penetration was measured at µg/cm2. The steady-state flux
(µg/cm2/h) was the slope of the steady part of the graph, and the
permeability coefficient was determined by dividing flux by
concentration in the donor compartment, as indicated in fig. 16,
table 15 [35, 39].
Formulation and optimisation of cilnidipine-loaded
nanoemulsion-gel
For this investigation, first, we prepared 20 different simple gel
formulations [(Coded as SG1-SG20)-Data Hidden] using HPMC K4M
and Carbopol 940 as gelling agents, with their percentages ranging
from 0.25 to 1.5 % with an increment of 0.25 %. Glycerin was used
5-10 % for ten simple gel formulations with HPMC K4M and ten with
Carbopol 940. A methodologically weighed amount of Carbopol 940
was added to 100 ml of distilled water to swell overnight. HPMC K4M
was separately dispersed in hot water at a slightly modified
temperature at 80 °C and stirred till room temperature. Other
excipients were slowly added with continuous stirring, and a
sonicator was used for 10 min to remove the trapped air. Finally,
triethanolamine was added to adjust pH 6 to 6.8. These simple gels
were optimised by checking viscosity and spreadability, and only
eight formulations (HPMC K4M and Carbopol 940 with 1 and 1.5 %)
were selected for further study, as reported in table 5 [40–43]. SG 19
was selected based on its characterisation for the nanoemulsion
gelling process. 40 g of Optimised nanoemulsion DOE_CIL_7 was
slowly and continuously stirred with 14 g of simple gel SG 19 to get
approximately 54 g of cilnidipine-loaded nanoemulsion gel, as
shown in table 6 [40, 41, 44].
Table 5: Simple gel (SG) formulations
Formulation
code
HPMC K4M
% w/w
Carbopol
940 % w/w
PEG 400 %
w/w
Glycerol
% w/w
Methylparaben
Peppermint
oil
Triethanolamine
w/w
Distilled
water
SG4
1
5
5
0.03
0.2
Q. S.
Q. S 100
SG5
1.5
5
5
0.03
0.2
Q. S.
Q. S 100
SG9
1
5
10
0.03
0.2
Q. S.
Q. S 100
SG10
1.5
5
10
0.03
0.2
Q. S.
Q. S 100
SG14
1
5
5
0.03
0.2
Q. S.
Q. S 100
SG15
1.5
5
5
0.03
0.2
Q. S.
Q. S 100
SG19
1
5
10
0.03
0.2
Q. S.
Q. S 100
SG20
1.5
5
10
0.03
0.2
Q. S.
Q. S 100
Table 6: Cilnidipine-loaded nanoemulsion gels
Ingredients
Cilnidipine-loaded optimized nanoemulsion
Formulation
code
Cilnidipine
%
Carbopol
940 gel 1 %
Carbopol 940
Gel 1.5 %
HPMC K4M
gel 1 %
HPMC K4M
gel 1.5 %
Mls of nanoemulsion
added
KOA
Smix
Water
NEn_CiL_1
0.201
14 gm
40
8
30
62
NEn_CiL_2
0.201
14 gm
40
8
30
62
NEn_CiL_3
0.201
14 gm
40
8
30
62
NEn_CiL_4
0.201
14 gm
40
8
30
62
Characterization of cilnidipine-loaded nanoemulsion-gel
Exterior appearance, pH, and viscosity
The prepared gel's exterior appearance, pH, and viscosity were
examined. Visual evaluation was done to determine the
nanoemulsion gel's clarity, consistency, and homogeneity. The pH of
the optimised gel was measured using a digital pH meter (Model
802, Systronics, India) at room temperature using a 1 percent
solution of nanoemulsion gel. A Brookfield RST rheometer
(Brookfield Engineering Laboratories, Mumbai) determined the
viscosity of cilnidipine-loaded nanoemulsion gel at 25±3 °C. Three
readings were taken [18, 35].
Spreadability
A lower glass slide turned into a constant in this block. An extra
prepared nanoemulsion gel (approximately 1 gm) underneath the
study was placed on this ground slide. The nanoemulsion gel
formulation was then inserted among this slide and some other glass
slides, which had the dimension of a fixed ground slide and turned
into provided with a hook. To release air and create a consistent gel
layer between the slides, a 100 g weight is placed on each slide for
five minutes. Excess of the nanoemulsion gel was removed from the
borers. The upper slide was then exposed to a tug of 25 g weight
with the help of a cord linked to the hanger, and the time (in sec)
required via the top slide to cover a distance of 7 cm was mentioned.
A briefer interval indicates higher spreadability [41].
Extrudability
The capped collapsible aluminium tubes were filled with
nanoemulsion gel formulations and sealed by crimping. We recorded
the tube weights. Clamps were used to position the tubes between
two glass slides. Afterwards, the cap was removed, and 50 g was
placed over the slides. We counted and weighed the amount of
extruded nanoemulsion gel [41].
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Int J App Pharm, Vol 17, Issue 1, 2025, 255-274
261
In vitro permeation study for cilnidipine-loaded nanoemulsion-
based gel
In vitro skin permeation experiments were carried out using Franz
diffusion cells. The dialysis membrane-60 separated the donor and
receptor compartments to simulate the skin. The Donar compartment
received 1.5 g of optimised nanoemulsion gel, equivalent to 0.2 % of
the drug. The diffusion medium (75% phosphate buffer 7.4 and 25%
isopropyl alcohol) was agitated using a Teflon-coated magnetic bead
to maintain a constant drug circulation at 37±2 °C. About 1 ml of
sample was taken from the receptor compartment at 0.5, 1, 2, 4, 6, 8,
12, and 24 h. These samples were replaced with equivalent medium
volumes of diffusion media to maintain sink conditions. The dialysis
membrane’s total drug penetration was measured in µg/cm2. Steady-
state flux (µg/cm2/h) is the slope of the linear part of the line, and the
permeability coefficient is calculated by dividing the steady-state flux
by the drug’s concentration in the donor compartment, as indicated in
fig. 17, table 16 [35, 39].
Acute dermal toxicity study of cilnidipine-loaded
nanoemulsion-based gel
In a study on acute dermal toxicity in female Wistar albino rats, the
procedures were conducted per OECD Guideline No. 402. Fifteen
healthy Wistar Albino rats weighing 200-250 g were divided into
three groups. The standard control and test groups were prepared,
each consisting of 5. Before the study commenced, the rats' backs
were clipped and individually caged for 24 h. On the test day, the
group received an o ptimised nanoemulsion gel of cilnidipine, which
was evenly applied to the exposed skin. Over the next 14 d, the rats
were observed twice daily for signs of irritation, changes in
behavior, and mortality. Water intake, diet intake, and body mass
were measured every day. The rats were sacrificed to examine and
weigh their organs on the fifteenth day. This study focused on rat
skin reactions, general health, and organ function to assess the acute
dermal toxicity of cilnidipine-loaded nanoemulsion gels. Every
animal in the study was observed daily to evaluate clinical indicators
such as irritability, behavioural changes, toxicity symptoms,
morbidity, and mortality. They were monitored closely for any
changes or reactions, and detailed daily records of their clinical
status were kept. Physiological parameters, including body weight,
food and water consumption, and haematological and serum
biochemical profiles, were assessed to fully comprehend the effect of
the test substances on the animals' health [45–49].
Pharmacokinetic study
15 Sprague Dawley rats were divided into five groups of 3 animals
each. Standard Control/Control group (G1) animals have received no
treatment. Animals of Group G2 and G3 were administrated orally
with Marketed formulations of cilnidipine tablets at the doses of 1.1
mg/kg. The doses were calculated according to the reported method.
Further, the skin on the abdominal side of groups G4 and G5 rats
was shaved with depilated cream. For the test formulations, a
cilnidipine-loaded nanoemulsion gel was applied topically. The rats
underwent isoflurane anesthesia, and 500 µl** of blood was
extracted from their retro-orbital plexus in EDTA tubes at various
time breaks, including 0.25, 0.5, 1, 2, 3, 6, and 12 h. The blood
samples were centrifuged at 3500-4000 rpm for 10-12 min. After
separation, the blood plasma was kept at-21 °C. Then, drug analysis
was carried out using a developed HPLC method [45–47, 49].
RESULTS AND DISCUSSION
Solubility study of cilnidipine
Based on the solubility of cilnidipine in various components, oils,
surfactants, and cosurfactants were selected. Results revealed that
cilnidipine has maximum solubility in oils Kollicream®OA38.2±2.36
and Ethyl Oleate 12.5±1.02. Maximum solubility found in surfactants
Kolliphor®RH40 51.72±2.36, Kollisolv®PEG400 19.45±2.31 and co-
surfactants like Ethanol 29.76±1.56 and in Transcutol®P 48.92±2.74.
All other excipients with more solubility were rejected due to their
less emulsification efficacy tested as per the protocol shown in table
1. All values represented in mg/ml are denoted in fig. 3.
Fig. 3: Solubility of cilnidipine in oils, surfactants, and co-surfa ctants, data represented as mean±SEM, n = 3 observations
Drug's chemical compatibility: excipients
Cilnidipine's FTIR spectroscopy and formulations with different
excipients were essential to the for mulation process. This study
confirmed the integrity of critical functional gro ups and offered
insightful information about molecular interactions. The
particular abso rption peaks s howed no interaction w ith
Kollicream®OA. Still, mild interactio ns with Kolipho r®RH40,
Transcutol®P, Kollisolv®PEG400, and ethyl o leate were show n,
as illustrated in fig. 4. The analysis verified cilnidipine's
molecular structure and purity , guaranteeing the creation of
stable nanoemulsions.
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262
Fig. 4: FTIR study of various components in 1:5 physical mixtures
Surfactant and co-surfactant testing: evaluation of the
emulsification efficiency
Emulsification efficacies research was carried out on
components selected based on the solubility of oils, surfactants,
and co-surfac tants. A visual appraisal system is em ployed.
Results showed that amongst Kollicream®OA, Ethyl Oleate and
Paceol w ere selected as oil phases and tested agai nst a series of
surfactants, Kolliphor®RH40, Ko lliphor®EL, Kollisolve®PEG 400
show ed that Kolliphor®RH40 can emulsify Kollicream® OA,
wher eas Kollisolve® PEG 400 can emulsify Ethy l Oleate in 1:3
w/w mass ratios. Paceol was not emulsified. Selected surfactants
were then studied against E thanol and T ranscutol®P as co-
surfactants efficiency against oil. Hence, only Transcutol®P was
able to em ulsify oil in combinatio n with 1% w/w oil. Results
were demonstrated in table 7 and table 8.
Table 7: Emulsification efficacy of emulsifier with selected oil (oil 1: surfactant 3) w/w
Emulsifier (3%
w/w)
Oils (1% w/w)
Kollicream®OA
Ethyl Oleate
Dispersibility and
presence
Self-emulsification
period (min)
Score
Dispersibility and
presence
Self-emulsification
period (min)
Score
Kolliphor®RH40
The transparent
spontaneous blue
emulsion formed
1-2 min
+++Very
good
Turbid
>2 min
-Poor
Kollisolve®PEG400
Turbid emulsion
>4 min
+Good
The clear spontaneous
blue emulsion formed
1-2 min
+++Very
good
Kolliphor® EL
Turbid
>3 min
-Poor
Turbid
>4 min
-Poor
Table 8: Emulsification efficiencies of Smix combinations with oil (oil 1: surfactant 2:co-surfactant 1) w/w
Co-surfactant
(1% w/w)
Oily phases (1% w/w) Kollicream®OA
Surfactant (2% w/w)
Kolliphor®RH40
Kollisolve®PEG400
Dispersibility and
presence
Self-emulsification
period (min)
Score
Dispersibility and
presence
Self-emulsification
period (min)
Score
Transcutol®P
Clear nanoemulsion formed
<1 min
++++Very good
Turbid
>2 min
-Poor
Ethanol
Turbid
>3 min
-Poor
Turbid
>3 min
-Poor
Smix mass ratio's impact on nanoemulsion region of pseudo-
ternary phase diagram
A pseudo-ternary phase diagram identifies the nanoemulsion region.
Kollicream®OA and ethyl oleate were utilised as the oil phase. In the
screening process, Transcutol®P was used as a co-surfactant, and
Kolliphor®RH40 and Kollisolve®PEG400 were used as surfactants.
Phase diagram investigation discovered that, as shown in fig. 2F to J,
the primary region for nanoemulsions was identified in Smix
proportions of 1:1 and 1:2. The maximum amounts of oil that could
be soluble can be seen in these diagrams. Fig. 2F reveals a 30%
wt/wt solubility at a 52.2 % wt/wt Smix ratio for 1:1 and 28% wt/wt
solubility of Kollicream ®OA at a 55.5 % wt/wt Smix ratio for 1:2.
Greater emulsification efficiency inside the system is shown by an
increased nanoemulsion area fig. 2, Tables 7 and 8 show that
Transcutol®P performed better than the other groups, probably
because of its high cilnidipine solubility. Increasing the surfactant
concentration at a 1:3 Smix ratio (fig. 2H) resulted in less region, and
a 3:1, 3:2 Smix ratio (fig.2I, J) resulted in no nanoemulsion area.
Therefore, experimenting with a 4:1 Smix ratio was pointless.
A decrease in the nanoemulsion region was noted after the
surfactant concentration of Smix was raised from 1:1 to 3:1 for
Kollicream ®OA. A low concentration of co-surfactant could explain
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this, as it would lessen interfacial tension and increase the flexibility
of the nanoemulsion region and interface. No nanoemulsion zone
was found in fig. 2 A–D or fig. 2E for Ethyl Oleate, with a smaller
nanoemulsion region [50, 51]. Our examination of the nanoemulsion
area has produced significant recommendations for the
effectiveness of drug delivery. The region shortened as the co-
surfactant concentration rose from Smix 1:1 to Smix 3:2. As the co-
surfactant concentration increased, the area shrank increasingly,
reaching 3:1 and 3:2 Smix ratios. Phase diagrams optically illustrate
how Smix declines interfacial tension and increases interfacial area.
Introducing Smix can significantly lower the free energy of the
nanoemulsion system to a minimum concentration, ensuring
thermodynamic stability and presenting a hopeful method.
Formulation of cilnidipine-loaded nanoemulsions
NEn_CiL_A1 to A4 were synthesised from Smix mass ratio 3: 1,
whereas Smix ratios 1:1, 1:2, one is to 3 were used to synthesise
NEn_CiL_B1 to B4, C1 to C4, and D1 to D3. D4 is not synthesised due
to gelling before water titration. NEn_CiL_C2 is selected for further
optimisation as it contains less Smix concentration than others, which
will justify the toxicity issues of Smix. Not o nly was this phase
separation observed in NEn_CiL_A1 and A2 after the Cycle of
Heating-Cooling, but NEn_CiL_A3 and A4 were unstable after the
Freeze-Thaw Cyc le. NEn_CiL_B2, B3 sho ws increased viscosity,
turbidity, and phase separation after the heating and c ooling
cyc le. NEn_CiL_B4 s hows a complete milky appearance.
NEn_C iL_C1 and C3 have slight t urbidity. C4 is slightly apparent,
wher eas NEn_CiL _B1 and C2 fo rmed clear and stable
nanoemuls ions even after thermo dynamic stability studies.
Hence, NEn_CiL_C2 is selected for further optimi zation, as
indic ated in fig. 5.
Optimisation of cilnidipine nanoemulsion
Investigation of optimization by 2-factor central composite
design
A comprehensive literature review was meticulously conducted
before the synthesis of cilnidipine nanoemulsions. This step was
crucial as it provided a solid foundation for selecting oils, a
combination of emulsifiers and co-emulsifiers, based on proper
emulsification analysis and the solubility of cilnidipine in each
excipient. Subsequently, trial nanoemulsions were synthesised
based on the nanoemulsion area in pseudo-ternary phase diagrams.
The NEn_CiL_C2 batch was then chosen for optimisation.
Fig. 5: Cilnidipine-loaded nanoemulsions (Trial batches)
Table 9: Optimisation by the central composite design of batch NEn_CiL_C2
Independent variables
Dependent variables
Batch code
Run
Smix (KOLRH40_TrP)
factor 1
Ultrasonic irradiation time
(min) factor 2
Particle size (nm)
response 1
Drug content
(%) response 2
PDI response
3
DOE_CiL_1
1
30
0.1715
151.9
99.5
0.341
DOE_CiL_2
2
35
5
215.2
100.1
0.321
DOE_CiL_3
3
37.07
3
350
101
0.485
DOE_CiL_4
4
25
5
105
99.2
0.125
DOE_CiL_5
5
22.92
3
120
98.3
0.179
DOE_CiL_6
6
30
5.8284
123.3
97.2
0.286
DOE_CiL_7
7
30
3
97.70
99.8
0.384
DOE_CiL_8
8
35
1
380
120
0.395
DOE_CiL_9
9
25
1
120
101
0.215
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264
Fig. 6: Cilnidipine-loaded optimised nanoemulsions
Cilnidipine nanoemulsion optimisation results
The response surface method approximates the correlation function
using a complete quadratic equation. The impact of independent
variables on particle size (Y1), drug content (Y2), and PDI (Y3) are
given in table 4. Polynomial equation coefficients were calculated
using experimental data to predict the response variable's values.
Regression equations for each response variable, obtained from
response surface methodology, are mentioned in
Y= β0+β1A+β2B+β3AB+β4 A2+β5 B2
Where Y is the designated response, β0– intercept, β1– β5 are the
regression coefficients, A and B are the factors studied,
The relationship between the response model equation and particle
size, Drug Content, and PDI is given below:
Particle size (Y1) =+97.60+86.10*A-28.23*B-
39.10*AB+73.68*A2+24.60*B2 ----------- (1)
Drug Content (Y2) =+101.00+0.6036*A-0.6250*B-0.1000*AB-
0.4644*A2-1.01*B2 ----- (2)
For PDI (Y3) =+0.0.3840-0.1001*A-0.0307*B+0.0055*AB-0.0397A2-
0.0493*B2 ------- (3)
The results of the ANOVA are shown in tables 10, 11, and 12. Overall,
each model was significant (p>0.05). Non-significant terms (p>0.05)
were removed from equations to simplify models, resulting in the
following data.
Independent variable's impact on the response
Optimisation by the central composite design and different levels of
independent variables were used to synthesise cilnidipine-loaded
nanoemulsions, as indicated in fig. 6. Table 9 shows the impact of
independent variables on particle size, drug content, and PDI. Table
13 summarises the regression coefficients for the independent
variables. The model F-values of 43.88, 5.89, and 19.94 for Particle
size, drug content, and PDI, respectively, imply that the model is
significant. 3-dimensional surface response plots of the separate
process constraints particle size, drug content, and PDI are indicated
in fig. 7, 8 and 9, as well as the cumulative effect of independent
variables on particle size, drug content and PDI are noted in fig. 10,
11 and 12. The findings indicated that as S mix concentration rises to
35 ml with an ultrasonic irradiation time of 1 min, particle size
increases to 382 nm. With an optimum concentration of Smix 30 ml
and ultrasonic irradiation time of 3 min, we will get particle size 97.7
nm. For drug content analysis, the concentration of Smix 25 ml with
an ultrasonic irradiation time of 5 min drug content was observed at
98.4 %, and Smix 30 and time 3 min will get drug content 101.1 %. As
we aimed to minimise the Particle dispersity index, Smix 25 with an
ultrasonic irradiation time of 5 min led to generating a PDI of a
minimum value of 0.127.
Table 10: ANOVA study for particle size
Source
Sum of squares
df
Mean square
F-value
p-value
Model
1.112E+05
5
22231.44
43.88
<0.0001
Significant
A-Smix
59309.23
1
59309.23
117.07
<0.0001
B-Ultrasonic irradiation time
6374.01
1
6374.01
12.58
0.0094
AB
6115.24
1
6115.24
12.07
0.0103
A²
37769.01
1
37769.01
74.55
<0.0001
B²
4209.38
1
4209.38
8.31
0.0236
Residual
3546.17
7
506.60
Lack of Fit
3546.17
3
1182.06
Pure Error
0.0000
4
0.0000
Cor total
1.147E+05
12
Table 11: ANOVA study for drug content
Source
Sum of squares
df
Mean square
F-value
p-value
Model
13.92
5
2.78
5.89
0.0189
significant
A-Smix
2.91
1
2.91
6.17
0.0420
B-Ultrasonic irradiation time
3.13
1
3.13
6.61
0.0369
AB
0.0400
1
0.0400
0.0847
0.7795
A²
1.50
1
1.50
3.17
0.1180
B²
7.05
1
7.05
14.93
0.0062
Residual
3.31
7
0.4725
Lack of Fit
3.31
3
1.10
Pure Error
0.0000
4
0.0000
Cor total
17.22
12
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265
Table 12: ANOVA study for PDI
Source
Sum of squares
df
Mean square
F-value
p-value
Model
0.1125
5
0.0225
19.94
0.0005
significant
A-Smix
0.0801
1
0.0801
70.99
<0.0001
B-Ultrasonic irradiation time
0.0075
1
0.0075
6.67
0.0363
AB
0.0001
1
0.0001
0.1072
0.7529
A²
0.0110
1
0.0110
9.74
0.0168
B²
0.0169
1
0.0169
14.95
0.0062
Residual
0.0079
7
0.0011
Lack of Fit
0.0079
3
0.0026
Pure Error
0.0000
4
0.0000
Cor total
0.1204
12
Fig. 7: 3-D surface response design showing the impact of separate process constraints on particle size
Fig. 8: 3-D surface response design showing the impact of separate process constraints on drug content
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266
Fig. 9: 3D surface response design showing the impact of separate process constraints on PDI
Fig. 10: Impact of Smix and ultrasonic irradiation time on particle size
Fig. 11: Impact of Smix and ultrasonic irradiation time on drug content
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267
Fig. 12: Impact of Smix and ultrasonic irradiation time on PDI
Table 13: Regression coefficient values for optimised cilnidipine nanoemulsion
Regression coefficients
Particle size (nm)
Drug content (%)
PDI
Intercept
97.60*
101.00*
0.3840*
A-Smix
86.10*
0.6036*
0.1001*
B-Ultrasonic irradiation time
-28.23*
-0.6250*
-0.0307*
AB
-39.10*
-0.1000*
0.0055*
A²
73.68*
-0.4644*
-0.0397*
B²
24.60*
-1.01*
-0.0493*
R2
0.9691
0.8079
0.9344
*Data represented indicates P<0.05
The experimental data could be well represented, according to the
results of statistical analysis (ANOVA), by a quadratic polynomial
model, with coefficient of determination (R2) values for particle
size (Y1), drug content (Y2), and PDI (Y3) being 0.9691, 0.8079,
and 0.9344 as shown in table X. Our model is statistically accurate
because lack of fit was non-significant (p<0.05) with pure error for
all variables. It indicates that the model fits the data better if the
R2 value is closer to unity. Conversely, response variables are
unsuitable for explaining the behavioral var iation, acco rding to
lower R2 values [52]. The influence of Smix concentration (X1) and
ultrasonicatio n irradiation time (X2) on response variables in our
study could be suitably described by a quadratic polynomial
model, as evidenced by the closeness to 1. Using analysis of
variance (ANOVA), the significance level fo r the quadratic
polynomial model's coefficients was established. Larger F-values
and smaller P-values suggest that any term significantly impacts
the response variable [37].
Thermodynamic stability studies
The cycle of heating-cooling, test fo r centrifugation, the freeze-
thaw cycle
As per the procedures discussed earlier, we found that NEn_CiL_B1
and C2 formed clear and stable nanoemulsions after a comprehensive
thermodynamic stability study [38]. Optimised formulations
DOE_CiL_2, 3, and 8 were found unstable after stress testing, while
other formulations were observed to have robust stability.
Characterization of cilnidipine-loaded nanoemulsions
Cilnidipine-loaded nanoemulsion’s Viscosity, pH, and refractive
indices were expressed in table 14. The outcomes of the particle
size, zeta potential, and Transmission Electron Microscopy of the
selected optimised cilnidipine-loaded nanoemulsions are reported
in fig. 13 and 14, 15 [35, 38].
Table 14: Characterisation of optimised cilnidipine-loaded nanoemulsion
Code/Design of experiments (DOE)
Viscositya (Pa*s)
pHa
Refractive indexa
DOE _CiL_1
0.120±0.00119
6.67±0.15
1.348±0.04
DOE _CiL_2
0.123±0.0061
6.16±0.03
1.432±0.07
DOE _CiL_3
0.137±0.002
6.65±0.03
1.411±0.04
DOE _CiL_4
0.100±0.0021
6.16±0.02
1.374±0.08
DOE _CiL_5
0.0861±0.0011
6.38±0.08
1.382±0.02
DOE _CiL_6
0.121±0.0023
7.01±0.13
1.401±0.03
DOE _CiL_7
0.117±0.00119
6.33±0.15
1.312±0.06
DOE _CiL_8
0.127±0.00144
6.79±0.01
1.417±0.07
DOE _CiL_9
0.0981±0.00283
6.28±0.02
1.368±0.09
aData represented as mean±SEM, n = 3 observations
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268
Fig. 13: Particle size analysis of cilnidipine-loaded nanoemulsion, DOE_CiL_7
Fig. 14: Zeta potential of cilnidipine-loaded optimised nanoemulsion, DOE_CiL_7
A B
Fig. 15: Transmission electron microscopy of cilnidipine-loaded optimised nanoemulsion DOE_CiL7 (A, B), Scale bar is 50 nm; (B) Scale
bar is 500 nm
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In vitro permeation study of optimised cilnidipine-loaded
nanoemulsions
The optimised formulations DOE_CiL_1 to 9 containing 10 mg
equivalent cilnidipine were tested for in vitro permeation. The
cumulative amount permeated from optimised DOE_CiL_1 to 9
nanoemulsions for 0.5 to 24 h is represented in fig.16. The steady-
state transdermal flux and permeation coefficient values are
displayed in fig. x. The maximum flux in DOE_CiL_7 was 107.7±2.04
µg/cm2/h, and the permeability coefficient was 1.07 cm/h × 10−2.
Fig. 16: Cumulative amount permeated from cilnidipine-loaded optimised nanoemulsions, data represented as mean±SEM, n = 3
observations
Table 15: Steady-state flux and permeability coefficients for cilnidipine-loaded optimised nanoemulsions
Cilnidipine-loaded optimised NEns
Jssa (µg/cm2/h)
Kpa(cm/h) × 10−2
Calculated flux
47.5±2.36
0.47±0.037
DOE_CiL_1
56.65±1.08
0.56±0.021
DOE_CiL_2
48.59±0.71
0.48±0.047
DOE_CiL_3
43.97±0.66
0.43±0.032
DOE_CiL_4
80.65±2.24
0.80±0.087
DOE_CiL_5
74.45±2.33
0.74±0.048
DOE_CiL_6
68.84±0.65
0.68±0.067
DOE_CiL_7
107.7±2.04
1.05±0.094
DOE_CiL_8
35.71±1.27
0.35±0.071
DOE_CiL_9
65.14±1.02
0.65±0.087
aData represented as mean±SD, n = 3 observations
Formulation and optimisation of cilnidipine-loaded
nanoemulsion-gel
Results showed that some formulations w ere discarded from simple
gel 1 to simple gel 20 (SG1_SG20) due to the low viscosity profiles of
simple gels. Only four formulations were decided to contain 1 and
1.5% of Carbopol 940 and four for HPMC K4M. Out of those,
Cilnidipine was loaded in Simple Gel 4,10,15,19 (SG 4,10,15,19) to
synthesise cilnidipine-loaded formulations re-coded as
NEn_CiL_Gel_01 to 04, showing excellent results [40, 41, 44].
Characterisation of cilnidipine-loaded nanoemulsion-gel
Exterior appearance, pH and viscosity
Results are expressed in table 17. pH of NEn_CiL_Gel_01_04 was
noted as 6.21±0.35 to 6.36±0.25 and viscosity values as
3.039±0.0038 to 2.193±0.0037 in Psa [18, 35].
Spreadability, extrudability
The table represents the spreadability and extrudability of
optimised cilnidipine-loaded nanoemulsion gels
(NEn_CiL_Gel_01_04). NEn_CiL_Gel_01_04 shows varied results, with
spreadability ranging from 9.52±0.34 to 13.84±0.51 g/cm/s, and
excludability is graded [41].
In vitro permeation study for cilnidipine-loaded nanoemulsion-
based gel
The developed optimised cilnidipine-loaded nanoemulsion gels
(NEn_CiL_Gel_01_04), which contain 1per cent Carbopol 940
(NEn_CiL_04), were selected as the optimised formulation since
they have a satisfactory viscosity profile and gelling capacity. The
cumulative amount of drug permeated for 0.5 to 24 h from the
diffusio n membrane was shown in fig. 17, and steady-state
transder mal flux and permeability coefficient were repor ted in
table 16. We found a maximum flux from 80.64±1.38 µm/cm2/h
for NEn_CiL_Gel_03 and a permeability coefficient of
0.267±0.0045×10−2 cm/h. Hence, it is reported that 1% Carbopol
940 gel had 26.703±0.459 maximum flux, and the permeability
parameter indicated that the nanoemulsion gel had improved
transder mal flux [35, 39].
Acute dermal toxicity study
The current study used female Wistar albino rats to investigate the
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acute dermal toxicity of cilnidipine-loaded nanoemulsion gel. The
results demonstrated that the treatment with cilnidipine-loaded
nanoemulsion gel did not cause any evidence of animal death.
Additionally, after applying the cilnidipine-loaded nanoemulsion gel,
there were no visible signs of erythema (redness) or oedema
(swelling) (table 18). The analysis shows no irritation or
inflammation on the skin when using the gel formulation. Compared
to normal animals, female animals that received topically applied
cilnidipine-loaded nanoemulsion gel demonstrated a significant
(P<0.05) decrease in body weight. Feed and water consumption was
not altered significantly ((P<0.05) by the treatment of cilnidipine-
loaded nanoemulsion gel (table 19 and20). Biochemical parameters,
including triglycerides, HDL, LDL, total cholesterol, SGOT, and SGPT,
were estimated after the experiment. The female animals'
biochemical parameters showed no significant (P<0.05) changes
upon treatment with the cilnidipine-loaded nanoemulsion gel; table
21 reports these findings. Similarly, table 22 indicates no
appreciable changes in the animals' absolute and relative organ
weights, including the weight of the liver, kidneys, pancreas, thymus,
lungs, spleen, and heart, compared to standard control animals.
When compared to standard control animals, blood parameters such
as haemoglobin, red blood cells (RBCs), white blood cells (WBCs),
platelets, and differential white blood cells (WBCs) like monocytes,
neutrophils, lymphocytes, eosinophils, and basophils did not
significantly change (P>0.05) when test compounds were
administered (table 23).
Fig. 17: Cumulative amount permeated from Cilnidipine-loaded optimised nanoemulsions gels, data represented as mean±SEM, n = 3
observations
Table 16: Steady-state flux and permeability coefficients for cilnidipine-loaded optimised nanoe mulsion gels
Formulation code
Jssa (µg/cm2/h)
Kpa (cm/h) × 10−2
DOE_CiL_Gel_1
52.81±0.938
17.486±0. 311
DOE_CiL_Gel_2
40.88±1.44
13.538±0.478
DOE_CiL_Gel_3
80.64±1.38
26.703±0.459
DOE_CiL_Gel_4
69.24±1.62
22.928±0.537
aData represented as mean±SD, n = 3 observations
Table 17: Evaluation of cilnidipine-loaded optimised nanoemulsion gels
NEn_CiL_gel formulation code
Clarity
Homogeneity
Consistency
Spreadabilitya
(g/cm/s)
Extrudabilitya
pHa
Viscositya (Pa*s)
NEn_CiL_Gel_1
++
Homogeneous
Better
9.52±0.34
+++
6.21±0.35
4.039±0.0038
NEn_CiL_Gel_2
++
Homogeneous
Better
5.82±0.62
++
5.84±0.21
6.185±0.0024
NEn_CiL_Gel_3
+++
Homogeneous
Excellent
18.39±0.43
++++
5.87±0.12
8.344±0.0023
NEn_CiL_Gel_4
+++
Homogeneous
Excellent
13.84±0.51
+++
6.36±0.25
9.193±0.0037
aData represented as mean±SD, n = 3 observations
Table 18: Effect of cilnidipine-loaded optimised nanoemulsion gel on clinical score of skin and animal mortality
Groups
Clinical scorea
Animal mortalitya
Erythema edema
Standard control (SC)
0.0±0.0
0.0±0.0
0.0±0.0
NEn_CiL_Gel
0.0±0.0
0.0±0.0
0.0±0.0
[aData represented as mean±SD, n = 5 observations]. *P<0.05 significantly different in comparison to Standard control. (Dunnett's multiple
comparison t-test is performed after a one-way analysis of variance) (Clinical score: 0: Normal; 1: Mild; 2: Minimal; 3: Moderate; 4 Severe);
Abbreviations: SC: Standard Control; NEn_Gel: Nanoemulsion gel
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Table 19: Effect of cilnidipine-loaded optimised nanoemulsion gel onanimal feed consumption
Groups
d-1
d-2
d-3
d-4
d-5
d-6
d-7
d-8
d-9
d-10
d-11
d-12
d-13
d-14
Female rats (g)
Standard control
(SC)
20.96
21.51
21.85
23.64
23.66
23.21
24.13
27.93
28.13
27.95
27.89
28.97
29.53
29.07
NEn_Cil_gel
20.25
22.34
20.80
24.22
24.17
24.29
24.85
27.68
27.72
25.84
28.29
28.07
28.09
27.96
Abbreviations: SC: Standard control; NEn_CiL_Gel: Cilnidipine-loaded nanoemulsion gel
Table 20: Effect of cilnidipine-loaded optimised nanoemulsion gel onanimal water consumption
Groups
d-1
d-2
d-3
d-4
d-5
d-6
d-7
d-8
d-9
d-10
d-11
d-12
d-13
d-14
Female rats (ml)
Standard control
(SC)
31.2
32.0
33.6
34.4
32.8
34.4
30.0
32.0
31.2
30.6
35.6
35.8
36.4
35.6
NEn_CiL_Gel
32.0
32.4
32.0
29.4
32.6
33.6
30.6
34.4
30.4
33.4
35.2
36.2
35.2
36.0
Abbreviations: SC: Standard Control; NEn_CiL_Gel: Cilnidipine-loaded nanoemulsion gel
Table 21: Effect of cilnidipine-loaded optimised nanoemulsion gel on biochemical parameters
Groups
Triglyceridesa (mg/dl)
HDLa(mg/dl)
LDLa(mg/dl)
Cholesterola(mg/dl)
SGOTa(U/l)
SGPTa(U/l)
Female rats
Standard Control (SC)
98.6±5.2
21.7±1.5
6.2±1.1
54.2±3.4
81.1±5.2
43.3±3.7
NEn_CiL_Gel
99.2±6.2
22.4±2.5
6.1±0.2
54.5±3.6
81.6±7.4
43.6±1.5
[aData represented as mean±SD, n = 5 observations]. *P<0.05 significantly different in comparison to Standard control. (Dunnett's multiple
comparison t-tests are conducted after one-way ANOVA); Abbreviations: SC: Standard Control; NEn_CiL_Gel: Cilnidipine-loaded Nanoemulsion Gel
Table 22: Effect of cilnidipine-loaded optimised nanoemulsion gel on organ weight of an animal
Groups
Liver (g)a
Kidney (g)a
Pancreas (mg)a
Thymus (mg)a
Lung (mg)a
Spleen (mg)a
Heart (mg)a
Female rats (Relative; Per 100 gm of animal)
Standard control
(SC)
5.70±0.58
1.30±0.19
462.64±40.73
248.88±22.87
724.82±43.70
307.37±22.02
442.37±42.83
NEn_CiL_Gel
5.69±0.62
1.29±0.10
445.52±43.46
249.40±12.42
701.88±95.51
304.96±40.74
434.26±71.5
[aData represented as mean±SD., n=5 observations]. *P<0.05 significantly different from Standard control. (Dunnett's multiple comparison t-tests
are conducted after one-way A NOVA); Abbreviations: SC: Standard Control; NEn_CiL_Gel: Cilnidipine-loaded Nanoemulsion Gel
Fig. 18: Effect of cilnidipine-loaded optimised nanoemulsion gel on animal body weight change (%) A females, [Data represented as
mean±SD., n=6 observations]; Two-way analysis of variance is used to analyse the data, and then Bonferroni's multiple comparison t-tests
are used.; *P<0.05, **P<0.01, ***P<0.001, significantly different in comparison to Standard control. Abbreviations: SC: Standard Control;
NEn_CiL_Gel: Cilnidipine nanoemulsion gel
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Table 23: Effect of cilnidipine-loaded optimised nanoemulsion gel on haematological parameters
Groups
WBCsa
(10^3/µl)
NEUa
(%)
LYMa
(%)
MONOa
(%)
EOSa
(%)
BASa
(%)
RBCsa
(10^6/µl)
HGBa
(g/dl)
MCVa
(fl)
MCHa
(pg)
MCHCa
(g/dl)
PLTsa
(10^3/µl)
Female Rats
Standard
control (SC)
15.2±1.2
2.5±0.3
6.4±0.8
0.6±0.1
0.4±0.1
0.2±0.1
7.3±0.4
15.4±0.4
51.7±2.7
20.8±1.2
38.9±1.5
909.2±54.2
NEn_CiL_Gel
15.2±2.0
2.5±0.3
6.4±1.2
0.6±0.1
0.4±0.1
0.2±0.0
7.3±0.2
15.4±0.7
51.7±2.5
20.3±1.1
39.8±2.1
910.8±67.5
[aData represented as mean±SD., n=5 observations]. *P<0.05 significantly different in comparison to Standard control. (Dunnett's multiple
comparison t-tests are conducted after ANOVA). Abbreviations: SC: Standard Control; NEn_CiL_Gel: Cilnidipine-loaded Nanoemulsion Gel; WBCs:
White blood cells, NEU: Neutrophils, MONO: Monocytes, EOS: Eosinophils, BAS: Basophils, RBCs: Red blood cells, HGB: Hemoglobin, MCV: mean
corpuscular volume, MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration, PLTs: Platelets
Pharmacokinetic study
The trapezoidal area under the curve (AUC) was computed in the
pharmacokinetic (PK)study to evaluate the total drug exposure.
Significant differences were observed in evaluating cilnidipine in
tablet and cilnidipine-loaded nanoemulsion gel formulations, as
shown in table 24. The Cmax for cilnidipine in the tablet was
332.3±14.2 ng/ml, but in the nanoemulsion gel, it increased
significantly (P<0.05) to 593.00±24.8 ng/ml. The Tmax for both
formulations stayed at 3 h despite this increase. Furthermore, from
1279±34.1 ng/ml, the AUC0-12 demonstrated a significant (P<0.05)
increase. To 1922.50±162.8 ng/ml·h with the tablet and the
nanoemulsion gel. Additionally, the nanoemulsion gel's AUC0-∞
increased from 1395.5±156.7 ng/ml·h to 1962.30±174.9 ng/ml·h.
These findings suggest that the nanoemulsion gel formulation offers
higher absorption than the cilnidipine tablet.
Table 24: Effect of cilnidipine-loaded optimised nanoemulsion gel on pharmacokinetic parameters of the drug
Formulations
Cmaxa (ng/ml)
Tmaxa (h)
AUC0-12a (ng/ml·h)
AUC0-∞a(ng/ml·h)
Cilnidipine Tablet
332.3±14.2
3.0±0.0
1279±34.1
1395.5±156.7
NEn_CiL_Gel
593.00±24.8*
3.0±0.0
1922.50±162.8*
1962.30±174.9*
[aData represented as mean±SD., n=3 observations ]; Student’s unpaired t-test data analysis; *P<0.05 significantly different compared to cilnidipine
Tablet. Abbreviations: Cmax: Peak of maximum concentration; Tmax: Time of maximum concentration; AUC0-12: Area under the curve until last
observation; AUC0-∞: Area under the curve from time 0 to infinity; NEn_CiL_Gel: Cilnidipine Nanoemulsion Gel
Fig. 19: Plasma concentration-time profile of cilnidipine-loaded optimised nanoemulsion gel, data represented as mean±SEM, n=3
observations, Abbreviations: CiL-TAB: Cilnidipine tablet; NEn_CiL_Gel: Cilnidipine nanoemulsion gel
The study demonstrates that cilnidipine-loaded nanoemulsion gel
significantly enhances the transdermal delivery of cilnidipine, as
evidenced by increased Cmax and AUC. Nanoemulsion delivery
through the transdermal route is promising for drugs with rapid
first-pass metabolism and very low aqueous solubility.
Nanoemulsions minimise interaction with different layers of the skin
and could significantly improve drug bioavailability. It also opens up
the possibility of terminating therapy with transdermal
nanoemulsion, a hopeful prospect. It's a promising development,
and studies have already demonstrated that nanoemulsions can
boost the bioavailability of poorly soluble medications. Recent
studies on enhanced solubility and dissolution rates support the
idea that the increased surface area of nanoemulsion droplets
facilitates better skin permeation, which accounts for the observed
improvements in drug delivery [18, 53, 54]. The cilnidipine-loaded
nanoemulsion gels' biocompatibility and stability (data hidden)
were verified, and tests on acute dermal toxicity revealed no adverse
effects, indicating their safety for use in clinical settings. This
discovery emphasises the possible effectiveness of nanoemulsion
drug delivery systems. Cilnidipine's dual-action mechanism aligns
with recent research highlighting nanoemulsions' benefits in
managing hypertension, offering improved drug solubility, sustained
drug levels, and enhanced therapeutic outco mes. Challenges such as
long-term stability and uniformity of nanoemulsion gels persist and
require ongoing optimisation. The smaller droplet sizes of
nanoemulsions improve drug absorption by increasing contact area
M. T. Gaikwad & R. P. Marathe
Int J App Pharm, Vol 17, Issue 1, 2025, 255-274
273
with the skin, which is crucial for drugs like cilnidipine with limited
solubility. The enhanced stability and good release properties of
nanoemulsion gels are beneficial for chronic conditions, reducing
dosing frequency and improving patient compliance. Reduced
systemic side effects due to localised drug delivery are
advantageous, especially for drugs with significant systemic effects.
This study highlights the need for further research to address
scalability and efficacy across diverse patient populations, stressing
the urgency and importance of this task. This study contributes to
the evidence supporting nanoemulsions and nanoemulsion gels for
improved transdermal drug delivery, emphasising the need for
further research to address scalability and efficacy across diverse
patient populations.
CONCLUSION
The study assessed cilnidipine's acute dermal toxicity and
pharmacokinetics in animal models while evaluating its efficacy in
nanoemulsion and cilnidipine-loaded nanoemulsion gel formulations.
The study's main conclusions showed that these formulations
considerably increased the drug’s bioavailability compared to
conventional tablets, with high Cmax and AUC values for
nanoemulsion gels indicating improved transdermal absorption. Acute
dermal toxicity tests demonstrated the safety of these formulations by
showing no appreciable adverse effects on organ weights, biochemical
parameters, or overall health. The formulations necessary for reliable
therapeutic impact also demonstrated stable physical and chemical
characteristics, like pH and viscosity. These findings strongly support
the safety and stability of nanoemulsion technologies, reassuring the
audience about their potential for clinical use and enhancing drug
delivery and treatment results.
ACKNOWLEDGEMENT
Authors are thankful to Gattefossé, India, for providing gift samples
of Paceol, Transcutol ® P, Lauroglycol™ FCC, Labrafil M1944, and
IMCD India Private Ltd., Bandra (East), Mumbai for gift samples of
Kollicream ® OA, Kolliphor ® EL, Kolliphor®RH 40. The authors also
thank the principal and staff of R. C. Patel Institute of Pharmaceutical
Education and Research, Shirpur, for their help. The authors express
deep gratitude towards the Principal, Govt. College of Pharmacy,
Chh. Sambhajinagar, for encouragement throughout the work.
FUNDING
Nil
AUTHORS CONTRIBUTIONS
Mahesh T. Gaikwad conducted the research, analysed the data, and
wrote the manuscript. Dr Rajendra P. Marathe supervised the
research project and reviewed and approved the manuscript.
CONFLICT OF INTERESTS
Declared none
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