Colloids and Surfaces B: Biointerfaces 228 (2023) 113399

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Colloids and Surfaces B: Biointerfaces

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Meloxicam emulgel potently suppressed cartilage degradation in knee

osteoarthritis: Optimization, formulation, industrial scalability and
pharmacodynamic analysis
Vaskuri GS Sainaga Jyothi a, Harithasree Veerabomma a, Rahul Kumar b,
Dharmendra Kumar Khatri b, Shashi Bala Singh b, Jitender Madan a, *
a
Department of Pharmaceutics, National Institute of Pharmaceutical Education and Research, Hyderabad, Telangana, India
b
Department of Biological Sciences, National Institute of Pharmaceutical Education and Research, Hyderabad, Telangana, India

A R T I C L E I N F O A B S T R A C T

Keywords: Background and objective: Meloxicam (MLX) is prescribed for the management of pain and inflammation allied
Emulgel with osteoarthritis (OA). However, MLX causes intestinal damage in long term administration. Hence, meloxicam
Meloxicam loaded emulgel (MLX-emulgel) was optimized, formulated and examined under stringent parameters in
Osteoarthritis
monosodium-iodoacetate (MIA) induced knee OA in Wistar rats.
Western blotting
Methods and results: Nanoemulsion of MLX was fabricated by ultrasonication and microfluidization method with
Inflammatory cytokines
a droplet size of 66.81 ± 5.31-nm and zeta potential of − 24.6 ± 0.72-mV. Further, MLX nanoemulsion was
optimized with centrifugation, heating-cooling cycles and transmittance parameters in addition to scale-up
feasibility with microfluidizer. Post optimization, MLX-nanoemulsion was tailored as emulgel with Carbopol
Ultrez 10 NF and assessed for pH, rheology, textural properties, assay and stability features. The in-vitro release
study revealed the Korsmeyer-Peppas release kinetics and ex-vivo skin permeation was improved by 6.71-folds.
The skin distribution of MLX-emulgel evinced the transfollicular mode of permeation. In-vivo study indicated the
protective action of MLX-emulegl expressed in terms of inflammatory cyctokines level, X-ray analysis of knee
joints of rats, histopathology and OARSI (Osteoarthritis Research Society International) scoring. MLX-emulgel
treated group displayed lower (P < 0.001) level of COX-2 intensity as compared to positive control group.
However, it was comparable (P > 0.05) to the normal control group, MLX oral dispersion, i.v. solution and
etoricoxib gel groups. MLX-emulgel showcased an alternative to the long term usage of analgesics for relieving
the symptoms of knee OA.
Conclusion: MLX-emulgel may be a potential candidate for translating in to a clinically viable dosage form in the
management of knee OA.

1. Introduction metalloproteinases (MMPs) [8,9], contributing to cartilage destruction

Osteoarthritis (OA), is a degenerative joint disease, that mainly af­
fects hip and knee joints [1] with worldwide prevalence >7 % [2,3]. OA
is triggered by both internal and external factors including ageing,
obesity, joint trauma, increased mechanical loading, and hereditary
predisposition [4]. Pathophysiologically, OA alters the equilibrium of
catabolic and anabolic process of articular cartilage where the catabolic
process predominates causing breakdown of cartilage leading to syno­
vial inflammation by inflammatory cytokines namely, TNF-α, IL-1β and
IL-6 [5–7]. IL-1β upregulates matrix-degrading enzymes called matrix
[10,11]. Conversely, TNF-α interferes with the chondrocytes’ produc­
tion of type II collagen, proteins that bind proteoglycans, and compo­
nents of proteoglycans [12,13] and IL-6 inhibits the formation of type II
collagen and enhances MMPs’ production [14]. Additionally, IL-1β and
TNF-α stimulates the expression of cyclooxygenase-2 (COX-2) enzyme
[15,16] leading to PGE2 (Prostaglanding) production which plays a
major role in pain and inflammation.
International and national guidelines recommended oral non-
steroidal anti-inflammatory drugs (NSAIDs) for managing pain in OA,
which inhibit COX-2 enzyme for those who do not respond to

* Corresponding author.
E-mail address: jitenderpharmacy@gmail.com (J. Madan).

https://doi.org/10.1016/j.colsurfb.2023.113399
Received 18 March 2023; Received in revised form 7 June 2023; Accepted 8 June 2023
Available online 10 June 2023
0927-7765/© 2023 Elsevier B.V. All rights reserved.
V.G.S. Jyothi et al.
paracetamol [17–21]. Hence, NSAIDs like diclofenac, naproxen and
meloxicam (MLX) are prescribed for oral administration to manage OA
[22]. However, MLX has anti-inflammatory effects similar to or better
than those of other NSAIDs in animal models, with a greater therapeutic
ratio [23,24]. MLX is an oxicam derivative recommended by US-FDA for
pain and inflammation management in OA. It is a selective COX-2 in­
hibitor and commercially available as an oral dosage form (7.5–15 mg)
[25,26]. However, long term oral use of MLX is associated with certain
side-effects like gastrointestinal ulceration, perforation, bleeding, and
colitis, contraindicated in patients having asthma [27] and increases the
risk of myocardial infarction by 38 % due to COX-2 inhibition activity
[28]. Hence, topical NSAIDs are generally recommended over oral
NSAIDs for pain management in OA [29].
Nevertheless, potential of topical application of MLX in coping pain
and inflammation in OA is still not explored. Recently, our group re­
ported MLX loaded solid lipid nanoparticles topical gel in the manage­
ment of OA [30]. MLX owing to low molecular weight (Mw~351.403
Da), BCS (Biopharmaceutical Classification System) Class-II, and Log P
of 3.02 is a suitable drug entity for topical delivery [31] in managing OA.
Lately, emulgel dosage form has been widely explored for topical
delivery of therapeutic entities in several pain disorders like sunburn
[32], anal fissure [33], post hemorrhoidectomy pain [34], and vulvar
pain [35]. Emulgel, an amalgamation of emulsion and gel [36], dem­
onstrates several desirable features like spreadable, thixotropic,
greaseless, pleasing appearance, emollient and long shelf-life. In addi­
tion, emulgel has exhibited great clinical success in delivery of topical
therapeutics [37–39].
Therefore, in present investigation, MLX was loaded in emulgel
(MLX-emulgel) for demonstrating a high degree of analgesic and anti-
inflammatory activity in managing knee OA. The therapeutic potential
of MLX-emulgel was evaluated in monosodium iodoacetate (MIA)
induced OA in Wistar rats under a set of stringent in vivo parameters
[40].
2. Materials and methods
2.1. Materials
MLX (99.5 %) was received as a gift sample from Swati Spentose
Private Limited, Gujarat, India; Castor oil was purchased from Sisco
Research Laboratories, Mumbai, India; Transcutol HP was obtained as a
gift sample from Gattefosse, France; Tween 80 was purchased from Avra
Laboratories Private Limited, Hyderabad, India. Carbopol®Ultrez 10 NF
was provided by Lubrizol, Mumbai, India. Meglumine was procured
from Sigma Aldrich, Bangalore, India. All the solvents and chemicals
used were of higher analytical grade. Water used was of Milli-Q purified.
2.2. Preparation and optimization of meloxicam nanoemulsion
2.2.1. Selection and solubility of drug in oil, surfactants and cosurfactants
Castor oil was selected as oil phase owing to its analgesic properties
in OA [41]. Preliminary solubility of MLX was determined in castor oil,
where 0.1 g of MLX was suspended in 1 g of castor oil in presence and
absence of 0.125 g of meglumine and shaken vigorously for 2 h by vortex
mixer, centrifuged and supernatant was collected, diluted appropriately
with ethanol and filtered through 0.22 µm membrane filter to determine
the drug content by HPLC method [42]. Similarly, the solubility of MLX
in selected surfactants and cosurfactants was also determined (Suppl.
Table 1).
2.2.2. Nanoemulsification capability screening of surfactants and
cosurfactants
Surfactants labrasol (HLB~14), tween 20 (HLB~16.7), and tween 80
(HLB~15) were screened where 2.5 mL of 15 % w/w of each surfactant
solution was tested for its emulsifying ability against castor oil, added in
a drop-wise fashion till the solution turns cloudy under vortexing [43].
2
Colloids and Surfaces B: Biointerfaces 228 (2023) 113399
Next, the screened surfactant was combined separately with different
cosurfactants, namely span 80 (HLB~4.3), span 20 (HLB~8.6) and
transcutol HP (HLB~4) at 7.5 % w/w concentration in 2.5 mL of water
and noticed for turbidity of the emulsion.
2.2.3. Preparation of nanoemulsion and construction of ternary phase
diagram
High energy emulsification method was adopted for the preparation
of nanoemulsion using probe sonicator [44,45]. The viscous nature of
castor oil hampered the preparation of self-emulsifying nanoemulsion,
leading to gelation of the nanoemulsion. Hence, the fraction of castor oil
was kept constant to 10 % w/w [46] while the surfactant ratio was
varied from 15 % to 20 % w/w. Surfactant ratio less than 15 % w/w did
not yield emulsion formation and higher than 20 % w/w yielded gela­
tion of nanoemulsion [47]. The surfactant and cosurfactant were taken
as Smix ratio in the range of 1:9–9:1 and weighed amount of water was
added drop-wise under continuous stirring at 700-rpm for 30 min.
Subsequently, the preformed emulsion was probe sonicated and the
sonication time was optimised (Table 1 and Fig. 1) for gaining desirable
droplet size [44]. Ternary phase diagram was constructed using Tri-plot
v4.1.2 by varying the concentration of surfactant (1.5–18 % w/w),
cosurfactant (1.5–18 % w/w) and water (70–75 % w/w).
2.2.4. Preparation of meloxicam loaded nanoemulsion
2.2.4.1. Ultrasonication method. Post optimization, nanoemulsion of
MLX was prepared by dissolving the drug (1 % w/w) and meglumine in
1:1.25 ratio in addition to surfactant, cosurfactant (Table 1) and oil
mixture under continuous stirring at 70 ◦ C followed by cooling at room
temperature. Water was added drop-wise with continuous stirring at
700-rpm to obtain an emulsion and ultrasonicated to reduce the droplet
size and stored for further characterization.
2.2.4.2. Microfluidizer method. Microfluidizer was used for MLX-
nanoemulsion fabrication as industrial scalable technique [48]. In
brief, 100 mL of coarse emulsion was processed through microfluidizer
(LM20, Microfluidics, USA), operated at different pressures of
20000–30000 psi and the samples were passed for 1–6 cycles and
assessed for droplet size [49].
2.3. Characterization of nanoemulsion
2.3.1. Droplet size and zeta potential
The droplet size of MLX-nanoemulsion (F1-F8; Table 1) was deter­
mined by diluting 1 mL of nanoemulsion to 10-folds with distilled water
and the droplet size, polydispersity index (PDI) and zeta potential were
measured using a Zetasizer (Zeta Sizer Nano NS, Malvern, UK) at 25 ◦ C
over a 90◦ angle.
2.3.2. Centrifugation and thermodynamic stability study
MLX-nanoemulsion (F1-F8; Table 1) was centrifuged at 25,000-rpm
for 20 min and observed for cracking, creaming and phase separation.
The stable samples were subjected to thermodynamic stability studies
[50] where MLX-nanoemulsion (F3-F6; Table 1) were subjected to
heating-cooling cycles, placed alternatively at 4 ℃ and 45 ℃ for a
period of 48 h for six cycles and observed for cracking, creaming and
phase separation [51].
2.3.3. Percentage transmittance
Percentage transmittance of the stable MLX-nanoemulsion (F3-F6;
Table 1) was determined using UV–VIS spectrophotometer (V-650
double beam UV-Visible spectrophotometer, Jasco Inc., Germany) by
diluting the sample 100-folds with distilled water and analyzed at
680 nm. The optimized ratio was selected based on percentage trans­
mittance [52].
 V.G.S. Jyothi et al.
Table 1
Ternary phase composition, characterization and optimizing parameters of meloxicam loaded nanoemulsion.
Formulation Cosurfactant to Cosurfactant Surfactant Water (% Region/ Droplet Polydispersity Zeta Observation
Code surfactant ratio (Transcutol, % w/w) (Tween 80; % w/ w/w) observation size (nm) index potential
Centrifugation Thermodynamic Percent
w) (mV)
stability transmittance

F1 01:09 1.5 13.5 75 Δ/transparent 60.71 ± 0.303 ± 0.08 -25 ± 3.25 Turbid _ _
8.23
F2 01:09 2 18 70 Δ/transparent 68.36 ± 0.222 ± 0.05 -25.9 ± 3.61 Turbid _ _
0.91
F3 01:07 1.875 13.125 75 Δ/transparent 61.84 ± 0.3 ± 0.05 -28.9 ± 3.76 Clear and Clear and 94.75 ± 0.92
3.86 transparent transparent
F4 01:07 2.5 17.5 70 Δ/transparent 66.81 ± 0.112 ± 0.03 -24.6 ± 0.72 Clear and Clear and 96.06 ± 0.63
5.31 transparent transparent
F5 01:05 2.5 12.5 75 Δ/transparent 65.06 ± 0.321 ± 0.05 -31.1 ± 1.82 Clear and Clear and 89.38 ± 1.36
3.33 transparent transparent
F6 01:05 3.33333 16.6667 70 Δ/transparent 55.79 ± 0.208 ± 0.01 -25.3 ± 3.26 Clear and Clear and 94.04 ± 1.6
3

1.38 transparent transparent

F7 01:03 3.75 11.25 75 Δ/transparent 75.6 ± 0.316 ± 0.1 -24.5 ± 2.2 Turbid _ _
14.16
F8 01:03 5 15 70 Δ/transparent 70.1 ± 0.084 ± 0.09 -24.5 ± 1.65 Turbid _ _
12.54
F9 01:01 7.5 7.5 75 +/ cracking _ _ _ _ _ _
F10 01:01 10 10 70 +/ cracking _ _ _ _ _ _
F11 03:01 11.25 3.75 75 +/ cracking _ _ _ _ _ _
F12 03:01 15 5 70 +/ cracking _ _ _ _ _ _
F13 05:01 12.5 2.5 75 +/ cracking _ _ _ _ _ _
F14 05:01 16.6667 3.33333 70 +/ cracking _ _ _ _ _ _
F15 07:01 13.125 1.875 75 +/ cracking _ _ _ _ _ _

Colloids and Surfaces B: Biointerfaces 228 (2023) 113399

F16 07:01 17.5 2.5 70 +/ cracking _ _ _ _ _ _
F17 09:01 13.5 1.5 75 +/ cracking _ _ _ _ _ _
F18 09:01 18 2 70 +/ cracking _ _ _ _ _ _

Note: All measurements were noted in triplicate (n = 3)

V.G.S. Jyothi et al.
Fig. 1. A) Optimization of sonication time of nanoemulsion and B) Ternary phase diagram of nanoemulsion (Δ/ transparent, +/cracking). Data repre­
sented ***p < 0.001.
2.3.4. Drug loading efficiency and capacity
The drug loading efficiency (DLE) and drug loading capacity (DLC)
of MLX-nanoemulsion (F4; Table 1) was performed by diluting the
sample with water and acetonitrile (1:1 ratio) mixture and analysed
using SHIMADZU HPLC over C18 column (250 × 4.6 mm, 5 µm) sup­
plied with Lab solutions software. The mobile phase was composed of
water (pH 3 adjusted with trifluoroacetic acid) and acetonitrile
(35:65 v/v) [30,42].
Weight of drug recovered
%DLE = × 100 (1)
Weight of drug added
%DLC =
Weight of drug recovered
× 100 (2)
Weight of nanoemulsion
2.3.5. Transmission electron microscopy (TEM)
The optimized MLX-nanoemusion (F4; Table 1) was stained with 2 %
phosphotungstic acid on copper grid and allowed to air dry for 1 min
and observed under TEM (Jeol India Private Limited, JEM 1400HR) with
an accelerating voltage of 120 kV.
2.4. Preparation of meloxicam loaded emulgel
Carbopol gel was used as gelling agent where 4 g of Carbopol®
Ultrez 10 NF was suspended in 100 mL of water with stirring at 1000-
rpm overnight and pH adjusted with triethanolamine to 6–6.5. MLX-
nanoemulsion was mixed with carbopol gel in 1:3 ratio with gentle
stirring at 1000-rpm under overhead stirrer for 3 h to obtain MLX-
emulgel [53].
2.5. Characterization of emulgel
2.5.1. Visual inspection, pH determination
The MLX-emulgel was inspected visually for its appearance, homo­
geneity and colour. Further, 1 g of emulgel was dispersed in 20 mL of
water and assessed by pH meter (Mettler Toledo, UK) [54].
2.5.2. Rheology and texture analysis
Rheological behaviour was measured by modular compact rheom­
eter (Anton Paar Private Limited, Germany) at 25 ◦ C. MLX-emulgel was
placed on the plate and measured by increasing the shear rate from 0.1
to 100 s− 1 and compared with the marketed gel. The spreadability was
evaluated by texture analyser (T.A. XT Plus, UK) where MLX-emulgel
was placed on the female cone and a graph was obtained by pene­
trating the male cone in female cone cavity and vice-versa.
4
Colloids and Surfaces B: Biointerfaces 228 (2023) 113399
2.5.3. Assay
Assay of the MLX-emulgel was performed by dispersing in 5 mL of
water and acetonitrile (1:1 ratio) mixture and suitably diluted to analyse
by RP-HPLC method [30,42].
Weight of drug recovered
%Assay = × 100 (3)
Weight of drug added
2.5.4. Accelerated and real time stability studies
MLX-emulgel was subjected to stability studies as per ICH guidelines
at accelerated stability conditions of 40 ± 2 ◦ C with 75 ± 5 % relative
humidity (RH) in stability chamber (Osworld Scientific Equipment Pri­
vate Limited, India) [55] for 6 months and at room temperature for 12
months and assessed for pH and assay at the time intervals of 1, 3, 6
and/or 12 months.
2.5.5. In vitro drug release
The release of drug from the MLX-emulgel was assessed by Franz
diffusion cell apparatus [56]. Dialysis membrane-110 (Mw
2000–14000 Da) was embedded between the donor and receptor
compartment. MLX-emulgel (~2.5 mg of MLX) and MLX-suspension
(2.5 mg) were separately dispersed in donor compartment while the
receiver compartment was filled with 28 mL of phosphate buffer saline
(pH~7.4) containing 30 % w/v polyethylene glycol 400 [57]. The buffer
in the receptor compartment was maintained at 32 ± 1 ◦ C with
continuous stirring at 200-rpm. 1 mL of sample was withdrawn at in­
tervals of 0.16, 0.33, 0.5, 0.75, 1, 2, 3, 4, 5, 6, 7 and 8 h and replaced
with equal volume of buffer. Samples were passed through 0.22-µm
syringe filter and analysed by RP-HPLC. Release model kinetics were
applied using the DDSolver® software to assess zero order, first order,
Higuchi, Korsmeyer-Peppas and Hixson-Crowell models.
2.5.6. Ex-vivo skin permeation studies
Ex-vivo skin permeation studies of MLX-emulgel were performed in
male Wistar rats which were approved (NIP/05/2021/PE/411) for study
by Institutional Animal Ethics Committee (IAEC). Animals were sacri­
ficed and skin of the dorsal abdominal region was shaved, excised and
the subcutaneous fatty tissue was removed, washed and stored at − 80 ◦ C
until further use [58]. The skin was mounted between the donor and
receptor compartment of Franz diffusion cell apparatus with the stratum
corneum facing towards the donor compartment and experiment was
performed as discussed in the release study session [57]. The slope of the
linear portion of the curve was contemplated as flux (Jss, μg/cm2/h) and
the enhancement ratio (ER) was calculated [59].
Flux of drug from emulgel
ER = (4)
Flux of drug from drug suspension
V.G.S. Jyothi et al. Colloids and Surfaces B: Biointerfaces 228 (2023) 113399

2.6.3. Western blotting

2.5.7. Skin retention studies
Animals were sacrificed on 14th day of induction and the synovial
For skin retention study, the skin was removed post ex-vivo skin
tissues were collected and stored at − 80 ◦ C till use. The synovial tissues
permeation study [60], washed to get rid of remaining content, minced,
were lysed using RIPA (Radioimmunoprecipitation assay) buffer con­
homogenized with 10 mL of acetonitrile, vortexed, incubated overnight
taining both protease and phosphatase inhibitors and homogenised at
and sonicated for 1 h. The sample was then centrifuged at 12,000-rpm
4 ◦ C. The lysates were centrifuged (13,000 rpm) for 15 min at 4 ◦ C and
for 15 min and the supernatant was filtered through 0.22-µm syringe
the supernatant was separated. The synovial tissues were assessed for
filter and analyzed by RP-HPLC [30,42].
protein estimation by Bicinchoninic Acid (BCA) protein assay kit and
tissue samples of 10-µg equivalent amount of protein were fractioned on
2.5.8. Confocal laser scanning microscopy (CLSM)
10 % SDS-PAGE gel electrophoresis and electroblotted on nitrocellulose
Visualization of emulgel permeation pathways in skin was achieved
membrane, treated with COX-2 primary monoclonal antibody (Santa
by loading coumarin-6 dye (0.4 % w/w). Coumarin-6 dye loaded
Cruz Biotechnology, Inc., Texas, USA) and incubated again with bio­
emulgel incubated with skin in Franz diffusion cell apparatus for 2 h and
tinylated secondary IgG antibody conjugated with horseradish-
compared with the coumarin-6 dye suspension [53]. The skin was
streptavidin (Santa Cruz Biotechnology, Inc., Texas, USA) for 1 h. The
sectioned to 20-µm by cryotome at − 25 ◦ C, stained with DAPI (4′ ,
membrane was envisaged by chemiluminescene reagent under chemdoc
6-diamidino-2-phenylindole) and observed under CLSM (Leica TCS SP8
and bands were quantified with Image-J software [40,63].
Laser Scanning Spectral Confocal).
2.6.4. Inflammatory cytokines
2.6. Therapeutic efficacy analysis
The level of inflammatory cytokines in synovial tissue homogenates
was estimated by commercially available ELISA kits [40] for TNF-α,
The therapeutic efficacy of MLX-emulgel was evaluated in MIA
IL-1β, IL-6 and IL-10. Briefly, 96-well plates were incubated with the
induced knee OA in male Wistar rats [61]. Studies were performed as per
corresponding capture antibody overnight and incubated with standard
Committee for the Purpose of Control and Supervision of Experiments on
or synovial tissue homogenates for 2 h, followed by incubation with
Animals (CPCSEA) guidelines, Ministry of Fisheries, Animal Husbandry,
detection antibody and streptavidin-horsereddish peroxidase B solution
and Dairying, Department of Animal Husbandry, and Dairying, Gov­
and colour reagents, followed by the addition of stop solution
ernment of India, New Delhi, India. The study was approved by the
(2 N H2SO4). The wells were immediately analysed by microplate reader
Institutional Animal Ethics Committee (IAEC) vide protocol #
to determine the optical density at 450 nm.
NIP/05/2021/PE/411. All animals were acclimatized for one week in
animal house and divided into 8 groups with 6 animals in each group.
2.6.5. Histopathology
Group A was retained as normal control group with no induction of OA
Histopathology of knee joints was assessed by fixing the knee joints
and injected with 50-µL of sterile normal saline to the right knee joint.
in 10 % v/v formalin solution and decalcified for 21 days in 20 % v/v
All the remaining groups were induced OA by injecting a single dose of
Ethylenediamine tetra-acetic acid and implanted in paraffin wax [64].
3-mg of MIA dispersed in 50-µL normal saline via intra-articular route to
The joints were sectioned sagittally, stained with toluidine blue and
the right knee joint [40]. Group B was assigned as disease or positive
visualised under microscope (Leica Microsystems DM750 microscope,
control group and given no treatment. Group C and D were treated with
Germany). OARSI (Osteoarthritis Research Society International)
oral dispersion of MLX (1-mg/kg) and intravenous (i.v.) injection of MLX
scoring ranging from 0 to 24 was given to the joint sections [65].
(0.2-mg/kg) once-a-day, respectively. Group E, F, G and H were treated
with 1 % w/w MLX-emulgel, commercial 1 % w/w etoricoxib (COX-2
2.7. Statistical analysis
inhibitor) gel, 1 % w/w commercial diclofenac (non-selective COX in­
hibitor) gel and blank emulgel twice-a-day, respectively. The treatment
All measurements were noted in triplicate (n = 3). Using GraphPad
was given for 14 days.
Prism version 5 software (GraphPad software, San Diego, California,
US), the data was statistically evaluated. "Bonferroni’s Multiple Com­
2.6.1. Thickness of knee joint, motor incoordination and thermal
parison Test" was used for a follow-up study, and results were deemed
hyperalgesia test
statistically significant at P < 0.05.
The swelling of knee joint following the administration of MIA was
assessed using a calibrated Vernier calliper (Mitutoyo ABSOLUTE digital
3. Results and discussion
caliper, USA) by measuring the thickness of right knee joint while the
left knee joint thickness served as control [61].
3.1. Ultrasonication and microfluidization assisted in scale-up of a stable
Motor incoordination was assessed by rota-rod device which reveals
MLX-emulgel
the ability of knee joint to coordinate moment. Rats were placed on
rotating cylinders with an acceleration speed of 5–35 rpm over 1 min
According to Association of the Scientific Medical Societies in Ger­
and the time taken for rats to fall-off the rod was noted with a cut-off
many guideline for knee OA, reducing risk should be the primary
time of 3 min [62].
approach to improve the quality of life while treating with analgesic and
Thermal hyperalgesia response of rats to heat was assessed by hot
anti-inflammatory drugs [66]. Hence, MLX-emulgel dosage form [67]
plate test. Rats were individually exposed to the hot plate heated to a
was explored as a vehicle for delivering therapeutic entity in OA topi­
temperature of 55 ◦ C and the pain threshold was measured as a response
cally to reduce systemic exposure [68]. Thus, MLX-nanoemulsion with
of jumping or paw licking [62]. All tests were performed at time in­
castor oil as an oil phase was customized owing to its clinical utility in
tervals of 1, 3, 7 and 14 days.
OA [41]. However, the solubility of MLX in castor oil was low (0.66
± 0.06 mg/mL) (Suppl. Table 1) and therefore meglumine was incor­
2.6.2. Radiographic analysis of knee joints
porated in the oil phase to enhance its solubility [63]. The 1.8-folds hike
The radiographic images of rat knee joints were taken on 14th day of
in solubility of MLX in presence of meglumine in castor oil may be
induction in antero-posterior and lateral view, captured at Versha Di­
attributed to the formation of molecular complex between MLX and
agnostics (Wipro GE - ELPRO X-ray machine), Hyderabad, India at a
meglumine (pH~11.0 and log P~− 2.509) in castor oil (Hydroxyl value:
voltage of 50-kV and a current of 100-mA with 10 s as exposure time.
≥160; Acidity value~1.5; pH~5.7–6.3) owing to the presence of alka­
line (-NH2) group in meglumine and -OH of MLX (Suppl. Fig. 1) [69].
However, the increased solubility was not sufficient to load MLX in

5
V.G.S. Jyothi et al.
nanoemulsion. Hence, the solubility of MLX in the selected surfactants
and cosurfactants was also determined (Suppl. Table 1).
Next, the critical parameter in nanoemulsion formulation was the
proper selection of surfactants and their concentration [70,71].
Non-ionic surfactants are relatively less toxic than their ionic counter­
parts [72]. As the intended formulation is o/w nanoemulsion, surfac­
tants with high HLB values and cosurfactants with low HLB values were
selected for screening [73]. Based on the emulsifying ability, tween 80
and transcutol HP was selected as surfactant and cosurfactant respec­
tively, based on the emulsifying ability. The solubility of MLX in sur­
factant and cosurfactant was found to be 115.91 ± 0.52 mg/g and
79.77 ± 0.54 mg/g (Suppl. Table 1) respectively, which were adequate
to load MLX in the nanoemulsion.
Nanoemulsion was prepared by ultrasonication and the droplet size
was decreased as a function of sonication time (Fig. 1A). Nevertheless,
there was no significant (One-way ANOVA test, P > 0.05) difference
between the droplet size at 6 and 9 min sonication time, hence; 6 min
was taken as optimized time (Fig. 1A). Ternary phase curve was plotted
by varying the concentration of surfactant, cosurfactant and water and
keeping the oil quantity as constant. The cosurfactant to surfactant ra­
tios were evaluated from 1:9–9:1 where a total of 18 (F1-F18, Table 1)
formulations were prepared of which only F1-F8 were found to be stable
and the rest were unstable (Fig. 1B).
The droplet size of nanoemulsion < 150 nm was preferred for the
topical delivery of therapeutic entity [74]. Lower PDI value (<0.3) in­
dicates the uniformity of droplets in nanoemulsion [75]. The droplet size
of F1 to F8 nanoemulsion formulations was found in the range of 60.71
± 8.22-nm to 75.6 ± 14.16-nm (Table 1) with a PDI between 0.321
± 0.05–0.084 ± 0.09. A high zeta-potential value of ≥ ± 25 mV confers
stability preventing the agglomeration of droplets of nanoemulsion [76,
Fig. 2. A) Average droplet size (66.81 ± 5.3 nm) and droplet size distribution (0.112 ± 0.03) of nanoemulsion by Zeta sizer; B) average zeta potential (− 24.6
± 0.72 mV) of optimised nanoemulsion; C & D) TEM images of nanoemulsion showed spherical droplets with a size range of 60–90 nm at the scale bar of 500 nm
and 200 nm.
6
Colloids and Surfaces B: Biointerfaces 228 (2023) 113399
77]. F1-F8 nanoemulsions (Table 1) showed zeta-potential in the range
of − 24.5 ± 1.65 mV to − 31.1 ± 1.82 mV. The negative zeta potential of
tailored nanoemulsions may be attributed to the hydroxide ions in
water, which reacted with ricinoleic acid, present in the castor oil and
carried to the interface and provided a negative charge at the hydro­
phobic/water interface [78].
Centrifugation was performed to evaluate the effect of physical stress
on nanoemulsion stability. F3-F6 nanoemulsions (Table 1) were found to
be stable post centrifugation with no sign of phase separation and sub­
jected to thermodynamic stability analysis. F3-F6 nanoemulsions were
found to be stable and clear with no sign of phase separation or creaming
(Table 1).
Percentage transmittance of F3-F6 was estimated between 89 % and
96 % (Table 1). A value of percentage transmittance closer to 100 %
indicates that the nanoemulsion is clear and transparent, hence; F4
nanoemulsion with 1:7 ratio (20 % w/w) of cosurfactant to surfactant
was selected for the further characterization. The droplet size, PDI and
zeta-potential of F4 nanoemulsion was found to be 66.81 ± 5.31-nm,
0.112 ± 0.03 % and − 24.6 ± 0.72-mV, respectively (Fig. 2 A-D and
Table 1). TEM was used to analyse the size and shape of F4 nano­
emulsion, where the droplets were spherical (60–90 nm range) and well
separated with absence of aggregation or coalescence (Fig. 2A-D) [79,
80]. The DLE of F4 nanoemulsion was found to be 102.7 ± 1.8 % and
DLC was found to be 1.37 ± 0.02 %, calculated from HPLC method [30,
42]. The retention time (RT) of MLX was found to be 6 min with 0.999 as
R2 value (Suppl. Fig. 2).
Microfluidizer [49], as industry scalable technology was explored for
tailoring nanoemulsion. The coarse emulsion was subjected to pressures
ranging from 20,000 to 30,000 psi for 6 cycles (Suppl. Fig. 3). The
droplet size of nanoemulsion was reduced significantly (two-way
V.G.S. Jyothi et al.
ANOVA test, P < 0.001) as a function of pressure and number of cycles.
At 20,000 psi, the average droplet size was noticed in the range of 203.8
± 2.4-nm to 141.5 ± 0.9-nm from cycle 1–6 and 166.2 ± 1.0-nm to
136.2 ± 1.9-nm for 1–6 cycles at 25,000 psi. At 30,000 psi, the droplet
size reduced to 68.3 ± 0.57-nm at 4th cycle which was similar (unpaired
t-test, P > 0.05) to the droplet size tailored by ultrasonication. More­
over, the droplet size noticed at 30,000 psi with cycle 5 was reduced
further to 54.45 ± 0.75-nm with PDI of 0.226 ± 0.01 (Suppl. Fig. 3).
Hence, 30,000 psi and cycle 5 was selected as the optimized process
parameters. In addition, DLE and DLC of microfluidizer based nano­
emulsion tailored with optimized process parameters were estimated to
be 101.4 ± 1.3 % and 1.35 ± 0.04 %, respectively. However, no sig­
nificant (Unpaired t test, P > 0.05) differences were noticed between the
DLE and DLC of F4 nanoemulsion formulated by ultrasonication method
and microfluidizer based nanoemulsion.
Next, F4 nanoemulsion prepared by ultrasonication was fabricated as
MLX-emulgel and found to be smooth and yellow in colour with absence
of grittiness with 6.4 ± 0.2 pH. Rheological parameters were assessed
by rheometer and the plot of shear rate vis-a-vis shear stress for MLX-
emulgel showed no linear correlation indicating non-Newtonian
behaviour (Suppl. Fig. 4). Similarly, in viscosity vis-a-vis shear rate
curve, the viscosity of MLX-emulgel was decreased as a function of the
shear rate (Suppl. Fig. 4) indicating pseudoplastic behaviour which is
anticipated for topical dosage forms [81]. Both the graph patterns were
similar to the commercial gel.
Textural properties were determined by texture analyzer and gel
strength, work of shear, stickiness and force of extrusion estimated to be
259.68 ± 5.07 g, 141.74 ± 6.25 g.sec, − 89.78 ± 1.17 g.sec and
− 208.92 ± 13.4 g, respectively (Suppl. Fig. 5). Gel strength, work of
shear, stickiness and force of extrusion of marketed gel was noted to be
98.28 ± 5.81 g, 31.45 ± 3.39 g.sec, − 16.90 ± 1.74 g.sec, and − 87.97
± 3.99 g, respectively. Gel strength and work shear indicate the
spreadability of the emulgel [82]. Compared to marketed gel,
MLX-emulgel exhibited low spreadability and therefore requires high
pressure for extrusion. The drug content of the MLX-emulgel was esti­
mated to be 98.4 ± 0.12 %. Moreover, MLX-emulgel was stable at both
room temperature (12 months) and accelerated stability conditions (6
months) in terms of appearance, pH in addition to assay values > 95 %
Fig. 3. A) In vitro drug release study of MLX from emulgel and MLX dispersion# with 3.6- folds improvement in drug release from the emulgel as compared to the
plain drug at 8 h; B) ex-vivo skin permeation profile of MLX from emulgel and MLX dispersion# with a flux of 9.23 ± 0.1 µg/cm2/h and 1.38 ± 0.7 µg/cm2/h
respectively, and C) skin distribution studies of coumarin-6 suspension# showed high fluorescence intensity in epidermis and appendages of hair follicles than
coumarin-6 dye loaded in emulgel where it is found in epidermis only (green arrow: epidermal layer; red arrow: appendages of hair follicles).#Adopted from [30]
with permission.
7
Colloids and Surfaces B: Biointerfaces 228 (2023) 113399
(Suppl. Table 2) with no significant (Unpaired t-test, p > 0.05) differ­
ence at selected time periods.
Release of drug from MLX-emulgel evaluated by Franz diffusion cell
apparatus was compared with MLX suspension where MLX-emulgel
demonstrated 3.6-folds improvement as compared to MLX suspension
at 8 h (Fig. 3A). MLX-emulgel showed 75.78 ± 2.8 % drug release at 8 h
with significant (two-way ANOVA test, P < 0.001) improvement in
comparison to MLX suspension exhibiting 21 ± 1.44 % release. Release
kinetic models were assessed where R2 for Korsmeyer-Peppas model
with the value of 0.9874 was selected as best fit (Suppl. Table 3),
following diffusion and erosion kinetics [83]. Non-Fickian (anomalous)
diffusion mechanism of drug permeation was revealed by n value
(0.621) as both diffusion through the gelling polymer and swelling of
gelling polymer [84].
Permeation of drug across the rat skin was evaluated for both the
MLX-emulgel and MLX suspension where emulgel displayed improved
permeation as compared to plain drug (Fig. 3B). Nevertheless, perme­
ation of drug from MLX-emulgel across the skin was improved (two-way
ANOVA test, P < 0.001) post 2 h. The ER was calculated to be 6.71-folds
higher for MLX-emulgel (9.23 ± 0.1 μg/cm2/h) as compared to the MLX
suspension (1.38 ± 0.7 μg/cm2/h). This may be attributed to the
nanodroplet size of MLX embedded in emulgel which augmented the
absorption of drug across skin [85]. In addition, retention of MLX in skin
layers was 1.97-folds higher for MLX-emulgel (21.7 ± 8.6 µg/cm2) as
compared to MLX suspension (11.0 ± 3.5 µg/cm2). Further, higher
retention of MLX within skin layers may act as a reservoir for controlled
release of drug entity required to maintain a continuous therapeutic
concentration at the target site [53].
In order to visualize the permeation pathway, coumarin-6 dye loaded
emulgel was formulated and penetration pathway was envisioned using
CLSM [86]. The fluorescence intensity of coumarin-6 dye under CLSM
was quite low and can be seen in the epidermal layer only. On the other
hand, the fluorescence intensity of coumarin-6 dye loaded emulgel was
noticed in epidermis and dermis layer of skin with high intensity at
appendages of hair follicles (Fig. 3C). The results revealed transfollicular
route of drug permeation for emulgel [53,85].
V.G.S. Jyothi et al.
3.2. MLX-emulgel suppressed pain sensation and cartilage degradation in
MIA induced knee OA
MIA is widely implemented for the OA model development as it in­
hibits glyceraldehyde-3-phosphate dehydrogenase which causes the
death of chondrocytes [87]. This also leads to the formation of osteo­
phyte and degradation of articular cartilage [88]. Injection of MIA
resulted in inflammation and swelling of knee joint [61]. Thickness of
knee joints was measured and difference between the thickness of right
and left knee joint was noted as change in thickness due to inflammation
(Fig. 4A). The positive control group showed significant difference
(two-way ANOVA test, P < 0.001) in swelling of knee joint as compared
to normal control group. MLX-emulgel significantly decreased the
swelling of knee joint as compared to positive control (two-way ANOVA
test, P < 0.001) group on all days and normal control group (two-way
ANOVA test, P < 0.001) on day 1. However, MLX-emulgel treated group
showed no significant (two-way ANOVA test, P > 0.05) difference with
normal control group on day 3, 7 and 14. MLX oral dispersion and i.v.
solution treated groups were comparable (two-way ANOVA test,
P > 0.05) to MLX-emulgel treated group. This showed that the
MLX-emulgel was efficient in reducing swelling of the knee joint.
Motor incoordination test was performed to determine incapability
of rats to sustain on rolling rods and measured by marking fall off time
from the rotating rod [89]. MLX-emulgel showed significant (two-way
ANOVA test, P > 0.05) difference with the positive control group
(Fig. 4B). However, it demonstrated significant (two-way ANOVA test,
P < 0.001) difference in fall off time with normal control for 1st and 3rd
day and comparable (two-way ANOVA test, P > 0.05) from day 7 on­
wards. This indicates that MLX-emulgel was effective post 3–7 days of its
application in alleviating the discomfort in the moment of joints.
However, MLX-emulgel demonstrated comparable (two-way ANOVA
test, P > 0.05) results with that of MLX oral dispersion and i.v. solution
treated groups.
Intra-articular injection of MIA also resulted in change in pain
threshold [61]. The pain threshold was measured by the latency to
withdrawal from heat where MLX-emulgel treated group was signifi­
cantly (two-way ANOVA test, P < 0.001) different from normal control
group at day 1 and 3 and comparable (two-way ANOVA test, P > 0.05)
since day 7. Nevertheless, it was significantly different from positive
control group (two-way ANOVA test, P < 0.05) (Fig. 4C). The results
were comparable (two-way ANOVA test, P > 0.05) to MLX oral disper­
sion and i.v. solution treated groups demonstrating noteworthy effect of
MLX-emulgel in reducing latency period.
Radiographic analysis of knee joints was performed at 14th day.
Normal control group showed intactness of the surface of cartilage and
Fig. 4. Therapeutic efficacy analysis measured in terms of A) thickness of knee joint, B) motor incoordination and C) heat hyperalgesia for normal control group#,
positive control group# depicting monosodium iodoacetate induced OA, MLX emulgel treated group, MLX-oral treated group#, MLX i.v. treated group#, etoricoxib
marketed gel treated group#, diclofenac marketed gel# treated group, and blank gel treated group. #Adopted from [30] with permission.
8
Colloids and Surfaces B: Biointerfaces 228 (2023) 113399
no joint distension (Fig. 5A, Anterior-Posterior view). Positive control
group pointed-out reduced joint space (Fig. 5B, red arrow, Anterior-
Posterior view) with erosions on joint surface [90]. Treatment with
MLX oral dispersion, MLX-i.v. injection, MLX-emulgel and etoricoxib gel
displayed no joint distension (Fig. 5C-F, Anterior-Posterior view),
whereas, blank emulgel and diclofenac gel treated groups demonstrated
narrowing of joint space (Fig. 5G-H, Anterior-Posterior view). The
joint distension was lesser in MLX-emulgel group as compared to the
positive control group. MLX oral, i.v. injection, MLX-emulgel and etor­
icoxib gel treated groups displayed mild erosion on the surface of
femoral condyles and proximal tibia (Fig. 5C-F, Lateral view). Blank
emulgel and diclofenac gel showed erosion at tibia and femur regions
(Fig. 5G-H, Lateral view). Hence, MLX-emulgel was quite effective and
comparable to the MLX oral dispersion, i.v. injection and etoricoxib gel.
Blank emulgel was better than positive control in terms of joint space
and erosion which could be attributed to the anti-inflammatory property
of castor oil in blank emulgel.
Intra-articular injection of MIA resulted in cartilage damage leading
to the release of proinflammatory cytokines, thereby elevated PGE2
production by upregulating COX-2 expression [91]. Inhibition of COX-2
enzyme activity alleviates discomfort and pain of knee joint. Positive
control group showed elevated COX-2 levels indicating OA disease
model development (Fig. 6). MLX-emulgel treated group displayed
lowered (one-way ANOVA test, P < 0.001) COX-2 intensity as compared
to positive control group and comparable (one-way ANOVA test,
P > 0.05) to the normal control group, MLX oral dispersion, i.v. solution
and etoricoxib gel groups. Nevertheless, it was significantly different
from diclofenac gel (one-way ANOVA test, P < 0.05) and blank emulgel
(one-way ANOVA test, P < 0.01) groups (Fig. 6). Thus MLX-emulgel
alleviated pain, inflammation and discomfort associated with knee OA
by decreasing COX-2 expression.
Degradation of articular cartilage leads to the release of proin­
flammatory cytokines [91–94]. MLX-emulgel showed insignificant
(one-way ANOVA test, P > 0.05) difference in IL-1β level as compared to
normal control, MLX oral dispersion, i.v. solution, etoricoxib and
diclofenac gels. However, IL-1β level in MLX-emulgel treated group was
significantly (one-way ANOVA test, P < 0.01) different from positive
control group with 1.32-folds reduction (Fig. 6). Moreover, high level of
TNF-α and IL-6 also modulates MMPs expression stimulating cartilage
degradation [95]. MLX-emulgel portrayed 2-folds and 2.23-folds
decrease in TNF-α and IL-6 levels, respectively as compared to positive
control group (Fig. 6). The level of TNF-α and IL-6 in MLX-emulgel
treated group was similar (one-way ANOVA test, P > 0.05) to MLX
oral dispersion, etoricoxib and diclofenac marketed gels. However, it
showed significant variation from positive control group (one-way
V.G.S. Jyothi et al. Colloids and Surfaces B: Biointerfaces 228 (2023) 113399

Fig. 5. Radiographic analysis of knee joint lateral view and anterior- posterior view: A) normal control# B) positive control# C) MLX oral dispersion#, D) MLX i.v.
solution#, E) MLX emulgel, F) etoricoxib (COX-2 inhibitor) marketed gel#, G) diclofenac (Non-selective COX inhibitor) marketed gel# and H) blank emulgel (N:
normal E: erosion and ME: mild erosion of femoral condyles and proximal tibia; Orange arrow: normal joint space, red arrow: reduced joint space with erosion, green
arrow: joint space similar to normal control and yellow arrow: narrow joint space).#Adopted from [30] with permission.

ANOVA test, P < 0.001), normal control group (one-way ANOVA test,
P < 0.01 and P < 0.05) and MLX i.v. solution group (one-way ANOVA
test, P < 0.01 and P < 0.05). These results showed that MLX- emulgel
was able to reduce the TNF-α and IL-6 levels preventing further pro­
gression of disease by attenuating the expression of MMPs [96]. The role
of castor oil in reducing inflammatory markers was evaluated as blank
emulgel. MLX-emulgel showed insignificant (one-way ANOVA test,
P > 0.05) difference in IL-1β level and significant (one-way ANOVA test,
P < 0.001 and P < 0.01) difference in TNF-α and IL-6 levels as compared
to the blank emulgel. Hence, castor oil had little to weak impact in
reducing proinflammatory cytokines [97].
Anti-inflammatory cytokine, IL-10 is involved in reversing the effect
of TNF- α and thereby reduces the production of prostaglandin [5,98].
This was reflected in elevated levels of IL-10 in all MIA induced groups
except normal control group (Fig. 6). These results were in-line with a
reported clinical study which justified that IL-10 expression was higher
in severe OA damage patients [99]. Thus, MLX-emulgel showed effec­
tiveness in decreasing COX-2 expression and proinflammatory levels.
Consequently, histopathology of rat knee joints was performed to
evaluate the cartilage surface. Normal control group patterned intense

9
V.G.S. Jyothi et al.
Fig. 6. Measurement of COX-2 expression in addition to level of IL-1β, IL-6, TNF-α and IL-10 in normal control group#, positive control group#, MLX oral
dispersion#, MLX i.v. solution#, MLX-emulgel, etoricoxib (COX-2 inhibitor) marketed gel#, diclofenac (non-selective COX- inhibitor) marketed gel# and blank
emulgel. Data represented * **p < 0.001, * *p < 0.01, *p < 0.05 vs MLX emulgel group.#Adopted from [30] with permission.
blue staining of cartilage surface with intact articular cartilage (Fig. 7A,
orange arrow). The positive control group displayed either a very weak
or absence of staining due to deformation of cartilage (Fig. 7A, red
arrow). This showed the induction of disease condition in the rat’s right
knee joint. MLX oral dispersion and i.v. solution showed staining of to­
luidine blue with profound discontinuity of the cartilage (Fig. 7A, yel­
low arrow). This indicated that both MLX oral dispersion and i.v.
solution were able to protect the cartilage in OA disease condition,
consistent to the previous report [100]. MLX-emulgel group showed
incorporation of stain in cartilage and the subchondral bone (Fig. 7A,
orange arrow) with erosion of > 10 % cartilage surface. Etoricoxib and
diclofenac gels displayed either absence or very weak staining of tolui­
dine blue dye due to denudation of cartilage (Fig. 7, black arrow)
indicating minimal protection against OA disease progression which
were in harmony with the reported literature [101,102]. Blank emulgel
also showed negligible staining and therefore, deformation of cartilage
(Fig. 7, blue arrow) was noticed. Castor oil present in the blank emulgel
highlighted no remarkable effect on decreasing the OA severity.
Accordingly, MLX-emulgel was found to be superior to etoricoxib and
diclofenac gels and comparable to MLX oral and i.v. groups in treating
and providing protection against OA.
OARSI scoring was depicted in Fig. 7B. The highest score was noticed
in the positive control group followed by blank emulgel. MLX oral and i.
v. groups showed scoring equality and lower than MLX-emulgel. How­
ever, score of MLX-emulgel was lesser than blank emulgel, etoricoxib
and diclofenac gels [64,88]. MLX-emulgel proclaimed effective action
against knee OA by preventing the degradation of cartilage.
Hence, evidences indicated a mechanistic pathway of cartilage pro­
tection in OA by MLX-emulgel. MLX-nanoemulsion droplets embedded
in emulgel dosage form might be diffused through the skin by following
Korsmeyer-Peppas model (Fig. 3A and Suppl. Table 3) post topical
application in knee OA that subsequently penetrated into the skin layers
via trans-follicular route as reflected in CLSM imaging (Fig. 3A-C) and
reached to the inflamed tissue surrounding the knee joint [103]. Upon
reaching to the inflamed synovial tissue in a therapeutic concentration,
MLX-emulgel decreased COX-2 expression and inflammatory cytokine
levels (IL-1β, TNF-α and IL-6), as supported by western blotting and
10
Colloids and Surfaces B: Biointerfaces 228 (2023) 113399
ELISA (Fig. 6) assay, respectively. In addition, protective action of
MLX-emulgel was demonstrated by X-ray radiographic analysis and
histopathology (Fig. 7A-B). Thus, evidences support and advocate the
topical application of MLX-emulgel for OA management as compared to
MLX oral and i.v. administration which eliminates the side-effects
associated with the long term systemic exposure of drug.
4. Conclusion
In conclusion, MLX-emulgel can be prepared by tailoring nano­
emulsion using ultasonication and microfludizer methods suitable for
scalability at industry level. Post examining the stringent pharmaceu­
tical features, MLX-emulgel showed an improvement in permeation flux
of 6.71-folds as compared to plain drug and assessed for its potential in
alleviating pain and inflammation associated with knee OA. X-ray
radiography of knee joint of rats revealed protective action of MLX-
emulgel against OA that was further supported by measured cytokines
level, western blotting, histopathology and OARSI scoring. Interestingly,
the results were comparable to MLX oral and i.v. groups and showed
superiority in terms of topical application to the knee joint exempting
the side-effects associated with systemic absorption. MLX-emulgel
showcased an alternative to the long term usage of NSAIDs for
relieving symptoms of knee OA. Therefore, MLX-emulgel may be a po­
tential candidate for translating into a clinically viable dosage form in
managing knee OA.
CRediT authorship contribution statement
Vaskuri G S Sainaga Jyothi: Conceptualizartion, Experimentation
and manuscript writing and editing; Harithasree Veerabomma, Rahul
Kumar: Experimentation; Dharmendra Kumar Khatri, Shashi Bala Singh,
Jitender Madan: Supervision.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
V.G.S. Jyothi et al. Colloids and Surfaces B: Biointerfaces 228 (2023) 113399

Fig. 7. A) Histopathology of representative Wistar rats from normal control group#, positive control group#, MLX oral dispersion#, MLX i.v. solution#, MLX emulgel,
etoricoxib (COX-2 inhibitor) marketed gel#, diclofenac (non-selective COX inhibitor) marketed gel# and blank emulgel. Orange arrow: Intact cartilage, red arrow:
deformation of cartilage, yellow arrow: discontinuity of cartilage, brown arrow: erosion of cartilage, black arrow: denudation of cartilage and blue arrow: defor­
mation of cartilage. B) Osteoarthritis Research Society International (OARSI) scores. #Note: Adopted from [30] with permission.

11
V.G.S. Jyothi et al.
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
Authors are highly thankful to Department of Pharmaceuticals,
Ministry of Chemicals and Fertilizers, Government of India, New Delhi,
India for providing financial assistance to carry out the research work.
Appendix A. Supporting information
Supplementary data associated with this article can be found in the
online version at doi:10.1016/j.colsurfb.2023.113399.
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