International Journal of Applied Pharmaceutics

ISSN- 0975-7058 Vol 14, Issue 2, 2022

Original Article
LOXOPROFEN NANOSPONGES: FORMULATION, CHARACTERIZATION AND EX-VIVO STUDY

MANAL Y. HAMZAa,b, ZEINAB RAGHEB ABD EL AZIZ*b, MOHAMED ALY KASSEMc, MOHAMED AHMED EL-NABARAWIc
aDepartment of Pharmaceutics, Faculty of Pharmacy, Egyptian Russian University, Cairo, Egypt, bDepartment of Pharmaceutics, Egyptian
Drug Authority (EDA) Formerly Known as National Organization for Drug Control and Research (NODCAR), Cairo, Egypt, cDepartment of
Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Cairo University, Cairo, Egypt
Email: [email protected]
Received: 23 Nov 2021, Revised and Accepted: 04 Feb 2022
ABSTRACT
Objective: The objective of this study is to optimize a nanosponge formulation for Loxoprofen and then incorporating it into a gel formulation
offering a controlled drug release, enhanced skin permeation and thus better bioavailability.
Methods: Loxoprofen nanosponges were prepared using the emulsion solvent diffusion method and formulated using Polyvinyl alcohol,
Ethylcellulose and Dichloromethane. The effect of the different formulation variables like ethyl cellulose: polyvinyl alcohol ratio, drug: ethyl
cellulose ratio, stirring time, stirring speed, internal phase volume and external phase volume on the particle size, entrapment efficiency, production
yield, polydispersity index and Zeta potential was investigated. The optimized nanosponge formulation was incorporated into a gel. The loaded gel
was evaluated by in vitro release and permeation studies and the results were compared to that of a marketed formulation (Loxonin® gel).
Results: The optimized formulation showed 67.29±1.19 % entrapment efficiency, 239.8±16.95 nm particle size and-8.32±0.87 mV Zeta potential. The
drug was released slowly from the nanosponge-loaded gel where the cumulative percentage of drug released was only 77.71±0.42 % in 8 h where it was
incorporated in the entrapped form while it was 99.31±0.64% from Loxonin® gel where it was in the unentrapped form. The cumulative percent of
drug permeated through the skin from the nanosponge-loaded gel was 98.66±0.14% for 24 h while it was only 60.38±0.18% from Loxonin® gel.
Conclusion: The nanosponge-loaded gel showed more sustained drug release and a better drug permeation when compared to a marketed gel
(Loxonin® gel).
Keywords: Loxoprofen sodium, Nanosponges, Ethylcellulose, Gel, Carbopol 934, Emulsion solvent diffusion method
© 2022 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.2022v14i2.43670. Journal homepage: https://innovareacademics.in/journals/index.php/ijap

INTRODUCTION systems. Several topical dosage forms are used to deliver NSAIDs.
Many widely used topical agents such as ointments, creams and
Osteoarthritis is a leading cause of disability among older adults lotions have many disadvantages. They are sticky in nature causing
worldwide. Treatment aims are to alleviate inflammatory pain and uneasiness to the patient when applied and have low spreadability
improve physical function through non-pharmacological and and they may also exhibit stability problems. The use of gels has
pharmacological interventions. Non-steroidal anti-inflammatory emerged both in cosmetics and pharmaceutical preparations because
drugs (NSAIDs) are recommended as first-line therapy [1]. NSAIDs of their unique array of features in terms of use and patient
are among the most frequently prescribed drug groups. These drugs acceptability. Gel is a dosage form formed by the entrapment of large
are used locally or systemically in the treatment of various chronic amounts of aqueous or hydroalcoholic liquid in a network of colloidal
inflammatory conditions, relieving pain, reducing fever, and solid particles, which may consist of inorganic substances, such as
preventing local inflammation [2], NSAIDs lead to unfavorable aluminum salts or organic polymers of natural or synthetic origin. The
effects on the stomach as a result of inhibition of prostaglandins, constitution of a high aqueous component permits greater dissolution
which play a role in the protection of the gastric mucosa. of drugs, and also permits easy migration of the drug through a vehicle
Furthermore, the acidic character of NSAIDs may lead to local that is essentially a liquid, compared with either the ointment or
irritation on the gastrointestinal mucosa which is known as NSAIDs cream bases. Moreover, using hydrogel topical formulation as a
gastropathy [3]. Therefore, some NSAIDs are administered delivery system can reduce irritation and improve retention on the
transdermally to achieve local or systemic effect as an alternative to skin compared to the other topical formulations [10]. It was reported
oral and parenteral administration [4]. Loxoprofen sodium is a that hydrogel increased drug skin absorption and permeation 10 times
phenyl propionic acid type non-steroidal anti-inflammatory drug higher than oil-based formulations [11]. Also, hydrogel unique
with excellent efficacy in treating inflammatory rheumatoid diseases property (porosity) provides beneficial sustained and controlled drug
and relieving acute pain. Conventional transdermal delivery systems delivery of hydrophobic drugs via a suitable release mechanism. The
such as ointments and creams are associated with side effects due to objective of the present study is to assess the applicability of
the uncontrolled drug release from the formulation. Therefore, formulating nanosponges of Loxoprofen sodium and incorporating
attention is shifted towards the development of particulate carrier them into a transdermal gel and comparing this nanosponge-loaded
systems such as nanosponges for drug-controlled delivery [5]. In gel to conventional gels to clarify the advantages of the nanosponges
recent years, more focus has been drawn towards nanoparticulate technology.
systems e. g. nanosponges, as they offer more precise control of drug
release [6]. Nanosponges are a class of polymer-based colloidal MATERIALS AND METHODS
structures having nanosized cavities. A wide variety of topical agents
Materials
can be safely incorporated into nanosponges for getting the benefits
of these systems [7]. They are non-irritating, non-mutagenic, non- Loxoprofen sodium dihydrate was obtained as a gift sample from
allergenic, non-toxic [8] and can serve as a local depot for sustained Egyptian Group for Pharmaceutical Industries (EGPI) (Cairo, Egypt),
drug release, facilitate drug permeation across the skin, improve the Ethylcellulose, dialysis bag (100KD cut off) and Dichloromethane
bioavailability, increase the stability, reduce drug toxicity and were obtained from Sigma-AlDrich (St. Louis, USA), Polyvinyl alcohol
irritation, decrease adverse effects and improve the patient was procured from LOBA chemie PVT. LTD (Mumbai, India),
compliance by prolonging the dosage intervals [9], and thus Propylene glycol and Triethanolamine were purchased from El Nasr
overcoming the limitations of conventional transdermal delivery pharmaceutical chemicals (Cairo, Egypt), Carbopol 934 was
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Int J App Pharm, Vol 14, Issue 2, 2022, 233-241

procured from Techno pharm Chem (India). Distilled water was Production yield % was calculated by using the following equation:
used throughout the study. All the other chemicals were of analytical
grade and were utilized without any further purification. Production yield (%) = (Practical mass of nanosponges/Theoretical
mass [polymer+drug]) × 100 …… (1)
Animals
Measurement of particle size, zeta potential and polydispersity
This study was conducted after approval from the ethical committee of index
animal care of the faculty of pharmacy, Cairo University, Egypt (Approval
number: serial no. of the protocol: PI (2118)). Nine male albino Wistar The Particle size, PDI and Zeta potential of the prepared nanosponge
rats weighing (200-250 g) were obtained from and acclimatized at formulations were measured using a Zeta sizer (Malvern Zeta sizer
the central animal house of the national organization for drug control Nano series Ver. 7.11, Serial Number: MAL1044595) [14]. For this,
and research (NODCAR). The animals were kept in individual cages aqueous dispersions of nanosponges were diluted to an appropriate
under well-defined and standardized conditions (humidity and scattering intensity (100 µl of the dispersions diluted with 20 ml
temperature-controlled room; 12-h light and 12-h dark cycle) and they distilled water) [15].
were fed with standard dry food and water ad libitum. The skin of the
Entrapment efficiency
rats was collected and used in the ex-vivo permeation study.
A UV spectrophotometric method was used to calculate the
Method
entrapment efficiency of the nanosponge formulations. UV-visible
Preparation and optimization of Loxoprofen sodium nanosponges spectrophotometer (Unicam, England) was used to determine the
maximum absorbance (λmax) of Loxoprofen sodium. A calibration
Emulsion solvent diffusion method curve was plotted in distilled water at the determined (λmax).
Accurately weighed 10 mg of the formulation was added to 60 ml
In this method, different proportions of ethyl cellulose (EC) and
distilled water and stirred using a magnetic stirrer (Magnetic Stirrer
polyvinyl alcohol (PVA) are used to prepare the nanosponges. Two
Hot Plate: Prolabo: France) at 1000 rpm for 15 min then filtered. The
phases are used in this method; the dispersed is organic and the
absorbance of the filtrate was measured spectrophotometrically at
continuous is aqueous. The dispersed phase consists of the drug and
the predetermined λmax and the concentration was calculated using
EC dissolved in 20 ml of dichloromethane and the required amount
the constructed calibration curve [16].
of PVA is added to 150 ml of distilled water (the continuous phase).
The organic phase was slowly added to the aqueous phase and the % Entrapment efficiency was calculated using the following equation
mixture was stirred for 2 h at 1000 rpm using a magnetic stirrer
[12]. The resultant nanosponge dispersion was centrifuged at 6000 % Entrapment efficiency = (Actual drug content in the
rpm and 25 °C for 60 min and the excess solvent was decanted. The nanosponges/Theoretical drug content) × 100 …… (2)
separated nanosponges were air dried at room temperature then
The optimized nanosponge formulation was selected for further studies.
packed in airtight vials for evaluation.
Surface morphological studies
Twenty formulations were prepared using six varying formulation
factors; polymer surfactant ratio (EC PVA ratio), drug polymer ratio, Surface morphology of the optimized nanosponge formulation was
stirring time, stirring speed, the volume of internal organic phase studied using scanning electron microscopy (SEM) [17], scanning
and volume of the external aqueous phase. The effect of these electron microscope (Quanta FEG 250) operated at an acceleration
variables on the production yield, particle size, polydispersity index voltage of 20 kV was used.
(PDI), Zeta potential, and entrapment efficiency was studied.
Initially, six nanosponge formulations were prepared. EC was used The optimized nanosponge formulation was selected for formulating
as entrapping agent, Dichloromethane as cross-linking agent and a gel using Carbopol 934 as a gelling agent. Drug-excipient
PVA as an emulsifying agent. Nanosponges were prepared using compatibility studies (Differential scanning calorimetric (DSC)
different EC: PVA ratios; 1:1, 1:2, 1:3, 1:4, 1:5 and 1:6 (F1 to F6 studies and Fourier Transform Infrared (FTIR) analysis) were
respectively) while the other variables were kept constant; distilled performed before formulation.
water volume was 150 ml, drug: EC ratio 1:2, stirring time 2 h, Differential scanning calorimetric studies
stirring speed 1000 rpm and Dichloromethane volume 20 ml.
The melting point of the pure drug was compared to the melting
Another six nanosponge formulations were prepared using the point of the drug in the physical mixture of the gel-forming polymer
chosen EC: PVA ratio 1:6 and different drug: EC ratios; 1:0.5 (F12), Carbopol 934 and the selected optimized nanosponge formulation
1:1 (F7), 1:3 (F8), 1:4 (F9), 1:5 (F10) and 1:6 (F11) using distilled (DSC-60, Shimadzu Corporation, Japan) was used after calibration
water volume of 150 ml, stirring time 2 h, stirring speed of 1000 with Indium and lead standards, samples (3-5 mg) were heated
rpm and Dichloromethane volume of 20 ml. (range 25-400 ⁰C, 10 ⁰C/min) in crimped Aluminum pans under a
To find out the optimum stirring time, two formulations were prepared nitrogen atmosphere at a flow rate of 10 ml/min [18]. The recorded
using stirring time 3 h (F13) and 1 h (F14) using distilled water volume thermograms were analyzed for any interaction between the drug
of 150 ml, EC: PVA ratio 1:6, a drug: EC ratio 1:5, a stirring speed of 1000 and the excipients.
rpm and a dichloromethane volume of 20 ml. Two more formulations Fourier transform Infrared analysis
were prepared using stirring speeds of 800 (F15) and 900 rpm (F16)
using stirring time of 3 h, the distilled water volume of 150 ml, EC: PVA Fourier transform infrared analysis was used to check any kind of
ratio 1:6, a drug: EC ratio 1:5 and dichloromethane volume of 20 ml, chemical interaction between Loxoprofen sodium and the excipients
another two formulations were prepared using dichloromethane volume that would be used in the formulation of the nanosponge-loaded gel.
of 10 ml (F17) and 30 ml (F18) using EC: PVA ratio 1:6, a drug: EC ratio Schimadzu FT-IR Affinity-1 Spectrometer was used. Samples of pure
1:5, stirring time of 3 h, stirring speed 900 rpm and distilled water Loxoprofen sodium and the physical mixture of the gel-forming
volume of 150 ml. Two final formulations were prepared using distilled polymer Carbopol 934 and the selected optimized nanosponge
water volume of 100 ml (F19) and 200 ml (F20) using EC: PVA ratio 1:6, formulation were mixed with IR grade KBr. Samples were scanned
a drug: EC ratio 1:5, stirring time 3 h, stirring speed of 900 rpm and in the range from 4000 to 400 cm-1 and carbon black reference. The
Dichloromethane volume 20 ml. detector was purged carefully by clean, dry helium gas to increase
the signal level and reduce moisture [19].
Evaluation of Loxoprofen soduim-loaded nanosponges
Design and preparation of Loxoprofen-loaded nanosponge gel
Determination of production yield
The optimized nanosponge formulation was selected for formulating
It was calculated using the weight of the formed nanosponges after hydrogel using Carbopol 934 as a gelling agent, propylene glycol (PG) as
drying and the initial total weight of the drug and polymer used for a permeation enhancer and Triethanolamine (TEA) as a pH neutralizer, a
the preparation of nanosponges [13]. conventional loxoprofen sodium 1% gel was also prepared [20].

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Preparation of Loxoprofen-loaded nanosponge gel Preparation of the conventional gel

Accurately weighed amount of Loxoprofen sodium nanosponge Carbopol 934 was dispersed in the solution of Loxoprofen sodium in
powder and the required quantity of Carbopol 934 polymer were part of the required amount of distilled water, then PG was added
dispersed in part of the calculated amount of distilled water then PG and they were homogenized, TEA was added slowly with constant
was added and they were homogenized, TEA was slowly added with stirring till a viscous gel was formed, the final weight was completed
by distilled water and final homogenization was made.
constant stirring for neutralization till a viscous gel was formed, the
final weight was completed by distilled water and final The prepared gels were stored in tightly closed screw-capped plastic
homogenization was made [14]. jars at 5 °C for further investigations.

Table 1: The composition of Loxoprofen-loaded nanosponge hydrogel and loxoprofen sodium conventional hydrogel
Component Loxoprofen-loaded nanosponge hydrogel Loxoprofen sodium conventional hydrogel
Carbopol 934 2g 2g
Loxoprofen sodium dihydrate nanosponge 2g -
Loxoprofen sodium dihydrate - 1g
PG 20 ml 20 ml
TEA 5 ml 3 ml
Distilled water (q. s) To 100 g To 100 g

Evaluation of gels Ex-vivo skin permeation studies

The prepared formulations and a marketed reference formulation A comparative study was carried out on the Loxoprofen-loaded
(Loxonin®) gel were evaluated. nanosponges gel and a marketed reference formulation Loxonin®
gel. Shaved rat skin was mounted between the donor and receptor
Physical examination compartments of a vertical Franz diffusion cell [25], with the
The prepared formulations and the reference formulation were first stratum corneum side facing the donor compartment and the dermal
inspected visually for their appearance, clarity, color and side facing the receptor compartment, which was 11 ml phosphate-
homogeneity [7]. buffered saline (PBS) pH 7.4, stirred by a magnetic bar at 100 rpm
and a temperature of 37 °C±1 °C maintained by water circulation
Viscosity determination throughout the experiment. 250 mg of each gel formulation was
applied uniformly to the dorsal side of the rat skin in the donor
The viscosity of the prepared hydrogels and the reference
compartment. Available surface area for permeation was 1.23 cm2, 1
formulation was measured using (Brookfield DVIII cone and plate
ml samples were withdrawn from the sampling port of the receptor
viscometer) with spindle no. 52 [21]. Viscosity was recorded at 25
compartment at predetermined time intervals and immediately
°C, at 10 and 100 rpm.
replaced with another 1 ml of equally warmed fresh buffer for 24 h
pH determination [26]. Samples were analyzed using a validated UV method and the
concentration of the permeated drug was determined at the
The pH was measured using a calibrated pH meter (HANNA predetermined λ max. A graph was constructed between the
Instruments, Portugal), 0.5 g gel was diluted with 4.5 ml distilled cumulative percentage drug permeated versus time [27]. The data
water then the pH was measured at 25 °C [14]. obtained were also subjected to mathematical models like zero-
In vitro release studies order, first-order, second-order, Higuchi, Hixson-Crowell and Baker
Lonsdale kinetic models [28].
In vitro drug release studies for the pure drug, the marketed product
Loxonin® gel, the conventional hydrogel, the optimized formulation N. B. all measurements in this study were in triplicates and average
nanosponge loaded gel and the optimized nanosponge formulation values were calculated and noted.
were performed. 100 mg of samples were suspended in 2 ml RESULTS AND DISCUSSION
phosphate buffer pH 5.5 and added to a dialysis bag (100KD cut off)
[22]. The dialysis bag was sealed properly from both the top and the Preparation and optimization of Loxoprofen sodium
bottom and inserted into 30 ml dissolution medium (phosphate nanosponge formulations
buffer pH 5.5) in a 50 ml falcon tube. The whole system was fixed in
a shaking water bath (Fischer Scientific, USA), rotating at 100 rpm Effect of formulation variables on the physicochemical
with temperature adjusted to 37±1 °C Temperature and pH were characteristics of nanosponge formulations
chosen to simulate that of human skin. At specified time intervals, 1 In order to assess the effect of EC: PVA ratio on the physicochemical
ml medium sample was withdrawn and immediately replaced with characteristics of nanosponges, six formulations with different EC:
another 1 ml of equally warmed fresh buffer to maintain sink PVA ratios were prepared. All formulations resulted in the formation
conditions for 8 h. Withdrawn samples were filtered and assayed at of nanosponges. The entrapment efficiency of the prepared
each time interval for the drug released at the predetermined λ max nanosponges was in the range of 7.99±2.11 to 33.9±3.28 %. The
[23] using UV-Visible spectrophotometer (Unicam, England) and highest value was found to be with formulation F4. The
phosphate buffer pH 5.5 as blank. A calibration curve of serial drug concentration of the surfactant needs to be optimized to avoid
dilutions was constructed to enable the calculation of the amount of foaming, particle aggregation and a decrease in entrapment
released drug in the sample. From this percentage drug released was efficiency. With respect to PVA, entrapment efficiency decreased
calculated and values of cumulative % drug released were plotted when the EC/PVA ratio exceeded 1:4, which was found to be
versus time. optimum concerning the entrapment efficiency. The most uniform
Kinetic analysis of drug release size distribution was obtained with EC/PVA ratio 1:1 in F1 which
showed the lowest PDI of 0.348±0.02. The surface charge of
To study the drug release mechanism from the optimized formulation nanosponges was determined by Zeta potential and it was found to
and the nanosponge-loaded hydrogel formulation, the release data be in the range of-9.85±2.56 to-40.7±0.87 mV. The formulation F5
was fitted to zero-order, first-order, second-order, Higuchi, Hixson- with EC/PVA ratio 1:5 showed Zeta potential value of-40.7±0.87 mV.
Crowell and Baker Lonsdale kinetic models [24]. The kinetic model At this high potential, nanosponge particles would exhibit a great
with the highest coefficient of correlation (R2) value was considered to repulsion with each other, which will significantly prevent their
be the best fit model for describing the drug release. agglomeration. It was observed that the above 1:5 ratio increasing

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the concentration of PVA decreased the production yield. This may the best values concerning entrapment efficiency and Zeta potential
be due to the increased foaming during the nanosponge preparation and this ratio was selected for further optimization.
with the increase in the amount of PVA hindering the formation of
the nanosponges [29]. Particle size did not follow any particular The third studied parameter was stirring time. It varied from 1 h to 3
pattern. Smallest particle size was obtained with F6 with EC/PVA h. In our study we have found that the stirring time of 3 h yielded
ratio 1:6. Particles of size range 100-200 nm are favored for topical nanosponges with the highest entrapment efficiency and production
formulations [30]; that is why this ratio was chosen for further yield with the least particle size which is favored but with the least
optimization. Zeta potential and highest PDI, which means the least stable
formulation and since 3 h resulted in nanosponges with the highest
The effect of the drug to polymer ratio on the physicochemical entrapment efficiency and production yield with the least particle size;
characteristics of the formulated nanosponges was examined for therefore this stirring time was selected for further optimization.
various ratios from 1:0.5 to 1:6. Nanosponges were successfully
formed with all formulations except F9 and F11 having drug to The effect of the stirring speed on the physicochemical
polymer ratios 1:4 and 1:6 respectively, they showed particle sizes characteristics of the formulated nanosponges was examined. The
out of the nano range (>1 μm) and hence did not meet the stirring speed varied from 800 rpm to 1000 rpm. Results indicated
requirements to be characterized as nanoparticles [12], for this that as the speed increased, the particle size and production yield
reason they were discarded from further studies. The entrapment increased. It was also observed that as the speed exceeds 900 rpm,
efficiency of the valid nanosponges was in the range of 12.15±1.76 to entrapment efficiency is decreased. Our results have also shown that
49.45±3.21 %. Maximum value was achieved with 1:5 ratio. The the formulation F16 with 900 rpm stirring speed showed the least
valid nanosponges mean particle size ranged from 62.89±7.69 to PDI value and the highest Zeta potential, which means that it was the
533.2±18.12 nm. The mean particle size was found to increase with most stable formulation. Therefore, this speed was selected for
an increase in polymer amount [31]. This may be due to the further optimization.
availability of a large quantity of polymer. It was observed that as Dichloromethane as internal phase varied in volume from 10 to 30
the polymer amount decreases, the particle size decreases. This may ml and the impact was studied. As the volume of internal phase
be due to the fact that at low polymer concentration and relatively increased, particle size and entrapment efficiency did not follow any
higher drug content, the amount of polymer available per particular pattern. 20 ml was found to be the optimum volume as it
nanosponge to encapsulate the drug becomes less. The thickness of yielded the most ideal formulation F16 showing promising results in
polymer wall is thereby reduced leading to small-sized nanosponges all parameters; a favorable least particle size, highest entrapment
[14]; moreover, large quantities of EC increased the viscosity of the efficiency and production yield, least PDI value and highest Zeta
system which created hindrances in the formation of smaller potential which means the most stable formulation. Therefore, this
droplets [32]. At low EC quantities, diffusion of the internal phase volume was used for further optimization.
(dichloromethane) into the external phase (aqueous phase) was
improved, reducing the time for droplet formation, which resulted in As the volume of distilled water was increased, entrapment
smaller particle sizes. It was observed that as the polymer amount in efficiency was increased. Although F16 with 150 ml distilled water
the formulation increases, the production yield also increases till showed the least particle size, the least PDI value and the highest
drug: EC ratio 1:2 above which the production yield decreased. This Zeta potential, which means the most stable formulation. The
may be due to that larger amounts of polymer increased the wall formulation having maximum entrapment efficiency and production
thickness of the nanosponge as indicated by a larger particle size yield was chosen to formulate a hydrogel. The entrapment efficiency
resulting in the formation of lesser number of nanoparticles [29]. of F20 formulation was found to be 67.29±1.19 %. Hence, the
F12 formulation with drug to polymer ratio 1:0.5 showed the least formulation F20 was considered the optimized Loxoprofen sodium
PDI value. Formulation F10 with drug to polymer ratio 1:5 showed nanosponge formulation and it was selected for further studies.

Table 2: Physicochemical characteristics of the valid nanosponge formulations

Formulation Particle size±SD* (nm) Zeta potential±SD* PDI±SD* Production yield Entrapment efficiency
(mV) (%)±SD* (%)±SD*
F1 380.7±5.65 -20.3±4.73 0.348±0.02 17.27±1.43 7.99±2.11
F2 237.8±11.85 -9.85±2.56 1 21.6±4.51 9.97±5.21
F3 231.9±8.52 -10.7±1.07 0.54±0.040 22.99±4.89 10.78±3.78
F4 465.7±9.13 -13.15±0.97 0.453±0.025 25.18±6.32 33.9±3.28
F5 248.7±7.48 -40.7±0.87 0.392±0.016 38.9±1.54 28.7±3.56
F6 202.1±5.67 -15.85±1.32 0.494±0.04 34.12±4.21 15.75±2.54
F7 75.6±3.78 -13±1.17 1 32.41±3.21 12.15±1.76
F8 219.7±14.76 -8.18±0.38 0.666±0.04 31.38±5.11 44.88±3.65
F 10 533.2±18.12 -28.75±0.87 0.351±0.033 21.1±2.54 49.45±3.21
F12 62.89±7.69 -16.75±0.34 0.3475±0.032 25.57±2.77 30.23±6.32
F13 248±17.89 -7.2±0.77 0.962±0.054 46.2±1.34 58.97±6.55
F14 292.2±10.76 -13.2±0.55 0.813±0.043 41.4±1.89 50.26±6.72
F15 16.35±10 -14.1±0.88 1 33.72±0.67 60.9±0.95
F16 53.42±20.26 -20.2±0.89 0.667±0.042 43.56±1.12 61.82±1.36
F17 191.6±25.19 -10.7±1.15 0.71±0.04 18.84±1.32 51.18±0.82
F18 288.4±28.93 -13.5±0.66 0.854±0.01 41.28±0.98 60.16±0.52
F19 542.5±30.09 -6.27±1.54 0.805±0.02 47.1±6.20 59.96±2.14
F20 239.8±16.95 -8.32±0.87 0.787±0.08 69.55±8.07 67.29±1.19
*Values are expressed as mean±SD, SD: Standard deviation, n=3.

Surface morphology inwards, which may be due to the diffusion of Dichloromethane from
the surface of the nanosponges during preparation.
The morphology of the selected optimized formulation F20
nanosponges was investigated by SEM. The representative SEM The optimized Nanosponge formulation (F20) was selected for
photograph of the nanosponges is shown in fig. 1. It was observed formulating a hydrogel using carbopol 934 as a gelling agent, PG as a
that the nanosponges were nanosized particles uniformly spherical permeation enhancer and TEA as pH neutralizer. Drug-excipient
in shape with a spongy and porous nature. The pores are tunneled compatibility studies (IR, DSC) were performed before formulation.

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Drug-excipient compatibility studies

Differential scanning calorimetric studies
DSC thermograms of pure Loxoprofen sodium dihydrate and the
physical mixture of Carbopol 934 polymer and the
optimized nanosponge formulation (F20) are shown in fig. 2. DSC
curve of pure Loxoprofen sodium showed a sharp endothermic peak
at 80.23 °C corresponding to its melting point, while DSC curve of
the physical mixture showed the absence of the drug melting peak
indicating the successful encapsulation of the drug within the
nanosponge cavities imparting higher thermal stability to the
Fig. 1: SEM image of F20 nanosponge formulation with Mag.6 KX formulation when compared to the pure unentrapped drug [33].

Fig. 2: DSC thermograms of pure loxoprofen sodium dihydrate and Loxoprofen sodium F20 nanosponge formulation-Carbopol 934 mixture

Fourier transform infrared analysis Evaluation of gels

FTIR spectroscopy was used to check any possible chemical The physical properties of the prepared hydrogels and the
interaction between the drug and the excipients which would be marketed formulation Loxonin® gel were evaluated. All the
used in the formulation. By comparing FT-IR spectra of the pure hydrogels were smooth and homogenous. The physicochemical
drug with that of a physical mixture of nanosponge F20 formulation properties like viscosity and pH were determined and
with Carbopol 934 polymer, it was found that all the characteristic tabulated (table 3). The viscosity affects the spreadability,
peaks of Loxoprofen sodium appeared at their respective wave extrudability and drug release. The gels with high viscosity may
number ranges in the FTIR spectrum of the physical mixture which not extrude from the tube easily, whereas low viscosity gels may
means the absence of any change in the chemical integrity of the
flow quickly. Hence, there should be an optimum viscosity. The
drug during preparation and thus the drug stability in the
viscosity values of the prepared formulations were near that of the
formulation was confirmed [34]. Results are shown in fig. 3.
marketed formulation at different rates of shear. The hydrogel pH
Gel formulations were prepared using Carbopol 934 as a gelling values were considered acceptable so there is no risk of skin
agent and evaluation studies were carried out. irritation upon application [35].

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Fig. 3: FTIR spectrum of pure loxoprofen sodium dihydrate and Loxoprofen sodium F20 nanosponge formulation-Carbopol 934 mixture

Table 3: Physical evaluation of hydrogels

Gel formulation Clarity and color Viscosity (cPs) at 10 rpm±SD* Viscosity (cPs) at 100 rpm±SD* pH±SD*
Loxonin® gel Transparent white 22146±2.5 3310±3.0 6.45±0.19
Conventional gel Opalescent white 36982±2.0 6299±1.1 7.77±0.05
Nanosponge-loaded gel Opaque white 39218±3.0 6070±1.0 8.28±0.02
*Values are expressed as mean±SD, SD: Standard deviation, n=3.

Fig. 4: The cumulative percentage drug release profile of loxoprofen sodium from nanosponge F20 formulation and pure Loxoprofen
sodium in phosphate buffer pH 5.5 (Values are expressed as mean±SD, SD: Standard deviation, n=3)

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In vitro release studies the polymer as shown in fig. 6 and fig. 7. This may be due to the fact
that the release of drug from the polymer matrix takes place after
The percentage cumulative drug released from the pure drug was the complete swelling of the polymer.
compared with the optimized nanosponge formulations F20, where
the percentage drug released, was 94.67±0.61 % in 6 h while the pure Kinetic data modeling studies
drug dissolved almost completely after 3 h due to its solubility in
phosphate buffer pH 5.5. The matrix of the nanosponges held the drug Different kinetic models were applied to find out the release
and controlled its release over a longer period of time as shown in behavior from the optimized formulation F20 and the nanosponge
fig. 4. The in vitro release profile of Loxoprofen sodium from the loaded hydrogel. The interpretation of data showed that maximum
optimized formulation F20 in fig. 4 showed a bi-phasic pattern with an linearity (highest R2 value) was found with Hixon Crowell model and
initial burst release for the initial 0.5 h followed by a slow and Baker Lonsdale model, respectively. The Baker and Lonsdale model
sustained release. Initial burst release may be due to the unentrapped indicates that the structure of the releasing matrix is spherical,
adsorbed drug on the surface, which was released at a faster rate because Baker and Lonsdale model is based on the Higuchi diffusion
compared to the entrapped drug inside the nanosponges core [36]. model, this proves that the matrix is porous, and with channels and
the drug can diffuse out of these pores and channels [37]. This
As shown in fig. 5, Loxoprofen sodium nanosponge-based gel diffusion is the principal mechanism of drug release which may be
formulation sustained the release for more duration when compared controlled by the porosity of the nanosponges.
to the other formulations where the drug was in the free
unentrapped form. Hence, it was clear that drug was released slowly Ex-vivo skin permeation studies
from the dosage form when it was incorporated in the entrapped The Loxoprofen-loaded nanosponges gel formulation showed higher
form rather than that in the unentrapped form [30]. This could be drug permeation through the skin within 24 h where the cumulative
due to the thickness of the nanosponges’ matrix wall which could percent of drug permeated through the skin was found to be
lead to a longer diffusional path, and consequently controlled drug 98.66±0.14 % for 24 h while it was only 60.38±0.18 % for Loxonin®
release. It is assumed that the possible mechanism behind the drug gel. The nanosponges being in nanosize can easily permeate the skin
release is a combination of dissolution, diffusion and erosion where without any need to permeation enhancers [38], in addition to the
the decrease in release pattern may be due to the difficulty of the
huge surface area available for dissolution, nanosponges are nano-
diffusion of the drug through the hydrophobic core.
sized colloidal bearer, so they easily pierce into the skin [39], that
When comparing the cumulative percent of released Loxoprofen explains why the ex-vivo skin permeation from the Loxoprofen
sodium from the conventional gel and pure Loxoprofen sodium and loaded nanosponges gel is higher. All these factors synergize and
also from the nanosponge F20 and the nanosponge F20 loaded gel, it improve the drug bioavailability. A graph was constructed by
was observed that the drug release decreased with the presence of plotting the cumulative percent drug permeated versus time (fig. 8).

Fig. 5: Comparative release profiles of the different gel formulations in phosphate buffer pH 5.5 (Values are expressed as mean±SD, SD:
Standard deviation, n=3)

Fig. 6: Comparative release profiles of the conventional gel and pure loxoprofen sodium in phosphate buffer pH 5.5 (Values are expressed
as mean±SD, SD: Standard deviation, n=3)

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Fig. 7: Comparative release profiles of the nanosponge F20 and the nanosponge F20 based gel in phosphate buffer pH 5.5 (Values are
expressed as mean±SD, SD: Standard deviation, n=3)

Fig. 8: The cumulative percent drug permeated versus time for Loxoprofen-loaded nanosponges gel and Loxonin® gel (Values are
expressed as mean±SD, SD: Standard deviation, n=3)

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