Transfersomal gel nanocarriers for enhancement the permeation of lornoxicam

Journal of Drug Delivery Science and Technology 56 (2020) 101540

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Transfersomal gel nanocarriers for enhancement the permeation of lornoxicam

T

Hesham M. Tawfeeka,∗, Ahmed A.H. Abdellatifb,c, Jelan A. Abdel-Aleema, Yasser A. Hassand,e, Dina Fathallaf a

Department of Industrial Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, Egypt Pharmaceutics and Industrial Pharmacy Department, Faculty of Pharmacy, Al-Azhar University, 71524, Assiut, Egypt c Pharmaceutics Department, College of Pharmacy, Qassim University, 51452, Buraydah, Saudi Arabia d Faculty of Pharmacy, Delta University for Science and Technology, Gamasa City, Dakhlia, Egypt e Department of Pharmaceutics, College of Pharmacy, Al-Bayan University, Baghdad, Iraq f Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut, Egypt b

A R T I C LE I N FO

A B S T R A C T

Keywords: Lornoxicam Topical hydrogel Transfersomes Hydroxypropyl methylcellulose Ex vivo permeation

In respect to the major gastrointestinal disorders associated with oral administration of Lornoxicam, LOR, a potent anti-inflammatory drug, topical delivery could be an alternative route of administration. The objective of this work was to formulate LOR in the form of topical hydrogel after encapsulation into deformable vesicles, transfersomes, TRSs for maximum penetration and activity. LOR TRSs were prepared through thin film hydration technique and characterized for their encapsulation efficiency, size, charge, morphology and stability. Furthermore, LOR transfersomal and non-transfersomal hydrogels were prepared using different gelling agents and characterized for their pH, contents, viscosity, homogeneity, skin irritation, in vitro release, skin permeation, and pharmacodynamic activity. Results revealed that optimum LOR TRSs had an encapsulation efficiency of 99.34 ± 0.2%, size of 233.5 ± 12.5 nm and zeta potential of −35.34 ± 0.78 mV. Furthermore, they showed higher chemical and physical stability when stored in the fridge. Transfersomal hydrogels stabilized with sodium deoxycholate showed higher drug permeation through rat skin. In addition, they have higher flux and apparent permeability coefficient and superior anti-inflammatory activity compared to non-transfersomal LOR hydrogel and indomethacin gel as a standard NSAID. These findings confirmed that LOR transfersomal hydrogel is a promising topical formulation for effective treatment of local inflammatory conditions.

1. Introduction Oral non-steroidal anti-inflammatory drugs (NSAIDs) are most widely used for the healing of acute and chronic pain disorders [1]. However, their chronic usage may be associated with serious systemic side effects, especially gastrointestinal disorders, such as nausea, diarrhea, and ulceration. In addition, some severe side effects including bronchospasms, bleeding, and the rare Stevens-Johnson syndrome [2]. One of the most potent and widely used NSAIDs is lornoxicam, LOR. It is a powerful inhibitor of both COX-1 and COX-2 enzymes [3]. In addition, LOR has confirmed higher clinical efficacy in reducing chronic pain accompanying with osteoarthritis [4], rheumatoid arthritis, and ankylosing spondylitis [5,6]. Moreover, for the healing of postoperative pain, lornoxicam has been shown to be as efficient as morphine [7]. It ∗

has a half-life ranged from 3 to 5 h and peak plasma concentration is attained within 2.5 h. LOR is ten times more potent than other oxime derivatives [8]. However, like other NSAIDs, LOR still has an issue associated with its oral delivery on the gastrointestinal tract as the only marketed route of administration. Previously, LOR was formulated into mini-tablets and rectal suppositories [9,10]. However, oral tablets still showed the gastric effect and rectal suppositories might be unsuitable for some patients. LOR was formulated in the form of sustained release buccal patches for treatment of patients suffering from post-operative pain and edema following maxillofacial operations [11]. However, the inconvenient nature associated with these patches administration, especially with elderly people and children, is considered a problem. Moreover, the patients cannot eat, drink or even speak when using these patches for prolonged period of time. Swallowing of saliva may

Corresponding author. Department of Industrial Pharmacy, Faculty of Pharmacy, Assiut University, Assiut, 71526, Egypt. Tel.: 002 0882411322. E-mail addresses: [email protected], [email protected] (H.M. Tawfeek).

https://doi.org/10.1016/j.jddst.2020.101540 Received 12 November 2019; Received in revised form 4 January 2020; Accepted 22 January 2020 Available online 27 January 2020 1773-2247/ © 2020 Elsevier B.V. All rights reserved.

Journal of Drug Delivery Science and Technology 56 (2020) 101540

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also lead to the loss of dissolved and suspended drugs, as well as the low permeability of buccal membrane when compared with sublingual one [10], this is why looking for another delivery route is an important issue. For minimizing the frequency of systemic events related to NSAIDs, topical delivery has been established and showed a promising system to manage different disorders long ago. However, conventional topical delivery showed some disadvantages such as minimum penetration and very low efficacy and permeation [12]. Nowadays, lipid vesicles exhibited a higher awareness in the field of transdermal drug delivery such as liposomes, transfersomes, ethosomes and niosomes [13–15]. These unique lipid vesicles especially the second-generation type, transfersomes and ethosomes, showed a characteristic lipid fluidity and elasticity enabling them to perform a superior penetration performance [16–18]. Previously, LOR has been studied for its topical delivery using ethosomes, niosomes and proniosomes and nanostructured lipid carriers [19–22]. Nevertheless, LOR transfersomes have been investigated rarely in literature [23]. So, the objective of this study was to formulate LOR transfersomes followed by addition in a suitable hydrogel base for maximum drug loading and higher penetration performance. LOR transfersomes, LOR TRSs, were prepared via thin film hydration method using phosphatidyl choline and two edge activators, tween 80 and sodium deoxycholate. Then, TRSs characterized for drug entrapment, size, charge, morphology as well as stability at two different conditions. LOR hydrogel was formulated using different gelling agents such as hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, and sodium alginate. In addition, the transfersomal hydrogel was prepared using HMPC and characterized for its drug content, pH, viscosity, skin irritation, in vitro release, ex vivo permeation through incised rat skin. Finally, pharmacodynamics activity of the optimum formulations was considered, using paw edema technique in rats, and compared with non-transfersomal LOR hydrogel and indomethacin gel as a standard NSAID.

Table 1 Composition of various LOR vesicular transfersomes (TRSs) per 1 ml of colloidal carrier solution. Ingredients (mg)

TRSs (1)

TRSs (2)

Lornoxicam Phospholipid Tween 80 Sodium deoxycholate Water up to (μL) as final volume

5 42.5 – 7.5 1000

5 42.5 7.5 – 1000

The vesicles were prepared by the thin film hydration method [24,25]. Briefly, drug, phosphatidylcholine and edge activators, sodium deoxycholate and tween 80, were dissolved in a mixture of chloroform and methanol (2:1, v/v). Then, the organic solvent was vaporized under reduced pressure at a temperature of 40 °C using rotary evaporator (Büchi, type R 110, Switzerland). A thin layer of lipid film was obtained on the walls of the flask and the residual organic solvents were let to completely evaporate by keeping overnight in a vacuum desiccator. Further, a thin lipid film was hydrated at 37 ± 1.0 °C using phosphate buffer (pH 6.8). Afterward, the obtained suspension was vortexed for approximately 2 min followed by incubation at room temperature for 2–3 h to permit complete hydration of the lipid film. 2.4. Characterization of LOR TRSs 2.4.1. Measurement of LOR entrapment efficiency To isolate the free drug from the formulated TRSs, the vesicles were centrifuged at 20,000 rpm for 30 min at 4 ± 0.5 °C in a cooled centrifuge (Hettich, Germany). Further, the supernatant was collected and analyzed by UV-VIS Spectrophotometer (Jenway, Japan) at λmax of 377 nm [26]. The pellet gained after centrifugation was dissolved using methanol then sonicated for 12 min. The concentration of LOR in the pellet was quantified spectrophotometrically at λmax of 377 nm. Percentage drug encapsulation efficiency, % EE, and loading capacity were estimated as follows using Equations (1) and (2), respectively [27]:

2. Materials and methods 2.1. Materials

%EE = Lornoxicam was obtained as a gift from Delta Pharma, (10th of Ramadan City, Cairo, Egypt). Carbopol 934 (BDH Chemicals LTD.UK). Hydroxypropyl methylcellulose, Hydroxypropyl cellulose, sodium carboxymethyl cellulose, sodium alginate (Aldrich Chemical Company, USA). Phosphatidylcholine (PC) phospholipon® 90G were purchased from Lipoid (Steinhausen, Switzerland). Tween 80, sodium deoxycholate and semipermeable cellulose membrane (12000–14000 MW cutoff) were purchased from Sigma Chemicals (St. Louis, MO, USA). Uranyl acetate (Polyscience Inc. Warrington, PA, USA). Chloroform, Triethanolamine, methanol, potassium dihydrogen phosphate, and disodium hydrogen phosphate were obtained from the United Company for Chem. and Med. Prep., (Cairo, Egypt). All other chemicals and solvents were of analytical grade and used as received.

T− C × 100 T

(1)

Where T is the initial amount of drug added and C is the amount of drug in the supernatant

Percent LOR loading (wt%) =

Weight of drug in the vesicles × 100 (Total weight of the tested vesciels ) (2)

2.4.2. Size, polydispersity, and zeta potential The size and polydispersity for the prepared plain and LOR loaded TRSs were measured using zetasizer-nano S90 (Malvern Instruments GmbH, Herrenberg, Germany). Samples were adjusted to 25.0 ± 2.0 °C, and laser light scattering analysis was adjusted with a laser beam of 633 nm at a scattering angle of 90°. Surface charge of drug loaded TRSs vesicles was measured using zetasizer (Malvern Instruments, Malvern, UK). Analysis time was retained at 60 s, and the average zeta potential of the vesicles was determined.

2.2. Fourier-transform infrared spectroscopy (FT-IR) FT-IR study was investigated to demonstrate any interaction between LOR and excipients used in TRSs formulation. Briefly, PC, sodium deoxycholate, LOR and corresponding physical mixtures as well as LORloaded TRSs (TRSs 1) were mixed separately with infrared (IR) grade KBr in a ratio of 1:100. Pellets were prepared by applying 15,000 lb of pressure using a hydraulic press, followed by scanning in an inert atmosphere over a wave number range of 4000–400/cm in a Hitachi 295 spectrophotometer (Tokyo, Japan).

2.4.3. Morphology For visualization of the transfersomal vesicles, Transmission Electron Microscopy (TEM) was used. Transfersomal dispersion was diluted 10 fold using deionized water, then a drop of the diluted vesicles was applied to a carbon coated 300 mesh copper grid and left for 1 min to allow some of vesicles to adhere to the carbon substrate. Excess dispersion was removed by a piece of filter paper followed by rinsing the grid twice in deionized water for 3–5 s. Next, a drop of 2% aqueous solution of uranyl acetate (Polyscience Inc. Warrington, PA, USA) was applied for 1 s. The remaining solution was removed by absorbing the

2.3. Preparation of plain and LOR loaded TRSs The composition of different TRSs formulations is shown in Table 1. 2

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Table 2 Characterization of LOR loaded transfersomes. LOR-loaded TRSs

EE%

Size (nm) & PDI

Charge (mV)

TRSs (1) TRSs (2)

99.34 ± 0.2 99.74 ± 0.3

233.5 ± 12.5 & 0.31 ± 0.02 270.0 ± 8.5 & 0.252 ± 0.01

−35.34 ± 0.78 −0.875 ± 0.30

TRSs (1); LOR loaded transfersomes prepared using sodium deoxycholate, TRSs (2); LOR loaded transfersomes prepared using tween 80, EE (%) percentage encapsulation efficiency, PDI; polydispersity index. Data present as a mean ± S.D. (n = 3).

then 1 ml of the filtrate was transferred to a 50 ml volumetric flask and finalized to volume with phosphate buffer. After suitable dilution absorbance was recorded via UV-VIS spectrophotometer (Jenway, Japan) at λmax of 377 nm. Drug content was then calculated from the linear regression analysis equation obtained from the LOR standard calibration curve. The pH of LOR hydrogel formulations was determined using pH meter (3500 pH meter, Jenway, UK). Regarding LOR hydrogel containing TRSs, a weighted amount of gel was dissolved first in methanol to dissolve the vesicles and extract the drug, then phosphate buffer was added and proceeded as mentioned before.

liquid with the tip of a piece of filter paper and the sample was air dried. Afterward, the sample was viewed under the microscope at 10–100 k magnification power using an accelerating voltage of 100 kV. (JEM-1230, Joel Japan). 2.5. Stability study TRSs were stored at 4.0 ± 1.0 °C and at room temperature of 25.0 ± 1.0 °C for 30 days [28]. Then the physical stability of the formulation was assessed by visual inspection of sedimentation and measuring the particles size. In addition, LOR loading was performed after 1, 2, 3 and 4 weeks.

2.7.2. Viscosity The viscosity of transfersomal and non-transfersoaml formulations was performed on a Brookfield Digital Viscometer (Model DV-II Brookfield Engineering Laboratories, Inc., Stoughton, MA) at room temperature. Viscosity was recorded using spindle no. S94 and at 15 rpm (n = 3) [25].

2.6. Preparation of LOR hydrogels The weighed amount of the investigated gelling agents namely; hydroxypropyl cellulose (15.0% w/v), hydroxypropyl methylcellulose (2.5% w/v), sodium carboxymethyl cellulose (2.0% w/v), sodium alginate (3.0% w/v) was dispersed in warm water with continuous stirring until plain gel is formed. These amounts of gelling agents were chosen based on previous experiment to give gel with reasonable viscosity and homogeneity. Then, air bubbles were removed via the assistance of sonication for 15 min. Finally, LOR solution (1.0%w/w), LOR powder dissolved in triethanolamine, was added as portion wise with stirring till a homogenous transparent LOR hydrogel is formed. The prepared LOR-loaded TRSs (equivalent to 1% w/w drug) were incorporated into HPMC plain gel 2.5% w/v to prepare transfersomal hydrogel. Briefly, the calculated amount of TRSs suspension containing 100 mg drug was centrifuged and the pellets obtained were incorporated into HPMC gel by vortex then the final weight of the gel was adjusted to 10 g with distilled water. Vortexing was continuous until a homogenous transfersomal gel is achieved, followed by sonication to become bubble-free. Finally, the prepared transfersomal and nontransfersomal LOR hydrogels were left in the fridge until further analysis.

2.7.3. Homogeneity test LOR hydrogel formulations were examined for their homogeneity via visual inspection after they have been dispensed into their containers. In addition, they were hard-pressed between the index finger and thumb, and the consistency was recorded as homogenous or nonhomogenous [25]. 2.7.4. Skin irritation study Both non-transfersomal and transfersomal LOR hydrogels were studied for their irritation test. The study was conducted on human volunteers within the age of 25–40 years mixed male and female after informing them with the nature of the formulation as well as singing a written informed consent. The study was approved by the Institutional Ethics Committee of Faculty of Medicine, Assiut University, Assiut, Egypt. Briefly, one gram of each formulation was applied to the hands of volunteers. Then, the volunteers were observed for the appearance of erythema, edema and or irritation as shown in Table 4 [25].

2.7. Characterization of LOR hydrogels

2.8. In vitro release study

The prepared LOR hydrogel preparations were examined visually for their clarity, homogeneity and phase separation.

The in vitro release experiment was performed as described in the previous study [25]. Briefly, 250 mg sample of both transfersomal and non-transfersomal LOR hydrogels were accurately weighed and placed on a semipermeable cellophane membrane formerly immersed in phosphate buffer of pH 6.8 for 24 h then was struggle over the lower open end of a glass tube with 2.5 cm diameter and made water tight by a rubber band. Tubes submersed in 125 ml phosphate buffer of pH 6.8.

2.7.1. LOR contents and pH measurements LOR contents in the prepared hydrogel preparations were measured by dissolving accurately weighed quantity of gels (0.5 g) in aqueous phosphate buffer (pH 6.8). The solution was filtered via a filter paper,

Table 3 Mean vesicle size, polydispersity index (PDI) and entrapment efficiency (EE%) of LOR loaded TRSs after 30 days storage at 4.0 °C and 25.0 °C. (Results presented as a mean value ± S.D of five different measurements, n = 5). Size (nm) (n = 5) Day 0 TRSs TRSs TRSs TRSs

(1), (2), (1), (2),

4 °C 4 °C 25 °C 25 °C

233.5 270.0 233.5 270.0

PDI (n = 5) Day 30

± ± ± ±

12.5 8.5 12.5 8.5

238.6 277.3 539.5 595.7

± ± ± ±

Day 0 6.2 3.5 4.5 2.4

0.311 0.252 0.311 0.252

EE% (n = 3) Day 30

± ± ± ±

3

0.02 0.01 0.02 0.01

0.319 0.261 0.422 0.622

± ± ± ±

Day 0 0.03 0.02 0.09 0.08

99.44 99.75 99.44 99.75

Day 30 ± ± ± ±

0.11 0.09 0.11 0.09

99.34 99.63 99.43 99.36

± ± ± ±

0.133 0.014 0.081 0.028

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Table 4 LOR contents, pH, viscosity, homogeneity and skin irritation of different LOR hydrogel formulations. (Results presented as a mean value ± S.D of three different measurements, n = 3). Formulation No.

Content

pH

F1 F2 F3 F4 F5 F6

HPC 15.0% Na CMC 2.0% Na alginate 3.0% HPMC 2.5% HPMC loaded TRSs (1) HPMC loaded TRSs (2)

7.2 7.1 7.0 7.0 7.0 7.1

± ± ± ± ± ±

0.02 0.03 0.04 0.03 0.01 0.02

Skin irritation

5800 ± 240 18500 ± 450.0 6500 ± 340.0 2750 ± 250 11400 ± 310 11380 ± 420

Good Good Good Good Good Good

null null null null null null

2.11. Statistical analysis Statistical evaluation was implemented using one-way analysis of variance (ANOVA) by means of Graph Pad Prism Statistical Software with the Tukey Kramer multiple assessments. In addition, student t-test was also used for analysis of TRSs size, charge, stability, permeation and flux values. All statistically significant differences were anticipated when p < 0.05. All values are conveyed as their mean ± standard deviation.

2.9. Ex vivo permeation study Skin permeation study of selected LOR hydrogel formulations was carried out using abdominal skin of male mouse as previously reported method [29]. Animals were sacrificed then the hairs which on the dorsal side was shaved with the aid of a 0.1 mm animal hair clipper in the direction of the tail to head. Then, the dermal part of male mouse skin was cleaned three times with a wet cotton wipe saturated in isopropanol to completely detach any sticking fat material. The dermal side was soaked into a phosphate buffer of pH 6.8 for 6 h, to equilibrate the membrane and just before starting the diffusion experiment. Additionally, the skin was stretched over one open-ended glass tube. Tubes, with a total base surface area of 4.91 cm2, were placed in a glass beaker, containing 125 ml of phosphate buffer, and kept in the vertical position, so the membrane was just under the surface of the solution. The tube (donor) and beaker (receptor) were kept at 37 ± 0.5 °C in a thermostatic shaker water-bath. Then, the donor compartment was filled with 0.25 g of the tested gel formulation. At predetermined time intervals (up to 4 h) samples of 2.0 ml were withdrawn from the receptor and analyzed spectrophotometrically at λmax of 377 nm, (n = 3 ± S.D.). The cumulative quantity of drug permeated per unit area was plotted as a function of time. Furthermore, flux was determined from the slope of the linear portion of the obtained curves. The apparent permeability coefficient (Kp) of LOR across rat skin was calculated using Fick's first law of diffusion as present in Equation (3).

J C

Homogeneity

received in non-transfersomal HPMC hydrogel containing 1% LOR and transfersomal HPMC hydrogel containing LOR-loaded TRSs prepared using sodium deoxycholate. Group 4 received indomethacin gel 1% as a standard NSAID. Then, 200 mg of the tested formulation was applied on the edematous paw after 30 min post induction. The growth in the paw thickness was determined using a dial micrometer. Moreover, the measurement of inflammation of the paw and the percent inhibition of edema were calculated. The data were recorded as mean ± SD (n = 4).

The system was maintained at 37 ± 0.5 °C in a thermostatic shaker water bath (WiseBath, WSP-45, korea) rotate at 75 rpm. Samples of 5 ml were withdrawn at time intervals of 15, 30, 45, 60, 90, 120, 180 and 240 min. The volume of each withdrawn sample was replaced by the same volume of same dissolution medium maintained at the same temperature to keep constant volume. The released amount of LOR was determined spectrophotometrically at λmax of 377 nm after appropriate dilution [26,29]. Furthermore, the kinetic of LOR release from the investigated gel formulations was studied by curve fitting method to different kinetic models of zero order, first order, Higuchi diffusion and Korsmeyer-Peppas models [30,31].

Kp =

Viscosity (cPs)

3. Results and discussion 3.1. FT-IR study Drug-excipient interaction was studied before developing the formulation by using FT-IR spectroscopy, which is one of the most important analytical tools to investigate the stability of formulation, and molecular interactions between drugs and the used excipients [34]. The IR spectra of LOR, PC, sodium deoxycholate, physical mixtures and drug-loaded TRSs (TRSs 1) are shown in Fig. 1. The IR spectrum of pure LOR (Fig. 1, trace A) showed characteristic peaks at 3443.22 cm−1 (aliphatic O–H stretch), 3100.96 cm−1 (N–H stretching), 3067.20 cm−1 (CH-Ar), 2926.11 cm−1 (aliphatic C–H stretch), 1647.03 cm−1 (C=N), 1595.51 cm−1 (C=O), 1425.35 cm−1 (C=C), 1039.53 cm−1 (S=O) and 737.66 cm−1 for (C–H bending). The IR spectrum of the PC (Fig. 1, trace B) showed characteristic peaks at 2925.10 cm−1 (C–H stretch), 2854.04 cm-1 (C–H stretch), and 1736.15 cm−1 (carbonylic C = O stretch of ester) and 1240.04 cm−1 (ester C–O stretching). Sodium deoxy cholate FT-IR spectrum (Fig. 1, trace C) showed its characteristic peaks at 3405.10 cm−1 (aliphatic O–H stretch), 2937.25 cm−1 (C–H stretch) and 1447.82 cm−1 (C=C). The physical mixture of LOR with sodium deoxycholate and lipid (Fig. 1, trace D) showed additive spectra. For LOR-loaded TRSs (TRSs 1), the IR spectrum showed minor shifting in some peaks compared with the individual excipients and LOR as depicted in (Fig. 1, trace E). For example, aliphatic alcoholic O–H stretch (3343.22–3416.87 cm−1), C–H stretch (2926.11–2925.77 cm−1), N–H stretching (3100.96–3102.29 cm−1), CH-Ar (3067.20 to 3066.02 cm−1), C=N (1647.03 to 1647.13 cm−1), C=O (1595.51–1590.62 cm−1), C=C (1425.35 to 1429.27 cm−1), C–H bending (737.66–718.79 cm−1) and S=O (1039.53–1035.67 cm−1). The observed shifts may be due to the formation of hydrogen bonds, Van der Walls attractive forces or dipole moment which are weak forces seen in the polar functional groups of drug and excipients. The shifts seen due to the above mentioned interaction may however support the

(3)

Where; J is the flux (mg/cm /h) and C is the drug concentration in the donor compartment. 2

2.10. Pharmacodynamics activity The acute anti-inflammatory activity for the optimized hydrogel preparations was performed using rat induced carrageenan paw edema method [32,33]. The study was also approved by the Institutional Ethics Committee of Faculty of Medicine, Assiut University, and Assiut, Egypt. Four groups of rats have participated in the study and each group has 4 rats. The approximate weight of rats was about 200 ± 10.0 g. One percent w/v carrageenan in saline solution was injected subcutaneously into the left hind paw of rats for induction of edema. Group 1 received placebo HPMC hydrogel. Groups 2 and 3 4

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Fig. 1. FT-IR spectra of (A) pure drug LOR, (B) pure PC, (C) pure sodium deoxycholate, (D) their physical mixture and (E) medicated transfersomes (TRSs.1).

efficiency of 99.43 ± 0.18 and 99.74 ± 0.3% and LOR loading of 33.11 ± 2.4 and 33.24 ± 3.2% for LOR loaded TRSs prepared using sodium deoxycholate and tween 80, respectively. Particle size analysis showed a size and polydispersity, PDI, of 233.5 ± 12.5 nm, 0.31 ± 0.02 and 270.0 ± 8.5 nm, 0.252 ± 0.01 using sodium deoxycholate and tween 80 as edge formers, respectively as depicted in Table 2. The lower polydispersity showed that the particles are uniform in size and good homogeneously. Similarly, Bragagni M et al., found that different edge activators did not markedly influence the particle size of the formulated ultradeformable vesicles [28]. Surface charge measurements showed that LOR loaded TRSs prepared using sodium deoxycholate and tween 80 had a charge of −35.34 ± 0.78 mV and −0.875 ± 0.3 mV, respectively. Generally, the entrapment of lipophilic drugs into lipid vesicles depends on the interaction between the edge formers and lipophilic drugs as well as the phospholipid bilayer. Both tween 80 and sodium deoxycholate could have solubilizing properties as well as being incorporated into the lipid bilayer which enhances the encapsulation and eventually the stability of TRSs [28,37]. Previously, it was reported that a higher encapsulation efficacy of meloxicam in TRSs using sodium cholate compared with other used

formation of favorable vesicle shape, structure with good stability and sustained drug release [35]. Furthermore, these results are in a good agreement with the results obtained with LOR in ethosomal preparations [21]. 3.2. Vesicles preparation and characterization Thin film hydration method was successfully employed for the preparation of LOR loaded TRSs. This method has shown a promising and effective method for effective preparation of TRSs as reported from other published studies [25,36]. Several drug concentrations were tested and it was found that 5 mg of LOR per ml was appropriate concentration for LOR entrapment. Hence higher concentrations led to LOR precipitation without noticeable encapsulation. Furthermore, several trials were performed using different concentrations of edge activators till it was observed that 15% of edge activator was considered an optimum for giving a good vesicle preparation. It was noticed that the produced TRSs had a higher encapsulation efficiency and LOR loading. TRSs encapsulation efficiency, size and charge are depicted in Table 2. Results showed that TRSs had encapsulation 5

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Fig. 2. Transmission electron micrograph of LOR loaded transfersome stabilized with sodium deoxycholate. Magnification of 58000 and 72000 (A & B), the scale bar represents 100 nm.

3.4. Preparation of LOR hydrogels

edge formers [38]. Jain S et al., showed a higher encapsulation efficiency for dexamethasone using both sodium deoxycholate and tween 80 compared to span 80 and there is no significant difference between both edge formers in drug encapsulation efficiency. The relatively smaller particle size of TRSs stabilized with sodium deoxycholate could be attributed to the higher flexibility and the reduced surface tension of respective vesicles [39]. TRSs stabilized using sodium deoxycholate showed a higher zeta potential value, which indicated the stability of vesicles, and a minimum attraction forces that resist their aggregation. Hence, more electrically stable colloid system [40]. Meanwhile, tween 80 showed a small negative value less than one which is related to its nature as a non-ionic surfactant. Fig. 2 a and b showed the TEM images of TRSs stabilized with sodium deoxycholate. Vesicles were spherical, peculiar, multinuclear structure, non-aggregated with uniform and homogenous distribution which is characteristic morphology for vascular systems. It is also worthy to note that the obtained TRSs were of smaller particle size as determined by TEM imaging compared to the light scattering analysis which could be clarified due to the fact that the attained size from light scattering technique is an indirect method which can only measure the mean size hydrodynamic diameter. It also adds surface structure concentration upon measurement leading to larger size compared to the TEM as found from different researchers [41–43].

Visual inspection of freshly prepared LOR hydrogel formulation indicated that all the prepared hydrogels were clear, translucent and homogenous. Similarly, LOR loaded TRSs hydrogels are translucent and homogenous without any appreciable lumps and uniform in consistency. 3.5. In vitro evaluation studies The LOR contents in the different prepared hydrogel formulations were ranged from 98.0 ± 2.5 to 101.0 ± 1.8%. This indicated a high uniformity of the prepared gel formulation. Furthermore, hydrogels containing LOR-loaded TRSs showed LOR contents of 9.69 ± 0.39 and 9.44 ± 0.99 mg/g gel for LOR gel loaded TRSs stabilized with sodium deoxycholate and tween 80, respectively. This higher content was found to be satisfactory and approved the preparation method. In addition, pH measurements showed that all the prepared gel formulations had pH values ranged from 7.0 to 7.2. The relatively higher pH values could be due to the addition of triethanolamine during gel formulation. Viscosity is a critical factor for describing the gels as it affects the extrudability and release of drugs [36]. Viscosity measurements as shown in Table 4 showed that HPMC had the lowest viscosity, 2750 ± 250 cPs compared with the other investigated gel bases. The highest viscosity was recorded for sodium CMC and sodium alginate, 18.500 ± 450.0 and 6.500 ± 340.0 cPs, respectively. Moreover, hydrogel containing LOR loaded TRSs showed a viscosity of 11.400 ± 310 and 11.380 ± 420 cPs for TRSs stabilized with sodium deoxycholate and tween 80, respectively. Volunteers involved in skin irritation study showed that LOR hydrogel formulations, as well as transfersomal gels, demonstrated zero scores (null irritation). Thus, confirmed the safety and compatibility of the formulations to the skin even though the recorded relatively high pH values.

3.3. TRSs stability Regarding TRSs stability, TRSs formulations stored at 4.0 ± 1.0 °C in refrigeration conditions showed no sedimentation and resist the aggregation and fusion. Furthermore, they showed a non-significant (ttest; Ρ > 0.05) change in size and polydispersity. However, those stored at 25.0 ± 1.0 °C showed slight sedimentation which could be easily redispersed on slight shaking. In addition, they showed a significant change (t-test; Ρ < 0.05) in particle size as shown in Table 3. Moreover, TRSs formulations stored at the investigated two temperatures showed an enhanced stability as the LOR contents did not change during and till the end of the storage study. Hence, higher physical stability of these kinds of vesicles. Increasing the particle size of TRSs formulations stored at 25 °C could be possibly attributed to the aggregation and swelling of vesicles at this temperature. Similar results were also recorded for papaverine hydrochloride and sertraline TRSs [44,45]. It could be concluded from the stability study that keeping vesicles at 4.0 ± 1.0 °C is the best to minimize problems associated with instability issues.

3.6. In vitro release performance LOR released from the different investigated hydrogel bases is presented in Fig. 3. It was observed that HPMC showed a significantly higher (ANOVA/Tukey; p < 0.05) percentage of LOR released of about (90.31 ± 3.08%) after 240 min followed by HPC (72.01 ± 1.74%), sodium alginate (71.01 ± 0.86%) and sodium CMC (63.38 ± 6.12%) after the same time interval. The difference in drug release could be attributed to the difference in structure and viscosity of the gel bases as 6

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Fig. 3. In vitro release of LOR from different hydrogel bases at phosphate buffer of pH 6.8, n = 3, error bars indicate ± S.D.

Fig. 4. In vitro release of LOR from transfersomal and non-transfersomal HPMC hydrogel at phosphate buffer of pH 6.8, n = 3, error bars indicate ± S.D.

LOR released compared to HPMC hydrogel without TRSs as shown in Fig. 4. The hydrogel containing TRSs showed 36.94 ± 1.66 and 32.17 ± 0.45% of LOR released for TRSs stabilized with sodium deoxycholate and tween 80, respectively after 240 min compared to 90.31 ± 3.08% of LOR released from non-transfersomal HPMC hydrogel. Moreover, there is no significant difference (p > 0.05) between

well as the difference in drug polymers interactions. As shown from the viscosity results, the gel bases having a higher viscosity lead to a lower percentage of the drug release and those with lower viscosity showed a higher LOR release as observed with HPMC hydrogel base. A similar observation was also reported by other researchers [46,47]. TRSs gels showed a significant (ANOVA/Tukey; p < 0.05) lower percentage of

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as shown in Table 5. The nature of transfersomal gel concomitant with its size and vesiculating could be the main cause of such observation. Flux values were 1.5 and 2.0 times higher from transfersomal gel compared with the corresponding non-transfersomal gel. The greatest mean apparent permeability co-efficient of LOR was recorded with transfersomal gel containing sodium deoxycholate (1.829 ± 0.102 cm/ h). The higher permeability and flux of LOR from transfersomal gel could be explained according to the nature of TRSs vesicles which have the ability to squeeze themselves in the tiny pores of stratum corneum and or fuse with it. Moreover, the intact vesicle can penetrate into and through the intact skin [34,49,50]. The non-significantly (t-test; p > 0.05) higher permeability of transfersomal gel containing sodium deoxycholate could be possibly attributed to its smaller size and its negative charge as well as the pronounced effect of sodium deoxycholate in increasing the gross fluidity of stratum corneum lipid bilayers. Several mechanism could explain the ability of vesicles to modulate drug transfer across the skin including, adsorption and diffusion of vesicles onto the surface of the skin which would facilitate drug permeation and the vesicles themselves act as a penetration enhancers to reduce the barrier properties of the stratum corneum [49,50].

both transfersomal gel formulations containing sodium deoxycholate and tween 80. Release pattern showed a continuous and sustained release of LOR which mainly due to the reservoir effect of TRSs [48]. The release of LOR from transfersomal gels is a combination of the release of LOR from transfersomes first followed by a diffusion through the polymer network channel structure of the hydrogel. The entrapped LOR molecules could leak out gradually from the vesicles into the surrounding gel [34]. Regarding the release kinetics, it was found that all the investigated hydrogels showed a Higuchi model of the drug release as the correlation of coefficient values higher than those obtained from zero and firstorder kinetic. This kinetic behavior illustrated that the drug penetration mechanism was by diffusion kinetic order which showed a slow and sustained penetration of the drug from the skin membrane. Moreover, the release data were fitted into a semi-empirical Korsmeyer-Peppas model Equation (4).

Mt = Ktn M∞

(4)

Where, Mt/M∞, is the fraction drug release at time t, K is the constant correlated to the structural and geometric characteristics of the method, and n is the exponential diffusion, which is indicative of the drug release mechanism. Using the least squares procedure, the value of (n) was estimated. It was found for all the investigated formulations that the best fitting was obtained with (n) values 0.5 < n < 1.0, which indicates a non-fickian model of drug release. It could be concluded from the studying of release kinetics that LOR diffusion from gel network structure and partitioning through TRSs vesicles are responsible for continuous delivery of LOR.

3.8. The anti-inflammatory activity The pharmacodynamics behavior of LOR hydrogels utilizing the anti-inflammatory effects on the hind paw edema of the rats is shown in Fig. 6. Generally, it was found that LOR hydrogel formulation and those containing LOR loaded TRSs have a significant (ANOVA/Tukey; p < 0.05) reduction in a percentage swelling of the induced edema compared to plain HPMC hydrogel. The percentage swelling of the induced paw edema was variable and ranged from 78.21 ± 13.31 to 18.30 ± 1.12% swelling over the study time (1–5 h). After 2 h of the study, transfersomal formulation containing TRSs stabilized with sodium deoxycholate showed 32.98 ± 8.43% swelling compared to 56.90 ± 5.2% from non transfersomal HPMC hydrogel. After 5 h the transfersomal gel showed a significant (ANOVA/Tukey; p < 0.05) reduction in the swelling of the induced edema compared to both nontransfersomal gel and indomethacin gel 1% which was used as a standard NSAID. The percentage swelling of the induced edema was 18.31 ± 1.12, 27.73 ± 7.79 and 26.97 ± 5.6%, respectively for

3.7. Ex vivo permeation study LOR permeated through rat skin from non-transfersomal, transfersomal HPMC hydrogel containing both edge formers is depicted in Fig. 5. It was clear that both transfersomal gel formulations had a significantly (t-test; p < 0.05) higher amount of LOR permeated 164.36 ± 4.11 and 144.68 ± 3.14 μg/cm2 compared to 2 111.47 ± 3.87 μg/cm obtained from non-transfersomal HMPC hydrogel. Furthermore, transfersomal gel showed a significantly (t-test; p < 0.05) higher flux values than those obtained from HPMC plain gel

Fig. 5. Cumulative amounts of LOR permeated through hairless rat skin from transfersomal and non-transfersomal HPMC hydrogel, n = 3, error bars indicate ± S.D. 8

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Table 5 Ex vivo permeation of LOR from non-transfersomal HPMC and transfersomal HPMC hydrogels through rat skin model. (Results presented as a mean value ± S.D of three different measurements, n = 3). Formulation

Amount permeated (μg/cm2)

Flux (mg/cm2/h)

Apparent Permeability co-efficient (cm/h)

Non-transfersomal HPMC gel HPMC gel loaded TRSs (1) HPMC gel loaded TRSs (2)

111.47 ± 3.87 164.36 ± 4.11* 144.68 ± 3.12*

18.98 × 10−3 ± 0.001 36.59 × 10−3 ± 0.002** 32.67 × 10−3 ± 0.001**

0.9152 ± 0.071 1.829 ± 0.102 1.633 ± 0.077

*Significantly higher amount permeated (P < 0.05) than non-transfersomal HPMC hydrogel. **Significantly higher flux (p < 0.05) than non-transfersomal HPMC hydrogel. Fig. 6. Percentage swelling of the induced paw edema in rats after application of A: plain HPMC hydrogel, B: non-transfersomal HPMC hydrogel, C: transfersomal HPMC hydrogel and D: indomethacin gel (1%). * Significantly different (p < 0.05) Vs plain HPMC hydrogel; ** significantly different (p < 0.05) Vs non-transfersomal HMPC hydrogel and indomethacin gel standard. Data presented as a mean of four different measurements n = 4, error bars indicate ± S.D.

transfersomal HPMC hydrogel, non-transfersomal HPMC and indomethacin gel 1% after 5 h, which revealed superior anti-inflammatory effect of transfersomal hydrogel. The rank order of edema inhibition was non-transfersomal HPMC hydrogel > indomethacin gel > transfersomal hydrogel. This order comes in accordance with LOR permeation which showed a strong correlation between the percentages of LOR permeated and the pharmacodynamics activity. A similar observation was found by El-Badry et al., 2015 who studied the anti-inflammatory activity of meloxicam niosomal gel. Such findings demonstrate the applicability of LOR transfersomal gel as an effective topical formulation for reducing the inflammation [47].

LOR transfersomal hydrogel. As well as wider patient acceptability especially for those having GIT disorders and consuming a lot of amount of NSAIDs.

4. Conclusion

Declaration of competing interest

LOR TRSs was efficiently formulated with higher encapsulation efficiency and drug loading using thin film hydration method. Sodium deoxycholate capable of producing TRSs within size optimum for higher skin penetration. Furthermore, they showed higher physical stability when they are stored at 4.0 ± 1.0 °C. The prepared hydrogels were homogenous and did not show any appreciable skin irritation. Moreover, HPMC base demonstrated higher amounts of LOR release compared with the other investigated polymers. Transfersomal hydrogels released lower amounts of the drug compared to the non-transfersomal HPMC hydrogel confirming sustained LOR release. In addition, a higher flux and permeability through rat skin which became more pronounced with a formulation containing sodium deoxycholate. It is also worth noting the superior anti-inflammatory activity of the transfersomal LOR hydrogel compared with commercial anti-inflammatory indomethacin gel and non-transfersomal LOR hydrogel. This gives a good indication about the potential applicability of topical

The authors report no potential conflicts of interest associated with the article.

CRediT authorship contribution statement Hesham M. Tawfeek: Conceptualization, Writing - review & editing, Supervision. Ahmed A.H. Abdellatif: Writing - original draft. Jelan A. Abdel-Aleem: Investigation, Formal analysis. Yasser A. Hassan: Writing - review & editing. Dina Fathalla: Data curation, Writing - review & editing, Methodology.

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