TOPICAL DELIVERY OF QUERCETIN LOADED TRANSFERSOMES FOR WOUND TREATMENT: IN VITRO AND IN VIVO EVALUATION

Objective: To design topical Quercetin (Qc)-loaded transfersomes (TFs) for wound treatment.
Methods: Qc-loaded TFs were prepared by thin-film hydration technique using 2241full factorial design and the optimum formula was selected. In vivo skin, deposition and cutaneous wound induction studies were performed for four groups of male wistar rats. At the end of the experiment, biochemical parameters were measured in the healed tissues (total proteins (TP), total antioxidant capacity (TAC), glutathione reductase (GSH), nitric oxide (NO), and malonaldehyde (MDA). Two in vivo histopathological experiments using male wistar rats were performed; the first study was done for the healed tissues of the above experiment and the second was to confirm the safety of formulations.
Results: Qc optimum TFs (F6) showed EE% of 91.1%, PS of 695.35 nm, PDI of 0.592, and ZP of-11.1 mV, and spherical shape. In vivo skin deposition study showed that drug percentage retained in the skin from Qc optimum TFs was significantly higher than that from Qc suspension and Qc liposomes (p<0.05). There was no significant difference in the values of TP, TAC and MDA between the treated groups (p>0.05). GSH in TFs treated groups was significantly higher than the other groups (p<0.05) while NO in TFs treated groups was significantly lower than the other treated groups (p<0.05). Histopathological experiments showed that wounds treated by TFs healed better than those treated by both liposomes and Qc suspension.
Conclusion: Qc-loaded TFs can be used as successful drug-delivery system for wound healing.


INTRODUCTION
Herbal drugs have been used in many diseases [1]. Plant pigments which are found in herbal drugs as flavonoids which are also found in fruits and vegetables, have many physiological activities [2]. Flavonoids are important drug candidates due to their free radical scavenging and anti-inflammatory properties [3,4]. There is a structure-activity relationship between several flavonoids (luteolin, kaempferol, and chrysin) and their antioxidant activity and the number of OH groups in their structure so they are used in inflammatory disorders such as psoriasis and arthritis [5].
Quercetin (Qc) has high antioxidant activity which reduces the expression of matrix metalloproteinase that causes wrinkling and decreasing of the elasticity of healthy and photoaged skin [6]. Qc has antioxidant and antiradical effects due to three functional groups in its structure [7][8][9]. Nanotechnology is urgently needed for herbal drugs due to the poor solubility of those drugs [10]. The topical application of Qc in various formulation approaches includes permeation enhancers [11] such as lecithin and chitosan nanoparticles [12] which increase the penetration of Qc through the skin to make its topical and transdermal delivery easier, targeting the drug towards the desired tissue and concentrating the drug in it while reducing its concentration in the non-target tissues. Hence, drug action increases and side effects decrease [13]. Topical dosage forms are superior drug delivery systems because they avoid firstpass hepatic metabolism, gastric degradation and improve penetration of the drug into skin layers [14,15]. Surface-active agent vesicles are an alternative drug delivery system to conventional liposomes. Transfersomes (TFs) are elastic nano-vesicles composed of lecithin and surface-active agent and they differ from the conventional liposomes by their ability to squeeze themselves into the intercellular region of the stratum corneum [16,17].
This study aimed to develop topical Qc-loaded TFs for wound treatment. Optimization of production parameters to produce stable, highly concentrated Qc vesicles with a small particle size was done using full factorial statistical design. In addition, in vivo skin deposition of Qc from optimum TFs was compared with Qc liposomes and Qc suspension using male Wistar rats. In addition, wound induction was done to male Wistar rats which were then treated with Qc optimum TFs, Qc liposomes, and Qc suspension by measuring the biological parameters in the treated skin tissues of rats. Furthermore, two in vivo histopathological experiments were performed; the first to compare the wound treatment effect of Qc optimum TFs, Qc liposomes, and Qc suspension. The second in vivo histopathological experiment was performed to assess the irritation potential of Qc optimum TFs on rat skin.

Experimental design
The design used was complete factorial 2 2 4 1, which studied the influence of different variables on Qc-loaded TFs [18]. One factor was assessed with 4 levels (X1: Type of the surface-active agent), while the others with two levels (X2: Lecithin to surface-active agent molar ratio) and (X3: Hydration volume). (Y1: EE %), (Y2: PS), (Y3: PDI) and (Y4: ZP) were selected as the dependent variables. Table 1 shows the composition of the prepared TFs suspensions. Design Expert ® software was used to calculate desirability for the selection of the optimum formula. Abbreviations: EE%, entrapment efficiency percent; PS, particle size; PDI, polydispersity index; and ZP, zeta potential.

Preparation by thin-film hydration technique
Preparation of TFs was performed by thin-film hydration technique [19] as follows: specified weights of Qc, lecithin from egg yolk, Span 60, Span 80, Tween 60, Tween 80 were dissolved in 20 ml (chloroform and ethanol, 1:1 v/v) then were put in a rotaryevaporator (Rotavapor, Heidolph VV 2000, Burladingen, Germany). The solvent was evaporated at 60 °C, then the thin layer formed was rehydrated by saline phosphate buffer at 60 °C with agitation using glass beads followed by sonication. To compare the permeation of the prepared TFs and other vesicles; Qc liposomes (composed of Qc and lecithin from egg yolk) were prepared in the same method and amounts as above.

Measurement of PS, PDI, and ZP
Measurement of PS (z-average), PDI, and ZP of the prepared TFs suspensions was performed by Photon Correlation Spectroscopy (PCS) using a Zetasizer Nano ZS-90 instrument (Malvern Instruments, Worcestershire, UK). TFs suspensions were diluted before the measurement. Measurements were performed in triplicate using a 90 °C scattering angle at 25 °C. Conversion of electrophoretic mobility to zeta potential was calculated by the Smoluchowski equation [20]. The viscosity of the samples was considered to be that of water [21]. Results are the average value±SD.

Determination of Qc EE% in TFs
The difference between the total amount of Qc added in the formulation and that remaining in the aqueous medium, after separating the TFs suspension by centrifugation at 8500 rpm for 30 min using a centrifuge (Biofuge primo Heraeus instruments, Germany), was calculated to determine the EE% of Qc TFs. TFs contents were determined spectrophotometrically at a wavelength of 372 nm. EE% was calculated using the equation: Total amount of Qc added − amount of free Qc Total amount of Qc added �� × 100

Formulation optimization
The optimum formula was obtained using the Design Expert ® software by applying constraints on encapsulation efficiency percent of the TFs to reach the maximum value, ZP to obtain the maximum as an absolute value and on particle size and PDI to obtain the smallest value.

TEM
The particle shape of the Qc optimum TFs suspension was visualized using JEM-2100 Electron Microscope, Japan. A drop of TFs suspension was placed onto a film-coated copper grid, to make a thin film, before the specimen was dried on the grid. A drop of 1% phosphotungstic acid was used to stain the above film. The grid was then viewed at an accelerating voltage of 200Kv [22].

DSC
DSC (Shimadzu DSC-50, Japan) calibrated with purified medium (99.9%) was used to perform thermal analysis of Qc, lecithin egg yolk, Tween 80, physical mixture of Qc with TFs components, and the Qc optimum TFs. Mounting the sample was done in standard aluminum pans and a temperature of range 10-35 °C was used to heat it at a scanning rate of 10 °C/min inert nitrogen flow (25 ml/min).

In vivo assessment of Qc loaded TFs
The ethical committee of the Faculty of Pharmacy, Cairo University (PT 1428) approved the in vivo experiment. Male Wistar rats weighing (150-200 g) were obtained from the farm of the National Organization for Drug Control and Research, Giza, Egypt. Rats were maintained on a basal diet. Animals were treated according to the guidelines of Association of Official Analytical Chemists (AOAC) [23].

In vivo skin deposition experiment
Seventy-two male Wistar rats were used in the experiment. Four groups of 18 rats each were used. Group (I) acted as a control group, group (II), (III), and (IV) received topical application of Qc suspension, Qc liposomes, and Qc optimum TFs, respectively. Drug pools were made by bottle caps with an area of 4.91 cm 2 and this area was shaved carefully 24 h before application of the sample [24]. Half ml of each suspension was added to the drug pool. Scarification was done to rats at the end of the experiment. Overdose anesthetic ether was used to sacrifice three rats from each group at intervals of (1, 2, 4, 6, 8, 10h) and excision of the dorsal skin section that was in contact with the formulation was performed and washed three times with 10 ml normal saline. Sonication with 5 ml of methanol was performed to the excised skin sections after cutting into pieces for 30 min. Filtration of the homogenized skin was done through a 0.45 µm filter membrane and an assay of Qc using HPLC was performed to determine its concentration. These data were used to calculate the skin deposition of Qc generated by the range of formulations tested. The animal carcasses were incinerated.

Assay of Qc by HPLC
A gradient HPLC method was chosen for the assay of Qc [25]. Waters 2690 Alliance HPLC system detector (America) was used, and the detector used was a UV detector. The type of column was C18 thermo column (4.6 mm x 250 mm). The temperature of the column was maintained at room temperature (25.0±2.0 °C). Mobile phase A was 0.1% Phosphoric acid and mobile phase B was acetonitrile. The flow rate was 0.5 ml/min and the UV detector was set at 262 nm. Data analyses were made using SAS, (1999). Comparison of means was done using Duncan's at 5% level of significance [26].

Cutaneous wound induction and treatment
Forty-eight male Wistar rats were used in the experiment. Anesthesia was made by intraperitoneal injection of ketamine and xylazine, and then shaving to the thoracodorsal part was done. A square-shaped wound 400 mm 2 thick was created on the back of the rats using a scalpel blade and forceps [27]. After recovery from anesthesia animals were grouped in cages into 4 groups with twelve rats in each group. Treatment was done daily as follows: Group (I), Group (II), Group (III), and Group (IV) were treated with specified amounts of buffer (control), Qc suspension, Qc liposomes, and Qc optimum TFs, respectively. At the end of the experiment after 15 d, the rats of each group were killed and the healing tissue was collected and divided into two portions. The first portion was stored at-20 °C and then homogenized in phosphate buffer saline (pH=7.4). The tissue homogenates were centrifuged for 20 min at 12000rpm and 4 °C and the following biological parameters were estimated in the supernatants: GSH [28], MDA [29,30], NO [31], TP [32], and TAC assay [33]. The second portion was used for the following histopathological examination.

In vivo histopathological study
The second portion from the above experiment was preserved in 10% formal saline and was used for the first histopathological study to compare the wound treatment effect of Qc suspension, Qc liposomes and Qc optimum TFs. Trimming of the samples was done and serial grades of ethanol were used for its processing, clearance in xylene, and then embedding them in paraffin beeswax blocks. Rotatory microtome was used to cut sections of 5 µm thick. The sections were stained with Harris Hematoxylin and Eosin as a general examination method for histological evaluation. Masson's Trichrome stain was used for the demonstration of collagen fibers [34].
A second in vivo histopathological examination study using 16 male Wistar rats was performed to explore the irritation potential and the ultrastructural changes in the skin after topical application of the Qc optimum TFs and Qc liposomes formulation onto the skin three times a day for one week. Random division of rats was made into 4 groups; each group contained 4 animals: Group (I), group (II), group (III), and group (IV) for control, Qc suspension, Qc liposomes, and the Qc optimum TFs, respectively. At the end of the week, scarification of animals was done and excision of the skin was performed for histopathological examination according to the above protocol [34]. and HLB 15, respectively. In addition, increasing hydration volume decreased the EE%. When the hydration volume increases, the drug entrapment in the lipid bilayers decreases, and its partition in the aqueous phase increases [35].

Formulation variables effect on PS
Small particles pass through skin more than larger ones [36,37]. The results of PS of TFs are presented as Z average diameter in table 2 [38]. The PS of TFs composed of Tweens ranged in size between 674.2 and 1323.6 nm. The influence of the type of surface-active agent, lecithin to surface-active agent molar ratio, and hydration volume is presented as 3D plots in fig. 1. Factorial analysis showed that only the type of surfaceactive agent had a significant effect on PS (P<0.0001).
Formulae from 9 to 16 containing Span 80 and Span 60 showed particle size higher than formulae from 1 to 8 containing Tween 60 and Tween80. This could be attributed to that Tweens have more deformability due to high flexibility and non-bulky hydrocarbon chains compared to Spans that have bulky hydrocarbon chains [39].

Formulation variables effect on PDI
Wholly monodispersed particles have PDI of 0, while highly polydispersed particles have PDI of 1 [40]. All TFs ranged from 0.335 to 1. The influence of the type of surface-active agent, lecithin to surface-active agent molar ratio, and hydration volume is presented as 3-D surface plots in fig. 2. Factorial analysis showed tha tno factor had a significant effect on PDI (P>0.1).

Formulation variables effect on ZP
ZP is the measure of the total charges acquired by vesicles. Any formula is considered stable when the zeta potential value is around±30 mV because of electrical repulsion between particles [41].
The obtained ZP values of TFs formulae ranged from-6.63 to-19.9 to mV. The values of ZP of TFs formulae are presented in table 2. Because ZP was negative in all the formulations in our study, the discussion of variation in ZP will be in terms of its absolute value. The influence of the type of surface-active agent, lecithin to surface-active agent molar ratio, and hydration volume is presented as 3-D surface plots in fig. 2. Factorial analysis showed that the type of surface-active agent and lecithin to surface-active agent molar ratio had a significant effect on zeta potential (P<0.0001 and P<0.1, respectively).
Decreasing lipophilicity of surface-active agents increased ZP due to the reduction of the surface free energy of the surface-active agent. Accordingly, formulae from 9 to 16 containing Span 60 and Span 80 with HLB 4.7and HLB 4.3, respectively, had higher ZP than formulae from 1 to 8 containing Tween 60 and Tween 80 with HLB 14.9 and HLB 15, respectively [42]. Also, the lecithin to surfaceactive agent molar ratio had a significant effect on ZP. The isoelectric point of lecithin is between 6-7; therefore, it is a zwitterionic compound since the pH of the saline buffer is 7.4, which is higher than the isoelectric point and since we use a nonionic surface-active agent, therefore, the net charge is negative therefore ZP is negative. The negativity of ZP was also due to the presence of hydroxyl ion on the surface of the vesicles [43]. When lecithin to surface-active agent molar ratio increased, ZP increased because the negative charge increased [44,45].

Optimum formulation
Using Design Expert ® software, the optimum formulation was found to be F6. Choosing the optimum formulation was according to the following criteria (achieving the maximum value of EE%, minimum values of PS and PDI, and maximum value of ZP as absolute value). F6 was prepared with Tween 80, lecithin: surface-active agent molar ratio of 10:1, and hydration volume of 10 ml. This formulation showed EE% of 91.1%, PS of 695.35 nm, PDI of 0.592, and ZP of-11.1 mV.

TEM
TEM analysis was used to verify the results of the Malvern analyzer of PS and also to examine the shape of the vesicular system [46]. Fig.  3 shows the TEM image of F6. The vesicles are non-aggregated, unilamellar, and spherical in shape. The size of the TEM agrees with that obtained from the Malvern particle size analyzer. Fig. 4 [48,49]. Tween 80 shows one endothermic peak at 64.34 °C [50]. In the peaks of the physical mixture of Qc with the TFs constituents, the endothermic peak of Qc appears but less sharp due to its dilution with the excipients [51,52]. In F6, the endothermic peak of Qc disappears due to inhibition of its re-crystallization and solubilization in TFs and its appearance in the more soluble and amorphous state, which leads to a high energy state which enhanced its solubility [53,54].

In vivo assessment of Qc loaded TFs
In vivo skin deposition experiment Fig. 5 shows the in vivo skin deposition profile from Qc suspension, Qc liposomes, and F6. Drug percentage retained in the skin from F6was significantly higher than Qc liposomes and Qc suspension at 2h, 4h, 6h, 8h, and 10h (p ˂ 0.05) (table 3). The percentage of Qc deposited from F6 in rat skin was 1.89 times higher than that from Qc liposomes and 4.3 times higher than Qc suspension. The higher Qc skin deposition from TFs could be due to the higher elasticity of TFs when compared to conventional liposomes as they have deformable properties, which can make them easily squeeze through the channels of the stratum corneum [55]. TFs are drug carrier systems that can penetrate the skin. The unimpeded moving of such carriers depends on two factors: the vesicle bilayers are very elastic and the presence of an osmotic gradient through the skin. Accordingly, TFs pass through the skin because their membrane is flexible, which prevents the rupture of the vesicles in the skin and permits the ultra-deformability of the TFs to change the composition of their membrane locally and reversibly when they are attracted to narrow pore. This decreases the percentage of membrane deformation and permits the flexible particles first to pass rapidly [56].  Means in the same column with small letters are significantly different (p<0.05), n=3±SD.

Biochemical parameters
Wound induction undergoes an imbalance between production and accumulation of reactive oxygen species (ROS) in cells and tissues and this phenomenon is called oxidative stress and occurs by neutrophils which are derived from oxidants and MPO due to inflammation of wounds [57]. Biochemical parameters are shown in table 4.
Protein content in the wounded tissue represented the protein levels of cellular proliferation [58]. The difference between groups in the assay of TP was not significant (p>0.05) [59]. TAC is an indication of wound healing. The difference in TAC between the control group and other groups was significant (p<0.05) as total antioxidant capacity increased in all treated groups. GSH is an antioxidant that captures free radicals; its increase is an indication for wound healing [60]. GSH decreased in the control group and increased in other groups. The difference between Qc liposomes and Qc suspension was not significant (p>0.05). However, GSH in F6 group was significant compared to Qc liposomes and Qc suspension (p<0.05). NO is a free radical and its decrease indicates healing of wounds [61,62]. NO increased in the control group and decreased in treated groups. The difference between Qc liposomes and Qc suspension was not significant (p>0.05). However, NO in TFs was significantly lower than both of them (p<0.05). MDA is a natural marker of oxidative stress [63]. An increase in wound tensile strength increases MDA levels and mast cells [64]. After treatment of the wound, MDA and mast cells levels decrease as histamine increases [65]. MDA was high in the control group and low in the treated groups. There was no significant difference between the treated groups (p>0.05).  Fig. 6 shows the histopathological findings of the second section of the wound induction experiment. H and E stained wound sections were magnified at different levels. Control group (I) demonstrated retarded wound healing process with the persistence of a wide wound gap. Incomplete re-epithelialization due to immature epithelial layer and the presence of ulceration (arrow) with scab formed from necrotic tissue depresses and inflammatory cells infiltration (dashed arrow). Highly cellular granulation tissue with less mature collagen fibers is found in the dermis (star). Group treated with Qc suspension(II) demonstrated the same records as a control group with retarded wound healing process under scab (dashed arrow) with incomplete re-epithelialization and granulation tissue formation in dermal layer (star) accompanied with severe inflamed cells infiltrations and several congested and dilated deeper blood vessels (arrows). Group (III) treated with Qc liposomes showed incomplete healing with the persistence of a wide wound gap surrounded with granulation tissue (arrow) under scab from necrotic tissue depress and severe inflammatory cells infiltrates (star). Group (IV) treated with F6revealed the best wound healing with full re-epithelialization of the epidermis (arrow) and mature collagen made of fibers in the dermis with several hair follicles which were active and small amounts of inflamed cells. Remnant of old granulation tissue could be detected in the sub-epithelial region of the epidermis. Table 5shows the difference between groups in collagen percentage. Collagen percentage in F6 was significantly higher than control, Qc suspension, and Qc liposomes. Fig. 7 shows histopathological examination for collagen. F6 showed a higher inflammatory response, accelerated.   Wound healing and collagen percentage than Qc liposomes and Qc suspension as TFs were able to squeeze into the inside region of the outer layer of the skin (stratum corneum) and exhibit the anti-inflammatory and antioxidant activity because TFs are deformable so that it can go to the pores which are smaller than their size. On the contrary, Qc suspension and Qc liposomes didn't squeeze in the intercellular layer of the stratum corneum so they showed retardation in wound healing and antioxidant activity [56,66].

Histopathological examination
For the second histopathological study, fig. 8shows microscopic pictures of rat skin sections that were stained: group (I), group (II), group (III), and group (IV) for control, Qc suspension, Qc liposomes, and F6, respectively. Microscopic examination of different skin samples from all groups revealed normal morphological features of different skin layers, including epidermis with intact keratinocytes, (Black arrows) dermis with mature collagen fibers, and many active hair follicles (red star) intact subcutaneous tissue muscles, and tissue (black star). No abnormal tissue alterations could be detected.

CONCLUSION
Qc-loaded TFs were used topically for wound treatment. Statistical optimization of formulation variables was performed by factorial design using Design-Expert ® software. The Qc optimum TFs (F6) formulation had a spherical shape, EE% of 91.1 %, the particle size of 695.35 nm, PDI of 0.592, and ZP of-11.1 mV. In vivo skin deposition experiment showed that F6was superior compared to Qc suspension and Qc liposomes. In addition, cutaneous wound treatment displayed that F6was superior to Qc suspension and Qc liposomes in wound treatment when assessed using biological parameters and histopathological studies. Furthermore, in vivo histopathological experiment confirmed that the topically applied F6 was not irritant. Therefore, the results proved that Qc-loaded TFs could be used as a successful drug delivery system to be used for wound healing.