Tailoring novel soft nano-vesicles ‘Flexosomes’ for enhanced transdermal drug delivery: Optimization, characterization and comprehensive ex vivo – in vivo evaluation

Accepted Manuscript Tailoring Novel Soft Nano-vesicles ‘Flexosomes’ for Enhanced Transdermal Drug Delivery: Optimization, characterization and comprehensive ex vivo - in vivo evaluation Hanaa A. Abdel-Messih, Rania A.H. Ishak, Ahmed S. Geneidi, Samar Mansour PII: DOI: Reference:

S0378-5173(19)30114-0 https://doi.org/10.1016/j.ijpharm.2019.01.072 IJP 18135

To appear in:

International Journal of Pharmaceutics

Received Date: Revised Date: Accepted Date:

24 October 2018 25 January 2019 31 January 2019

Please cite this article as: H.A. Abdel-Messih, R.A.H. Ishak, A.S. Geneidi, S. Mansour, Tailoring Novel Soft Nanovesicles ‘Flexosomes’ for Enhanced Transdermal Drug Delivery: Optimization, characterization and comprehensive ex vivo - in vivo evaluation, International Journal of Pharmaceutics (2019), doi: https://doi.org/10.1016/j.ijpharm. 2019.01.072

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Tailoring Novel Soft Nano-vesicles ‘Flexosomes’ for Enhanced Transdermal Drug Delivery: Optimization, characterization and comprehensive ex vivo - in vivo evaluation

Hanaa A. Abdel-Messiha, Rania A.H. Ishak*a, Ahmed S. Geneidia, Samar Mansoura,b aDepartment

of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams

University, Cairo, Egypt bPharmaceutical Technology Department, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt.

*Corresponding author: Rania A. H. Ishak, Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; Postal Code: 11566, Phone: +201222930214; E-mail: [email protected], [email protected]

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Abstract The transdermal route is a convenient non-invasive way for drug delivery, however, the hydrophobic compact nature of stratum corneum (SC) forms an obstacle hindering the diffusion of drugs particularly hydrophilic ones. Hence, the purpose of this study was to develop novel soft nano-vesicles, entitled Flexosomes, amalgamating two penetration enhancers, ethanol and one edge activator (EA) from various types and different hydrophilic-lipophilic balances. The tailored vesicles were loaded with tropisetron hydrochloride (TRO), a potent highly-soluble anti-emetic, and compared with ethosomes. Aiming to preclude the formation of rigid non-deformable mixed micelles, all critical parameters; EA type, phosphatidylcholine-to-EA molar ratio, and cholesterol concentration, were optimized proving their influences on vesicle-to-micelle transitions. The prepared formulations were characterized in terms of visual inspection, particle size, polydispersity, zeta potential, turbidity measurements, entrapment efficiency, and vesicle morphology. The permeation mechanisms were assessed by differential scanning calorimetry on isolated SC. The modified vesicles, based on ethanol and either vitamin E or PEGylated castor oil derivatives exhibited the highest transdermal fluxes confirmed by a deeply tracking to dermis using confocal laser microscopy. Both vesicles demonstrated higher bioavailability relative to ethosomes, topical and oral aqueous solutions. The findings endorsed the effectiveness of tailored nano-vesicles in boosting TRO skin transport suggesting their applicability with various drug entities for enhanced transdermal delivery.

Keywords: nano-vesicles; ethosomes; micelles; edge activator; transdermal delivery; ex vivo permeation; in vivo pharmacokinetics

Chemical Compounds Chemical compounds studied in this article Phosphatidylcholine (PubChem CID: 24779388), Cholesterol (PubChem CID: 5997), Ethanol (PubChem CID: 702), Tropisetron hydrochloride (PubChem CID: 656664), Cetyl trimethyl ammonium bromide (PubChem CID: 5974), Sodium cholate (PubChem CID: 23668194), Sodium 2

deoxycholate (PubChem CID: 23668196), Tween 80 (PubChem CID: 443315), D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS) (PubChem CID: 71406), Cremophor RH 40 (CASRN: 61788-85-0), Span 85 (PubChem CID: 9920343).

Abbreviations AUC: Area Under the concentration Vs time curve, CLSM: Confocal laser scanning microscope, Cmax: Maximum plasma drug concentration, CMC: critical micelle concentration, CPP: Critical packing parameter, CREM: Cremophor RH 40, CTAB: Cetyltrimethylammonium bromide, D: Diffusion coefficient, DSC: Differential scanning calorimetry, EA: Edge activator, EE: Entrapment efficiency, EPC: Egg phosphatidylcholine, ER: Enhancement ratio, Eth: Ethosomes, Flex: Flexosomes, Frel: Relative Bioavailability, FT-IR: Fourier-transform infrared, HLB: Hydrophilic-lipophilic balance, HPLC: High performance liquid chromatography, HR-TEM: High resolution transmission electron microscope, IS: Internal standard, Jss: Steady state transdermal flux, Kp: Permeability coefficient, LC-MS/MS: Liquid Chromatography/Mass Spectrometry, Mw: Molecular weight, MWCO: molecular weight cut off, n: Diffusional release exponent, NaC: Sodium cholate, OS: Oral solution, PBS: Phosphate buffer saline, PC: Phosphatidylcholine, PDI: Polydispersity index, PE: Penetration enhancer, PS: Particle size, Q: Cumulative amounts of drug permeated per unit area, Q24: Cumulative amounts of drug permeated per unit area over 24 h, SC: Stratum corneum, SD: Standard deviation, SDC: Sodium deoxycholate, SEM: Standard error of the mean, T80: Tween 80, Tmax: Time to reach maximum drug concentration, TPGS: D-αTocopherol polyethylene glycol 1000 succinate, TRO: Tropisetron hydrochloride, TS: Topically applied drug solution, UV: Ultra-violet, ZP: Zeta potential.

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1. Introduction The transdermal route is a promising way for drug administration through the skin barrier, being alternative to parenteral and oral delivery. It is considered convenient, non-invasive, and efficacious in terms of evading first pass effect for orally administered metabolized drugs, eluding the pain and safety concerns related with injections, reducing the dosing frequency and hence the drug side effects (Vyas and Khar, 2002). Additionally, this route improves compliance with special treatments viz. the antiemetic therapy, with potential benefits to quality of life, and overall treatment satisfaction. Despite the various benefits of transdermal delivery, the skin exhibits a prominent barrier against effective drug transport, counting on the hydrophobic compact nature of its outermost layer, the stratum corneum (SC). This layer consists of multi-layered dead flattened cells, known as corneocytes entrenched in a lipid matrix, with filamentous keratin in their cytoplasm. Therefore, the physiochemical properties of permeants, for instance partition coefficient, ionization state, solubility, and molecular size, are considered crucial factors affecting their diffusion through SC. For that reason, limited number of drugs have been approved by FDA for such administration. These drugs share common characteristics –small molecular weight (Mw) (<400 Da), high lipophilicity (log P up to 10,000), and low daily dose (< 10 mg) (Vinod et al., 2010, Choy and Prausnitz, 2011). Substantial efforts have been consumed on the development of innovative strategies to enhance the transdermal delivery for a higher number of drugs. The use of penetration enhancers (PE) is a common approach for this purpose, from which surfactants, as edge activators (EA), are commendable examples counting on its effect on modifying the drug thermodynamic activity, and 4

solubilizing it in the SC lipids (Thong et al., 2007). However, due their amphiphilic properties, EA tend to self-assemble into nano-sized aggregates called ‘micelles’ above their critical micelle concentration (CMC). The rigidity and low deformability of these nanoconstructs make their transdermal effectiveness very limited (El Zaafarany et al., 2010). Therefore, EA molecules have been integrated with phospholipids forming flexible vesicles, known as transferosomes, their effectiveness was confirmed in improving the drug transport across animal and human skin delivery compared to conventional liposomes (Cevc and Blume, 1992). However, some reports limited their efficiency to the enhancement of skin deposition only, hence considered suitable for dermal drug delivery (El Maghraby et al., 1999, El Maghraby et al., 2001, Trotta et al., 2002). Afterwards, the alcohols e.g. ethanol as PE were chosen to be included into the vesicle structures, due to their known effect on SC lipid disorganization (Thong et al., 2007), forming the ultra-flexible carriers ‘ethosomes’ (Eth) (Touitou et al., 2000). These nanostructures showed superior elasticity, deformability, in vitro/in vivo skin deposition and penetrability, when compared with both liposomes and transferosomes. Hence, the purpose of the present study was to develop a novel combinatorial system through the integration of both ethanol and EA merging the advantages of both transferosomes and ethosomes, assuming the superiority in terms of skin permeability, and enhanced transdermal drug delivery compared to all former vesicles. The presence of both PE in the same construct triggers the vesicle-to-micelle transitions forming the rigid non-deformable ‘mixed micelles’. This raises the importance to preclude this transition during the development of PE-based nano-vesicles through careful investigating and optimizing the critical process parameters. To our best 5

knowledge, no systematic empirical research has been reported yet in this concern. Very few reports were recently noticed merging both ethanol and an EA in the same construct, however, none of them discuss the anticipated structural transitions, nor optimize process parameters, nor explored in depth the ex vivo and in vivo performances of the developed carriers (Song et al., 2012, Shaji and Garude, 2014, Ascenso et al., 2015, Ma et al., 2015, Alomrani and Badran, 2017, Chen et al., 2017, Garg et al., 2017, Habib et al., 2018). Nausea and vomiting are considered inevitable adverse effects of chemotherapy directing 20% of cancerous patients to postpone or decline potentially beneficial treatment. Tropisetron hydrochloride (TRO) is a highly potent and selective antagonist of serotonin (5-hydroxy tryptamine type 3, 5-HT3) receptors, particularly indicated for management and treatment of emesis in patients suffering from chemotherapy-induced nausea and vomiting (de Bruijn, 1992). It is a hydrochloride salt of high water solubility (>5%) and a molecular weight (Mw) of 320.8 daltons. Its absolute oral bioavailability is 60% due to first pass effect, however, its hepatic metabolism greatly varies due to a genetic polymorphism of the cytochrome P450-2D6 isoenzyme. The metabolites have a greatly reduced potency for serotonin receptors and do not contribute to the pharmacological action of the drug (de Bruijn, 1992). TRO is currently available at 2 and 5mg doses in the form of hard gelatin capsules and ampoules for oral and parenteral administration, respectively. Based on all listed-above attributes in addition to its non-irritating and nonsensitizing property, TRO is considered a promising candidate for transdermal delivery. Refer to literature, no previous works were noticed delivering TRO with the aid of any vesicular systems, only a transdermal patch loaded with TRO was the sole attempt. It was designed and evaluated for its efficacy against emesis induced by anticancer agents, in comparison with the 6

effect of traditional TRO injection, in rats (Jeong et al., 2005). It was suggested that the novel TRO patch could be a promising regimen for the relief of emesis, based on the long-term antiemetic effects on the diverse anticancer agents and the convenience to use the transdermal delivery system for the cancer patients who have difficulty in taking drugs due to surgical operation or gastrointestinal dysfunction. Hence, the aim of the present work was to fabricate the novel soft nano-vesicles, entitled as Flexosomes (Flex), optimize their critical formulation parameters to control the vesicle-tomicelle transitions, characterize them, and pursue their ex vivo- in vivo performances for transdermal TRO delivery. Different EA types with different hydrophilic-lipophilic balances, varying from cationic, anionic, and non-ionic, phophatidylcholine to EA molar ratios, and cholesterol concentrations were carefully and systematically optimized. Amongst EA types integrated with ethanol, cetyl trimethyl ammonium bromide, bile salts (sodium cholate and sodium deoxycholate), Tween 80, Span 85, D-α-tocopherol polyethylene glycol 1000 succinate, and Cremophor RH 40, were explored. An Eth formulation was also developed for comparison purpose. The prepared formulations were fully characterized in terms of visual inspection, particle size (PS), polydispersity index (PDI), zeta potential (ZP), turbidity measurements, drug entrapment efficiency percent (EE%), vesicle morphology, and ex vivo drug permeation. Differential scanning calorimetry (DSC) technique was applied on isolated SC to explore the enhanced permeation mechanisms of both Eth and Flex formulations. After being fluorescently labeled, the chosen vesicles were tracked through the rat skin layers using confocal laser scanning microscopy (CLSM). Finally, a pharmacokinetic study was developed for assessing the in vivo performance of the Eth and selected Flex, in comparison with orally administered and topically applied aqueous 7

solutions. The safety of the prepared formulations was also evaluated by dermato-histopathology study.

2. Materials and Methods 2.1. Materials Tropisetron hydrochloride (TRO) was purchased from Shijiazhuang Aopharm Import & Export Trading Co., Ltd. (Shijiazhuang, China). Lipoid® E PC S (egg phosphatidylcholine, EPC) with 96% PC was kindly provided by lipoid GMBH (Braunschweig, Germany). Absolute ethanol and acetonitrile, both of HPLC grade: purchased from Fisher Co. (London, UK). Cholesterol, stabilized cholesterol 95%, nitrogen flushed, was supplied from Acros Organics N.V. (Geel, Belgium). Sodium cholate (NaC) (assay >97%) and Sodium deoxycholate (SDC) (assay >99%) were provided from Honeywell Fluka (USA). Tween 80 (T80), phosphotungstic acid, Nile red, paraformaldehyde, hematoxylin-eosin stain, xylene, formic acid, EDTA and torsemide as the internal standard were obtained from Sigma-Aldrich (USA). Cetyl trimethyl ammonium bromide (CTAB), potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, and potassium chloride were purchased from ADWIC, El-Nasr Pharmaceutical Co. (Cairo, Egypt). Cremophor RH 40 (CREM) under the trade name EMAROL H 40®, was purchased from CISME (Italy). Dα-Tocopherol polyethylene glycol 1000 succinate (TPGS) was generously supplied by Isochem (France). All other chemicals used throughout this study were of analytical grade. Deionized water used for experiments was produced using a Milli-Q Gradient A10 System (Millipore, Billerica, MA). Spectra/Por® dialysis membrane, 12000–14000 MWCO was purchased from Spectrum Medical Industries (Houston, TX). Nanosep® centrifuge tubes, fitted with an ultra-filter of 100 8

kDa MWCO was provided from Pall Life Sciences (USA). Disposable syringe membrane filters of pore size 0.45 μm were obtained from Thermo Fisher Scientific (Waltham, MA, USA).

2.2. Methods 2.2.1. Preparation of TRO-loaded Flex The Flex loaded with TRO were prepared by the ‘hot’ method as previously described (Abdel Messih et al., 2017) with some modifications. Briefly, in a water bath adjusted at 50°C on a magnetic stirrer (Heidolph, Schwabach, Germany), the drug (0.1% w/v) was dissolved in deionized water, each of the hydrophilic EAs viz. CTAB, NaC, SDC, T80, and CREM was then dispersed in the drug solution. Both EPC and cholesterol (if present) were then added until a colloidal dispersion was obtained. Ethanol was finally added with continuous stirring for 30 min in a closed system. Due to their higher solubility in ethanol, either Span 85 or TPGS was solubilized in alcohol when included. For comparison purpose, the drug-loaded Eth was prepared deprived from EA and cholesterol by the same technique described above. This formulation was subjected to homogenization for size reduction using a high shear homogenizer (Heidolph Diax900, Heidolph, Schwabach, Germany) adjusted at 8000 rpm for 30 sec (Abdel Messih et al., 2017). The concentration of EPC and ethanol were maintained in all preparations at 3%(w/v) and 40%(v/v), respectively.

2.2.2. Optimization Study The effect of EA type, phosphatidylcholine (PC):EA molar ratio, and cholesterol concentration are the critical formulation parameters that were systematically optimized at different levels for the development of Flex. Different variables and levels are summarized in Table 1. 9

Table 1. Formulation variables and their levels studied during the systematic optimization of TROloaded Flex.

Formulation Variables EA type

Levels CTAB, NaC, SDC, T80, TPGS, CREM, Span 85 5:1, 2:1, 1:1

PC:EA molar ratio

0, 0.1, 0.2, 0.3

Cholesterol concentration (%w/v)

2.2.3. Characterization of the prepared formulae 2.2.3.1. Visual inspection All prepared formulae were examined by visual observation for their physical appearance. They were described as either clear transparent or turbid cloudy systems.

2.2.3.2. Turbidity measurements The turbidities of some formulations were determined by measuring the percent transmittance at 500 nm using a UV-visible spectrophotometer (Shimadzu UV visible 1601 PC, Kyoto, Japan) using deionized water as blank. The individual components had no absorption at the studied wavelength (Mondal et al., 2016).

2.2.3.3. Determination of PS, PDI, and ZP Size and size distribution measurements were performed using dynamic light scattering (DLS) on a Malvern Zetasizer Nano-ZS instrument (Malvern Instruments, Malvern, UK), the intensity 10

fluctuations of the scattered light were detected at a backscattering angle of 173° and then analyzed to obtain an autocorrelation function. The surface charges of all prepared formulae were measured using laser Doppler anemometry (LDA) technique using the same instrument. The samples were measured at 25 ± 0.5°C after being suitably diluted with deionized water till their count rates fall within 200-500 Kcps (Hashad et al., 2017).

2.2.3.4. Determination of drug EE% Two techniques were adopted for the determination of drug EE% in Flex; the minicolumn centrifugation (Abdel Messih et al., 2017) using Nanosep® centrifuge tubes and the dialysis technique (Zhang et al., 2014) using Spectra/Por® dialysis membrane (12000–14000 MWCO). For the mini-column centrifugation technique, the prepared nanosuspensions were processed as previously described (Abdel Messih et al., 2017); they were placed in Nanosep® after suitable dilution, and then separated by a cooling micro-centrifuge (Hermle Labortechnik GmbH, Model Z216 MK, Wehingen, Germany) adjusted at 10,000 rpm, 4°C for 90 min. The unentrapped drug content in the supernatant was analyzed at λmax 285 nm by UV-Visible spectrophotometer (Shimadzu UV visible 1601 PC, Kyoto, Japan) after being diluted when necessary using the subsequent equation. In case of the dialysis method, a specified volume of the prepared nanosuspension was placed in a hydrated dialysis bag closed using dialysis tubing closures and then immersed in a closed container containing 50 ml of deionized water. The whole experiment set was placed in shaking water bath (Abbota 110X, USA) adjusted at 100 rpm at room temperature. After a specified time, the drug in

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the receiver medium was then assayed at λmax 285 nm using UV-Visible spectrophotometer. The EE% was calculated using the following equation: 𝑬𝑬(%) =

(𝑫𝒕 − 𝑫𝒅 ) 𝑫𝒕

× 𝟏𝟎𝟎

Eq. (1)

where Dt is the total amount of TRO added in the nanosuspension and Dd is the amount of free drug detected in the supernatant or the amount of the drug that diffused into the receiver medium.

2.2.3.5. High resolution-transmission electron microscope (HR-TEM) imaging of the selected Flex For vesicle imaging, few drops of the selected samples were dropped on a carbon-coated copper grid, and stained by 1% phosphotungstic acid, and then allowed for drying at room temperature. The prepared grids were placed in a holder and loaded into HR-TEM (JEM-2100, JEOL, Tokyo, Japan) for visualization (Hashad et al., 2016). 2.2.3.6. Drug–excipient interaction study using Fourier transform-infrared (FT-IR) spectroscopy Drug–excipients compatibilities were studied using FT-IR instrument (Nicole 6700, Thermo Scientific, Waltham, MA). The study was carried out on single formulation components; drug, EPC, cholesterol and selected EAs, their physical mixtures, as well as their corresponding formulations.

2.2.4. Ex vivo drug permeation study through excised rat skin The ex vivo permeation of TRO through the rat skin from the selected Flex as well as Eth was performed as previously described (Abdel Messih et al., 2017). Briefly, male albino rats (250 ± 30 g) were humanely killed by chloroform inhalation. The dorsal full-thickness skin was carefully excised after hair removal from which the connective tissue and adhering subcutaneous fats were 12

only teased off, placed on aluminum foil, and cautiously observed for any scratches or holes. The ex vivo drug permeation study was performed using our own-designed glass diffusion cells of permeation area of about 5.7 cm2. The receptor compartment constituted of 50 ml PBS containing 20%(v/v) ethanol acting as a preservative for the skin from putrefaction as the experiment lasted for 24 h. The excised skin was mounted between the donor and the receptor compartment with the stratum corneum (SC) facing upwards. After equilibrium for 1 h, each formulation, equivalent to 2 mg of the drug, was applied to the SC surface of skin under non-occlusive condition. The whole diffusion cell was then placed in a shaking water bath adjusted at 37 ± 0.5°C. Samples of 2 ml volume were withdrawn from the receptor compartment at 1, 2, 4, 6, 8 and 24 h time intervals, with replacing them with the receptor medium of equal volumes. The amounts of drug diffused across the excised rat skin were analyzed by high performance liquid chromatography (HPLC) after filtrating the samples using 0.45 µm disposable filters. The cumulative amounts of drug permeated through the skin (µg/cm2) were plotted as a function of time (t) for each formulation. Drug transdermal flux (permeation rate) at steady state (Jss) (µg/cm2/h) was obtained from the slope of the linear portion of graph. The permeability coefficient (Kp) (cm/h) was also determined by the following equation: Kp = Jss/Co

Eq. (2)

where Jss is the steady-state flux and Co is the initial TRO concentration in the donor cell (Abdel Messih et al., 2017). The diffusion coefficient (D) (cm2/h) was determined after plotting the cumulative amount of drug permeated versus the square root of time and then substituting in Eq. (3) (Ahmed and El-Say, 2014): D= (slope / Co)2 ×π 13

Eq. (3)

The enhancement ratio (ER) was also determined by comparing the transdermal flux of each Flex to that of Eth formula, and was calculated as follows (Takmaz et al., 2009): ER = Jss of the selected Flex / Jss of Eth

Eq. (4)

The kinetics of the ex vivo drug permeation profile was determined by applying the following Korsmeyer-Peppas equation and then calculating the diffusional release exponent (n). Mt / M∞ = K tn

Eq. (5)

Where Mt/M∞ is the cumulative drug permeated at time (t) and K is the release rate constant. The (n) exponent was determined from the slope of the graph plotted between log cumulative amounts of drug permeated versus log time. A (n) value of 0.5 indicates normal concentration-controlled Fickian diffusion (case I). The 0.5
2.2.5. Quantitative determination of TRO by HPLC assay The TRO concentrations were quantitatively determined in samples of the ex vivo permeation study by a validated analytical assay using HPLC (Agilent Technologies 1200 Series, Waldbronn, Germany) with a LOQ and LOD of 5 and 1.67 µg/ml (Abdel Messih et al., 2017). The samples were injected with a 20-µl, loop and rheodyne sample injector onto C18 reversed-phase analytical column (Phenomenex®- 5-µm particle size; 250 x 4.6 nm ID, Torrance, CA, USA) adjusted at a temperature of 30°C. The mobile phase consisted of potassium dihydrogen phosphate buffer (pH 4.5) and acetonitrile (75:25, v/v) at a flow rate of 1 mL/min. The wavelength of UV detector was

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set at 285 nm. No interference from any formulation components was observed during the elution of TRO.

2.2.6. Ex vivo assessment of the enhanced permeation mechanism of the selected vesicles 2.2.6.1. SC isolation from rat skin For isolation of SC from the excised dorsal rat skin, the method adopted elsewhere (Vidlářová and Doležal, 2012) was performed with some modifications. Full-thickness skin punches of about 25 mm in diameter were isolated from frozen skin and then incubated in warm water adjusted at 60°C for 1 min. Thereafter, the skin samples were removed from water, placed on filter paper and the dermis was peeled off with forceps. For SC isolation, the obtained epidermis was incubated in 0.1% trypsin solution in PBS for 24 h at room temperature. The isolated SC from each epidermal piece was then separated, washed with copious amounts of deionized water and then placed in ependorffs. 2.2.6.2. SC treatment with selected formulations Each isolated SC section was incubated with 1 ml of loaded formulations either Eth, TPGS-Flex, or CREM-Flex for 24 h at room temperature. After that, all treated SC samples as well as untreated one (just incubated in PBS) were removed, washed 3 times with deionized water, and then allowed to dry completely in a desiccator for 48 h. 2.2.6.3. DSC study on SC samples For investigating the thermal properties of the SC samples, DSC technique was adopted. For each analysis, about 10 mg of dried SC samples were placed into hermetically sealed aluminum pans

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after being lightly grinded and then analyzed in the temperature range of 20 – 250°C with a heating rate of 10°C/min under nitrogen flow.

2.2.7. Colloidal stability study The stability of the vesicles was determined by storing the vesicles under refrigeration at 5 ± 3°C. The drug EE%, PS, PDI, and ZP were measured initially and after 1, 3 and 6 months of storage as described earlier. 2.2.8. Tracking the fluorescently-labeled vesicles in the rat skin layers using confocal laser microscopy (CLSM) The CLSM (Zeiss LSM 710, Germany coupled with ZEN 2009 software) was employed to visualize the localization and distribution of the selected vesicles within the skin layers. The drug in the selected formulations (Eth, TPGS-Flex and CREM-Flex) was replaced by the red fluorescent dye Nile Red at a concentration of 0.05%, adopting the same preparation technique described before. The excised dorsal side of rat skin was treated with the fluorescent vesicles for 4 h using our modified diffusion cells, just as established in the ex vivo permeation studies. After the permeation period, the skin sections were removed from the cells, washed three times with deionized water, and dried with filter papers. The skin samples were vertically cross-sectioned into thin microscopic sections using a microtome (the Hacker MR-2 Series Microtome, USA) blade, and then placed onto the slide. The full skin thickness was optically scanned and observed using CLSM. Images were captured using a 10× objective lens of an inverted CLSM at 543 and 633 nm as the excitation and emission wavelengths, respectively. 16

2.2.9. Pharmacokinetic studies 2.2.9.1. Administration of the selected TRO-loaded formulations to rats The study protocol was reviewed and approved by the Experiments and Advanced Pharmaceutical Research Unit (EAPRU), Faculty of Pharmacy, Ain Shams University on the use of the animals. All the animals used in the study were caged and preserved according to the principles documented for care and use of laboratory animals in the “Guiding Principle in Care and Use of Animals” (DHEW Publication No. (NIH) 80-23). The selected formulations (Eth, TPGS-Flex, and CREM-Flex) in addition to an aqueous drug solution (TS) were applied transdermally, from which the pharmacokinetics of TRO was studied and compared to an orally administered solution (OS). The drug dose was maintained at 10 mg/Kg (Huang et al., 1999). Thirty male albino rats of 250 ± 30 g weight were divided into 5 groups each containing six animals (n=6). The OS has been administered to group I. The rats of this group were fasted overnight with the allowance of water only before drug administration via an oral tubing. The food was then allowed 4 h after drug administration. The rats of the other four groups (Group from II to V) had the skin shaved from the dorsal area using a depilatory cream for 5-min application, washed well with copious amounts of water to remove any cream residues, followed by a one-day recovery period to facilitate the re-establishment of skin barrier function (Choi et al., 2012, McCrudden et al., 2014). A flat cylindrical cup of 11.34 cm2 was glued to the shaved area of the rats after being anesthetized using thiopental intraperitoneal injection. A formula volume equivalent to the stated dose was then 17

inserted into the cup through a small orifice, which was well sealed after that. The groups of rats encoded II, III, IV, and V have been administered the TS and the selected formulations (Eth, TPGS-Flex, and CREM-Flex), respectively. 2.2.9.2. Blood sampling The blood samples of 0.5 ml were withdrawn from the retro-orbital venous plexus puncture at different time intervals; 1, 2, 4, 6, 8, 12, 24 and 48 h; for all groups except for Group I where an additional sample at 0.5 h was collected. Samples were retained in tubes containing EDTA as an anticoagulant and the plasma was separated by centrifugation at 5000 rpm for 15 min. The plasma samples were then stored at -20°C until drug analyzed using liquid chromatography-mass spectrometry (LC-MS/MS). 2.2.9.3. Sample preparation for analysis The plasma samples were analyzed for their drug content by extraction of TRO with ethyl acetate. Stock solution of torsemide as an internal standard (IS) was prepared and then serially diluted with the mobile phase to give a final working concentration of 100 ng/ml. At time of analysis, 500 µl of each plasma sample was added in glass centrifuge tubes, and then spiked with 100 µl of IS solution. Four milliliters of ethyl acetate were added onto plasma-IS mixture for extraction. The samples were vortexed for 5 min. then centrifuged at 3500 rpm for 10 min. The organic supernatant was then collected and evaporated to dryness under vacuum. The obtained residue was reconstituted in 500 µl of mobile phase used for the quantitative estimation of TRO by LC-MS/MS. 2.2.9.4. Quantitative determination of TRO in plasma using LC-MS/MS assay A LC system (Shimadzu Prominence, Shimadzu, Japan) was used to inject 5 µl aliquots of the processed samples on Waters® SunfireTM (Milford, USA) C18 column (50 × 4.6 mm ID, 5 µm 18

particle size). The isocratic mobile phase consisted of acetonitrile and 0.1% formic acid in water at a ratio 80:20 (v/v), respectively, was delivered at a flow rate of 1 ml/min into the mass spectrometer’s electrospray ionization chamber. All analyses were carried out at room temperature. Quantitation was achieved by MS/MS detection in negative ion mode for both TRO and IS, using a mass spectrometer (AB Sciex Instruments-API 4000TM Mass spectrometer, Model 1034067S, Ontario, Canada), equipped with a Turbo ion spray interface at 450°C. The ion spray voltage was set at 5500 V. The nebulizer gas was set at 30 psi, curtain gas at 30 psi, auxillary gas at 45 psi and collision gas at 5 psi. The compound parameters, viz., declustering potential, collision energy, entrance potential and collision exit potential were 100 V, 52 V, 10 V, 12 V for TRO and 15 V, 14 V, 10 V, 4 V for torsemide (IS), respectively. The ions were detected in the multiple reaction-monitoring mode, monitoring the transition of the m/z 284.916 precursor ion to the m/z 124.1 for TRO and m/z 349 precursor ion to the m/z 264.3 for IS. Quadrupoles Q1 and Q3 were set on unit resolution and the analytical data were processed using Mass Analyst software (Version 1.6). The LC-MS/MS assay was developed and validated according to ICH guidelines for the determination of TRO concentrations in plasma, in terms of accuracy, precision, linearity, specificity, limit of detection, and limit of quantitation. 2.2.9.5. Pharmacokinetic analysis The data of plasma TRO concentrations versus time were analyzed by extravascular noncompartmental estimations using PK solver, the add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Different pharmacokinetic parameters; Cmax (the maximum plasma concentration) and Tmax (time to reach maximum plasma concentration) 19

were directly detected from the plasma concentration versus time profile. The areas under the curve from time zero to last sampling time (AUC0−t), and the area under the curve from time zero to infinity (AUC0−∞) were determined. The relative bioavailability (Frel), defined as the ratio of AUC0−∞ of each transdermal system to that of the OS administered at the same doses, was also calculated (Ishak et al., 2017).

2.2.10. In vivo skin-vesicle interaction study Autopsy samples, taken from the skin of different groups of rats (Group II, III, IV, and V) categorized in the earlier pharmacokinetic studies, were fixed in 10% paraformaldehyde in PBS for 24 h. Washing was done in tap water then serial dilutions of absolute ethyl alcohol were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in hot air oven (DSO-D / DSO-DF, Taiwan) for 24 h. Paraffin bees wax tissue blocks were prepared for sectioning at 4 microns thickness by sledge microtome. The obtained tissue sections were collected on glass slides, de-paraffinized, stained by Hematoxylin-Eosin stain for examination via an optical microscope (Axioskop, Zeiss, Jena, Germany) coupled with a photographic camera, Axiocam, Model ICc3 (Jena, Germany) .

2.2.11. Statistical analysis Each in vitro or ex vivo experiment was performed in triplicates; the average data and their standard deviations (SD) were calculated. Results of the in vivo pharmacokinetic studies were expressed as means of 6 determinations ± standard error of the mean (SEM). For statistical comparisons, a paired t-test was used; a p value of less than 0.05 was considered statistically significant. 20

3. Results and discussion 3.1. Preparation of TRO-loaded Flex and Eth The Flex formulations were prepared by applying the hot technique, with a basic composition similar to that of Eth; EPC and ethanol adjusted at 3%(w/v) and 40%(v/v), respectively (Abdel Messih et al., 2017). The size of vesicles was targeted to attain less than 300 nm, reported suitable for transdermal administration (Abdulbaqi et al., 2016). For that reason, the Eth formulation was subjected to a size reduction by homogenization kept constant at 8000 rpm for only 30 sec using a high shear homogenizer, being the lowest rotor speed and the shortest time required preventing excessive alcohol loss. The formed vesicles attained PS, PDI, and ZP values of 142 ± 0.84 nm, 0.166 ± 0.011, and +15.8 ± 0.53 mV, respectively. Without homogenization, a coarse emulsionlike dispersion was obtained, characterized by a micrometric PS and a heterogeneous size distribution ranging from 0.5-2 µm and 0.7-1, respectively. The homogenization process was excluded during the preparation of all Flex, relying on the effect of EA in minimizing surface free energy and hence reducing PS without the need to external energy supply. 3.2. Optimization study Different formulation variables (Table 1) were systematically optimized aiming to form a colloidal vesicle dispersion characterized by its well-known cloudy turbid appearance, a PS < 300 nm convenient for transdermal administration, a fairly homogenous size distribution (PDI <0.5), and a relatively high ZP values warranting the stability of the prepared formulae. 3.2.1. Effect of EA type

21

For the preparation of Flex, various EAs, commonly employed in topical and transdermal formulations, were investigated varying from anionic, cationic and non-ionic types. Among EA types tried, CTAB (HLB = 10) as a cationic quaternary ammonium surfactant, bile salts; NaC (HLB = 18) and SDC (HLB = 16) as examples of anionic EAs, and T80 (HLB = 15), TPGS (HLB = 13), CREM (HLB = 14-16), and Span 85 (HLB = 1.8) representing the non-ionic types, were chosen. Both PC to EA molar ratio and cholesterol concentration were maintained constant at 2:1 and 0%w/v, respectively. The effect of different EA types on PS, PDI, ZP, and turbidity percent of the prepared formulations was studied. The different EAs under study showed variable influences on PS. Span-modified Eth acquired the highest size, where it exceeds the nanometer range (4.169 µm), compared with the other vesicles. This could be elucidated taking into consideration the HLB of EAs. An inverse relationship was reported between vesicle size and HLB of surfactant; attributed to the decrease in surface free energy associated with the increase in surfactant hydrophilicity. Thus, the hydrophobic EA would interact with phospholipids head groups in the membrane bilayers increasing the layer packing density, leading to a high surface free energy which, in turn, caused a fusion between lipid bilayers and hence larger size (Mikulcová et al., 2017). This could clarify the extremely large size of Span 85-based vesicles ascribed to its strongest hydrophobicity denoted by its exceptionally low HLB (= 1.8). Consequently, Span 85 was excluded from further experiments. Interestingly, Flex prepared with both types of bile salts (NaC and SDC) showed higher PS compared with the remaining formulae. The fact could be due to their rigid bulky steroid-like structures, in conjunction with their highly negative charges creating a repulsion between the bilayers, hence augmenting PS of their vesicles. The NaC-based Eth were much bigger (1.28 ± 22

0.19 µm) than SDC-Flex (383.90 ± 26.16 nm), this could be due to the difference in their chemical structures where NaC carries three hydroxyl (-OH) groups while SDC has only two reflecting higher repulsive forces in case of NaC-based vesicles. Therefore, NaC was omitted from further studies. All other EAs investigated (CTAB, T80, TPGS, and CREM) formed nanosystems with promising sizes and PDI ranging from 49.01–82.29 nm and 0.114 – 0.609, respectively. The effect of EA charge was found prominent on the net surface charge of the formed structures. The nanoconstructs based on the cationic and anionic EAs (CTAB and SDC) carried the highest positive and negative ZP (+72.70 and -45.30 mV), respectively, while the magnitudes of surface charges were the least for all nano-vesicles prepared with the non-ionic EAs. Unexpectedly, the latter nanosystems appeared clear transparent except SDC-Flex when visually inspected. Therefore, investigating the effect of different PC:EA molar ratios on the nanosystem characteristics was considered crucial. 3.2.2. Effect of PC to EA molar ratio Aiming to evaluate the effect of EA concentration, different PC to EA molar ratios ie, 5:1, 2:1 and 1:1 were tried, and the results are illustrated in Fig. 1. Irrespective to EA type, increasing EA proportion ie, decreasing PC:EA ratio from 5:1 to 1:1 resulted in a significant reduction in PS (p<0.05) from >200 nm to <100 nm, as observed in Fig. 1(A). The decrease in PC:EA molar ratio showed increasing the particle homogeneity in the prepared formulations, as illustrated in Fig. 1(B). Also, the elevation in EA concentration seemed causing an augmentation in the positivity, negativity, or neutrality of the systems prepared with cationic, anionic, and non-ionic EAs, 23

respectively, however, the effect was considered non-significant (p>0.05) in the majority of the prepared formulae, as obvious in Fig. 1(C). The physical appearance of the prepared formulae is fundamentally considered as a tool to anticipate the type of nano-dispersions formed. Curiously, the prepared formulations acquired different physical appearances altering from the well-known cloudy colloidal feature of the vesicles (Sun et al., 2016), particularly observed at 5:1 PC:EA molar ratio, to a clear transparent appearance at lower ratios, as clearly seen in Fig. 1(D). The turbidities of CTAB-based formula as a model were assessed, where the percent transmittances (%T) were found to increase from 14.90 to 91.52 to 95.68% when reducing PC:EA molar ratio from 5:1 to 2:1 to 1:1, respectively, confirming the clarity afforded by EA when increased its concentration. The interaction of surfactants with phospholipids in vesicles is assumed to cause the disintegration of the bilayer membranes and hence the formation of different non-lamellar structures ie, dynamic small sized aggregates known as ‘micelles’ when EA concentration increases above its CMC. Actually, these structures are transparent (Sun et al., 2016), thermodynamically stable with size less than 100 nm (López et al., 1998). Indeed, the so-called ‘mixed micelles’ are actually formed due to the presence of lecithin together with EA (López et al., 1998), being known as rigid less deformable structures insensitive to water activity gradient than the vesicles (El Zaafarany et al., 2010, Ahad et al., 2017). Moreover, the presence of alcohol could add to the aforementioned transition process, as it was reported to be surface-active, reducing the CMC of EA (Sidim and Acar, 2013), and hence enhancing its micellization. Consequently, this arises the importance of elucidating the underlying mechanism of such vesicleto-micelle transition. 24

The alteration from vesicle to micelle could be elucidated by the phospholipid critical packing parameter (CPP) that is equal to V/ (Ao×L). The phospholipids tend to form planar lamellar structures when CPP is close to 1 .i.e. its molecular volume (V) is almost equal to the surface crossarea (Ao) of the polar head and the length (L) of its hydrophobic chain (Sun et al., 2016). The insertion of EA within the PC vesicles would increase Ao, hence resulting in CPP reduction, turned into the formation of highly curved micelles. The vesicle-to-micelle transition induced by EA could be assumed to occur in two stages; (1) The EA molecule partitions itself at water/outmost bilayer interface, then re-arranges to penetrate deeply into the bilayers, peeling them off and forming smaller bilayer fragments, (2) after that, both PC and EA molecules self-assemble into micelles via hydrophobic interactions (Sun et al., 2016). Although the estimated vesicles were produced at 5:1 PC:EA molar ratio, such formulations suffered from either an extremely large PS (in case of CTAB and SDC) or a size heterogeneity reflected by a high PDI ranging from 0.454 – 0.727 (in case of T80, TPGS, and CREM). This could be ascribed to the extremely low amount of EA incorporated in case of 5:1 PC:EA ratio considered ineffective to reduce the initial size of the vesicles especially the homogenization step was annulled during the preparation. Moreover, it could be assumed the co-existence of both vesicles and mixed micelles in equilibrium, hence forming intermediate structures at this stage resulting in lack of size homogeneity. Further, such small EA proportion could be presumed nonbeneficial to ameliorate the transdermal drug permeability. Therefore, this molar ratio was omitted from further works. 3.2.3. Effect of cholesterol concentration 25

As explained above, the increase in EA proportion triggers the disintegration of vesicle membrane and thus lecithin-EA micellization. Therefore, the preservation of the membrane wall could be the key strategy. Cholesterol was reported to act as a fluidity buffer in the bilayer membrane improving its rigidity and stability, therefore its inclusion has been explored. The influence of cholesterol was investigated at different concentration levels (0.1, 0.2, and 0.3%w/v of the nano-dispersion). The study was performed on the representative CTAB-based formulation prepared at 2:1 PC:EA molar ratio, described earlier as clear transparent, and characterized by a small size <100 nm, heterogenous size distribution (PDI=0.609), and a positive surface charge (ZP=+72.70 mV). Increasing cholesterol concentration linearly enlarged PS, as demonstrated in Fig. 2(A). Similar findings were previously reported (Ahmed et al., 2016). The size homogeneity of the vesicles obtained was also improved after cholesterol addition, manifested by lower PDI values ranging from 0.077 to 0.399 compared to the cholesterol-deprived formula (PDI=0.609), as shown in Fig. 2(B), though, raising cholesterol content was associated with a relative increase in size heterogeneity. The positive surface charge of the formed vesicles was maintained with a nonsignificant change in ZP values (p>0.05) relative to the cholesterol-free formula, as represented in Fig. 2(C). The visual observation of the prepared formulae, illustrated in Fig. 2(D), revealed that the inclusion of cholesterol at all concentrations incredibly provoked an alteration in the physical appearance from transparent to cloudy appearance. Such conversion was readily monitored by observing the turbidity of the samples, and measuring the %T of scattered light where they recorded 91.52, 0.72, 0.67, and 0.54%, for formulae prepared with 0, 0.1, 0.2, and 0.3%w/v cholesterol, respectively. 26

Cholesterol is among the most common excipient incorporated in vesicle membrane walls. It acts as a cement agent providing an enhanced stability and mechanical strength and stiffness to the lipid bilayer; by raising the gel liquid transition temperature of the phospholipids, and increasing the lipid packing density via “ordering and condensing” effect (Lawrence et al., 1996). It has been reported that the addition of cholesterol is mandatory to form bilayer vesicles in presence of EA when its HLB is higher than 6 aiming to compensate for their large head groups (Lawrence et al., 1996). The presence of cholesterol molecules in the phospholipid bilayers could intermingle with surfactant molecules disrupting micellar aggregates (Woodford, 1969). Based on the results obtained, the least cholesterol concentration (0.1%w/v) was considered appropriate, and was thus maintained in further formulations. 3.2.4. Preparation of different EA-modified formulations using the selected cholesterol concentration. Cholesterol was incorporated at a concentration 0.1%(w/v) in different EA-based formulations prepared at 2:1 and 1:1 PC:EA ratios. The results are represented in Table 2. It is obvious that the transparent feature was maintained in all systems prepared at the lowest ratio (1:1) ie, higher EA content. Surprisingly, viscous or gel-like clear systems were detected in case of CTAB, SDC and CREM-based formulations. The solubilization of the lecithin-cholesterol mixtures might occur in presence of higher EA content (1:1 PC:EA molar ratio) assuming the formation of stable mixed micelles ‘cholesterol-PC-EA’ characterized by a viscous gel-like system. The fact might be due to a sphere-to-rod micellar transition or even more tangled worm-

27

Table 2. The critical quality attributes of formulations prepared with different EAs at 2:1 and 1:1 PC:EA molar ratios after the inclusion of 0.1%(w/v) cholesterol. Formula Type Eth#

PC:EA molar

Expected System (based on

ratio

physical appearance)

---

Vesicles (C)

PS (nm)* ± SD

PDI* ± SD

ZP (mV)* ± SD

142.00 ± 0.84

0.166 ± 0.011

+15.80 ± 0.53

Cationic CTAB-Flex**

2:1

Vesicles (C)

184.40 ± 2.19

0.077 ± 0.010

+81.10 ± 3.40

1:1

Viscous mixed micelles (T)

NA

NA

NA

Anionic SDC-Flex**

2:1

Vesicles (C)

4197.50 ± 0.71

1.00 ± 0.00

-30.00 ± 1.48

1:1

Jellified mixed micelles (T)

NA

NA

NA

Non-ionic 2:1

Vesicles (C)

139.00 ± 7.28

0.435 ± 0.002

+14.00 ± 0.38

1:1

Mixed micelles (T)

54.90 ± 0.02

0.294 ± 0.005

+7.36 ± 0.37

2:1

Vesicles (C)

111.90 ± 4.95

0.402 ± 0.027

+16.60 ± 0.61

1:1

Mixed micelles (T)

20.29 ± 1.70

0.386 ± 0.009

-3.28 ± 0.56

2:1

Vesicles (C)

272.50 ± 6.28

0.328 ± 0.033

+17.70 ± 0.29

1:1

Jellified mixed micelles (T)

NA

NA

NA

T80-Flex**

TPGS-Flex**

CREM-Flex**

*Average of 3 determinations C: Cloudy T: Transparent NA: Not Applicable **All Flex formulations were prepared with 3%(w/v) EPC, 40%(v/v) ethanol, 2:1 PC:EA molar ratio, and 0.1%(w/v) cholesterol without homogenization. # The Eth formula was prepared with 3%(w/v) EPC and 40%(v/v) ethanol with homogenization.

28

like networks, often known as liquid crystalline structures, with the increase in EA concentrations (Guo and Colby, 2001). The presence of lower EA proportion (at 2:1 PC:EA ratio) was capable to maintain the formation of vesicles, accredited to cholesterol in the phospholipid bilayers which slows down the lipid solubilization and micellization. These vesicles attained nanometric sizes ranging from 111.90 to 272.50 nm. Only the SDC-based ethanolic vesicles exhibited a micrometric size (4.197 µm), hence they were excluded. From all EA types studied, only CTAB, T80, TPGS, and CREM were able to form the novel nanovesicles (Flex) when prepared at 2:1 PC:EA molar ratio merely after the inclusion of 0.1%(w/v) cholesterol. These formulations were considered satisfactory as they displayed an acceptable PS ranging from 111.9 to 272.5 nm, an almost homogenous size distribution (PDI = 0.077 – 0.435), and a positively charged surface ranging from +14 to +81.1 mV warranting good stability.

3.3. Determination of TRO EE% in the selected Flex The mini-column centrifugation technique using Nanosep® was initially tried for the determination of free TRO in the supernatant in case of Flex and Eth formulations. Surprisingly, this technique was found inefficient for the separation of Flex where the supernatants always appeared cloudy turbid, mostly owing to the high deformability of Flex hindering their efficient retention onto the membrane filter of the centrifugal tubes. Using the same technique, the Eth formula was well separated, and the drug EE% was calculated and found to reach 27.45 ± 0.92%. Further, the drug entrapment in the selected Flex was determined by the dialysis bag technique aiming to analyze the amount of free unentrapped TRO passing through the dialysis membrane to the receptor medium, the actual amounts of loaded drug were then computed by subtraction from 29

their theoretical quantities added. Based on the drug EE% in case of Eth obtained by the centrifugation technique, the 1-h dialysis period was found equivalent to eliminate a corresponding amount of free drug. Applying the dialysis method for 1 h, the EE% of TRO were determined for CTAB, T80, TPGS and CREM-based Flex, and found to attain 5.04 ± 7.13, 43.21 ± 3.66, 45.87 ± 2.23 and 38.50 ± 1.33%, respectively. The EE% data of all prepared Flex were found significantly higher (p<0.05) than that of the Eth (27.45 ± 0.92%) except for CTAB-based Flex. The cationic CTAB produced a highly positive charge on the vesicles which, in turn, could make an electrostatic repulsion with the positively charged drug TRO hindering its efficient entrapment into the Flex. Though, higher values obtained with the other EAs might rely on the presence of cholesterol in the vesicle membrane wall acting as a hydrophobic barrier which impeded the diffusion of the hydrophilic drug to the external aqueous medium. Moreover, the exclusion of homogenization phase during the preparation of Flex might add in preventing the drug loss, and hence improving the EE%.

3.4. Ex vivo permeation study of the selected Flex The ex vivo permeation study was performed on the selected Flex across the excised rat skin for 24 h. The cumulative amounts of TRO permeated per unit area (Q) at different time intervals were calculated for the various formulations and their profiles are illustrated in Fig. 3. The data of permeation parameters, Q24, Jss, Kp, and D are represented in Table 3. It has been reported that the PE performs his function in improving the drug penetrability through the skin by different mechanisms; either by increasing the thermodynamic activity, reducing the

30

convoluted inter-cellular pathway within the skin or generating a high concentration gradient across the skin (Vaddi et al., 2002). By inspecting the permeation profiles in Fig. 3, it is obvious that both CTAB and T80-based Flex unexpectedly showed the least Q values at different time intervals compared with TPGS and CREM-based Flex as well as the Eth formula. This is translated by significant lower values of permeation parameters (Q24, Jss, Kp and D) for both formulations (p<0.05) in comparison with the

Table 3. The permeation parameters of the selected Flex in comparison with the Eth formula as a control. Permeation Parameters Q24 (µg/cm2) Jss (µg/cm2/h) Kp (cm/h) ×10-2 D

(cm2/h)

ER

×10-2

Data* ±SD Eth 256.87 ± 28.38

CTAB-Flex

T80-Flex

222.25 ± 35.27 189.97 ± 8.16

TPGS-Flex

CREM-Flex

256.83 ± 17.73

318.62 ± 21.14

16.05 ± 2.33

11.46 ± 0.85

12.53 ± 0.62

24.87 ± 0.13

25.50 ± 0.83

1.605

1.146

1.250

2.487

2.550

1.440

0.739

1.150

2.400

3.100

---

0.71

0.78

1.55

1.59

*Average of 3 determinations Eth: ethosomes Flex: flexosomes Q24: Cumulative drug amount permeated per unit area after 24 h. Jss: drug transdermal flux at steady state Kp: permeability coefficient D: diffusion coefficient ER: enhancement ratio SD: standard deviation

other flexosomal systems (TPGS and CREM) and even more relative to the Eth control formula, as depicted in Table 3. These findings could be elucidated to the highly positive CTAB-based vesicles (ZP = +81.10 mV) which are adsorbed onto the skin attracted by the native negative charge of the SC, compensating it by reversing the skin charge, and hence reducing the electro-osmotic flow of the vesicles (Pandey

31

et al., 2014), consequently hindering the partitioning and permeation of the positively charged TRO into the rat skin. Moreover, the low entrapment of TRO into CTAB-based vesicles could add to its low transdermal flux. Besides, the hydrophilicity of EA head groups has been claimed to affect the penetration of polar molecules through the skin according to Laughlin’s hypothesis. The EAs with hydrophilic head groups are capable to effectively enhance the percutaneous penetration of polar molecules, more than those of lesser hydrophilicity. Our results could be in agreement with this hypothesis based on the lower hydrophilicity of CTAB compared with the other EAs studied, expressed by the HLB value of CTAB found be the least (HLB=10) (Laughlin, 1978). Surprisingly and contrary to the aforementioned hypothesis, the drug permeation from T80-based Flex was found to be low, as depicted from Table 3, noting a non-significant difference in its Q24 and Jss values when compared with those obtained with CTAB-vesicles (p>0.05). This was inconsistent with several works (Song et al., 2012, Pandey et al., 2014). However, Tween was reported to significantly decrease the drug Jss and Kp by reducing its thermodynamic activity owing to micellar complexation, and hence dropping the driving force for drug absorption (Ghafourian et al., 2015). Therefore, a possible explanation of our results could rely on the CMC of this EA, reported to attain an extremely low value approaching 0.0014% compared with the other EAs as their CMC values > 0.02%. Therefore, the co-existence of higher number of mixed micelles closely packed with the vesicles could be expected within T80-based formula assuming the rigidity and low deformability of the former structures. These findings are in agreement with published data (El Maghraby et al., 2000, Hiruta et al., 2006). Both TPGS and CREM-based Flex showed the highest enhancement of TRO transdermal flux among all studied Flex, reflected by higher Q24, Jss, Kp and D values, as obvious in Table 3. Both 32

formulations were also found to enhance the drug permeation 1.5-fold relative to the EA-free vesicles (Eth). Although the significant higher Q24 in case of CREM-vesicles, a non-significant difference in Jss and Kp values (p>0.05) was observed compared to that based on TPGS. The penetration enhancement of vitamin E, the main component of TPGS, has been previously confirmed, its mechanism was elucidated triggered by the increase in the gloss fluidity of the SC lipids through the intercalation of vitamin E with the lipid bilayers, the increase in lipid lamellae disorder within SC, and hence the formation of a looser and more permeable structure (Trivedi et al., 1995). Moreover, it has been reported that both ethanol and TPGS synergistically enhanced the drug flux by favorably altering the interfacial barrier function of the SC, thus reducing the resistance to drug permeation (Aggarwal et al., 2012). The percutaneous permeation triggered by EA was demonstrated to be influenced by their hydrocarbon chain length. The longer the chain, the more drug permeation will be. In the same context, the hydrogenated castor oil derivative, CREM, bearing three long C18 alkyl chains per triglyceride molecule pertaining to its large content of fatty acids mainly hydrogenated ricinoleic acid (>85%), might have its impact on permeability enhancement. Moreover, the branched structure of CREM could have a positive effect on permeation because its incorporation into the SC lipid lamellae would mandate more space causing more prominent disturbance of the barrier (Klimentová et al., 2006) . Therefore and based on the previous results, both TPGS and CREM-based Flex were selected for further investigations. The korsmeyer-Peppas equation was applied to define the drug permeation kinetics through the skin. The n values of the permeation profiles were computed in case of TPGS and CREM-based

33

Flex, and found to be 0.646 and 0.981, respectively. These data indicate an anomalous (nonFickian) diffusion-controlled drug permeation for TPGS formulation, while a zero-order release in case of CREM-vesicles comparable to Eth formula where their n values were almost 1, providing a constant drug permeation rate over an extended period of time.

3.5. Ex vivo assessment of the permeation enhancing mechanism of the selected vesicles The DSC is a conventional technique adopted to determine the phase transitions of different skin constituents. Before measurement, all SC samples were subjected to complete dryness in a desiccator for 48 h to exclude any discrepancies in their hydration levels. The thermograms obtained from various SC samples are collected and illustrated in Fig. 4. Untreated SC exhibits four main endothermic peaks at 43.67, 78.45, 87.76, and 121.84°C. This is in consistency with previous data in literature (Van Duzee, 1975, Al-Saidan et al., 1998). The first thermal event (T1) around 40°C was accredited to the melting of sebaceous lipids or ascribed to the gel-liquid crystalline phase transition of skin lipid bilayers. The two small successive peaks (T2-T3) were described to the steady disorganization and fluidization of lipids in the bilayer lamellae occurred with the increase in temperature. The last thermal event (T4) around 120°C was attributed to the denaturation of keratin in keratinocytes. After being incubated with SC for 24 h, the vesicles under study showed varied impacts on SC thermal patterns (Fig. 4). The first three thermal peaks which have been related to lipid lamellae, completely disappear in all treated SC samples. This could stand for the penetration enhancing effect of ethanol reported to cause a liquefaction and extraction of lipids within SC (Lane, 2013). The thermal denaturation peak of keratin filaments was maintained with slight shift to 125°C in treated SC with Eth, while it completely vanished in SC incubated with both types of Flex. This 34

could be ascribed to the reported interaction of EAs with keratin filaments and their denaturation effect on skin proteins (Williams and Barry, 2004). This endorses the additive transdermal permeation enhancing effect of EA in ethanolic vesicles.

3.6. Characterization of the selected Flex 3.6.1. Particle morphology using HR-TEM imaging The morphologies of the selected Flex based on TPGS and CREM were determined using HRTEM, and their images are represented in Fig. 5 (A and B), respectively. The TEM pictures of the Eth formulation is also illustrated in Fig. 5 (C) for comparison. All EA-based vesicles appear as lipid stacked particles with almost spherical shape and defined edges Fig. 5 (A and B), while the Eth particles show hazy vesicles with no clear sharp edges (Fig. 5 C). The fact might be due to the presence of the wall forming material (cholesterol) in the former structures. The size of the Eth is observed smaller (Fig. 5C) than the hydrodymanic diameter measured by DLS, attributed to the process of sample drying just before TEM imaging. However, the images of both Flex show rather comparable sizes to those measured by DLS, the fact might also be accredited to the presence of cholesterol as the membrane wall cement preserving the shape and size of the vesicles. 3.6.2. Drug-excipient interaction study using FT-IR spectroscopy FTIR spectra of the Flex constituents; TRO, EPC, cholesterol and EA either TPGS or CREM, as well as their physical mixtures are illustrated in Fig. 1S and interpreted in Supplementary file. The FTIR spectra confirmed the presence of TRO in its native form without any chemical interaction with the vesicle constituents, and hence its compatibility with all components. 35

3.7. Colloidal stability study of the selected formulations The selected Flex were stored under refrigeration (5 ± 3°C) for a period of 6 months. The effect of storage on PS, PDI, ZP, and EE% of TPGS-flex and CREM-Flex was assessed initially and at 1, 3, and 6-month intervals, and the results are represented, interpreted and illustrated in Fig. 2S (Supplementary file), in comparison with the stability data of Eth formula, confirming a reasonable stability for the developed vesicles. 3.8. Tracking the fluorescently-labeled vesicles in the rat skin layers using CLSM The selected Flex as well as Eth were loaded with a fluorescent dye, Nile Red, instead of the drug. Contrary to TRO character, Nile Red is a lipophilic dye employed per se taking advantage of the high loading capacity the lipid bilayers afford for lipophilic molecules, hence endorsing the tracking of vesicles and their distributions across the different skin layers. All experimental conditions; including the dose of the fluorescent dye in vesicles, were entirely kept constant in order to allow for precise comparison. Confocal laser scanning micrographs, represented in Fig. 6, show the fluorescence intensity and depth of Nile Red within the rat skin layers after application of the fluorescently-labeled formulae; TPGS-based Flex and CREM-based Flex, relative to Eth. Noting the thicknesses of SC and epidermis in rats are about 5 and 20 µm, respectively (Monteiro-Riviere et al., 1990). Fig. 6 (A) show the distribution of dye-loaded Eth within the rat skin layers. It is clearly seen that the fluorescence intensity is observed accumulated onto the skin surface ie, SC, associated with a discrete localization in deeper skin layers ie, the epidermis and the dermis. In contrast, the fluorescent dye is observed deeper and higher in intensity in case of both Flex formulations, as

36

shown in Fig. 6 (B and C). This could reflect the enhanced permeation of TPGS- and CREMbased Flex across the rat skin compared to Eth. These results also deduced the high elasticity and flexibility of Flex, attributed to the presence of both ethanol and EA. It could be assumed that the fluorescence appeared within skin layers is due to the permeation of either dye-containing vesicles and/or free dye. Therefore, we can propose that the prepared Flex would be able to enhance the skin permeation by a variety of mechanisms: (a) the penetration enhancement of their components (ethanol, EA) via lipid solubilization and keratin denaturation, (b) the penetration of intact vesicles, owing to their high flexibility, across the intact skin, and/or (c) the fusion of the vesicles with the skin components eg, lipids.

3.9. Pharmacokinetic studies A high performance liquid chromatographic method coupled with mass spectrometric detection (LC–MS/MS) was developed, optimized and validated for the determination of TRO in rat plasma using torsemide as IS. For transdermal TRO delivery, the selected ethanolic vesicles, either Eth or Flex, as well as TS, at a drug dose of 10 mg/kg, were applied onto the skin of rats by means of our own-designed cups. A drug solution was also administered orally (OS) as a reference formula alternative to the marketed capsules. The plasma TRO concentrations at different time intervals for the different formulations were analyzed by LC-MS/MS technique, their data profiles are illustrated in Fig. 7. It was obvious that the EA-based ethanolic vesicles (TPGS-Flex and CREM-Flex) showed the highest drug plasma concentrations at different time intervals, in contrast to Eth, as observed in Fig. 7. However, the aqueous solution applied transdermally (TS) displayed the least drug levels

37

in plasma as expected. After oral administration, the TRO plasma concentrations exhibited a gradual decline being on its highest level at 0.5 h next to administration. Aiming to analyze in depth the obtained concentration-time profiles, a non-compartmental model was applied to determine the various pharmacokinetic parameters for each formulation investigated, the data are summarized in Table 4. After transdermal application of the ethanolic vesicles, the Cmax could be arranged in an ascending order as follows; 342.50 < 458.33 < 571.00 ng/ml, reached after 12, 7.33, 5.33 h, for Eth, TPGS-Flex, and CREM-Flex, respectively. It is quite evident that the highest Cmax and the shortest Tmax are relevant to CREM-Flex, this could be attributed to the synergistic effect of both CREM and ethanol in enhancing the transdermal permeation of TRO (Karande and Mitragotri, 2009, Ahmed et al., 2016). These findings confirmed the results obtained from the ex vivo permeation study described earlier. The Tmax values were found extended for transdermal administration than for the oral route with a statistically significant difference (p< 0.05). This could be indicative of the delay in the drug permeation across the rat skin (El-Nabarawi et al., 2013), attributed to the barrier property of SC and the lengthy diffusional pathway through the underlying skin layers as well (Kamran et al., 2016). The transdermal administration of TRO in the form of aqueous solution (TS) showed a constrained permeation flux translated by an extremely lower value of Cmax, 41.10 ng/ml, compared with the vesicular formulations. This might be due to the hydrophobic nature of skin phospholipid bilayers, impeding the simplistic penetrability of the aqueous formula into the skin. Compared with OS, the various ethanolic vesicles showed significant increase in AUC0-∞ (p<0.05), as obvious in Table 4. Although the Cmax obtained with Eth was found comparable to OS, yet the

38

AUC value was significantly higher in the former. This indicates the improved systemic drug bioavailability by transdermal route, which could be due to the evasion of first-pass hepatic TRO metabolism. In addition, this could suggest that the vesicles were retained within the skin layers acting as a drug reservoir, hence achieving an extended drug plasma level for a longer period of time (Fahmy, 2015). For better elucidating the impact of transdermal vesicles on the systemic TRO availability, the relative bioavailability (Frel) as the ratio of transdermal AUC0-∞ to oral AUC0-∞, was computed and collected in Table 4. They attained 2.27, 1.92, 1.77, and 0.67, for CREM-Flex, TPGS-Flex, Eth, and TS, respectively. This confirms the superiority of transdermal ethanolic vesicles, particularly CREM-based ones, in improving the bioavailability of the water-soluble drug, hence suggesting them as a better alternative to the oral route.

Table 4. Pharmacokinetic parameters of TRO following oral and transdermal administration in rats at 10 mg/kg dose.

PK Parameters

Data# ± SEM OS

TS

Eth

TPGS-Flex

CREM-Flex

Cmax (ng/ml)

326.50 ± 20.34

41.10 ± 19.46*

342.50 ± 34.60NS

458.33 ± 52.57*

571.00 ± 107.48*

Tmax (h)

0.88 ± 0.38

7.00 ± 1.00*

12.00 ± 0.00*

7.33 ± 0.67*

5.33 ± 1.77*

AUC 0-48 (ng/ml/h) AUC 0-∞ (ng/ml/h)

2011.50 ± 290.26

618.22 ± 433.74*

3910.52 ± 131.16*

4169.27 ± 285.58*

4084.20 ± 626.60*

2534.85 ± 576.23

1705.50 ± 299.60NS

4482.41 ± 8.53*

4874.48 ± 167.01*

5750.56 ± 1760.74*

Frel

---

0.67

1.77

1.92

2.27

# Average of 6 determinations OS: oral aqueous solution TS: transdermal aqueous solution Eth: ethosomes Flex: flexosomes *Significant difference at p<0.05 compared with oral TRO solution NS non-significant difference at p>0.05 compared with Oral TRO solution PK: pharmacokinetic, SEM: Standard error of the mean, Cmax: Maximum plasma drug concentration, Tmax: Time to reach maximum drug concentration, AUC: Area Under the concentration vs time Curve, Frel: Relative Bioavailability = Ratio of AUCs (transdermal formulation to oral solution).

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3.10. In vivo skin-vesicle interaction study The dermato-histopathologic examinations were assessed for the skin treated for 24 h with the selected formulations (Eth, TPGS-Flex, and CREM-Flex) as well as an aqueous drug solution as a control using an optical microscope with a 10x objective lens, the images are illustrated in Fig. 8. It is obvious, from Fig. 8 (A) representing the control skin, the well-defined uniformly layered SC, epidermis, and dermis with the appearance of sebaceous glands and hair follicles, without any histological alteration. Fig. 8 (B-D) show the histology of rat skin, after being in contact with Eth, TPGS-Flex, and CREM-Flex, respectively. A detachment of SC from the epidermal layer was observed in all treated skin sections. No structural alterations in the underlying dermis were detected, except for the infiltration of few inflammatory cells. Similar results were obtained in previous works (Patel et al., 2012, Subongkot et al., 2014). Ethanol is able to extract and liquefy the lipids leading to the formation of microcavities within SC, thereby increasing the water volume between layers for drug diffusion (Lane, 2013). Therefore, it could be assumed that ethanol is the primary cause for the alteration obtained in the outermost epidermal layer. It is worthy to mention that TPGS and CREM were extensively applied for topical/transdermal use in different dosage forms, such as microemulsions and nanoemulsions, reporting their safety and compatibility with the skin (Soliman et al., 2010, Aggarwal et al., 2012). From all results obtained, Flex are suggested to be promising nanocarriers for TRO systemic delivery via a more convenient non-invasive alternative route than peroral and parenteral ways of administration.

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4. Conclusions In the present study, a novel soft nano-vesicular carrier (Flex) has been developed, optimized, fully characterized and evaluated after being loaded with the highly water-soluble anti-emetic drug (TRO). Both ethanol and one EA from different types and HLB, varying from cationic, anionic, non-ionic, have been integrated in the same constructs. The EA type, PC:EA molar ratio as well as the cholesterol concentration were found to impose substantial impacts on the critical quality attributes of the developed nano-carriers with the propensity to control vesicle-to-micelle transitions. The findings of this study emphasized the superiority of the novel TPGS-Flex and CREM-Flex, in terms of ex vivo transdermal flux, and in vivo bioavailability, over the ethanolic vesicles (Eth), oral and topical aqueous solutions. The modified nano-vesicles are considered promising carriers for enhanced transdermal TRO delivery. Further investigations are recommended to assess the effectiveness of the tailored vesicular carriers in boosting the transdermal delivery of large Mw molecules. Declaration of Interest The authors declare that there is no conflict of interest. References Abdel Messih, H.A., Ishak, R.A., Geneidi, A.S. & Mansour, S., 2017. Nanoethosomes for transdermal delivery of tropisetron HCl: multi-factorial predictive modeling, characterization, and ex vivo skin permeation. Drug Dev Ind Pharm, 43, 958-971. Abdulbaqi, I.M., Darwis, Y., Khan, N.A., Assi, R.A. & Khan, A.A., 2016. Ethosomal nanocarriers: the impact of constituents and formulation techniques on ethosomal properties, in vivo studies, and clinical trials. Int J Nanomedicine, 11, 2279-2304. Aggarwal, N., Goindi, S. & Mehta, S.D., 2012. Preparation and evaluation of dermal delivery system of griseofulvin containing vitamin E-TPGS as penetration enhancer. AAPS PharmSciTech, 13, 67-74. Ahad, A., Al-Saleh, A.A., Al-Mohizea, A.M., Al-Jenoobi, F.I., Raish, M., Yassin, A.E.B. & Alam, M.A., 2017. Formulation and characterization of novel soft nanovesicles for enhanced transdermal delivery of eprosartan mesylate. Saudi Pharmaceutical Journal, 25, 1040-1046.

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44

(A)

(B)

(D)

(C)

Fig. 1.

45

(B) 0.8

(A)

PDI

0.6 0.4 0.2 0.0

0

0.1

0.2

0.3

Cholesterol Concentration(%w/v)

(D)

Fig. 2.

46

Fig. 3.

47

Fig. 4.

(A)

(B)

(C)

Fig. 5.

48

Merge Bright Field

Nile Red

(A) (B)

Fig. 6.

49

Fig. 7.

(A)

SC

(B) EP

HF

SG

D

50

(D)

(C)

Fig. 8.

Figure Captions: Fig. 1. Effect of PC:EA molar ratio on (A) PS, (B) PDI and (C) ZP of the nano-formulations prepared with various EA types. (D) Physical appearance of the representative formulations based on CTAB prepared at (a) 5:1, (b) 2:1, and (c) 1:1 PC:EA molar ratios. Fig. 2. Effect of cholesterol concentration on (A) PS, (B) PDI, (C) ZP, and (D) physical appearances (a) with and (b) without cholesterol of CTAB-based formulations prepared at 2:1 PC:EA molar ratio. Fig. 3. The ex vivo drug permeation profiles of the selected Flex compared to that of Eth as control. Fig. 4. DSC thermograms of untreated and treated SC with Eth and selected Flex formulations. Fig. 5. HR-TEM images of (A) TPGS-Flex, (B) CREM-Flex, and (C) Eth.

51

Fig. 6. CLSM images showing cross-sectional views of rat skin treated with (A) Nile red-labeled Eth, (B) Nile red-labeled TPGS-Flex, and (C) Nile red-labeled CREM-Flex after 4-h application. The arrow points out the SC of the skin. Fig. 7. Plasma TRO concentrations versus time profiles after oral administration of a drug solution (OS), and transdermal administration of the selected ethanolic vesicles and an aqueous solution (TS) to rats at 10 mg/kg drug dose. Each point represents mean ± SEM (n = 6). Fig. 8. Light microscopic photographs (100×) of rat skin sections stained with hematoxylin-eosin and treated with (A) control aqueous drug solution, (B) Eth, (C) TPGS-Flex, and (D) CREM-Flex for 24 h. SC: stratum corneum, EP: epidermis, D: dermis, HF: hair follicles, SG: sebaceous glands.

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53