Cilostazol niosomes-loaded transdermal gels: An in vitro and in vivo anti-aggregant and skin permeation activity investigations towards preparing an efficient nanoscale formulation

2.1 Materials

CLZ was donated by SEDICO Pharmaceuticals, Egypt. Methanol, KH₂PO₄, and Na₂HPO₄ were obtained from El Nasr Pharmaceutical Chemical Co., Cairo, Egypt. Cholesterol, Span 20, 40, and 60, and Tween 60, 80, Carbopol-934, Pluronic-F-127, HPMC, and triethanolamine were purchased from Sigma Chem. Co., St. Louis, MO, USA.

2.2 Preparation of CLZ-loaded niosomes by ether injection method

By using an ether injection approach, drug-loaded niosomes, with the properties shown in Table 1, were produced. Accurately weighed quantities of nonionic surfactant, i.e., Span 20, Span 40, Span 60, Tween 60, and Tween 80, and cholesterol (different cholesterol/surfactant molar ratios) were used in diethyl ether (6 mL). The preceding solution was mixed with CLZ in methanol (2 mL), and the resulting combination was carefully injected using a micro syringe at a rate of 1 mL/min into 10 mL of phosphate buffer saline (PBS, pH 6.8). The dispersion was heated to 60 ± 5°C with continuous stirring on a magnetic stirrer [32]. Under the conditions, the organic solvents were evaporated, resulting in the formation of CLZ-loaded niosomes.

2.3 Characterization of the prepared CLZ niosomes

2.4 In vitro drug release test

The in vitro release experiments of CLZ from niosomal formulations were carried out using a standard cellophane membrane. In brief, an open-ended glass tube with a piece of wetted cellophane membrane (2.65 cm², surface area), which was wrapped over one end and secured with a rubber band, was used. In a 0.5 L beaker, the tube was submerged in PBS (200 mL) that had a pH of 6.8 and was kept under shaking (50 rpm) at 37°C [36]. The cellophane membrane was located slightly below the surface of the buffer in the tube and held vertically inside the beaker. The tube contained niosomal preparation equal to 5 mg of CLZ. Free CLZ, 5 mg, was suspended in 1 mL distilled water, fixed inside the release tube, and acted as a control for drug release. At predetermined time intervals, and to maintain a consistent volume, 5 mL aliquots of the released media were taken out and replaced with an equivalent volume (5 mL) of fresh PBS. Using a UV spectrophotometer, at a wavelength of 258 nm, the absorbance of the samples was measured in comparison to the blank, and the quantity of the released drug was calculated. The mean value of three releases of the same experiment were recorded.

2.5 Fourier transform infrared (FT-IR) spectroscopic analysis

Infrared spectroscopic analyses for CLZ, excipients, and selected niosomal formula were recorded by FT-IR- 476 (Shimadzu Kyoto, Japan) instrument. KBr was added to the dry test sample before being compacted into a solid disc. Spectra were recorded in the IR region 400–4,000 cm⁻¹.

2.6 Differential scanning calorimetry scanning

DSC analyses were conducted using a differential scanning calorimeter calibrated with indium (DSC-50, Shimadzu Co., Japan). Samples of CLZ, surfactant, cholesterol, their physical mixtures, and CLZ-loaded niosomal formulations were analyzed. The thermograms were taken at a 10°C/min rate. The temperature range for the scanning of each sample was 20–380°C.

2.7 SEM scanning

Surface morphology of CLZ niosomal formulations was observed through scanning electron micrograph, using the SEM instrument (JSM-5400LVSEM, Jeol, Japan). One drop of niosomal formulation, placed on a transparent glass stub, was air dried and coated with Polaron-E-5100 sputter coater, and SEMs were obtained.

2.8 Stability studies

For 6 months, stability investigations of selected CLZ niosomal formulations were conducted at 4 and 25°C to observe the changes within the chosen parameters. To prevent any possible interactions between the niosomal formulations and the glass of the utilized vials, which may impair the study, the formulations were stored in boron glass vials. Each month, the evaluated CLZ niosomal formulations were examined for any physical alterations in parameters, including color changes, vesicular size change, DEE, and any in vitro drug release.

2.9 Preparation of CLZ niosomal gels

The following gel bases, Carbopol-934 (G1), Pluronic-F-127 (G2), and HPMC (G3) were selected to observe the delivery of CLZ at concentrations of 1, 30, and 3%, respectively. The gelling ingredients were dissolved in distilled water to create the bases, which were then given a 24-h hydration time. The selected CLZ-loaded niosomal dispersion (F10) was centrifuged at 10,000 rpm at 4°C for 45 min to obtain the niosomal vesicles. These vesicles (F10) were then incorporated into aqueous dispersions of Carbopol-934 (CLZ-G1-F10), Pluronic-F-127 (CLZ-G2-F10), and HPMC (CLZ-G3-F10). The resultant semi-solid dispersions were slowly triturated and handled to avoid entrapment of air bubbles. In the case of the G1 gel base (Carbopol-934), the dispersion was neutralized by triethanolamine (5%, w/v) to obtain the viscous, translucent gel.

2.10 Characterization of the prepared CLZ niosomal gel drug contents

In 100 mL of PBS (pH 6.8), 500 mg of prepared CLZ niosomal gel, precisely weighed and shaken, was taken, and a clear solution was produced when the mixture was mechanically shaken for 30 min. After filtering the resultant solution, the quantity of the medication was measured at 258 nm on a UV instrument using PBS (pH 6.8) as a reference.

2.11 Viscosity determination

By using a Brookfield viscometer (DV-III ultra-viscometer, Brookfield Engineering Laboratories, INC, Stoughton, USA), the viscosity of the generated CLZ gel compositions was assessed. The samples’ viscosities were measured using spindle number 4, which revolved at 50 rpm, with 20 g of each gel charged in a beaker [37].

2.12 In vitro release of CLZ from niosomes-loaded gels

The effects of compositional variation on release rates were examined under in vitro conditions for various formulations. The tests were conducted by placing 3 g of each gel into the dialysis bag, bags closed with clips and immersed in 250 mL PBS (pH 6.8), which were kept at 37°C. A magnetic stirrer was used to stir the beaker’s contents at ∼100 rpm. Aliquots of samples were taken at predetermined intervals and were promptly replaced with an equivalent quantity of fresh fluid. The withdrawn samples were analyzed by UV spectrophotometer at 258 nm wavelength for determination of the quantity of the released drug, CLZ.

2.13 Kinetic studies of the in vitro drug release

All the prepared niosome gel formulations’ drug release mechanisms were examined. In vitro, release data were subjected to regression analysis using zero-order, first-order, and Higuchi-diffusion models [38].

2.14 Ex vivo permeation of CLZ from niosomal gel

CLZ penetration from the prepared gels was examined through a 3.65 cm² patch of rat abdomen skin using a Franz diffusion cell. The receptor cells containing 20 mL of diffusion medium (PBS, pH 6.8) were kept at 37°C and were shaken constantly at 300 rpm using a horizontal water bath shaker. On the shaved rat abdomen skin in the donor area, CLZ niosomal gel preparations containing niosomal formulations corresponding to 5 mg of CLZ, or pure drug, were applied. For 12 h, 1 mL sample was taken at regular intervals and subjected to spectrophotometric analysis to determine the quantity of the CLZ released. To maintain optimal sink conditions and maintain a consistent volume of the diffusion, an equivalent volume of fresh diffusion medium was introduced into the receptor cells. The mean was obtained after each experiment was run in triplicate.

2.15 Pharmacokinetics of CLZ from drug-loaded niosomal gel

The procedure, in brief, included loading Carbopol-934 gel preparation with CLZ niosomal formulation equivalent to 1% drug (CLZ-G1-F10) and pure drug (1%, w/w) of a Carbopol-934 gel formulation (CLZ-G0). The ethical committee, national legislation, and university codes of conduct regulations for the care of experimental animals were followed. The animal experiments were approved by the Zagazig University, with approval No. ZU-IACUC/3/E/861/2021. The rats were divided into three groups (n = 3): group 1 was treated with CLZ niosomal gel (CLZ-G1-F10), group 2 was treated with pure CLZ gel (CLZ-G0), and group 3 did not receive any treatment (control group). The rats fasted during the night with free access to water till the drug was administered [39]. On the next day, groups 1 and 2 of the rats, CLZ niosomal gels, and pure CLZ gels, respectively, were treated with 100 mg gel containing 1 mg of CLZ (equivalent to 3 mg/kg). Blood samples were taken after 1, 2, 3, 4, 6, 8, 10, and 12 h from the ear vein of the animal under light ether anesthesia and were analyzed for drug content using HPLC.

2.16 Blood samples’ treatment and HPLC assay

Collection of blood samples (1 mL) was performed from the rat’s ear vein in heparinized tubes, which were centrifuged at 5,000 rpm for 15 min. A 5 mL polypropylene centrifuge tube with a flattened tip was filled with 0.5 mL of methyl tertiary butyl ether, and 0.2 mL of the separated plasma before the tube was vortexed for 1 min at a high speed. Immediately afterward, the sample was agitated on a revolving shaker at 50 revolutions per minute for 3 min. The transparent layer was transferred with a measured pipette to a temporary glass tube and evaporated over a moderate flow of dry nitrogen gas after the sample was centrifuged for 20 min @2,000 rpm, at 4°C temperature. Dried residue, 20 µL, in mobile phase combination, was injected into the HPLC. The mobile phase for HPLC analysis was composed of 50 portions of CH₃CN, 20 portions of acetate buffer (50 mM, pH 5.0), and 30 portions of H₂O (ratio of 50:20:30, v/v). On the C18 column (Phenomenex, 250 4.60 mm, 0.5 mm particle size), filtered samples of CLZ were analyzed at a rate of 1.5 mL/min. The CLZ detection wavelength was 258 nm. The extraction recovery was calculated by comparing the assay results of plasma, spiked with CLZ, at three concentrations of 0.5, 1, and 1.5 μg/mL, with the same concentrations dissolved in methanol. CLZ’s pharmacokinetic characteristics, C _max (the maximum plasma concentration), and T _max (the time to reach the peak concentration) were observed using the plasma concentration plotted against time. The absorption rate constant (K _abs), elimination rate constant (K _el), and t _1/2 (half-life) were also calculated.

2.17 Coagulation measurements

The rats were fasted overnight, and the blood samples were drawn in EDTA-tubes 2, 6, 12, 18, 24, 36, and 48 h after the application of the pure drug, and the niosomal CLZ formulations for PT and APTT (prothrombin time and activated partial thromboplastin time) [40].

2.18 Bleeding time (BT) and clotting time (CT)

The BT was measured according to the described procedure [41]. The rat’s tail was heated for 1 min in a water bath at 40°C, and before it was dried, with a knife, a tiny incision was created in the tail’s center. When the first drop contacted the circular filter paper, the bleeding process was counted to begin. Up until the bleeding stopped, it was checked every 30 s.

For measuring CT, a glass microhematocrit capillary was used to hold a 25 μL sample of capillary blood. Once the blood initially came into touch with the glass capillary tube, the chronometer was activated. When the blood stopped flowing (the endpoint), the capillary tube was tilted alternatively to +60° and −60° angles concerning the horizontal plane. The two tube markings were left to flow by gravity between them [42].

2.19 Prothrombin time and activated partial thromboplastin time (PTT and APTT)

Partial thromboplastin time (PTT), a frequently used test for coagulation, and APTT, quantifying the combined activities of the majority of coagulation components in intrinsic and extrinsic blood coagulation cascade pathways, were performed. The PTT measured the time it took to clot after the tissue factor was added (PT and APTT). A Systemex CA-5000 Coagulation Analyzer was used to measure CTs [43].

2.20 Statistical analysis

Using SPSS v.24 software, the data were subjected to analysis of variance at the 0.05 level of statistical significance. The information was displayed as mean ± SE. Every experiment was run in triplicate.

3.1 CLZ structure and characterization of the prepared CLZ niosomes: size and polydispersity indices

The CLZ chemical structure possessed a quinolinone alkaloid molecular skeleton, with cyclohexyl moiety attached to the tetrazole ring, which bridged to the quinolinone through a butoxy group (Figure 1). The common structural features for the activity elicitation needed to possess a strong dipole producer, adjacent acid proton, short alkyl chain attached to the heterocyclic ring, an electron-rich center with a hydrogen bond acceptor, and a nearly flat molecular geometry. These structural features contributed to the structural stability of the CLZ molecule under different bio-conditions and made the molecule susceptible to hydroxylation and dehydrogenative transformations under the hepatic and other metabolites forming conditions [6,7,44]. The niosomal preparation using different types and ratios of the surfactants with the drug and cholesterol (both components’ weight ratios were kept constant) produced niosomes of different sizes. The increase in the niosome sizes was observed for certain types of surfactants. The use of nonionic surfactant’s physico-chemical properties, the lipophilic/hydrophilic character, molecular size, the presence of polar and non-polar groups, together with the alkyl chain length and their disposition in the molecular structure of the surfactant, methods of preparation, and the niosome formation parameters, including the surfactants’ HLB values, are among the main factors in the niosomal size variations during their preparations. Prepared formulations, F1–F9, had progressively increased in size. For the formulations F10–F15, there was a drop in the size of the formulation F10 as compared to the formulation F9, but the sizes increased gradually from formulations F10 to F15, thereby suggesting the roles of the molecular and physico-chemical characteristics of the surfactant, the interactions of the preparative components of the formulating niosomes, lamellar layers’ formation, and the lipophilic nature of the surfactants playing their respective parts in size determinations. Changes in surfactants and the ratio of their use were also among the deciding factors in the niosome size determinations. Surfactants, Span 20, Tween 60, and 80 in different but constant ratios of 0.5, 1, and 1.5 were utilized for the preparations of F1–F9 formulations, while Span 60 and 40 were used for preparations of F10-F15 formulations of niosomes. For these preparations, the surfactant–cholesterol ratios were kept constant at 0.5, 1, and 1.5 for all the used surfactant types. For both the size increment trends, F1–F9, and F10–F15, the size increase from F1–F9, and F10–F15 was attributed to the use of different span surfactant types, and the most hydrophilic surfactant, Tween 60, was primarily the major cause. The size differences were also thought to be a culmination of unilamellar and multilamellar niosome formations, the multilamellar vesicles being larger. However, current preparations had provided over 100 nm-sized niosomes, all being multilamellar in their built. The surfactant effect played its main part in deciding the size increments of the niosomal formulations. The observations suggested that the hydrophobicity of the surfactant, and the HLB values had a significant impact on the vesicle size. The hydrophobicity of the surfactant increased with an increase in the alkyl chain length, which lowered the HLB score. The lowest vesicle sizes were observed in niosomes that were prepared using Span 60, and that may be due to the lowest level of HLB value of the employed surfactant [45]. Niosome size and PDI of the prepared CLZ-loaded niosomal vesicles are presented in Table 2 and Figure 2. The particle size of the obtained vesicles averaged from 102 to 550 nm for all niosomal preparations. The HLB value of the utilized surfactant determined the size of the vesicles. The vesicle surface energy and water absorption into the niosomal bilayer increased as the hydrophilic monomers of the surfactant increased, which may have also contributed to the vesicle’s growth in size. These findings also indicated that the size of the niosomal vesicles decreased with the decreasing HLB values. The size of the developed CLZ-loaded niosomal formulations also grew as the cholesterol content arose. The niosomal preparations with CLZ revealed a limited peak in their size distribution, which indicated that the currently used technique for the niosome preparation (Ether injection method) produced comparatively homogeneous niosomal vesicles. The PDI for the most tested niosomal formulations was around 0.3, suggesting a monodisperse vesicle condition [46,47,48].

Figure 2

Sizes of different CLZ-loaded niosomal formulations (inset figure: size distributions of CLZ niosomal formula, F10).

3.2 Drug entrapment efficiency of CLZ-loaded niosomes

The DEE results of the prepared CLZ-loaded niosomal formulations are presented in Figure 3. The results showed that the DEE for the prepared niosomes using Span^® (especially Span 60) was higher than those prepared using the Tween^® as a surfactant, which could be due to the lipophilic nature of both the Span (low HLB) used. In contrast, due to the hydroxyl groups and ether oxygen-based functional groups in their chemical structures, Tweens^® behaved as hydrophilic surfactants (with high HLB, and had high aqueous solubility, as compared to the hydrophobic Span^® surfactants). As a result, in an aqueous media, the Tweens^® did not properly form the niosomal vesicular structures. However, since Span^® surfactants are lipophilic (low HLB), they formed the niosomal vesicles and entrapped higher concentrations of the drug, CLZ [49]. An increase in the Spans^® and Tweens^® concentrations resulted in lowering the entrapment efficiency, which was attributed to the decreased effects of cholesterol ratio in the preparative constituents of the niosomes, and played its role in stabilizing the prepared niosomal vesicles. According to previous reports, the quantity of lipophilic drug that is integrated into the niosomal bilayers would increase in entrapment with a lower HLB value surfactant [50]. Niosomal formulation, F10, containing Span 60 displayed maximum DEE (96.4 ± 2.6%), an impressive higher DEE than other formulations containing Span 40 and Span 20. Span 60 had a longer alkyl chain moiety in its structure as compared to the Span 40 and Span 20, which provided higher entrapment efficiency for the drug, which was also demonstrated earlier [51].

Figure 3

Entrapment efficiency (%) of the CLZ-loaded niosomes.

3.3 In vitro release of CLZ from niosomes

The in vitro release of CLZ from niosomal formulation was carried out by the diffusion method using a semi-permeable cellophane membrane (molecular cut-off range of ∼12,000). The results are illustrated in Figures 4 and 5. The highest drug, CLZ, release rate after 24 h (84.11 ± 4.06%) was observed in the case of F10 containing Span 60, with a ratio equal to (1:0.5) cholesterol/surfactant presence, while the slowest release was noticed from the niosomal formulation, F9 (27.58 ± 2.55%). The ongoing results indicated that the CLZ niosomal formulations had sustained release patterns, which were extended for a comparatively longer period. It was detected that in vitro CLZ release from the niosomal formulations containing different Spans^® (20, 40, and 80) was sharply increased, and it was higher than the formulations containing CLZ from different formulations containing Tweens^® (Tween 60 and 80). In addition, it was noted that the drug release rate slowed down (with an increase in size) when the surfactant’s contents were also higher. These results demonstrated the impact of Spans’ alkyl chain length on the niosomes’ stability and quantity of the CLZ released [52,53]. The stability and size quotients of the niosomes and the use of the surfactants, i.e., non-ionic and highly lipophilic, in nature provided comparatively higher drug entrapments as well as higher drug release [54]. The drug release from the niosomes prepared from Tween^® surfactants fell behind the Span^® surfactants-based niosome formulations.

Figure 4

Cumulative percent of CLZ quantities released from niosomes prepared from different types of Spans^®.

Figure 5

Cumulative percent of CLZ quantity released from niosomes containing different types of Tweens^®.

3.4 FT-IR spectroscopic analysis

FT-IR spectra of the ingredients of CLZ-loaded niosomes formulations were recorded and are illustrated in Figure 6. The characteristic peaks in the FT-IR spectrum of the CLZ, cholesterol, and Span^® 60 were comparable to those described in the literature, which indicated the purity of the samples suitable for niosomal formulations. The CH stretching at 2950 cm⁻¹, aromatic ether peak at 1,196 cm⁻¹, and NH peak of the quinolinone moiety showed between 3100 and 3400 cm⁻¹. These exhibited peaks were assigned for the drug, CLZ [53]. FT-IR spectrum of the physical mixture of components showed a nearly similar peak pattern (spectrum D). The FT-IR spectra of CLZ niosomal formulations (F10) (spectrum E) showed major absorption peaks with nearly similar peak patterns, thereby indicating the niosomal preparation (Figure 6).

Figure 6

FT-IR spectra of (A) CLZ, (B) cholesterol, (C) Span 60, (D) physical mixture of the ingredients, and (E) CLZ-loaded niosomal formulation (F10).

3.5 DSC scanning

DSC thermograms of CLZ, cholesterol, Span 60, nonloaded drug niosomes, and CLZ-loaded niosomal formulation (F10) are presented in Figure 7. The thermogram of CLZ (Scan A) revealed a clear endothermic peak at 159–161°C, which is the melting point of the drug. The thermal transitions of the cholesterol monohydrate are known to be below 100°C (Scan B). The conversion of cholesterol monohydrate to anhydrous cholesterol is known as cholesterol transition, which happens at 70°C. The endothermic peak (Scan C) for Span 60 was seen at 35°C, which matches the transition temperature for Span-60 hydrate [55]. The DSC thermograms of drug-free niosomal formula (Scan D) indicated a shift in the transition endotherm of Span 60–86°C. The melting endotherm of the cholesterol also appeared to have diminished in intensity and shifted on the thermogram. On the other hand, the melting endotherm of CLZ was shifted to 98°C on the thermogram of the niosomal formulation (Scan E). In addition, it demonstrated a reduced intensity of the cholesterol endotherm, showing that the medication was distributed uniformly in the niosomal formulation [56].

Figure 7

DSC thermograms of (A) CLZ, (B) cholesterol, (C) Span 60, (D) nonloaded drug niosomes, and (E) CLZ-loaded niosomal formulation (F10).

3.6 Scanning electron microscopy

SEM image of the selected niosomal formulation loaded with CLZ (F10) showed a nearly spherical shape. The prepared niosomes also indicated a closed vesicular structure with smooth surfaces and no aggregation. The SEM image demonstrated differently sized CLZ-loaded niosomal vesicles, which were produced using the ether injection method (Figure 8).

Figure 8

SEM of the (a) empty niosomes and (b) CLZ-loaded niosomes, formulation (F10).

3.7 Physical stability of the selected niosomal formulation (F10)

Physical stability studies of the selected CLZ-loaded niosomes formulation were conducted at two different temperatures, 4°C and 25°C. In the refrigerator (4°C), the stored CLZ-loaded niosomes (formulation, F10) prepared with Span 60 surfactant (at a ratio of 0.5:1, surfactant: cholesterol) containing 10 mg CLZ were stable during the storage period (6 months). During this storage time, the drug was not leaked from the stored niosomes. The color of the niosomal dispersion also did not change, and the vesicles particle sizes were also not observed to be changed noticeably. At room temperature (25°C), the stored niosomes preparation (F10) was not stable during the time (6 months). The instability was caused due to drug leaking from the stored CLZ-loaded niosomes, and the niosomal suspension’s color was also noticed to have changed with the size increment of the vesicles. The hydrated bilayer niosomal vesicles are thermodynamically unstable and present a metastable condition [57]. The niosomal vesicular systems kept in aqueous dispersion tend to clump and fuse, and as a result of these changes, the encapsulated drug leaks during storage. The cholesterol’s capacity to stabilize membranes may explain the CLZ niosomal formula (F10) stability, whereas the breakdown and/or clumping of the prepared niosomal vesicles can be related to the loss of niosomal vesicles’ density in the suspension media. A minimal loss of CLZ was seen during the cold storage (4°C), which may have happened because of the rigidification of the niosomal vesicles at low temperature, which decreased the permeability of the drug, CLZ, across the membrane [58]. Accordingly, the stability study results indicated that the prepared niosomal vesicular systems were stable at 4°C as compared to the storage at 25°C in terms of mean vesicle size, the entrapped concentration of the drug, and percent cumulative drug released after 24 h (Table 3).

3.8 Preparation of different CLZ gel formulations

Three types of gels were prepared, namely, Carbopol-934, 1% gel (CLZ-G1-F10), Pluronic F-127, 30% gel (CLZ-G2-F10), and HPMC-based 3% gel (CLZ-G3-F10). The results of the drug content assays confirmed nonsignificant variations between the theoretical and actual CLZ contents in the prepared niosomal gels (94.16–97.54%). The gel formulations were kept in brown-colored glass vials (120 mL) in the refrigerator until further investigation. The viscosities of the prepared CLZ gel formulations ranged between 7945.34 and 5097.53 cps (Table 4), which was considered ideal since they offered excellent in vitro drug release from the gel.

3.9 In vitro CLZ release from gel bases containing CLZ-loaded niosomal formula through cellophane membranes

The in vitro CLZ release profile obtained from various niosomal gel formulations is shown in Figure 9. It was observed that only about 54.44 to 70.90% of the encapsulated CLZ was released during 12 h from different niosomal gel formulations. The in vitro release of CLZ was significantly impacted by the changes in the gelling agent/gelling points. As a result, the increased viscosity of CLZ-G1-F10, as compared to other gel formulations, showed in vitro CLZ release slower for the Carbopol 934 gel. In contrast, the niosomal HPMC gel formulation (CLZ-G3-F10) exhibited the highest drug release (70.90 ± 2.22%) as compared with the other two gel formulations. The highest drug release from HPMC gel may be linked to lower viscosity of CLZ-G3-F10 [59]. The quantity of CLZ released from different gel formulations was, for CLZ-G3-F10 > CLZ-G2-F10 > CLZ-G1-F10. Accordingly, the transdermal gel formulation CLZ-G1-F10 was selected for further skin permeation study in comparison to other gel formulations containing pure CLZ with similar levels of the gelling agent.

Figure 9

Percent cumulative CLZ quantity released through cellophane membrane from different gel formulations.

3.10 Kinetic studies of in vitro drug release

To validate the model that described the CLZ release, an in vitro release of CLZ from various gel bases loaded with the drug niosomal formula (F10) was conducted using zero-order, first-order, and Higuchi diffusion models, together with the Korsmeyer-Peppas equation. The correlation coefficient (R2) value was used to determine the suitable release model. The release profiles of CLZ from all studied gel formulations in the consecutive release profile corresponded best to the Higuchi-diffusion model (Table 5).

3.11 Ex vivo skin permeation studies

Figure 10 represented the ex vivo skin permeation of the drug, CLZ, from the CLZ niosomal gel-based formulation, (CLZ-G1-F10), as compared to the plain CLZ gel (CLZ-G0). The percentage quantity of the CLZ permeated after 12 h from the CLZ-G0 was found to be at 60.47 ± 3.44%, which was smaller than the percentage of CLZ that seeped from the CLZ niosomal gel (CLZ-G1-F10) (71.34 ± 3.12%). The Transdermal flux (J) values for the CLZ-G1-F10 and CLZ-G0 were found to be at 27.107 and 26.318 μg/cm² h⁻¹, respectively. The obtained data revealed the potential of CLZ niosomal gel (CLZ-G1-F10) to remain at the targeted site for a considerable duration of time, which may increase the patients’ compliance [60].

Figure 10

Percent cumulative CLZ released through abdominal rat skin after 12 h of duration from CLZ-G0 and CLZ-G1-F10.

The kinetics of the CLZ permeated from the niosomal gel-based formulation (CLZ-G1-F10) across the skin showed a Higuchi diffusion release profile by which it can be inferred that the CLZ niosomal gel combined with hydrophilic Carbopol-934 gel shall release adequate quantities of the drug [61].

3.12 In vivo pharmacokinetics parameters study

Figure 11 illustrated the CLZ plasma concentration profiles versus the time after topical application of the CLZ-G0 and CLZ-G1-F10 gel formulations. Table 6 lists the corresponding pharmacokinetic parameters (C _max, T _max, AUC_0–12, AUC_0–∞, CL_T, and t _1/2) of CLZ from these formulations. The peak plasma CLZ concentration (C _max), after applying the gel base containing CLZ-loaded niosomal formulation, CLZ-G1-F10 (3850.92 ± 207.50 ng/mL), was significantly (p < 0.005) higher than that obtained after the topical administration of the CLZ-G0 preparation (1250.46 ± 99.56 ng/mL). The investigated pure CLZ gel showed a shorter T _max (3 h) than that obtained for CLZ niosomal gel formulation (4 h). The stratum corneum layer of the rat skin’s barrier capacity and the continuous release of CLZ from the prepared niosomal transdermal gel formulations were the reasons for the variations in the T _max values. The CLZ was released instantly from the pure drug gel formulation [62]. Moreover, the AUC_0–12 and AUC_0–end values of CLZ-G1-F10 formulation were 28992.16 ng h/mL and 35508.72 ng h/mL, respectively, which, when compared with the same parameters, were observed for CLZ-G0 to be at 8488.825 and 5312.3 ng h/mL, respectively, and these values were substantially different (p < 0.005).

Figure 11

Mean plasma concentration-time profiles of CLZ after topical administration of CLZ-G0 and CLZ-G1-F10 gel formulations.

These results confirmed that the enhanced CLZ bioavailability could be obtained from topically routed niosomal gel formulations, as observed throughout the evaluation period (12 h). The considerably enhanced CLZ bioavailability through transdermal niosomal gel formulations may be a result of the hepatic enzymes’ avoidance of CLZ metabolism. The elimination half-lives (t _1/2) of CLZ-G1-F10 (4.0306 h) were significantly higher than that of the pure CLZ gel (CLZ-G0) (3.171 h). This occurred as a result of the niosomal gel formulations’ longer-lasting CLZ release profile.

3.13 Coagulation profile measurements

The effects of CLZ on bleeding time (BT), and on ex vivo APTT and PT anti-platelet effects could be accompanied by adverse effects, such as, prolonged BT, and impaired blood coagulation. First, the effects of gel base containing pure CLZ (CLZ-G0) formulation, and niosomal CLZ formula (CLZ-G1-F10), on bleeding and CT, were determined under in vivo conditions, using male Wister rats.

A significant increase in bleeding and CTs was observed at all the tested time durations and compared with the control group. The observations are presented in Table 7. Pure CLZ gel (CLZ-G0) and the niosomal CLZ gel (CLZ-G1-F10) showed no effects on the PT and on the APTT, as they did not increase the risk of platelet inhibitor-induced bleeding effects in the experiments.