Development of Dermal Films Containing Miconazole Nitrate

This study aims to develop new antifungal dermal films based on their mechanical properties (elongation, adhesion, behaviour towards vapour moisture) and the in vitro availability of miconazole nitrate, used as a pharmaceutical active ingredient in various concentrations. The three polymeric films prepared were translucent or shiny, with the surface of 63.585 cm2, 0.20–0.30 mm thickness, and content of miconazole nitrate of 3.931 or 15.726 mg·cm2. The mechanical resistance and elongation tests demonstrated that the two films based on hydroxyethyl cellulose (HEC) polymer were more elastic than the one prepared with hydroxypropyl methylcellulose (HPMC). The vapour water absorption and vapour water loss capacity of the films revealed that the HPMC film did not dry very well in the process of preparation by the evaporation of the solvent technique, unlike the HEC films that jellified more evenly in water and had higher drying capacity at 40 °C. The in vitro availability of miconazole nitrate from dermal films was evaluated using the Franz diffusion cell method, through a synthetic membrane (Ø 25 mm × 0.45 µm) and acceptor media with pH 7.4 (phosphate buffer and sodium lauryl sulphate 0.045%), resulting a release rate of up to 70%.


Introduction
The development of pharmaceutical forms begins with the preformulation process, which establishes the physical and chemical, pharmaceutical and technological, pharmacological and toxicological features of substances [1]. As an active pharmaceutical ingredient, miconazole nitrate (MN) is a broad spectrum antifungal agent which has already been studied in the form of mucoadhesive tablets, polymeric matrices (chitosan, gelatine, gum arabic, alginate, carbopol or acrylic resins), microgels, nano-lipid gels or nano-vesicles formulated to treat oral or vaginal candidiasis [2][3][4][5][6][7]. In addition to its antifungal activity, MN also has an antimicrobial action-the imidazole class, which makes it frequently applied on skin mucosa to heal fungal infections [8]. It inhibits the synthesis of ergosterol, a key component of fungal cell membranes [9]. For external use, MN is characterized by very good action as it easily penetrates the skin, and because of its efficient concentration the therapeutic effects last more than four days from the time of application [10,11]. Therefore, the development of MN-dermal films can bring various benefits to patients resistant to classical antifungal pharmaceutical forms because this active substance lasts longer in the skin layers, acting as a slow release product. Repeated use multiplies the dose in the corneous layer, leading to the disappearance of the mycosis infection. Initially, it can be started with a smaller dose and afterwards tripled depending of the

Tensile Strength and Elongation Capacity
The mechanical strength of dermal films (FI-FIII) was evaluated by testing their elongation capacity (E) under the action of descending traction forces, of increased intensity ( Figure 1). The tension that led to the rupture of the film (TS break ) had the smallest value for the HPMC-based film (FI-0.0083 N·mm −2 ) and the highest for the film prepared with 2% HEC (FIII-0.2906 N·mm −2 ). At a concentration of 3% HEC, the film resistance to breaking decreased by approximately 4× (FII-0.0673 N·mm −2 ). Such a behaviour indicates that the plasticizer (polyethylene glycol 400 (PEG 400 )) in concentration of 1% is insufficient to elasticize the structure of the polymeric matrix; this is also suggested by the maximum values of elasticity (E max -elongation recorded due to the tension applied before the film breaking), where the size relation was maintained (FII 63% and FIII 180%).

Tensile Strength and Elongation Capacity
The mechanical strength of dermal films (FI-FIII) was evaluated by testing their elongation capacity (E) under the action of descending traction forces, of increased intensity ( Figure 1). The tension that led to the rupture of the film (TSbreak) had the smallest value for the HPMC-based film (FI-0.0083 N·mm −2 ) and the highest for the film prepared with 2% HEC (FIII-0.2906 N·mm −2 ). At a concentration of 3% HEC, the film resistance to breaking decreased by approximately 4× (FII-0.0673 N·mm −2 ). Such a behaviour indicates that the plasticizer (polyethylene glycol 400 (PEG400)) in concentration of 1% is insufficient to elasticize the structure of the polymeric matrix; this is also suggested by the maximum values of elasticity (Emax-elongation recorded due to the tension applied before the film breaking), where the size relation was maintained (FII 63% and FIII 180%). The value of Emax determined in the case of the HPCM film ( Figure 1) does not in fact indicate the elongation capacity, but rather the plasticity level correlated to the low cohesion in the film mass, its elongation being determined almost exclusively by the value of the applied tension (Pearson r-  The value of E max determined in the case of the HPCM film ( Figure 1) does not in fact indicate the elongation capacity, but rather the plasticity level correlated to the low cohesion in the film mass, its elongation being determined almost exclusively by the value of the applied tension (Pearson r-0.9973), the film breaking at a tension up to 8-35× times (respectively) smaller compared to the films with HEC (FII, FIII). In these latter cases, HEC elasticizes the film almost to the same extent as it did when having a 2% concentration (Pearson r FIII-0.9005) or 3% (Pearson r FII-0.8995). Considering the mechanical strength and elongation, the most appropriate HEC concentration used at films preparation was that of 2%.
The results of FI were not as good as those of FII and FIII, fact that can be attributed to HPMC matrix which retained too much water, with a negative influence on elongation and mechanical strength. Although FI also contains other excipients for improving elongation and strength features, the HEC polymer matrix was demonstrated to be more elastic.

Adhesive Capacity and Behaviour towards Vapour Moisture
The adhesive capacity of the films was determined and expressed by the value of the vertical ascendant force (S t ), which caused a smooth and rigid surface to detach from the film surface. The values recorded at environmental temperature and humidity ( Figure 2) indicated that FIII (S t = 1.93 N·mm −2 ) had twice the adhesive capacity of FII, which suggests that 1% PEG 400 was more suitable for HEC 2% concentration than 3% concentration.
when having a 2% concentration (Pearson r FIII-0.9005) or 3% (Pearson r FII-0.8995). Considering the mechanical strength and elongation, the most appropriate HEC concentration used at films preparation was that of 2%.
The results of FI were not as good as those of FII and FIII, fact that can be attributed to HPMC matrix which retained too much water, with a negative influence on elongation and mechanical strength. Although FI also contains other excipients for improving elongation and strength features, the HEC polymer matrix was demonstrated to be more elastic.

Adhesive Capacity and Behaviour towards Vapour Moisture
The adhesive capacity of the films was determined and expressed by the value of the vertical ascendant force (St), which caused a smooth and rigid surface to detach from the film surface. The values recorded at environmental temperature and humidity ( Figure 2) indicated that FIII (St = 1.93 N·mm −2 ) had twice the adhesive capacity of FII, which suggests that 1% PEG400 was more suitable for HEC 2% concentration than 3% concentration.
The water vapours absorption (Aw) caused by the very humid atmosphere (80% RH) was favoured by the use in the formulation of 2% HEC (Aw FIII-2.67%), compared to 3% HEC (Aw FII-0.83%). In the case of the film prepared with 1% HPMC (FIII) both the water vapours absorption capacity test (Aw of-1.56%), and the amount of water lost as vapours by desiccation (Lw of 19.46%) suggests this polymer, given its property to withhold water without dehydrating the contact surface-which could be skin or mucosa, provided the film a certain humidity since it still had a high content of volatile constituents.
The volatile content was much reduced-by approximately 3%-for the films prepared with HEC (FII, FIII) which suggests the preparation method and the formulation were more suitable for HEC as film former. The water vapours permeability (Pw) of films ranged between 0.0012 and 0.0021 g·cm −2 ·h −1 , FII (with 2% HEC) manifesting an occlusive effect of double intensity with respect to the other two (0.0012 vs. 0.0021).  The water vapours absorption (A w ) caused by the very humid atmosphere (80% RH) was favoured by the use in the formulation of 2% HEC (A w FIII-2.67%), compared to 3% HEC (A w FII-0.83%). In the case of the film prepared with 1% HPMC (FIII) both the water vapours absorption capacity test (A w of-1.56%), and the amount of water lost as vapours by desiccation (L w of 19.46%) suggests this polymer, given its property to withhold water without dehydrating the contact surface-which could be skin or mucosa, provided the film a certain humidity since it still had a high content of volatile constituents.
The volatile content was much reduced-by approximately 3%-for the films prepared with HEC (FII, FIII) which suggests the preparation method and the formulation were more suitable for HEC as film former. The water vapours permeability (P w ) of films ranged between 0.0012 and 0.0021 g·cm −2 ·h −1 , FII (with 2% HEC) manifesting an occlusive effect of double intensity with respect to the other two (0.0012 vs. 0.0021).

Analysis of In Vitro Release Profiles
The ability of MN to be released from the polymeric matrix of the films was evaluated using the Franz cell diffusion method (Figure 3a). The HPMC film (FI) profile shows that after 3-4 h a plateau appeared that lasted up to 24 h; this behaviour could be attributed to the structural disintegration of the film under the moisture action. The amount of MN almost constantly released (approximately 30% of the film dose) seems more likely determined by the dissolution process-MN having a moderate solubility in water (26.3 µg·mL −1 in buffer at pH 7.4) but also positively influenced by the sodium lauryl sulphate included in the aqueous media used as acceptor compartment, rather than the diffusion of the substance among the macromolecules of the polymer that relaxes and swells [38] in the absorbed water. The ability of MN to be released from the polymeric matrix of the films was evaluated using the Franz cell diffusion method (Figure 3a). The HPMC film (FI) profile shows that after 3-4 h a plateau appeared that lasted up to 24 h; this behaviour could be attributed to the structural disintegration of the film under the moisture action. The amount of MN almost constantly released (approximately 30% of the film dose) seems more likely determined by the dissolution process-MN having a moderate solubility in water (26.3 µg·mL −1 in buffer at pH 7.4) but also positively influenced by the sodium lauryl sulphate included in the aqueous media used as acceptor compartment, rather than the diffusion of the substance among the macromolecules of the polymer that relaxes and swells [38] in the absorbed water. On the other hand, the two films prepared with HEC 3% (FII) and 2% (FIII), respectively, released MN with similar profiles: at 12 h 45.58-50.31% and at 24 h 63.56-73.31% of the 40 mg-the calculated dose of the film samples. FII film released MN at a lower rate than FIII, the releasing capacity expressed by the area under the curve (AUC) having quite close values after 24 h: FII-1.816 mg·h·mL −1 , while FIII-2.094 mg·h·mL −1 .
The comparative analysis of release profiles (mg·cm 2 vs. h) linearized by simple regression of the segments corresponding to the first 12 h (Figure 3b) shows that the deviation from zero is significant for both lines (p < 0.0001) while the deviation from linearity is not significant (FII-p = 0.6429, FIII-p = 0.5000), and there are no significant differences between the two curves, neither for the slopes (p = 0.37) nor for the intercepts as measure of elevations (p = 0.74). Thus, there was the possibility of calculating the average value of the slopes (pooled slope = 0.61), and also of the intercepts (pooled intercept = 0.5305). The R square coefficient indicates a good determination (of 96-98%), with the release/diffusion rate of 0.59-0.64 mg·cm −2 ·h −1 MN through the synthetic membrane.
Although a wide range of oral pharmaceutical drugs use as polymer HPCM with high molecular mass, in case of the studied miconazole nitrate films this excipient makes the release of the antifungal more difficult, probably due to MN's low solubility in water. The use of HEC as film forming polymer behaves better than HPMC in terms of the release/diffusion processes. Thus, a concentration of 2% HEC determines a film matrix which releases up to 70% miconazole nitrate, unlike the matrix film prepared with 3% HEC which releases only up to 60%. Practically, when the concentration level The comparative analysis of release profiles (mg·cm 2 vs. h) linearized by simple regression of the segments corresponding to the first 12 h (Figure 3b) shows that the deviation from zero is significant for both lines (p < 0.0001) while the deviation from linearity is not significant (FII-p = 0.6429, FIII-p = 0.5000), and there are no significant differences between the two curves, neither for the slopes (p = 0.37) nor for the intercepts as measure of elevations (p = 0.74). Thus, there was the possibility of calculating the average value of the slopes (pooled slope = 0.61), and also of the intercepts (pooled intercept = 0.5305). The R square coefficient indicates a good determination (of 96-98%), with the release/diffusion rate of 0.59-0.64 mg·cm −2 ·h −1 MN through the synthetic membrane.
Although a wide range of oral pharmaceutical drugs use as polymer HPCM with high molecular mass, in case of the studied miconazole nitrate films this excipient makes the release of the antifungal more difficult, probably due to MN's low solubility in water. The use of HEC as film forming polymer behaves better than HPMC in terms of the release/diffusion processes. Thus, a concentration of 2% HEC determines a film matrix which releases up to 70% miconazole nitrate, unlike the matrix film prepared with 3% HEC which releases only up to 60%. Practically, when the concentration level increased by 1% HEC, the amount of MN released decreased; this makes us conclude that HEC should not exceed 2% in the fluid casted phase for optimal release of MN from the HEC-based matrix films.
The jellification capacity of HEC-a non-toxic thickening agent-responded better to the requirements of a MN dermal or mucosal film, unlike HPMC which, although having the advantage of not dehydrating the skin or the mucosa, decreases the MN release processes significantly, probably due to its great capacity of absorbing and retaining the water.
Regarding the published data, Saudagar R.B. (2017) obtained about a 82-99% MN release (24 h) when studying film formulations prepared with Eudragit RS-PO/hydroxypropyl cellulose (HPC), using egg membrane for the in vitro study [39]. Ofokansi K.C. (2015) obtained values of in vitro permeation flux of about 5 µg·cm −2 ·h −1 MN permeated through rat skin with, using in formulations mixtures of film forming polymers Eudragit RS-100/hydroxypropyl methylcellulose (HPMC) [40]. However, the different experimental conditions used in these studies do not allow comparisons in terms of delivery efficiency.

Analysis of In Vitro Release Profiles
In order to determine the kinetics and mechanism of MN release, the release profiles of the two HEC films (FII-prepared with 2% and FIII-with 3% polymer) were fitted using the first order kinetic equation in four variants: the simple equation; the equation considering the latency time (T lag ); the equation considering the maximum fraction that can be released using the gradient created by the initial dose of MN in the film (F max ); and the equation that considers both T lag and F max parameters, respectively (Table 3). The analysis of the calculated data indicates for both studied films (FII and FIII) that the highest value of R sqr_adj (the adjusted coefficient of determination) was obtained by fitting the release profiles-% (m/m) vs. time (h) with the model no. 4-first order kinetic with T lag and F max , this proving to be the closest model to the studied time-dependant curves (of the four models used) [41]. The kinetic parameters k 1 , T lag and F max quantify the release differences and allow the comparisons of the kinetic release processes of MN from the two HEC films matrix, which differ only by the concentration of polymers used in the film preparation: 2% for FIII and 3% for FII. The results show that the additional use of 1% HEC in the film composition generates the modification of k 1 (the constant of the release process kinetic) by 0.06 −1 (as the difference between k 1 -FII and k 1 -FIII), accompanied by a tripling (from 11.57 to 35.99%) of the amount of MN that could be released from a film of 2.54 cm 2 × (0.30 − 0.23) mm.

Films Preparation Technique
MN was dissolved in alcohol by stirring (500 rpm) for 5 min. PEG 400 , distilled water and in the end the film forming polymer (HPMC or HEC) are successively added under constant stirring, continuing the stirring for another 25 min after dispersion preparation, avoiding evaporation. The air was then eliminated from the structured gel by maintaining the fluid phase for 25 min in the ultrasound bath. The resulting composition was then poured onto circular plates (diameter of 9.8 cm) and left to dry at 40 • C (24 h) [42]. The films obtained were used in the study after 48 h of preservation at 20 • C, protected from light and moisture.

Measurement of Physical Parameters
The appearance of the dermal films was visually analysed. The mass of each film was established by weighing. The thickness (Tick) was determined using a micrometre, by measuring it at five different points [43].

Mechanical Resistance Test
The film sample of 3.5 cm long (L i ) and 1.5 cm wide (W) was fixed at one of the ends to a fixed support, while at the other end were placed heavier weights-m (g) until the film broke (M f ), measuring each time the length of the film-L f (cm).
The film elongation-E was calculated as percentage of the initial length, using the Equation (1): The tensile strength-T s (N·mm −2 ) was calculated using the Equation (2): where S = W (mm)·Tick (mm) [43].

Stickiness (Adhesion) Test (S t )
The weight-m (g) which, with the use of a certain lever, generated a vertical ascendant force which lead to the separation of a smooth plate from the surface-S (cm 2 ) of the film sample, onto which it was previously pressed and attached, was determined for each film sample.
The calculation considered the Equation (3):

Water Vapour Absorption Test (A w )
The film sample weighing m i (g) was maintained in a sealed compartment, in the presence of KCL 2 saturated solution (80% relative humidity-RH), up to a constant weight-m f (g).
The calculation used the Equation (4):

Water Vapour Loss by Desiccation Test (L w )
The film sample weighing m i (g) was maintained in a sealed compartment, in the presence of CaCl 2 anhydrous (0% RH), up to a constant weight-m f (g). The calculation used the Equation (5): 3.2.6. Water Vapour Permeability Test (P w ) The film sample with the determined surface of s (cm 2 ) was attached so as to close a glass containing CaCl 2 anhydrous (0% RH), resulting an ensemble with the mass-m i (g), which afterwards was kept in a sealed compartment in the presence of KCl 2 saturated solution (80% RH), up to the constant mass-m f (g). The calculations considered the Equation (6): P w g·cm −2 ·h −1 = (m f − m i ) × 100/s cm 2 /t(h)

Determination of In Vitro Release Profiles
Franz cells of 14 mL capacity and 1.8 cm diameter were used. The diffusion membrane (Teknokroma Ø 25 mm × 0.45 µm) was prepared before each determination by maintaining it in a phosphate buffering solution pH 7.4.
The samples analysed consisted of disk shaped films with diameter of 1.8 cm (surface of 2.54 cm 2 ). The media consisted in a buffer solution pH 7.4 to which was added 0.045% sodium lauryl sulphate.
During the determination: the diffusion cells were maintained at 32 ± 0.5 • C, by heating in recirculated water, with oscillating (electromagnetic) stirring. The samples taken (5 mL) were replaced each time with fresh media, and the MN content was assessed by ultraviolet (UV)-spectrophotometric method, at the wave length of λ = 273 nm (Spectrometer HP 8451A UV-VIS, Hewlett Packard, Palo Alto, CA, USA).
The calculations considered Equation (7): where MN (mg)-mass of miconazole nitrate released at moment n (collection n), C pn -the concentration of miconazole nitrate expressed in mg/mL for sample collected at moment n, 14-the volume of Franz cell expressed in mL, 9-the volume expressed in mL of the acceptor media that remained in Franz cell after the collection of the 5 mL sample [44,45].