Colored Polymeric Nanofiber Loaded with Minoxidil Sulphate as Beauty Coverage and Restoring Hair Loss

Polymeric nanofibers fabricated by electrospinning either blank (PVA) or loaded with minoxidil sulphate have yielded optimum fibers with an average diameter 273 nm, and 511 nm, respectively. Thermal analysis of nanofibers indicated no chemical interaction. The NMR spectrum confirmed stability of nanofiber as there were no interactions between functional groups. Prepared nanofibers showed a 47.4% encapsulation efficiency and 73% yield. In vitro drug release of minoxidil sulphate from nanofiber exhibited an initial burst release followed by a slower release pattern. Stability studies revealed that minoxidil nanofiber was stable if stored at room temperature and protected from light with only loss of 9.6% of its nominal concentration within 6 months. As a result, the prepared solid/colored formula serves as an ideal formulation for such instable drug in liquid formula taking the advantage of the attractiveness of beauty colored coverage, and the simple, and non-tousled application.

Nanofibers specifications. The chemical structure of minoxidil sulfate and the NMR spectra of pure minoxidil sulphate, PVA polymer and electrospun nanofibers both blank (PVA nanofibers) and loaded with minoxidil sulphate are shown in Fig. 2(A-F, respectively). NMR spectroscopy was used to confirm the encapsulation of the drug by PVA nanofiber. 1 H and 13 C NMR spectra of pure minoxidil sulphate Fig. 2(B,C) and PVA nanofiber Fig. 2(D,E) were obtained and compared with the minoxidil-loaded PVA nanofiber. The following 1 H NMR peaks of minoxidil sulphate were observed in the loaded 1 13 C NMR spectrum appeared in the loaded nanofiber spectrum. All of these findings confirmed that minoxidil sulphate was encapsulated successfully by PVA nanofiber.
DSC thermograms of PVA-minoxidil sulphate are depicted in Table 1. The loaded nanofibers prepared by electrospinning. DSC provides information about decomposition and changes in heat capacity, melting, or crystallization of the drug. Distinguished peaks were observed at 100.63 °C, as a characteristic peak of PVA, and minoxidil sulphate showed characteristic peak observed at 169.63 °C.
The DSC of loaded nanofiber depicted the presence of minoxidil sulphate in the nanofibers; indicating stable nanofibers with no interaction between the drug and the PVA. Furthermore, the peaks have also shown not only a stable polymer, but also confirmed the stability of the drug in the nanofibers. On the other hand, the minor decrease in the melting point of the drug in the electrospun nanofibers may be ascribed to the amorphous state of the drug. The distinguished peak of the drug and polymer further indicate that there is no chemical interaction between the drug and polymer.
HpLc analysis of minoxidil sulphate. Selectivity is the ability of the method to distinguish and quantify the analyte in the presence of endogenous interferences. A representative chromatogram of spiked minoxidil sulphate 0.5 µg/ml is depicted in Fig. 3. The peak of minoxidil sulphate was well resolved, with retention times of approximately 1.62 min for minoxidil sulphate. The total chromatographic run time was 5.0 min.
The calibration curves were all linear with a coefficient of determination (r 2 ) > 0.99 over the concentration range of 0.25-0.75 µg/ml on all tested concentrations. The calibration curve was Y = 6864.9 × −753.84 with correlation coefficient r = 0.9996, where X represents the concentration of minoxidil sulphate and Y is the area under the peak area ratio of the drug. The results implied that the method developed was linear over the specified range. Figure 4 is showing the representative chromatograms of pure sample (minoxidil sulphate (A)), sample spiked with 0.5 µg/ml minoxidil sulphate (B), while the chromatogram for formulated minoxidil sulphate nanofiber is shown in Fig. 4(C). The peak of minoxidil sulphate was well resolved, with retention times of nearly 3.28 ± 0.14 min for the drug. The total chromatographic run time was 5 min. Compared to spiked released samples, Fig. 4(A,B) showed no significant peaks, at the retention times of the drug; which proves the assay specificity. It is well observed also that the representative chromatogram of real formulation sample showed similar chromatographic behavior to quality control samples.
Drug entrapment efficiency. The %EE of the minoxidil sulphate in the nanofiber showed minimum EE for loaded minoxidil sulphate of 47.4% which is considered suitable for delivering a therapeutically active dose. However, the yield of minoxidil sulphate-nanofiber was 73%.
In vitro release study. The in vitro release study of minoxidil sulphate was conducted in phosphate buffer 14 due to its ability to maintain sink conditions. The drug release pattern for 24 h is shown in Fig. 5. The profile indicated biphasic release of minoxidil sulphate from the nanofiber. The release pattern indicated that in the initial phase (5 h, r > 0.9), there was a rapid release of about 48% according to the formulation followed a slow phase from 7 to 24 h where about 20% was released. From the results obtained, it was found that there no lag time which indicates a burst in drug release during the first 15 min (about 4-10%) of the adsorbed drug on the nanofiber.
Stability study of the prepared formulations. Minoxidil sulphate was stable in the processed samples held in the autosampler at 25 °C for 24 h with mean calculated values within 3.2% of the nominal concentration (Fig. 6). However, the samples lost only 9.6% (RSD of 4.2%) of its nominal concentration within 6 months www.nature.com/scientificreports www.nature.com/scientificreports/ conclusion Based on several previous studies, the applicability and efficacy of minoxidil sulphate in restoring hair growth in both male and female who suffer from alopecia is well established and proved. However, the available formulation in-use are all in liquid unstable formula, and vastly expensive. Knowing the advantage of nanofibers prepared  www.nature.com/scientificreports www.nature.com/scientificreports/ by electrospinning and raw materials polyvinylpyrrolidone (PVP) and minoxidil are inexpensive, and solid formula generally exhibit high stability. It is believed that this work will be valuable to produce solid nanoformula of easy use and inexpensive. The resultant minoxidil sulphate nanofibers were successfully prepared with water  www.nature.com/scientificreports www.nature.com/scientificreports/ soluble and a biocompatible polymer, polyvinyl alcohol with desirable characteristics. The fabricated nanofibers revealed an acceptable average diameter for both unloaded and loaded fibers. Additionally, the physicochemical compatibility studies of nanofibers specified compatibility between minoxidil and used polymer and the prepared nanofibers were stable at room temperature. Therefore, the designed PVA-minoxidil sulphate loaded nanofiber serves as a potential stable solid nanoformula to be used for hair restoring as previously prov and it's shading color as beauty coverage upon application.
preparation of polymer solutions. The polymer solutions were prepared using a modified version of a previously reported research work in 2016 by Sharma et al. 15 . In brief, PVA solutions (aqueous) of 5%, was made by dissolving of PVA in 100 ml of deionized water, with stirring for 3 h using magnetic stirrer (PTFE, SterliTech Co., North Chesterfield, VA, USA). Once PVA clear solutions achieved the result mixture of polymer was named blank nanofiber. The solution was then subjected to electrospinning (NaBond Technologies Co., Ltd., ShenZhen, Guangdong, China) 2,3 . Minoxidil sulphate (5%) was prepared by dissolving the calculated amount in ethanol that was then added dropwise and mixed by stirring with the above polymeric solution and the result formula was named loaded nanofiber.

Preparation of nanofiber.
Two different subsets of solutions were prepared and subjected to electrospinning, (a) 5% PVA, and (b) 5% PVA to be loaded with 5% minoxidil sulphate as reported 15 . The solutions were put into a 5-ml syringe fitted to a needle with a tip diameter of 22 gauges, and the syringe was then placed in the electrospinning apparatus. The polymeric solution at flow rate of 25 µl/min (voltage of 15 kV) was delivered using a syringe pump. The collector was covered with an aluminum foil of (23 cm * 24 cm) on which the nanofibers were collected. After collection, the yielded nanofibers were dried overnight at room temperature.
Coloring the nanofiber. The food color used does not contain any nuts products and was choose as it is safe and approved for human use. The color agent was mixed with polymeric solution and also added to the final solid fibers.
Electrospun nanofibers characterization. Morphological evaluation. The size and shape of the electrospun fibers were determined by scanning electron microscopy (SEM) JSM-5510 (Jeol Ltd., Tokyo, Japan) equipped with a digital camera, at 15 kV accelerating voltage. Fibers were examined in different positions to estimate the electrospun fibers diameters 15 . A segment of the fibers was positioned on the sample holder, then sputter-coated with platinum palladium (Au/Pd) using a vacuum evaporator (Edwards) 15,16 .  www.nature.com/scientificreports www.nature.com/scientificreports/ Physicochemical compatibility studies of nanofibers. 1 H and 13 C and NMR spectra were recorded in deuterated dimethyl sulfoxide (DMSO-d6) which was obtained from Cambridge Isotope Laboratories on a Bruker 700 MHz NMR spectrometer (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA) using tetramethylsilane as an internal standard. Bruker Topspin software (Bruker BioSpin Corporation, Billerica, MA, USA) was used to analyze the NMR spectra generated 17 .
Differential scanning calorimetry of minoxidil-loaded nanofibers. Minoxidil-loaded polymeric nanofibers were thermally analyzed using differential scanning calorimetry (DSC). The DSC was performed using a Perkin Elmer Diamond hyper differential scanning calorimetry (Perkin Elmer, USA). The samples were loaded in the DSC pan and sealed, then heated from 25 to 350 °C at a rate of 10 °C/min under the flow of nitrogen gas. Melting temperature was determined from the heating curve 15,18 . High performance liquid chromatography (HPLC) assay for minoxidil sulphate. A modified HPLC method for minoxidil published by Rudrapal et al. 19 was utilized in this study. Minoxidil sulphate concentration was quantified using a Waters HPLC system. The system consists of a Waters 2707 autosampler delivery system (Waters Inc., Bedford, MA, USA), and a symmetry C 18 column (4.6 ×1.0 cm) packed with 5-µm spherical particles (Waters Inc., Bedford, MA, USA). The mobile phase contains of methanol:phosphate buffer pH 3.0 (60:40, v/v) at a flow rate of 1.0 ml/min. The mobile phase was prepared daily during the study, filtered through a 0.22-µm Millipore filter and degassed under vacuum. The volume injection was 10 µl, and the detection was performed at 250 nm at a run time of 4 min, and the HPLC system was operated at 40 °C. Data were analysed using an Empower Pro chromatography manager data collection system (Waters Corporation, Milford, MA, USA).
Standard and sample solutions. Stock solutions containing 250 mg of drug in mobile phase were stored in 4.0 ml glass vials at −20 °C. Standard calibration curve (n = 3) ranging from 0.25, 0.357, 0.5, 0.635, and 0.75 µg/ml was prepared on a daily basis to estimate the unknown minoxidil sulphate concentration for the determination of drug entrapment efficiency and drug release. Standards were transferred to glass autosampler vials with pre-slit septum, where 10 µl was injected into the HPLC system for analysis.
Drug entrapment efficiency and polymeric nanofiber yield determination. Minoxidil sulphate drug content in the nanofibers were precisely determined. Weighed nanofiber (10 mg) dissolved in mixture of methanol:water (1:20 v/v). Then, the solution was stirred for 60 min to assure complete dissolution of the samples. An aliquot (1.25 ml) of the stock solution was transferred into a glass autosampler vial with pre-slit septum and injected (30 µl) into the HPLC instruments using a developed validated HPLC method 20 . The minoxidil sulphate concentration was calculated using the following equations: In vitro drug release study. The in vitro release test was performed using US Pharmacopeia XXXII dissolution apparatus 1 (basket). The dissolution was performed in 900 ml of phosphate buffer pH 7.2 ± 0.1 at 75 rpm and the temperature was maintained at 37 ± 0.5 °C. A sample of Minoxidil beads equivalent to 10 mg of Minoxidil was used. At appropriate time intervals (0.5, 1, 3, 5, 7 and 24 h), 2.0 ml samples were withdrawn from each vessel, mixed with 5.0 ml of methanol: water (75:25) 14,[20][21][22] . The solution was filtered through a 0.22 µm Millipore membrane filter and analyzed using HPLC assay 20 . The volume was replaced each time with 2 ml of fresh medium and was kept at 37 ± 0.5 °C to maintain a sink condition 21 .
Stability study of released minoxidil sulphate from the formulation. Stability studies (freeze-thaw) of the quality control samples were assessed by exposing samples to different cycles, namely: three freeze (−20 °C) and thaw (room temperature) after 0, 72 h, 1 week, 2 weeks, 4 weeks, 3 months as well as after 6 months of preparation (nanofibers), which were stored at −20 °C. The processed samples were kept in vials sealed with parafilm at 25 °C. Similarly, minoxidil sulphate was exposed to a drastic condition, in an amber volumetric glass, by diluting it in water, 1 M solution of NaOH and 2 M HCl solution (n = 6). The area under the peak (AUP) of minoxidil sulphate was measured as the zero AUP. Each solution was carefully heated to boiling and left to cool down and minoxidil sulphate AUP after boiling was measured 15,19 .