Nanoclay-Based Composite Films for Transdermal Drug Delivery: Development, Characterization, and in silico Modeling and Simulation

Purpose Halloysite nanotubes (HNTs) are a versatile and highly investigated clay mineral due to their natural availability, low cost, strong mechanical strength, biocompatibility, and binding properties. The present work explores its role for retarding and controlling the drug release from the composite polymer matrix material. Methods For this purpose, nanocomposite films comprising propranolol HCl and different concentrations of HNTs were formulated using the “solution casting method”. The menthol in a concentration of 1% w/v was used as a permeation enhancer, and its effect on release and permeation was also determined. Quality characteristics of the nanocomposite were determined, and in vitro release and permeation studies were performed using the Franz diffusion system. The data was analyzed using various mathematical models and permeation parameters. Optimized formulation was also subjected to skin irritation test, FTIR, DSC, and SEM study. Systemic absorption and disposition of propranolol HCl from the nanocomposites were predicted using the GastroPlus TCAT® model. Results The control in drug release rate was associated with the higher concentration of HNTs. F8 released 50% of propranolol within 8 hours (drug, HNTs ratio, 1:2). The optimized formulation (F6) with drug: HNTs (2:1), exhibited drug release 80% in 4 hours, with maximum flux of 145.812 µg/cm2hr. The optimized formulation was found to be a non-irritant for skin with a shelf life of 35.46 months (28–30 ℃). The in silico model predicted Cmax, Tmax, AUCt, and AUCinf as 32.113 ng/mL, 16.58 h, 942.34 ng/mL×h, and 1102.9 ng/mL×h, respectively. Conclusion The study demonstrated that HNTs could be effectively used as rate controlling agent in matrix type transdermal formulations.


Introduction
A pharmaceutical system that offers the passage of the drug through the skin to produce a systemic effect is termed a transdermal drug delivery system (TDDS). 1 It is one of the most promising drug conveying means due to its various advantages, such as offering continuous therapeutic effect from hours to days, avoiding frequent dosing, providing painless and easy administration, and ensuring minimal chances of toxic reactions. 2,3 These properties of TDDS provide improved patient compliance in long-term treatment especially for chronic pain and discontinuation of smoking. Therefore, a large number of TDDS are becoming commercially available day by day throughout the world. 4 Halloysite nanotubes (HNTs) are nanosized clay materials consisting of nanotubes. Each nanotube is further comprised of 10 to 15 rolls of double layers (600 to 900 nm) of alumina-silica having 50 nm and 15 nm inner and outer diameters, respectively. 6 Structurally, they consist of a 1:1 ratio of tetrahedral siloxane (Si-O-Si) and octahedral aluminum hydroxide (Al-OH) layers. [6][7][8] The presence of the siloxane layer on the outer surface is responsible for their negative charge, while the aluminum hydroxide layer on the inner surface is responsible for their positive charge. 6 They possess significant loading and adsorption capabilities. Hence, they could interact with different molecules and help to modify their physicochemical characteristics. Moreover, their tubular structure is beneficial for improving the rheology of certain polymers and enhancing their mechanical strength. 9,10 They are considered economical with abundant natural availability, non-toxic, biocompatible, and capability of binding with the drug particles. [6][7][8]11 These properties make HNTs an attractive carrier for the delivery of pharmaceutical drug substances. They have been utilized successfully as a drug carrier excipient in different pharmaceutical dosage forms. 9,12,13 A slow drug release rate of more than 100 h has been achieved using the material, and their drug loading efficiency has been observed between 10% and 30%. 9 They have been utilized as nanofillers in polymers such as polyvinyl alcohol (PVA) composite to modify their physicochemical characteristics. 9 Similarly, the polymer is low-cost, biocompatible, soft, and biodegradable. It acts as a reservoir for drug substances and releases the drug as it degrades. Therefore, HNT-based composites are becoming the material of choice as a drug carrier for the development of drug delivery systems. 14 Numerous studies have been conducted for the design and characterization of HNTs/PVA-based nanocomposites. 15 The modified polymeric material has been used to improve the release profile and bioavailability of several drug substances including antihistamine, anti-inflammatory, and antibiotics. 14,16 Propranolol hydrochloride (HCl) is a non-selective beta-blocker generally used in the management of hypertension, angina, cardiac arrhythmias, and pheochromocytoma. 17 It has a molecular weight of 259.35 g/mol, possesses a biological half-life of 2-6 h with an oral bioavailability of about 25%, and is used in an adult dose of 10-30 mg taken 3-4 times per day. [18][19][20] The properties of propranolol HCl, such as low molecular weight, low oral bioavailability, and short halflife, suggest that it is a potential candidate for the design of a transdermal delivery system.
In the present study, different nanocomposite films of HNT and PVA as a drug carriers were developed using a facile and convenient method for the controlled transdermal delivery of propranolol HCl (see Figure 1; diagrammatic illustration). Permeation enhancer such as menthol was incorporated in the formulations. The physicochemical characteristics were evaluated, including weight and thickness variation, folding endurance, moisture uptake, moisture content, and drug content. The release and permeation behavior of the drug from the system were determined using the Franz diffusion cell. Skin sensitivity, chemical interaction, morphology, thermal and mechanical properties were determined. In vivo characteristics were predicted using an in silico model, and stability was also determined at different temperatures.

Preparation of Transdermal Formulations
The formulations (containing HNTs or without HNTs) were prepared by utilizing the solvent casting method. 23 The composition of each formulation is shown in Table 1. A 2% w/v solution of propranolol HCl was prepared in deionized water. HNTs in different concentrations were incorporated in the propranolol solution with continuous agitation for 5 minutes. PVA (film former) along with or without menthol (permeation enhancer) in a fixed concentration was added in the drug and HNTs mixture, and it was agitated overnight at 250 rpm using a flat orbital shaker (KS 260 B, Industrial and Automotive Equipment, Germany). The mixture was later poured into a petri dish and dried at 60 °C for 6 hours in a hot air circulating oven (YCO-N01, Gemmy Industrial Corporation, Taiwan).

Evaluation of Formulations Organoleptic Evaluation
The formulations were examined visually as well with the sense of touch for smoothness, flexibility, and clarity. 24

Weight Variation and Thickness Evaluation
The prepared transdermal nanocomposite films (F1-F8) were subjected to uniformity of weight and thickness variation tests. For this purpose, the films, each of 1cm 2 , were weighed separately on an analytical balance (Sartorius CP 224 S, Gottingen, Germany), and the readings were then recorded for performing statistical analysis. The thickness of the formulations was also determined with the help of a digital vernier caliper (Seiko Brand, China). The thickness and weight variation of the formulations were determined statistically by calculating the mean and standard deviation. 24,25 Folding Endurance Test The folding endurance of the formulations was determined to measure the capability of a transdermal film system to resist rupture or maintain the physical integrity with general skin folding. 26 In this test, the nanocomposite films of each formulation were repeatedly folded manually at the same place, and the number of folds required to break the film was recorded. 25,26 Moisture Content Test The moisture content within the nanocomposite films helps in maintaining physical stability. The moisture content of formulations was determined by weighing each film and keeping it in a desiccator (containing silica gel as a desiccant) at normal room temperature. Their weight was measured after every 24 hours until a constant weight was attained. The percentage moisture content was calculated by using equation 1. 27 % Moisture content ¼ initial weight of the film À final weight of the film initial weight of the film x 100 (1)

Moisture Uptake Test
The capability of a polymeric nanocomposite film to uptake moisture content from the environment is considered to affect the release rate of the drug from the system. 28 The formulations were subjected to moisture uptake test by weighing each film initially and keeping it in a desiccator containing a saturated solution of sodium chloride. The weight of each film was measured after every 24 hours until a constant weight was achieved. Equation 2 was used to calculate the percentage moisture uptake. 29 % Moisture uptake ¼ final weight of the film À initial weight of the film initial weight of the film

Content Uniformity
All the nanocomposite formulations (F1-F8) were subjected to content uniformity test for determining the percent amount of drug in the formulation. The content of the drug in each formulation was determined by following the HPLC technique as described in British Pharmacopoeia (BP 2020). The chromatographic separation was performed by using High-Pressure Liquid Chromatograph (LC-20A Shimadzu, Kyoto, Japan). The mobile phase (100 mL) was prepared by mixing 1 mL aqueous sulfuric acid (10% v/v), 2 mL of tetra-butylammonium dihydrogen orthophosphate (1.7% w/v), acetonitrile (60% v/v), sodium dodecyl sulphate (0.115% w/v) and 37% v/v distilled water. The mobile phase was pumped (isocratic) at a rate of 1.8mL/min through a C18 column (20 × 4.6cm, Mediterranean Sea 5µm) maintained at an ambient temperature by a column oven (CTO-20 A, Shimadzu, Kyoto, Japan). The peak response of propranolol HCl was recorded at 289 nm using a UV detector (SPD-20A, Shimadzu, Kyoto, Japan). The standard solution was prepared in a 100 mL volumetric flask by dissolving 3.33 mg of propranolol HCl in phosphate buffer B.P. having a pH of 7.4. Similarly, the test solution was prepared by dissolving a 1cm 2 -sized film in the same buffer solution. The solution was agitated overnight on a flat orbital shaker at a speed of 250 rpm and sonicated using Elmasonic D-78224 (Elma, Germany) for 15 minutes.

In vitro Drug Release Study
The release rate of the drug from the nanocomposite films was determined by using Franz Diffusion Cell (V9-CB Manual diffusion system, PermeGear Incorporated, USA). A film of 1 cm 2 size was selected from each formulation. A fiber net of appropriate size carrying the transdermal formulation was placed between the donor and acceptor compartment. The acceptor compartment was loaded with 5 mL of phosphate buffer B.P., having a pH of 7.4. The temperature of the water jacket was held at 37 °C. 29 And, 0.5 mL aliquots of the drug samples were collected in triplicate at different time intervals (0, 5, 10, 15, 30 min, and 1, 2, 3, 4, 5, 6, and 8 h) and diluted separately with an appropriate volume of the phosphate buffer. The drug concentration in each sample was determined by using a double beam UV-Vis. Spectrophotometer (UV-1800, Shimadzu Corporation, Kyoto, Japan) at a wavelength of 289 nm. The drug release kinetics was determined by using different drug release kinetic models such as zero order, first order, Higuchi, and Korsmeyer-Peppas (Equation 3-6) using the Microsoft Excel add-in program DDSolver. 30 where Q 1 and Q 0 are the amounts of drug dissolved at a particular time t and the amount of the drug present at time zero in the dissolution media, and K 0 is the zero-order rate constant.
where Q 1 and Q 0 are the amounts of the drug in the dissolution media at time t and zero, and K 1 is the first-order rate constant.
where Q 1 is the dissolved amount of the drug in the dissolution media at time t, t 1/2 is the square root of time (t), and K H is the rate constant for the Higuchi model.
where a is a constant that depends on geometric and structural characteristics of the dosage form and n is the release exponent indicating the mechanism of drug release.

In vitro Permeation Study
The permeation test was conducted by Franz Diffusion Cell using dialysis tubing (500 g/mol cut-off molecular weight) as a barrier membrane. 31 Sample nanocomposite films of 1 cm 2 size were placed between the donor and acceptor compartment. The acceptor compartment was filled with 5 mL of phosphate buffer pH 7.4. The temperature of the water jacket was held at 37 °C. 32 And, 0.5mL aliquots of the drug samples were collected in triplicate at a different time intervals (0, 0.25, 0.5, 1, 2, 4, 6, 8, 10, and 12 h) and diluted with an appropriate volume of the same buffer. The absorbance of the drug in each sample was determined by a UV-Vis spectrophotometer (UV-1800, Shimadzu, Japan) at a wavelength of 289 nm, and the amount of the drug diffused through the barrier membrane at different time intervals was calculated. Permeation profiles were obtained by plotting a graph between the amount of drug permeated (µg/cm 2 ) versus time. Various permeation parameters such as flux, permeation coefficient, lag time, and diffusion coefficient were also determined using the method stated by Ubaidulla in 2007. 33 The flux (J) was calculated as the slope of the graph plotted between the linear portion of the cumulative amount of drug permeated and time. Whereas the lag time (L) was determined by extrapolating the line of the same graph to the abscissa. The partition coefficient (P) and the diffusion coefficient (D) were calculated by using equations 7 and 8. 34,35 where P is the partition coefficient, J is the flux and C d is the amount of drug in the donor compartment.
where D is the diffusion coefficient, h is the thickness of the membrane and L is the lag time.

Fourier Transform Infrared (FTIR) Spectroscopy
The compatibility of the drug with the excipients in the optimized formulation was determined by FTIR spectroscopy (Nicolet Avatar 330, ThermoFisher Scientific, USA) study. The raw FTIR data of the was used to construct a composite plot in MS Excel 365 for all the samples, whereas the images generated from the OMNIC software ver. 7.3 (ThermoFisher Scientific, USA) are presented in Figures S1 and S2. The obtained spectra were compared for any chemical incompatibility of the samples with the drug present in the composite transdermal film.

Differential Scanning Calorimetry (DSC)
The thermal behavior of propranolol HCl, PVA, HNT, PVA/HNT nanocomposite film and the optimized formulation were determined using DSC (DSC 250, TA Instruments, DE, USA) method to study the mechanical properties of the samples. The heating of the samples was carried out at a rate of 10 °C/min in a temperature range of 30 to 240 °C. The raw data of the thermogram were used to construct a composite plot using MS Excel 365 for all the samples, whereas the images generated from the TRIOS software (TA Instruments, New Castle, DE, USA) are presented in the Figures S1 and S2. The obtained thermograms were compared for any change in thermal and mechanical properties of the samples in the composite transdermal film.

Scanning Electron Microscopic (SEM) Analysis
The surface morphology of the prepared transdermal polymeric films with or without HNTs was analyzed by scanning electron microscope (SEM, JEOL JSM 6380-A, Tokyo, Japan) at an accelerating voltage of 20 kV. Sputter coating of the samples was carried out with gold up to 250°A using an automated sputter coater (JEOL FC-1500). The images obtained were compared for the difference in surface characteristics between the films.

Skin Irritation Test
The optimized nanocomposite transdermal film formulation (F6) was subjected to skin irritation test, using mice as subjects. Six mice were taken. The animals were kept at normal room temperature with free access to food and water. The National Research Council and the ARRIVE guidelines for the use and care of laboratory animals were properly followed. 21,36 A particular area of the skin was shaved using an electric hair clipper (KM-6330, Kemei, China) a day before the test. The optimized formulation was applied on three of the animals (marked as test) on the area with the help of CoTran 9697 Tape (3M, Saint Paul, USA) for 24 hours. It was then evaluated for any signs and symptoms of edema or erythema. In addition, an aqueous solution of 0.8% formalin was applied on the remaining three animals (marked as control). 37 The site of applications was rated based on the method described earlier by Vlasses et al 1985. 38 In

silico Modeling and Simulation
The systemic disposition of propranolol HCl from optimized transdermal nanocomposite film was predicted using the "Transdermal Compartmental Absorption and Transit" (TCAT®) model embedded in GastroPlus TM software version 9.8 (Simulations Plus Inc., Lancaster, CA, USA). Several formulations, physiological and pharmacokinetic parameters obtained from the literature, 17,18,[39][40][41] and ADMET TM predictor (in GastroPlus TM ) module were incorporated as shown in Table 2. The simulation was performed using 'transdermal: reservoir patches' as dosage form under human-fed conditions and the human arm was selected as the dosing application region. Diffusivity values in various layers of skin were used as predicted by the default in silico method present in GastroPlus®. The pharmacokinetic parametric values of propranolol HCl were calculated after processing and modeling (compartmental modeling) the reported in vivo pharmacokinetics data of extended-release (ER) propranolol HCl 80 mg tablets in the PKPlus TM module of GastroPlus TM . 42 The predicted plasma profiles obtained from single and multiple doses of propranolol HCl were cross-matched with the reported in vivo profile and various pharmacokinetic parameters of optimized formulation were compared.

Stability Study
The stability study of the optimized nanocomposite film formulation (F6) was conducted according to the method reported by Puri et al. 43 In the stability testing, the samples of the optimized formulation packed separately in an

Organoleptic Evaluation
The transdermal nanocomposite film formulations were physically examined to determine texture, flexibility and clarity. All the formulations were found to possess satisfying clarity, texture and brittleness as shown in Table 3. An inverse relation was found between the concentration of HNTs and the film's clarity and smoothness. The formulations without HNTs (F1 and F2) were found to have the greatest smoothness and clarity. The smoothness and clarity of formulations were observed to decrease with an increase in the proportion of HNTs. The formulations F3 and F4 were found to have

Weight Variation and Thickness Variation
The mean weight and thickness of the nanocomposite film formulations were calculated along with the standard deviation. The results shown in Table 4 indicate that both the weight and thickness of all formulations did not deviate vastly from their mean value. A low SD value and closeness of results among the films of the same formulation indicate uniformity. 44 Furthermore, it was also noticed that as the concentration of HTNs was increased in the formulations, the weight and thickness were also enhanced.

Folding Endurance
The folding endurance test determined the flexibility and physical stability of all the transdermal film formulations, as shown in Table 4. The value of folding endurance (>300-fold) indicates that all the formulations can withstand the skin's folding and retain its physical stability. 26

Moisture Content
The results of the moisture content are shown in Table 4. The results indicated the highest moisture content in formulations without HNTs (F1 and F2). Moisture content was found to decrease with an increment in HNTs amount (F3-F8). High moisture content in the polymer was supposed to be due to the hydrophilic nature of the compound. 27 The presence of SiO 2 and a small number of hydroxyl groups in HNTs make the clay hydrophobic, resulting in strong hydrogen bonding with polymer matrices and reducing the interaction of the composite with water molecules. 45

Moisture Uptake
The average percent moisture uptake by the nanocomposite film formulations is shown in Table 4. It was observed that moisture uptake capability was increased with a reduction of HNTs amount in the formulations. Low moisture content in the formulation is considered favorable as it reduces the chances of microbial contamination and enhances stability. 46

In vitro Drug Release Study
The in vitro drug release profiles of the nanocomposite film formulations are presented in Figure 2. The dissolution profile of formulations indicated an initial burst effect within 10 minutes. The burst effect of the drug from the formulations was supposed to occur due to the presence of adsorbed drug molecules on the outer surface of the HNTs. 47 The dissolution rate of the drug in formulations without HNTs (F1 and F2) was the highest (>80% drug was

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International Journal of Nanomedicine 2022:17 released in 30 min) among all. A rapid drug release from the formulations was observed due to the presence of hydrophilic polymer prepared without HNTs. 48 The formulations (F3 and F4) containing drug and HNTs in the same amount (1:1) showed about 60% drug release in 8 h (480 min). The nanocomposite films (F5 and F6) containing drug and HNTs in a ratio of 2:1 showed about 80% drug release in the first 4 h (240 min). About 50% of the drug was released from the transdermal films (F7 and F8) containing drug and HNTs in a ratio of 1:2 within 8 h (480 min). However, a desired controlled release profile of propranolol HCl was obtained by using drug and HNTs in a ratio of 2:1 (F6). In a study conducted by Levis and Deasy, a similar sustained released effect of propranolol HCl was observed from the halloysite nanotubes, and about 80% of the drug was released in 8 h. 49 These release profiles indicated that with an increment of HNTs ratio in the transdermal films, the drug release rate was retarded. Levis and Deasy in 2003 reported that the release rate of propranolol HCl was observed to decrease by loading into halloysite and increasing the concentration of halloysite in the formulation. A similar inverse relationship of HNTs' concentration with drug release was also reported by many researchers. 8,50 The decrease in drug release rate in the presence of HNTs is considered due to loading of drug into the material lumen or strong ion interaction between drug and HNTs as well as improvement in mechanical properties and enhancement in the thermal stability of the polymer composite. 8,15,51 The dissolution data of the formulations were subjected to different release kinetic models, including zero order, first order, Higuchi, and Korsmeyer-Peppas, and the results are presented in Table 5. Korsmeyer-Peppas model was best fitted in the dissolution data with an r 2 value of 0.9655 to 0.9835. The n values of the formulations were less than 0.5, representing Fickian diffusion as the drug release mechanism. 52 The same model was observed fitted by Abdouss et al, where HNT-methionine nanocomposite was formulated for the controlled delivery of phenytoin, and by Mohebali et al, where HNT/PVA nanocomposite was developed for the controlled delivery of minocycline. 47,53 The Korsmeyer-Peppas release constant (K KP ) value was highest in formulations without HNTs (F1 and F2). The K KP was found to decrease proportionally with an increase in the amount of HNTs. 54 The dissolution profile of optimized formulation (F6) after the Korsmeyer-Peppas model (r 2 = 0.979) was found to best fit in the first-order release model (r 2 = 0.979), indicating concentration-dependent drug release.

In vitro Permeation Study
The permeation profile of all the transdermal film formulations is presented in Figure 3 along with permeation parameters as shown in Table 6. The amount of permeation per unit area was found to be improved with the incorporation of HNT. However, the flux of the drug from the formulations was found to be inversely proportional to the amount of HNTs in the formulation. Furthermore, the rate of permeation was observed improved in the formulations (F2, F4, F6, F8) containing menthol with a flux of 56.819 µg/cm 2 hr, 103.225 µg/cm 2 hr, 145.812 µg/cm 2 hr, and 87.724 µg/cm 2 hr as compared to the formulations (F1, F3, F5, F7) without menthol with a flux of 33.021 µg/cm 2 hr, 85.763 µg/cm 2 hr, 128.080 µg/cm 2 hr and 78.484 µg/cm 2 hr respectively. It has been reported that menthol in a concentration of 1% w/v is a suitable permeation enhancer. It is believed that terpenes such as menthol form permeable boundary regions that enhance the drug permeation rate from the formulation. In one of the reported studies, Jeevan et al observed a flux of 122 µg/cm 2 hr. 55  The permeation rate of propranolol HCl was observed slowest from the film formulation (F1) without HNTs. The formulation F6 containing drug and HNTs in a ratio of 2:1 showed the highest amount (about 463 µg/cm 2 ) of propranolol HCl permeated per unit square within 12 hours. In a study by Krishna and Pandit, about 1248 µg/cm 2 propranolol was permeated in 24 hours from the optimized patch of size 2.5 cm 2 . 56 An increase in the amount of drug permeation with a reduction in the ratio of HNTs can be related to a faster dissolution rate of the drug. This results in a more significant amount of drug available at the site of permeation and that too in the presence of menthol. It has been revealed that the nanotubes also enhance the permeation rate of the drug molecules. This might be due to the nano-size structure of the HNTs, enhancing the penetration power of the drug molecules across the barrier membrane. 7,57 Similarly, the highest lag time was observed from the formulation (F1) without HNTs or the permeation enhancer, while the lag time was found reduced in the presence of the permeation enhancer or HNTs. Whereas, the lowest lag time was observed from the formulation F6, 0.336 h among the all. Similarly, a lag time of 0.50 h was observed by Jeevan et al. They reported a decrease in the lag time of propranolol in the presence of menthol. 55 Based on the above results, film formulation, F6, was selected as the optimized formulation. It was therefore further evaluated for skin irritation, chemical interaction, surface morphology and stability.

Drug Content
The content uniformity test results of all the transdermal formulations are given in Table 4. The results indicated that the mean amount of drug in all formulations was within a limit of 90-100%. The propranolol HCl content in the F6 was found to be 94.60% ± 1.74% (see Figure 4), meeting the official compendia limit (90.0-110.0%) for the drug content. 58

Fourier Transform Infrared (FTIR) Spectroscopy
The FTIR spectra of the film (containing the propranolol HCl, HNTs, PVA, and menthol) and pure propranolol HCl were recorded at 4000 to 400 cm −1 scanning range. The spectra results were then compared (see Figure 5 and Figure S1). The spectra of propranolol HCl showed naphthalene stretch at 796 cm −1 , O-CH bond stretch at 1240 cm −1 , C=C stretching at 1579 cm −1 , C-H bending at 2983 cm −1 and O-H bending vibrations at 3691 cm −1 . 59 The equivalent FTIR peaks in the formulated film were at 796 cm −1 , 1240 cm −1 , 1579 cm −1 , 2921 cm −1 and 3676 cm −1 . A slight change in bending vibration at 2983 cm −1 and 3691 cm −1 was observed. It might be due to the C-H bond of PVA and O-H group present at HNTs' surface. 51,60 It can be concluded that there is no chemical interaction between the film components.

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Differential Scanning Calorimetry (DSC) The DSC plots of propranolol HCl, PVA, HNT, PVA/HNT nanocomposite and the optimized formulation F6 are presented in Figure 6 and Figure S2). Halloysite nanotubes have a very stable structure, and apart from the removal  of surface and intercalated moisture, which is evident by a broad endothermic event from ~45-140 °C, there is no other thermal phenomenon observed. 61 Since PVA is a partially crystalline polymer with varying levels of hydrogen bonding with the surface -OH groups of nanotubes, the characteristic endothermic phenomena (~173 °C) were found to shift to higher temperature values in the HNT-PVA composites. 15 In the case of optimized formulation (F6), however, the sharp endothermic drop at ~44 °C is attributed to the melting of menthol in a small amount in the formulation (permeation enhancer 1%) along with its thermal oxidation at around ~138-150 °C. 62 This thermal event in the F6 (optimized formulation) is fused with the melting of the propranolol HCl in the formulation, which would be present in the amorphous form as reported for various drugs loaded onto HNTs and propranolol HCl adsorption on clays. [63][64][65] Moreover, the melting of the drug was followed by volatilization at around ~179 °C. 63 As confirmed by the FTIR and HPLC analysis, the drug was found chemically stable in the films. The composite's high mechanical and thermal stability is largely responsible for slow release of the drug for the formulation. 23,66

Scanning Electron Microscopy (SEM)
The surface morphology of the transdermal PVA film with or without the HNTs was analyzed on SEM. The presence of HNTs increased the roughness of the film comparable with PVA without HNT (see Figure 7). The PVA film can be seen properly covered with the HNTs. The good bonding between PVA and HNTs enhances the mechanical stability of the composite. The nanotubes wrapped with the polymer also increase the stability of the film. 66 The absence of agglomeration or fractured surfaces on the film indicates a uniform distribution of the film components.

Skin Irritation Study
The transdermal films were applied to the mice and evaluated for skin irritation such as edema or erythema. A moderate erythema reaction was observed in control group mice ( Figure 8). There was almost no skin irritation observed in the test group mice compared to the control group, and the films were found safe. The transdermal delivery systems containing propranolol HCl were observed non-irritant to the skin and suggested to be safe in other studies also. 67,68 In silico Modeling and Simulation The systemic absorption and disposition prediction for optimized propranolol HCl formulation (F6) was carried out using the TCAT® model. Contemplating the oral administration of 80 mg propranolol and to simulate the systemic disposition into individuals with an average weight of 75 kg as a comparison parameter, the three-compartmental model was selected with the help of the PKPlus® module. 69 Different formulations, pharmacokinetics, and physiological parameters are illustrated in Table 2. The mean pharmacokinetic parametric values (Table 7) for F6 were calculated as 32.113 ng/mL (C max ), 16.58 h (T max ), 942.34 ng/mL×h (AUC t ) and 1102.9 ng/mL×h (AUC inf ). After selecting different application areas and dosage strength, the model integrated pharmacokinetic parameters and TCAT® model for F6 using values of a dose of 20 mg and 9 cm 2 as the application surface area. The predicted plasma concentration profiles after 80 mg oral and 20 mg transdermal administration of propranolol HCl are shown in Figure 9A. A similarity between bioavailability profiles can be readily observed from both the curves though having different dosage strengths. This may be explained by the fact that propranolol HCl undergoes more than  75% first-pass metabolism via the oral route. 18 A cumulative increase in the plasma concentration of propranolol administered by a transdermal route was a bit slower when compared with the oral route. However, it was found to be equal after 9 hours of transdermal application. However, continuous therapy with multiple dosing suggests that the plasma profile is maintained in the therapeutic window (see Figure 9B) and the plasma levels are retained above the minimum therapeutic concentration (20 ng/mL). 70 This 20 mg dose of propranolol HCl, which is four-fold lesser than the oral route would suggest that the transdermal system embedded with halloysite nanotubes would be implicit in promoting drug diffusion across the skin compartments DovePress and effectively delivering therapeutic concentrations of propranolol HCl. This is an important feature of the transdermal route as the release kinetics, and cutaneous absorption can provide controlled plasma drug concentrations. 71 Based on the results obtained from in silico pharmacokinetic modeling, it is observed that the viability of halloysite nanotubes in the transdermal route as an alternative to oral administration for hypertension therapy diminishes hepatic first-pass load and possible adverse effects.

Stability Study
The stability study of the optimized transdermal film formulation (F6) was carried out at the room and at accelerated temperature. The physical characteristics, drug content, release, and permeation of the propranolol HCl were determined at different time intervals during the study, as shown in Table 8. The nanocomposite was observed to be satisfied physically, and no substantial change in the physical appearance was observed. The content of propranolol HCl was found to be 96.13 ± 0.42% at room temperature and 95.56 ± 0.51% at accelerated temperature. Although no significant difference in the drug content was observed, a slight decrease at higher temperatures might be due to the effect of certain environmental factors such as heat, moisture, and oxygen that could influence the stability of the drug and reduce the shelf life of the formulation. 72 Similarly, the permeation of the drug was observed to decrease after prolonged storage, especially at accelerated temperature. The permeation parameters were calculated, and it was revealed that the flux rate decreased (140.878 µg/cm 2 h and 138.299 µg/cm 2 h) while the lag time increased (0.363 hr and 0.370 hr) after 3 months of storage. The loss of permeation enhancers after prolonged storage might be responsible for the reduction in permeation of the drug substance through the barrier membrane. 72 The shelf life of propranolol HCl transdermal nanocomposite films was calculated and found to be 35.46 months at 28-30 °C and 19.66 months at 40-45 °C. Therefore, it can be proposed that the transdermal formulation should be stored at room temperature. The same has also been suggested for the propranolol HCl oral formulation. 73

Conclusion
Halloysite nanotubes-based transdermal nanocomposite films of propranolol HCl with different ratios of HNTs were formulated and evaluated. The nanocomposite films have a satisfactory appearance and mechanical stability, insignificant variation in weight and thickness and sufficient drug content. It was revealed that the drug release from the formulation was controlled with the increase in HNTs, and the permeation was enhanced. The menthol 1% concentration also enhanced the permeation of propranolol HCl through the barrier membrane. The drug release was found following the Korsmeyer-Peppas model with an n value of less than 0.5, representing Fickian diffusion. The formulation F6, which

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International Journal of Nanomedicine 2022:17 contained the 2:1 ratio of drug to HNTs, was selected as optimized due to a suitable release and permeation profile. The optimized formulation was considered safe and non-irritant to the skin. Moreover, the formulation was found mechanically stable, while the components were chemically non-reacting to the drug product, and the surface morphology revealed uniform distribution of HNTs in the composite. A maintained therapeutic window was also predicted from the in silico model. Furthermore, the formulation was found more stable at room temperature. Based on the data obtained, the HNTs could be a potential candidate for developing a transdermal drug delivery system, and the PVA/HNTs nanocomposite can be used for the design of a cost-effective and commercially viable transdermal drug delivery system with fewer regulatory challenges.

Disclosure
The authors report no conflicts of interest in this work.