In vitro evaluation and characterization of tolvaptan/cyclodextrin complex loaded electrospun nanofibers

Kara, Adnan Altuğ; Tort, Serdar; Yücel, Çiğdem; Acartürk, Füsun

doi:10.1590/s2175-97902025e24314

Abstract

Tolvaptan is a BCS IV group compound that is insoluble in water and slightly soluble in all pH ranges. Therefore, it is important to increase its solubility and dissolution rate, thereby increasing its bioavailability. For this purpose, tolvaptan containing nanofiber formulations have been prepared to increase the dissolution rate and solubility. Hydrophilic polymers such as polyvinyl pyrrolidone, polyethyleneoxide, and Soluplus; solubilizing agents such as Labrafil M 1944-CS, Solutol HS15, Gelucire varieties, and cyclodextrin (CD) derivatives were used. SEM analyses, contact angle, wettability studies, porosity, and solubility studies were performed on the prepared formulations. The HPβCD-NF2 formulation prepared with polyvinyl pyrrolidone and Solutol HS-15 increased the water solubility of tolvaptan by 7.96 times, and it was chosen as the final formulation. More than 90% of tolvaptan in the HPβCD-NF2 formulation was dissolved within 20 min in different buffers (distilled water, pH 1.2, 4.5, 6.8) with and without sodium lauryl sulfate. The HPβCD-NF2 formulation did not have any cytotoxic effects on the Caco-2 cell line and increased permeability compared to the commercial product, according to Caco-2 cell permeability studies. An innovative tolvaptan nanofiber formulation with increased solubility and dissolution rate compared to pure tolvaptan was developed, and the HPβCD-NF2 formulation has the potential to be an alternative to a commercial product.

Keywords:
Electrospinning; Nanofiber; Tolvaptan; Cyclodextrin; Solubility.

INTRODUCTION

The active compound tolvaptan is a vasopressin V2 receptor antagonist that controls renal fluid excretion. It is used to help adults with diseases including heart failure and specific hormonal imbalances and raise the low sodium content of their blood. Tolvaptan, a BCS IV group active ingredient, is a compound practically insoluble in water and has poor solubility in all pH ranges. Its bioavailability is also low due to poor solubility and poor permeability. The estimated bioavailability of tolvaptan after oral administration in humans is approximately 56%. For these reasons, it is essential to increase the water solubility and dissolution rate of tolvaptan, thereby increasing its bioavailability (Kim et al., 2021). As a general approach, it is known that bioavailability increases as solubility increases (Varma, Panchagnula, 2005; Bonthagarala, Dasari, Kotra, 2015).

In the pharmaceutical field, nanofibers are produced by the electrospinning method, which is one of the innovative technologies used for the development of new drug forms and solubility enhancements (Ozemre et al., 2023; Tugcu-Demiroz et al., 2021). Thanks to their large surface area and high pore volume, nanofibers are frequently used to increase the solubility, dissolution rate, and bioavailability of various drugs. Nanofibers are porous solid materials with microand nano-sized diameters with a very large surface area (Islam et al., 2019; Yildiz, Kara, Acarturk, 2020). The properties of nanofibers, such as high surface/volume ratio, adjustable pore size and pore volume, suitable mechanical properties, and flexibility in surface functionalities, make them suitable materials in different scientific fields (Yao et al., 2022).

Nanofibers are used in tissue engineering (He, Wan, Yu, 2004), wound care (Lee, Lee, 2020), enzyme immobilization (Lee et al., 2005), production of artificial organs (Huang et al., 2015), bone repair (Wang et al., 2018), and various drug delivery system (Hakkarainen et al., 2019; Tort, Acarturk, Besikci, 2017). In a study, a nanofiber formulation was prepared using the electrospinning method to increase the solubility of curcumin, which has poor solubility. In the solubility study conducted in distilled water and pH 1.2 buffers, the solubility of curcumin increased from zero to 7.66 mg/L and 1.57 mg/L, respectively. It has been stated that electrospinning is a method for enhancing the solubility of poorly soluble pharmaceuticals such as curcumin (Rüzgar et al., 2013). In another study, polyvinyl pyrrolidone (PVP) nanofibers containing piroxicam were prepared and compared with the pure drug in terms of dissolution rate. At the end of 30 min, approximately 92% of piroxicam in the nanofibers was dissolved, and approximately 62% of the pure drug was dissolved. It was observed that the dissolution rate of piroxicam from nanofibers was higher than that of the pure drug (Begum et al., 2012). In the study, a nanofiber formulation of curcumin with PVP was prepared and evaluated for its efficacy in vivo and in vitro with pure curcumin. In the in vitro dissolution study, 90% of the curcumin in the nanofiber formulation dissolved within 15 min in buffer at pH 7.4, while pure curcumin did not dissolve. In the pharmacokinetic study conducted on mice, in plasma taken after the application of pure curcumin and nanofiber formulations; T_max values were 120 and 60 min, C_max values w ere 167±19 and 14±3 ng/mL, and AUC_0-t values were 31,238±563 and 2720±124 ng.min/ mL, respectively. When nanofibers and curcumin were compared in terms of tumor growth inhibition in the in vivo anticancer test, nanofibers were found to be more effective due to increased bioavailability (Wang et al., 2015).

Various conventional formulation development strategies, such as salt formation (Patrick, 2014), complexation (Lee et al., 2007), particle size reduction (Khadka et al., 2014), prodrug (Jornada et al., 2015), micellization (Seedher, Kanojia, 2008), and solid dispersion (El-Badry, Fetih, Fathy, 2009) are used to increase the solubility, dissolution rate, and oral absorption of active substances with low water solubility. Cyclodextrins (CD) are used as complexing agents in the complexation method, a formulation-development strategy that forms inclusion complexes. The dissolution rate and oral absorption of active substances with low water solubility can be increased by the formation of inclusion complexes of active substances and CD. The outer surface of CDs is hydrophilic, and the inner surface is lipophilic. Lipophilic drugs interact with the internal environment and dissolve in the water thanks to the hydrophilic outer surfaces of CDs. Thus, by acting as hydrophilic carriers for lipophilic drugs, CDs can increase drug solubility and play an important role in the formulation of poorly water-soluble drugs (Saokham et al., 2018).

The properties of the obtained nanofibers change according to the properties of the polymers used in the electrospinning process. The release rate of the active substance from the nanofiber formulations prepared with hydrophilic polymers was high, and the solubility of the active substance increased. For this purpose, hydrophilic polymers such as PVP, polyethylene oxide (PEO), hydroxypropyl cellulose (HPC), and polyvinyl alcohol (PVA) have been used (Kajdič et al., 2019). Additionally, it has been observed that using hydrophilic polymers with CDs improves the efficiency of complexation and dissolution (Bashir et al., 2020).

There are studies in the literature in which solubility and dissolution rate were increased by preparing nanofiber formulations (Adeli, 2015) and forming complexes with CDs (Halder et al., 2023); however, to the best of our knowledge, there are no studies in the literature in which nanofiber tolvaptan formulations with or CD free were prepared and evaluated in terms of solubility, dissolution rate, and Caco-2 cell permeability of these formulations. By preparing nanofiber formulations of tolvaptan, an expensive drug, the efficacy of the drug will be increased, and the cost will be reduced by preparing the nanofiber formulation as a dosage form in future studies. Our study aimed to prepare nanofiber formulations with and CD-free using electrospinning, an innovative approach, and to compare these formulations in terms of solubility, dissolution rate, and permeability. Characterization studies were carried out on electrospinning solutions and produced nanofibers. According to the results of the characterization studies, the optimum nanofiber formulation was selected. Finally, the solubility, dissolution rate, and Caco-2 cell permeability of the optimal formulation were determined.

MATERIAL AND METHODS

Material

Tolvaptan was donated by Abdi İbrahim Otsuka (Turkey). PVP and Solutol HS-15 were obtained from BASF (Germany). PEO (Polyox™ WSR N80) was provided by ChemPoint. Gelucire varieties were obtained from Gattefosse (France). Beta-cyclodextrin (βCD) was purchased from Central Drug House (P) Ltd (India). 2-hydroxypropyl-beta-cyclodextrin (2-HPβCD) was obtained from Roquette (France). Sodium lauryl sulfate (SLS) and methanol were purchased from Sigma Aldrich (Germany). Dulbecco’s Modified Eagle’s Medium (DMEM) was obtained from Cegrogen Biotech GmbH (Germany).

METHODS

Nanofiber production by electrospinning method

Electrospinning was performed using a laboratoryscale electrospinning unit (NE-300, Inovenso Ltd., Turkey). The electrospinning solutions were placed into 10 mL plastic syringes. A drape was placed on a rotary cylinder to collect the fibers. Electrospinning was performed at room temperature and humidity. PVP, PEO, and Soluplus as hydrophilic polymers and Solutol HS-15, Gelucire varieties (44/14, 50/13, 48/16), and Labrafil M 1944 CS as solubilizers were used in pre-formulation studies on the production of nanofibers without tolvaptan. Distilled water and methanol were used as solvents to prepare polymer solutions. In total, 45 formulations were prepared (Table I).

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TABLE I
Components of the different nanofiber formulations

Preliminary characterization studies such as contact angle, dispersion time, and microscopic images were performed (S1) to determine the suitable formulations from which nanofibers can be obtained. Wetting occurred on the nanofiber surfaces in the electrospinning process of formulations coded 2, 4, and 5. In formulations coded 32-42, nanofibers could not be obtained despite the experiments over a wide voltage range. Since residue was formed in the dispersion test of formulations coded 9 and 10 containing Labrafil M 1944 CS, these formulations were not selected. As a result of the contact angle, disintegration studies, and evaluation of the microscope images in the nanofiber formulations, five formulations (Formulations coded 2, 8, 12, 22, and 44) were selected; the active substance was loaded; new formulations were constituted (Table II). Optimized process parameters, such as feed rate, distance from the needle tip to the collector, and applied voltage, are listed in Table II.

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TABLE II
The composition of tolvaptan loaded nanofiber formulations and electrospinning parameters

Preparation of Tolvaptan-CD complexes

βCD and 2-HPβCD are CDs that can be used frequently and are effective in increasing solubility. Phase-solubility studies were performed with these CDs (Mihajlovic et al., 2012). A phase-solubility study, was performed to form the inclusion complex, determine the drug: CD molar ratio, and calculate the association constant (Higuchi, Connors, 1965). First, solutions of β-CD or 2-HPβCD in different molar ratios (mM) (0, 2, 3, 4, 6, 8, and 10) were prepared. Tolvaptan was added in excess to each CD solution. To form complexes, the mixture was stirred at room temperature for 24 h on a magnetic stirrer, and each mixture was filtered through a 0.45 μm membrane filter. The phase-solubility diagram was drawn by quantifying tolvaptan from the supernatant. Based on the graphs obtained from the phase-solubility study, the solubility types of the β-CD: tolvaptan and 2-HPβCD: tolvaptan complexes and their molar ratios were determined.

Preparation of CD-free, βCD, and HPβCD nanofiber formulations

Polymer solutions with (β-CD or 2-HPβCD) and CD-free were prepared. In the production of CD-free nanofibers in preformulation studies, PVP and PEO were used as hydrophilic polymers, and Solutol HS-15 and Gelucire 44/14 were used as solubilizers (Table III). A mixture of distilled water and methanol was used to prepare the PEO solutions. Thus, PEO was completely dissolved, and a continuous electrospinning process was carried out. To produce CD-free nanofibers, tolvaptan was first dissolved in methanol, and the polymers were added and mixed in a magnetic stirrer for 1 h. A solubility enhancer was added, and mixing was continued until homogeneity was achieved. In the production of nanofibers with CD, in addition to hydrophilic polymers and solubility enhancers used in CD-free nanofibers, complexing agents (βCD and HPβCD) were used. Tolvaptan was first dissolved in methanol, and CDs were added and mixed with a magnetic stirrer for 24 h. After 24 h, the polymers were added, and mixing was continued for 1 h. Then, a solubility enhancer was added, and mixing was continued until homogeneity was achieved. After mixing, the solutions were drawn into a 10 mL syringe, and nanofibers were produced by electrospinning. To select the optimum formulations, solubility studies in distilled water at 37°C were performed for five nanofiber formulations CD-free containing βCD, or 2-HPβCD.

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TABLE III
Components of CD-free, βCD and HPβCD nanofiber formulations

Characterization of electrospinning solutions

Viscosity measurements

A stress-controlled cone/plate rheometer (Brookfield, DV-III Rheometer with Spindle Type CPE41, USA) was used to measure the rheology of polymer solutions. Approximately 1 mL of the polymer solution was added to the cone using a plastic syringe. All the samples were measured at room temperature, and the measurements were repeated three times.

Conductivity measurements

A conductivity meter (Mettler-Toledo Inc, Seven2Go S3 Portable Conductivity Meter, USA) was used to measure the conductive properties of the polymer solutions. The polymer solutions were added to the glass bottle of the device, and the measuring probe was immersed in the solution. Values were expressed as µS/cm.

Surface tension measurements

The surface tensions of the polymer solutions were measured using an optical tensiometer (Attension-Theta Lite, Biolin Scientific, Finland) according to the pendant drop method. The polymer solution was formed into drops at the needle tip. Then the surface tensions were calculated according to the Young-Laplace equation (Zhang et al., 2022), and the values were recorded as mN/m.

Characterization of the nanofibers Morphologies studies and the mean diameter of the fibers

Electrospun fibers were imaged using fieldemission SEM (FEI Company, Quanta 400 F, USA). The fibers were covered with gold/palladium before imaging. The images were taken at different parts of the fibers at 5000x, 10000x, and 20000x magnification. The average fiber diameters were determined by measuring randomly selected 20 different fibers from the SEM images using ImageJ software.

Porosity of nanofibers

The porosity values of the nanofibers were calculated using the ImageJ program and the SEM images of the nanofibers (Hou et al., 2023). In this method, the area of both colors was calculated after the nanofibers were white and the pores were black. The percentage of the black area relative to the total surface area in each image was defined as % porosity.

Differential Scanning Calorimetry (DSC), X-Ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy (FT-IR) analyses

DSC (Shimadzu, DSC-60, Japan) analysis was carried out on the drug substance, excipients, and nanofibers. The samples were heated from 25 to 360 C° at the heating rate of 10^º C/min under a nitrogen atmosphere. XRD patterns were taken to evaluate the solid-state structural properties of tolvaptan, excipients, and nanofibers. In XRD analysis, an X-ray diffractometer (Rigaku ULTIMA IV) with Cu Kα radiation generated at 30 mA and 40 kV was used. Scanning was performed between the angular ranges of 3-60°(2θ) and with the speed of 1°/min. For FT-IR analysis, tolvaptan, excipients, and nanofibers were scanned at 650-4000 cm^-1 wavelengths (Spectrum 400, PerkinElmer, USA).

Mechanical properties

The mechanical properties of the nanofibers were analyzed using a texture analyzer (TA.XT. PlusTexture Analyzer, UK) (Lin et al., 2022). For this purpose, nanofibers were cut into dimensions of 3x1 cm and placed on the mini tensile grip apparatus of the device with a tension area of 1 cm². The elongation at break (%) and tensile strength (MPa) values were calculated from the strain-stress graphs.

Wettability studies

Contact angle measurements were performed using an Attension Theta Lite optical tensiometer (Tugcu-Demiroz et al., 2021). The prepared nanofibers were stretched on a convex sample holder to obtain a smooth surface. Five µL of distilled water was dropped on different areas, and the angle of the drop with the surface was calculated using the software of the device.

Solubility studies

The solubility study of tolvaptan in nanofibers was carried out at 37°C in SLS-free distilled water, pH 1.2, 4.5, 6.8, and all media prepared by adding 0.22% (w/v) SLS. The nanofiber formulations were weighed to contain excess tolvaptan, placed in vials, and mixed in a water bath for 1 h. Samples were collected at the end of the experiment and analyzed using a UV/Vis spectrophotometer at 260 nm in SLS-free media and 270 nm in SLS-containing media.

In vitro dissolution test and release kinetics studies

The in vitro dissolution studies of tolvaptan from the nanofiber formulations were investigated in 900 mL of SLS-free distilled water, pH 1.2, 4.5, 6.8, and media prepared by adding 0.22% (w/v) SLS at 37°C using the USP paddle method (50 rpm) specified by the FDA for tolvaptan. In this study, samples were collected for 2, 5, 10, 15, 20, 25, 30, 45, and 60 min, and an equal volume of fresh medium was added. After the samples were filtered through a 0.45 μm membrane filter, absorbances were measured using the validated UV spectrophotometric method, and the concentrations were calculated.

Drug dissolution data were placed to zero-order, first-order, Higuchi model, Weibull, Hixson-Crowell, and Korsmeyer-Peppas kinetics to determine the possible mechanisms of tolvaptan dissolution from nanofibers using DDSolver Software (An Add-In Program for Modeling and Comparison of Drug Dissolution Profiles) (Zhang et al., 2010).

Determination of drug content

Three fiber samples were cut from the midhorizontal lines of the nanofibers and weighed around 10 mg. The fiber sections were mixed until they were dissolved in 20 mL of distilled water (1 h). After the fibers were dissolved, the solution was filtered through a 0.45 μm membrane filter, and the amount of tolvaptan was determined spectrophotometrically.

Cytotoxicity and permeability studies

Cytotoxicity Studies

The cytotoxic effects of tolvaptan and its nanofiber formulation on Caco-2 cells and their passage through the Caco-2 cell line were investigated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) cytotoxicity test was performed to determine the amount of active substance to be used during formulation development and the maximum amount of the developed formulations that can be used safely while maintaining the viability of the cells. This test is based on the conversion of the yellow soluble MTT dye into a dark blue insoluble formazan product by the Succinate Dehydrogenase (SDH) enzyme found in the mitochondria of metabolically active cells. The formed formazan product was solubilized, and the resulting purple color was determined spectrophotometrically. The Caco-2 cell line used for testing was grown in 96well tissue culture dishes. A hundred µL of cell culture medium, DMEM was added to the dishes and incubated on a CO₂ incubator at 37°C for 24 h. After determining the control groups that did not contain the active substance, solutions of the samples (100 µL) in DMEM at varying concentrations were added to the dishes on the plates and incubated in a CO₂ incubator at 37°C for 24 h. After 24 h, the contents of the plates were emptied, and 100 µL of fresh DMEM and 13 µL of MTT solution (5 mg/mL in phosphate buffer) were added to all dishes. The culture dishes were tightly wrapped with aluminum foil and maintained in a CO₂ incubator for 4 h at 37°C. After four hours, the plates in the incubator were emptied, and 100 µL of dimethyl sulfoxide (DMSO) was added to the dishes. The resulting purple color was spectrophotometrically determined at a wavelength of 570 nm. Based on the different absorbance values obtained from the substances at different concentrations, the percentage viability was calculated by comparing them with the control group.

Permeability studies

In cell permeability studies, cells were grown in a monolayer of 6-well special cell dishes (TranswellCorning Cat. No. 3412) with a pore diameter of 0.4 µm. The formulation sample was placed on the apical part, the cell monolayer, and the cell medium was placed on the receptor phase. Samples were taken at certain time intervals, and active substance permeability was determined. The cell culture medium at 37°C was added to the receptor phase at the same volume as the sample so that the medium volume did not change after sample collection. At the end of the experiment, the cumulative amount of active substances accessed from the apical part to the basal part was calculated using a UV spectrophotometer at 285 nm.

RESULTS AND DISCUSSION

Selection of nanofiber formulations

The five nanofiber formulations listed in Table II were selected from 45 formulations after examining the contact angle, disintegration time, and microscope images of nanofiber formulations (S1). After adding CD-tolvaptan complexes to these five nanofiber formulations, characterization studies were carried out on the new nanofiber formulations.

Preparation and characterization of Tolvaptan-CD complexes

According to the results of the phase solubility study of βCD and 2-HPβCD, both CD molecules showed A_L-type phase-solubility diagram with tolvaptan due to a linear increase in solubility depending on CD concentration and a 1:1 (drug:CD) inclusion complex was formed between the drug molecule and the CD (Figure 1A) (Miyazawa et al., 1995).

FIGURE 1
A Tolvaptan-CD-phase solubility diagram and solubility results of pure tolvaptan and B five nanofiber formulations containing CD-free (NF), βCD (βCD-NF), and 2-HPβCD (HPβCD-NF) (n=3).

For 1:1 complexes, the association (stability) coefficient (K) was calculated using the following equation (Londhe, Pawar, Kundaikar, 2020):

Eq. 1

K_{1 : 1} = \frac{Slope}{(So (1 - Slope))}

So = Solubility of the tolvaptan

The association coefficient K_1:1 varies between 0-100,000 M^-1 for the drug: CD complexes (Higuchi, Connors, 1965). The K value of βCD and 2-HPβCD were 55.85 M^-1 and 40.19 M^-1, respectively, and stable complexes were formed (Figure 1A). Similar to our results, in a study, they formed a 1:1 inclusion complex with fluconazole and βCD in their study, and the association coefficient K_1:1 value was found to be 68.7 M^-1 (Upadhyay, Kumar, 2009). In the literature, complexes were formed between lidocaine and 2-HPβCD at a ratio of 1:1. The association coefficient K_1:1 value was found to be 35.7±4.7 M^-1, and it was stated that stable complexes were formed (Moraes et al., 2007).

Solubility Studies

The CD-free, βCD, and 2-HPβCD-containing nanofiber formulations were evaluated for the solubility of tolvaptan (Figure 1A). The nanofiber formulations were compared to determine its solubility.

CDs have a cyclic structure with a hydrophobic interior and hydrophilic exterior. The hydrophobic core can interact with low-solubility compounds, encapsulating them and making them more soluble in a solvent medium. βCD-containing oligosaccharides and 2-HPβCD are cyclic oligosaccharides with similar structural properties. In the solubility study of βCDcontaining nanofibers, an increase in solubility was expected because of the high porosity and large surface area of the nanofibers and the formation of an inclusion complex between tolvaptan and CD.

When the five nanofiber formulations CD-free, five nanofiber formulations containing βCD, and five nanofiber formulations containing 2-HPβCD were compared in terms of solubility, nanofibers containing 2-HPβCD showed better solubility and HPβCD-NF2 (containing 2% 2-HPβCD, 10% PVP, and 5% Solutol HS 15) had a solubility of 0.2118 mg/mL, making the formulation with the highest solubility. It is thought that HPβCD-NF2 shows higher drug solubility than HPβCD-NF4 because of the polymers used in these two formulations. PEO N80 used in HPβCD-NF4 had a lower molecular weight than PVP K90 used in HPβCD-NF2; therefore, its gelation was also lower (Qin et al., 2014; Chaudhari, Dave, 2016). This may explain the lower solubility of tolvaptan in HPβCDNF4. It was thought that the reason why HPβCD-NF2 showed higher drug solubility than HPβCD-NF5 was due to Solutol HS-15. Solutol HS-15 is a solubilizing agent that effectively increases the solubility of active substances with low solubility (Swain, Subudhi, Ramesh, 2019). In addition, HPβCD-NF4 contains PEO N80, PEO N80 has a lower molecular weight than PVP K90; therefore, its gelation is lower and can be thought to cause lower drug solubility. 2-HPβCD increased the drug solubility more than βCD because βCD has limited water solubility and 2-HPβCD has a hydroxypropyl group and is more hydrophilic than βCD (Patel et al., 2018). In formulations containing 2-HPβCD, solubility may have increased because of both the high porosity and large surface area of the nanofibers and the formation of an inclusion complex between tolvaptan and CD. In a study, it was shown that nanofiber formulations containing 2-HPβCD increase the solubility of different active substances (Celebioglu, Uyar, 2017). Therefore, 2-HPβCD was thought to be superior to βCD in increasing solubility, and 2-HPβCD was chosen as the complexing agent. Subsequent studies were continued with a fivenanofiber formulation containing 2-HPβCD.

Characterization of electrospinning solutions

Viscosity measurements

The viscosity of the polymer solution affects the diameter of the nanofibers and fiber formation. The jets formed during electrospinning are kept in a continuous form to produce fibers instead of splitting them into droplets and forming a fiber structure. High or low viscosity of the solution may cause negative effects, such as stopping the electrospinning process, formation of a wet surface, and formation of nanofibers in the droplet structure. In addition, the polymer concentration affects the viscosity and surface tension of the solution, and both parameters can affect the morphology and size of the obtained fibers. In general, lowering the viscosity and surface tension can produce finer fibers (Xue et al., 2019). The viscosities of the polymer solutions of the selected five nanofiber formulations are listed in Table IV.

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TABLE IV
Different parameters of electrospinning solutions (mean ± SD)

The viscosity values of HPβCD-NF2 and HPβCD-NF3 were lower than those of the HPβCDNF1, HPβCD-NF4, and HPβCD-NF5 in the viscosity measurements made in nanofiber formulation solutions. However, the low viscosity did not cause any problems in the electrospinning process, and uniform nanofibers were obtained via continuous production. In addition, owing to the increase in the viscosity of the produced nanofibers, thicker nanofibers were produced (Table IV). In the literature, findings support the increase in the fiber diameter depending on the increase in the viscosity of the polymer solutions used for nanofiber production (Lee, Song, Yoon, 2010).

Conductivity measurements

In the electrospinning process, an electric charge transfer is required from the electrode to the polymer droplet at the end of the injection needle. Generally, the net charge density is proportional to the conductivity of the solution. The higher the net charge density, the smaller the beads and the finer the fibers. The decrease in charge density facilitates bead formation (Zheng et al., 2006). The conductivity measurements for the polymer solutions of the selected 2-HPβCD containing nanofiber formulations are listed in Table IV. The conductivity values of nanofiber formulations containing 2-HPβCD were found to be between 44.10-10.36 µS/cm (Table IV). These values were suitable for all formulations.

Surface tension measurements

In the production of nanofibers by electrospinning, surface tension must be overcome by the charges in the polymer solution. Different surface tensions can be obtained by using different solvents. Generally, the surface tension of the polymer solution is expected to be low for jet formation. The surface tensions of the polymer solutions of the selected 2-HPβCD containing nanofiber formulations are listed in Table IV.

Viscosity and net charge density affect bead formation in the fibers. Therefore, by decreasing the surface tension, the formation of beaded fibers can be reduced (Fong, Chun, Reneker, 1999). The surface tensions of the nanofiber solutions containing 2-HPβCD were found to be similar, between 19.94-27.98 mN/m (Table IV). These values were also suitable for nanofiber formulations containing 2-HPβCD and did not cause any problems in terms of morphology.

Characterization of the nanofibers Morphologies studies and the mean diameter of the fibers

SEM images of HPβCD-containing nanofiber formulations were obtained, and the mean fiber diameter was calculated from these images (Figure 2).

FIGURE 2
SEM images of HPβCD-NF1- HPβCD-NF5 (mag. A 5000x, B 10000x, C 20000x).

The CD-tolvaptan complex containing nanofiber compositions did not exhibit any bead formation (Figure 2). Crystal structures were observed on the surface of the fibers in the HPβCD-NF4 and HPβCDNF5 formulations, and deformation was observed on the surfaces of the fibers in the HPβCD-NF4 formulation due to the viscosity and conductivity values which were higher than those of the other three formulations. In addition, similar to the literature, an increase in the diameter of the fibers was observed, depending on the increase in the viscosity and conductivity (Table IV) (Jian et al., 2018).

Porosity of nanofibers

The porosity of nanofibers is important in terms of its effect on the mechanical properties of the fibers. After the nanofibers were colored white and the blank part black, the areas of the two colors were calculated. The area of the nanofibers was proportional to the total area, and this ratio was determined as the percentage of porosity. The porosities of all nanofiber formulations were 50-62%. The highest porosity was found for the HPβCD-NF2 formulation with 61.92% (Table V). In general, the porosity of nanofibers can vary between 5090% (Širc et al., 2012; Wei et al., 2005). Porosity can affect the solubility and dissolution rate. The solubility of active substances with low water solubility can increase depending on the increase in porosity. The higher solubility of the HPβCD-NF2 formulation compared to the other formulations may be due to its higher porosity.

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TABLE V
Diff erent physical properties of nanofi ber formulations (mean ± SD)

XRD analyzes

XRD analyses were performed to examine changes in the physical and chemical structures of tolvaptan after electrospinning (Figure 3). Thus, the effect of amorphous crystalline transformation on solubility was investigated.

FIGURE 3
XRD diffractograms; A Tolvaptan, PVP, PEO, Gelucire 44/14 and 2-HPβCD; B nanofiber formulations containing 2-HPβCD; C DSC thermograms of HPβCD-NF2 formulation, PVP, solutol, tolvaptan, HPβCD and HPβCD-tolvaptan complex; D FTIR spectra of HPβCD-NF2 formulation and excipients.

Tolvaptan, PVP, and 2-HPβCD did not exhibit sharp peaks because they were amorphous. Gelucire 44/14 showed sharp XRD peaks at 19.14º and 23.36º regions (Figure 3A). These sharp peaks indicate that Gelucire 44/14 had a crystalline structure. XRD analysis findings, in which Gelucire 44/14 has a crystal structure and exhibits sharp peaks, are also available in the literature (Sethia, Squillante, 2004). PEO N80 exhibited sharp XRD peaks at 19.140º and 23.340º (Figure 3A). These sharp peaks indicate that PEO N80 had a crystalline structure. XRD analysis findings, in which PEO N80 has a crystal structure and exhibits sharp peaks, are also available in the literature (Sethia, Squillante, 2004). No peaks were observed for the HPβCD-NF1, HPβCD-NF2, and HPβCD-NF3 formulations. These formulations appeared in amorphous form. In HPβCD-NF4 and HPβCD-NF5 formulations, the characteristic sharp XRD peaks of PEO N80 decreased, and it was thought that it turned into a semi-amorphous structure (Figure 3).

Mechanical properties

The mechanical properties of the nanofibers affect fiber diameter and morphological structure. In the study of elongation at break in which the mechanical properties were examined, the % elongation at break values of HPβCD-NF1, HPβCD-NF2, HPβCD-NF3, and HPβCD-NF5 formulations were found to be close to each other, while these values of the HPβCD-NF4 formulation were found to be higher than the others. In the SEM images of HPβCD-NF4 (Table V), the deformation of the morphological structure of the fibers may be related to the high rupture value owing to the elastic structure of the fibers. It has been reported in the literature that fiber morphology can affect breaking value (Vargas et al., 2010). The tensile strength of HPβCD-NF2 was found to be the highest. The tensile strength affects the diameter, size, and morphological structure of the fibers. In general, the tensile strength decreased with an increase in fiber diameter. SEM analysis revealed that the HPβCD-NF2 formulation with the lowest fiber diameter had the highest tensile strength. The fact that HPβCD-NF4 and HPβCDNF5 formulations have larger fiber diameters and lower tensile strengths than HPβCD-NF2 supports the relationship between fiber diameter and tensile strength. The literature has reported that there may be a decrease in tensile strength depending on the increase in fiber diameter (Baji et al., 2010).

Wettability studies

The contact angle is an important parameter that provides information on wettability. If the contact angle was <90º, the structure of the fiber was considered hydrophilic. The rapid wettability of the fibers affects the dissolution rate. The active substance transition from easily wetted fibers to the dissolution medium was also rapid. Wettability and contact angle measurements of the nanofiber formulations containing 2-HPβCD were performed, and the results are shown in Table V.

The contact angles of all five fiber formulations were found to be below 90º, and all formulations were found to be very hydrophilic. The contact angles of the HPβCD-NF2, HPβCD-NF3, and HPβCD-NF4 formulations were 0º, and they disintegrated in a short time when they came into contact with water. The contact angles of the HPβCD-NF1 and HPβCD-NF5 formulations were 46.95 °and 61.44°, respectively, and the wetting time was 20 s (Table V). It was observed that the wetting time increased with the increase of the contact angle. In the literature, there are studies in which a nanofiber formulation with a contact angle of 0º gets wet very quickly in distilled water (Tort et al., 2019).

Characterization studies of the selected nanofiber formulations

In SEM analyses of selected 2-HPβCD containing nanofiber formulations, fiber diameters were found to be nanometer-range for HPβCD-NF1 and HPβCD-NF2, micrometer-range for HPβCD-NF3, HPβCD-NF4, and HPβCD-NF5 (Table V). When the contact angle and wettability are examined, HPβCD-NF1 and HPβCDNF5 formulations were wetted in 20 s, while HPβCDNF2 was wetted as quickly as <1 s when contacted with water. In terms of porosity, the HPβCD-NF2 formulation had the highest porosity value with 61.92% (Table V). When evaluated in terms of solubility, HPβCD-NF2 had a higher solubility than the other formulations. Therefore, as a result of our studies on these five formulations, HPβCD-NF2 formulation containing 2% 2-HPβCD, 10% PVP K90, and 5% Solutol HS-15 was chosen as the final formulation, and further studies were continued with this formulation (Table III).

DSC measurements of the active substance, excipients, and the nanofiber formulation were performed (Figure 3C). Pure tolvaptan exhibited an amorphous distogram in the XRD pattern and a melting peak in the DSC curve (Figure 3A). The melting peaks of pure tolvaptan and 2-HPβCD were observed between 300-350 ºC. According to the literature, tolvaptan gave a melting peak at 225.9 °C (Cordero-Vargas, QuicletSire, Zard, 2006). The DSC curve of tolvaptan at temperatures above 300 °C (Figure 3C) may be related to drug degradation. Pure tolvaptan and 2-HPβCD peaks disappeared with the formation of the inclusion complexes. No peaks corresponding to pure substances were observed for the HPβCD-NF2 formulation (Figure 3C). The reason for this might be the low amount of tolvaptan and the formation of the inclusion complex. There are studies in the literature in which the DSC melting peak of the pure substance disappeared after the formation of the inclusion complex (Ficarra et al., 2000). Similarly, there are studies in which the melting peak of the pure substance was lost in the nanofiber formulation (Adeli, 2015).

FTIR analyses of HPβCD-NF2 and the excipients used were performed (Figure 3D). Pure PVP formed a wide band between 3700-3000 cm^-1 (Tantishaiyakul, Kaewnopparat, Ingkatawornwong, 1999) and a C=O tension band at 1648 cm^-1 (Adeli, 2015). C=O stretching is observed at 1732 cm^-1 in Solutol HS-15 (Varshosaz, Ghassami, 2015). In pure 2-HPβCD, a wide band of OH stretching is observed in the range of 3000-3600 cm^-1 (Mihajlovic et al., 2012). While the PVP peak was observed in the HPβCD-NF2 formulation, the peaks of tolvaptan could not be distinguished due to the low amount of tolvaptan in nanofibers.

Solubility studies of the selected nanofiber formulation

Before the dissolution and solubility studies, the amount of loaded drug in the HPβCD-NF2 formulation was determined. The amount of tolvaptan in the HPβCDNF2 formulation was recovered as 99.44±4.57%. Solubility studies of HPβCD-NF2 formulation were performed in SLS-free and 0.22% SLS-containing media (Figure 4).

FIGURE 4
Solubility results of HPβCD-NF2 formulation in A SLS-free buffer, B 0.22% SLS buffers (n=3).

In the solubility study performed in SLS-free media, a significant increase in the solubility of tolvaptan was observed compared to that of pure tolvaptan. The HPβCD-NF2 formulation dissolved 0.2118 mg/mL in distilled water, indicating an increase of approximately 7.97 times compared to the solubility of pure tolvaptan in distilled water (Figure 4A).

This may be due to the use of a hydrophilic polymer (PVP), a solubility enhancer (Solutol HS-15), and the formation of an inclusion complex between tolvaptan and CD (2-HPβCD). Previous studies have shown that hydrophilic polymers, solubility enhancers, and CDs are effective in increasing the solubility (Hirlekar, Sonawane, Kadam, 2009; Rajebahadur et al., 2006). In the solubility study of the HPβCD-NF2 formulation in media containing 0.22% SLS, the solubility of tolvaptan in the formulation was higher than that of pure tolvaptan. It dissolved 1.6996 mg/mL in distilled water containing 0.22% SLS, indicating an approximately 8.12-fold increase in the solubility of pure tolvaptan in distilled water containing 0.22% SLS (Figure 4B). The reason for this is the use of hydrophilic polymers, solubility enhancers, and an inclusion complex between tolvaptan and CD. It also increases solubility in SLS, an anionic surfactant (Granero, Ramachandran, Amidon, 2005). In the study, PVP and 2-HPβCD nanofibers were prepared to improve the solubility and physicochemical properties of resveratrol. As a result of the study, the solubility of pure resveratrol was 0.041 ± 0.001 μg/ mL, while the solubility of resveratrol in nanofiber formulations was found between 900.6 ± 31.4 μg/mL and 855.50 ± 35.20 μg/mL (Lin et al., 2020).

Tolvaptan has been reported to exhibit pHindependent solubilities (Shoaf, Bricmont, Gordon, 2021). The results obtained in our study showed that solubility in SLS-free buffers was relatively independent of pH. By adding SLS to the dissolution medium, the solubility of active substances with low water solubility increased. This increase is related to the formation of micelles by SLS, which reduces the surface tension of the dissolution medium and increases the wettability of the active substance (Saydam, Takka, 2020). The solubility of tolvatan in distilled water and pH 1.2 buffers containing SLS was found to be relatively higher than its solubility at pH 4.5 and 6.8 containing SLS. Due to disodium hydrogen phosphate at pH 4.5 and 6.8, Na⁺ ions ionized by dissolving SLS and disodium hydrogen phosphate in buffer solutions will increase the Na⁺ ion concentration. As the number of Na⁺ ions increases, a common ion effect occurs. Owing to the common ion effect, the solubility of active substances may be lower than expected (Serajuddin, Sheen, Augustine, 1987). The relatively lower solubility of tolvaptan in pH 4.5 and 6.8 buffers containing SLS compared to its solubility in distilled water and pH 1.2 buffers containing SLS, may be due to the common ion effect.

In vitro dissolution test and release kinetics studies

The dissolution rate of tolvaptan from the HPβCDNF2 formulation was determined in buffers with and SLS-free (Figure 5). Tolvaptan dissolved rapidly in all the buffers. In buffers containing SLS, 55-73% of tolvaptan was dissolved in the first 2 min, whereas in SLS-free media, 25-35% of tolvaptan was dissolved. At least 90% of the tolvaptan in the formulation dissolved within 20 min in all media with a SLS-free (Figure 5A-B). The presence of SLS in the buffer accelerated the disintegration of the nanofiber formulation and increased the dissolution rate of tolvaptan. SLS, a surfactant substance, ensures the dissolution of the active substance and a high dissolution rate. The rapid dissolution is due to the high porosity and large surface area of the nanofibers (Kajdič et al., 2019); thus, they are easily and quickly wetted by buffers. In a study on nanofibers, PVP/2-HPβCD fast-dissolving nanofiber drug delivery systems for meloxicam were developed. When the release profiles were compared, it was observed that the nanofibers produced had a faster release profile than the commercial product (Samprasit et al., 2018).

FIGURE 5
In vitro dissolution test of HPβCD-NF2 formulation in A SLS-free media, B 0.22% SLS media (n=3)

As a result of the dissolution studies, r²adj values were selected as criteria for the kinetic models. Among these values, as the r²adj approaches 1, the accuracy of the kinetic model increases. Based on these mathematical values, the Weibull model was the most appropriate kinetic model for the dissolution profiles of the HPBCDNF2 formulation in distilled water, pH 1.2, pH 4.5, and pH 6.8 buffers. The most appropriate kinetic model determined for the dissolution profiles of the HPBCDNF2 formulation in 0.22% SLS-distilled water, 0.22% SLS-pH 1.2, 0.22% SLS-pH 4.5, and 0.22% SLS-pH 6.8 buffers was the Korsmeyer-Peppas model (Table VI).

In the Weibull model, ß constants can provide information about the drug delivery mechanism in the polymeric fiber. ß < 0.75 Fickian diffusion is the dominant release mechanism, while 0.75 < ß < 1 the contribution of Fickian diffusion and swelling is estimated, and ß > 1 is considered a complex release mechanism (Tugcu-Demiroz et al., 2021). The ß values of HPBCD-NF2 formulation in distilled water, pH 1.2, pH 4.5, and pH 6.8 buffers in the Weibull model were 0.8755, 0.8195, 0.7655, and 0.5933, respectively. The HPBCD-NF2 formulation in distilled water, pH 1.2, and pH 4.5 buffers released the drug by the Fickian diffusion and swelling release mechanism while the drug release in the pH 6.8 buffer occurred by the Fickian diffusion mechanism. The study found that the release mechanism of benzydamine from the two nanofiber formulations was based on the Weibull model. ß values of the formulations were found to be 0.84 and 0.60, respectively. It was stated that the formulation with a value of ß = 0.84 released the drug by a combined release mechanism, and the formulation with a value of ß = 0.60 released the drug by a Fickian mechanism (TugcuDemiroz et al., 2021). In the Korsmeyer-Peppas model, the n value can provide information about the release exponent, indicating the drug release mechanism. For n < 0.5, a Pseudo-Fickian diffusion mechanism; for n = 0.5, a Fickian diffusion mechanism; for 0.5 < n < 1, an abnormal diffusion mechanism; and for n = 1, a nonFickian diffusion mechanism is stated to control the drug release mechanism. The n values of HPBCD-NF2 formulation in 0.22% SLS-distilled water, 0.22% SLSpH 1.2, 0.22% SLS- pH 4.5, and 0.22% SLS-pH 6.8 buffers in the Korsmeyer-Peppas model were 0.1745, 0.1738, 0.1567, 0.2457, respectively. The HPBCDNF2 formulation in 0.22% SLS-distilled water, 0.22% SLS-pH 1.2, 0.22% SLS-pH 4.5, and 0.22% SLS-pH 6.8 buffers released the drug by the Pseudo-Fickian diffusion mechanism (Rezaei, Nasirpour, 2019). In the study, the release mechanisms of nanofiber formulations containing curcumin in the gastrointestinal environment and nanofibers containing curcumin-CD complexes in the simulated saliva environment were examined, and it was found that drug release occurred according to Korsmeyer-Peppas. The n value in nanofibers containing curcumin was found to be higher than 0.5, and it was reported that the release of curcumin was due to anomalous diffusion in gastrointestinal conditions. In the nanofiber formulation containing curcumin-CD complexes, the n value was found to be 0.47, indicating that drug release occurred by pseudo-Fickian diffusion (Rezaei, Nasirpour, 2019).

Cytotoxicity and permeability studies

Cytotoxicity Studies

Cytotoxicity studies were performed using the selected HPβCD-NF2 formulations. To be equivalent to a commercial product containing 15 mg of tolvaptan, an amount of nanofiber equivalent to 15 mg of the active substance was weighed (142.5 mg of nanofiber containing 15 mg of tolvaptan). The mean viability values were determined after applying 5, 7.5, 10, 15, and 20 mg solutions of tolvaptan in 30% (v/v) ethanol, 30% ethanol, and nanofiber formulations. In solutions containing pure tolvaptan, a decrease in cell viability was observed because of an increase in the amount of tolvaptan. The 30% ethanol without tolvaptan showed 76.70% cell viability, less than that of the solution and nanofiber formulations containing 15 mg of tolvaptan. The solution containing 15 mg tolvaptan and nanofibers containing 15 mg tolvaptan showed similar cell viability (Figure 6). The cytotoxicity values of the prepared solutions and formulations were greater than 80% (Zhao et al., 2012), which indicates that the prepared nanofibers have high cell viability and are safe in terms of cytotoxicity.

FIGURE 6
Cytotoxicity study results (n=3) and permeability study results from Caco-2 cells of suspension, solution, nanofi ber, and commercial product; 285 nm, according to a oneway ANOVA test (p < 0.05).

Permeability studies

The permeabilities of the solution, nanofiber, and commercial product containing the same amount of tolvaptan through Caco-2 cells were compared. The absorbance values of tolvaptan were obtained from the samples taken in the permeability study at 285 nm, and the P_app (cm/s) values were calculated (Figure 6). The permeability coefficients of the suspension, solution, HPβCD-NF2, and commercial product of tolvaptan ranged from 1,187×10^-5 to 1,897×10^-5 cm/s at 285 nm were found. Although the commercial product showed the lowest permeability compared to the other forms, HPβCD-NF2 showed the highest. In the literature, it has been stated that the absorption will be between 70%-100% and higher for compounds with P_app values higher than 1 × 10^-5 cm/s (Yee, 1997). As a result of the analyses performed at 285 nm, it was found that the HPβCD-NF2 formulation had a statistically higher permeability coefficient than the commercial product (p<0.05). Passive transition through lipophilic cell membranes is related to the lipophilicity of penetrating molecules. CDs can penetrate biological membranes via passive diffusion. While water solubility can be increased by preparing complexes of a lipophilic active substance with CDs, the transition through biological membranes may be limited. Additionally, CDs can increase drug penetration through cell membranes; if the amount of CDs is excessive, drug penetration into the cell membrane may decrease (Loftsson, Brewster, 2011). It is thought that the reason why the permeability of the HPβCD-NF2 formulation is relatively higher than that of the solution, suspension, and commercial product may be due to CDs increase in the permeability of drugs (Kratz et al., 2012; Corazza et al., 2020) or formulation related factors. In addition, Solutol HS15, included in the HPβCD-NF2 formulation, has a permeability increasing effect as well as a solubility increasing effect (Brayden et al., 2012; Alani et al., 2010). Although CDs may have positive or negative effects on the permeability of tolvaptan, this negative effect may have been tolerated by the permeability increasing effect of Solutol HS15. Therefore, HPβCD-NF2 may have been relatively more effective in increasing permeability than other formulations.

CONCLUSION

In this study, nanofibers were produced using electrospinning technology, an innovative approach to increase the solubility and dissolution rate of tolvaptan, a BCS Class IV active substance, and the solubility and dissolution rate of the obtained nanofiber formulations were evaluated. hydrophilic polymers such as PVP, PEO, and Soluplus in formulations, solubility enhancers such as Solutol HS-15 and Gelucire varieties, and complex-forming agents such as βCD and 2-HPβCD have been used, and formulations that can act quickly and has an increased solubility and dissolution rate profile compared to pure tolvaptan have been developed . Detailed characterization of these formulations was performed, and the most suitable formulation (HPβCDNF2) was selected. The produced nanofibers were examined in terms of cytotoxicity and permeability, and a formulation with higher permeability than pure tolvaptan was obtained. Further, in vivo studies are needed for the developed HPβCD-NF2 formulation to be an alternative to commercially available products.

ACKNOWLEDGEMENTS

This study was supported by The Scientific Technological Research Council of Turkey (Project no: 221S450, TUBITAK). The authors would like to thank Abdi Ibrahim Pharmaceuticals, Roquette, and Gattefossé for providing tolvaptan, HPβCD and Gelucire 44/14, respectively. The authors would like to thank Gazi University Academic Writing Application and Research Center for proofreading the article.

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Vargas EA, do Vale Baracho NC, de Brito J, de Queiroz AA. Hyperbranched polyglycerol electrospun nanofibers for wound dressing applications. Acta Biomater. 2010;6(3):1069-78.
Varma MV, Panchagnula R. Enhanced oral paclitaxel absorption with vitamin E-TPGS: effect on solubility and permeability in vitro, in situ and in vivo. Eur J Pharm Sci. 2005;25(4-5):445-453.
Varshosaz J, Ghassami E. Enhancement of Dissolution Rate of Fenofibrate by Spray Drying Technique: Comparison of Eudragit E-100, Solutol (R) Hs15 and Hydroxypropyl Cellulose as Carriers. Farmacia. 2015;63( 3):433-45.
Wang C, Ma C, Wu Z, Liang H, Yan P, Song J, et al. Enhanced Bioavailability and Anticancer Effect of Curcumin-Loaded Electrospun Nanofiber: In Vitro and In Vivo Study. Nanoscale Res Lett. 2015;10:1-10.
Wang T, Zhai Y, Nuzzo M, Yang X, Yang Y, Zhang X. Layer-by-layer nanofiber-enabled engineering of biomimetic periosteum for bone repair and reconstruction. Biomaterials. 2018;182:279-88.
Wei HJ, Liang HC, Lee MH, Huang YC, Chang Y, Sung HW. Construction of varying porous structures in acellular bovine pericardia as a tissue-engineering extracellular matrix. Biomaterials. 2005;26(14):1905-13.
Xue J, Wu T, Dai Y, Xia Y. Electrospinning and Electrospun Nanofibers: Methods, Materials, and Applications. Chem Rev. 2019;119(8):5298-415.
Yao X, Zou SZ, Fan SA, Niu QQ, Zhang YP. Bioinspired silk fibroin materials: From silk building blocks extraction and reconstruction to advanced biomedical applications. Mater Today Bio. 2022;16.
Yee S. In vitro permeability across Caco-2 cells (colonic) can predict in vivo (small intestinal) absorption in man-fact or myth. Pharm Res. 1997;14:763-6.
Yildiz A, Kara AA, Acarturk F. Peptide-protein based nanofibers in pharmaceutical and biomedical applications. Int J Biol Macromol. 2020;148:1084-97.
Zhang P, Ren G, Tian L, Li B, Li Z, Yu H, et al. Environmentally Friendly Waterproof and Breathable Nanofiber Membranes with Thermal Regulation Performance by One-Step Electrospinning. Fibers Polym. 2022;23(8):2139-48.
Zhang Y, Huo M, Zhou J, Zou A, Li W, Yao C, Xie S. DDSolver: an add-in program for modeling and comparison of drug dissolution profiles. AAPS J. 2010;12( 3):263-71.
Zhao P, Wang L, Sun C, Jiang T, Zhang J, Zhang Q, et al. Uniform mesoporous carbon as a carrier for poorly water soluble drug and its cytotoxicity study. Eur J Pharm Biopharm. 2012;80(3):535-43.
Zheng J, He A, Li J, Xu J, Han CC. Studies on the controlled morphology and wettability of polystyrene surfaces by electrospinning or electrospraying. Polymer. 2006;47( 20):7095-102.

Edited by

Associated Editor: Silvya Stuchi Maria-Engler

Publication Dates

Publication in this collection
05 Dec 2025
Date of issue
2025

History

Received
20 May 2024
Accepted
07 Sept 2024

This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Formulation Code	Tolvaptan (%)	βCD (%)	HPβCD (%)	PVP K90 (%)	PEO N80 (%)	Solutol HS-15 (%)	Gelucire 44/14 (%)
NF1	2	-	-	10	-	-	-
NF2	2	-	-	10	-	5	-
NF3	2	-	-	10	-	-	5
NF4	2	-	-	-	10	5	-
NF5	2	-	-	5	10	-	-
βCD-NF1	2	2	-	10	-	-	-
βCD-NF2	2	2	-	10	-	5	-
βCD-NF3	2	2	-	10	-	-	5
βCD-NF4	2	2	-	-	10	5	-
βCD-NF5	2	2	-	5	10	-	-
HPβCD-NF1	2	-	2	10	-	-	-
HPβCD-NF2	2	-	2	10	-	5	-
HPβCD-NF3	2	-	2	10	-	-	5
HPβCD-NF4	2	-	2	-	10	5	-
HPβCD-NF5	2	-	2	5	10	-	-

Formulation code	Mean viscosity (mPas) ± SD (n=3)	Mean conductivity (µS/cm) ± SD (n=3)	Mean surface tension (mN/m) ± SD (n=3)
HPβCD-NF1	169.43 ± 0.37	14.03 ± 0.03	19.94 ± 0.22
HPβCD-NF2	71.23 ± 0.65	26.23 ± 0.12	21.45 ± 0.06
HPβCD-NF3	67.56 ± 0.37	10.36 ± 0.02	20.43 ± 0.10
HPβCD-NF4	430.80 ± 0.37	44.10 ± 0.06	25.89 ± 0.05
HPβCD-NF5	507.00 ± 0.00	41.34 ± 0.10	27.98 ± 0.06

Formulation code	Elongation at break (%) ± SD (n=3)	Tensile strength (MPa) ± SD (n=3)	Wetting time (s)	Contact angle (º) ± SD (n=3)	Mean fiber diameter (nm) ± SD (n=20)	Porosity (%)
HPβCD-NF1	13.72 ± 0.40	1.22 ± 0.28	20	46.95 ± 6.79	917.95 ± 152.15	54.09
HPβCD-NF2	14.95 ± 1.43	1.64 ± 0.15	<1	0	757.95 ± 88.46	61.92
HPβCD-NF3	11.86 ± 1.31	0.74 ± 0.08	1	0	1143.85 ± 153.34	53.72
HPβCD-NF4	74.18 ± 13.06	0.48 ± 0.06	1-2	0	1593.45 ± 352.28	56.72
HPβCD-NF5	5.36 ± 1.46	0.92 ± 0.08	20	61.44 ± 12.83	1814.05 ± 326.12	51.00

FORMULATION	CONTACT ANGLE (⁰)	DISINTEGRATION TIME (Sec)	MICROSCOPE IMAGE (40X)
1	0	12-13	Fiber formation was observed. Beads have formed in the fibers.
3	0	<1	The fibers are in good condition. Very little impurities and bead formation was observed.
6	0	14-15	The fibers are in good condition, there is no bead formation.
7	0	34-35	The fibers are in good condition, there is no bead formation.
8	0	<1	The fibers are in good condition, there is no bead formation.
9	0	<1	The fibers are in good condition. The fiber density was high. No bead formation was observed.
10	0	<1	The fibers are in good condition, there is no bead formation.
11	0	<1	The fibers are in good condition, there is no bead formation.
12	0	<1	The fibers are in good condition, there is no bead formation.
13	0	4-5	Fiber formation was observed. Beads have formed in the fibers.
14	0	20	The fibers are not in good condition. There is bead formation.
15	0	60	The fibers are not in good condition. There is bead formation.
16	0	3-4	The fibers are in good condition, there is no bead formation.
17	0	6-7	The fibers are in good condition, there is no bead formation.
18	0	5-6	The fibers are in good condition, there is no bead formation.
19	0	8-9	The fibers are in good condition, there is no bead formation.
20	0	60-65	The structure of the fibers is bad, there is no bead formation.
21	0	9-10	The fibers are in good condition, there is no bead formation.
22	0	1-2	The fibers are in good condition, there is no bead formation.
23	0	6-7	The structure of the fibers is not good, Fibers are very tight, No bead formation.
24	0	<1	The structure of the fibers is not good, Fibers are very tight, No bead formation.
25	0	<1	The structure of the fibers is not good, Fibers are very tight, No bead formation.
26	0	60	The structure of the fibers is not good, Fibers are very tight, No bead formation.
27	0	8-9	There are polymeric impurities in the fibers, there is no bead formation.
28	0	11-12	The fibers are in good condition, There is little bead formation in the fibers
29	48,37	50	Fiber formation is observed, the condition of the fibers is bad, there are beads and impurities.
30	0	2-3	The fibers are in good condition, with very little beading.
31	0	4-5	The fibers are in good condition, there is no bead formation.
43	0	4-5	The fibers are in good condition, there is no bead formation.
44	0	1-2	The fibers are in good condition, there is no bead formation.
45	0	5-6	The fibers are in good condition, there is no bead formation.

Code	PVP K90 (%)	PEO N80 (%)	Soluplus (%)	Solutol HS-15 (%)	Labrafil M 1944 cs (%)	Gelucire 44/14 (%)	Gelucire 50/13 (%)	Gelucire 48/16 (%)
1	5	-	10	-	-	-	-	-
2	5	10	-	-	-	-	-	-
3	10	-	-	-	-	-	-	-
4	10	5	-	-	-	-	-	-
5	10	10	-	-	-	-	-	-
6	10	-	5	-	-	-	-	-
7	10	-	10	-	-	-	-	-
8	10	-	-	5	-	-	-	-
9	10	-	-	10	-	-	-	-
10	10	-	-	-	5	-	-	-
11	10	-	-	-	10	-	-	-
12	10	-	-	-	-	5	-	-
13	10	-	-	-	-	10	-	-
14	10	-	-	-	-	-	5	-
15	10	-	-	-	-	-	10	-
16	10	-	-	-	-	-	-	5
17	10	-	-	-	-	-	-	10
18	-	5	10	-	-	-	-	-
19	-	10	-	-	-	-	-	-
20	-	10	5	-	-	-	-	-
21	-	10	10	-	-	-	-	-
22	-	10	-	5	-	-	-	-
23	-	10	-	10	-	-	-	-
24	-	10	-	-	5	-	-	-
25	-	10	-	-	10	-	-	-
26	-	10	-	-	-	5	-	-
27	-	10	-	-	-	10	-	-
28	-	10	-	-	-	-	5	-
29	-	10	-	-	-	-	10	-
30	-	10	-	-	-	-	-	5
31	-	10	-	-	-	-	-	10
32	-	-	10	-	-	-	-	-
33	-	-	10	5	-	-	-	-
34	-	-	10	10	-	-	-	-
35	-	-	10	-	5	-	-	-
36	-	-	10	-	10	-	-	-
37	-	-	10	-	-	5	-	-
38	-	-	10	-	-	10	-	-
39	-	-	10	-	-	-	5	-
40	-	-	10	-	-	-	10	-
41	-	-	10	-	-	-	-	5
42	-	-	10	-	-	-	-	10
43	10	5	-	-	-	-	-	-
44	5	10	-	-	-	-	-	-
45	10	10	-	-	-	-	-	-

Formulation Code	Tolvaptan (%)	PVP K90 (%)	PEO N80 (%)	Solutol HS-15 (%)	Gelucire 44/14 (%)	Solvent	Feed rate (mL/h)	Voltage (kV)	Tip-to- -collector distance (cm)
1	2	10	-	-	-	100% Methanol	4.5	16	10
2	2	10	-	5	-	100% Methanol	2	16	10
3	2	10	-	-	5	100% Methanol	4	16	10
4	2	-	10	5	-	80% Metha- nol-20% distilled water	8	16	15
5	2	5	10	-	-	80% Metha- nol-20% distilled water	7.5	16	15

Code	PVP K90 (%)	PEO N80 (%)	Soluplus (%)	Solutol HS-15 (%)	Labrafil M 1944 cs (%)	Gelucire 44/14 (%)	Gelucire 50/13 (%)	Gelucire 48/16 (%)
1	5	-	10	-	-	-	-	-
2	5	10	-	-	-	-	-	-
3	10	-	-	-	-	-	-	-
4	10	5	-	-	-	-	-	-
5	10	10	-	-	-	-	-	-
6	10	-	5	-	-	-	-	-
7	10	-	10	-	-	-	-	-
8	10	-	-	5	-	-	-	-
9	10	-	-	10	-	-	-	-
10	10	-	-	-	5	-	-	-
11	10	-	-	-	10	-	-	-
12	10	-	-	-	-	5	-	-
13	10	-	-	-	-	10	-	-
14	10	-	-	-	-	-	5	-
15	10	-	-	-	-	-	10	-
16	10	-	-	-	-	-	-	5
17	10	-	-	-	-	-	-	10
18	-	5	10	-	-	-	-	-
19	-	10	-	-	-	-	-	-
20	-	10	5	-	-	-	-	-
21	-	10	10	-	-	-	-	-
22	-	10	-	5	-	-	-	-
23	-	10	-	10	-	-	-	-
24	-	10	-	-	5	-	-	-
25	-	10	-	-	10	-	-	-
26	-	10	-	-	-	5	-	-
27	-	10	-	-	-	10	-	-
28	-	10	-	-	-	-	5	-
29	-	10	-	-	-	-	10	-
30	-	10	-	-	-	-	-	5
31	-	10	-	-	-	-	-	10
32	-	-	10	-	-	-	-	-
33	-	-	10	5	-	-	-	-
34	-	-	10	10	-	-	-	-
35	-	-	10	-	5	-	-	-
36	-	-	10	-	10	-	-	-
37	-	-	10	-	-	5	-	-
38	-	-	10	-	-	10	-	-
39	-	-	10	-	-	-	5	-
40	-	-	10	-	-	-	10	-
41	-	-	10	-	-	-	-	5
42	-	-	10	-	-	-	-	10
43	10	5	-	-	-	-	-	-
44	5	10	-	-	-	-	-	-
45	10	10	-	-	-	-	-	-

Code	PVP K90 (%)	PEO N80 (%)	Soluplus (%)	Solutol HS-15 (%)	Labrafil M 1944 cs (%)	Gelucire 44/14 (%)	Gelucire 50/13 (%)	Gelucire 48/16 (%)
1	5	-	10	-	-	-	-	-
2	5	10	-	-	-	-	-	-
3	10	-	-	-	-	-	-	-
4	10	5	-	-	-	-	-	-
5	10	10	-	-	-	-	-	-
6	10	-	5	-	-	-	-	-
7	10	-	10	-	-	-	-	-
8	10	-	-	5	-	-	-	-
9	10	-	-	10	-	-	-	-
10	10	-	-	-	5	-	-	-
11	10	-	-	-	10	-	-	-
12	10	-	-	-	-	5	-	-
13	10	-	-	-	-	10	-	-
14	10	-	-	-	-	-	5	-
15	10	-	-	-	-	-	10	-
16	10	-	-	-	-	-	-	5
17	10	-	-	-	-	-	-	10
18	-	5	10	-	-	-	-	-
19	-	10	-	-	-	-	-	-
20	-	10	5	-	-	-	-	-
21	-	10	10	-	-	-	-	-
22	-	10	-	5	-	-	-	-
23	-	10	-	10	-	-	-	-
24	-	10	-	-	5	-	-	-
25	-	10	-	-	10	-	-	-
26	-	10	-	-	-	5	-	-
27	-	10	-	-	-	10	-	-
28	-	10	-	-	-	-	5	-
29	-	10	-	-	-	-	10	-
30	-	10	-	-	-	-	-	5
31	-	10	-	-	-	-	-	10
32	-	-	10	-	-	-	-	-
33	-	-	10	5	-	-	-	-
34	-	-	10	10	-	-	-	-
35	-	-	10	-	5	-	-	-
36	-	-	10	-	10	-	-	-
37	-	-	10	-	-	5	-	-
38	-	-	10	-	-	10	-	-
39	-	-	10	-	-	-	5	-
40	-	-	10	-	-	-	10	-
41	-	-	10	-	-	-	-	5
42	-	-	10	-	-	-	-	10
43	10	5	-	-	-	-	-	-
44	5	10	-	-	-	-	-	-
45	10	10	-	-	-	-	-	-