AUC ₀ ^∞ : Total area under the curve, AUMC₀^∞: Total area under the first moment curve, BCS: Biopharmaceutics classification system, cGMP: Cyclic guanosine monophosphate, CL/F: Apparent total clearance, C_max: Maximum plasma concentration, DSC: Differential scanning calorimetry, F: Bioavailability, FDA: Food and Drug Administration, FTIR: Fourier transform infra-red spectroscopy, HPLC: High-performance liquid chromatography, IS: Internal standard, LTZ: Letrozole, NCs: Nanocrystals, NO: Nitic oxide, PAH: Pulmonary arterial hypertension PDE5: Phosphodiesterase 5, PDI: Polydispersity index, PKG: protein kinase G, PVA: Polyvinyl alcohol, SEM: Scanning electron microscope, TDA: Tadalafil, t_max: Time to maximum concentration, t_0.5: Terminal half-life, λ_z: Terminal elimination rate constant, XRPD: X-Ray powder diffraction.

Tadalafil (TDA) pure standard (Polpharma; Poland) was a kind donation from Tabuk Pharmaceuticals Co.; Jordan. Letrozole (LTZ) internal standard (Sigma-Aldrich, Germany) HPLC-grade acetonitrile and dimethyl sulfoxide (DMSO) were purchased from Fisher, China. Potassium dihydrogen phosphate (AZ chem; Spain), Tween 80 (polyoxyethylene sorbitan monooleate) (BBC Chemical; China), polyvinyl alcohol 125 KDa (PVA) (Alpha Chemika; India), glucose anhydrous (Sigma-Aldrich; USA), Pluronic F68 (polyoxy-ethylene-polyoxypropylene block copolymer) (Oakwood Chemical; US), Trehalose (Combi Blocks; USA), and phosphate buffered saline (PBS) were obtained from Al Takamul Company and deionized (DI) water.

TDA NCs were prepared by the sonoprecipitation technique. The experimental procedure starts initially with preparing the organic phase by dissolving 200 mg of TDA in 10 mL DMSO, and the resulting solution of TDA (20 mg/mL) was filtered through a syringe filter (nylon membrane; 0.45 μm). The aqueous (antisolvent) phase was prepared by dissolving 2 g of the cryoprotectant in 25 mL DI water, where glucose or trehalose was used by dissolving. A reference formulation was prepared without using any cryoprotectant. Then different amounts of stabilizer were added according to the ratio of TDA: stabilizer (1:2) and (1:4) by dissolving 1 g and 2 g of the stabilizer, respectively. Tween, PVA, or Pluronic F68 were the stabilizers tested in this work, and they were added to the 25 mL of DI water and stirred at 500 rpm at room temperature for 30 min, except PVA was stirred at 60 °C for one hour. The solutions were filtered through a syringe filter with a 0.45 μm nylon membrane. Then the organic phase was added dropwise at a rate of 0.5 mL/min to the aqueous phase in a volume ratio of 1:5 under magnetic stirring at 500 rpm. The formed nanosuspension was ultrasonicated for 10 min at an amplitude of 70% and a cycle of 1 rpm. The nanosuspension was stirred overnight to evaporate DMSO. The obtained nanosuspensions were centrifuged at 16000 rpm for 30 min and washed three times with DI water. The pellets were collected and lyophilized using a freeze dryer. The dry powder was collected and stored in a desiccator for further use. The control formula (F0) is a raw TDA prepared in the same process without any stabilizers or cryoprotectants. The contents of each formula are summarized in Table 1.

The prepared nanocrystals were characterized in terms of encapsulation efficiency%, mean particle size (PS), polydispersity indices (PDI), and zeta potential. The stability of the drug was also investigated. Further, the formation of the nanocrystals was confirmed using FTIR, XRD, DSC, and SEM. Finally, the drug solubility and release in vitro were assessed.

The mean particle size, polydispersity indices, and zeta potential of the prepared nanocrystals were analyzed using a Malvern zeta sizer (Malvern Instruments, Malvern, UK). The particle size and polydispersity indices were determined by dispersing lyophilized nanocrystals in DI water. The zeta potential was calculated via the zeta sizer-nano software. All measurements were carried out in triplicate.

The saturation solubility of pure TDA and formulated NCs was assessed in DI water at 37 °C for 48 h. Excess amounts of pure TDA and from each formulation were added to 10 mL of DI water using an Erlenmeyer flask, then mechanically shaken using a shaking water bath. The supernatant was filtered by a syringe filter (nylon membrane; 0.45 μm) and analyzed for TDA concentration. Each formula was assessed in triplicate. The concentration of TDA was measured using high-performance liquid chromatography coupled with a UV detector (HPLC-UV) at 285 nm wavelength. A C18 column (150 mm Å~ 4.6 mm, 5 μm) (Fortis; United Kingdom) was used for separation at room temperature. The mobile phase is composed of potassium dihydrogen phosphate buffer and HPLC-grade acetonitrile at a volume ratio of 50:50 v/v at a flow rate of 1.3 mL/min (Bojanapu et al. 2015). The calibration curve was constructed in the range of 0.39–100 µg/mL with good linearity (r² = 0.9999).

An accurately weighed amount of the lyophilized powder of each formula was dissolved in 10 mL DMSO. The concentration of dissolved TDA was determined using the HPLC-UV method described previously. The encapsulation efficiency% was calculated using equation (1) (Shen et al. 2017).

$Encapsulationefficiency\%=\frac{Actualamount}{Theorticalamount}\times 100\%$ Eqt. (1)

In vitro dissolution of pure TDA and TDA NCs was determined using apparatus II (paddle) according to the USP dissolution testing method of TDA with some modifications. Pure TDA (5 mg) and selected TDA NCs equivalent to 5 mg were added to 900 mL of dissolution media. The dissolution media was 0.156 M phosphate buffered saline (PBS) (pH 7.4), the stirring speed was 50 rpm, and the bath temperature was 37 ± 0.5 °C. Then, 1.5 mL samples were withdrawn automatically at 0.16, 0.33, 0.5, 0.75, 1, 1.5, 2, 4, 6, 8, 24, and 48 h and immediately replenished with the same volume of PBS. All samples were filtered through a syringe filter (nylon membrane, 0.45 μm) and analyzed via the HPLC-UV method described previously. The release profile was constructed between the percentage drug release and time for both pure TDA and selected TDA NCs. All measurements were performed in triplicate.

The FTIR spectra of TDA, selected stabilizer, selected nanocrystals, and their physical mixture were recorded using the FTIR spectrometer (Shimadzu, Kyoto, Japan). The physical mixture was prepared by mixing a specific amount of TDA with a stabilizer using the same ratio as in the formula preparation. The analysis was conducted using the KBr disc technique. A small amount of each sample was mixed with KBr, pressed into disks, and scanned from 400 to 4000 cm^-1. FTIR spectroscopy tests were conducted to ensure the stability and compatibility of TDA with other formulation components.

TDA, selected stabilizer, selected nanocrystals, and their physical mixture were tested using an X-ray powder diffractometer (Ultima IV X-ray diffractometer, Rigaku, Japan). The powders were analyzed using cobalt radiation at a voltage of 40 kV and a current of 40 mA. Diffraction angles (2θ) were varied from 3° to 60°. Step scan mode was used in the analysis with a step size of 0.02° at a speed of 3.0 deg/min. An XRPD test was conducted to study the crystallinity of TDA and the stability of the formula.

The thermal analysis was performed using a differential scanning calorimeter (DSC-204, Netzsch; Germany). Approximately 3 mg of TDA, selected stabilizer, selected nanocrystals, and their physical mixture were placed in an aluminum pan. The heating scans were performed from room temperature to 400 °C at heating and cooling rates of 10 °C/min in a dry N₂ atmosphere. An empty aluminum pan was used as a reference. DSC tests were conducted to ensure the stability and compatibility of TDA with other formulation components.

In this work, scanning electron microscopy (SEM) on a FEI Quanta 450 FEG SEM (FEI) was used to examine the surface morphology of the NCs. Before being examined, samples of lyophilized TDA NCs were placed on metal stubs and vacuum-coated with gold.

The selected TDA nanocrystal formula was stored at a room temperature of about 25 ± 0.5 °C and accelerated conditions of 4 ± 0.5 °C and 40 ± 0.5 °C in constant climate chambers. Then the particle size, polydispersity, and saturation solubility were evaluated on days 1, 30, 60, and 90 under the same procedures mentioned above.

All experiments were carried out according to the guidelines of the animal care and use committee (ACUC) of Jordan University of Science and Technology (JUST, Jordan). Male Sprague Dawley (SD) rats (n = 8; 300–350 g) were obtained from the animal house at JUST, Jordan. Rats were kept in a clean place at a room temperature of 25 ± 2 °C with a 12-hour light/dark cycle and ~50% relative humidity. The animals were acclimatized to laboratory conditions for a week before the beginning of the experiments, and they were provided with water and a standard rat chow diet ad libitum. All the procedures were followed according to the guidelines of the ACUC of JUST. Rats were randomly divided into two groups (n = 4). The first group received the chosen TDA formulation intranasally, and the second group received pure TDA intranasal. Tadalafil dose is calculated according to the following equation (2):

Animal dose (mg/kg) = HED × (Km_human ÷ Km_rat). Eqt. (2)

Where HED is the human equivalent dose in mg/kg, the Km_human factor for human adults equals 37, and the Km_rat factor for rats equals 7 (Nair and Jacob 2016). Accordingly, the dose administered in all groups will be equivalent to 4 mg/kg of TDA.

The first group of rats (n = 4) was given a single dose of TDA NCs (F11). The dose administered is equivalent to 4 mg/kg of TDA; the powder was suspended in 100 μL of 2% PEG400 and 0.02% Tween 80, and 50 μL of the resulting suspension was given in each nostril using a micropipette tip (Acharya et al. 2013; Erdő et al. 2018). Blood samples (150–200 μL) were collected using EDTA-treated plasma tubes at different time points of 1, 2, 4, 5, 6, 6.5, 7, 7.5, 8, and 24 h, while the second group of rats (n = 4) was given a single dose of pure TDA at a dose of 4 mg/kg suspended in the same vehicle used in the preparation of TDA NCs dose, and the sampling points were at 1, 2, 3.5, 4.5, 5, 6, 7, 8, and 24 h. Plasma samples were prepared by centrifugation (600 ×g) of the blood samples for 15 min, which then was stored at -80 °C until analyzed. Sample analysis was performed using HPLC-UV at 285 nm wavelength. A C18 column (150 mm Å~ 4.6 mm, 5 μm) (Agilent; US) was used at 35 °C. The mobile phase is composed of potassium dihydrogen phosphate buffer and HPLC-grade acetonitrile at a volume ratio of 63:37 v/v and was used at a flow rate of 1.3 mL/min. The drugs were extracted from the plasma using the direct precipitation method. In this extraction process, HPLC-grade acetonitrile (120 μL) containing the IS (LTZ, 40 µg/mL) was added to each tube of plasma. After that vortexing for 30 seconds, the samples were centrifuged for 5 minutes at 16000 rpm. After which, 150 μL of the supernatant was removed and reconstituted using 50 μL of DI water. This analysis method was validated in terms of specificity, selectivity, recovery, precision, accuracy, and linearity over the range between 10 and 10000 ng/mL according to US Food and Drug Administration guidelines (FDA 2022).

Statistical analysis was performed using GraphPad software (Prism 9.4.3); an unpaired t-test was done, and a p-value < 0.05 was considered significant. Data were presented as mean ± SD, unless otherwise specified (n = 3–4).

The particle size, polydispersity index, and zeta potential of the control formula (F0) and different TDA NC formulations are summarized in Table 1. The prepared formulas showed a wide variation in particle size and zeta potential. F11 had the smallest particle size (196.50 ± 3.30) with PDI (0.21 ± 0.01), while F15 had the largest particle size (1089.00 ± 73.00) with a PDI of (0.26 ± 0.01). The control formula (F0) had a particle size of 688.80 ± 52.60 and a PDI of 0.34 ± 0.10. All formulas were nanosized (less than 1000 nm) with a range between 196.50 ± 3.30 and 779.60 ± 9.00 nm and had PDI in the range of 0.19 ± 0.01–0.42 ± 0.00, except F15 had a particle size of (1089.00 ± 73.00). Unpaired t-test showed that all formulas have significantly lower sizes than F0, except F9, F15, and F16, which had significantly higher particle sizes than the control formula at p-value < 0.05. Moreover, the results showed that increasing the concentration of the stabilizer significantly increases the particle size. PVA-stabilized particles have significantly lower particle sizes than those stabilized by tween 80, as F2 compared to F8, F3 compared to F9, and F5 compared to F11. In addition, all PVA-stabilized NCs have a significantly lower particle size than those stabilized by Pluronic F68. Fig. 2. shows the effect of stabilizers on TDA NCs’ particle size. This difference in particle size among different formulations was related in literature to many factors that are mostly related to the efficiency of the stabilizer in stabilizing the NCs. It is well known that the interaction forces between the drug surface and the stabilizer (hydrogen binding, hydrophilic/hydrophobic or ionic interaction), the steric barrier created by the stabilizers’ chain length, and the percentage of stabilizer that covers the NC’s surface are all affecting the stabilizer performance (Liu et al. 2015; Tuomela et al. 2016). PVA is rich in hydroxyl groups within its structure, which engage in hydrogen bonding with the carbonyl group in the TDA structure. Such interactions enhance the adsorption of PVA polymer on the hydrophobic surface of TDA, resulting in further electrostatic and steric stabilization that prevent crystal growth and agglomeration (Raghavan et al. 2001; Douroumis and Fahr 2007; Lee et al. 2010). On the other hand, tween 80 acts as an electrostatic stabilizer, stabilizing the nanoparticles by countering the Vander Waals attractions between drug particles, which might be less effective in stabilizing TDA (Jassim and Hussein 2014). In addition, the higher viscosity of PVA compared to Tween 80 also plays a role in determining the particle size. The increased viscosity restricts the mobility of particles, thereby reducing the frequency of particle collisions, which contributes to a reduction in the final particle size (Dalvi and Dave 2009). While the Pluronic F68 is a block co-polymer of the hydrophobic polypropylene oxide group (PPO). The hydrophobic part of the polymer makes interaction with the drug surface, while hydrophilic polyethylene oxide (PEO) provides steric hindrance against aggregation (Cerdeira et al. 2013). Despite this interaction, larger particles were obtained in comparison to the PVA-stabilized NCs (Sinha et al. 2013; Jassim and Hussein 2014).

Figure 2. 

Effect of stabilizers on the particle size of tadalafil nanoparticles (TDA NCs). The y-axis represents: [(TDA: stabilizer); (cryoprotectant)]. Glu: glucose, Tre: trehalose. Data: mean ± SD (n = 3).

An increase in the concentration of the stabilizer resulted in increased particle size and a subsequent decrease in solubility. This is due to the flocculation phenomenon of nanoparticles at high stabilizer concentrations reported and confirmed by (Terayama et al. 2004; Dalvi and Davé 2009). This phenomenon also explains why the stabilizers failed to stabilize nanoparticles when used at high concentrations (Terayama et al. 2004; Dalvi and Davé 2009). Fig. 3 shows the effect of stabilizer concentration on TDA NC particle size.

Figure 3. 

Effect of stabilizer concentration on the particle size of tadalafil nanoparticles (TDA NCs). The y-axis represents: [Stabilizer; (cryoprotectant)]. Glu: glucose, Tre: trehalose. Data: mean ± SD (n = 3).

In addition, it was found that most formulas prepared using cryoprotectants have lower particle sizes than those prepared without using cryoprotectants. The cryoprotectant’s role in decreasing particle size is decreasing the aggregation of NPs that results from the dehydration process during freeze-drying. It can be noticed that formulas prepared using trehalose as a cryoprotectant have significantly lower particle sizes than those prepared using glucose. Trehalose is more efficient than other sugars as a cryoprotectant due to its lack of internal hydrogen bonding, low hygroscopicity, low chemical reactivity, and high glass transition temperature (Tg) (Crowe et al. 1996). This can be translated as glucose failing to protect the TDA NCs from aggregation, as there was a distinct increase in average particle size observed at the same concentration of stabilizer. However, the competition between cryoprotectants and stabilizers for particle surfaces provides an additional rationale for the inability of stabilizers to stabilize nanoparticles in the presence of either glucose or trehalose in some formulas.

The PDI of the nanocrystals range was found to be between 0.19 ± 0.01 and 0.42 ± 0.00, which indicates a narrow size distribution. Narrow size distribution is essential to ensure the physical stability and prevention of particle growth (Kabalnov 2001). The zeta potential of F0 is -15.00 ± 2.40 mV, while the zeta potential of prepared nanocrystals ranged from -2.99 ± 0.17 to -21.80 ± 1.41 mV. Finally, formulas with cryoprotectant glucose or trehalose have significantly lower zeta potential than those not containing cryoprotectant. Even though the non-ionic stabilization effect of PVA, Tween 80, and Pluronic F68 results in masking the negative charge of TDA, the adsorption of the cryoprotectant on the surface of nanocrystals shielded the TDA negative charge more (Verma et al. 2009).

The solubility of pure TDA in DI water at 37 °C (1.71 ± 0.04 μg/mL) was insignificantly different from the control formula’s solubility (1.93 ± 0.02 μg/mL). The solubility of prepared NCs is summarized in Table 1. F11 had the highest solubility (9.37 ± 0.36 μg/mL), about 5.5-fold greater than pure TDA. F15 has the lowest solubility (1.8 ± 0.19 μg/mL). In general, PVA-stabilized NCs have a significantly higher solubility when compared to tween 80 and Pluronic F68-stabilized NCs (Khadka et al. 2014).

The enhancement in the solubility of NCs could be related to the reduction of particle size that increases the surface area. Increasing surface area increases the wettability and the contact between the NCs and the dissolving medium (Sun et al. 2012). In addition, this enhancement in solubility may be partially related to the wetting and solubilizing effects of used stabilizers. This phenomenon also may explain the difference in saturation solubility for nearly similar-sized particles. Further, this reduction in particle size may be related to the partial decrease in crystallinity of TDA NCs. Therefore, it can be noticed that as the concentration of the stabilizer increases, the solubility decreases, which is related to the increase in the particle size (Dalvi and Dave 2009). Terayama et al. (2004) also observed the flocculation phenomenon, where the micelles formed at high concentrations of stabilizer leave the drug particles unprotected, which leads to a decrease in particle solubility (Terayama et al. 2004). Furthermore, it was observed that all formulas with trehalose have a higher solubility than others, and this may be due to the re-dispersibility of NCs, which is the ability of dried nanoparticles to return to their original state in an aqueous system, in which the type and concentration of the cryoprotectants have a major role in the re-dispersibility process (Lee et al. 2009). F11 had the lowest particle size and the highest solubility and therefore was selected for further characterization. Fig. 4. shows the effect of stabilizers on TDA NC saturation solubility.

Figure 4. 

Effect of stabilizers on tadalafil nanoparticles’ (TDA NCs) saturation solubility. The y-axis represents: [(TDA: stabilizer); (cryoprotectant)]. Glu: glucose, Tre: trehalose. Data: mean ± SD (n = 3).

The encapsulation efficiency of TDA NCs was measured in triplicate. Mean encapsulation efficiency % values ranged from 98.90 ± 2.8% to 103.1 ± 2.7% for the prepared formulas. The encapsulation efficiency of F11 was 99.16 ± 4.91%. t-test showed no significant differences between the mean actual and theoretical amount of drug in all formulas.

The dissolution profiles of pure TDA and the selected formula (F11) are shown in Fig. 5. F11 had 50.11 ± 1.69% in vitro release after 6 h and 57.70 ± 4.6% after 48 h in PBS, while pure TDA exhibited 30.90 ± 2.65% and 45.30 ± 2.75% release after 6 and 48 h, respectively. A significant increase in the drug release from F11 over all time points when compared to pure TDA. Further, 1.66 and 1.3-fold improvement of drug release for F11 over the pure drug at 6 and 48 h, respectively.

Figure 5. 

Dissolution profiles of pure TDA and TDA NC (F11) in PBS at 37 °C for 48 h. Data: mean ± SD (n = 3).

FTIR spectroscopy was used to examine pure TDA, PVA, physical mix, and optimized nanocrystal (F11) to evaluate if there is any chemical interaction or incompatibility between TDA and the stabilizer. The FTIR spectrum is shown in Fig. 6. For pure TDA, the main characteristic peaks were detected at 748 cm^-1 for the benzene ring, 1041 cm^-1 for (C-O-C) stretching, 1435 cm^-1 for (C-N) stretching, 1651 cm^-1 for (C=C) aromatic, 1682 cm^-1 for (C=O) carbonyl stretch, 2904 cm^-1 for (C-H) stretching, and 3327 cm^-1 for (N-H) stretching (Sharma et al. 2018). For PVA, the broadband in the 3000–3700 cm^-1 region is related to (O-H) stretching, 2360 cm^-1 is related to (CH₂) stretching, 1700 cm^-1 is related to (C=O) carbonyl stretch, 1650 cm^-1 is related to (C=C) stretching, 800 cm^-1 is related to (C-C) stretching, and 486 cm^-1 is related to (C-O) bending (Omkaram et al. 2007). The physical mixture spectra represented the peak of the functional groups of both TDA and PVA. The decreased intensity of the peaks of TDA may be due to the dilution effects of PVA. This could emphasize the lack of interaction between the TDA and PVA in the physical mixture. The FTIR spectra of TDA NCs (F11) showed the presence of all TDA main peaks. The peak at 3327 cm^-1 of (N-H) amine slightly shifted to 3325 cm^-1 with a decrease in its intensity; these changes may be due to hydrogen bond formation between the amine in TDA and hydroxy group (OH) in PVA. This type of interaction between TDA and polymers was illustrated by Bhokare et al. (Bhokare et al. 2015). Furthermore, the peak at 1041 cm^-1 was shifted to 1044 cm^-1 in NCs; this shifting might also be due to hydrogen bonding between the (C-O-C) group of TDA and the (OH) group of PVA; this effect of hydrogen bonding on FTIR spectra was previously proven by Athokpam et al. (Athokpam et al. 2017). No new peaks were observed in F11 spectra, which indicates that there is no chemical interaction between TDA and PVA.

Figure 6. 

FTIR spectra of pure TDA, PVA, physical mixture (PM), and tadalafil nanocrystal (TDA NC; F11).

The DSC thermographs of pure TDA, PVA, physical mixtures, and nanocrystals (F11) are shown in Fig. 7. The DSC thermogram of pure TDA showed a sharp endothermic peak at 302 °C, representing the TDA crystal melting point as reported by (Butarbutar et al. 2020). While PVA showed an endothermic peak at 194 °C, representing the PVA melting point as reported by (Zhu et al. 2020). The physical mixture showed two endothermic peaks at 194 and 302 °C representing the PVA and TDA melting points, respectively, indicating good stability and the absence of incompatibility between the TDA and the stabilizer. The F11’s DSC thermogram showed an endothermic peak at 270 °C that corresponds to the melting point of TDA NC; this reduction in the melting point may be due to the combined effect of the decrease in particle size and decrease in the crystallinity degree of TDA (Bhokare et al. 2015).

Figure 7. 

DSC thermograms of pure TDA, PVA, physical mixture (PM), and tadalafil nanocrystal (TDA NC; F11).

X-ray diffraction was performed to determine the crystalline structure of the prepared NCs. The XRPD patterns of pure TDA, PVA, physical mixtures, and NCs (F11) are presented in Fig. 8. The XRPD pattern of pure TDA shows sharp and intense diffraction peaks at 7, 10, 14, 16, 18, 22, and 24 2θ. These diffraction peaks are characteristic peaks of TDA in the crystalline form (Rad et al. 2017). While PVA showed broad diffraction peaks, which indicates the polymers’ amorphous nature (Aziz et al. 2017). The main TDA’s diffraction peaks are detected in a physical mixture with lower intensity due to the dilution effect of the stabilizer. The XRPD patterns of F11 showed all main diffraction peaks of TDA with lower intensity. This change in peak intensity is most probably attributed to the decrease in the particle size and/or the change in crystallinity of TDA. The change in crystallinity might be due to the proportion of PVA and its interaction with TDA in the formulation. These results are in good agreement with the DSC results and the changes in structure noticed on SEM below.

Figure 8. 

XRD patterns of pure TDA, PVA, physical mixture (PM), and tadalafil nanocrystal (TDA NC; F11).

SEM images of the pure TDA and selected TDA NCs are shown in Fig. 9. The pure TDA exhibited a mixture of needle-like and cubic crystals with a wide particle size distribution, and the smaller particles are shown to form aggregates. On the other hand, the formulated TDA NCs using PVA (F11) showed uniformly distributed needle-like crystals that are smaller in size in comparison to the pure TDA.

Figure 9. 

Scanning electron micrographs of (A) pure TDA and (B) TDA NCs F11.

The stability of TDA NCs (F11) was determined by examining the particle size, PDI, and solubility at days 1, 30, 60, and 90 at three different temperatures. The formula was stored at 25 ± 0.5 °C, 4 ± 0.5 °C, and 40 ± 0.5 °C. The particle size and PDI of F11 on day 1 were 181.6 ± 12 nm and 0.23 ± 0.014, respectively. The saturation solubility of F11 at day 0 was 9.63 ± 0.37 μg/mL. Table 2 shows the results of the particle size, PDI, and solubility of TDA NCs (F11) at different times and conditions.

Long-term stability study data indicated that F11 could retain its stability for 3 months upon storage at 4 ± 0.5 °C; the unpaired t-test shows no significant difference in size, PDI, or solubility.

While at 25 ± 0.5 °C, the size of the stored formula significantly increases after 60 and 90 days of storage. Further, the PDI of these formulations was not affected at days 30 and 60 but significantly increased after 90 days. The solubility of the stored formula starts to decrease but insignificantly till day 90, where the decrease was significant.

Finally, the storage of F11 at 40 ± 0.5 °C significantly affected the particle size, PDI, and solubility at all-time points; the size was increased by 1.58, 1.63, and 1.7-fold at days 30, 60, and 90, respectively. Moreover, the solubility was decreased by 14, 15.5, and 15.8% at days 30, 60, and 90, respectively. In addition, the PDI of the stored formula was increased over the 3 months of storage.

Although the stored nanoparticles of formula F11 suffered initial flocculation during the second and third months of storage at room temperature and the first month under stress conditions, the flocculated particles retained their particle size within the nano-range over the 3-month storage period and can maintain their enhancement in solubility over this period.

TDA plasma concentration versus time profiles of both the pure TDA and TDA NCs (F11) after intranasal administration in SD rats are shown in Fig. 10. The pharmacokinetic parameters for pure TDA and TDA NCs are summarized in Table 3.

Figure 10. 

Tadalafil (TDA) plasma concentration vs. time profiles of pure TDA (in black squares) and TDA nanocrystals (TDA NCs; F11) (in red circles) following intranasal administration. Data: mean ± SE (n = 4).

The pharmacokinetic parameters of TDA NCs (F11) showed a significant increase (p < 0.05) in C_max, AUC₀^∞, AUMC₀^∞, MRT, t_max, and t_0.5 with 1.4, 2.3, 3.3, 1.4, 1.3, and 1.93-fold, respectively, when compared to the pure TDA. The apparent clearance (CL/F) of TDA NCs significantly decreased (p < 0.05) by 57.14% when compared to TDA. The in vivo PK parameters show higher C_max, and AUC₀^∞ for TDA NCs, this could be a result of the previously shown enhancement in TDA’s solubility and dissolution when formulated as NCs, thus its extent of absorption. This could be caused by the nanosized and uniformed TDA NCs particles when compared to pure TDA. As mentioned earlier, when the particle size is smaller, more solubilization of particles in aqueous media is happening, and a predicted higher absorption of nanoparticles through the thin endothelial layer of the nasal cavity to the systemic circulation (Clementino et al. 2021). In addition to the special characteristic of PVA, as it is a mucoadhesive polymer, that would increase the residence time in the nasal cavity and enhance the contact between the drug and nasal mucosa, thus increasing the drug concentration at the site of deposition (Chowdary and Rao 2004). Furthermore, the PVA has a permeation enhancer effect, which facilitates drug absorption through the mucosa by opening the tight junctions of the epithelial layer, therefore increasing the extent of absorption of TDA NCs through the nasal mucosa when compared to pure TDA (Abd El-Hameed and Kellaway 1997).

In the current study, a longer t_max was observed for TDA NCs compared to pure TDA. The precise reason for this discrepancy warrants further investigation, as different studies have reported varying times to reach maximum concentration. For instance, Zode et al. (2020) examined the pharmacokinetic parameters of celecoxib nanosuspension administered intranasally in rats, finding a significantly faster t_max with the nanosuspension compared to micron-sized particles. However, parameters such as AUC and C_max remained unchanged between the two formulations (Zode et al. 2020). Similarly, another study prepared rufinamide NCs and incorporated them into a thermoresponsive nasal in situ gel. Intranasal administration in rats showed that the rufinamide NCs in situ gel had a substantially shorter t_max than the rufinamide in situ gel without nanocrystallization (Dalvi et al. 2022).

Conversely, other studies reported delayed t_max similar to the current study’s findings, attributing this to factors like the slow release of the drug from the NC formulation and lower free drug levels, influenced by the pH of the nasal cavity and the pH of both the pure drug and NCs (Morgen et al. 2012). Several studies have also demonstrated that nanoparticles can enhance the bioavailability and plasma concentration of drugs compared to their pure forms. They exhibit delayed t_max, increased intranasal retention time, or delayed absorption when administered intranasally in animal models. For example, Ahmad et al. (2022) studied L-Dopa-loaded chitosan nanoparticles and found that these NPs demonstrated higher bioavailability and plasma concentration compared to the pure drug, with a delayed t_max (Ahmad et al. 2022). This delay was attributed to the positive charge of the chitosan nanoparticles, enhancing their interaction with the negatively charged nasal mucosa and the larger size of the nanoparticles compared to the free drug, resulting in increased intranasal residence time and more sustained drug release (Ahmad et al. 2022). Additionally, Al Asmari et al. (2016) investigated the intranasal administration of donepezil in liposomal formulation versus free drug forms and found that the liposomal formulation had significantly higher plasma concentrations and a longer t_max, indicating a more sustained release and prolonged absorption period (Al Asmari et al. 2016). This difference was attributed to the liposomal formulation, which allows for controlled and extended drug release compared to the rapid absorption of the free drug (Al Asmari et al. 2016). It was also demonstrated that solid lipid nanoparticles and nanostructured lipid carriers used for nose-to-brain drug delivery have shown improved drug bioavailability by increasing solubility and permeation, extending drug action, and reducing enzyme degradation. These nanoparticles can also display prolonged nasal cavity residence time and minimize mucociliary clearance, maintaining a prolonged presence and gradual absorption into the brain (Nguyen and Maeng 2022). As mentioned earlier, the presence of PVA in the TDA NCs formulation in the current study likely increases the residence time in the nasal cavity and enhances the contact between the drug and nasal mucosa (Chowdary and Rao 2004), similar to other polymers like HPMC, chitosan, and its derivatives, which exhibit mucoadhesive properties that increase drug retention time and reduce mucociliary clearance (Nguyen and Maeng 2022). Sattar et al. (2017) studied the pharmacokinetic profile of cyadox nanosuspension and compared it to bulk cyadox after oral administration in rats, finding that the t_max for the nanosuspension was unexpectedly delayed, attributed to nanoparticles being absorbed by intestinal lymph vessels and lymph nodes, resulting in extended drug absorption and longer t_max (Sattar et al. 2017). Although this lymphatic interaction was observed following oral administration, its significance in altering pharmacokinetics following intranasal administration cannot be ignored (Lee et al. 2023). The interaction between nanoparticles and the nasal lymphatic system is well documented and used as a lymphatic delivery system (Lee et al. 2023). However, the specific cause of the delayed t_max in our study remains to be determined, necessitating further investigation to pinpoint whether this difference is due to the components of the NCs, their interaction with nasal anatomical and physiological features, or different uptake and transport pathways compared to pure TDA.

Although the delayed t_max observed with TDA NCs compared to pure TDA presents a drawback in the context of treating erectile dysfunction, where rapid onset of action is preferred by patients. However, the prolonged duration of action post-dose with tadalafil could be advantageous (Huang and Lie 2013). The prolonged period of effectiveness of tadalafil could also be advantageous in the treatment of conditions such as pulmonary artery hypertension and benign prostate hyperplasia (Van Driel 2006), which are approved indications of TDA. On the other hand, the extended residence time of TDA at a sustained high concentration offers potential benefits in the treatment of erectile dysfunction, as it helps maintain efficacy over a longer period, potentially leading to increased satisfaction for patients and their partners (Coward and Carson 2008). The increased MRT of TDA NCs is likely due to the prolonged adhesion and attachment of TDA NC particles to the nasal mucous layer. This effect may be attributed to the presence of PVA, which facilitates the attachment to the mucous layer and enables sustained particle release over an extended period, leading to a higher MRT of TDA NCs compared to pure TDA (Leone and Cavalli 2015).

When comparing the results with oral administration of the aqueous suspension of pure TDA in the literature, the C_max and AUC₀^∞ were 410 ± 120 ng/mL and 4000 ± 1800 ng.h/mL, respectively, which showed values that are higher for oral administration of TDA when compared to nasal TDA. This could be related to the higher dose (1.25-fold) that was used in the oral group, while the t_0.5 (3.9 ± 1.2 h) and MRT (8.8 ± 2.2 h) were reported to agree with the result of this study (Krupa et al. 2016; Krupa et al. 2017). The t_max (5.5 ± 1 h) of the oral administration study was longer than the nasal administration; this is related to the property of the nasal administration route, which has faster drug absorption with less time needed to reach the plasma, when compared to the oral route (Krupa et al. 2016; Krupa et al. 2017; Keller et al. 2021). To the author’s best knowledge, no studies are showing the pharmacokinetic parameters of pure TDA administered via intravenous or inhalation route. Teymouri Rad et al. (2019) studied the pharmacokinetics parameter of TDA nanocomposites as a dry powder formulation at a dose of 10 mg/kg administered by inhalation; the C_max and AUC₀^∞ were 721.7 ± 222.4 ng/mL and 11.9 ± 3.2 μg.h/mL, respectively. In addition, the t_0.5, MRT, and t_max were 4.1 ± 0.9 h, 19.2 ± 4.5 h, and 19.7 ± 6.7 h, respectively (Teymouri Rad et al. 2019; Zhenlei et al. 2019). However, the comparison between the results of this study and the current study is not valid due to many different factors related to the formulation, dose, and route of administration of TDA. The safety of intranasal TDA is not evaluated in this study, but we expect lower side effects in TDA NCs compared to pure TDA since lower doses of TDA NCs could be required for the same effect as the higher doses of pure TDA. Furthermore, the nasal administration of TDA is expected to cause lower gastrointestinal side effects compared to the oral administration since the nasal administration of the drug bypasses the gastrointestinal tract (Van Driel 2006). To our best knowledge, no studies have been published so far showing the pharmacokinetic characteristics of TDA after intranasal administration; thus, this study suggests a new strategy for possible improvement in the efficacy of TDA in the treatment of erectile dysfunction.

While it is premature to make definitive conclusions at this stage. However, assuming success in both the manufacturing of the dosage form, clinical studies, and regulatory approvals, many clinical advantages would be expected for administering TDA via the nasal route compared to the traditional oral route. Administering TDA via the nasal route presents several potential advantages over traditional oral administration. Nasal drug delivery offers high permeability through the nasal mucosa, allowing for efficient drug absorption and rapid onset of action (Ghori et al. 2015). This route bypasses the first-pass effect of the liver, leading to increased bioavailability and systemic exposure to drugs like tadalafil (Amponsah and Adams 2023). Studies have demonstrated that nasal delivery can reduce the risk of gastrointestinal issues associated with oral administration and provide faster therapeutic effects (Ghori et al. 2015; Amponsah and Adams 2023). Additionally, nasal drug delivery allows for direct drug delivery to target tissues, including the central nervous system via the olfactory nerve, potentially enhancing therapeutic outcomes (Dhuria et al. 2010). Overall, nasal administration of TDA may offer improved bioavailability, reduced systemic adverse effects, and targeted delivery, making it a promising alternative to oral administration for certain therapeutic applications (Pires et al. 2009; Kim et al. 2018).