Solubility Enhancement of Tadalafil Drug by Nano-Cocrystallization Technique

A major difficulty in developing new chemical or new molecular entities and existing pharmaceutical products of nearly 40% possess poor water solubility, which creates the possibility of low systemic absorption and decreased bioavailability. The key factor that needs to be considered during the development of dosage forms is improving the solubility and rate of drug dissolution. Enhanced solubility and dissolution rate are defined to achieve increased systemic absorption of the drug and its bioavailability.^[1] Currently, methods such as grinding, milling, micronization, hot melt extrusion, self-emulsification, solid dispersion, nanoparticles, salt formation, etc., have been used commonly to enhance the solubility to increase the absorption and bioavailability of medications. The important aspect to be considered while creating a new product successfully is making solubility improvements without altering the molecular structure.^[2] The active pharmaceutical ingredients (APIs) exist in two primary morphological forms either crystalline or amorphous. Crystalline materials are most frequently chosen for product development due to their excellent stability, reproducibility, and ease of purification. Amorphous forms are less used because they are less stable and eventually cause re-crystallization over time.  In the future, there is a tremendous prospect for improving the solubility, dissolution, and absorption of poorly soluble drug molecules using pharmaceutical techniques, such as crystal engineering innovations or absorption-enhanced formulation techniques.^[3,4] To increase solubility, stability, dissolution rate and resistance to humidity stress pharmaceutical nano-cocrystallization is a better alternative and its aim is to provide superior desirable drug release for higher systemic drug absorption with enhanced bioavailability.^[5.6] Also to keep all improved pharmaceutical features like compressibility, flow characteristics, etc., without altering the physicochemical or pharmacological characteristics of the API. Cocrystals and nano-cocrystals are the substances that perform the best at overcoming solubility issues.^[7,8] Pharmaceutical nano-cocrystallization is the process of merging cocrystallization with nano-sizing of a drug.^[9] Combining the benefits of cocrystal and nanocrystal technologies, nano-cocrystal formulations were proposed as a novel approach to achieve improved dissolution rate and oral bioavailability.^[10] The characteristics of nano-cocrystals, which are smaller than 1μm, alter significantly as their particle size reduces to 100 nm. This enhances drug molecule’s surface area and solubility, which increases the bioavailability of drugs that aren't very soluble.^[11] However, the particle size also affects the saturation solubility, which enhances the dissolution rate. It can greatly improve the delivery properties of poorly soluble drugs, which was a big challenge faced by the pharmaceutical industry for a long time.^[12,13] The techniques used for preparing nano-cocrystals can be classified as bottom–up and top–down processes according to differences in the production principles. Solvent-antisolvent precipitation is one of the best-established methods of manufacturing. However, the mean particle size in the nanometer range obtained by this procedure mainly depends on the hardness of the drug itself. The design of nano-cocrystals begins with the selection of the appropriate components such as co-formers, stabilizers, surfactants and solvents. Co-formers are the molecules that co-crystallize with the API to form the crystal lattice. Stabilizers and surfactants are the agents that stabilize the nanoparticles and prevent aggregation. ^[14-16] Tadalafil is a BCS Class II drug, is used in the treatment of erectile dysfunction, and is a potent, reversible, competitive inhibitor of phosphodiesterase 5 (PDE5), an enzyme that inactivates cyclic guanosine monophosphate (cGMP). Inhibition of PDE5 in the corpus cavernosum of the penis increases intracellular cGMP levels, thereby facilitating relaxation of smooth muscle leading to penile erection. However, insolubility and poor dissolution of this molecule, delay its rate of absorption and, finally the onset of action and reduced oral bioavailability.^[17] It was hypothesized to develop nano-cocrystals of tadalafil using solvent-antisolvent precipitation followed by a spray drying technique to improve its solubility into many folds. Further, the nano-cocrystals prepared can be characterized for solubility, melting point, microscopy, X-ray diffraction (XRD), particle size distribution and scanning electron microscopy. The developed nano-cocrystallization method should be easily scalable, cost-effective and feasible for commercial.

MATERIALS AND METHODS

Materials

Tadalafil drug, Poloxamer-188 and PVP K-30 were obtained from Yarrow chemical products (India). Benzoic acid and Citric acid from Loba Chemie Pvt.Ltd., Maharashtra; Ethyl alcohol from Rankem Chemicals Pvt.Ltd., Haryana; Vanillic acid Gallic acid and Tweens-80 from Lark Chemicals Pvt.Ltd., Mumbai; were purchased to conduct various experiments for the development of nano-cocrystals of tadalafil.

Pre-Formulation Studies

Necessary pre-formulation study on tadalafil drug was conducted before initiating the nano-cocrystals preparation as per below.

Solubility of Pure Drug

An excess quantity of tadalafil was added to ethanol in stoppered glass test tubes, which were kept on a mechanical shaker at room temperature for 48 hours. The saturated solutions were filtered through Whatman filter paper, suitably diluted and the drug concentration was estimated spectrophotometrically on a double-beam UV/Vis spectrophotometer (UV-1800, Shimadzu). The solubility of tadalafil pure drug in ethanol is found to be 4.096 ± 0.25 mg/mL.

Melting Point Determination

The melting point of the tadalafil was determined by using the capillary method. One end of the capillary tube was sealed on flame. From another open-end small amount of drug was placed at the bottom of the capillary. The capillary was then tied with a thermometer and immersed in Thiele tube containing liquid paraffin which is then heated. When the drug sample melted, the temperature was noted. The melting point of tadalafil pure drug is found to be 301.67°± 0.58°.

Determination of λ max for Tadalafil Drug

Tadalafil was dissolved in ethanol, diluted with SLS in water and scanned in the range of 200 to 400 cm^-1 spectrophotometrically on a double-beam UV/Vis spectrophotometer (UV-1800, Shimadzu). The absorbance values are plotted against the wavelength. In Table 1, the wavelength and the peak absorbance values is noted to determine the λmax

Table 1: Absorbance frequency of tadalafil in UV spectrophotometer

In the middle absorbance region of the UV spectral range of 400-200 nm, the maximum absorbance was observed at 284.2 nm and hence this value was noted as λmax of tadalafil pure drug.

Standard Curve Preparation Using λ max

The tadalafil standard curve was generated by plotting absorbance values against the concentration of tadalafil in the range of 1 to 50 μg/mL. Different concentrations of tadalafil solutions 1, 2.5, 5, 10, 20, 30, 40 and 50 µg/ml were prepared from an ethanolic stock solution in SLS in water and the absorbance of each solution at determined lambda max was recorded. The linear equation obtained from the curve is y = 0.0073x + 0.0046, with a correlation coefficient R² = 0.9994. R² value indicates the goodness of fit for the linear regression. An R² of 0.9994 is very close to 1, which suggests that the absorbance values are highly correlated with the tadalafil concentrations and the linear relationship is almost perfect. This means that the experimental data closely follows a straight line, indicating excellent consistency and reliability in the measurements. The absorbance value with respect to drug concentration of 1 to 50 μg/ml is recorded in Table 2.

Table 2: UV absorbance of tadalafil at different concentration

S. No.	Concentration (μ g/ml)	Absorbance at 284.2 nm
1	1.0	0.016
2	2.5	0.021
3	5.0	0.044
4	10.0	0.076
5	20.0	0.148
6	30.0	0.220
7	40.0	0.295
8	50.0	0.375

The calibration curve for tadalafil drug is shown in Figure 1.

Figure 1: Calibration curve of tadalafil drug

The powdered mixture of tadalafil and KBr was pressed in a 1:9 ratio to form a pellet. FTIR spectrum was recorded by using an FTIR spectrophotometer (Bruker) in the wavelength region of 4000 to 400 cm^-1. The absorption spectrum of the tadalafil drug is shown in Figure 2.

Figure 2: FT-IR spectrograph of tadalafil drug

Figure 3a: FT-IR spectrograph of drug with benzoic acid

Figure 3b: FT-IR spectrograph of drug with citric acid

Figure 3c: FT-IR spectrograph of drug with vanillic acid

Figure 3d: FT-IR spectrograph of drug with gallic acid

Figure 3e: FT-IR spectrograph of drug with poloxamer-188

Figure 3f: FT-IR spectrograph of drug with tweens-80

Accurately weighed quantity of tadalafil drug (500 mg) dissolved with coformers such as benzoic acid, citric acid, vanillic acid and gallic acid at various ratios of 1:1, 1:1.5 and 1:2 in ethanol (100 mL). Mix thoroughly using a magnetic stirrer to ensure completely dissolved. Separately dissolved poloxamer-188 (250 mg) which acts as a stabilizer for the nanoparticles and tweens-80 (150 mg) which act as a surfactant to improve wetting and dispersibility in distilled water (150 ml). Slowly add the ethanol solution containing drug with coformer drop-wise into the anti-solvent aqueous solution containing water with poloxamer-188 and tweens-80 under continuous magnetic stirring at around 500-700 rpm to ensure uniform mixing. Observed the formation of a turbid solution or precipitate, indicating the crystallization process. Then, used a probe sonicator for 5-10 minutes to reduce the particle size and produce nano-scale cocrystals. Transferred the sonicated suspension to a rotary vacuum evaporator and set the temperature to 40-50⁰C (below the boiling point of ethanol). Removed the ethanol under reduced pressure until a concentrated suspension was obtained. Dried the resulting suspension in a vacuum oven at 40⁰C to remove residual water and solvents.  Collected the dried nano-cocrystals and stored them in an airtight container to prevent moisture absorption. The composition nano-cocrystal formulation trials prepared is shown in Table 4.

Evaluation Of Nano-Cocrystals

The quality characterizations of the prepared nano-cocrystals done by testing of solubility, melting point, assay content, microscopy, particle size distribution, X–ray diffraction and scanning electron microscopy.

Saturation Solubility

The solubility of prepared tadalafil nano-cocrystals was measured by dissolving the prepared crystals in ethanol-filled 10 ml volumetric flask. Then, it was shaken and left for 24 hours to allow for equilibrations. After 24 hours, the crystals were filtered, diluted with ethanol and analyzed at 284.2 nm by using UV-visible spectrophotometer. This allowed us to determine the solubility of tadalafil nano-cocrystals.

Determination of Melting Point

A melting point apparatus is used to determine the melting point of prepared all tadalafil nano-cocrystals. In the determination of the melting point, a small amount of the tadalafil nano-cocrystals sample was placed into a capillary tube or melting point capillary. The capillary is then inserted into the melting point apparatus, which allows for controlled heating of the sample. As the temperature rises, the solid sample begins to melt, and the transition from solid to liquid phase occurs. The temperature at which this transition occurs is recorded as the melting point.

Drug Content of the Prepared Nano-Cocrystals

An accurately weighed amount of nano-cocrystals equivalent to 10 mg of the drug was taken in 100 ml volumetric flask, 20 ml of ethanol was added and shaken to dissolve the drug. After dissolving, the volume was made up to 100 ml with ethanol. These were filtered and 1 ml of aliquot of the above solutions was taken and diluted to 10 ml with ethanol. The absorbance of these solutions was determined at 284.2 nm against blank. The percentage drug content was calculated from the standard curve. The acceptance criteria based on the limit desceibeded in the pharmacopoeia.

Microscopic Study

Microscopic study provides information on the magnified size and morphology of the drug tadalafil and tadalafil nano-cocrystals using an optical microscope. The microscope allows for magnification and visualization of the samples under 10X magnification levels, enabling detailed observation.

Particle Size Analysis

The particle size distribution of the nano-cocrystal powder was determined using a Shimadzu SALD-2300 laser diffraction particle size analyzer equipped with a flow cell and sampler unit (Wing SALD II software, Version 3.1.1). Briefly, 5 mg of the sample was dispersed in 50 mL of deionized water and sonicated for 20 minutes to achieve a homogeneous suspension. The suspension was filtered through a 1 µm mesh and loaded into the sampler unit. Measurements were performed at a flow rate of 100 mL/min, and the particle size distribution was calculated using Mie theory. The results are reported as the mean ± standard deviation of triplicate measurements, including the D10, D50, and D90 values

X-Ray Diffraction Study

The crystalline structure and phase composition of the synthesized nano-cocrystal powder were investigated using a Bruker Eco D8 Advance X-ray diffractometer equipped with Cu Kα radiation (λ = 1.5418 Å). The instrument was operated at 40 kV and 40 mA, with a scan range of 5° to 80° (2θ), a step size of 0.02°, and a scan speed of 1° per minute. The powdered sample was finely ground and uniformly loaded onto a zero-background holder to ensure a smooth surface for analysis. The obtained XRD pattern revealed distinct diffraction peaks, indicating the crystalline nature of the nano-cocrystals.

Scanning Electron Microscopy

The morphology and particle size of the nano-cocrystal powder were analyzed using a scanning electron microscope (ZESS Model). Briefly, 1 mg of the sample was dispersed in the solvent and sonicated for 10 minutes to achieve a homogeneous suspension. A drop of the suspension was deposited onto a clean aluminum stub and allowed to dry at room temperature. The sample was sputter-coated with a 10 nm layer of gold to enhance conductivity. SEM imaging was performed at an accelerating voltage of 15 kV and a working distance of 10 mm. High-resolution images were captured at magnifications ranging from 5000x to 50000x. The particle size and morphology were analyzed using Image J software, and the results are reported as the mean ± standard deviation of at least 100 particles.

RESULT AND DISCUSSION

Solubility Study

The solubility of tadalafil nano-cocrystals showed significant improvement in solubility of about 2 to 2.5 times higher compared to pure drug. Tadalafil pure drug as it is showing a solubility of 4.096 mg/mL, compared with the nano-cocrystal formulation (F3) containing tadalafil:Benzoic acid at the ratio of 1:2,  exhibiting the highest solubility of 10 mg/mL. Increasing the co-former ratio consistently enhanced solubility, suggesting optimized molecular interactions. All formulations maintained low standard deviations and confirming reproducibility. Nano-cocrystallization with coformers like benzoic acid, citric acid, vanillic acid and gallic acid maximizes solubility and hence potentially improves bioavailability. The solubility results of all the formulations are described in Table 5.

Melting Point Determination

The melting point of pure tadalafil drug was found to be 301.67±0.58°C and significantly decreased in all nano-cocrystal formulations of 155-222°C, confirming successful cocrystal formation. The most stable formulation was gallic acid-based formulation (F10-F12), showing melting point of  218-222°C, while the citric acid-based system (F4-F6) showed the lowest melting point  of 155-158.5°C. This depression indicates modified crystal packing without drug degradation, as evidenced by the narrow melting ranges of less than 1°C variation in most formulations. The consistent melting behavior across triplicates suggests excellent batch reproducibility. Refer Table 5, showing the melting point of all the nano-cocrystal formulations.

Drug Content

The drug content analysis revealed a high uniformity of 95-98.4% across all nano-cocrystal formulations (F1-F12). Formulation containing Tadalafil:Vanillic acid, 1:2 ratio (F9) showing the highest drug content of 98.4%. The variation in drug content between all the formulations was found to be less than 3.5%, which confirms precise formulation control and homogeneous drug distribution in all the trials. These results align with pharmacopeial standards, typically requiring 95-105% drug content, validating the robustness of the nano-cocrystallization process. The drug assay results are shown in Table 5.

The microscopic study revealed distinct morphological differences between pure tadalafil and its nano-cocrystals. Pure tadalafil showed large, irregular crystals, while all nano-cocrystal formulations exhibited significantly smaller and more uniform particles. Among the coformers, benzoic acid produced needle-shaped crystals, citric acid formed spherical aggregates, and vanillic/gallic acid created plate-like structures. These morphological changes directly correlate with the enhanced solubility and dissolution profiles observed in previous studies. Notably, the vanillic acid nano-cocrystals showed the most favorable combination of small particle size and uniform shape, supporting their superior drug release performance (98.4%) compared to other formulations. The consistent reduction in particle size across all cocrystals confirms the successful nano-crystallization process and explains the improved bioavailability characteristics of these formulations.  In Figure 4, images captured in a microscope were presented.

Figure 4: Comparative microcopy of tadalafil drug with nano-cocrystal formulations

In Figure 5, the particle distribution curve for the tadalafil nano-cocrystals prepared is presented. The graph shows the cumulative percentage of particle sizes of nanoparticles.

Figure 5: Particle size distribution curve of tadalafil nano-cocrystals

 Note: D10, D50 and D10 - Diameter at which 10%, 50% and 90% respectively of the particles are smaller. Span: A measure of the width of the particle size distribution, calculated as Span = D90-D10 / D50

X-Ray Diffraction Analysis

In Figure 6, the XRD patterns of tadalafil and its nano-cocrystals formulations prepared in this study are presented. The diffraction peaks of pure tadalafil were observed at specific 2θ values, which are consistent with its known crystalline structure. In contrast, the nano-cocrystals exhibited distinct diffraction patterns, indicating the formation of new crystalline phases due to the interaction between tadalafil and their respective co-formers benzoic acid, citric acid, vanillic acid and gallic acid used in the formulation trials. The major diffraction peaks for tadalafil were observed at 2θ values of 12.5°, 18.7°, 25.3°, 30.8°, and 34.2°, corresponding to its characteristic crystalline planes. For the nano-cocrystals, the diffraction peaks were shifted or new peaks appeared, confirming the formation of cocrystals.

Figure 6: X-Ray diffraction graph of tadalafil drug and nano-cocrystals

In Table 7, XRD peak positions and corresponding crystallographic planes for tadalafil and its nano-cocrystals were presented.

Scanning Electron Microscopy (SCM)

The SEM images reveal distinct morphological differences between pure tadalafil and its nano-cocrystals prepared using four co-formers. In Figures 7a to 7e, the SEM analysis of pure tadalafil and its nano-cocrystals at 20 µm and 10 µm scales are presented. This revealed significant differences in morphology, particle size, and surface characteristics, which are critical for understanding the impact of cocrystallization on the physicochemical properties of the drug.

Figure 7a: SEM image of tadalafil

Figure 7b: SEM of tadalafil nano-cocrystals having benzoic acid

Figure 7c: SEM of tadalafil nano-cocrystals having citric acid

Figure 7d. SEM of tadalafil nano-cocrystals having vanillic acid

Figure 7e: SEM of tadalafil nano-cocrystals having gallic acid