Candesartan cilexetil powder was purchased from Wuxi Hexia Chemical Company, Chin. HPMC E5 and PXM-188 were purchased from Hyperchem (China). Disodium hydrogen phosphate (Na2HPO4) was purchased from CDH (India). Potassium dihydrogen phosphate (KH2PO4) was purchased from Himdia (India). Sodium chloride (NaCl) was purchased from LAD (India). The PVP K-30 was purchased from Alpha Chemika (India), and ethanol was purchased from HaymanKimia (U.K.).

The current optimisation study used various response surface methodology (RSM) computations using Design-Expert^® software (Version 13, Stat-EaseInc., Minneapolis, MN). Three factorial designs of the factorial models were generated for all responses. Also, 3D plots were constructed using Design-Expert^® software. Analysis of variance (ANOVA, 2-way) was used to assess the significance of the selected parameters on the variables. The desirability function was employed after fitting the mathematical model for the optimisation. During the optimisation process, the responses were combined to find a product with high desirability. The desirability function combined all responses into one variable to predict the optimum levels gained from the independent variables. A desirability value ranged from zero to one. Thus, zero is an unacceptable value for the responses, while one is the most desired value. The selected optimised formulation by the design was prepared, and a comparison was carried out between predicted and observed values given by the design.

Candesartan cilexetil nanosuspensions were prepared using the solvent evaporation technique. The candesartan cilexetil powder, 6 mg, was dissolved in 2 ml of ethanol (solvent) to create the drug solution. The candesartan cilexetil solution was injected at a rate of 1 ml.min^-1 into 20 ml of distilled water (anti-solvent). The stabilisers were solubilised in distilled water 5 min pre-injections and involved combinations in various concentrations (A, B and C). Next, the prepared nanosuspensions were exposed to sonication for 20 min in a water bath. As a result, nanoparticles started to form simultaneously. A one-hour magnetic stirrer homogenised the produced nanosuspensions and evaporated the organic solvent. Ultimately, a lyophilised Labconco freeze drier (USA) has been employed to remove any traces of solvent.

The method of determining the melting point of candesartan cilexetil was adapted from Karar et al. (2020) (Albo Hamrah et al. 2020). A small amount of candesartan cilexetil powdered was mounted into a capillary glass tube. The glass tube was opened from one side and closed from the other. The temperature inside the glass tube was gradually raised and inspected visually. The temperature was recorded when the solid powder melted completely. This analysis would be replicated three times independently to obtain mean ± standard deviations.

The maximum λmax of candesartan cilexetil was determined according to our previous published work (Albo Hamrah et al. 2020). Stock solutions of candesartan cilexetil were prepared in HCl buffer (pH 1.2) and phosphate buffer (pH 6.8) at a concentration of 30 μg per ml. The range of UV-visible was 200–400 nm; hence, these solutions were scanned using a UV-visible instrument (Shimadzu, Japan). This analysis would be replicated three times independently so that mean ± S.D. would be obtained.

The method of construction of a calibration curve for the quantification of candesartan cilexetil was obtained from our previous published work (Albo Hamrah et al. 2020). This method was based on preparing a stock solution from dissolving candesartan cilexetil in HCl buffer (pH 1.2) and phosphate buffer pH (6.8) at 50 µg per ml. Next, serial dilutions of 5, 10, 20, 30 and 50 concentrations were prepared. Spectrophotometrically, these solutions were analysed at λmax 254 nm and plotted against concentrations to get the calibration curve. The calibration curve was ultimately constructed to gain the calibration curve equation and regression coefficient (R2) value. This analysis would be replicated three times independently so that mean ± S.D. would be obtained.

The method of determining the saturated solubility of candesartan cilexetil was based on our previous published work (Albo Hamrah et al. 2020). An excess candesartan cilexetil powder was added to a 10 ml tube containing 5 ml HCl buffer (pH 1.2). Next, these tubes were shaken at 25 ± 0.5 °C in an isothermal shaking water bath for 72 h. Then, they were centrifugated for 10 min at 2000 rpm (R LABNCO, USA) to remove the supernatant. The supernatants were filtered using a filter membrane (0.45 µm) and scanned at maximum absorption wavelength using a UV-visible spectrophotometer (Shimadzu, Japan).

Particle size parameters were determined using the ABT-9000 Nano Laser particle size analyser at a constant temperature of 25 °C and a scattering angle of 90°. The prepared nanosuspensions’ R1 (particle size) and R2 (PDI) were measured. The sample with a low polydispersity index means monodisperse, while a high level means wide-speared particle distribution. The average level of PDI values is 0–0.05, which means monodisperse standard, 0.05–0.08 refers to nearly monodisperse, 0.08–0.7 indicates mid-range PDI and more than 0.7 means very polydisperse (Satyajit et al. 2014; Alhagiesa and Ghareeb 2021).

The method used to determine EE% was based on the work of Hao (2011) with minor modifications (Hao 2011). The sample (1 mg) was placed in the dialysis membrane and dialysed in 100 ml of phosphate buffer (pH 6.5) for 12 h. Then, the entrapped candesartan cilexetil in the dialysis membrane was quantified. The EE% was measured by dividing the trapped amount by the total used amount (Abdalla et al. 2015; Huang et al. 2020).

Among nine runs, the Design-Expert^® software has developed an optimal formula based on maximising EE% and minimising particle size and polydispersity index. A desirable index was generated and compared with the resulting suggestions, with a value between 0 and 1. These criteria were established, and the best formula was chosen (Madan et al. 2015).

Samples of Candesartan cilexetil-loaded nanosuspensions were solidified by lyophilisation technique by water removal. This technique used the principle of sublimation and desorption with a negative vacuum (Powar and Hajare 2020). Nanosuspensions were frozen at - 30 °C for 12 h and then lyophilised using a freeze dryer (Christ, Osterode, Germany). The operating conditions of the freeze dryer were a pressure of 20 mbar and a temperature of -50 °C (Fonte et al. 2016).

Morphological analysis using AFM is a powerful tool to investigate surfaces of samples. It provides high-resolution images to examine nanoparticles precisely. Histograms of particle size distribution, particle size, and 3D surface morphology of Candesartan cilexetil-loaded nanosuspensions were obtained (Dolenc et al. 2010).

A mica disc was mounted on a metal base, and 10 µl of nanosuspensions were deposited on the mica disc (Al-Edresi et al. 2020). Samples were washed five times using filter-sterilised Milli-Q water and then air dried at room temperature. Furthermore, images were taken using a Picoforce Nanoscope V Multimode atomic force microscope (Bruker). The mode was tapping, the scanning rate was between 0.5 and 1.5 Hz, and the resonant frequency range was 270–460 kHz. Representative areas were first specified, and then images were captured. The amplitude error mode (5 mm × 5 mm) and the height were determined using Nanoscope software v7.2 after flatting, while the determined resolution of the images was 512 × 512 points.

The compatibility of candesartan cilexetil and other additives in the nanosuspensions was analysed by DSC. The crystal state of the model drug, particularly when incorporated into nanosuspension, was significant (Jassim and Hussein 2014). The thermal characteristics of the pure candesartan cilexetil, A (PVP K30), B (HPMC E5), and C (PXM 188) were examined by an automatic thermal analyser system (Perkin-Elmer DSC 4000) instrument (Rizal et al. 2020). The thermos-grams of the control (candesartan cilexetil free) nanosuspensions and candesartan cilexetil-loaded nanosuspensions were recorded. Nanosuspensions were first frozen and lyophilised using a freeze-dryer (Martin Christ alpha 1–4 L.D. 92 plus, Germany) at 0.01 bar and -55 °C. Then, 5 mg was sealed by crimping an aluminium lid in an aluminium pan with a crimper press (Perkin-Elmer, U.K.). Next, the temperature of the DSC was raised from 0 °C to 280 °C at a scan rate of 10 °C. min^-1. The used reference was an empty pan with a lid. The plots were generated by StarE 9.10 software (Isailović et al. 2013).

The FTIR analysis was carried out to gain spectra of the Candesartan cilexetil-loaded nanosuspensions, nanosuspensions-free candesartan cilexetil, pure candesartan cilexetil, A (PVP K30), B (HPMC E5), C (PXM 188). Samples were mixed with crushed potassium bromide and compressed into a thin tablet. The range of the resulting spectra was from 4000 to 400 cm^-1 with a resolution of 2 cm⁻¹ and was gained using a Nicolet Avatar 370 instrument (Thermo Nicolet Corporation, USA) (Ahmed and Aljaeid 2017).

A study of the in vitro candesartan cilexetil release was adapted from Jigar Shah and his colleagues (2020) with minor modifications (Shah et al. 2020). A modified Franz diffusion cell, which has a receptor and donor compartment separated by a membrane, has been used. The receptor volume was 15 ml, and the diffusion area was 1.4 cm². The cut-off of the dialysis membrane was 8000–14000 (Sigma- Aldrich Corp. St. Louis, MO, USA). The membrane was soaked in phosphate buffer (pH 6.8) for 24 hours before use. On one face, the dialysis membrane was in contact with the receptor medium, composed of phosphate buffer (pH 6.8). The other face of the membrane contains samples having candesartan cilexetil. Both compartments’ temperatures were kept constant at 37 ± 2 °C by submerging them in the water bath. The same experiment was repeated for HCl buffer (pH 1.2). Homogenisation inside the receptor compartment was maintained throughout the experiment using a magnetic stirrer bar rotated at 100 rpm using a hot plate magnetic stirrer (aLFA, china). A 0.5 ml sample was withdrawn at predetermined intervals for one hour and immediately replaced with fresh phosphate buffer (pH 6.8). The withdrawn samples were assayed spectrophotometrically (Shimadzu, Japan) at 254.

The stability study was conducted according to the guidelines of ICH. The Candesartan cilexetil-loaded nanosuspensions were exposed to three different temperatures, which were 32 ± 2 °C (high), 25 ± 2 °C (average) and 4 ± 2 °C (low). Samples were analysed (quantification of payload drug) monthly for three months to measure particle size and EE % (Sambhakar et al. 2017; Mittal et al. 2020).

The t-test and ANOVA were conducted using IBM SPSS version 20 and Design-Expert® software version 9. A significant p-value was obtained at ≤ 0.05. Standard deviations and means were measured by Microsoft Excel 2020, and data are expressed as mean ± standard deviation. Where necessary, data were normalised to percentages. Comparisons are always made according to a control condition.

The recorded melting point of candesartan cilexetil was 171 °C to 172 °C. Results from the following experiment were consistent with the finding of Al-Shaibani and his colleagues (2019), who indicated that the pure powder of candesartan cilexetil would melt at 171–172 °C (Al-Shaibani et al. 2019).

It has been found that the maximum λmax for candesartan cilexetil in phosphate buffer (pH 6.8) and HCl buffer (pH 1.2) appeared at 254 nm. Results from this experiment were consistent with the findings published by Pradhan and his colleagues (2011) (Pradhan et al. 2011).

Two calibration curves were constructed at pH 1.2 and 6.8 with regression coefficients of 0.9991 and 0.9998, respectively. Absorbencies against concentrations were taken, and a straight line was drawn using Excel software. The generated curves from the selected concentrations agreed with Beer-Lambert’s law at λ max 254 nm, as shown in Figs 1, 2.

Figure 1. 

Calibration curve of candesartan cilexetil in HCl buffer (pH 1.2). Candesartan cilexetil was dissolved in HCl buffer (pH 1.2) at a concentration of 5, 10, 20, 30 and 50 µg.ml^-1, and the U.V. absorbance at λmax 254 nm was gained. Data are represented as mean ± standard deviation of 3 independent experiments.

Figure 2. 

Calibration curve of candesartan cilexetil in phosphate buffer (pH 6.8). Candesartan cilexetil was dissolved in phosphate buffer (pH 6.8) at a concentration of 5, 10, 20, 30 and 50 µg.ml^-1, and the U.V. absorbance at λmax 254 nm was gained. Data are represented as mean ± standard deviation of 3 independent experiments.

Results revealed that the saturated solubility of candesartan cilexetil was 63 ± 6 µg.ml^-1 in phosphate buffer at pH 6.8 and 7 ± 0.35 µg.ml^-1 in HCl buffer at pH 1.2. Hoppe, K. and M. Sznitowska (2014) have presented similar findings (Hoppe and Sznitowska 2014). It has been suggested that candesartan cilexetil has weak acidic properties as its solubility was higher in primary mediums (Ardiana et al. 2012). The saturated solubility of the candesartan cilexetil-loaded nanosuspension was 344.7 ± 16 µg.ml^-1, revealing a more than five times increase in solubility than phosphate buffer (pH 6.8) and a 9-time increase in solubility than HCl buffer (pH 1.2). The results of the increase in solubility of candesartan cilexetil-loaded nanosuspension were consistent with the findings of Detroja et al. (Detroja et al. 2011), who reported a 20 times increase in solubility through loading candesartan cilexetil into nanosuspension.

The results of the particle size ranged from 15.6 to 155 nm. The variations in the particle sizes could be attributed to the polymer concentrations and affinities of molecules for drug particles (Liu et al. 2015). The largest particle size was revealed in Run 12 (155 nm), while the smallest was realised in Run 18 (15.6 nm), as illustrated in Table 2. The variation in particle size might be due to the high percentage of stabilisers. An increased stabiliser ratio would reduce surface tension, stabilising the new particles during precipitation. As a result, smaller particle sizes would be generated as stabilisers would wrap the surface of the particles properly. Bernard and his colleagues have come up with similar findings (Van Eerdenbrugh et al. 2009), as he reported that increasing stabilisers would decrease particle size.

The PDI ranged from 0 to 0.12 for runs 5 and 12, respectively (Table 2). Run 9 revealed a monodispersed PDI, while Run 17 revealed a mid-range PDI (Gadad et al. 2012; Abbas et al. 2017).

Results of the EE% of candesartan cilexetil in nanosuspension revealed 91.9% in Run 11, which was the highest EE%. On the contrary, Run 1 has only 51.6%, as listed in Table 2. Such variations in the EE% could be attributed to the differences in the concentration of HPMC E5; hence, increasing HPMC concentration would decrease EE% because of the increased viscosity of the internal phase. The highly viscous internal phases hindered the migration of its payload, resulting in reduced EE% (Sharma et al. 2015).

Results of the DL% ranged from 5.37% (Run 1) to 36.76% (Run 11) as listed in Table 2. The DL% was shown to increase as polymer concentrations increased, and this result was consistent with the finding of Dora and his colleagues (2010), who found that an increase in the polymer ratio and EE % increased the DL % (Dora et al. 2010).

The Design-Expert^® software and fit statistics analysed the resulting data listed in Table 3. Parameters were generated, such as predicted determination coefficient (pred. R²), p-value, adjusted determination coefficient (adj. R²) and ANOVA for all responses. A p-value of (ABC) was 0.785, 0.843, 0.037 (i.e., ≤ 0.05) and 0.172 for R₁, R₂, R₃ and R₄, respectively. This indicates that only the R₃ model was significant. Thus, the three polymers have a significant effect on this response.

Factorial design for three factors at three levels coded as -1, 0 and +1 was equivalent to an 18 run and was chosen as the experimental design. This is considered an effective first-order design with a minimum number of experiments. Thus, the influence of individual variables was estimated to be the main effect. Besides, the response surface was determined, adding additional advantages to this design. Therefore, a full factorial design was employed to investigate the factors systematically.

The effect on particle size (R₁) was observed to be non-significant by ANOVA, as shown in Equation 2.

R₁= 72.56111 + 3.60625A - 5.55625B + 8.03125C + 10.15625AB + 13.49375AC - 23.99BC + 2.79375ABC E.q. 2

The negative sign shown in Equation 2 related to the coefficient of factor B (HPMC) indicates that the particle size decreases as the concentration of B increases. The findings from this analysis agreed with the findings from Mandlik and Ranpise (2017) (Mandlik and Ranpise 2017), who reported that chitosan concentration on nanoparticle size was more pronounced than STPP. The response surface 3D plots revealed that the particle size headed toward the upper level at low concentrations, as shown in Fig. 3a. The reduction in the particle size resulted from increasing HPMC concentrations due to supersaturation, which led to rapid precipitation during diffusion (Sahu and Das 2013). Agglomeration or aggregation might be induced on a smaller concentration of the stabiliser; however, adding stabiliser in too much concentration could promote Oswald’s ripening (Tadros 2017).

Figure 3. 

Response surface 3D plots showing the effect of independent variables on the dependent variables via (a) particle size, (b) PDI, (c) EE%, and (d) DL% by Design-Expert^® software.

The effect on PDI (R₂) was observed to be non-significant by ANOVA, as shown in Equation 3.

R₂=0.0440 + 0.0020A - 0.0070B+0.0094C + 0.0058AB + 0.0019AC - 0.0091BC + 0.0081 ABC E.q. 3

The PDI, which reflects the uniformity of size, was more dependent on stabiliser concentration. Results indicated that PDI decreases as the stabiliser concentration increases, as shown by the negative charge in Equation 3. As PDI decreased, better homogeneity was gained; however, less homogeneous particles were obtained at very high stabiliser concentrations. The response surface 3D plots revealed that the PDI is heading toward the upper level at low concentrations, as shown in Fig. 3b.

The effect on EE% (R₃) was observed to be significant by ANOVA, as shown in Equation 4.

R₃=72.70 + 1.86A - 2.19B - 7.26C + 11.03AB - 3.23AC + 0.7000BC - 0.4125ABC E.q. 4

A stabiliser A revealed a positive sign for the coefficient, indicating that the EE% increases as the stabiliser concentration increases. The response surface 3D plots (Fig. 3c) showed a linear ascending pattern for EE% with increasing stabiliser concentration.

The effect on DL% (R₄) was observed to be non-significant by ANOVA, as shown in Equation 4.

R₄=12.36 + 0.3181A + 0.0344B - 0.1606C + 5.14AB - 3.80AC - 3.77BC - 0.6944ABC E.q. 5

The DL% (R₄) analysis revealed that the coefficient has a positive sign for stabilisers A and B, while stabiliser C possesses a negative charge. The stabiliser possesses a negative charge on DL%, indicating that increasing stabiliser concentration would decrease DL%. The response surface 3D plots revealed that the DL% is heading toward the upper level at low stabiliser concentration, as shown in Fig. 3d.

Optimisation aims to find the variables that could dramatically affect the chosen responses and specify the level of variables that a high-quality and robust product might produce (Zidan et al. 2007). The measured responses affecting the product’s quality should be considered during optimisation. The criteria of the responses were: particle size ≤ 100, PDI ≤ 0.1, EE% ≥ 50 and DL% ≥ 10. Factorial formulation responses suggested 150 mg, 180 mg, and 82 mg of PVP K-30, HPMC E5 and PXM-188, respectively. The predicted particle size values were 72.56 nm, PDI was 0.049, EE% was 73.34% and DL% was 12.04%. The selected optimised formulation was prepared, and the observed values were found to be quite comparable to the predicted values. The experimental (found) particle size values were 64.65 nm, PDI was 0.059, EE% was 86.73% and DL% was 10.17%. Relative errors of experimental and predicted values are calculated in Equation 6.

$\mathit{\text{Relative error}}=\frac{(predicted-experimental)}{predicted}$ E.q. 6

The relative error of particle size was 0.1, PDI was 0.2, EE% was 0.18, and DL% was 0.16, reflecting an agreement between predicted and experimental values. Results from this analysis demonstrate a model’s ascertained viability (Rane et al. 2007).

The morphological analysis and particle size of Candesartan cilexetil-loaded nanosuspensions performed by AFM show regular to spherical-shaped nanoparticles with a size of 30.4 nm and approved by the particle size distribution histogram, as shown in Fig. 4. In contrast, the ABT-9000 particle size analyser estimated the particle size was 64.65 nm. The particle size of the optimised Candesartan cilexetil-loaded nanosuspensions obtained by AFM was smaller than that measured by the ABT-9000 particle size analyser, and this difference was because a particle size analyser could only provide data on the volumetric mean diameter of a large number of particles; it is challenging to get findings that reflect actual size distribution.

Figure 4. 

The AFM images of the Candesartan cilexetil-loaded nanosuspensions. A. 3D AFM image of candesartan cilexetil-loaded nanosuspensions; B. 2D AFM image of candesartan cilexetil-loaded nanosuspensions.

Pure candesartan cilexetil revealed peaks at 2950.5 cm⁻¹ due to aromatic C-H stretching and 2870.2 cm⁻¹ due to O-H stretching, as shown in Fig. 5A. Peaks were revealed at 1752.13 cm⁻¹ and 1718.01 cm⁻¹ due to ester C=O stretching vibration, whereas peaks at 1271.12 cm⁻¹ and 1315.83 cm⁻¹ for C-O stretching of the carbonyl group of aromatic esters. Peaks were revealed at 1030–1281 cm^-1 for N-C stretching of the drug.

Figure 5. 

FTIR spectrum of A. Pure candesartan cilexetil compound; B. PVP K-30 polymer; C. HPMC E5 polymer; D. PXM 188 polymer; E. Candesartan cilexetil-free nanosuspensions; and F. Candesartan cilexetil-loaded nanosuspensions.

For PVP k30, there is a strong absorbance band at 1675.25 cm^-1 due to the C=O of tertiary amide and at 2358.17 cm^-1 due to C-H stretching. A vast band was shown at 3264.15 cm^-1 due to O-H stretching vibrations of absorbed water (Fig. 5B). The broadband was confirmed by DSC analysis as the broad endothermic peak. Results from these experiments were consistent with the findings of Wegiel and his colleagues (2014), who had similar FTIR peaks (Wegiel et al. 2014). HPMC E5 analysis showed a peak at 3312 cm^-1 due to O-H stretching vibration, 1170.13 cm^-1 due to C-O-C stretching vibration, and 1103.07 cm^-1 (Fig. 5C) due to C-O stretching. The findings from this analysis agreed with the findings of Oh and his colleagues (2012) (Oh et al. 2012). The FTIR spectrum of PXM 188 revealed a principal absorption peak at 3287.44 cm^-1 due to aliphatic C-H stretching (Fig. 5D). Also, the O-H bending appeared at 1344.03 cm^-1, and C-O stretching at 1121 cm^-1. Sharma and his colleagues (2013) have found the same results (Sharma et al. 2013).

The FTIR peaks of nanosuspensions-free candesartan cilexetil revealed peaks at 3435.22 cm^-1, 2347.37 cm^-1, 1645.26 cm^-1 and 1093.64 cm^-1 for PVP K-30 and HPMC E5 (O-H stretching), PXM 188 (O-H bending), PVP K-30 (C=O of tertiary amide) and HPMC E5 and PXM 188 (C-O stretching) (Fig. 5E). The C=O stretching vibration peak of candesartan cilexetil disappeared in the nanosuspensions complex of candesartan cilexetil/polymers (Fig. 5E). The candesartan cilexetil revealed a C=O stretching vibration peak, indicating an H-bonding between the drug and polymer, as shown in Fig. 5F. There was no significant shift in the FTIR spectrum, indicating no interaction or complexation between payload and polymers during the preparation of candesartan-cilexetil nanosuspensions. The characteristic peaks of the Candesartan cilexetil-loaded nanosuspensions in the region of 2870.2 cm⁻¹–2950.5 cm⁻¹ were observed in the loaded nanosuspensions, revealing that the drug was entrapped physically inside the nanosuspensions.

The thermal behaviour of candesartan cilexetil and polymers is depicted in Fig. 6A–F. The classic application of the DSC analysis is determining any possible interactions between the entity of a drug and its excipients within a formulation. The formation of candesartan cilexetil inclusion complexes and host-guest interactions lead to boiling, glass transition, melting, and sublimation points disappearing or shifting to different temperatures. The polymers’ effect on the candesartan cilexetil’s inner structure was investigated using DSC analysis.

Figure 6. 

The DSC thermogram of A. pure candesartan cilexetil; B. Candesartan cilexetil-loaded nanosuspensions; C. Candesartan cilexetil-free nanosuspensions; D. PVP K-30; E. HPMS E5; F. PXM 188.

The pure Candesartan cilexetil DSC curve revealed a sharp endothermic peak at 165 °C, corresponding to its melting followed by an exothermic peak, indicating its crystalline nature (Fig. 6A). This result was consistent with the results of Gurunath et al. (Gurunath et al. 2014). However, the endothermic peak of the candesartan cilexetil was shifted (towards lower temperature), slightly broadened and reduced in intensity in Candesartan cilexetil-loaded nanosuspensions (Fig. 6B). This behaviour could be due to the uniform distribution of the candesartan cilexetil in the polymer’s crust, causing the molten drug’s entire miscibility in polymers. Also, the DSC results revealed no interactions between the lipophilic excipients and the drug (Kamalakkannan et al. 2013). A broad endotherm peak was also realised at 55 °C due to the water.

The candesartan cilexetil-free nanosuspensions revealed a vast, broad endotherm peak at 140–170 °C (Fig. 6C). The DSC thermogram of PVP K30 polymer revealed a broad endotherm peak ranging from 20 to 90 °C due to its highly hygroscopic nature (Fig. 7D) (Sethia and Squillante 2004). An amorphous characteristic of PVP K30 polymer was suggested from the results above. A broad endothermic peak, ranging from 15 to 100 °C, was also seen in the DSC thermogram of HPMC E5 due to the possible dehydration of water molecules (Zaini et al. 2017). The PXM 188 revealed an endothermic melting peak at 51.73 °C, as shown in Fig. 6F.

Figure 7. 

The in vitro release profile of Candesartan cilexetil-loaded nanosuspensions and Candesartan cilexetil powder at A. Dissolution medium phosphate buffer (pH 6.8) and B. Dissolution medium HCl buffer (pH 1.2). Data are represented as mean ± standard deviation of three independent experiments.

The dissolution rate of the payload has a significant effect on the absorption and, consequently, bioavailability. Therefore, comparing the dissolution profiles of different formulations is essential. The in vitro results of the following study for Candesartan cilexetil-loaded nanosuspensions and Candesartan cilexetil powder revealed > 95% release in PBS (pH 6.8) (Fig. 7A) and HCL buffer (pH 1.2) (Fig. 7B) within one hour with a steep profile. The release from candesartan cilexetil powder revealed 62% in PBS (pH 6.8) (Fig. 7A) and 36% in HCl buffer (pH 1.2) within one hour with the slow graduate profile. The in vitro release study revealed a significant (p-value ≤0.05) increase in the dissolution rate of the candesartan cilexetil. In general, the Candesartan cilexetil powder was released slowly in HCl buffer (pH 1.2) compared to PBS buffer (pH 6.8), which could result from the solubility difference of candesartan cilexetil in these media.

On the other hand, the release profile of Candesartan cilexetil-loaded nanosuspensions exhibited a biphasic pattern with an initial rapid phase followed by a slow phase in PBS and in HCl buffers. The fast release phase might be due to the burst release of the payload. A possible explanation is the enrichment of the payload in the nanosuspension’s outer region. Thus, a short diffusion path would be carried out (zur Mühlen et al. 1998).

Stability studies included analysis of the particle size and EE % over three months at 32 °C, 25 °C and 4 °C. Analysis of particle size after three months at 32 °C, 25 °C and 4 °C was 75.5, 69.3 and 66.4 nm, respectively, as shown in Fig. 8A. No significant differences (P ≤ 0.5) in nanosuspension particle sizes reflected a stable formulation over the studied period. On the other hand, the EE % of the Candesartan cilexetil after three months at 32 °C, 25 °C, and 4 °C was 60, 80, and 86, respectively, as shown in Fig. 8B. There was a significant difference (p > 0.05) in the EE % of the candesartan cilexetil at 32 °C, reflecting the inability to stand the same concentration at this temperature. However, no significant differences (P ≤ 0.5) were found at 25 °C and 4 °C.

Figure 8. 

Particle size and EE % analysis of the Candesartan cilexetil-loaded nanosuspensions. A. Particle size and B. EE % over three months at 32 °C, 25 °C and 4 °C. Data are represented as mean ± standard deviation of three independent experiments. * p-value > 0.05.