Statistical analysis

PTX novasomal vesicles were prepared adopting CCD generated by Design expert®13 to evaluate the effects of the different formulation variables on novasomes’ characteristics. CCD is an experimental statistical design used in response surface methodology (RSM) [56]. CCD is known for its superior analyzing ability in minimizing experimental errors, while maximizing estimation of quadratic effects [57], and facilitating the optimization process. It is more effective than other RSM designs at predicting the nonlinearity of the selected responses with fewer experimental runs. Additionally, it is characterized by having fixed variance predictions at all experimental points that are present at equal distances from the design center [58]. CCD uniquely provides a robust statistical framework through the integration of axial, factorial, and central points: axial points allow for the precise estimation of design curvature, factorial points enable the analysis of main effects and interactions between formulation variables, and central points are explicitly used to assess model adequacy and potential experimental errors [59]. CCD was previously used in statistical evaluation and optimization of various drug delivery systems, such as nanosuspension [60] and solid lipid nanoparticles [61].

Based on the calculated statistical parameters, the software suggested reduced quadratic and reduced cubic models to analyze the PS and PDI values, respectively. Whereas the reduced 2FI model was selected for the analysis of ZP values, and the reduced linear model was proposed to evaluate EE%. Model reduction is a statistical technique employed to enhance model performance by minimizing the mathematical difference between adjusted and predicted R2 values through removal of insignificant factors to potentiate model predictive ability [31]. The suggested models were perfectly aligned as indicated by the mathematical difference between adjusted and predicted R2 (less than 0.2 in all models), validating the reliability of the optimization process [62]. Additionally, the adequate precision values were higher than 4 (Table 3), indicating the discriminating analyzing power of the suggested models [63]. Furthermore, all models exhibited the lowest predicted residual error sum of squares (PRESS) values, whereas the p-values of the ANOVA analysis across all studied responses were less than 0.05, designating their statistical significance.

Table 3 Summary of central composite design statistics for the selected responses of PTX-loaded novasomesEvaluation of PTX novasomal vesiclesEffect of formulation variables on particle size (PS) and polydispersity index (PDI)

The PS of the novasomal vesicles is a crucial factor in pulmonary drug delivery because it influences how the vesicles deposit in lung tissues. It has been shown that vesicles with a PS less than 500 nm enable deeper lung penetration, reaching the distal airways and alveoli, and also extend drug retention [64]. In this study, the PS of the formulated PTX novasomal vesicles ranged from 178.43 ± 0.75 nm to 392.70 ± 0.96 nm, as detailed in Table 2. ANOVA analysis revealed that the PS of the novasomes was significantly affected only by the aqueous phase volume (p-value = 0.0051). As depicted in Fig. 1A, increasing aqueous phase volume results in vesicles with smaller PS. This reduction in PS may be due to better dispersibility of the novasomal ingredients in larger aqueous phase volumes, leading to smaller PS [65]. Conversely, the higher PS observed at lower aqueous phase volumes can be explained by supersaturation of the novasomal dispersion during preparation, which promotes nucleation and particle growth. At higher concentrations associated with lower aqueous phase volume, particles tend to grow faster, dominated by particle growth; whereas, at lower concentrations, nucleation exceeds particle growth, resulting in significantly smaller novasomal vesicles [66]. These findings are consistent with those reported by Laouini et al. [67], who observed that increasing the aqueous phase volume during liposomal formulation led to vesicles with notably smaller PS. This is likely due to increased dilution of the lipid ingredients after mixing with the aqueous medium, which promotes the immediate formation of smaller vesicles and reduces the chance of fusion into larger ones.

Fig. 1

Line plot for the effect of aqueous phase volume on the PS (A), ZP (B), and a 3D surface plot for the effect of aqueous phase volume and drug amount on EE% (C) of the prepared PTX-loaded novasomes

PDI is a critical parameter that is used to evaluate the size uniformity of the vesicular dispersion. It is particularly vital for localized pulmonary delivery, as a narrow size distribution ensures predictable aerodynamic behavior and uniform deposition across pulmonary tissue [68]. It typically ranges from 0 to 1, with values approaching 1 demonstrating heterogeneous dispersion with a wide range of particle size distribution [69]. Furthermore, PDI serves as a preliminary indicator of physical stability, as PDI values less than 0.5 suggest a diminished susceptibility to Ostwald ripening, where small particles tend to dissolve and redeposit onto large ones, ultimately compromising formulation stability, dissolution rates, and overall bioavailability [70]. In this study, the PDI values of all the formulated PTX-loaded novasomes ranged from 0.123 ± 0.01 to 0.470 ± 0.02 (Table 2), indicating homogeneous, stable novasomal dispersions with a narrow particle size distribution.

Effect of formulation variables on zeta potential

The ZP of the formulated novasomal vesicles is used to evaluate their physical stability and tendency to aggregate [71]. Colloidal dispersions with ZP values more than 20 mV (absolute value) are physically stable with low tendency for aggregation [72]. In this study, the ZP values of PTX-novasomal formulations ranged from − 29.80 ± 0.15 mV to -52.96 ± 0.94 mV as shown in Table 2, reflecting excellent physical stability with a high repulsion force preventing novasomal aggregation. The observed negative charge is probably attributed to the ionization of the carboxylic acid group in stearic acid [73]. Statistical analysis of ZP results revealed that the ZP values of novasomal dispersions were significantly affected by the aqueous phase volume only (p-value = 0.0031). As observed in Fig. 1B, increasing the aqueous phase (buffer) volume reduces the ZP values of the novasomal vesicles. This reduction could be attributed to the surface hydration dynamics and steric masking. Specifically, the larger aqueous volume facilitates the maximum hydration of the hydrophilic headgroups of the non-ionic SAA on the vesicle surface. This extensive hydration induces steric swelling, which physically displaces the hydrodynamic slipping plane further away from the negatively charged vesicle surface. An outward shift of the slipping plane results in a lower measured ZP, even though the absolute structural charge remains intact [74]. Similar results were reported by Kumar et al. [75], who observed a significant reduction in the ZP of several investigated nanosystems, specifically liposomes, niosomes, and nanoemulsion upon dilution.

Effect of formulation variables on entrapment efficiency

Given that PTX-loaded novasomes were designed to enhance the aerodynamic properties and improve drug deposition in deep lung tissues, the evaluation of PTX encapsulation efficiency is of critical importance. However, from a formulation perspective, the encapsulation of hydrophilic drugs frequently presents a challenge. Because hydrophilic drugs are restricted to the limited volume of the aqueous core of the nanovesicles [76], a drug portion naturally partitions into the external aqueous phase during the formulation of lipid-rich systems [77]. This physical distribution inherently limits entrapment capacity, often resulting in a relatively low EE%. In this study, the dialysis method was used to quantify the unentrapped drug and indirectly estimate the EE%. The nanoformulation was dialyzed against fresh water for 2-hours to allow the complete diffusion of the unentrapped drug. The 2-hour threshold was previously verified by estimating the time required for the complete diffusion of free PTX solution. The EE % of PTX-loaded novasomal vesicles ranged from 31.24 ± 0.91% to 75.76 ± 0.00% as shown in Table 2. Statistical analysis of the data showed that the EE% of the formulated novasomes was significantly affected by drug amount (p-value < 0.0001) and aqueous phase volume (p-value = 0.0003). As shown in Fig. 1C, the EE % of PTX vesicles was increased by increasing the amount of PTX added during the preparation. This effect could be attributed to the formation of a saturated solution that acts as a driving force, causing PTX to diffuse into the novasomal vesicles [78]. On the contrary, increasing the aqueous phase volume reduced PTX concentration in the aqueous phase, resulting in a lower PTX EE% [79]. Same results were reported by Suwanpitak et al. [80], who explained this based on the lower concentration gradient obtained with large aqueous phase volume, and hence, less driving force for PTX to be entrapped within the novasomal nanovesicles. These results also align with those obtained by Maiti et al. [81], who observed that increasing aqueous phase volume from 100 mL to 140 mL reduced the EE% of microspheres by almost twenty times.

Optimization of PTX-loaded novasomes

The formulation of PTX-loaded novasomes was optimized by adopting the desirability factor (DF) approach using Design Expert®13. DF is a numerical value ranging from 0 to 1, with values approaching 1 indicating an optimal alignment of the independent variables to produce nanovesicles with desired characteristics [82]. In this study, the formulation optimization process aimed to prepare optimized PTX-novasomes with the smallest PS, lowest PDI, highest ZP value, and EE%. PTX-novasomal dispersion prepared using Span®80 as SAA with 30 mg of PTX and an aqueous phase volume of 22.618 mL, was suggested by the software as the optimized nanoformulation with a DF of 0.728 as shown in Fig. 2 and Table S2. The optimization process was verified by preparing the suggested optimized novasomes, which were further characterized and compared to the observed values, as shown in Table 4. The actual values of the PS, PDI, ZP, and EE% of the optimized novasomes were perfectly aligned with the predicted values, as indicated by the low residual values and bias% (less than 10%). These values signify the marked predictive power of the statistical model [83].

Fig. 2

Contour plot (A) and 3D cubic plot (B) for the desirability data from the central composite statistical design, indicating the optimal formulation parameters for the PTX-loaded novasomes. PTX = pentoxifylline

Table 4 Predicted and observed values of optimized PTX novasomal systemCharacterization of optimized novasomesTransmission electron microscopy

The optimized novasomes were morphologically visualized using transmission electron microscopy to ascertain the vesicles’ size as well as the shape of the formed vesicles. As shown in Fig. 3A-C, TEM micrographs revealed well-defined, discrete spherical vesicles. Notably, these vesicles exhibit a multi-lamellar structure, as highlighted in Fig. 3A, and display a clear boundary characteristic of true vesicular morphology at higher magnification power (Fig. 3C). The PS of the vesicles observed with TEM aligns with the values previously determined by the Zetasizer (~ 240 nm). Furthermore, the absence of any aggregation in the TEM images corresponds with the high ZP values previously measured (-46.9 mV), indicating their high colloidal stability and diminished tendency for aggregation.

Fig. 3

Transmission electron micrographs of the optimized PTX-loaded novasomes (A: magnification power = 15000x, B: magnification power = 12000x, and C: magnification power = 120000x)

In-vitro release study

The in-vitro release of PTX from the optimized novasomal dispersion in PBS (pH 7.4) was investigated and compared to PTX solution to characterize its behavior and provide a preliminary insight into its release characteristics under physiological conditions. As illustrated in Fig. 4, the free PTX solution exhibited a rapid release of approximately 70% within the first hour, reaching nearly complete release (~ 85%) by 2 h. In contrast, PTX-loaded novasomes demonstrated a modified biphasic release profile characteristic of vesicular systems encapsulating highly hydrophilic drugs, where it showed an initial burst release of ~ 38% within the first hour. This fraction corresponds to the un-encapsulated PTX molecules that are loosely attached to vesicles surface and rapidly diffuse into the outer release compartment [35, 84]. Following this initial phase, the fully encapsulated PTX was released gradually from the aqueous core of the nanovesicles, reaching 88% by 4 h.

Fig. 4

In-vitro release profile of PTX from the optimized novasomal vesicles and PTX solution in phosphate-buffered saline (pH = 7.4) using dialysis bag technique. PTX = pentoxifylline

This subsequent gradual release is governed by novasomal structural integrity. By integrating cholesterol and stearic acid, the novasomal vesicles form a cohesive lipid matrix that regulates the diffusion of the encapsulated hydrophilic PTX, preventing its immediate escape [85]. Cholesterol is known to reduce membrane fluidity, forming a rigid outer membrane with minimal transient pores [86]. Furthermore, LCFAs such as stearic acid enhance chain tightness with structural remodeling of the vesicles’ layers [87]. Consequently, the inclusion of both components within the lipid matrix controls PTX diffusion from the novasomes, dictating its modified biphasic release pattern.

This biphasic-release profile is consistent with previous investigations evaluating hydrophilic drugs encapsulated into novasomal nanovesicles. For instance, studies evaluating hydrophilic terbutaline sulfate (TBN) and zolmitriptan (ZT) reported an immediate burst release within the first two hours. Specifically, about 40% of TBN was rapidly released, which is attributed to its hydrophilicity and the rapid liberation of un-encapsulated molecules near the surface. A similar mechanism was documented for ZT-loaded novasomes, where an instant rapid release was driven by the rapid dissolution of surface-adsorbed ZT molecules. Following this phase, both systems transitioned into a more gradual release governed by the structural integrity of novasomes, which forced the drug to diffuse slowly through the lipid-bilayer matrix towards the external medium [25, 48].

Furthermore, the kinetic modeling of the release data revealed that the Korsmeyer-Peppas model provided the best fit with the highest R2 (0.991), as shown in Table 5. The calculated release exponent (n) of 0.864 indicates non-Fickian anomalous transport, suggesting that the drug release is driven by a combination of diffusion and erosion mechanisms [88]. This is consistent with the previous findings by Kamboj et al. [89], who reported that the release kinetics of tenofovir disoproxil fumarate from niosomes also followed the Korsmeyer-Peppas model, via a coupling mechanism of membrane diffusion and nanovesicular carrier erosion.

Table 5 Kinetic analysis of PTX release date from optimized PTX-loaded novasomes and PTX solution PTX = pentoxifylline

From a therapeutic perspective, the biphasic release pattern could be beneficial for providing adequate prophylaxis against PF. The initial burst allows for rapidly established, high concentrations of PTX within the lung tissues, while the subsequent gradual release helps maintain localized drug exposure [68].

Aerodynamic size characterization

The aerodynamic behavior of the optimized novasomes was investigated to evaluate the potential deposition of PTX within deep pulmonary tissues. As the assessment of aerodynamic performance using an ACI strictly requires the use of a dry powder, the optimized formulation was first lyophilized. As shown in Table 6, the key physicochemical properties (i.e., PS, PDI, ZP, and EE%) of the formulation remained stable, with no statistically significant differences observed between the pre-lyophilization and post-reconstitution values. These data confirm that freeze-drying did not compromise the structural integrity of the optimized PTX formulation, ensuring consistent performance before aerosolization.

Table 6 Characterization of optimized PTX-loaded novasomes before and after lyophilization and reconstitution

Subsequent analysis of the ACI results using CITDAS facilitated the determination of TED and the estimation of nanoparticles’ aerodynamics after inhalation. As presented in Table 7, the optimized novasomes showed a higher TED% than free PTX powder, reflecting the ability of PTX-loaded novasomal vesicles to deliver higher doses via inhalation compared to free PTX powder. Additionally, FPD and FPF were measured to indicate the fraction of PTX novasomes with PS less than 5 microns that deposit into deep pulmonary tissues [90]. In this context, PTX novasomes had significantly higher FPD (87.600 µg, p-value ˂ 0.001) and FPF (43.819%, -value ˂ 0.001) than free PTX powder (9.724 µg and 5.353%, espectively). This was evident by the higher levels of PTX detected after administration of inhalable PTX novasomal vesicles in comparison to free PTX (Fig. 5), especially in the last stages (3–5), suggesting the possible deposition of PTX into deep lung tissues [91]. Furthermore, MMAD was estimated to predict the deposition of novasomes within the pulmonary tissues. The MMAD of PTX novasomes was 1.612 μm, significantly lower (p-value ˂ 0.001) than the MMAD of free PTX powder (6.380 μm). It is well reported in the literature that particles with an MMAD ˂ 3 μm have an 80% probaility of reaching the lower respiratory tract and a 50–60% chance of alveolar deposition; while those with MMAD ˃ 5 μm are primarily trapped in the oropharyngeal area and barely reach minute airways [92]. Consequently, these results strongly support their ability to successfully traverse distal airways and deposit into deep pulmonary tissues, thereby enhancing the localized bioavailability of PTX.

Table 7 The aerodynamic parameters of the inhaled PTX novasomes and free PTX powder using Andersen Cascade ImpactorFig. 5

Total amounts of PTX trapped into different stages of Andersen Cascade Impactor after inhalation of PTX novasomes and free PTX. PTX= pentoxifylline. * = p-value ˂ 0.05, ** = p-value ˂ 0.01, *** = p-value ˂ 0.001, **** = p-value ˂ 0.0001, and n.s = non-significant

While the MMAD ensures deep lung deposition, the physical particle size of the novasomes (~ 240 nm) minimizes potential systemic translocation. Literature indicates that vesicles smaller than 100 nm are the most likely to cross the air-blood barrier following inhalation [93]. Therefore, maintaining a particle size around 240 nm represents a significant advantage for a restricted localized pulmonary delivery. This is firmly supported Patlolla et al. [94], who investigated inhaled celecoxib-loaded nanostructured lipid carriers (Cxb-NLC). Interestingly, they prepared an optimized formulation with aerodynamic and physical parameters that align closely with our data (MMAD: 1.6 ± 0.13 μm; PS: 217 ± 20 nm). Their corresponding pharmacokinetic analysis revealed that the systemic clearance of the Cxb-NLC (0.93 L/hr) was 22-fold slower than that of the free Cxb solution (20 L/hr). These findings strongly support the hypothesis that nanocarriers within this size range can effectively deposit in lung tissues while successfully limiting systemic absorption.

Fourier-transform infrared spectroscopy (FTIR)

The FTIR spectra of PTX, stearic acid, cholesterol, blank novasomes, and PTX-loaded novasomes are shown in Fig. 6. The FTIR spectrum of PTX (Fig. 6A) exhibited a characteristic band at 2945.30 cm−1attriburted to C-H stretching in addition to distinct peaks at 1701.22 cm− 1 and 1660.71 cm− 1 representing the vibrational stretching of the amide and ketone groups, respectively. Additionally, PTX demonstrated a peak at 1546.91 cm− 1 for the vibrational bending of amide (N-H) in addition to a characteristic peak that appears distinctly at 1354.03 cm− 1 related to amide elongation (C-N) [95]. Simultaneously, the FTIR spectrum of stearic acid (Fig. 6B) showed characteristic bands appearing at 2914.44 cm− 1 and 2848.86 cm− 1 (CH2) [96], while those specific bands of cholesterol (Fig. 6C) appeared at 3396 cm− 1 [97] and 3421.72 cm− 1, corresponding to the vibrational stretching of the hydroxyl group [26].

Fig. 6

FTIR spectra of (A)PTX, (B) stearic acid, (C) cholesterol, (D) blank novasomes, and (E) PTX-loaded novasomes. PTX = pentoxifylline

The FTIR spectrum of the blank novasomes (Fig. 6D) retained the individual peaks of both stearic acid and cholesterol, confirming high compatibility among the vesicular components. Furthermore, the PTX-loaded novasomes (Fig. 6E) displayed the characteristic peaks of PTX, stearic acid, and cholesterol, signifying the absence of any chemical interaction between PTX and excipients [35]. However, the reduced intensity of the characteristic PTX characteristic peaks (1701.22 cm− 1 and 1660.71 cm− 1) indicates the successful encapsulation of PTX within the novasomes [98].

Differential scanning calorimetry (DSC)

DSC analysis was performed to exclude any possible incompatibility [99], assess the physical form (amorphous or crystalline) of the drug within the optimized formulation, and investigate the thermal stability of optimized PTX-novasomes [100]. The DSC thermographs for PTX, physical mixture, physical mixture: PTX (1:1), blank novasomes, and PTX-loaded novasomes are represented in Fig. 7. The DSC isotherm of free PTX exhibited a sharp endothermic peak at 103.04 °C (Fig. 7A) corresponding to the melting point of its crystalline form [101]. Simultaneously, the physical mixture of the novasomes components (Fig. 7B) displayed a sharp endothermic peak at 55.06 °C. On the other hand, the DSC thermogram of PTX and physical mixture (1:1) (Fig.