Particle size analysis
Figure 1 shows two cumulative particle size distribution (PSD) diagrams that illustrate each of the carrier’s PSD when using the (A) dry system and when using the (B) wet system. Moreover, given the use of the Mini Spray Dryer B-290, it was expected to obtain particle sizes that were below that of the 63–90-μm range (Fig. 1a), given that the spray-drying process produces particles below such range .
Likewise, Fig. 1b shows that the particle sizes of each of the carriers, when using the wet system, fell within the 63–90-μm range. The known occurrence of agglomeration was exploited in such a manner that allowed for it to be used as a carrier within this overall study. Such focus offered the opportunity to investigate the agglomerates on the basis of parameters that are used in the characterization and analysis of single particles. Particle size measurement in the dry system was able to break the aggregates of spray-dried particles (due to applying a pressure of 3 bar during the measurement) whereas in the wet system the spray-dried particles stayed as aggregates, although the ultrasound was applied during the measurement.
Relatedly, Table 1 highlights each of the carrier’s distinct characteristics such as volume mean diameter (VMD) and span of the RODOS dry system and the CUVETTE wet system comparing each characteristic side by side. Table 1 shows that all of the carriers experienced a significant difference in their VMDs when comparing each system to one another. For the dry system, the VMD ranged from 8.39 ± 0.40 to 37.97 ± 0.08 μm whereas the range for the wet system was 79.31 ± 2.19 to 87.95 ± 1.91 μm due to the presence of aggregated particles. The dry system experienced a particle diameter range of 1.56 ± 0.05 μm (D10%) to 75.46 ± 7.22 μm (D90%) whereas the particle diameter range for the wet system fell between 20.77 ± 11.02 μm (D10%) and 147.87 ± 170.11 μm (D90%). Due to the aggregation of particles, samples that were measured through the wet system showed smaller span values (narrower distribution) compared to that of the dry system, coinciding with the possibility of the dry system containing mixtures of aggregated and de-aggregated spray-dried particles during the measurement (Table 1).
Such outcomes, then, allowed for the carriers to be implemented and further studied to determine their physicochemical properties and particle morphology given that they underwent spray-drying, known to alter such characteristics, while also introducing L-leucine as an excipient. VMD (obtained via wet method) of the formulation after mixing for 30 min with salbutamol sulfate showed that the mixing process was unable to break down the agglomerates as the VMD was similar to the VMD of particles before mixing. For example, the VMD of formulations containing 0.1 and 10% leucine after 30 min of mixing with SS was 88.0 ± 9.62 and 71.97 ± 0.16 μm, respectively.
Figure 2 highlights the electron micrographs of each of the carriers making it evident that all of the formulations, with respect to their carrier, experienced some agglomeration giving way to their larger particle size, thereby supporting the results presented in Fig. 1 and Table 1. Moreover, the SEM micrographs also indicate, and account for, each of the carrier’s morphology as they show that all of them contain spherical particles with some agglomerates, particularly in the cases of 0.5% leucine (some of these agglomerated particles for each formulation are shown by red arrows). Such irregularity has previously been shown to be more effective in the delivery of salbutamol sulfate when compared to particles that are classified as being more spherical and regular in shape . The morphology of the spray-dried lactose, with increasing concentrations of leucine, is supported by data published by Aquino et al. , where they showed that more irregular and corrugated particles were obtained in the presence of high concentrations of leucine. Generally, corrugated particles disperse better than spherical ones as this kind of particle reduces contact areas and decreases inter-particulate cohesion. Therefore, it was expected that the formulation composition of the leucine carrier would result with an enhanced aerosolization performance, when compared to the carrier without leucine, which, in essence, would deliver salbutamol sulfate more poorly.
Solid-state characterization of spray-dried samples
Figure 3 shows DSC traces of L-leucine, original lactose monohydrate, and spray-dried lactose containing 0, 0.1, 0.5, 1, 5, and 10% L-leucine indicating where water evaporation, amorphous lactose recrystallization (Hc), α-lactose melting (Hα), and β-lactose melting (Hβ) took place. It is obvious from the figure that commercial lactose monohydrate shows an endothermic peak around 149 °C, which corresponds to the evaporation of water, followed by an exothermic peak around 171 °C, indicating the amorphous state in the sample; moreover, the endothermic peak around 220 °C corresponds to the melting of α-lactose  whereas any peak around 238 °C is an indication of β-lactose in the sample.
Pure L-leucine was also tested to determine whether or not any thermal events arose between the 25–300 °C range, which would rule out whether such events were due to the presence of leucine or not. No thermal events were seen within the range where lactose thermal events occurred and the endothermic peak around 300 °C corresponds to the melting of leucine. Spray-dried lactose showed three main thermal events with the first being an exothermic peak around 170 °C, attributed to the recrystallization of amorphous lactose to both α-lactose and β-lactose, which was then followed by the melting of α-lactose at around 220 °C; furthermore, the third endothermic peak around 238 °C was an indication of β-lactose in the sample. Moreover, spray-dried lactose did not show any DSC traces for the water evaporation which was a similar pattern that was observed for spray-dried lactose containing 0.1, 0.5, and 1% leucine, but with different intensities when compared to the spray-dried carrier with no leucine. Spray-dried formulations containing 5 and 10% L-leucine did not show any sharp or obvious peaks for water evaporation, the transition of amorphous lactose to crystalline lactose, and melting of lactose. On the basis of this information, all spray-dried carriers were considered to be in their amorphous state as the data that was collected indicates given that a definite crystalline structure was not present prior to their analysis which would have been depicted through the emergence of the amorphous lactose recrystallization enthalpy. Neither a recrystallization nor a melting peak was observed in the 5 and 10% L-leucine carrier, which serves as an indicator of their higher stability against recrystallization. Such results follow similar patterns that have been presented elsewhere [31,32,33,34] where amorphous drug carriers were formulated in such a way as to increase amorphous stability. To make sure spray-dried lactose was in the amorphous state, a more reliable technique (PXRD) was used.
Figure 4 contains the x-ray diffraction peaks for spray-dried lactose monohydrate containing 0, 0.1, 0.5, 1, 5, and 10% L-leucine, which provides an insight into the polymorphic state of each of the carriers. A carrier’s morphology plays an integral role in the drug delivery process that dictates whether a formulation is deemed effective in the delivery of the API of interest .
Looking at Fig. 4 more closely, it becomes evident that all of the carriers, from each of the formulations, were classified as being in their amorphous state given the absence of peaks (halo structure). In addition, all of the carriers showed two distinct peaks each (2θ = 12.2° and 18.6°) that are broad and distributed over a wide range of degrees on the 2θ plane, which also characterizes them as being of amorphous state. Likewise, given that all of the carriers exhibited irregular diffraction of electromagnetic radiation when compared to pure L-leucine (XRD not shown), it correspondingly catalogs them as amorphous as well .
To further assess the solid state of each carrier within this study and identify any interaction between lactose and leucine at the molecular level, FT-IR spectroscopy was implemented with the understanding that amorphous lactose displays a distinct frequency at 1260 and at 900 cm−1, α-lactose monohydrate at 920 cm−1, and β-lactose at 950 cm−1 . Figure 5 presents the results for the FT-IR spectra of L-leucine, spray-dried lactose monohydrate with its different concentrations of L-leucine (0, 0.1, 1, 5, and 10%), and further supports the fact that the carriers are in their amorphous state as the aforementioned peaks were present. In addition, Fig. 5 also reveals that, with the increasing concentration of L-leucine, each formulation underwent a phenomenon known as Fermi resonance where a shift in the vibrational energy causes the spectra to have a change in its intensity and resolution [36, 37].
Such variation within the spectra explains why the frequencies that are associated to key functional groups like aromatic C-H, alkanes, aldehydes, hydroxyl, carbonyl, ethers, and primary amines (which have frequencies at 2900, 3100–3400, 800–1400, and 3500 cm−1) become broadened or eliminated completely.
In vitro analysis of DPI formulations
Salbutamol sulfate assessment
Performance of the drug delivery profile of salbutamol sulfate, with respect to each of the formulations within this overall study, is defined in Fig. 6 where the amount of salbutamol sulfate recovered from each individual section within the MSLI is looked with a narrower focus: capsules (C), inhaler (I), mouthpiece (M), induction port (IP), stage 1, stage 2, stage 3, stage 4, and filter (stage 5). All of the formulations experienced minimal salbutamol sulfate deposits in the capsules with 5 and 10% L-leucine having the least amount after their actuation due to the lubrication effect of leucine, which makes particles flow more easily from the capsule to the inhaler device (Cyclohaler). The lubrication effect of the spray-dried leucine has been reported where increasing amounts of L-leucine show good lubricating properties . As particles maneuver through the respiratory tract, spray-dried lactose monohydrate along with 0.1% L-leucine experienced the highest amounts of salbutamol sulfate (43.63 ± 23.48 and 49.89 ± 27.80 μg, respectively) in the inhaler device when compared to the concentrations above 0.5% L-leucine which experienced the least amount at 13.79 ± 11.47 μg; the lubrication effect of leucine can also be observed here, as described previously. Furthermore, all of the formulations showed about the same amount of salbutamol sulfate in the mouthpiece (Fig. 6) but begin to differ at the IP as spray-dried lactose monohydrate had the highest amount (65.24 ± 4.26 μg) when compared to the other formulations, which had a range of 12.66 ± 5.66 to 29.02 ± 18.56 μg.
Moreover, 0.1% L-leucine had the highest salbutamol sulfate recovered from within stage 1 (176.06 ± 50.94 μg), but where it began to change was with stage 2 onward as 0.5% L-leucine experienced the highest salbutamol sulfate amounts in stage 2, stage 3, stage 4, and filter (81.89 ± 50.20, 145.58 ± 88.08, 87.45 ± 48.49, and 29.25 ± 20.16 μg, respectively) indicative of it being the most successful at delivering salbutamol sulfate to the targeted area that correlates to the alveoli, found in the lower respiratory tract. In other words, the formulations ranked in the following order 0.5% L-leucine > 0.1% L-leucine > 1% L-leucine > spray-dried lactose monohydrate > 5% L-leucine > 10% L-leucine.
Table 2 shows the aerosolization performance and deposition data for all formulations studied. The authors in the present study were interested in looking at, namely, FPF (fine particle fraction), DL (drug loss), impaction loss (IL), and effective inhalation efficiency (EI). All of the formulations differed remarkably from one another with respect to DL and percent emission (Table 2) given that they all undertook a high number of actuations (n = 10) per run, with each being filled with a consistent weight of around 33 ± 1 mg. The table shows that the performance of DPI formulations containing spray-dried leucine is much better than when leucine was excluded from the formulation (the lowest drug loss belonged to spray-dried lactose containing 0.5% L-leucine).
Impact loss (IL) within the formulations varied from 34.61 ± 12.38%, attributed to 0.5% L-leucine, to 52.49 ± 2.81%, belonging to spray-dried lactose monohydrate. Such variation between the formulations could be attributed to their aerodynamic diameter given that impaction is a flow-dependent mechanism governed by particle size .
Effective inhalation index (EI) ranged from 10.87 ± 0.22 (spray-dried lactose monohydrate) to 11.98 ± 0.37 (0.5% L-leucine) aligning with other data suggesting that 0.5% L-leucine has a high-drug aerosolization efficiency.
DS and FPD also showed a variation among the formulations with ranges of 29.31 ± 0.36 to 48.94 ± 10.78% and 262.28 ± 156.60 to 110.41 ± 4.77 μg, respectively. Such variation was attributed to the formulation’s particle size given that the phenomenon of inertial impaction becomes prevalent for large particles .
When it came to MMAD and GSD, however, all of the formulations gave similar results with MMAD being 3.12 ± 0.10 μm and GSD being 2.12 ± 0.03 μm. Such results mean that all of the SS was, theoretically, delivered to the area of keen interest. It also correlates with obtaining a FPF of 100%, which was not the case, as other factors came into play like impaction onto the upper respiratory tract where particles greater than or equal to 10 μm are removed by the mucociliary escalator and subsequently swallowed [40, 41].
The results showed that spray-dried lactose-leucine (containing 0.5% L-leucine) exhibited the highest FPF of 47.11 ± 9.94% suggesting that such formulation was the most efficient at delivering the most SS to the lower respiratory tract. This is because of the correlation that is seen between FPF and amount of SS delivered; that is to say, when FPF increases, the expected amount of SS that is delivered to the lower respiratory tract also increases . Such values, when compared to those obtained by Kaialy et al.  (FPF of 44.85 ± 1.76%) and Kaialy and Nokhodchi  (FPF of 46.9 ± 3.6%), prove to be an increase in the efficacy of salbutamol sulfate’s aerosolization performance. This formulation also had the highest percent emission of 96.41 ± 1.23%, when compared to the other formulations (Table 2), suggesting that SS was able to detach itself from the carrier easier when compared to the other formulations. This means that optimal physicochemical properties were attained such that a complementary system emerged between SS and the 0.5% L-leucine carrier. On the other hand, spray-dried lactose monohydrate showed the lowest percent emission (87.02 ± 3.79%) and consequently the lowest FPF (25.51 ± 1.23%). Such results infer that SS had a more difficult time detaching itself from the spray-dried lactose monohydrate carrier during inhalation when compared to 0.5% L-leucine.
Assessing the homogeneity of each of the formulations was an essential phase of this overall study given that a uniform formulation will give rise to a more effective drug delivery profile with a consistent dose to the patient. Table 3 eludes the homogeneity profile of each of the formulations (spray-dried lactose monohydrate, samples spray-dried with 0.1, 0.5, 1, 5, and 10% L-leucine) under investigation showing the potency of each and also presents the percent content homogeneity, which is expressed as the percent coefficient of variation (%CV), of each of the aforementioned formulations. The drug content of all formulations was within 75–125%, and the smallest %CV of 5.48% belonged to 0.5% L-leucine, which was the formulation that showed the best aerosolization performance. Such results indicate that 0.5% L-leucine had the best salbutamol sulfate content homogeneity among all of the formulations followed by 0.1% L-leucine with a %CV of 7.15%. In addition, the results showed that it is a bit difficult to obtain a very low CV% for DPI formulation containing salbutamol sulfate in the DPI formulation studied in the current research. This should be investigated more in the future ongoing research.