3.2. Physicochemical Characterization of HA-VAN25 in the Solid State
The solid-state integral characterization of HA-VAN25 microparticles was carried out using several characterization techniques including FTIR analysis, PXRD, TGA and DSC.
Figure 1 shows the comparative FT-IR spectra, where the bands that could serve as indicators of acid-base interactions were identified in order to analyze the changes attributable to the ionic association between HA and VAN. Regarding to FT-IR spectrums of the raw materials, HA showed the characteristic bands related to carboxylic groups, such as those at 3357 cm
-1 corresponding to the O-H tensile vibration of the COOH group, at 1732 cm
-1 attributable to the C=O carbonyl stretching vibration of the COOH group and at 1314 cm
-1, corresponding to the C-O bond of the carboxyl group of COOH [
12,
24]. In the VAN spectrum, a broad band at 3280 cm
-1 corresponding to the overlapping O-H and N-H tensile vibrations of the acid and amino groups was observed. In addition, the C=O stretching vibration at 1647 cm
-1 overlapped with the N-H bending vibration of the amino groups present, and the signal at 1225 cm
-1 was attributed to the C-O bond stretching [
25].
Comparing the HA-VAN25 complex with the raw materials and its PM, notable differences were observed. The band at 1732 cm-1, corresponding to the C=O of the COOH group, evident in both HA and the PM, was absent in the complex. Furthermore, a band emerged at 1401 cm-1, attributed to the stretching of the C-O bond in the carboxylate group (COO-), which is not present in the PM. These findings suggest a potential ionic interaction between HA and VAN, as only the bands corresponding to the ionized groups are evidenced.
The DSC and TGA thermograms, depicted in
Figure 2, illustrate the thermal characteristics of the raw materials (HA and VAN), the HA-VAN
25 complex, and the PM. The TGA analysis reveals two significant weight loss events in VAN. The initial extensive event, starting from room temperature (25 °C) to 100 °C, is attributed to material dehydration resulting from the loss of water adsorbed on the solid particle surfaces, with a weight percent loss of (9.8 ± 0.7) %. This event aligns with the DSC thermogram, represented by a broad and nonspecific endotherm within the same temperature range. The second TGA event likely corresponds to the decomposition temperature, initiating at 208 °C, as evidenced by the sustained weight loss in the TGA curve. Additionally, it is noteworthy that the melting temperature of pure VAN could not be observed under the assay conditions, conducted up to 215 °C. This is consistent with the findings of other authors who investigated the thermal properties of VAN [
26].
On the other hand, the DSC thermogram of HA exhibits an initial endothermic peak followed by an exothermic peak at approximately 180 °C, coinciding with mass loss in TGA, indicative of the initiation of its decomposition. The glass transition of pure HA could not be observed at the test temperatures. This aligns with prior findings, as reported by others authors, who similarly demonstrated a multi-stage decomposition process for HA where the initial degradation step involves dehydration from the compound structure, followed by a second stage near 200 °C, consistent with our results, and two additional peaks observed at 320 °C and 415 °C [
27].
The thermograms of the HA-VAN25 complex exhibit a weight loss in TGA and an endothermic dehydration peak in DSC between room temperature and 100 °C, mirroring the behavior of the precursors. However, unlike the PM, which decomposes at the same temperature as HA (the precursor that decomposes the first), the TGA spectra of the HA-VAN25 complex reveal a shift in the decomposition temperature towards higher temperatures compared to HA. Consequently, the formation of a complex with VAN appears to shift HA decomposition at temperatures higher than 180 °C, thereby enhancing the polymer's stability. This observation suggests the presence of intermolecular forces between HA and VAN, influencing their thermal behavior, which aligns with the previously presented FT-IR results.
The p-XRD patterns of VAN, HA and HA-VAN
25 were performed. In
Figure 3, it can be observed that both precursors, the PM, and the complex are amorphous, evidenced by the absence of diffraction peaks, a consistent observation reported by previous researchers and in alignment with the earlier mentioned DSC spectra [
28].
It has been reported that binary PE-D complexes, obtained by mixing a PE with a drug of opposite charge in a convenient medium capable of dissolving one or both components, result in amorphous solid materials where the drug is ionically bound to the polymeric carrier [
28].
Generally, amorphous powders are favored over crystalline counterparts for pulmonary drug delivery due to the numerous advantages they offer [
29]. For example, the deposition of crystalline particles can induce an inflammatory response, as reported by other authors who studied the inhalation of crystalline rifapentine particles [
30]. Currently, TOBI®, an inhalable antibiotic containing tobramycin used in the treatment of
Pseudomona aeruginosa infections associated with CF, possesses an amorphous structure, akin to our HA-VAN
25 system [
31].
Despite the p-XRD patterns obtained, revealing amorphous structures in both HA-VAN25 and its precursors, HA and VAN, the comprehensive information provided by FT-IR, DSC, and TGA suggests an ionic interaction between the carboxylic groups of HA and the basic groups of VAN.
3.3. Powder Characterization
There are different physical factors that can affect the aerosolization and therefore the deposition in the deep lung of dry powders, such as particle size distribution, flowability of the formulation as well as particle density and shape [
32,
33,
34].
As it’s known, particles having a geometric size range between 1-5 µm are, in principle, suitable to reach the deep lung since particles with sizes higher than 5 µm can be deposited in the upper airways, whereas particles smaller than 1 µm can get exhaled [
32].
SEM images were taken after the spray-drying process of the complex as well as the VAN as the raw material at different magnification values. According to the micrographs observed in
Figure 4A,B (magnification 1,000×), VAN showed a flat and lamellar structure with irregular shape and size of the particles between 20-40 µm, which is considered too large for inhalation purposes. On the contrary, when the HA-VAN
25 complex was observed, much smaller particles were created (4B) and in the image with a magnification 10 times higher (4 C, 10,000×) it was noticed that most of the particles presented a size between 3-4 µm. Regarding the morphology of HA-VAN
25, the particles showed a characteristic hemispherical hollow shape with smooth surface and edges, as was previously described by Martinelli
et al. [
35] and Ceschan
et al. [
36] for similar hyaluronate sodium salts-based spray dried particles. The formation of agglomerates was noticeable, as it was evident also macroscopically, and it could also be seen structures of one-inside-other particles in some cases.
The use of spray-drying as a process for the obtention of the solid can be beneficial in the morphology of the particles as already demonstrated by the spray dried inhalation powder of colistimethate sodium for lung infections in CF [
37]. Advantageous properties can be associated with the use of this technique as the shape and diameter uniformity of the particles which can provide good flowability, while the formation of hollow particles allow low-density structures, reducing the aerodynamic diameter and improving the aerosolization performances [
38,
39].
In this context, the determination of bulk density and flowability properties of inhalation dry-powders provides important material quality attributes directly related to the aerosolization behavior [
40,
41]. Taken all into consideration, it was measured the bulk and tapped density of the complex HA-VAN
25 showing a value of (0.13 ± 0.02) g/mL and (0.18 ± 0.03) g/mL, respectively. These values are reasonable for inhalable powders since tapped densities below 0.4 g/mL have been reported in many works as cut-off for determining good aerodynamic characteristics [
42].
However, since the smaller the size of particles, the higher is the interaction particle-particle related to Van der Waals forces concerning high contact area, these cohesive forces can lead to poor flow properties and formation of agglomerates [
40]. Particle agglomerates and poor flowability can impact the aerosolization performance of the powder formulation that ultimately can remain in the inhaler after patient inhalation, resulting in low emitted dose [
34]. On the other hand, it is well-established that aerosolization performance in terms of fine particle dose of the emitted powder is optimal with smaller particle sizes, therefore the particle size and density of inhaled powders has to be engineered to balance their properties in order to provide the aerosolization performance as well a proper flowability.
In order to study the flow properties of the complex HA-VAN
25, Carr's index (CI) and Hausner ratio (HR) were used according to Equation 2 and 3, obtaining values of 26.9 ± 5.8 and 1.4 ± 0.1, respectively. The high CI and HR values indicate a powder with poor flow characteristics based on the scale of powder flowability according to USP [
20].
As regards to flowability, as less compressible is the powder, less cohesive it will be and for that reason better flow it will have [
42,
43]. In the case of HA-VAN
25, the differences between bulk and tap densities evidenced a high compressibility, leading to a poor flow property, which has been also seen previously for sodium hyaluronate spray-dried particles by Martinelli
et al. [
35].
In addition, in a preliminary determination of the capability of the powder to reach the deep lung, the particle size distribution was measured by laser diffraction, which is correlated with the aerodynamic particle size. HA-VAN25 presented a Dv90 = (6.37 ± 0.07) µm, Dv50 = (2.90 ± 0.02) µm and Dv10 = (1.19 ± 0.02) µm, meaning that 90 %, 50 % and 10 % of the particle population, respectively, has a particle size below those respective values. Besides, a SPAN value of (1.79 ± 0.03) was measured, which indicates a monodisperse distribution of particle size. These are promising results, where more than half of the population have a geometric size below 5 µm within the acceptable range being suitable for deep lung deposition using only the HA-VAN25 complex, without other pharmaceutical excipients.
3.4. In Vitro Biopharmaceutical Performance of HA-VAN25 Complex
The
in vitro deposition distribution study after the aerosolization of complex powder loaded in rigid HPMC capsules using the NGI equipment was performed.
Table 1 summarized the parameters measured, including ED, EF, FPF, Extra-FPF and MMAD, that can be related to the capability of the powder to be transported through the patients’ airways. Despite the poor flowability evidenced, the powder provided a good emitted fraction (above 80%), when aerosolized with RS01 device. It appears evident that the agglomerates of HA-VAN
25 spray dried microparticles highlighted by SEM analysis and the cohesive forces providing poor flow were disrupted during the capsule spinning, powder extraction and subsequent impaction on the walls and de-agglomeration grid of the RS01 device. The FPF value showed that about 43% of the powder has the potential to reach the deep lung since the particles have an aerodynamic diameter lower than 5 µm, while the 26% of the particles can go deeper until the alveolus. The MMAD exhibited that 50% of the population have a size lower than 4.29 µm which is in good agreement with the PSD obtained by laser diffraction.
The drug’s aerodynamic particle size distribution reported in
Figure 5 revealed that approximately 16% of the VAN was not emitted from the capsule and the inhaler device, while the 22% was retained in the IP, which simulates the throat. Those percentages, besides the amount of VAN deposited at stage 1 (9%) correspond to the quantity of drug that will not reach the deep lung. The presence of a high amount of drug from stage 2 onwards is considered desirable for lung deposition [
44] since this stage has a cut-off diameter of 4.46 µm at the flow rate of 65 L/min, almost half of particles of HA-VAN
25 are in these stages. The higher values of VAN at stage 2 and 3 in comparison with the following is consistent with the MMAD value obtained.
Despite the absence of vehicle excipients frequently used in dry powder inhaler formulations, such as lactose or mannitol, the HA-VAN
25 powder presented by itself a satisfactory efficiency for pulmonary administration. Moreover, as demonstrated by the optimal value of VAN emitted fraction, the formation of weak agglomerates could result in the easy dis-aggregation either for the inspiratory force generated by the patients and/or the collisions between the particles or particles-wall inside the inhaler device leading to a proper EF [
45]. The suitable aerosolization performance observed is consistent with the results earlier informed where the powder showed a low bulk density, the morphology presented a hollow hemispherical shape and the Dv50 value was about 2.9 µm; and all these together influence the aerodynamic performance which would lead to an adequate pulmonary deposition [
46].
In a previous study by Sullivan
et al. (2015), the
in vitro and
in vivo performance of dry powder Vancomycin hydrochloride (VAN) without further processing was investigated in intubated rabbits, revealing promising results. Intubated rabbits administered a 1 mg/kg dose of VAN via inhalation demonstrated a comparable AUC to those receiving the same dose through a single bolus IV infusion. Notably, inhaled VAN exhibited reduced C
max and increased T
max, indicating a more sustained pulmonary level of the drug. However, the physicochemical and flow properties obtained by these authors were not the most suitable for inhalation therapy, for example, the VAN showed different particle sizes and shapes; presented high bulk and tap densities of (0.35 ± 0.01) g/cm
3 and (0.51 ± 0.01) g/cm
3, respectively; FPF value was no more than 26%, and MMAD value was close of 7 μm, accompanied by a substantial geometric standard deviation [
26]. For this reason, our dry powder formulation, based on HA-VAN
25 with better physicochemical and flow properties mentioned before, is expected to overcome these results in terms of
in vivo VAN performance, emphasizing both efficacy and safety.
Following the impaction process, dry powders deposited within the lungs are required to undergo complex drug absorption processes that include wetting, dissolution, and diffusion. Since no established a specific dissolution method for inhalation products is defined by pharmacopoeias and guidelines, it was used the traditional basket apparatus dissolutor (USP Apparatus 1), which has a relatively large volume of dissolution medium where the powder is dispersed. The dissolution profiles of VAN and the HA-VAN
25 complex are depicted in
Figure 6. It was evident that the complete amount of pure VAN dissolved within 5 min, while the dissolved proportion of VAN from HA-VAN
25 was less than 40% at the same time. The comparison between VAN and HA-VAN
25 dissolution profiles showed
f1 = 40.6 denoting significant differences (
f1 > 15). Given the hydrophilic nature of VAN, a prompt drug dissolution was anticipated. Conversely, the formation of an ionic complex with HA leads to a slower dissolution rate. Additionally, it is noteworthy that the complex, when in contact with the dissolution medium, undergoes gelation and slight swelling due to the presence of the polymer, potentially contributing to the observed reduction of the dissolution rate.
Beyond the differences in the dissolution profiles between VAN and HA-VAN
25, it is noticeable that more than 85% of the loaded VAN dissolves within the initial 30 min. This aspect is crucial, as inadequate drug dissolution could potentially induce lung irritation, local side effects, and trigger processes like macrophage phagocytosis or mucociliary clearance of solid particles, ultimately leading to a rapid reduction in lung dose [
47].
In addition, bicompartmental Franz cells were used in order to study the release behavior of the aqueous HA-VAN25 complex dispersion in comparison with a solution of pure VAN, in two different media: water and PBS pH 7.4.
It can be seen in
Figure 7, when water was used as a receptor medium, a fast diffusion of VAN across the dialysis membrane from its solution was observed. However, the release rate of VAN from the aqueous dispersion of its complex was substantially slower. This response can be attributed to the reservoir behavior usually described to PE-D complexes, where the dissociation of ionic complex between HA and VAN is the determining step in the VAN release toward the semipermeable membrane [
12,
48]. In addition, the electrostatic attraction between the oppositely charged macroions present in the donor compartment, makes the diffusion of ionic species more restricted [
14].
When water was replaced by PBS pH 7.4 solution (as a simulated physiological receptor medium), a significant increase of VAN diffusion from the HA-VAN
25, in comparison with its release profile in water, was observed, reflected by a
f1 value of 72.6. It is known that the presence of dissolved ions in the receptor medium (from PBS solution) promotes ion exchange from the macromolecular complex microenviroment, increasing the proportion of free ionic species of VAN and consequently significant increase of drug release rates [
16].
On the other hand, the similarity of VAN release profiles from pure VAN solution and from the complex, both in PBS medium, (
f1 = 15), could be related with the diffusion of ions from the receptor compartment to the donor compartment, promoting the dissociation of VAN HCl into VAN and HCl separately, shifting the acid base equilibrium towards the non-ionized VAN (VAN base) increasing their proportion in solution. The VAN base presents a lower solubility (0.225 mg/mL) than VAN HCl in water (> 100 mg/mL),
i.e., the solubility decreases over 400 times [
49]. This could be the reason why VAN diffuses faster in water than PBS 7.4, and, at the same time, with a similar release profile than the complex in the saline solution. However, additional experiments assay would be necessary to confirm this observation.
Finally, kinetic analysis of the release profiles showed a strong fit to the power equations (R
2 0.96 and 0.99) above the Higuchi diffusion model. The values of diffusional exponent
n between 0.5 and 1, suggest an anomalous or non-Fickian release behavior (
Table 2). Consequently, the dissociation of ionic pairs between HA and VAN and the later diffusion of VAN from HA-VAN
25 seems to be the principal control mechanisms of drug delivery. The polymer chains reorganize slowly, whereas the diffusion process proceeds very rapidly and leads to anomalous time-dependent effects as it was observed in the majority of PE-D systems [
50].
3.5. Antibacterial Activity Tests
The agar diffusion test was carried out to assess the susceptibility against
Staphylococcus aureus reference strains, specifically strains 29213, 25923, and 43300. The results, presented in
Table 3, indicate that HA does not exhibit inhibition, displaying a halo of 8 mm corresponding to the diameter of the original well. HA itself is not considered inherently antimicrobial. In contrast, VAN demonstrates an inhibition halo ranging between 29-31 mm, depending on the strain, and the complex exhibits a similar inhibition pattern. Notably, none of these samples demonstrates reduced inhibition against the MRSA strain.
The antibacterial efficacy of HA-VAN
25 was assessed by examining MIC and MBC against
Staphylococcus aureus.
Table 4 reveals that not only does VAN exhibit antibacterial activity, but so does HA-VAN
25 against MRSA and MSSA. Remarkably, HA alone does not demonstrate inhibitory or bactericidal activity in concordance with the absence of halo inhibition previously mentioned. In contrast, VAN exhibits the same MIC and MBC values, consistent with its recognized bactericidal nature and these values are between 4 and 8 µg/mL in agreement with previous findings [
51,
52].
Notably, HA-VAN25 demonstrates comparable antimicrobial activity with VAN, underscoring that the formation of the complex preserves the well-established antibacterial efficacy of VAN against both MSSA and MRSA strains while simultaneously offering the advantages of being complexed with HA.