3.1.1. Preparation and Characterization of the Freeze-Dried Inserts Containing VA
The freeze-drying technique allows for the better handling of thermo-labile active pharmaceutical ingredients (API) by eliminating water at low temperatures to ensure long-term stability of the final product [
33,
34] and production of matrices with a rapid hydration/solubilization when they come in contact with the tear fluid of the eye cul-de-sac.
Hydroxypropyl methylcellulose (HPMC) and hyaluronic acid (HA) were chosen as mucoadhesive polymers, as they are widely used in the development of ophthalmic dosage forms. All the dispersions contained 1 wt % of total polymer as HPMC or a mixture of HPMC and HA (ratio 9:1). Mannitol was used as the crystalline bulking agent to obtain a uniform cake appearance [
35]. Moreover, trehalose (ratio 1:2) was introduced as a cryoprotectant and lyoprotectant agent to stabilize the peptide during the freeze-drying process, replacing a part of the mannitol, since it is known that the crystalline modification of mannitol during the lyophilization process might negatively impact the storage stability of proteins [
36,
37].
The integrity of the components of freeze-dried matrices was visually evaluated in terms of cake collapse, volume, melt-back, puffing, texture, cracking, and color changes [
38]. As shown in
Figure 1, good cake quality was obtained for all developed matrices (diameter: 7.88 mm, thickness: 2.30 mm, area: 48.73 mm
2), with no fractures or cake collapse.
Coefficients of variation [CV = (SD/mean) * 100] were calculated for the weights of the matrices belonging to two different batches for each developed formulation (
Table 3). CV ranged between 1.37% and 2.04% for a mean weight of the matrices between 5.67 and 5.85 mg. These low CV values, in accordance with the indications in the European Pharmacopoeia, suggest a good reproducibility of the preparation method.
To understand the influence of the freeze-drying conditions on the stability of the final product, the rheological characteristics were checked before and after lyophilization. All formulations showed pseudoplastic behavior and
Table 4 summarizes the apparent viscosity values, in mPa*s at D = 1 s
−1, taking into account the weaker interactions of the macromolecules when the fluid is at rest, such as during the formation of adhesive bonds [
32].
The HPMC/VA
disp starting dispersion showed a viscosity of 227.7 ± 11.82 mPa*s. The presence of high molecular weight HA (1000–1800 kDa) produced a predictable increase in viscosity: HPMC/HA/VA
isp gave the highest viscosity value (528.3 mPa*s) [
39]. The addition of trehalose instead of a part of mannitol showed a small increase in viscosity, which became more marked when hyaluronic acid was present (HPCH/T2/HA/VA
disp, 321.2 mPa*s). After freeze-drying, the viscosity of the dispersions obtained from the reconstituted matrices tended to decrease, but only in the case of HPMC/HA/VA
fd was the difference statistically significant (351.7 ± 23.76 vs. 528.3 ± 2.00 mPa*s after and before freeze drying, respectively). This behavior could be attributable to a partial depolymerization of HA during the lyophilization process [
40]. This phenomenon seemed to disappear after partial replacement of mannitol with trehalose (HPCH/T2/HA/VA
fd), directing the choice towards this formulation.
The role of the lyophilization process on the stability of drugs was verified by determining the VA content of the final matrices. Drug recovery values (%), summarized in
Table 5 as the mean of three determinations ± SE, ranged between 91.39% and 100.2%, depending on the formulation. Drug recovery from the matrices containing mannitol was about 92%; the partial replacement of mannitol with trehalose, in a ratio 2:1:1 (trehalose/polymer(s)/mannitol), proved to be an optimal strategy to stabilize protein-like drugs with an increase of drug recovery ranging from 96% to 100.2% for HPCH/T2/VA and HPCH/T2/HA/VA formulations, respectively. It is known that sugars should be able to interact with the protein-like drug during the freeze-drying process to maintain the protein’s native conformation and reduce both local and global mobility during the storage of the final product. In particular, small sugars, such as disaccharides, should be able to reduce the local mobility, since they are less inhibited by steric hindrance due to their better ability to form interactions with proteins [
41].
The ability of the matrices to rehydrate once applied in vivo was evaluated by the determination of WST. Trehalose-based matrices showed shorter WST between 60 and 120 sec for HPMC/T2/HA/VA
fd and HPMC/T2/VA
fd, respectively. On the other hand, matrices HPMC/VA
fd (WST: 12 min) and HPMC/HA/VA
fd (WST: 14 min) needed 10 times longer than trehalose-based matrices to rehydrate. The replacement of mannitol with trehalose, highly soluble in water (50.8 vs. 23.8 wt % at 20 °C [
42]) appeared to facilitate the interaction with the aqueous medium. In addition, mannitol and sugars (as the sucrose, structurally similar to trehalose) led to different porosity in the freeze-dried material: The former led to large and elongated clusters, while the latter produced small and circular cavities. This can result in a slightly quicker water uptake over time for sugar-based lyophilized polymers than those containing only mannitol [
43]. Moreover, increasing the additives amount in the formulation with respect to the polymer quantity, as in this study in which the additive(s)/polymer(s) ratio was 3:1, caused faster water medium permeation into the matrix [
44]. Clearly, the WST value can be used as an indicator of the in vivo matrix rehydration time, even if the experiment in vitro was performed in stagnant conditions [
31].
3.1.3. DSC Analysis
Influence of the lyophilization process on the components of the formulations under study was verified by DSC analysis. The thermal transitions are graphically reported in
Figure 2. At about 173 °C, HPMC
pow and HA
pow polymers showed a broad endothermic peak (more intense for HA
pow). In the case of HA, next to the transition at 172.54 °C ascribable to the dehydration process, there appeared an exothermal transition at 223.78 °C for the degradation phenomenon [
45]. DSC of pure mannitol exhibited a melting peak at 163.74 °C, a value very close to that reported in the literature [
44].
An interaction between HPMC and mannitol seemed to exist, since a widened peak to 158.36 °C, and a new endothermic transition onset at 208.35 °C were observed when the HPMC physical mixture was evaluated. In the case of freeze-died matrices (HPMCfd), the lyophilization process appeared to modify the amorphous state, a typical feature of HPMC, since another thermal peak appeared at 145.28 °C for HPMCfd. The replacement of a part of HPMC with HA gave similar results for both the physical mixture and freeze-dried matrix.
When part of mannitol had been replaced with trehalose in the physical mixture (HPMC/T2/HA
phmix), a transition peak at 93.31 °C appeared. Thermogram of pure trehalose revealed two peaks at 102.21 °C (sharp) and at 183.14 °C (wider); the first peak is attributable to the melting of the crystalline part of the dehydrated trehalose, while the latter peak can be associated with the melting of its anhydrous counterpart, which corresponds to the β-form of anhydrous trehalose [
46]. The presence of trehalose appeared to influence the interaction with polymers more markedly than mannitol, probably in term of polymer structure order [
43]. Intermolecular interactions between sugars and polymers could be the cause of this trend, as demonstrated by Imamura et al. (2010) who studied how Tg values changed when sucrose was combined with, for example, polysaccharides [
47].
HPMC/T2/HAfd matrices showed a thermal transition at 164.38 °C, as was the case of the same matrix without trehalose (HPMC/HAfd), while the introduction of trehalose appeared to modify the peak at 147.44 °C. The change in the thermal profile could suggest a close interaction among all components of the formulation.
3.1.4. In Vitro Drug Release Studies
The matrices under study were subjected to in vitro release studies using vertical diffusion cells (VDC); the amount of released drug through the period of the experiment (4 h) was determined by RP-HPLC. All the formulations showed the same drug-release profile (
Figure 3, drug released % vs. time): an initial linear trend followed by a slight decrease in the amount of released glycopeptide. In the matrices containing trehalose, this phenomenon was more markedly visible. At the end of the experiment, the percentage of VA released from the matrices was 70.54% ± 8.45% for the reference matrix HPMC/VA
fd, 79.11% ± 17.02% for HPMC/HA/VA
fd, 59.79% ± 1.51% for HPMC/T2/VA
fd, and 59.54% ± 0.94% for HPMC/T2/HA/VA
fd. Trehalose-containing matrices initially appeared to have a greater release, probably due to faster hydration as demonstrated by the value of WST (1–2 min vs. 12–14 min for the formulation with and without trehalose, respectively); afterwards they showed a slower release.
Mannitol appeared to lead to a faster dissolution, promoting the release rate of the glycopeptide; channel formation into the matrix due to the solubilization of mannitol could facilitate the entry of medium, matrix polymer(s) swelling, and consequent drug dissolution [
44]. When in contact with the hydration medium, matrices produced a viscous gel through which the peptide had to diffuse; a delay of drug release could depend on the type of polymeric structure. The addition of trehalose could change the polymer gel structure, by further entrapping the protein-like drug and slightly reducing its diffusion from the donor (matrix) to the receiving phase.