Fabrication and morphological characterization of the μPL
Two configurations of RSP-loaded μPL – short μPL and tall μPL – were fabricated following a multi-step, top-down approach and previously described by the authors [25, 26]. In brief, μPL are realized using a template-based fabrication strategy involving a silicon master template, whose wells’ geometry dictate the particle morphology, replicated in an intermediate PDMS template and eventually into a sacrificial PVA template (Fig. 1a). The wells in the PVA template are accurately filled with a polymeric paste (PLGA) dissolved in organic solvent (acetonitrile) carrying the therapeutic molecule (RSP). The PVA template is dissolved in water releasing the RSP-μPL in solution.
The two μPL configurations were characterized via scanning electron microscopy (SEM), Multisizer Coulter counter, and profilometric analyses for their morphological properties. The SEM images of Fig. 1b, c show well-defined short and tall μPL with a common edge size length of 20 μm and a height of 10 and 20 μm, respectively. The different μPL heights can be readily appreciated in the lateral magnified pics of the microparticles laying on their sides. No porous structure was observed on the particle surface at this magnification level. Moreover, as expected, no significant difference in morphology was documented when comparing empty μPL and RSP-μPL (Fig. S1a, b). The Multisizer analysis showed single peaks around 20 μm and 30 μm for the short and tall μPL, respectively, with again no appreciable difference between empty and RSP-μPL (Fig. 1d, e). Additional topographical analyses carried out with an optical profilometer confirmed the μPL geometrical features as documented by the cross-section profiles of Fig. 1f, g and in the false-coloring 3D reconstruction in Fig. S1c − f, for both the short and tall μPL. Indeed, one major advantage of the template-based fabrication strategy is the accurate control in particle morphology, which cannot be generally achieved with bottom-up fabrication strategies.
Degradation of the µPL
To investigate the biodegradation of the PLGA polymer matrix, both short and tall μPL were incubated in PBS at 37 °C, and their morphology was assessed over time to document any possible alterations. Figure 2a, b show SEM images of the μPL acquired at different time points, from 0 up to 42 days, post-incubation.
On day 0, both short and tall μPL appeared as well-defined, squared particles as from Fig. 1b − g. On day 7, however, both short and tall μPL started to present some preliminary sign of biodegradation as manifested by the rounding of the originally sharp corners and the progressive transition from a square base to an overall rounded shape. At longer time points, the process started to affect also the core of the short μPL with a degeneration of their structure into a round microparticle already on day 42. The tall μPL followed overall the same behavior but at lower degradation rates, likely due to the larger amount of PLGA used to realize the tall versus short μPL. Nonetheless, even for the tall μPL, the microparticles tended to progressively lose their original squared shape to become more globular towards the end of the observation period (Fig. S2). This behavior was quantified via a Multisizer Coulter counter analysis returning the overall size spectrum distribution for the short and tall μPL over time (Fig. 2c, d). This shows a progressive shift of the characteristic peak towards a larger average size and a reduction of the peak value and overall area under the curve. The shift of the peak has to be associated with the change in microparticle structure and possible coalescence with other particles or debris. The reduction in peak value is directly correlated with the number of particles. Multisizer data are referred to the degradation of particles from day 0 to day 21. Note that longer time point measurements were not possible due to the change in μPL morphology that made the sample unsuitable for the analysis.
Biopharmaceutical characterization of the μPL
Short and tall μPL loaded with RSP were characterized in terms of RSP loading (LE%), defined as the ratio between the loaded mass of RSP and the total particle mass; encapsulation efficiency (EE%), defined as the ratio between the loaded mass of RSP and the initial RSP input; as well as for their ability to control the release of RSP over time. A direct comparison with PLGA microspheres was also performed. Specifically, ~ 10 μm PLGA spherical particles (μS) were realized using a standard homogenization protocol [34, 35]. Figure S3 shows electron microscopy images, DLS, and Multisizer analysis for the ~ 10 µm microspheres.
No statistically significant differences were observed in terms of loading among the three different microparticles, with 1.52 ± 0.20% for the short RSP-μPL; 1.73 ± 0.59% for the tall RSP-μPL; and 1.84 ± 0.18% for the μS, respectively (Fig. 3a – one-way ANOVA: p = 0.685 short µPL vs tall µPL, p = 0.837 short µPL vs µS, p = 0.799 tall µPL vs µS). On the other hand, significant differences were documented for the encapsulation efficiency. The drug encapsulated within the RSP-μS (27.57 ± 0.20%) was significantly higher than for the tall RSP-μPL (6.55 ± 0.13%) and the short RSP-μPL (2.21 ± 0.43%), respectively (Fig. 3b – one-way ANOVA, ***p < 0.001). Moreover, the encapsulation efficiency of the tall µPL was significantly higher than the encapsulation efficiency of the small µPL (Fig. 3b – one-way ANOVA: **p < 0.01).
The RSP release kinetics was determined under physiological conditions (PBS, pH 7.4, 37 °C) in a 500 μl volume to better mimic the confined space for intratissue deposition. The release profiles for the two μPL configurations and the μS are shown in Fig. 3c in terms of percentage of drug released over the first 40 days of observation. Tall RSP-μPL showed the slowest release profile with less than 30% (26.85 ± 1.50%) of the loaded drug being released within the first 5 weeks of observation. In the same time period, short RSP-μPL and RSP-μS released over 50% (57.81 ± 0.85% and 55.69 ± 2.28%, respectively) of the loaded drug (one-way ANOVA: #p < 0.00001 short µPL vs tall µPL, §p < 0.0001 µS vs tall µPL). Given the similarity between the release profiles for the short RSP-μPL and RSP-μS (p = 0.21 – no significant differences), more details are acquired for the short and tall RSP-μPL in terms of cumulative and instantaneous drug released, as shown in Fig. 3d, e, respectively. Within the first 24 h of incubation, about 12% of RSP was released from the short μPL as opposed to 4% for the tall μPL (one-way ANOVA: ***p < 0.001), while at 50 days, about 60% and 29% of RSP was released from the short and tall μPL, respectively (one-way ANOVA: #p < 0.00001). For the tall μPL, the drug molecules were continuously delivered over time to reach just about 33% of release at 100 days (Fig. 3d). The daily release of RSP from short and tall μPL is shown in Fig. 3e. For the short RSP-μPL, the drug is rapidly released within the first week reaching a peak of 4000 ng/ml at day 1 that drops rapidly to 1000 ng/ml at day 5 and continuously decreases down to about 50 ng/ml around day 60. A similar trend is documented for the tall RSP-μPL but with a more moderate variation in daily release rate. Specifically, a peak of only 2000 ng/ml is detected at day 1 followed by a drop to 1000 ng/ml at day 7 that stays quasi-constant till day 25. The daily release rate continuously decreases till 50 ng/ml at day 100.
The release kinetics data were interpolated using the Weibull equation to extrapolate the RSP release mechanisms for both short and tall μPL. The Weibull model parameters (a, b) and the corresponding fitting accuracy R2 for the in vitro release profiles are reported in Table 1. In this work, the fitting was successful for the entire set of data, with a R2 of 0.9960 and 0.9979 for short and tall µPL, respectively. The values of b obtained for the best fit were 0.6557and 0.6766 for short and tall µPL, respectively. These values suggest that µPL provide a sustained release of RSP based on a diffusion-controlled mechanism through the PLGA matrix.
This implies that drug release from the μPL into the surrounding aqueous environment would depend on the molecular weight of the drug as well as on the size of the pores in the PLGA matrix rather than its biodegradation. These results are also in line with the release mechanism of other small molecules from similar microparticles . In comparing the short and tall μPL, one can observe a doubling in volume (μPL height increasing from 10 to 20 μm) and a three-time increase in PLGA mass (from 20 to 60 mg) used during the fabrication process. Therefore, it can be argued that the slower release kinetics associated with the tall μPL should be associated with their more compact structure.
Temporal order object recognition (TOR) test
Single injection of short RSP-μPL
In order to investigate if a single injection of RSP-μPL could ameliorate dysbindin-induced deficit, Dys ± mice received either free RSP (or vehicle only) or RSP-μPL (or empty μPL). The free drug or vehicle were administered intraperitoneally (0.1 mg/kg) on a daily basis for 2 weeks, whereas the RSP-μPL or empty μPL were deposited i.p. once at the beginning of the experiment for a total equivalent RSP dose of 1.4 mg/kg, to match the total amount of free drug received in 2 weeks. TOR tests are conducted at 2- and 4-week post-treatment initiation, as described in Fig. 4a.
At all the time points, Dys ± mice treated with short empty μPL were not able to discriminate between the two objects presented at different time, confirming previously found recency memory cognitive impairment (Fig. 4b, c). This suggests that the short μPL per se had no effect. At 2 weeks, the treatment with both free RSP and RSP-μPL did not affect the total time (Ttot) mice spent to explore the two objects (F(3,22) = 1.276, p = 0.3070) as shown in Fig. S4a, but it is effective in selectively improving TOR cognitive performances (F(3,22) = 7.943, p = 0.0009). Specifically, at 2 weeks, Dys ± mice treated with daily injections of free RSP showed a significantly higher DI when compared to the vehicle-treated Dys ± mice (**p < 0.01) (light vs dark gray bars – Fig. 4b). Notably, also Dys ± mice treated with a single injection of short RSP-μPL improved their TOR performances, showing an increased DI that was significantly higher (*p < 0.05) than that of the Dys ± mice receiving a single injection of empty μPL (black vs white bars – Fig. 4b). Importantly, there were no significant differences between the TOR performance of Dys ± mice treated with daily injections of free RSP and Dys ± mice treated with a single injection of short RSP-μPL (black vs light gray bars – Fig. 4b). This result suggests that the μPL-based system helped ameliorate the dysbindin-induced cognitive deficits similarly to the free drug, with one single injection of particles releasing a lower amount of drug than the conventional dose. TOR was also performed at 4-week post-treatment initiation. Similar to the 2-week post-treatment, there was no significant difference in the time spent by the mice to explore the two objects (p = 0.5358) (Fig. S4b). However, even if Dys ± mice treated with short RSP-μPL showed improvement in the TOR performances over Dys ± mice treated with short empty μPL (Fig. 4c), the difference between the two groups was not significant (p = 0.6099).
Single injection of tall RSP-μPL
In these studies, Dys ± mice and their wild-type (Dys + / +) littermates are injected intraperitoneally on day 0 with tall empty μPL or tall RSP-μPL (1.4 mg/kg drug equivalent dose), according to the timeline shown in Fig. 5a.
The mice were subjected to the TOR tests at 2-, 4-, 8-, and 12-week post-treatment initiation. At all the time points, treatment with tall RSP-μPL did not lead to any difference in the total time spent by the mice to explore the two objects during the 5-min test trial (Fig. S5a − d), but it was effective in rescuing the cognitive impairment. Specifically, Dys ± mice treated with the RSP-μPL showed an increased DI when compared to Dys ± mice treated with the empty μPL (Fig. 5b − e). However, the difference between the two was statistically significant at 2 (Newman-Keuls post-hoc: **p < 0.001 vs empty μPL), 4 (Newman-Keuls post-hoc: ***p < 0.0001 vs empty μPL), and 8 weeks (Newman-Keuls post-hoc: *p < 0.05 vs empty μPL) only, and not significant at 12 weeks. Second, the therapeutic effect of the tall RSP-μPL on the Dys ± mice was compared to the effect of both the tall empty μPL and the tall RSP-μPL on the Dys + / + mice. No significant difference was observed at all four times points (Fig. 5b − e), thus implying that the RSP-μPL alone can rescue the animal behavior and that one single administration of RSP-μPL at day 0 returns Dys ± mice behaving just like the Dys + / + mice up to 12 weeks.
To assess if the tall RSP-μPL would progressively lose their efficacy over time or if there was any kind of habituation of the animals to the TOR tasks, Dys ± mice and Dys + / + mice were injected with tall RSP-μPL or empty μPL on day 0. Then, their behavioral performances are tested only at 8- and 12-week post-injection, according to the schematic of Fig. 6a.
As previously shown, treatment with tall RSP-μPL did not lead to any difference in the total time spent by the mice to explore the two objects during the 5-min test trial (Fig. S6a, b). Importantly, a single injection of tall RSP-μPL was effective in restoring the recency memory impairment in Dys ± mice, with a DI that was significantly higher than that of Dys ± receiving tall empty μPL, at both 8 (Bonferroni post-hoc: ***p < 0.0001 vs empty μPL) and 12 weeks (Bonferroni post-hoc: ***p < 0.0001 vs empty μPL) (Fig. 6b, c). Notably, at both time points, the cognitive performance was impacted by the treatment (8 weeks – treatment effect: F(1,28) = 17.76, p = 0.0002; 12 weeks – treatment effect: F(1,28) = 16.66, p = 0.0003) and by the interaction between treatment and genotype (8 weeks – treatment × genotype effect: F(1,28) = 11.29, p = 0.0023; 12 weeks – treatment × genotype effect: F(1,28) = 12.90, p = 0.0009). These results suggest that RSP-μPL can have a prolonged effect up to 3 months upon single administration, leading to an overall improvement of the cognitive performances of mice carrying dysbindin-induced deficit.
It should be here highlighted that for all the TOR tests (Figs. 5 and 6), Dys ± mice receiving a single injection of tall empty μPL showed no discrimination, indicating the presence of a cognitive impairment and confirming that the tall μPL too per se had no effect. Notably, for all the TOR tests, the administration of tall empty µPL had no negative outcome on the cognitive performances of Dys + / + mice, reinforcing the fact that the µPL per se did not lead to any side effect.
Finally, the RSP concentration was assessed through HPLC MS/MS quantitative determination 12 weeks after a single injection of tall RSP-μPL. The analysis revealed a serum concentration of 6.40 ± 1.26 ng/ml and 4.90 ± 0.77 ng/mL in RSP-μPL treated Dys + / + and Dys ± mice, respectively (Fig. S7), which appears to be comparable to the daily RSP release measured in vitro at 100 days (Fig. 3e). Non-detectable RSP concentration was confirmed for the empty μPL treated mice.