The surface and cross-section morphologies of Formulations B50-L, B75-L, C50-L, and B50-S (Table I) are shown in Fig. 1. For all formulations, the surface of microparticles had a combination of wrinkled and smooth surfaces. This is likely due to the high MW (120 kDa and 180 kDa) at a low concentration (3%) of PLGAs used. Some microparticles may have relatively lower PLGA concentration to produce a highly wrinkled surface, while others may have relatively higher polymer density (and thus, viscosity), resulting in a smoother surface (26). When the PLGA L:G ratio increased from 50:50 to 75:25 (Fig. 1-a and b), the crater-like porous structures formed (red arrow). Solvent molecules might have been assembled in local regions to form oil droplets which were eventually removed from the surface. When the theoretical drug loading was increased from 40% to 50%, no significant difference was observed in the morphology of the microparticles (Fig. 1-a and c), as the PLGA concentration remained the same. Additionally, the change of the stator diameter-speed of the homogenizer-sieve size from 10 mm-5000 RPM-25 μm to 25 mm-3000 RPM-10 μm resulted in smaller microparticles by increasing the shear force at the interface and breaking the oil phase into smaller droplets (Fig. 1-a and d). The cross-sections of the naloxone-PLGA microparticles made with the different formulation- and processing-parameters were similar and solid with minimum observable porosity, probably due to the low concentration (3%) of PLGA and naloxone (below 5%), which allowed continuous dispersion of polymers in the core region (a2-d2 in Fig. 1) (18).
Figure 2 shows the size distributions of the naloxone-PLGA microparticles with different theoretical drug loadings (30%–60%), L:G ratios (50:50 and 75:25), and stator diameter-speed-sieve size of 10 mm-5000 RPM-25 μm and 25 mm-3000 RPM-10 μm. D-Values (D10, D50, D90) and span are provided in Table II, which provides a better understanding of the importance of different parameters on the size distribution of the resulting particles. Figure 2-a and Table II show that the size of the microparticles was decreased when the naloxone drug loading was lower. The D90 of the microparticles with the T-DL of 30% reduced by 15.3% compared to the microparticles with the T-DL of 60%. This is understandable, as the total weight was reduced when the naloxone concentration was lower. The microscopic images of the microparticles for Formulations A50-L (T-DL = 30%) and D50-L (T-DL = 60%) are shown in Fig. 2 (d) and (e), respectively. However, because most of the particles have the size of 50–60 μm (Fig. 2 (a)), the difference between the size of the microparticles is not significant. The L:G ratio of the PLGA had no significant influence on the size distribution of the microparticles (Fig. 2-b and Table II). Figure 2-c demonstrates the strong effect of the homogenization-sieving conditions, particularly stator diameter, on the size distribution of the particles, even for the same formulation. For example, the D10, D50, and D90 of Formulation B50 were decreased by 61.7%, 54.7%, and 49.5%, respectively, by changing the stator diameter-speed-sieving conditions of 10 mm-5000 RPM-25 μm to 25 mm-3000 RPM-10 μm.
The encapsulation efficiency (EE) of naloxone and the actual drug loading (A-DL) in the microparticles made with PLGA 50:50 and 75:25 are shown in Fig. 3 as a function of theoretical drug loading (T-DL%). The particles characterized for Fig. 3 were made with the homogenization condition of 10 mm-5000 RPM. Table III provides a complete list of A-DL and EE values for all of the microparticles made in this study with both of the stator diameter-speed-sieve size values of 10 mm-5000 RPM-25 μm and 25 mm-3000 RPM-10 μm. As shown in Fig. 3, the EE was improved significantly by increasing the T-DL for both PLGAs. For instance, the increase of the T-DL from 30% to 60% enhanced the EE by 11%. A linear relationship between the T-DL and A-DL was also observed in Fig. 3. Table III shows that the EE and A-DL improved slightly by about 3% when the homogenizer stator diameter-speed-sieve size was 25 mm-3000 RPM-10 μm compared with 10 mm-5000 RPM-25 μm.
Figure 4 shows the in vitro cumulative release profiles in percentage (%) (Fig. 4-a1, b1, and c1) and the actual amount (mg) of released naloxone (Fig. 4-a2, b2, and c2) per mg of naloxone-PLGA microparticles. The parameters varied were the naloxone loading ranging from 21.08% to 48.54% (a1 and a2), the L:G ratio of 50:50 and 75:25 (b1 and b2), and homogenizer stator diameter-speed-sieve size of 10 mm-5000 RPM-25 μm and 25 mm-3000 RPM-10 μm (c1 and c2). Table IV lists the parameters of the in vitro drug release profiles from the microparticles: the initial drug release rate (I-DRR) and steady-state drug release rate (SS-DRR), their ratios (I-DRR/SS-DRR), drug release duration (in days), and the zero-order kinetics models.
When the A-DL increased from 21.08% to 39.44%, the drug release duration was the same for 28 days, and the steady-state naloxone release rate slightly increased from 0.009 mg naloxone/day/mg particles to 0.013 mg naloxone/day-mg particles (Fig. 4, a1 and a2). While the drug release kinetics of the microparticles with the A-DL in the 21.08–39.44% range were similar, the 48.54% A-DL accelerated the drug release rate and reduced the release duration to 10 days with the 220% increased steady-state release rate of 0.032 mg naloxone/day/mg particles. Fig. 4 (b1 and b2) show that the increase of the lactide content (i.e., change of the L:G ratio from 50:50 to 75:25) resulted in a longer drug release duration. When the T-DL was 60%, the increase of the L:G ratio from 50:50 (Formulation D50-L) to 75:25 (Formulation D75-L) increased the A-DL by only 2.56% (from 48.54% to 51.10%) but reduced the I-DRR and SS-DRR by 37% and 69%, respectively, resulting in longer duration from 10 days to 35 days. For the T-DL of 30%, however, the I-DRR and SS-DRR did not change significantly when the L:G ratio was changed (Table IV-Formulations A50 and A75). In this case, the extension of the drug release was possibly due to the 10-day lag phase for Formulation A75 between days 4–14 (Fig. 4 (b1 and b2)). When the homogenizer stator diameter-speed-sieve size was changed from 10 mm-5000 RPM-25 μm to 25 mm-3000 RPM-10 μm, the naloxone release rate became faster. For example, in Formulation C50, the drug release duration was reduced from 28 days to 4 days, and the SS-DRR was increased by 8.2 fold from 0.013 mg naloxone/day-mg particles to 0.107 mg naloxone/day-mg particles. This is most likely due to the smaller microparticle sizes.
Figure 5 shows the SEM images of the microparticles of Formulations B50-L, B75-L, D50-L, and B50-S, at Days 0 (before the in vitro test), 2, 4, 10, 14, 21, and 28. In Formulation B50-L, the wrinkles on the surface of the microparticles increased until Day 10, indicating microparticle swelling and the drug release through the swelling skin layer, like a reservoir system. As PLGA degrades, the formed acidic oligomers lowered the pH and increased the osmotic pressure, causing further swelling on Day 14. The continued PLGA degradation and increased osmotic pressure eventually ruptured the membrane (27,28,29), as observed around Day 21. Similar behavior was observed for Formulation B50-S, but the morphology change occurred faster than Formulation B50-L due to their smaller sizes. On Day 2, the microparticles exhibited irregular shapes with crater-like pores, and many of them appeared as deflated balls (30). The microparticles swelled to large sizes at Day 4 and degraded and disintegrated at Day 10. Formulation B75-L had a smooth surface at Day 2, and the continued swelling resulted in the shallow wrinkled surface at Day 4 and large crater-like holes sine Days 10. The microparticles started to collapse on Day 21 due to swelling-shrinking, thermal expansion, and mechanical stress (26, 31). Formulation D50-L has about 50% (48.5% w/w) naloxone loading, and the drug release was faster than other formulations with lower drug loading. The swelling was not as pronounced as Formulation B50-L, probably due to the faster release of naloxone and other components from the microparticles. No significant changes in the surface morphology were observed during Days 2–14. The microparticles started to disintegrate on Day 21.
DAMGO is a μ-opioid receptor-selective agonist which efficaciously recruits β-arrestin 2 when activating human μ-opioid receptors (pEC50 = 6.9 ± 0.1, n = 6, Fig. 6-a). β-arrestin 2 is critical for developing tolerance to opioids (25). Naloxone by itself does not recruit β-arrestin 2, but inhibits 1 μM (EC80) DAMGO-induced β-arrestin 2 recruitment (pIC50 = 8.8 ± 0.3, n = 7, Fig. 6-a) (Control group 1). Additionally, the samples from bare PLGA microparticles without naloxone (Control group 2) did not induce β-arrestin 2 recruitment (data not shown). Naloxone release from Formulations B50-L, D50-L, and B50-S were monitored over time. Except for the samples collected on Day 28 (Formulations B50-L) and Day 6 (Formulations D50-L and B50-S), Panels B-D of Fig. 6 show that the average pIC50 for the samples was 8.2 ± 0.1 (n = 11) (Table V). The pIC50 for Formulations B50-L at Day 28, Formulations D50-L at Day 6 and Formulations B50-S at Day 6 were 9.6 ± 0.4 (n = 3) (Fig. 6 (b)), 9.2 ± 0.5 (n = 4) (Fig. 6-c), and 8.9 ± 0.3 (n = 4) (Fig. 6-d), respectively.
Naloxone released from all three formulations was biologically active as it potently blocked β-arrestin 2 recruitment by the μ-opioid receptor agonist DAMGO. The potency for naloxone in these samples was slightly lower than that of the control, naloxone hydrochloride (pIC50 8.8 vs. 8.2). There could be two explanations for this difference: either the buffer used for the formulations had a negative quenching impact on the readout, or the naloxone being released lacked 100% integrity, leading to an overestimation of the concentration. Less naloxone is released from the formulations as time passes, making it harder to measure the naloxone being released accurately. The higher pIC50 determined for Formulations B50-L at Day 28 and Formulations D50-L and Formulations B50-S at Day 6 may be explained by the increased error in calculating the naloxone concentration for those days that increases the variability when creating the dilution series for these samples. This is supported by the observation of large standard errors of the mean for these sample pIC50 values.