Sustained Release of Levobupivacaine, Lidocaine, and Acemetacin from Electrosprayed Microparticles: In Vitro and In Vivo Studies

Abstract

In this study, we explored the release characteristics of analgesics, namely levobupivacaine, lidocaine, and acemetacin, from electrosprayed poly(D,L-lactide-co-glycolide) (PLGA) microparticles. The drug-loaded particles were prepared using electrospraying techniques and evaluated for their morphology, drug release kinetics, and pain relief activity. The morphology of the produced microparticles elucidated by scanning electron microscopy revealed that the optimal parameters for electrospraying were 9 kV, 1 mL/h, and 10 cm for voltage, flow rate, and travel distance, respectively. Fourier-transform infrared spectrometry indicated that the analgesics had been successfully incorporated into the PLGA microparticles. The analgesic-loaded microparticles possessed low toxicity against human fibroblasts and were able to sustainably elute levobupivacaine, lidocaine, and acemetacin in vitro. Furthermore, electrosprayed microparticles were found to release high levels of lidocaine and acemetacin (well over the minimum therapeutic concentrations) and levobupivacaine at the fracture site of rats for more than 28 days and 12 days, respectively. Analgesic-loaded microparticles demonstrated their effectiveness and sustained performance for pain relief in fracture injuries.

Keywords: acemetacin; electrospraying; levobupivacaine; lidocaine; poly(D,L-lactide-co-glycolide); sustained drug release.

Figure 1

Chemical structures of (A) lidocaine, (B) levobupivacaine, and (C) acemetacin.

Figure 2

Field emission scanning electron microscopy images of analgesics-loaded microparticles subjected to various processing conditions after being gold-sputtered. Conditions were: (A) 9 kV, 1 mL/h, and 5 cm; (B) 9 kV, 1 mL/h, and 10 cm; (C) 9 kV, 1 mL/h, and 20 cm; (D) 6 kV, 1 mL/h, and 10 cm; (E) 15 kV, 1 mL/h, and 10 cm; and (F) 9 kV, 2 mL/h, and 10 cm for voltage, flow rate, and travel distance, respectively. Scale bar = 10 µm.

Figure 3

Fourier-transform infrared spectra of empty (black line) and analgesics-loaded (red line) electrosprayed microparticles.

Figure 4

Cell viability of human fibroblasts treated with the eluents from analgesics-loaded microparticles incubated in phosphate-buffered saline for different periods. The asterisks denote significant differences from the control group. (N = 3, * p < 0.05).

Figure 5

Release curves of analgesics from the electrosprayed microparticles during incubation in vitro. (A) Daily releases of levobupivacaine (black squares), lidocaine (red circles), and acemetacin (blue triangles). The dashed lines indicate the minimum therapeutic concentration (MTC) for each analgesic. (B) Cumulative values over the 30-day period.

Figure 6

In vivo release curves of analgesics to the area surrounding the bone fracture site treated with analgesics-loaded microparticles. The levels of levobupivacaine (black squares), lidocaine (red circles), and acemetacin (blue triangles) were measured in the tissues 1, 3, 7, 14, 21, and 28 days after the treatment. The dashed lines indicate the minimum therapeutic concentrations (MTC) for each analgesic.

Figure 7

Rat locomotor activity recorded in the activity cages. Group A (red columns) was the control untreated group. Group B (green columns) was subjected to the experimental bone fracture procedure without any other treatment. Group C (blue columns) was subjected to the experimental bone fracture procedure followed by the administration of analgesics-loaded microparticles to the bone fracture site. The asterisks denote a significant difference (p < 0.01) between groups B and C.

Figure 8

Femur shaft fracture surgical procedure. The lateral part of the middle-thigh region was disinfected, and a 2 cm skin incision was made (left panel). The femur shaft was exposed by blunt dissection of the surrounding musculature. The osteotomy was performed in a short oblique direction with a bone cut (middle panel). The analgesics-incorporated microparticles were directly sprayed on the fracture site (right panel).

Figure 9

Schematic representation of the animal activity cage.