Preparation and characterization of liposomal bupivacaine

The overall study processes are presented in Fig. 3.

Schematic representation of osmotically balanced, large unilamellar liposomes (~ 6 μm in diameter) in which highly concentrated bupivacaine (up to 30 mg/mL) was encapsulated for sustained bupivacaine release and subsequent prolonged pain relief. Bupivacaine binds to the voltage-gated sodium channels and blocks sodium influx, preventing the conduction of pain signals. To evaluate the analgesic duration of liposomal bupivacaine in vivo, male Sprague–Dawley rats were used to measure the mechanical withdrawal threshold (MWT) with von Frey filaments.

1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC), cholesterol, and bupivacaine hydrochloride were purchased from Sigma (St Louis, MO, USA), and 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG) was purchased from Cayman (Ann Arbor, MI, USA).

The bupivacaine liposomes were prepared based on the reverse-phase evaporation methods developed by Szoska et al. 100 mg of DOPC, 100 mg of cholesterol, and 20 mg of DPPG were dissolved in 20 mL of chloroform/ethanol (1:1, v/v) in a 100 mL round-bottom flask. 5 mL of bupivacaine-HCl solution (0.1X PBS, 30 mg/mL bupivacaine-HCl) was added to this solution. The resulting two-phase system was sonicated briefly for 5 min in a bath-type sonicator. The ethanol and chloroform were removed at 40 °C via rotary evaporation under reduced pressure to form the final liposome aqueous dispersion. The liposomes were washed with 0.94 × phosphate-buffered saline (PBS) and harvested via centrifugation at 2000 g to separate the free drug from the vesicles. After washing, the liposomes were resuspended in 4 mL of 0.94 × PBS to yield a liposome suspension with an approximate bupivacaine concentration of 5 mg/mL.

The morphology of the bupivacaine liposomes was estimated using an optical microscope (Leica DM 4000 M). The particle size was measured using a particle size analyzer (Malvern Zetasizer).

The drug encapsulation efficiency was determined by comparing the amount of the encapsulated bupivacaine (D_en) with the total amount of bupivacaine in the preparations (D_tot). A total of 0.1 mL of the liposome suspension was dissolved in 10 mL of the extraction solution (0.2% Triton X-100, 28% ethanol, 71.8% water, v/v) to extract the bupivacaine from the liposome. The total drug content (D_tot) in the suspension was quantified using HPLC. The free drug (D_free) was separated from the pellet via centrifugation (2000 × g, 10 min) and quantified using HPLC. The encapsulation efficiency was estimated using the following equation:

The in vitro drug release of the bupivacaine liposome was determined using dialysis bags (MW cutoff 10 K). Two milliliters of the liposome suspension was transferred to the dialysis bag placed in a flask containing 200 mL of 0.94 × PBS. The flasks were incubated at 37 °C under constant stirring. Samples were collected at time points of 3, 8, 24, 72, 120, and 144 h and analyzed using HPLC.