Preparation and characterization of liposomal bupivacaine
The overall study processes are presented in Figure 3.
Materials
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).
Preparation of bupivacaine liposomes
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.
Characterization of bupivacaine liposomes
The morphology of the bupivacaine liposomes was estimated using an optical microscope (Leica DM 4000M). The particle size was measured using a particle size analyzer (Malvern Zetasizer).
Determination of encapsulation efficiency
The drug encapsulation efficiency was determined by comparing the amount of the encapsulated bupivacaine (Den) with the total amount of bupivacaine in the preparations (Dtot). 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 (Dtot) in the suspension was quantified using HPLC. The free drug (Dfree) was separated from the pellet via centrifugation (2000 × g, 10 min) and quantified using HPLC. The encapsulation efficiency was estimated using the following equation:
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In vitro release
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.
In vivo application of liposomal bupivacaine
The experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee at Chung-Ang University (2020–00131). All the experiments were conducted following the guidelines established by the National Institutes of Health, the policies of the International Association for the Study of Pain for the Use of Laboratory Animals, and the guidelines recommended in the Animal Research Reporting In Vivo Experiments (ARRIVE) statement 27.
Animal preparation
Adult male Sprague-Dawley rats weighing 250–300 g (Coretec Laboratories, Seoul, Korea) were used in this study. The rats were habituated in a colony room for a week before the experimental study. Two rats were housed in each cage at 22 ± 0.5 °C with a 12:12 h light-dark cycle. Food and water were available ad libitum. Female rats were not included in this study to avoid the impact of hormonal fluctuations on the pain threshold 28. Rats with any abnormalities were excluded.
Group allocation and blindness
The rats were randomly divided into groups to assess the analgesic effects of liposomal bupivacaine and bupivacaine. Random assignment was based on a table generated by a computer-applied Wei’s Urn model using PASSTM 11 software (NCSS, Kaysville, UT, USA). The randomization code was generated by a statistician, not otherwise involved in the study.
For allocation concealment, another investigator, not involved in this study, prepared the syringes containing the study drugs for the experiments. For intra-plantar application, 1 mL syringes containing 0.2 mL of normal saline or study drugs were prepared. The syringes were covered with opaque tape and numbered sequentially according to a randomized list of the respective experiments. The prepared syringes were delivered to the researcher in charge of the surgery, who participated only in the surgery and was blind to the group assignment.
Surgical procedure
All surgical procedures were performed under sterile conditions. The rats received general anesthesia as an anesthetic, induced with 6% isoflurane in 100% oxygen inside a sealed clear plastic chamber until the rats became immobile. It was then maintained on a non-rebreathing anesthetic circuit mask using 1% to 3% isoflurane in 100% oxygen until the end of the surgery to prevent the rats from suffering during the surgical procedure. Before incision, cefazolin (20 mg/kg; Chong Kun Dang Pharmaceutical Co., Korea) was administered subcutaneously. The plantar surface of the left hind paw of each rat was prepared aseptically for surgery. The incisional pain model was created, as previously described,13 with minor modifications in the reported technique. At a point, approximately 0.5 cm distal to the tibiotarsal joint on the plantar surface of the left hind paw, a 1 cm longitudinal skin incision, extending towards the digits, was made with a blade (Figure 4A). The plantaris muscle was isolated, elevated slightly, and incised longitudinally (Figure 4B and 4C). Study drugs in the prepared syringes were administered to the incision site over the desiccated area. The incision was closed with two interrupted horizontal mattress sutures of 5–0 nylon (Figure 4D) All the rats were allowed to recover, and their sutures were removed on the third postoperative day.
Sample size calculation
The primary outcome measured was the threshold of withdrawal by mechanical stimuli using von Frey filaments. A pilot study was conducted to measure the MWT in six incisional pain model rats (group C) to estimate the group size for the study assessing the antinociceptive activity of bupivacaine and liposomal bupivacaine. The averages of the natural log-transformed MWT at the base, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 24 h, 48 h, and one week after surgery were 4.68, 1.49, 2.05, 2.05, 2.04, 2.52, 2.49, 2.41 3.04, and 3.70 ln(mN), respectively. The standard deviations of the natural log-transformed MWT ranged from 0.06 to 0.89, and the autocorrelation between adjacent measurements on the same individual was 0.7. For the power calculations, we assumed that a first-order autocorrelation adequately represented the autocorrelation pattern. The Geisser-Greenhouse Corrected F test for the repeated measures of ANOVA was used to analyze the differences between groups. We aimed to detect increments of 50% and 60% in MWT in groups B and LB over group C. It was determined that, with α=0.05 and power=80%, ten rats were required per group. Considering a 20% follow-up loss, we utilized a total of 36 rats in the study.
Statistical analysis
The Shapiro-Wilk test was used to test the normality of variables. As MWT did not pass the Shapiro-Wilk test, natural log-transformation was tested. The natural log-transformed variables passed the Shapiro-Wilk test. Therefore, we assumed that the normal distribution assumption for the parametric test was not violated and decided to apply repeated measures ANOVA: the within-subjects factors of times (at the base, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 24 h, 48 h, and one week after surgery) and between-subjects factor of groups (group C, group B and group LB). As Mauchly’s sphericity test indicated that the assumption of sphericity was violated in the MWT test (χ2 (44) = 179.32, P < 0.001, Mauchly’s W = 0.002), we used one-way Wilk’s lambda multivariate analysis of variance (MANOVA): each group (group C, group B and group LB) and each time point (at the base, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 24 h, 48 h, and one week after surgery) were independent factors, and the MWT at each time point (at the base, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 24 h, 48 h, and one week after surgery) was the dependent variable. Univariate analysis of variance (ANOVA) using Bonferroni correction (α = 0.05/10 = 0.005) was used to compare the MWT at each time point.
The between-group differences for Ln(MWT) were analyzed using an LMEM, with time (at the base, 30 min, 60 min, 90 min, 120 min, 180 min, 240 min, 24 h, 48 h, and one week after surgery) and group as independent fixed factors and individual patients as random effects.
Individual measurements were expressed as the mean ± standard error and analyzed with SPSS 23.0 (IBM Corp., Armonk, NY, USA). A P value of 0.05 was considered statistically significant.