Enantiomeric Excess Bupivacaine in a Lavender Oil NLC Tested in a Melanoma Model: Prolonged Release and Anticancer Effect

Abstract

Recent studies have highlighted the potential of local anesthetics (LA) as adjuvants in cancer treatment, specifically by increasing survival rates when used in surgical excisions. However, the clinical use of LA is restricted due to their systemic toxicity. The development of drug delivery systems could address this issue and advance the utilization of these molecules. In this research, we explored the pharmacokinetics (using microdialysis probes) and antitumor properties of a nanostructured lipid carrier (NLC) formulation containing the commercially available enantiomeric excess form of bupivacaine (BVC_S75). This NLC was prepared with lavender oil (NLC-L-BVC), an excipient with inherent antitumor properties. We compared this formulation to a control (NLC-BVC) using synthetic lipids. Pharmacokinetic assessments of the NLCs confirmed the sustained release of BVC_S75 within the tumor, characterized by a reduced elimination rate constant and longer half-life (∼6×). The encapsulation of BVC_S75 within nanoparticles (whether natural or synthetic) enhanced its effectiveness in treating the primary tumor, resulting in the inhibition of tumor growth (70% with NLC-L-BVC and 72% with NLC-BVC), outperforming free BVC (17% inhibition). However, the association of lavender oil with BVC_S75 in an NLC did not yield synergistic properties. Furthermore, all BVC_S75 treatments (whether free or encapsulated) improved animal survival rates. These findings confirm that encapsulation of bupivacaine in NLC can prolong drug action at the local site, contributing to improved local antitumor therapy while mitigating systemic effects.

Keywords: bupivacaine; drug delivery; lavender oil; melanoma; nanostructured lipid carriers.

1

Local unbound concentration inside the tumor as a function of time. Data represent mean ± SD (n = 5). Free BVC = unbound solution of 0.5% bupivacaine S75:R25; NLC-L-BVC = nanostructured lipid carrier with lavender oil and 0.5% of BVC_S75; NLC-BVC nanostructured lipid carrier with 0.5% of BVC_S75; and no lavender oil.

2

(A) Residual posteuthanasia intratumoral BVC_S75 concentration after 8 h. Fluorescence microscopy images of tumors treated with free BVC (B), NLC-L-BVC (C), or NLC-BVC (D). The NLC formulations were labeled with red fluorescent rhodamine-PE. Statistical analysis carried out by unpaired Student’s t-test; * p < 0.05.

3

(A) Timeline of the experimental design: T1, T2, T3, and T4 = treatments. (B) Evolution of the primary tumor during and after treatment of animals with negative control = 0.9% NaCl, positive control = dacarbazine, free BVC (0.5%), lavender oil formulation (NLC-L), or the formulations containing 0.5% BVCS75 (NLC-L-BVC and NLC-BVC).

4

Analysis of post-treatment tumor growth. (A) Comparison of tumor volume at day 16 (last day of treatment) and 1 week after (day 22) the end of treatment with negative control (0.9% NaCl), positive control (dacarbazine), free BVC (0.5%), lavender oil formulation (NLC-L), or formulations containing 0.5% BVC_S75: NLC-L-BVC and NLC-BVC. (B) Representative examples of tumors from each treatment, excised at day 22 (same scale). Statistical analysis: one-way ANOVA plus posthoc Tukey. * p < 0.05; *** p < 0.001; n.s. = nonsignificant. No statistical analysis was applied to the negative control group because only one animal in this group survived until day 22.

5

Histopathological sections of excised tumors (H&E staining): naive (A), negative control (B), dacarbazine (C), free BVC (D), NLC-L-BVC (E), NLC-BVC (F), and NLC-L (G). Scale bar = 100 μm, magnification: 5× and 10×. The black arrows point to areas of necrosis, asterisks represent areas of edema, and circled areas show inflammatory infiltrate.

6

Weight analysis and feed consumption during the experiment. Average weight (A) and feed consumption (B) by animals without treatment (naive) and treatment with negative control (0.9% NaCl), positive control (dacarbazine), free BVC (0.5%), lavender oil formulation (NLC-L), or formulations containing 0.5% BVC_S75: NLC-L-BVC and NLC-BVC. Statistical analysis: two-way ANOVA plus posthoc Tukey. a = in comparison to the naive group. *** p < 0.001; **** p < 0.0001.

7

Biochemical parameters: serum ALT, AST, CK-MB activity, and urea levels in the post-treatment period of animals treated with 0.9% NaCl, dacarbazine, free BVC, NLC-L, NLC-L-BVC, or NLC-BVC. ALT (A), AST (B), CK-MB (C), and urea (D). Statistical analysis: one-way ANOVA plus posthoc Tukey. *p < 0.05; **p < 0.01; ***p < 0.001.

8

Results of metabolic profiling of the liver. Multivariate analysis (left): (A) principal component analysis (PCA) and (B) sparse partial least-squares discriminant analysis (sPLS-DA). Univariate analysis (right): (C) heatmap of significant metabolites (ANOVA, p < 0.05) and (D) violin plot for the metabolite hypoxanthine.

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(A) Animal survival after melanoma induction. The survival rate was 100% in the naive, free BVC, and NLC-L-BVC groups; 80% in the NLC-BVC group (1 animal died); 40% in the dacarbazine and NLC-L groups (3 animals/group died); and 20% in the negative control group (4 animals/group died). (B) Percentage of survival across treatments, indicating higher survival odds for animals treated with BVC_S75 (free or encapsulated).