Articaine in functional NLC show improved anesthesia and anti-inflammatory activity in zebrafish

Abstract

Anesthetic failure is common in dental inflammation processes, even when modern agents, such as articaine, are used. Nanostructured lipid carriers (NLC) are systems with the potential to improve anesthetic efficacy, in which active excipients can provide desirable properties, such as anti-inflammatory. Coupling factorial design (FD) for in vitro formulation development with in vivo zebrafish tests, six different NLC formulations, composed of synthetic (cetyl palmitate/triglycerides) or natural (avocado butter/olive oil/copaiba oil) lipids were evaluated for loading articaine. The formulations selected by FD were physicochemically characterized, tested for shelf stability and in vitro release kinetics and had their in vivo effect (anti-inflammatory and anesthetic effect) screened in zebrafish. The optimized NLC formulation composed of avocado butter, copaiba oil, Tween 80 and 2% articaine showed adequate physicochemical properties (size = 217.7 ± 0.8 nm, PDI = 0.174 ± 0.004, zeta potential = - 40.2 ± 1.1 mV, %EE = 70.6 ± 1.8) and exhibited anti-inflammatory activity. The anesthetic effect on touch reaction and heart rate of zebrafish was improved to 100 and 60%, respectively, in comparison to free articaine. The combined FD/zebrafish approach was very effective to reveal the best articaine-in-NLC formulation, aiming the control of pain at inflamed tissues.

Figure 1

(A) Representative TEM micrographs of the nanostructured lipid carriers prepared for articaine; magnification: 100000x (images 2–5), 129,300× (1, 6). (B) In vitro release curves of articaine at 37 °C, free (in solution) or encapsulated into the optimized NLC formulations (n = 6). The images in (A) were edited with ImageJ software v.1.52a (https://imagej.nih.gov/ij/).

Figure 2

Absorption of NLCs in the larval body. (A) TgBAC(cldn15la:GFP)^pd1034 larvae, at 5 days post fertilization (dpf), exposed to NLCs loaded with rhodamine-PE, by the immersion method. The larvae intestine is demarked with a black dotted line. (B) Timeline: fluorescence was monitored at 1, 12 and 24-h post incubation (hpi). Representative images of larvae examined under a fluorescence stereomicroscope at 1 hpi (C), 12 hpi (E) and 24 hpi (G). A region of the mid-intestine of TgBAC(cldn15la:GFP)^pd1034 larvae, demarked by a red dotted line in (C,E,G) was examined under confocal microscope at 1, 12 and 24 hpi (D,F,H, respectively). NLC-R: NLCs labeled with red fluorescent rhodamine-PE. Scale bar 200 μm. The images in A, C–H were edited with ImageJ software v.1.52a (https://imagej.nih.gov/ij/).

Figure 3

NLC excipients anti-inflammatory activity in zebrafish larvae. (A,B) Experimental strategy and representative image of a Tg(BACmpx:GFP)ⁱ¹¹⁴ larvae before damage. (C–I) Representative images of the damaged area of nontraded larvae (C) or treated (D–I) with different NLCs. (J) Quantification of the number of neutrophils present at the damaged zone (Region of interest (ROI)) at 3 h post damage (hpd) from 3 independent experiments (n = 24). Scale bar 200 μm. Statistical analysis: one-way ANOVA and Tukey post-hoc test. Statistical significance: ***p < 0.001, ****p < 0.0001; n.s. not significant. The images (B) and (C–I) were edited using ImageJ software v.1.52a (https://imagej.nih.gov/ij/).

Figure 4

(A) Experimental strategy. (B) Bradycardia after 1 h of incubation of free articaine (ATC) and articaine-loaded nanostructured lipid carriers at concentrations of 0.5 and 1 mM and (C) their respective controls without articaine. (D) Touch reaction after 1 h incubation of ATC and articaine-loaded nanostructured lipid carriers at concentrations of 0.5 and 1 mM and (E) their respective controls without articaine. Touch reaction scale: Score 0 means larva did not react, score 1 means larva react between 1 and 3 s after touch, and score 2 means larva react in less than 1 s after touch. n = 24. E3 = medium only, no treatment (control). Statistical analysis: one-way ANOVA and Tukey post-hoc test. Statistical significance: ***p < 0.001, ****p < 0.0001; n.s. non-significant.

Figure 5

Bradycardia recovery. (A) Experimental strategy. (B) Heartbeats counted after 1, 2 and 3 h post removal of treatment with 1 mM articaine, free or in NLC formulations. (C) Comparison of the effect of copaiba oil-NLC formulations, with and without articaine on the heartbeat. n = 24. Statistical analysis performed by one-way ANOVA and Tukey post-hoc test. Statistical significance: **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s. non-significant.