Fabrication and characterization of Agarwood extract-loaded nanocapsules and evaluation of their toxicity and anti-inflammatory activity on RAW 264.7 cells and in zebrafish embryos

Figures & data

Table 1. Particle size and PDI of B-PNCs by DLS technique in different formulations.

Formula	Homogenizer	Overhead stirrer	Magnetic stirrer	% of TG (w/v) (%)	% of Triton X-100 (w/v) (%)	% of AlCl3 (w/v) (%)	Mean diameter (nm)	PdI
F1	+	−	–	1	1	2	8409.33 ± 382.51	0.60 ± 0.15
F2	−	+	−	1	1	2	1475.67 ± 67.26	1.00 ± 0.00
F3	−	−	+	1	1	2	846.20 ± 14.77	0.54 ± 0.02
F4	−	−	+	0.5	1	2	804.73 ± 105.8447	0.99 ± 0.02
F5	−	−	+	2	1	2	8720.00 ± 648.77	0.29 ± 0.05
F6	−	−	+	3	1	2	1943.33 ± 1177.17	0.35 ± 0.06
F7	−	−	+	1	1	1	1795.33 ± 543.31	1.00 ± 0.00
F8	−	−	+	1	1	3	1171.33 ± 113.80	0.96 ± 0.07
F9	−	−	+	1	2	2	2483.33 ± 241.83	1.00 ± 0.00
F10	−	−	+	1	0.5	2	806.40 ± 66.33	0.56 ± 0.01
F11	−	−	+	1	0.25	2	533.13 ± 17.05	0.71 ± 0.22
*F12	−	−	+	1	0.1	2	187.46 ± 1.76	0.40 ± 0.01
F13	−	+	−	1	0.1	2	217.63 ± 13.57	0.71 ± 0.06
F14	+	−	−	1	0.1	2	1738.33 ± 275.43	1.00 ± 0.00
F15	−	−	+	1	0.1	1	368.83 ± 4.25	0.41 ± 0.00
F16	−	−	+	0.5	0.1	2	359.63 ± 20.08	0.80 ± 0.06
F17	−	−	+	1.0	0.50	1	433.56 ± 46.9	0.77 ± 0.16

*Represents the optimized formulation.

Figure 1. Particle size distribution by intensity of ALEX-M-PNCs measured by DLS technique.

Figure 2. Zeta Potential distribution of ALEX-M-PNCs measured by DLS technique.

Figure 3. TEM images of ALEX-M-PNCs at different magnifications.

Figure 4. In-vitro release of ALEX-M from ALEX-M-PNCs in 48 hours.

Figure 5. Thermogravimetry diagram of ALEX-M, ALEX-M-PNCs and B-PNCs.

Figure 6. FTIR spectra of (a) ALEX-M, (b) ALEX-M-PNCs and (c) B-PNCs.

Figure 7. Concentration dependent cytotoxicity effect of LPS/INFγ, ALEX-M, ALEX-M-PNCs, B-PNCs, and Ibuprofen against RAW 264.7 cell line. Cell cytotoxicity was determined using MTT assay. Data are the means ± standard deviation (SD) of three independent experiments.

Figure 8. Effect of ALEX-M, ALEX-M-PNCs, and B-PNCS on LPS/IFNγ-induced NO in RAW 264.7 cells. Data are the means ± standard deviation (SD) of three independent experiments, p < 0.05. Student’s t test was used to compare EC₅₀ values at p < 0.05.

Figure 9. Signs of lethality in zebrafish embryos: (a) coagulation (b) non-detachment of tail (c) lack of somite formation (d) normal embryo showing no signs of toxicity at 24 hpf. Images were captured under magnification of 40×.

Figure 10. Dose response to mortality of zebrafish embryos at 96 hpf, exposed to ALEX-M (6.25–800 µg/ml) and ALEX-M-PNCs and B-PNCs at concentrations (6.25–100 µg/ml) (n = 12 embryos/group) of three independent replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Different letters (a–d) indicate significant difference at p < 0.05.

Figure 11. Effect of different concentrations of the tested samples on hatchability percentage in zebrafish embryo. Values are expressed as mean ± SD (n = 12 embryos/group) of three independent replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Different letters (a–e) indicates significant difference at p < 0.05.

Figure 12. Effect of different concentrations of the tested samples on the heart rate in zebrafish embryo. Values are expressed as mean ± SD (n = 3 embryos/group) of three independent replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Different letters (a–b) indicates significant difference at p < 0.05.

Figure 13. Malformations in zebrafish larva: (a) normal larva at 72 hpf, (b) spinal curvature, (c) scoliosis, (d) pericardial edema. Images were captured under magnification of 40×.

Figure 14. Dose response of embryonic zebrafish mortality at 96 hpf, exposed to ALEX-M (6.25–100 µg/ml) and ALEX-M-PNCs and B-PNCs at concentrations (0.625–2.5 µg/ml), (n = 12 embryos/group) in three replicates. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. No significant difference occurred between all groups at p < 0.05.

Figure 15. Fluorescence micrographs of LPS-stimulated NO generation in zebrafish embryos showing the effect of ALEX-M, ALEX-M-PNCs and B-PNCs on LPS-induced NO production. The images were visualized using 40× magnification power. (a) control, (b) ALEX-M 100 µg/ml, (c) LPS 5 µg/ml, (d1) ALEX-M 100 µg/ml + LPS 5 µg/ml, (d2) ALEX-M 50 µg/ml + LPS 5 µg/ml, (d3) ALEX-M 25 µg/ml + LPS 5 µg/ml, (d4) ALEX-M 6.25 µg/ml + LPS 5 µg/ml, (e1) ALEX_M_PNCs 2.5 µg/ml + LPS 5 µg/ml, (e2) ALEX_M_PNCs 1.25 µg/ml + LPS 5 µg/ml, (e3) ALEX_M_PNCs 0.625 µg/ml + LPS 5 µg/ml, (f1) B-PNCs 2.5 µg/ml + LPS 5 µg/ml, (f2) B-PNCs 1.25 µg/ml + LPS 5 µg/ml, (f3) B-PNCs 0.625 µg/ml + LPS 5 µg/ml.

Figure 16. Quantitative analysis of fluorescence intensity of zebrafish embryos after staining with DAF-FM-DA using imageJ program. Data are represented as means ± SD from three independent experiments (n = 9 embryos/group). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Values with different letters (a–j) are significantly different in comparison to negative and positive control at p < 0.05.