Formulation, characterization, and geno/cytotoxicity studies of galbanic acid-loaded solid lipid nanoparticles

Figures & data

Figure 1. Chemical structure of galbanic acid.

Figure 2. SEM images of (A) blank SLNs (F10) and (B) GBA-SLNs (F19), and zeta potential of (C) F10 and (D) F19. (F10) and (F19) represent the formulation code of 10 and 19 for blank SLNs and GBA-SLN, respectively.

Table 1. Composition, particle size, and encapsulation efficiency (EE) of galbanic acid-loaded SLNs formulation.

Formulation code	Lipid type and concentration (g)	Drug concentration (g)	Oil phase surfactant type and concentration (g)	Aqueous phase surfactant concentration^a (g)	Particle size (nm)	Poly-dispersity	Encapsulation efficiency (EE %)	Drug loading (DL, mg/g)	Drug content (%)
Blank SLNs
F1	Precirol (0.55)	–	Tween 20 (0.01)	0.52	183 ± 8.13	0.558 ± 0.131	–	–	–
F2	Precirol (0.55)	–	Tween 20 (0.05)	0.68	164 ± 6.24	0.482 ± 0.142	–	–	–
F3	Precirol (0.60)	–	Tween 20 (0.07)	0.75	132 ± 4.14	0.332 ± 0.121	–	–	–
F4	Precirol (0.58)	–	Tween 20 (0.10)	0.83	101 ± 3.03	0.227 ± 0.128	–	–	–
F5	Precirol (0.58)	–	Tween 20 (0.13)	0.88	98 ± 1.88	0.189 ± 0.109	–	–	–
F6	Precirol (0.62)	–	Tween 20 (0.15)	0.92	83 ± 2.04	0.115 ± 0.089	–	–	–
F7	Precirol (0.60)	–	Tween 80 (0.01)	0.43	203 ± 9.91	0.449 ± 0.79	–	–	–
F8	Precirol (0.61)	–	Tween 80 (0.06)	0.59	173 ± 7.68	0.332 ± 0.203	–	–	–
F9	Precirol (0.59)	–	Tween 80 (0.08)	0.68	140 ± 4.56	0.204 ± 0.194	–	–	–
F10	Precirol (0.59)	–	Tween 80 (0.10)	0.79	93 ± 2.78	0.115 ± 0.023	–	–	–
GBA-loaded SLNs
F11	Precirol (0.59)	0.043	Tween 20 (0.01)	0.48	162 ± 7.01	0.444 ± 0.109	77.13 ± 3.34	34.21 ± 0.45	3.02
F12	Precirol (0.62)	0.039	Tween 20 (0.06)	0.55	155 ± 5.34	0.502 ± 0.151	82.22 ± 2.98	32.01 ± 0.23	2.52
F13	Precirol (0.65)	0.049	Tween 20 (0.09)	0.69	125 ± 3.89	0.253 ± 0.105	88.42 ± 1.88	42.74 ± 0.12	2.90
F14	Precirol (0.60)	0.047	Tween 20 (0.10)	0.78	94 ± 1.83	0.177 ± 0.221	91.55 ± 4.43	43.1 ± 0.36	2.75
F15	Precirol (0.63)	0.044	Tween 20 (0.13)	0.81	90 ± 1.33	0.142 ± 0.231	94.39 ± 3.99	42.68 ± 0.45	2.54
F16	Precirol (0.59)	0.045	Tween 20 (0.15)	0.88	78 ± 1.54	0.121 ± 0.091	96.69 ± 2.65	43.28 ± 0.25	2.58
F17	Precirol (0.61)	0.039	Tween 80 (0.01)	0.58	151 ± 7.34	0.331 ± 0.171	68.25 ± 5.31	26.61 ± 0.46	2.09
F18	Precirol (0.63)	0.040	Tween 80 (0.05)	0.62	122 ± 6.52	0.373 ± 0.274	86.11 ± 5.24	34.44 ± 0.33	2.53
F19	Precirol (0.58)	0.045	Tween 80 (0.08)	0.64	92 ± 3.302	0.135 ± 0.041	98.89 ± 3.67	44.5 ± 0.28	4.13
F20	Precirol (0.63)	0.039	Tween 80 (0.12)	0.88	77 ± 3.44	0.101 ± 0.108	96.09 ± 4.43	37.4 ± 0.37	1.37

Data are expressed as mean ± SD (n = 3).

^aPoloxamer 407 was used as the aqueous phase surfactant in all formulations.

Figure 3. Cumulative release of GBA from SLNs under medium containing 0.3% Tween 80, at different temperatures (24 and 37 °C) and pHs (7.4 and 5.6) after 16 h (384 h). All data represent the mean of at least three-recorded OD (error bars represent mean ± SD).

Table 2. The kinetics models used to fit the release data.

		Coefficient of determination (R^2)
		24 ± 2 °C		37 ± 0.5 °C
Kinetic model	Equation	pH 5.6	pH 7.4	pH 5.6	pH 7.4
Zero order		0.763	0.721	0.795	0.773
First order		0.831	0.792	0.873	0.829
Higuchi		0.918	0.901	0.928	0.906
Weibull		0.978	0.974	0.983	0.977
Hixson–Crowell		0.858	0.838	0.862	0.854

Parameters of models were obtained by linear regression. F represents fraction of drug released up to time t. The k₀, kf, k_H, and k_1/3 are parameters of the models. Ln, natural logarithm.

Table 3. Physical stability and drug leakage studies of the GBA-SLNs.

	Size volume average (nm)	Polydispersity index (PI)	Encapsulation efficiency (%)	Drug leakage (%)
Fresh GBA-SLNs	92 ± 4	0.135 ± 0.04	98.89 ± 0.19	–
GBA-SLNs in 4 °C after 3 month	93.1 ± 5	0.147 ± 0.03	93.21 ± 0.26	5.68
GBA-SLNs in 24 °C after 3 month	99.3 ± 6	0.214 ± 0.05	90.7 ± 0.29	8.19

Error bars represent mean ± standard deviation (n=3).

Figure 4. The differences in formulated SLNs size between the initial (A) and 3 months after storage at (B) 4 °C and (C) 24 °C were not found to be statistically significant.

Figure 5. Inhibition properties of plain GBA (black) and GBA-SLNs (gray) on A549 human lung epithelial carcinoma after (A) 24 h, (B) 48 h, and (C) 72 h. (D), (E), and (F) MTT result of treatment of plain GBA (black) and GBA-SLNs (gray) on HUVEC after 24 h and 48 h and 72 h, respectively. Error bars represent SD (n=3).

Figure 6. Light and fluorescent microscopy images of A549 cells and HUVEC stained with DAPI; (A) the HUVEC and (B) A549 cell line are shown. The arrows show fragmented nucleus in the treated cells.

Figure 7. Photographic illustrations of comet assay after 48 h incubation. (A) Untreated A549 cells, (B) treated A549 cells by 200 µM H₂O₂ (positive control), (C) treated A549 cells by GBA, and (D) treated A549 cells by GBA-SLNs. (E) Box-plot graph of DNA tail/DNA head% is shown; the DNA cleavage level in GBA and GBA-SLN-treated cells is not as high as treated cells with 200 mM H₂O₂ but is significantly (p<0.001) higher than untreated cells.

Figure 8. DNA ladder formation through gel electrophoresis in different treatments, and A549 cells and HUVEC. M, molecular marker.

Figure 9. FITC-labeled annexin V flow cytometric detection of apoptosis in A549 cells. (A) Untreated control cells, (B) treated cells with 200 µM H₂O₂ as a positive control, (C) treated cells with GBA, and (D) treated cells with GBA-loaded SLNs.

Figure 10. Differential expression levels of Casp 9 and Bcl-xL genes in plain GBA, GBA-SLNs, and doxorubicin (as positive control). * and ** represent significant differences (p < 0.05).