Folic acid functionalized long-circulating co-encapsulated docetaxel and curcumin solid lipid nanoparticles: In vitro evaluation, pharmacokinetic and biodistribution in rats

Figures & data

Figure 1. Schematic diagram of FA–SA conjugate synthesis.

Table 1. FC-CCD design with factors with corresponding levels and responses.

		Levels
Critical factors	Symbol	−1	0	+1
Homogenization time (min)	X1	5	10	15
Homogenization speed (rpm)	X2	16 000	20 000	24 000
Stabilizer concentration (%)	X3	0.5	1	1.5
Lipid amount (%)	X4	1	3	5
Responses	Symbol	Constraints
Particle size (nm)	Y1	Minimize
Zeta potential (mV)	Y2	Maximize
PDI	Y3	Minimize
Entrapment efficiency (EE %)	Y4	Maximize

PDI, polydispersity index.

Figure 2. Cytotoxicity of DTX and CRM in MDA-MB-231 cells. Results are mean ± SEM (n = 3). p value significant at ***0.01.

Figure 3. Solubility study of DTX (A), CRM (B) and 1:1 mixture of DTX and CRM (C) in solid lipids. Values are mean ± SD (n = 3).

Table 2. Results of FC-CCD experiment trials.

Run	Factor: X1 H. Time (min)	Factor: X2 H. Speed (rpm)	Factor: X3 Stabilizer conc. (%)	Factor: X4 lipid amount (%)	Response Y1 Particle size (nm)	Response Y2 Zeta potential (mV)	Response Y3 PDI	Response Y4 EE (%)
1	10	20 000	1	1	159	−33	0.347	80.03
2	5	16 000	0.5	1	240	−25	0.473	98.12
3	10	20 000	1	3	130	−10	0.296	96.71
4	15	16 000	1.5	5	144	−17	0.251	96.37
5	5	24 000	0.5	5	482	−15	0.858	85.94
6	5	20 000	1	3	172	−18	0.320	97.74
7	15	24 000	1.5	1	107	−27	0.307	85.92
8	5	24 000	1.5	1	175	−18	0.317	96.44
9	15	24 000	1.5	5	114	−22	0.351	96.96
10	1	20 000	1	3	134	−20	0.291	64.76
11	15	24 000	0.5	1	127	−19	0.326	64.76
12	15	16 000	0.5	5	201	−28	0.418	86.07
13	5	24 000	0.5	1	214	−17	0.288	77.65
14	5	24 000	1.5	5	238	−21	0.306	59.15
15	10	20 000	1	3	163	−19	0.390	80.88
16	10	20 000	1	3	145	−20	0.292	73.58
17	10	20 000	1.5	3	134	−20	0.272	85.24
18	10	20 000	1	3	137	−21	0.387	98.12
19	5	16 000	1.5	1	492	−27	0.590	96.57
20	15	20 000	1	3	118	−19	0.328	98.19
21	10	24 000	1	3	188	−17	0.589	97.31
22	5	16 000	0.5	5	624	−23	0.597	78.40
23	5	16 000	1.5	5	371	−16	0.451	42.16
24	10	16 000	1	3	198	−19	0.596	92.99
25	10	20 000	0.5	3	148	−18	0.437	95.70
26	10	20 000	1	3	180	−21	0.307	98.42
27	10	20 000	1	5	167	−25	0.294	98.86
28	15	16 000	1.5	1	417	−18	0.776	95.59
29	15	16 000	0.5	1	250	−24	0.058	94.01
30	15	24 000	0.5	5	185	−27	0.219	98.91

Figure 4. TEM image of F-DC-SLN.

Table 3. Particle size, PDI and EE of SLN before and after freeze drying.

	Before			After
Formulation	Particle size (nm)	PDI	EE (%)	Particle size (nm)	PDI	EE (%)	Ratio (Sf/Si)
Blank SLN without mannose	212 ± 2.05	0.219 ± 0.09	–	5771 ± 20.01	1.000	–	27.22
DC-SLN	221.3 ± 2.59	0.297 ± 0.03	69.70 ± 0.72	305 ± 12.49	0.163 ± 0.090	61.05 ± 0.70	1.38
F-DC-SLN	247.5 ± 3.40	0.211 ± 0.12	73.88 ± 1.08	348 ± 18.35	0.353 ± 0.009	69.43 ± 8.40	1.40

Sf/Si ratio of particle size after freeze drying to particle size before freeze drying.

Figure 5. DSC characterization of SLN. (A) CRM, (B) DTX, (C) GMS, (D) Compritol, (E) blank SLN, (F) DC-SLN, (G) F-DC-SLN.

Figure 6. PXRD characterization of SLN.

Figure 7. Polarized light microscopy image shows absence of birefringence in SLN dispersion.

Figure 8. FTIR characterization of folic acid on surface of SLN. (A) Folic acid, (B) blank SLN, (C) DC-SLN, (D) F-DC-SLN.

Figure 9. In vitro release of F-DC-SLN in PBS (0.01 M, pH 7.4, 50% v/v ethanol). Values are mean ± SD (n = 3).

Figure 10. Cell uptake efficiency of SLN formulations. Values are mean ± SEM (n = 3). p value significant at *0.05.

Figure 11. Fluoroscence microscope images shows internalization of free coumarin (a), DC-SLN (b) and F-DC-SLN (c) in MCF-7 cells.

Figure 12. In vitro cytotoxicity of SLN in MCF-7 cells. Values are mean ± SEM (n = 3). p value significant at **0.01; ***0.001.

Figure 13. In vitro hemolysis test of SLN formulations. Values are mean ± SD (n = 3). p value significant at ***0.001.

Figure 14. Plasma concentrations versus time profile of DTX after i.v. administration of DTX formulations. Values are mean ± SD (n = 5).

Table 4. Pharmacokinetic parameters of DTX after i.v. administration of Taxotere, DC-SLN and F-DC-SLN in rats.

Parameters	Taxotere	DC-SLN	F-DC-SLN
Cmax (ng/mL)	551.33 ± 92.79	331.98 ± 72.42	418.86 ± 139.06
Cl (L/h/kg)	6.14 ± 1.20	1.99 ± 0.57***	0.96 ± 0.44***
t1/2 (h)	10.95 ± 8.61	8.95 ± 4.93	21.34 ± 13.66
MRT (h)	7.24 ± 4.51	13.19 ± 6.80	31.14 ± 19.94*
AUC0–∞  (μg/mL h)	39.05 ± 7.41	138.26 ± 40.19**	594.21 ± 64.34***

Data represent mean value ± SD, n = 5. C_max, maximum plasma concentration; Cl, clearance; t_1/2, elimination half-life; MRT, mean residence time; AUC_0–∞, area under the curve.

*p < 0.05.

**p < 0.01.

***p < 0.001 significant difference from Taxotere.

Figure 15. Biodistribution of DTX after i.v. administration of DTX formulations. Stastitical diffrence between Taxotere 7 min and F-DC-SLN 7 min is shown that values are mean ± SD (n = 6). p value significant at *0.05, **0.01 and ***0.001.