In Vitro and In Vivo evaluation of a novel folate-targeted theranostic nanoemulsion of docetaxel for imaging and improved anticancer activity against ovarian cancers

Figures & data

Figure 1. Schematic representation of receptor mediated endocytosis of NMI-500 and mechanism of docetaxel cellular uptake and efflux responsible for MDR.

Table 1. Characterization of NEs.

Formulations	Size (nm)	PDI	Zeta Potential (mV)
NT-RhNE	146.4	0.093	−42.3 ± 12.5
FA-RhNE (100)	145.2	0.026	−46.6 ± 10.5
FA-RhNE (300)	133.2	0.044	−37.4 ± 10.9
FA-RhNE (1200)	148.1	0.036	−45.8 ± 10.5
FA-RhNE (3600)	153.9	0.074	−40.7 ± 9.68
NT-NMI-500	137.2	0.091	−50.8 ± 12.4
FA100-NMI-500	140.5	0.064	−50.1 ± 13.7
FA1200-NMI-500	140.1	0.041	−48.6 ±14.5

Figure 2. FA targeted nanoemulsions significantly enhances cellular uptake in FR-α positive cells. IHC staining of FR- α in (A) A2780 (B) SKOV-3 (C) KB-WT and (D) KB-PR10 cells. Flow cytometry analysis of cellular uptake of the Rh-labeled NEs with varying amount of FA in (E) KB-WT cells, (F) KB-PR10 cells with or without pretreatment with FA (1 mM).

Table 2. IC₅₀ of docetaxel against KB-WT and KB-PR10 cells.

IC50 (nM)	KB-WT	KB-PR10
NT-NMI-500	1.26 ± 0.23	12.85 ± 11.5
FA-100-NMI-500	1.58 ± 0.56	3.05 ± 1.35
FA1200-NMI-500	0.62 ± 0.14	9.33 ± 1.29
DTX in DMSO	1.00 ± 0.19	1.35 ± 0.26
PTX in DMSO	2.95 ± 0.07	33.46 ± 10.7

Figure 3. NCI-60 Human Cancer Cell Screen of NMI-500. NMI-500 was incubated with 60 different cell lines for 72 hrs and GI₅₀ was determined. GI₅₀ of NMI-500 was compared to GI₅₀ of DTX for each cell line. Bars on the left of central line represent lower GI₅₀ and bars on the right of central line represents higher GI₅₀ for NMI-500 compared to DTX.

Figure 4. Pharmacokinetic profile of Taxotere, NT-NMI-500 and NMI-500. Balb/c mice were injected with formulation at 10 mg/kg of DTX intravenously via tail vein. Blood was collected at various time points and DTX concentration was measured in plasma using LC-MS. Pharmacokinetic parameters were calculated using Non-compartmental analysis using Phoenix-WinNonlin 6.2 version.

Table 3. Pharmacokinetic parameters of docetaxel administered in various formulations.

Plasma Pharmacokinetic Profile of Docetaxel
Groups	Tmax (h)	Cmax (ng/L)	t1/2 (h)	AUC0-∞ (h*ng/L)	Vd (L/kg)	Cl (L/h/kg)	MRT (h)
Taxotere	0.083	4207 ± 670	1.4 ± 0.3	3195 ± 412	6.4 ± 1.8	3.2 ± 0.4	0.9 ± 0.4
NMI-500 (NT)	0.083	2930 ± 1266	3.2 ± 0.7	2328 ± 614	20.8 ± 6.6	4.5 ± 1.2	1.9 ± 1.0
NMI-500	0.083	3660 ± 433	6.1 ± 3.8	2840 ± 55	31 ± 19	3.5 ± 0.1	3 ± 2.1

Figure 5. NMI-500 treatment inhibits ovarian tumor growth and ascites production in TgMISIIR-TAg transgenic mice. Spontaneous tumor development and growth in TgMISIIR-TAg mice was monitored by weekly magnetic resonance imaging (MRI) and drug treatment initiated when tumor volume reached ∼100 mm3. Mice were randomized into four groups (n = 20–21 mice/group) and treated with blank NE, NT-NMI-500, NMI-500 or DTX (Taxotere). (A) Tumor volumes calculated from MRI datasets at baseline (start) and after 8 weeks of treatment. (B) Tumor growth rates (slope) computed from longitudinal MRI data. (C) Total tumor volume calculated by caliper measurement of ovaries at necropsy. (D) The presence or absence of malignant ascites determined at necropsy. The MRI data for tumor growth rate were analyzed by the Wilcoxon signed-rank test, final tumor volumes by the Mann-Whitney t test and ascites fraction by the Fisher's exact two-sided test. P values less <0.05 were considered significant (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).

Figure 6. Body weight and average MRI tumor volume. NMI-500 treatment results nominal weight loss and decreased ovarian tumor growth in TgMISIIR-TAg transgenic mice. (A) Tumor weights in mice weighed weekly. (B) Average tumor volumes calculated from MRI datasets in drug and vehicle treated mice.

Figure 7. Cleaved caspase 3, phosphorylated H2AX and Ki-67 protein levels detected by immunohistochemistry in ovarian tumors in TgMISIIR-TAg mice.