Uptake and transport of a novel anticancer drug-delivery system: lactosyl-norcantharidin-associated N-trimethyl chitosan nanoparticles across intestinal Caco-2 cell monolayers

Figures & data

Table 1 Uptake of Lac-NCTD on Caco-2 cells with different preparations (n = 3)

	Preparations
	Lac-NCTD	Lac-NCTD-CS-NPs	Lac-NCTD-TMC-NPs
Uptake (mg/g protein)	0.80 ± 0.15	1.54 ± 0.32**	1.73 ± 0.17**,#

Notes:

** P < 0.01 versus Lac-NCTD;

# P < 0.05 versus Lac-NCTD-CS-NPs.

Abbreviations: Lac-NCTD, lactosyl-norcantharitin; Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells.

Table 2 Papp of Lac-NCTD at different concentrations in Caco-2 cells (n = 3)

Lac-NCTD (μg/mL)	Papp (×10^−6)		Ratio PB-A/PA-B
	AP-BL	BL-AP
100	3.29 ± 0.34	6.29 ± 0.43	1.91
250	3.67 ± 0.17	6.76 ± 0.28	1.845
400	3.34 ± 0.22	4.58 ± 0.15	1.37

Abbreviations: Lac-NCTD, lactosyl-norcantharitin-associated; Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells.

Table 3 Papp of Lac-NCTD on Caco-2 cells with different preparations (n = 3)

Preparations (250 μg/mL)	Papp (×10^−6)		Ratio PB-A/PA-B
	AP-BL	BL-AP
Lac-NCTD solution	3.67 ± 0.68	6.76 ± 1.12	1.85
Lac-NCTD-CS-NPs	5.38 ± 0.23**	6.01 ± 0.45*	1.11**
Lac-NCTD-TMC-NPs	5.73 ± 0.44**,#	6.12 ± 0.67*	1.07**

Notes:

* P < 0.05 versus Lac-NCTD;

** P < 0.01 versus Lac-NCTD;

# P < 0.05 versus Lac-NCTD-CS-NPs.

Abbreviations: Lac-NCTD, lactosyl-norcantharitin; Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells.

Figure 1 Particle size distribution analysis using a Zetasizer Nano particle analyzer.

Figure 2 TEM micrograph of Lac-NCTD-TMC-NPs.

Abbreviations: TEM, transmission electron microscopy; Lac-NCTD-TMC-NPs, lactosyl-norcantharitin (Lac-NCTD)-associated N-Trimethyl chitosan nanoparticles.

Figure 3 Time course of Lac-NCTD uptake in Caco-2 cell monolayers.

Note: Error bars represent standard error of the mean value for three determinations.

Abbreviations: Lac-NCTD, lactosyl-norcantharitin; Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells.

Figure 4 Effect of Lac-NCTD concentration on Caco-2 cells uptake.

Note: Error bars represent standard error of the mean value for three determinations.

Abbreviations: Lac-NCTD, lactosyl-norcantharitin; Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells.

Figure 5 Effect of CyA and MK-571 on Caco-2 cells uptake.

Notes: **P < 0.01 versus control group; ^##P < 0.01 versus control group. Error bars represent standard error of the mean value for three determinations.

Abbreviations: CyA, cyclosporine; MK-571, Sigma-MK-571 sodium salt hydrate; Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells.

Figure 6 Effect of CyA, MK-571 on the transport of Lac-NCTD.

Notes: *P < 0.05 versus control group; **P < 0.01 versus control group; ^##P < 0.01 versus control group. Error bars represent standard error of the mean value for three determinations.

Abbreviations: CyA, cyclosporine; MK-571, Sigma-MK-571 sodium salt hydrate; Lac-NCTD, lactosyl-norcantharitin.

Figure 7 Effect of oxophenylarsine and SDCh on the transport of Lac-NCTD.

Notes: ^#P < 0.05 versus control group. Error bars represent standard error of the mean value for three determinations.

Abbreviations: SDCh, sodium deoxycholate; Lac-NCTD, lactosyl-norcantharitin.

Figure 8 Laser scanning confocal microscopy images showing uptake and subcellular localization of FITC-labeled Lac-NCTD-CS-NPs and FITC-labeled Lac-NCTD-TMC-NPs. Panel 1. Green fluorescence shows the location of FITC-labeled Lac-NCTD-CS- NPs or FITC-labeled Lac-NCTD-TMC-NPs. Panel 2. Blue fluorescence shows nuclear staining with Hoechst33258. Panel 3. Overlaid image of Panel 1 and Panel 2. (A, C, E): Lac-NCTD-CS-NPs groups. (B, D, F): Lac-NCTD-TMC-NPs groups. (A and B): 15 min. (C and D): 30 min. (E and F): 60 min.

Abbreviations: FITC-labeled, fluorescein isothiocyanate-labeled; Lac-NCTD-CS-NPs, lactosyl-norcantharitin (Lac-NCTD)-associated N-Trimethyl chitosan nanoparticles; Lac-NCTD-TMC-NPs, lactosyl-norcantharitin (Lac-NCTD)-associated N-Trimethyl chitosan nanoparticles.

Figure 9 Atomic force microscope micrograph of Caco-2 cells. A: Caco-2; B: Caco-2 after treatment with Lac-NCTD-TMC-NPs. (10 μm × 10 μm).

Abbreviations: Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells; Lac-NCTD-TMC-NPs, lactosyl-norcantharitin (Lac-NCTD)- associated N-Trimethyl chitosan nanoparticles.

Figure 10 Size distribution histogram of protrusions on Caco-2. A: Caco-2; B: Caco-2 after treatment with Lac-NCTD-TMC-NPs.

Abbreviations: Caco-2, continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells; Lac-NCTD-TMC-NPs, lactosyl-norcantharitin (Lac-NCTD)-associated N-Trimethyl chitosan nanoparticles.