174
Views
43
CrossRef citations to date
0
Altmetric
Original Research

Enhanced oral absorption of pemetrexed by ion-pairing complex formation with deoxycholic acid derivative and multiple nanoemulsion formulations: preparation, characterization, and in vivo oral bioavailability and anticancer effect

, , , &
Pages 3329-3351 | Published online: 06 Jun 2018

Figures & data

Figure 1 (A) Structural diagram of the ion-pairing complex between PMX and DCK. (B) Schematic representation of HP-beta-CD/PMX/DCK/P188-NE. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion; PMX/DCK, ion-pairing complex between PMX and DCK.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; NE, nanoemulsion; PMX, pemetrexed; P188, poloxamer 188.

Figure 1 (A) Structural diagram of the ion-pairing complex between PMX and DCK. (B) Schematic representation of HP-beta-CD/PMX/DCK/P188-NE. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion; PMX/DCK, ion-pairing complex between PMX and DCK.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; NE, nanoemulsion; PMX, pemetrexed; P188, poloxamer 188.

Figure 2 (A) PXRDs of PMX, DCK, PMX/DCK, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and a physical mixture of HP-beta-CD, PMX, DCK, and P188. (B) DSC thermograms of PMX, DCK, PMX/DCK, HP-beta-CD/PMX/DCK, and HP-beta-CD/PMX/DCK/P188, and a physical mixture of HP-beta-CD, PMX, DCK, and P188.

Notes: HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; PMX/DCK, ion-pairing complex between PMX and DCK.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; DSC, differential scanning calorimetry; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; P188, poloxamer 188; PMX, pemetrexed; PXRD, powder X-ray diffractogram.

Figure 2 (A) PXRDs of PMX, DCK, PMX/DCK, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and a physical mixture of HP-beta-CD, PMX, DCK, and P188. (B) DSC thermograms of PMX, DCK, PMX/DCK, HP-beta-CD/PMX/DCK, and HP-beta-CD/PMX/DCK/P188, and a physical mixture of HP-beta-CD, PMX, DCK, and P188.Notes: HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; PMX/DCK, ion-pairing complex between PMX and DCK.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; DSC, differential scanning calorimetry; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; P188, poloxamer 188; PMX, pemetrexed; PXRD, powder X-ray diffractogram.

Figure 3 (A) Droplet size distribution and (B) TEM image of HP-beta-CD/PMX/DCK/P188-NE.

Notes: Scale bar represents 100 nm. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; PMX, pemetrexed; P188, poloxamer 188; TEM, transmission electron microscopy.

Figure 3 (A) Droplet size distribution and (B) TEM image of HP-beta-CD/PMX/DCK/P188-NE.Notes: Scale bar represents 100 nm. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; PMX, pemetrexed; P188, poloxamer 188; TEM, transmission electron microscopy.

Figure 4 In vitro cytotoxic effects of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE on (A) LLC and (B) A549 cells.

Notes: Data are presented as mean ± SD (n=6 for each group). *P<0.05, **P<0.01, ***P<0.001 compared with the PMX treatment group at the same concentration equivalent to PMX. A549, human lung carcinoma; HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK, HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; LLC, Lewis lung carcinoma; PMX, pemetrexed; P188, poloxamer 188.

Figure 4 In vitro cytotoxic effects of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE on (A) LLC and (B) A549 cells.Notes: Data are presented as mean ± SD (n=6 for each group). *P<0.05, **P<0.01, ***P<0.001 compared with the PMX treatment group at the same concentration equivalent to PMX. A549, human lung carcinoma; HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK, HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; LLC, Lewis lung carcinoma; PMX, pemetrexed; P188, poloxamer 188.

Figure 5 In vitro effects of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE on the proliferation/migration of LLC cells.

Notes: (A) Wound closure at 48 h of treatment (**P<0.01, ***P<0.001 compared with the PMX treatment group at the same concentration equivalent to PMX). (B) Time course of closure of the wounded areas after the cells had been treated with drug equivalent to 0.5 μg/mL PMX (***P<0.001 compared with the control group; ##P<0.01 compared with the PMX treatment group). (C) Representative images at different time points after the cells were treated with drug equivalent to 0.5 μg/mL PMX. Data are presented as mean ± SD (n=6 for each group). Scale bar represents 300 μm. Magnification ×10. HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; LLC, Lewis lung carcinoma; PMX, pemetrexed; P188, poloxamer 188.

Figure 5 In vitro effects of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE on the proliferation/migration of LLC cells.Notes: (A) Wound closure at 48 h of treatment (**P<0.01, ***P<0.001 compared with the PMX treatment group at the same concentration equivalent to PMX). (B) Time course of closure of the wounded areas after the cells had been treated with drug equivalent to 0.5 μg/mL PMX (***P<0.001 compared with the control group; ##P<0.01 compared with the PMX treatment group). (C) Representative images at different time points after the cells were treated with drug equivalent to 0.5 μg/mL PMX. Data are presented as mean ± SD (n=6 for each group). Scale bar represents 300 μm. Magnification ×10. HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; LLC, Lewis lung carcinoma; PMX, pemetrexed; P188, poloxamer 188.

Figure 6 In vitro effects of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE on the proliferation/migration of A549 cells.

Notes: (A) Wound closure at 48 h of treatment (**P<0.01, ***P<0.001 compared with the PMX treatment group at the same concentration equivalent to PMX). (B) Time course of closure of the wounded areas after the cells were treated with the drug equivalent to 1 μg/mL PMX (***P<0.001 compared with the control group). (C) Representative images at different time points after the cells were treated with drug equivalent to 0.5 μg/mL PMX. Data are presented as mean ± SD (n=6 for each group). Scale bar represents 300 μm. Magnification ×10. A549, human lung carcinoma; HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; PMX, pemetrexed; P188, poloxamer 188.

Figure 6 In vitro effects of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE on the proliferation/migration of A549 cells.Notes: (A) Wound closure at 48 h of treatment (**P<0.01, ***P<0.001 compared with the PMX treatment group at the same concentration equivalent to PMX). (B) Time course of closure of the wounded areas after the cells were treated with the drug equivalent to 1 μg/mL PMX (***P<0.001 compared with the control group). (C) Representative images at different time points after the cells were treated with drug equivalent to 0.5 μg/mL PMX. Data are presented as mean ± SD (n=6 for each group). Scale bar represents 300 μm. Magnification ×10. A549, human lung carcinoma; HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; PMX, pemetrexed; P188, poloxamer 188.

Table 1 Partition coefficient, effective permeability, and apparent permeability of PMX, PMX/DCK complexes, and HP-beta-CD/PMX/DCK/P188-NE

Figure 7 CLSM images of the cellular uptake of (A) FITC-PMX, FITC-PMX/DCK, FITC-PMX-NE, and FITC-PMX/DCK-NE in Caco-2 cells, (B) FITC-PMX and FITC-PMX/DCK in MDCK or ASBT-transfected MDCK cells, and (C) FITC-PMX-NE and FITC-PMX/DCK-NE in MDCK or ASBT-transfected MDCK cells.

Notes: Scale bar represents 20 μm. Magnification ×40. FITC-PMX, FITC-conjugated PMX; FITC-PMX/DCK, ion-pairing complex between FITC-PMX and DCK; FITC-PMX-NE, FITC-PMX-loaded nanoemulsion; FITC-PMX/DCK-NE, FITC-PMX/DCK-loaded nanoemulsion.

Abbreviations: ASBT, apical sodium bile acid transporter; CLSM, confocal laser scanning microscope; DAPI, 4′,6-diamidino-2-phenylindole; DCK, Nα-deoxycholyl-l-lysyl-methylester; FITC, fluorescein isothiocyanate; MDCK, Madin–Darby canine kidney; PMX, pemetrexed.

Figure 7 CLSM images of the cellular uptake of (A) FITC-PMX, FITC-PMX/DCK, FITC-PMX-NE, and FITC-PMX/DCK-NE in Caco-2 cells, (B) FITC-PMX and FITC-PMX/DCK in MDCK or ASBT-transfected MDCK cells, and (C) FITC-PMX-NE and FITC-PMX/DCK-NE in MDCK or ASBT-transfected MDCK cells.Notes: Scale bar represents 20 μm. Magnification ×40. FITC-PMX, FITC-conjugated PMX; FITC-PMX/DCK, ion-pairing complex between FITC-PMX and DCK; FITC-PMX-NE, FITC-PMX-loaded nanoemulsion; FITC-PMX/DCK-NE, FITC-PMX/DCK-loaded nanoemulsion.Abbreviations: ASBT, apical sodium bile acid transporter; CLSM, confocal laser scanning microscope; DAPI, 4′,6-diamidino-2-phenylindole; DCK, Nα-deoxycholyl-l-lysyl-methylester; FITC, fluorescein isothiocyanate; MDCK, Madin–Darby canine kidney; PMX, pemetrexed.
Figure 7 CLSM images of the cellular uptake of (A) FITC-PMX, FITC-PMX/DCK, FITC-PMX-NE, and FITC-PMX/DCK-NE in Caco-2 cells, (B) FITC-PMX and FITC-PMX/DCK in MDCK or ASBT-transfected MDCK cells, and (C) FITC-PMX-NE and FITC-PMX/DCK-NE in MDCK or ASBT-transfected MDCK cells.Notes: Scale bar represents 20 μm. Magnification ×40. FITC-PMX, FITC-conjugated PMX; FITC-PMX/DCK, ion-pairing complex between FITC-PMX and DCK; FITC-PMX-NE, FITC-PMX-loaded nanoemulsion; FITC-PMX/DCK-NE, FITC-PMX/DCK-loaded nanoemulsion.Abbreviations: ASBT, apical sodium bile acid transporter; CLSM, confocal laser scanning microscope; DAPI, 4′,6-diamidino-2-phenylindole; DCK, Nα-deoxycholyl-l-lysyl-methylester; FITC, fluorescein isothiocyanate; MDCK, Madin–Darby canine kidney; PMX, pemetrexed.

Figure 8 In vitro cumulative percentage release profiles of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE in (A) pH 1.2 and (B) pH 6.8 media.

Notes: Each value represents the mean ± SD (n=6 for each group). HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; PMX, pemetrexed; P188, poloxamer 188.

Figure 8 In vitro cumulative percentage release profiles of PMX, HP-beta-CD/PMX, HP-beta-CD/PMX/DCK, HP-beta-CD/PMX/DCK/P188, and HP-beta-CD/PMX/DCK/P188-NE in (A) pH 1.2 and (B) pH 6.8 media.Notes: Each value represents the mean ± SD (n=6 for each group). HP-beta-CD/PMX, HP-beta-CD containing PMX; HP-beta-CD/PMX/DCK, ion-pairing complex between HP-beta-CD/PMX and DCK; HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; PMX, pemetrexed; P188, poloxamer 188.

Table 2 Pharmacokinetic parameters of PMX in rats after IV injection of PMX and oral administration of PMX, HP-beta-CD/PMX/DCK/P188, or HP-beta-CD/PMX/DCK/P188-NE

Figure 9 The mean plasma concentration–time profiles of PMX after (A) single IV administration of PMX (10 mg/kg) and (B) oral administration of PMX in aqueous solution (20 mg/kg), HP-beta-CD/PMX/DCK/P188 (equivalent to 20 mg/kg PMX), or HP-beta-CD/PMX/DCK/P188-NE (equivalent to 20 mg/kg PMX) to rats.

Notes: Each value represents the mean ± SD (n=4 for each group). HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; IV, intravenous; PMX, pemetrexed; P188, poloxamer 188.

Figure 9 The mean plasma concentration–time profiles of PMX after (A) single IV administration of PMX (10 mg/kg) and (B) oral administration of PMX in aqueous solution (20 mg/kg), HP-beta-CD/PMX/DCK/P188 (equivalent to 20 mg/kg PMX), or HP-beta-CD/PMX/DCK/P188-NE (equivalent to 20 mg/kg PMX) to rats.Notes: Each value represents the mean ± SD (n=4 for each group). HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; IV, intravenous; PMX, pemetrexed; P188, poloxamer 188.

Figure 10 In vivo tumor growth inhibition efficacy in LLC tumor-bearing mice after treatment with once-weekly IV administration of PMX (20 mg/kg) and once-daily oral administration of PMX in aqueous solution (20 mg/kg) or HP-beta-CD/PMX/DCK/P188-NE (equivalent to 20 mg/kg PMX) for 21 days.

Notes: (A) Tumor volume in mice. (B) Variation of body weight in mice during treatment. (C) Tumor weight in LLC tumor-bearing mice. (D) Isolated tumors from each group on day 21. Each value represents the mean ± SD (n=10 for each group). Scale bar represents 10 mm. **P<0.01, ***P<0.001 compared with the control group; #P<0.05, ##P<0.01, ###P<0.001 compared with the oral PMX group. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; IV, intravenous; LLC, Lewis lung carcinoma; PMX, pemetrexed; P188, poloxamer 188.

Figure 10 In vivo tumor growth inhibition efficacy in LLC tumor-bearing mice after treatment with once-weekly IV administration of PMX (20 mg/kg) and once-daily oral administration of PMX in aqueous solution (20 mg/kg) or HP-beta-CD/PMX/DCK/P188-NE (equivalent to 20 mg/kg PMX) for 21 days.Notes: (A) Tumor volume in mice. (B) Variation of body weight in mice during treatment. (C) Tumor weight in LLC tumor-bearing mice. (D) Isolated tumors from each group on day 21. Each value represents the mean ± SD (n=10 for each group). Scale bar represents 10 mm. **P<0.01, ***P<0.001 compared with the control group; #P<0.05, ##P<0.01, ###P<0.001 compared with the oral PMX group. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; IV, intravenous; LLC, Lewis lung carcinoma; PMX, pemetrexed; P188, poloxamer 188.

Figure 11 Representative cross-sectional images of isolated tumor tissues stained with (A) anti-CD31 antibody for microvessels (brown), (B) PCNA for proliferating cells (brown), and (C) TUNEL for apoptosis (green fluorescence) in the tumor tissues taken 21 days after treatment with once-weekly IV administration of PMX (20 mg/kg) and once-daily oral administration of PMX in aqueous solution (20 mg/kg) or HP-beta-CD/PMX/DCK/P188-NE (equivalent to 20 mg/kg PMX) for 21 days.

Notes: Scale bar represents 50 μm. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion. Magnification for images in (A) and (B) ×20. Magnification for images in (C) ×40.

Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; IV, intravenous; PCNA, proliferating cell nuclear antigen; PMX, pemetrexed; P188, poloxamer 188; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Figure 11 Representative cross-sectional images of isolated tumor tissues stained with (A) anti-CD31 antibody for microvessels (brown), (B) PCNA for proliferating cells (brown), and (C) TUNEL for apoptosis (green fluorescence) in the tumor tissues taken 21 days after treatment with once-weekly IV administration of PMX (20 mg/kg) and once-daily oral administration of PMX in aqueous solution (20 mg/kg) or HP-beta-CD/PMX/DCK/P188-NE (equivalent to 20 mg/kg PMX) for 21 days.Notes: Scale bar represents 50 μm. HP-beta-CD/PMX/DCK/P188, ion-pairing complex between PMX and DCK containing HP-beta-CD and P188; HP-beta-CD/PMX/DCK/P188-NE, HP-beta-CD/PMX/DCK/P188-loaded nanoemulsion. Magnification for images in (A) and (B) ×20. Magnification for images in (C) ×40.Abbreviations: DCK, Nα-deoxycholyl-l-lysyl-methylester; HP-beta-CD, 2-hydroxypropyl-beta-cyclodextrin; IV, intravenous; PCNA, proliferating cell nuclear antigen; PMX, pemetrexed; P188, poloxamer 188; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.