Liquid crystalline assembly for potential combinatorial chemo–herbal drug delivery to lung cancer cells

Figures & data

Table 1 Composition and physicochemical characterization of placebo and PMX-RSV-loaded LCNPs

Formula	Hydrotrope (% w/v)	P407^a (% w/v)	PMX (mg)	RSV (mg)	Size (nm)	PDI	Zeta potential (mV)	RSV %EE	PMX %EE
F1a	1.72	0.5	–	–	190±0.83	0.35±0.01	−30.42±2.91
F2a	1.76	0.5	–	–	220±0.91	0.42±0.04	−32.01±3.21
F3	1.75	0.5	–	–	167±0.25	0.22±0.01	−34.85±2.03
F4	1.25	0.5	–	–	190±1.50	0.26±0.01	−32.45±2.94
F5	2.5	0.5	–	–	155±0.93	0.18±0.05	−31.69±1.57
F6	1.75	0.25	–	–	218±1.02	0.35±0.01	−31.44±2.03
F7	1.75	1	–	–	150±1.22	0.14±0.01	−33.91±3.12
F8	1.75	0.5	10	10	168±0.51	0.22±0.01	−39.34±1.15	90%±2.03%	50%±1.65%
F9b	1.75	0.5	10	10	176±0.16	0.20±0.01	+48.05±3.12	98%±2.01%	80%±1.01%
F10b	1.75	0.5	10	10	173±0.26	0.19±0.01	+58.03±1.12	98%±2.01%	95%±3.01%

Notes: All formulations were prepared using GMO 2.5% (w/v) with respect to total dispersion volume.

a F1 and F2 contain propylene glycol and PEG 400, respectively.

b F9 and F10 contain ion-paired PMX at molar ratio of PMX:CTAB (1:2 and 1:4, respectively).

Abbreviations: CTAB, cetyltrimethylammonium bromide; %EE, % entrapment efficiency; GMO, glyceryl monooleate; LCNPs, liquid crystalline nanoparticles; PDI, polydispersity index; PMX, pemetrexed; P407, poloxamer-407; RSV, resveratrol.

Figure 1 Physicochemical characterization of optimized LCNPs.

Notes: (A) Transmission electron micrograph displaying morphology of ion-paired PMX-RSV-LCNPs (F10). (B) In vitro release of PMX and RSV from PMX-RSV-LCNPs (F8) and ion-paired PMX-RSV-LCNPs (F10) in comparison to free PMX and RSV solution in PBS (pH 7.4) at 100 rpm and 37°C using the dialysis bag diffusion method. (C) The influence of different CTAB:PMX molar ratios on the release of PMX from ion-paired LCNPs in PBS (pH 7.4) at 100 rpm and 37°C using the dialysis bag diffusion method.

Abbreviations: CTAB, cetyltrimethylammonium bromide; LCNPs, liquid crystalline nanoparticles; PMX, pemetrexed; RSV, resveratrol.

Table 2 IC₅₀ of free drugs and optimized LCNPs after 24 and 48 hours using Compusyn^® software

IC50 (µg/mL)	Free PMX	Free RSV	Free RSV/PMX	Ion-paired PMX-RSV-LCNPs
After 24 hours	10.10	12.66	7.96	5.50
After 48 hours	7.94	8.43	4.51	4.0628

Abbreviations: LCNPs, liquid crystalline nanoparticles; PMX, pemetrexed; RSV, resveratrol.

Figure 2 In vitro cytotoxicity of ion-paired PMX-RSV-LCNPs (F10) compared to blank LCNPs, free PMX, free RSV, and free PMX/RSV cosolvent on A549 NSCLC cell line at the concentration of 0.468–30 µg/mL expressed as % cell viability after (A) 24 hours and (B) 48 hours.

Abbreviations: LCNPs, liquid crystalline nanoparticles; NSCLC, non-small-cell lung cancer; PMX, pemetrexed; RSV, resveratrol.

Figure 3 Confocal microscopy images demonstrating cellular uptake of coumarin-6-loaded LCNPs relative to free coumarin-6 and control cells after incubation with A549 lung cancer cell line for (A) 4 hours and (B) 24 hours in which the first channel of nuclei is represented by blue stain of Hoechst, the second channel of free coumarin-6 or coumarin-6-loaded LCNPs is represented by green fluorescence, third channel of merge is represented by overlay between two channels, and finally the fourth channel of DIC is represented by the light microscopic image of A549 cells.

Abbreviations: LCNPs, liquid crystalline nanoparticles; DIC, differential interference contrast.

Figure 4 In vivo antitumor activity.

Notes: (A) Average weight of excised lungs of mice measured at the end of treatment course. (B) Representative images of excised lung tumors displaying differences among mice groups after 3 weeks of exposing to different treatments; (a) negative control, (b) positive control, (c) free PMX-RSV mixture, and (d) ion-paired PMX-RSV-LCNPs (F10). The level of the tumor biomarkers measured using ELISA in tumor homogenate including (C) VEGF and (D) CASP-3 in urethane-induced lung cancer-bearing mice treated with free PMX-RSV and ion-paired PMX-RSV-LCNPs (F10) compared to untreated positive control. *P<0.05 vs negative control, ^#P<0.05 vs positive control, ^%P<0.05 vs free PMX/RSV mixture.

Abbreviations: CASP-3, caspase 3; LCNPs, liquid crystalline nanoparticles; PMX, pemetrexed; RSV, resveratrol; VEGF, vascular endothelial growth factor.

Figure 5 (A) Histopathological analysis of H&E-stained excised lung sections of mice treated with free PMX-RSV and ion-paired PMX-RSV-LCNPs (F10) in comparison to positive control and negative control groups. (B) Average number of foci in the excised lungs, (C) The diameter of lung foci (mm).

Notes: ^#P<0.05 vs positive control, ^%P<0.05 vs Free PMX/RSV mixture.

Abbreviations: LCNPs, liquid crystalline nanoparticles; PMX, pemetrexed; RSV, resveratrol.

Figure 6 Immunohistochemical analysis: (A) Ki-67 staining. (B) % expression of Ki-67 of excised lung sections of urethane-induced lung cancer-bearing mice received free PMX-RSV and ion-paired PMX-RSV-LCNPs (F10) in comparison to untreated positive control.

Note: *P<0.05 vs negative control, ^#P<0.05 vs positive control, ^%P<0.05 vs free PMX-RSV mixture.

Abbreviations: LCNPs, liquid crystalline nanoparticles; PMX, pemetrexed; RSV, resveratrol.

Figure 7 Assessment of safety index of optimized LCNPs.

Notes: (A) Average body weights of urethane-induced lung cancer-bearing mice treated with free PMX-RSV and ion-paired PMX-RSV-LCNPs (F10) for 3 weeks compared to untreated positive control. (B) Survival rate curve among treated groups in comparison to positive and negative control groups. H&E-stained kidney (C) and liver (D) of different treatment groups.

Abbreviations: IV, intravenous; LCNPs, liquid crystalline nanoparticles; PMX, pemetrexed; RSV, resveratrol.

Figure S1 HPLC calibration curve of PMX and RSV in methanol at λ_max 225 and 306 nm, respectively.

Abbreviations: PMX, pemetrexed; RSV, resveratrol.

Figure S2 Selected chromatogram of (A) PMX and (B) RSV displaying retention time at their corresponding λ_max.

Abbreviations: PMX, pemetrexed; RSV, resveratrol.

Figure S3 Live uptake of ion-paired LCNPs in A549 cells in which three-dimensional time laps images distinguishing between cystolic and perinuclear accumulation of 178 nm LCNPs of 30 z-stacks inside the cells in the z-direction along the total depth measured (z-stack =30 slices at 25 µm per slices).

Note: The individual stacks were assembled as video displaying the oscillation of green fluorescence along the cross-sectional internalization of LCNPs from the apical to basolateral cellular membrane represented from z-0 to z-29.

Abbreviation: LCNPs, liquid crystalline nanoparticles.

Table S1 Intra- and inter-day precision and accuracy of PMX and RSV (n=5 for each concentration)

Concentration (mg%)	Drug	Intra-day (n=5)			Inter-day (n=5)
		Mean ± SD	RSD	% error	Mean ± SD	RSD	% error
0.6	PMX	0.611±0.001	0.262	1.940	0.604±0.011	1.903	0.733
	RSV	0.611±0.011	1.864	1.916	0.588±0.003	0.619	−1.933
0.8	PMX	0.801±0.001	0.127	0.140	0.805±0.012	1.600	0.625
	RSV	0.787±0.016	2.084	−1.507	0.790±0.012	1.553	−1.201
1	PMX	0.999±0.001	0.154	−0.043	0.998±0.004	0.459	−0.201
	RSV	0.996±0.018	1.823	−0.401	0.990±0.015	1.613	−0.980
1.8	PMX	1.797±0.001	0.080	−0.133	1.814±0.017	0.989	0.777
	RSV	1.806±0.020	1.107	0.350	1.799±0.017	0.957	−0.055
2	PMX	1.999±0.001	0.052	−0.007	1.974±0.035	1.776	−1.301
	RSV	1.994±0.019	0.994	−0.270	1.999±0.009	0.463	−0.050

Abbreviations: PMX, pemetrexed; RSV, resveratrol.