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Abstract
Background
Lung cancer is the most common cancer and the leading cause of total deaths worldwide. Its classified into two major types including non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC) based on the origin of abnormal lung cells as well as the smoking status of the patient. NSCLC is the most common and aggressive type of lung cancer representing 80%–85% of all cases.
Purpose
The aim of the study was to present lyotropic liquid crystalline nanoparticles (LCNPs) as promising carriers for co-delivery of the chemotherapeutic agent, pemetrexed (PMX) and the herbal drug, resveratrol (RSV) for effective lung cancer management.
Methods
The proposed PMX-RSV-LCNPs were prepared by hydrotrope method. Hydrophobic ion pairing with cetyl trimethyl ammonium bromide (CTAB) was implemented to increase the encapsulation efficiency of the hydrophilic PMX up to 95%±3.01%.
Results
The tailored PMX-RSV-LCNPs exhibited a particle size of 173±0.26 nm and biphasic release pattern with a relatively initial burst release within first 3–4 hour followed by sustained release up to 24 hours. Moreover, PMX-RSV-LCNPs manifested superior concentration and time dependent cytotoxicity profile against A549 lung cancer cells with IC50 4.0628 µg/mL. Besides, the enhanced cellular uptake profile based on bioadhesive properties of glyceryl monoolein (GMO) as well as energy independent (cholesterol dependent) pattern. In-vivo evaluations against urethane induced lung cancer bearing mice demonstrated the potentiality of PMX-RSV-LCNPs in tumor growth inhibition via inhibition of angiogenesis and induction of apoptosis. The results were supported by histopathological analysis and immunohistochemical Ki67 staining. Moreover, PMX-RSV-LCNPs displayed a promising safety profile via attenuating nephro- and hepatotoxicity.
Conclusion
PMX-RSV-LCNPs elaborated in the current study hold a great promise for lung cancer treatment.
Supplementary materials
Methodology
HPLC method for the assay of pemetrexed (PMX) and resveratrol (RSV)
A novel, accurate, precise, and sensitive HPLC method was developed and validated for simultaneous determination of both RSV and PMX using Agilent 1260 Infinity HPLC system equipped with a quaternary pump, an autosampler, vacuum degasser, diode array detector, and Agilent Chemstation data-processing system. The HPLC analysis was carried out with an Inertsil® ODS-3 reversed-phase column (250 × 4.6 mm, 5 µm; GL Sciences Inc, Torrance, CA, USA). The column was maintained at room temperature. For chromatographic elution, the injection volume was 20 µL. A step gradient was utilized for elution in which the mobile phase consisted of acetonitrile: dibasic phosphate buffer (pH adjusted to 4.9 using orthophosphoric acid) with the following ratio 15:85 to 6 minutes then converted into 50:50 gradually over 0.5 minutes, and the flow rate was 1.5 mL/min. Total run time was 10 minutes; PMX was eluted at 4.8 minutes and RSV at 9.8 minutes. PMX was detected at 225 nm, while RSV was detected at 306 nm.
Evaluation of in vitro cytotoxicity
In vitro cytotoxicity of free PMX, free RSV, free combined PMX/RSV solution, blank liquid crystalline nanoparticles (LCNPs), and ion-paired PMX-RSV-LCNPs was evaluated in A549 lung cancer cells using MTT assay.Citation1 Briefly, 1,000–5,000 cells/well were seeded in 96-well culture plates containing 100 µL of cell line-specific medium and incubated at 37°C in a 5% CO2 atmosphere. The cells were then treated with each formulation at concentrations ranged from 0.468 to 30 µg/mL of each PMX and/or RSV and incubated at 37°C for 24 and 48 hours. Then, cells were subjected to the MTT analysis for cell viability determination. The optic density was measured at 490 nm on a microplate reader with control wells containing only cell culture medium. The viability data were analyzed in GraphPad Prism, and the IC50 value for each system was calculated. Each IC50 value was calculated from n=6 viability measurements per concentration. IC50 was calculated after 24 and 48 hours.
In vitro cellular uptake
A549 lung cells were seeded on CELL view™ slide at a density of 3,000 cells/well and left overnight to allow enough time for cell attachment. Cells were incubated with fresh medium containing free coumarin-6 dye and coumarin-6-loaded LCNPs at the concentration of 0.08% w/w coumarin-6 (with respect to lipid). After incubation for 4 and 24 hours, the medium was removed and cells were rinsed twice using PBS to eliminate any extracellular residues. Then, cells were fixed using 4% paraformaldehyde solution in dark. The nuclei were stained using 2-(4-ethoxyphenyl)-6-[6-(4-methylpiperazin-1-yl)-1H-benzimidazol-2-yl]-1H-benzimidazole (Hoechst AG, Frankfurt, Germany) containing DPX (DePeX [Distrene 80: A commercial polystyrene, a plasticizer, e.g., dibutyl phthalate and xylene]) as mounting medium. Finally, the intracellular uptake of optimized LCNPs into A549 cell line was visualized via confocal laser scanning microscopy (DMi8; Leica Microsystems, Wetzlar, Germany) using the following filter set: excitation wavelength, 450 nm; emission wavelength, 505 nm. All obtained confocal images were analyzed using Leica AF software. Moreover, the internalization of particles within each section of the cells at various cell depths was visualized using three-dimensional cellular images per time (z-stacks), where serial sections were taken across 30 fields and stacked with cross-sectional slices (25 µm) perpendicular to the plane of the cell monolayer midpoint (z-axis).Citation2,Citation3
Results and discussions
HPLC method for the assay of PMX and RSV
Several attempts were carried out for simultaneous quantitation of PMX and RSV in mixture. Isocratic elution using different proportions of mobile phases incluwding methanol, acetonitrile, acidified water, and buffer at different ratios was not enough to provide satisfactory separation for the mixture’s components. Therefore, gradient elution was applied to separate peaks of both drugs.Citation4 In the literature, various methods were reported for individual quantitation of PMX and RSV in formulations and biological fluids.Citation4–Citation8 The optimized mobile phase composed of HPLC grade acetonitrile: dibasic phosphate buffer (pH adjusted to 4.9 using orthophosphoric acid) with the following ratio 15:85 to 6 minutes and then converted into 50:50 gradually over 0.5 minutes; flow rate was 1.5 mL/min. A rapid, sensitive, simple, and efficient method was developed for simultaneous determination of RSV and PMX in stock solution and nanoformulations. The principal peak obtained with the standard solution was at 225 nm which was selected as the λmax of the PMX, whereas the major peak of RSV was detected at λmax 306 nm. A good linearity was shown by the calibration curve obtained from 0.4 to 2 mg% PMX and RSV concentration (). The coefficient of determination (R2) of 0.9997 was obtained from the linear regression analysis of the data (peak area vs concentration). Selected chromatograms were displayed (), in which retention time of PMX was 4.427 minutes and of RSV was 11.87 minutes so that the total run time was 12 minutes.
As summarized in , the HPLC method was validated according to International Conference on Harmonization (IHC) guidelines for intra- and inter-day precision and accuracy. The low values of relative standard deviation (RSD) as well as % error of mean <2 confirmed the precision and accuracy of the developed HPLC method. Limit of detection was 0.2 mg%, while the limit of quantitation was 0.4 mg%. The proposed assays were also successfully applied to evaluate the entrapment efficiency (EE) of the novel LCNPs for the co-delivery of PMX and RSV for lung cancer.
Figure S1 HPLC calibration curve of PMX and RSV in methanol at λmax 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 λmax.
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)
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Acknowledgments
This work was supported by the research grant (No 5731) from Science and Technology Development Fund (STDF), Ministry of Scientific Research, Egypt.
Disclosure
The authors report no conflicts of interest in this work.