Selective Targeting of Lung Cancer Cells with Methylparaben-Tethered-Quinidine Cocrystals in 3D Spheroid Models

Abstract

The development and design of pharmaceutical cocrystals for various biological applications has garnered significant interest. In this study, we have established methodologies for the growth of the methylparaben-quinidine cocrystal (MP-QU), which exhibits a well-defined order that favors structure-property correlation. To confirm the cocrystal formation, we subjected the cocrystals to various physicochemical analyses such as powder X-ray diffraction (PXRD), single-crystal X-ray diffraction (SCXRD), Raman, and IR spectroscopy. The results of the XRD pattern comparisons indicated no polymorphisms, and density functional theory (DFT) studies in both gaseous and liquid phases revealed enhanced stability. Our in silico docking studies demonstrated the cocrystal's high-affinity binding towards cancer-specific epidermal growth factor receptor (EGFR), Janus kinase (JAK), and other receptors. Furthermore, in vitro testing against three-dimensional (3D) spheroids of lung cancer (A549) and normal fibroblast cells (L929) demonstrated the cocrystal's higher anticancer potential, supported by cell viability measurements and live/dead assays. Interestingly, the cocrystal showed selectivity between cancerous and normal 3D spheroids. We found that the MP-QU cocrystal inhibited migration and invadopodia formation of cancer spheroids in a favorable 3D microenvironment.

Figure 1

Photograph of (a) MP–QU cocrystals and (b) BFDH morphology diagram of the MP–QU cocrystal.

Figure 2

Optimized structure of a MP–QU cocrystal in gas phase. The O–H···N intermolecular interaction is shown as a dotted line with a bond length in Å.

Figure 3

(Left) FT-IR and (right) Raman spectra of MP–QU cocrystals in comparison with their starting materials.

Figure 4

DSC thermogram of the grown MP–QU cocrystal.

Figure 5

Binding interaction of MP–QU with (a) 5D41, (b) 2ITO, (c) 4F09, (d) 3FUP, and (e) 5XRA protein.

Figure 6

Cell viability on adherent format upon the MP–QU cocrystal treatments for 48 h. (a) A549 lung cancer cells and (b) L929 normal connective tissue/fibroblast cells (*indicates p-value < 0.05 and ** indicates p-value < 0.01).

Figure 7

Cell viability of 3D spheroids upon MP–QU cocrystal treatments on day 01 and day 02. (a) A549 lung cancer cell spheroid and (b) L929 normal connective tissue/fibroblast cell spheroids (* indicates p-value < 0.05 and ** indicates p-value < 0.01).

Figure 8

Live/dead staining of 2D adherent cells and 3D spheroids: Treatments with the MP–QU cocrystal with A549 cells and L929 in 2D adherent and 3D spheroids.

Figure 9

(A) Spheroid invasion assay: control A549 spheroids (a and d) at 0 and 48 h showing an increase in area with a significant number of invadopodia and A549 spheroid under cocrystal treatment. (B) Quantified surface areas of the control and cocrystal-treated spheroids.

Figure 10

(A) Cocrystal (10 μg/mL)-treated spheroid completely died in 24 h and lost its integrity, observed with an increase in the surface area and (B) control spheroid (representative image) after 48 h showed multiple invadopodia formations (cyan arrows) from the periphery of the spheroid.