Biomimetic polycaprolactone-chitosan nanofibrous substrate influenced cell cycle and ECM secretion affect cellular uptake of nanoclusters

Abstract

Biomimetic cell culture substrates are developed as an alternative to the conventional substrates. They provide necessary biochemical and biophysical cues to the cells from their surrounding environment for their optimal growth, behaviour and physiology. Changes in physiology of cells growing on biomimetic substrate can essentially affect results of in vitro biological experiments such as drug cytotoxicity, nanoparticle internalization or signalling pathways. As majority of ECM proteins are fibrous in nature, nanofibrous scaffolds have more biomimicking properties. Therefore, in this study, we developed ECM mimicking polycaprolactone-chitosan nanofiber substrate and evaluated its effect on cell morphology, proliferation, cell cycle and ECM production. Further, cellular uptake of BSA-AuNCs has been assessed on conventional and biomimetic substrate in order to demonstrate the effect of these events on cellular properties. It was observed that the cells that were grown for 15 days on the nanofibers, had majority of cells in the proliferative phase of cell cycle compared to TCPS. Moreover, these cells showed extensive collagen and fibronectin production. Due to these conditions C3H10T1/2 cells displayed higher cell internalization of BSA-AuNCs. Overall, this study indicates that the nano-topographical and biochemical environment could alter the cell proliferative behaviour and ECM production, which affects the cell internalization of BSA-AuNCs. Also, PCL-chitosan nanofibrous substrate could be a better alternative to TCPS for cell culture studies.

Keywords: BSA-AuNCs internalization; Biomimetic substrate; Cell cycle; ECM production; Polycaprolactone-chitosan nanofibers.

Graphical abstract

Fig. 1

SEM of PCL-CHT nanofibers show the morphology and random distribution of fibers.

Fig. 2

Characterization of BSA-AuNCs. (A) UV–Visible spectra measured from BSA solution (black curve), BSA-AuNCs (No incubation-blue curve), BSA-AuNCs (overnight incubation-red curve). Inset images demonstrate the color of BSA-AuNCs before and after UV light exposure (280 nm). (B) Excitation and emission spectra of BSA-AuNCs. (C) The representative transmission electron micrograph of BSA-AuNCs. Inset: Mean particle size distribution of BSA-AuNCs. (D) FTIR spectra of BSA and BSA-AuNCs showing specific transmission bands for amide – I, II and III.

Fig. 3

Scanning electron micrographs of C3H10T1/2 cells grown on nanofibers. White arrow shows the nanofibrous mat (a) on which cells (shown by yellow arrows) are grown. The interaction between two adjacent cells can also be observed (b). After 14 days, the cells had started producing their own extracellular matrix (c), shown by blue arrows.

Fig. 4

Confocal microscopic images of cells grown on nanofibrous scaffold after 7 days. Figures (a) and (b) are cells stained with DAPI and Phalloidin-Alexa fluor 488, respectively. Figure (c) is the merged image of both a and b.

Fig. 5

Alamar Blue assay showing cell proliferation of C3H10T1/2 cells grown on nanofibers (grey) and TCPS (black) up to 15 days.

Fig. 6

Cell cycle analysis of C3H10T1/2 cells grown on TCPS and nanofibers for 7 days. (a) shows the representative DNA histograms, (b) shows percentage of cells residing in G0/G1, S and G2/M phases, along with proliferation index (PI) and (c) shows the quantitative expression of the same.

Fig. 7

Immunostaining of ECM proteins fibronectin, collagen IV and laminin produced by C3H10T1/2 cells grown on TCPS and nanofibers for 7 and 14 days. (a–p) are confocal microscopic images of same (Scale bar represents 50 μm); whereas (q, r) are quantitative measurement of fibronectin and collagen production, respectively. p value < 0.05.

Fig. 8

Fluorescence microscopy images of cells treated with BSA-AuNCs on TCPS (a) and nanofibers (b) (Images were captured at 20X magnification); whereas (c) is the quantitative measurement of the BSA-AuNCs internalization by cells grown on TCPS and nanofibers, respectively. p value < 0.05.