(178e) Investigating the Impact of Cell-Substrate Interactions on Nanoparticle Internalization

Nanoparticle delivery is crucial for a variety of biomedical applications such as drug delivery, nucleic acid delivery, diagnostic imaging, regenerative medicine, tissue engineering, and cancer treatment. The delivery of therapeutic nanoparticles from substrates localizes and controls delivery at the target site. This substrate-based delivery depends on cell-material interactions, which have been demonstrated to influence nanoparticle internalization efficiency [1]. The capacity of therapeutic nanoparticles to be endocytosed into cells when delivered from a substrate depends on substrate surface properties including mechanical properties [2]. However, the regulation of cellular responses to substrate mechanical properties involves integrin binding, focal adhesion complex formation, and cytoskeletal rearrangement, which are also involved in cell-extracellular matrix (ECM) interactions [3]. Thus, with the long-term goal of developing substrate-based therapeutic nanoparticle delivery systems, this study investigated the impact of cell-ECM interactions on nanoparticle internalization using ECM and ECM-mimetic surface coatings.

Material and Methods:

The effect of substrate coatings composed of Collagen I (Col I), fibronectin (FN), laminin, hyaluronic acid (HA), and poly-L-lysine (PLL) on cellular uptake of fluorescently-labeled poly(lactic-co-glycolic acid) (PLGA) nanoparticles was investigated in NIH3T3 fibroblasts, primary rat adipose-derived stem cells (ASCs), and RAW264.7 macrophages. Coatings were formed on glass (or silica for ellipsometry) substrates at a concentration of 100 mg/mL. Coating thickness, mechanical properties and morphology were analyzed via ellipsometry, quartz crystal microbalance with dissipation (QCMD) and atomic force microscopy. Cell adhesion and/or proliferation on coatings was analyzed via the live/dead assay and manual cell counting. Nanoparticle internalization was investigated via flow cytometry and fluorescence microscopy. Expression of adhesion and endocytosis related genes will be analyzed via qRT-PCR. Nanoparticle endocytosis pathways on coatings will be assessed via use of specific pathway inhibitors, including chlorpromazine, genistein, and cytochalasin D.

Results, Conclusions, and Discussion:

Preliminary data suggest that fibronectin coatings were the thickest and displayed the highest adhesion and/or proliferation of NIH3T3 and RAW264.7 cells, while HA coatings were the thinnest. The effects of ECM coatings on nanoparticle internalization are currently being studied via flow cytometry and fluorescence microscopy. Future work will examine the effect of coatings on specific endocytosis pathways as well as the effect of ECM coatings on nanoparticle surface adsorption, surface-based delivery, and cellular phenotype.

References

[1] S. hui Hsu, T. T. Ho, and T. C. Tseng, “Nanoparticle uptake and gene transfer efficiency for MSCs on chitosan and chitosan-hyaluronan substrates,” Biomaterials, vol. 33, no. 14, pp. 3639–3650, May 2012, doi: 10.1016/J.BIOMATERIALS.2012.02.005.

[2] C. Huang, P. J. Butler, S. Tong, H. S. Muddana, G. Bao, and S. Zhang, “Substrate stiffness regulates cellular uptake of nanoparticles,” Nano Lett., vol. 13, no. 4, pp. 1611–1615, Apr. 2013, doi: 10.1021/NL400033H.

[3] A. Dhaliwal, M. Maldonado, Z. Han, and T. Segura, “Differential uptake of DNA-poly(ethylenimine) polyplexes in cells cultured on collagen and fibronectin surfaces,” Acta Biomater., vol. 6, no. 9, pp. 3436–3447, 2010, doi: 10.1016/J.ACTBIO.2010.03.038.