Screening a chemically defined extracellular matrix mimetic substrate library to identify substrates that enhance substrate-mediated transfection

Abstract

Nonviral gene delivery, though limited by inefficiency, has extensive utility in cell therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery (SMD) increases efficiency and allows transfection at a cell-biomaterial interface, by immobilizing and concentrating nucleic acid complexes on a surface. Efficient SMD generally requires substrates to be coated with serum or other protein coatings to mediate nucleic acid complex immobilization, as well as cell adhesion and growth; however, this strategy limits reproducibility and may be difficult to translate for clinical applications. As an alternative, we screened a chemically defined combinatorial library of 20 different extracellular matrix mimetic substrates containing combinations of (1) different sulfated polysaccharides that are essential extracellular matrix glycosaminoglycans (GAGs), with (2) mimetic peptides derived from adhesion proteins, growth factors, and cell-penetrating domains, for use as SMD coatings. We identified optimal substrates for DNA lipoplex and polyplex SMD transfection of fibroblasts and human mesenchymal stem cells. Optimal extracellular matrix mimetic substrates varied between cell type, donor source, and transfection reagent, but typically contained Heparin GAG and an adhesion peptide. Multiple substrates significantly increased transgene expression (i.e. 2- to 20-fold) over standard protein coatings. Considering previous research of similar ligands, we hypothesize extracellular matrix mimetic substrates modulate cell adhesion, proliferation, and survival, as well as plasmid internalization and trafficking. Our results demonstrate the utility of screening combinatorial extracellular matrix mimetic substrates for optimal SMD transfection towards application- and patient-specific technologies.

Impact statement: Substrate-mediated gene delivery (SMD) approaches have potential for modification of cells in applications where a cell-material interface exists. Conventional SMD uses ill-defined serum or protein coatings to facilitate immobilization of nucleic acid complexes, cell attachment, and subsequent transfection, which limits reproducibility and clinical utility. As an alternative, we screened a defined library of extracellular matrix mimetic substrates containing combinations of different glycosaminoglycans and bioactive peptides to identify optimal substrates for SMD transfection of fibroblasts and human mesenchymal stem cells. This strategy could be utilized to develop substrates for specific SMD applications in which variability exists between different cell types and patient samples.

Keywords: Mimetic peptides; extracellular matrix; glycosaminoglycans; human mesenchymal stem cells; substrate-mediated gene delivery; transfection.

Figure 1.

ECM mimetic substrate library formulation. One of five different glycosaminoglycans (GAGs) was combined with one or more peptide conjugates in well plates and incubated for 12 h before drying to form biomatrices for SMD transfection experiments. (A color version of this figure is available in the online journal.)

Figure 2.

ECM mimetic substrates enhance LF2K SMD transfection of NIH/3T3 murine fibroblasts. pEGFP-Luc was complexed with LF2K and adsorbed to substrates prior to seeding of NIH/3T3 cells. Cells were assayed 24 h after SMD transfection. (a) Hep + RGD and Hep + RGD + R9 substrates significantly increased cell luciferase transgene expression, as measured by relative light units per mg of protein (RLU/mg Protein), and (b) Hep + RGD significantly increased the proportion of cells positive for EGFP fluorescence (transfection efficiency). (c) Cell count and (d) cellular metabolic activity was not significantly affected by substrate. All data from three independent experiments run on different days in triplicate was normalized to FBS conditions and graphed (n = 9). Asterisks (*) denote significance to FBS conditions (*P ≤ 0.05; **P ≤ 0.01). (A color version of this figure is available in the online journal.)

Figure 3.

ECM mimetic substrates enhance PEI SMD transfection of NIH/3T3s. pEGFP-Luc was complexed with PEI and adsorbed to substrates prior to seeding of NIH/3T3 cells. Cells were assayed 24 h after SMD transfection. (a) Hep + RGD + F2A and Hep + AG73 + F2A substrates significantly increased cell luciferase transgene expression, as measured by relative light units per mg of protein (RLU/mg Protein), and (b) Hep + RGD + F2A significantly increased the proportion of cells positive for EGFP fluorescence (transfection efficiency). (c) Cell count was significantly increased by seven different ECM mimetic substrates, and (d) cellular metabolic activity was significantly increased by Dext+RGD substrates. All data from three independent experiments run on different days in triplicate were normalized to FBS condition and graphed (n = 9). Asterisks (*) denote significance to FBS conditions (*P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001). (A color version of this figure is available in the online journal.)

Figure 4.

ECM mimetic substrates do not increase pDNA complex immobilization. pDNA complex adsorption to substrates was quantified by measuring radiolabeled pDNA. Substrates did not significantly affect the amount of pDNA complexes adsorbed, relative to FBS controls, for (a) LF2K complexes, (b) PEI complexes, or (c) LF3K complexes (P > 0.05) (n = 3). A total of 2.0 µg of complexed pDNA was added to each well, in 48-well plates. (A color version of this figure is available in the online journal.)

Figure 5.

Normalized results of all hAMSC donors SMD transfected on different ECM mimetic substrates. Heat map represents means of (a) transgenic luciferase expression, as measured by relative light units per mg of protein (RLU/mg Protein), and (b) total cell count, for each hAMSC donor on all ECM mimetic substrates with immobilized pDNA LF3K complexes, normalized to FBS-coated control substrates, 48 h after seeding cells. (A color version of this figure is available in the online journal.)