Differential uptake of DNA–poly(ethylenimine) polyplexes in cells cultured on collagen and fibronectin surfaces

Abstract

Genetically modified bone marrow derived mesenchymal stem cells (MSCs) have proven to be efficient cell carriers for local or systemic delivery of therapeutics as well as for growth factors to augment tissue formation. However, efficient non-viral gene transfer to these cells is limiting their applicability. Although most studies focus on designing more efficient condensation agents for DNA, our focus in this manuscript is to study the role of two extracellular matrix proteins on the ability of MSCs to become transfected, collagen I (Col I) and fibronectin (Fn). Here we report that plating MSCs on Col I-coated surfaces inhibits transfection, while plating MSCs on Fn-coated surfaces enhances transfection. The mechanism by which these ECM proteins affect non-viral gene transfer involves the endocytosis pathway used for polyplex uptake and intracellular tension. We found that Fn promoted internalization through clathrin-mediated endocytosis and that this pathway resulted in more efficient transfection than caveolae-mediated endocytosis and macropinocytosis. Further, the disruption of actin-myosin interactions resulted in an enhancement of gene transfer for cells plated on Fn coated surfaces, but not for cells plated on Col I. We believe that the cellular microenvironment can be engineered to enhance the ability of cells to become transfected and that through understanding the mechanisms by which the ECM affects non-viral gene transfer, better materials and transfection protocols can be realized.

Cellular Cytoskeleton Dynamics Modulates Non-Viral Gene Delivery through RhoGTPases

“…As mentioned we previously observed that gene transfer to mMSCs plated on collagen I was inhibited [6]. Thus, we investigated if the activation of RhoGTPases could rescue the transfection ability of mMSCs plated on collagen I.…”

“…While recent studies show that cellular microenvironment regulates gene delivery [4], [7], the mechanisms activated upon ECM binding that influence effective gene transfer have not been elucidated. Previously we found that mMSCs plated on fibronectin coated surfaces resulted in enhanced gene transfer, while cells plated on collagen I resulted in inhibited gene transfer compared to cells seeded on uncoated surfaces [6]. Here we aimed to determine if RhoGTPase activation was one of the reasons for the observed enhancement in transgene expression for cells plated on fibronectin-coated surfaces.…”

Utilizing Cell–Matrix Interactions To Modulate Gene Transfer to Stem Cells Inside Hyaluronic Acid Hydrogels

“…The lack of efficient gene transfer on stiff hydrogels is attributed to decreased migration rates, spreading, and actin polymerization, all of which have been implicated with enhanced transfection 28–31, 48 . Further, to achieve stiff hydrogels higher HA percentages were used to form the hydrogels, which decreased the pore size.…”

Dynamic analysis of DNA nanoparticle immobilization to model biomaterial substrates using combinatorial spectroscopic ellipsometry and quartz crystal microbalance with dissipation

“…SE/ QCM-D demonstrates the specific DNA-NP adsorption characteristics that are influenced by different substrates and by modifications to those substrates, which is an important consideration when optimizing SMD systems for nonviral gene delivery, since the transfection efficiency relies on DNA-NP loading and release to/from the substrate. Additionally, these SE/QCM-D studies suggest that while FBS may not be increasing the amount of adsorbed DNA to certain substrates, for example Au, FBS may be more important in facilitating favorable cell-material interactions for the endocytosis of immobilized DNA-NPs as suggested by previous studies [11,34]. This investigation demonstrates that SE/QCM-D can be used as a quick, reliable, and noninvasive tool to accurately monitor DNA-NP adsorption processes and to standardize or optimize substrate immobilization strategies in real time.…”