Gene expression and internalization following vector adsorption to immobilized proteins: dependence on protein identity and density

Abstract

Background-Gene delivery by non-specific adsorption of non-viral vectors to protein-coated surfaces can reduce the amount of DNA required, and also increase transgene expression and the number of cells expressing the transgene. The protein on the surface mediates cell adhesion and vector immobilization, and functions to colocalize the two to enhance gene delivery. This report investigates the mechanism and specificity by which the protein coating enhances gene transfer, and determines if the protein coating targets the vector for internalization by a specific pathway.

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

“…In addition, when analyzing the influence of FBS protein coatings on substrate immobilization of DNA-NPs, the SE/QCM-D data suggest that DNA-NP immobilization efficiency is substrate dependent, since different DNA-NP immobilization trends are observed between different substrates coated with FBS ( Table 2). The two previous observations, together, suggest that although similar amounts of protein are adsorbing to the substrate with similar packing characteristics, different proteins within the FBS mixture, including different protein functional groups and binding motifs for DNA-NPs, could be presented on different substrates, as indicated by differences in detected DNA-NP quantities and porosities between the three substrates, as well as reported differences in transfection efficiencies [9,11,32]. Previous work regarding fibronectin protein adsorption to different SAM surfaces has shown that different surface characteristics, such as charge and hydrophobicity, influences the conformation of adsorbing proteins [33], which could influence subsequent adsorption of biomolecules, such as DNA-NPs, due to the exposure of different protein functional groups between surfaces.…”

“…For example, Bengali et al [9], reported that increasing concentrations of DNA-PEI NPs added to FBS-coated polystyrene surfaces did not influence the adsorbed mass of DNA, an observation that contradicts the trend from the SE/QCM-D results shown in Figure 1. However, SE/QCM-D provides a more precise perspective of DNA-NPs associated with a surface, since loosely bound, passively adsorbed DNA-NPs are also evaluated with SE/QCM-D. For previously reported radioactivity measurements, loosely bound DNANPs would have been washed off the surface during rinse steps before even conducting admeasurement, thus measuring only tightly bound DNA-NPs, which explains why QCM-D detects an increased mass of adsorbed and loosely associated DNA-NPs relative to previous studies [7][8][9]11]. Another important difference to note is that previous DNA radiolabeling studies reported the adsorbed mass of DNA instead of the entire DNA-NP complex since DNA was the labeled component that was measured.…”

“…Previous studies on substrate immobilization of DNA-NPs for SMD found that coating substrates with either extracellular matrix proteins or mixtures of serum proteins found in FBS can increase DNA-NP immobilization to polystyrene surfaces, and also increases the efficiency of DNA-NP delivery to cells [9,11]. In addition, studies have demonstrated that surface charge and presence of PEG groups on surfaces enhance DNA-NP immobilization and nonviral SMD of genes to cells [8].…”

“…In comparison to bolus methods, SMD is a particularly advantageous gene delivery technique, as immobilization of DNA to surfaces places it directly within the cell's microenvironment, therefore overcoming diffusion and mass transport limitations associated with trafficking of nonviral complexes to cells [9]. In addition, the immobilization of DNA complexes to a substrate enhances gene transfer, since surface immobilization of DNA-NPs has the ability to preserve NP size observed in solution and inhibit complex aggregation, while cytotoxicity is reduced because less DNA is required to achieve gene transfer [7][8][9][11][12][13]. Finally, SMD offers the ability to pattern the immobilization of nonviral complexes on surfaces, which can lead to patterned transgene expression, which is particularly pertinent to tissue engineering applications [7].…”

“…For example, previous work has investigated surface chemistry as a variable to improve SMD, employing self assembled monolayers (SAMs) of alkanethiols on gold to influence electrostatic interactions between the surface and DNA-NP complex [7,8]. In addition, the roles of nonspecific interactions in DNA-NP adsorption and enhanced transfection have been investigated on surfaces coated with extracellular matrix and serum proteins, such as fetal bovine serum (FBS) [9,11]. However, existing SMD studies focus primarily on substrate biocompatibility and the effectiveness of cellular transgene expression, where the dynamic processes involved in DNA-NP adsorption to substrates have not yet been thoroughly evaluated.…”