Substrate-mediated Gene Delivery Research Articles

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

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 statementSubstrate-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.

Free Polyethylenimine Enhances Substrate-Mediated Gene Delivery on Titanium Substrates Modified With RGD-Functionalized Poly(acrylic acid) Brushes.

Substrate mediated gene delivery (SMD) is a method of immobilizing DNA complexes to a substrate via covalent attachment or nonspecific adsorption, which allows for increased transgene expression with less DNA compared to traditional bolus delivery. It may also increase cells receptivity to transfection via cell-material interactions. Substrate modifications with poly(acrylic) acid (PAA) brushes may improve SMD by enhancing substrate interactions with DNA complexes via tailored surface chemistry and increasing cellular adhesion via moieties covalently bound to the brushes. Previously, we described a simple method to graft PAA brushes to Ti and further demonstrated conjugation of cell adhesion peptides (i.e., RGD) to the PAA brushes to improve biocompatibility. The objective of this work was to investigate the ability of Ti substrates modified with PAA-RGD brushes (PAA-RGD) to immobilize complexes composed of branched polyethyleneimine and DNA plasmids (bPEI-DNA) and support SMD in NIH/3T3 fibroblasts. Transfection in NIH/3T3 cells cultured on bPEI-DNA complexes immobilized onto PAA-RGD substrates was measured and compared to transfection in cells cultured on control surfaces with immobilized complexes including Flat Ti, PAA brushes modified with a control peptide (RGE), and unmodified PAA. Transfection was two-fold higher in cells cultured on PAA-RGD compared to those cultured on all control substrates. While DNA immobilization measured with radiolabeled DNA indicated that all substrates (PAA-RGD, unmodified PAA, Flat Ti) contained nearly equivalent amounts of loaded DNA, ellipsometric measurements showed that more total mass (i.e., DNA and bPEI, both complexed and free) was immobilized to PAA and PAA-RGD compared to Flat Ti. The increase in adsorbed mass may be attributed to free bPEI, which has been shown to improve transfection. Further transfection investigations showed that removing free bPEI from the immobilized complexes decreased SMD transfection and negated any differences in transfection success between cells cultured on PAA-RGD and on control substrates, suggesting that free bPEI may be beneficial for SMD in cells cultured on bPEI-DNA complexes immobilized on PAA-RGD grafted to Ti. This work demonstrates that substrate modification with PAA-RGD is a feasible method to enhance SMD outcomes on Ti and may be used for future applications such as tissue engineering, gene therapy, and diagnostics.

Reverse transduction can improve efficiency of AAV vectors in transduction-resistant cells.

Reverse transduction, also known as substrate-mediated gene delivery, is a strategy in which viral vectors are first coated onto a surface that subsequently comes into contact with mammalian cells. The cells internalize the surface-attached vectors, resulting in transgene expression. We hypothesized that forcing the interaction between cells and adeno-associated virus (AAV) vectors through a reverse transduction format would increase in vitro gene delivery efficiencies of the vectors in transduction-resistant cells. We tested this hypothesis by comparing the gene delivery efficiencies of three AAV serotypes using either standard or reverse transduction approaches. Our study reveals reverse transduction of AAV7 and AAV9 can significantly improve their delivery efficiencies. In contrast, AAV2 does not perform better under the reverse transduction format. Interestingly, increased vector uptake by cells does not provide a complete explanation for the increased transduction efficiency. Our findings offer a simple and practical method for improving transduction outcomes in vitro in cell types less permissive to a particular AAV vector.

Gene immobilization on alginate/polycaprolactone fibers through electrophoretic deposition to promote in situ transfection efficiency and biocompatibility

Alginate and polycaprolactone (PCL) were coelectrospun as composite nanofibers for in situ transfection, in which anionic alginate fibers were used to adsorb polyethyleneimine (PEI)/DNA polyplexes and biocompatible PCL fibers were applied to promote cell adhesion. To improve gene immobilization, direct-current electric field (DCEF) was applied to guide cationic polyplexes toward nanofibers on cathode. Fluorescent labeling experiments suggested that the applied DCEF not only accelerated but also increased the saturation levels of gene immobilization. Interestingly, these DCEF also increased the degradation of nanofibers. The water contact angle and Fourier-transform infrared spectrometry results indicated that the degraded component was mainly alginate. It suggested that the DCEF treatment may cause the electrophoresis of calcium ions to destabilize alginates fibers, and thus the degradation rates increased with the applied voltages. This alginate degradation increased the ratio of PCL in composite fibers, so the cell adhesion, viability, and proliferation were improved. Finally, these DCEF-treated fibers were used for substrate-mediated gene delivery. The transfection efficiency highly increased with DCEF when the voltages were lower than 1.5 V. This dynamic scaffold system not only provided a suitable microenvironment for cell ingrowth, but also improved gene immobilization and transfection, and thus promised its therapeutic effect for tissue regeneration.

The control of alginate degradation to dynamically manipulate scaffold composition for in situ transfection application

In this study, nanofibrous scaffolds were used for in situ transfection application. Polyethylenimine (PEI)/DNA complexes adsorbed to alginate nanofibers, so the more alginate fibers resulted in the higher transfection efficiency. However, alginate was not favorable for cell adhesion. Therefore, poly (ε‑caprolactone) (PCL) nanofibers were electrospun with alginate to improve biocompatibility. The in situ transfection results demonstrated that although the incorporated PCL fibers effectively improved cell morphology, the bioactivity and proliferation rates of surface cells were not significantly increased due to the high ratio of alginate fibers. However, the reduction of the alginate ratio may decrease transfection efficiency because the immobilization of nonviral vectors linearly depended on the density of alginate fibers. To maintain transfection efficiency and increase biocompatibility, the stability of alginate fibers were manipulated by adjusting the concentrations of calcium ions during crosslinking. These partially crosslinked alginate fibers were initially intact to allow nanoparticle adsorption for cell uptake, and then gradually degraded in days to create an appropriate environment for cell survival. This dynamic system successfully fulfilled the requirements of both gene delivery and biocompatibility. To our knowledge, this study may be the first one which dynamically regulates scaffold composition for substrate-mediated gene delivery.

Nanostructured Lipid-based Films for Substrate Mediated Applications in Biotechnology.

Amphiphilic in nature, lipids spontaneously self-assemble into a range of nanostructures in the presence of water. Among lipid self-assembled structures, liposomes and supported lipid bilayers have long held scientific interest for their main applications in drug delivery and plasma membrane models, respectively. In contrast, lipid-based multi-layered membranes on solid supports only recently begun drawing scientists' attention. New studies on lipid films show that the stacking of multiple bilayers on a solid support yields interestingly complex features to these systems. Namely, multiple layers exhibit cooperative structural and dynamic behavior. In addition, the materials enable compartmentalization, templating, and enhanced release of several molecules of interest. Importantly, supported lipid phases exhibit long-range periodic nano-scale order and orientation that is tunable in response to a changing environment. Herein, we summarize current and pertinent understanding of lipid-based film research focusing on how unique structural characteristics enable the emergence of new applications in biotechnology including label-free biosensors, macroscale drug delivery, and substrate-mediated gene delivery. Our very recent contributions to lipid-based films, focusing on the structural characterization at the meso, nano, and molecular-scale, using Small-Angle X-ray Scattering, Atomic Force Microscopy, Photothermal Induced Resonance, and Solid-State NMR will be also highlighted.

Correlating cell transfectability and motility on materials with different physico-chemical properties

Gene delivery into cells can be facilitated by adding plasmid DNA/transfection reagent complexes in culture medium or pre-adsorbing the complexes on the substrate before cell seeding. Using transfection reagents, however, often causes cytotoxicity. Effective delivery of naked plasmid without any transfection reagent remains a challenge. In this study, we cultured human umbilical cord derived mesenchymal stem cells (hMSCs) on different biomaterial substrates with different physico-chemical properties and examined the transfectability of naked plasmid. Specifically, we synthesized a negatively charged polyurethane (PU) to mimic the hyaluronan-modified chitosan (CS-HA) membranes previously found to promote the transfection of naked plasmid. We observed that the PU membranes were as effective as CS-HA membranes in substrate-mediated delivery of naked plasmid into hMSCs. PU membranes with surface microgrooves further increased the gene delivery efficiency to a similar level as the commercial transfection reagent but without the harmful effect. The gene delivery efficiency was associated with the extent of activation of cellular integrins β1 and α5 on different substrates. Moreover, the delivery efficiency was positively correlated with the cell migration rate on various substrates. The substrate-mediated gene delivery by synthetic polymeric substrates supports that integrin activation and cell behavior (e.g. migration and transfectability) changes can be modulated by synthetic polymer surface with microfeatures. The transfection by PU microgrooves is easy, nontoxic, and as effective as the commercial transfection reagent.

Electrophoretic deposition to promote layer-by-layer assembly for in situ gene delivery application

In this study, layer-by-layer (LbL) assembly of polyelectrolyte was facilitated using electric field assistance (EFA). To elucidate the EFA effects on polyelectrolyte multilayers (PEMs), the electric fields were solely administrated to either chitosan or DNA adsorption. Both DNA and chitosan adsorptions can be augmented under low electric field due to the electrophoretic deposition. However, the ensuing electrochemical reactions on electrode interfered with interactions between multilayers when the bias was larger than 0.5V, leading to retard deposition. Subsequent delivery experiments indicated that EFA during DNA deposition demonstrated superior DNA release. In contrast, no obvious improvements were observed for the groups using the EFA during chitosan deposition. Moreover, water contact angle experiments revealed different effects of electric fields on multilayer structure. Using EFA during DNA deposition led to a layered-form composition, whereas interpenetration of electrolytes was enhanced with the application of the electric field during chitosan deposition. For in vitro experiments, EFA during DNA deposition significantly enhanced in situ transfection performances of PEMs that the transgene expression levels were increased and the periods were extended, suggesting this method is potential to quantitatively and temporally improve substrate-mediated gene delivery.

Vascular gene transfer from metallic stent surfaces using adenoviral vectors tethered through hydrolysable cross-linkers.

In-stent restenosis presents a major complication of stent-based revascularization procedures widely used to re-establish blood flow through critically narrowed segments of coronary and peripheral arteries. Endovascular stents capable of tunable release of genes with anti-restenotic activity may present an alternative strategy to presently used drug-eluting stents. In order to attain clinical translation, gene-eluting stents must exhibit predictable kinetics of stent-immobilized gene vector release and site-specific transduction of vasculature, while avoiding an excessive inflammatory response typically associated with the polymer coatings used for physical entrapment of the vector. This paper describes a detailed methodology for coatless tethering of adenoviral gene vectors to stents based on a reversible binding of the adenoviral particles to polyallylamine bisphosphonate (PABT)-modified stainless steel surface via hydrolysable cross-linkers (HC). A family of bifunctional (amine- and thiol-reactive) HC with an average t1/2 of the in-chain ester hydrolysis ranging between 5 and 50 days were used to link the vector with the stent. The vector immobilization procedure is typically carried out within 9 hrand consists of several steps: 1) incubation of the metal samples in an aqueous solution of PABT (4 hr); 2) deprotection of thiol groups installed in PABT with tris(2-carboxyethyl) phosphine (20 min); 3) expansion of thiol reactive capacity of the metal surface by reacting the samples with polyethyleneimine derivatized with pyridyldithio (PDT) groups (2 hr); 4) conversion of PDT groups to thiols with dithiothreitol (10 min); 5) modification of adenoviruses with HC (1 hr); 6) purification of modified adenoviral particles by size-exclusion column chromatography (15 min) and 7) immobilization of thiol-reactive adenoviral particles on the thiolated steel surface (1 hr). This technique has wide potential applicability beyond stents, by facilitating surface engineering of bioprosthetic devices to enhance their biocompatibility through the substrate-mediated gene delivery to the cells interfacing the implanted foreign material.

Molecular Recognition Enables Nanosubstrate-Mediated Delivery of Gene-Encapsulated Nanoparticles with High Efficiency

Substrate-mediated gene delivery is a promising method due to its unique ability to preconcentrate exogenous genes onto designated substrates. However, many challenges remain to enable continuous and multiround delivery of the gene using the same substrates without depositing payloads and immobilizing cells in each round of delivery. Herein we introduce a gene delivery system, nanosubstrate-mediated delivery (NSMD) platform, based on two functional components with nanoscale features, including (1) DNA⊂SNPs, supramolecular nanoparticle (SNP) vectors for gene encapsulation, and (2) Ad-SiNWS, adamantane (Ad)-grafted silicon nanowire substrates. The multivalent molecular recognition between the Ad motifs on Ad-SiNWS and the β-cyclodextrin (CD) motifs on DNA⊂SNPs leads to dynamic assembly and local enrichment of DNA⊂SNPs from the surrounding medium onto Ad-SiNWS. Subsequently, once cells settled on the substrate, DNA⊂SNPs enriched on Ad-SiNWS were introduced through the cell membranes by intimate contact with individual nanowires on Ad-SiNWS, resulting in a highly efficient delivery of exogenous genes. Most importantly, sequential delivery of multiple batches of exogenous genes on the same batch cells settled on Ad-SiNWS was realized by sequential additions of the corresponding DNA⊂SNPs with equivalent efficiency. Moreover, using the NSMD platform in vivo, cells recruited on subcutaneously transplanted Ad-SiNWS were also efficiently transfected with exogenous genes loaded into SNPs, validating the in vivo feasibility of this system. We believe that this nanosubstrate-mediated delivery platform will provide a superior system for in vitro and in vivo gene delivery and can be further used for the encapsulation and delivery of other biomolecules.

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

Gene expression within cells can be altered through gene delivery approaches, which have tremendous potential for gene therapy, tissue engineering, and diagnostics. Substrate-mediated gene delivery describes the delivery of plasmid DNA or DNA complexed with nonviral vectors to cells from a surface, with the DNA immobilized to a substrate through specific or nonspecific interactions. In this work, DNA-nanoparticle (DNA–NP) adsorption to substrates is evaluated using combinatorial, in situ spectroscopic ellipsometry and quartz crystal microbalance with dissipation (SE/QCM-D), to evaluate the basic dynamic processes involved in the adsorption and immobilization of DNA–NP complexes to substrates. The concentration of DNA–NP solutions influences the adsorbed DNA–NP surface mass, which increases by a factor of approximately 6 (detected by SE) and approximately 4.5-fold (detected by QCM-D), as the DNA concentration increases from 1.5μg/mL to 15μg/mL, with an increase in layer porosity. In addition, SE/QCM-D analysis indicates that DNA–NP adsorption rates, surface coverage densities, and volume fractions are dependent on the type of substrate: gold (Au) and silicon dioxide substrates, protein-coated and uncoated substrates, and surfaces modified with alkanethiol self assembled monolayers (SAMs). These studies also demonstrate that the influence of an adsorbed protein layer on resulting DNA–NP immobilization efficiency is substrate dependent. For example, Au surfaces coated with fetal bovine serum (FBS) resulted in two-fold greater mass of adsorbed DNA–NPs, compared to DNA–NP adsorption to FBS-coated SAM substrates. This investigation offers insights into dynamic DNA–NP surface adsorption processes, characteristics of the immobilized DNA–NP layer, and demonstrates substrate-dependent DNA–NP adsorption.

Substrate-mediated nanoparticle/gene delivery to MSC spheroids and their applications in peripheral nerve regeneration

Substrate-derived mesenchymal stem cell (MSC) spheroids show greater differentiation capacities than dispersed single cells in vitro. During spheroid formation, nanoparticles (NPs)/genes may be delivered into the cells. In this study, MSCs were conveniently labeled with superparamagnetic Fe3O4 NPs, or transfected with brain-derived neurotrophic factor (BDNF) gene, by the substrate-mediated NP/gene uptake. With the promising in vitro data showing the beneficial effect on neural development and neurotrophic factor expression, MSCs were combined with a polymeric nerve conduit to bridge a 10 mm transection gap of rat sciatic nerve. High-resolution (7-T) magnetic resonance imaging (MRI) was used to track the transplanted cells. Nerve regeneration was assessed by functional recovery and histology. Results revealed that Fe3O4 NP-labeled MSCs were successfully visualized by MRI in vivo. Animals receiving BDNF-transfected MSC spheroids demonstrated the shortest gap bridging time (<21 days), the largest regenerated nerve, and the thickest myelin sheath at 31 days. Compared to MSC single cells, the pristine or BDNF-transfected MSC spheroids significantly promoted the functional recovery of animals, especially for the BDNF-transfected MSC spheroids. The transplanted MSCs were incorporated in the regenerated nerve and differentiated into non-myelinating Schwann cells after 31 days. This study suggests that the substrate-mediated gene delivery and NP labeling may provide extra values for MSC spheroids to carry therapeutic/diagnostic agents in cell-based therapy.

The regulation of DNA adsorption and release through chitosan multilayers

To sustain transgene expression, chitosan was studied to immobilize DNA using layer-by-layer assembly to form polyelectrolyte multilayers (PEMs). Higher DNA concentrations and longer deposition periods demonstrated more DNA adsorptions to PEMs. By adjusting pH and the molecular weight of chitosan, PEM structures were manipulated. Chitosan molecules adsorption to PEMs increased when they were at pH 6 because of their low protonation. Furthermore, the configuration of chitosan favored a coiled-form when the pH was high, as the intramolecular repulsion decreased. Therefore, interdiffusion of polyelectrolytes in PEMs was promoted to increase DNA adsorption, especially for chitosan with high molecular weight. For the release experiments, because PEMs fabricated by lower pH chitosan owned less chitosan molecules, DNA release was enhanced. However, this phenomenon did not happen to chitosan with high molecular weight, which should be due to the entanglement between polymer chains. This comprehensive approach should be beneficial to substrate-mediated gene delivery applications.

Use of Biotinylated Chitosan for Substrate-Mediated Gene Delivery

To improve transfection efficiency of nonviral vectors, biotinylated chitosan was applied to complex with DNA in different N/P ratios. The morphologies and the sizes of formed nanoparticles were suitable for cell uptake. The biotinylation decreased the surface charges of nanoparticles and hence reduced the cytotoxicity. The loading capacities of chitosan were slightly decreased with the increase of biotinylation, but most of the DNA molecules were still complexed. Using different avidin-coated surfaces, the interaction between biotinylated nanoparticles to the substrate may be manipulated. The in vitro transfection results demonstrated that biotinylated nanoparticles may be bound to avidin coated surfaces, and the transfection efficiencies were thus increased. Through regulating the N/P ratio, biotinylation levels, and surface avidin, the gene delivery can be optimized. Compared to the nonmodified chitosan, biotinylated nanoparticles on biomaterial surfaces can increase their chances to contact adhered cells. This spatially controlled gene delivery improved the gene transfer efficiency of nonviral vectors and could be broadly applied to different biomaterial scaffolds for tissue engineering applications.

The identity of the cell adhesive protein substrate affects the efficiency of adeno-associated virus reverse transduction

Delivering genes from surfaces, called substrate-mediated gene delivery or reverse transduction, is a useful method to achieve spatial localization of gene delivery. We tested the compatibility of adeno-associated virus (AAV) vectors with various cell adhesive proteins to mediate gene delivery from surfaces. Our studies demonstrate that AAV vectors can be successfully adsorbed on collagen I, elastin, and laminin substrates leading to robust gene delivery to overlying cells. Notably, AAV immobilization on laminin yields the highest efficiency of gene expression. This increased gene expression cannot be explained by increases in the levels of virus deposition, transcriptional activity of cells, or virus vector uptake into cells. Further refinement of our knowledge of AAV interactions with extracellular matrix proteins may have important implications in a variety of applications ranging from tissue engineering to in vivo gene therapy.

Surface Immobilization of Plasmid DNA with a Cell-Responsive Tether for Substrate-Mediated Gene Delivery

The advancement of nonviral gene therapy hinges on the ability to exert highly specific spatial and temporal control of gene delivery systems to enable localized release of DNA. In this work, we have developed a system capable of promoting localized delivery of a plasmid by utilizing peptide nucleic acid (PNA) technology to bind DNA to a substrate via an enzymatically labile peptide sequence. The successful immobilization of the DNA to the model substrate as well as the specificity of the binding was confirmed with atomic force microscopy (AFM) and AFM-confocal overlay imaging. Fluorescence-based quantification revealed that surfaces treated with the conjugates had 49 ± 22 ng of DNA/cm(2), while there were 4.2 ± 2.1 ng of DNA/cm(2) on surfaces treated with unfunctionalized DNA. When NIH/3T3 cells were grown on the modified substrates, a significantly higher percentage of cells were transfected when the peptide tether was protease-sensitive as compared with when it was not labile. These results indicated that the peptide must be cleaved to release the DNA. In addition to providing cell-triggered release, this system decouples the properties of the complexation agent and the substrate from the method of immobilization/release to provide a model system that can be tailored to specific applications.

Microcontact printing for co-patterning cells and viruses for spatially controlled substrate-mediated gene delivery

Spatial organization of gene expression is a crucial element in the development of complex native tissues, and the capacity to achieve spatially controlled gene expression profiles in a tissue engineering construct is still a considerable challenge. To give tissue engineers the ability to design specific, spatially organized gene expression profiles in an engineered construct, we have investigated the use of microcontact printing to pattern recombinant adeno-associated virus (AAV) vectors on a two dimensional surface as a first proof-of-concept study. AAV is a highly safe, versatile, stable, and easy-to-use gene delivery vector, making it an ideal choice for this application. We tested the suitability of four chemical surfaces (–CH3, –COOH, –NH2, and –OH) to mediate localized substrate-mediated gene delivery. First, polydimethylsiloxane stamps were used to create microscale patterns of various self-assembled monolayers on gold-coated glass substrates. Next, AAV particles carrying genes of interest and human fibronectin (HFN) were immobilized on the patterned substrates, creating a spatially organized arrangement of gene delivery vectors. Immunostaining studies reveal that –CH3 and –NH2 surfaces result in the most successful adsorption of both AAV and HFN. Lastly, HeLa cells were used to analyze viral transduction and spatial localization of gene expression. We find that –CH3, –COOH, and –NH2 surfaces support complete uniform cell coverage with high gene expression. Notably, we observe a synergistic effect between HFN and AAV for substrate-mediated gene delivery. Our flexible platform should allow for the specific patterning of various gene and shRNA cassettes, resulting in spatially defined gene expression profiles that may enable the generation of highly functional tissue.

Localized Cell Uptake of His-Tagged Polyplexes Immobilized on NTA Self-Assembled Monolayers

A new method to study substrate-mediated gene delivery was developed using a hexahistidine-tagged polymeric gene delivery vehicle (His-tagged polyplex) and nickel nitrilotriacetic acid (Ni-NTA) self-assembled monolayers (SAMs) on gold surfaces. The His-tagged polyplexes showed specific interaction with Ni-NTA surfaces compared to control surfaces, with increasing NTA content in SAM formulations corresponding to increasing amounts of immobilized His-tagged polypexes on the surface. Cells seeded on NTA SAMs demonstrated uptake of His-tagged polyplexes in the presence of imidazole and EDTA with low cytotoxicity. Cells seeded on NTA SAMs without imidazole and EDTA showed minimal a amount of His-tagged polyplex uptake. This showed that the release of polyplexes from the surface by imidazole and EDTA was necessary for cell uptake. Thus, this system provides potential spatial specificity for polyplex delivery controlled by location of NTA surfaces and temporal specificity for polyplex delivery controlled by the addition of imidazole and EDTA.

Enhanced gene transfection with addition of a cell‐penetrating peptide in substrate‐mediated gene delivery

To overcome extracellular barriers in gene therapy, controlled release technology has been adapted to protect DNA from degradation and maintain elevated DNA concentration. Although cell-penetrating peptides (CPPs) have been widely utilized to enhance the transfection efficiency of solution-based transfections, as far as we are aware, the effect of these peptides on substrate-mediated transfection has not yet been reported. In the present study, the effect of a CPP, KALA, on the substrate-mediated gene delivery was studied. A fast degrading cholic acid functionalized star poly((DL)-lactide) was used to fabricate Ca-P/DNA/KALA co-precipitates-deposited films and Ca-P/DNA/KALA co-precipitates-encapsulated films for mediating substrate-mediated transfection. The in vitro gene transfections in HEK293T and HepG2 cells mediated by the different films were investigated. The effect of KALA content on gene transfection was studied. Gene expression of film-mediated transfections could be significantly enhanced by the addition of KALA at a quite low content with a DNA/KALA ratio of 10 : 1. As a result of the surface erosion mechanism of the functionalized star poly((DL)-lactide), our substrate-mediated transfection system could rapidly release Ca-P/DNA/KALA to mediate efficient gene expression. During the transfection, the degradation of the polymer films could be observed and the degradation did not show any unfavorable effects on gene expression. The presence of a CPP could significantly enhance the gene expression of substrate-mediated transfections.

Engineering Surfaces for Substrate-Mediated Gene Delivery Using Recombinant Proteins

Immobilized fibronectin and other natural proteins have been utilized to enhance substrate-mediated gene delivery, with apparent contributions from the intrinsic bioactivity and also physical properties of the immobilized proteins. In this report, we investigated the use of recombinant proteins, compared to the full-length fibronectin protein, as surface coatings for gene delivery to investigate the mechanisms by which fibronectin enhances gene transfer. The recombinant fibronectin fragment FNIII(7-10) (FNIII) contains the alpha(5)beta(1) binding domain of fibronectin and supports cell adhesion, whereas the recombinant protein polymer PP-12 is also negatively charged and has a molecular weight similar to FNIII, but lacks cell binding domains. Transfection was compared on surfaces modified with FNIII, full-length fibronectin, or PP-12. The full-length fibronectin provided the greatest extent of transgene expression relative to FNIII or PP-12, which was consistent with the amount of DNA that associated with cells. FNIII had 4.2-fold or 4.7-fold lower expression levels relative to fibronectin for polyplexes and lipoplexes, respectively. PP-12 produced expression levels that were 317-fold and 12.0-fold less than fibronectin for polyplexes and lipoplexes, respectively. Although expression was greater on FNIII relative to PP-12, the levels of DNA associated per cell with FNIII were similar to or less than those with PP-12, suggesting that the bioactive sequences may contribute to an enhanced intracellular trafficking. For lipoplexes delivered on FNIII, the efficiency of intracellular trafficking and levels of caveolar DNA were greater than that observed with either the full-length fibronectin or PP-12. For polyplexes, fibronectin fragment resulted in greater intracellular trafficking efficiency compared to PP-12 protein polymer. Recombinant proteins can be employed in place of full-length extracellular matrix proteins for substrate-mediated gene delivery, and bioactive sequences can influence one or more steps in the gene delivery process to maximize transfection.