Surface-mediated Gene Delivery Research Articles

Novel Platform for Surface-Mediated Gene Delivery Assisted with Visible-Light Illumination

Surface-mediated gene delivery has attracted more and more attentions in biomedical research and applications because of its characteristics of low toxicity and localized delivery. Herein, a novel visible-light-regulated, surface-mediated gene-delivery platform is exhibited, arising from the photoinduced surface-charge accumulation on silicon. Silicon with a pn junction is used and tested subsequently for the behavior of surface-mediated gene delivery under visible-light illumination. It is found that positive-charge accumulation under light illumination changes the surface potential and then facilitates the delivery of gene-loaded carriers. As a result, the gene-expression efficiency shows a significant improvement from 6% to 28% under a 10 min visible-light illumination. Such improvement is ascribed to the increase in surface potential caused by light illumination, which promotes both the release of gene-loaded carriers and the cellular uptake. This work suggests that silicon with photovoltaic effect could offer a new strategy for surface-mediated, gene-delivery-related biomedical research and applications.

Bioactive nanocomposite coatings under visible light illumination promoted surface-mediated gene delivery.

Gene delivery based on bioactive coatings on collagen has great potential for applications in bone repair. Meanwhile, controlled gene delivery at specific times/regions is essential for an efficient and complete bone reconstruction process. However, spatio-temporal regulation of gene release and delivery remains a great challenge. In this paper, we used visible light illumination to effectively regulate gene release and subsequent delivery into biological cells. A visible light responsive and bioactive nanocomposite coating (based on collagen/gold nanoparticles, e.g., Col/AuNPs) was prepared through hydrothermal and sol-gel processes and was used as a loading platform for complexes of enhanced green fluorescent protein and Lipofectamine2000 (LF/GFP). The results showed that the amount of immobilized LF/GFP was increased on Col/AuNPs and the release of pre-adsorbed LF/GFP was significantly enhanced in a spatio-temporal and controlled manner under visible light illumination. Moreover, the cellular intake of the released genes was improved, thus enhancing the gene expression efficiency of the cells. The mechanism of enhanced controlled gene delivery was attributed to the changes in collagen structures and rearrangement of cytoskeletal structures induced by the photothermal effect. The developed Col/AuNP composite coating is effective for both controlled surface-mediated gene delivery and gene-mediated bone repair.

Surface-Mediated Stimuli-Responsive Gene Delivery Based on Breath Figure Film Combined with Matrix Metalloproteinase-Sensitive Hydrogel.

Surface-mediated gene delivery appears to be potential gene delivery modes for various applications. Still, controlled and smart delivery manners are required especially considering the need for gene therapy to deliver gene with selectivity. A surface that can effectively payload DNA, promote cell adhesion, and stimuli response is an important prerequisite. Here, we report a matrix metalloproteinase (MMP)-responsive surface-mediated gene delivery system by combining MMP-degradable hydrogel with a breath figure (BF) porous film. The MMP-degradable hydrogel containing plasmid DNA was loaded into the surface pores of the BF film as DNA reservoirs. The uppersurface without hydrogel on the BF film served as footholds of integrin adhesions. MMP is one of the important endogenous signals in tumor-related pathologic changes, and MMP expressions in cancer cells are significantly higher than those in normal cells. Consequently, our surface-mediated gene delivery locally and rapidly released the payload DNA in response to cancer cells and transfected them. This work highlights the importance of the combination of stimuli-response and surface-mediated gene delivery to functional materials, showing good potential applications in the field of gene therapy.

Dendrimer-grafted bioreducible polycation/DNA multilayered films with low cytotoxicity and high transfection ability

Controlled release of incorporated foreign DNA from multilayered films plays an important role in surface-mediated gene delivery. Herein, multilayered polyelectrolyte complex thin films, composed of dendrimer-grafted bio-reducible cationic poly(disulfide amine) and plasmid DNA, were fabricated via layer-by-layer (LBL) assembly for in vitro localized gene delivery. The UV absorbance and thickness of the LBL films were found to have linear correlation with the numbers of poly(disulfide amine)/DNA bilayers. Although LBL films were stable in PBS buffer, their degradation could be triggered by reducing agents (i.e. glutathione, GSH). The degradation rate of the films is directly proportional to the GSH concentration, which in turn affected the corresponding gene expression. All poly(disulfide amine)/DNA films exhibited lower cytotoxicity and higher transfection activity in comparison with PEI/DNA multilayered films. Moreover, LBL films showed the highest transfection efficiency in the presence of 2.5 mM GSH when cultured with 293T cells, with ~36% GFP-positive 293T cells after 5-days of co-culture. These DNA-containing reducible films could potentially be useful in gene therapy and tissue engineering by controlling the release of incorporated DNA.

Photothermal-assisted surface-mediated gene delivery for enhancing transfection efficiency.

The development of gene therapy puts forward the requirements for efficient delivery of genetic information into diverse cells. However, in some cases of transfection, especially those for transfecting some primary cells and for delivering large size plasmid DNA (pDNA), the existing conventional transfection methods show poor efficiency. How to further improve transfection efficiency in these hard-to-achieve issues remains a crucial challenge. Here, we report a photothermal-assisted surface-mediated gene delivery based on a polydopamine-polyethylenimine (PDA-PEI) surface. The PDA-PEI surface was prepared through PEI-accelerated dopamine polymerization, which showed efficiency in the immobilization of PEI/pDNA polyplexes and remarkable photothermal properties. Upon IR irradiation, we observed improved transfection efficiencies of two important hard-to-achieve transfection issues, namely the transfection of primary endothelial cells, which are kinds of typical hard-to-transfect cells, and the transfection of cells with large-size pDNA. We demonstrate that the increases of transfection efficiency were due to the hyperthermia-induced pDNA release, the local cell membrane disturbance, and the polyplex internalization. This work highlights the importance of local immobilization and release of pDNA to gene deliveries, showing great potential applications in medical devices in the field of gene therapy.

Surface-mediated transfection of a pDNA vector encoding short hairpin RNA to downregulate TGF-β1 expression for the prevention of in-stent restenosis.

In-stent restenosis is one of the most serious modes of failure of cardiovascular stent implant. Although drug-eluting stents have been proven to reduce in-stent restenosis, the nonspecific inhibitory effects of anti-proliferative drugs, such as rapamycin, result in delayed re-endothelialization and fatal late stent thrombosis. Although many studies have focused on promoting rapid re-endothelialization, a feasible method of reducing excessive extracellular matrix (ECM) production and cell proliferation might provide a promising way to efficiently inhibit the restenosis invivo. In this study, we constructed a surface-mediated gene delivery system through a layer-by-layer assembly of protamine sulfate (PrS) and a functional plasmid DNA (pDNA) encoding short hairpin RNA to downregulate the expression of transforming growth factor-β1 (TGF-β1), aiming to inhibit cell proliferation and reduce excessive ECM production. We demonstrated that (PrS/pDNA) films were successfully constructed with good stability under physiological conditions. The (PrS/pDNA) films were able to transfect fibroblasts, thus reducing the secretion of fibronectin and collagen and inhibiting cell proliferation invitro. Further invivo experiments showed that the transfection of arterial tissue led to significant local downregulation of TGF-β1 and ECM proteins and inhibited neointimal hyperplasia. These functional gene delivery films avoid the use of non-specific drugs and may serve as part of a new strategy for targeting in-stent restenosis in the field of cardiovascular disease.

Controlled superficial assembly of DNA–amorphous calcium phosphate nanocomposite spheres for surface-mediated gene delivery

Surface-mediated gene delivery systems have many potential applications in tissue engineering. We recently fabricated an assembly consisting of DNA–amorphous calcium phosphate (DNA–ACP) nanocomposite spheres on a polymer substrate via coprecipitation in a labile supersaturated calcium phosphate (CaP) solution and demonstrated the assembly’s high gene delivery efficacy. In this study, we conducted a detailed investigation of the coprecipitation process in solution and revealed that the negatively charged DNA molecules were immobilized in the ACP spheres during the initial stage of coprecipitation and functioned as both sphere-dispersing and size-regulating agents. As a result, the DNA–ACP nanocomposites grew into size-regulated submicrospheres in solution and assembled onto the substrate via gravity sedimentation. The assembled nanocomposite spheres were chemically anchored to the substrate surface through an intermediate layer of CaP-based nanoparticles that was formed heterogeneously at the substrate surface. The coprecipitation conditions, i.e., coprecipitation time and Ca and P concentrations in solution, greatly affected the state of assembly of the nanocomposite spheres, thereby influencing the gene expression level of the cells cultured on the substrate. Increasing the number density and decreasing the size of the nanocomposite spheres did not always increase the assembly’s gene delivery efficacy (per surface area of the substrate) due to adverse effects on cellular viability. As demonstrated herein, controlling the coprecipitation conditions is important for designing a cell-stimulating and biocompatible scaffold surface consisting of an assembly of DNA–ACP nanocomposite spheres.

High-strength smart hydrogels as a versatile platform for surface-mediated gene delivery

High-strength smart hydrogels as a versatile platform for surface-mediated gene delivery

High-strength photoresponsive hydrogels enable surface-mediated gene delivery and light-induced reversible cell adhesion/detachment.

In the present study, high-strength photoresponsive hydrogels were prepared by the photoinitiated copolymerization of acrylamide (AAm, hydrophilic hydrogen bonding monomer), 2-vinyl-4,6-diamino-1,3,5-triazine (VDT, hydrophobic hydrogen bonding monomer), and spiropyran-containing monomer (SPAA) in the presence of cross-linker poly(ethylene glycol) diacrylate (PEG575DA, Mn = 575). The double hydrogen bondings from AAm-AAm and diaminotriazine-diaminotriazine contributed to the considerable enhancement in tensile and compressive properties of the hydrogels, which showed an excellent ability to resist a variety of external forces. Fifteen minutes of UV (365 nm) irradiation led to the detachment of adhered cells due to the increased surface hydrophilicity caused by the isomerization of spiropyran moieties. Furthermore, repeated attachment/detachment of cells was realized by the alternate illumination of visible and UV light. Reverse gene transfection was carried out successfully by anchoring the PVDT/pDNA complex nanoparticles on the gel surface through hydrogen bonding between diaminotriazine motifs prior to cell seeding. Importantly, fibronectin (FN) modification combined with supplementing PVDT/pDNA complex nanoparticles after the first cycle of reverse gene transfection, so-called sandwich gene transfection, further increased the gene transfection level. A short time of UV light exposure could result in the nonharmful detachment of gene-modified cells from the gel surface. This high-strength photosensitive hydrogel holds potential as a reusable soft-wet platform for cell harvesting as well as gene transfection operation at higher efficiency.

Characterization of degradable polyelectrolyte multilayers fabricated using DNA and a fluorescently-labeled poly(β-amino ester): shedding light on the role of the cationic polymer in promoting surface-mediated gene delivery.

Polyelectrolyte multilayers (PEMs) fabricated from cationic polymers and DNA have been investigated broadly as materials for surface-mediated DNA delivery. One attractive aspect of this "multilayered" approach is the potential to exploit the presence of cationic polymer "layers" in these films to deliver DNA to cells more effectively. Past studies demonstrate that these films can promote transgene expression in vitro and in vivo, but significant questions remain regarding roles that the cationic polymers could play in promoting the internalization and processing of DNA. Here, we report physicochemical and in vitro cell-based characterization of DNA-containing PEMs fabricated using fluorescently end-labeled derivatives of a degradable polycation (polymer 1) used in past studies of surface-mediated transfection. This approach permitted simultaneous characterization of polymer and DNA in solution and in cells using fluorescence-based techniques, and provided information about the locations and behaviors of polymer 1 that could not be obtained using other methods. LSCM and flow cytometry experiments revealed that polymer 1 and DNA released from film-coated objects were both internalized extensively by cells and that they were colocalized to a significant extent inside cells (e.g., ~58% of DNA was colocalized with polymer). Fluorescence anisotropy measurements of solutions containing partially eroded films were also consistent with the presence of aggregates of polymer 1 and DNA in solution (e.g., after release from surfaces, but prior to internalization by cells). Our results support the view that polymer 1, which is incorporated into these materials as "layers" rather than as part of optimized, preformed "polyplexes", can act to promote or enhance surface-mediated DNA delivery. More broadly, our results suggest opportunities to improve the delivery properties of DNA-containing PEMs by incorporation of additional "layers" of other conventional cationic polymers designed to address specific intracellular barriers to transfection, such as endosomal escape, more effectively.

Supramolecular Assembly of Cyclodextrin-Based Nanoparticles on Solid Surfaces for Gene Delivery

In this work, a new approach for surface-mediated gene delivery based on inclusion complex formation between the solid surface and delivery vehicles is presented. beta-Cyclodextrin (CD) molecules form high-affinity inclusion complexes with adamantane. This complexation ability was used to specifically immobilize beta-CD-modified poly(ethylenimine) (CD-PEI) nanoparticles on adamantane- (AD-) modified self-assembled monolayers. To investigate the nanoparticle/surface interaction, CD-PEI-based and PEI-based nanoparticles were passed through a surface plasmon resonance flow cell containing the monolayers. CD-PEI nanoparticles are specifically immobilized on the chip surface by cyclodextrin-adamantane inclusion complex formation. Minimal nanoparticle adsorption was detected with PEI-based nanoparticles or on control surfaces. Competition studies with free cyclodextrins reveal that the multivalent interactions between CD-PEI nanoparticles and the adamantane-modified surface results in significantly higher binding affinity than single cyclodextrin-adamantane complexes. Immobilized nanoparticles were characterized by atomic force microscopy and quantified by fluorescence assay. Thus, the ability of CD-PEI nanoparticles to form inclusion complexes can be exploited to attain specific, high-affinity loading of delivery vehicles onto solid surfaces.

172. Spatially-Controlled Delivery of Cyclodextrin-Based Polyplexes from Solid Surfaces

Localized, nucleic acid delivery from solid surfaces is an important technology for biomedical research, with applications ranging from gene therapy to tissue engineering to functional genomics. The spatially-controlled delivery of growth factor is required for creating a well-organized, complex cellular architecture by tissue engineering. Non-viral gene delivery vehicles have been immobilized to solid surfaces by physical adsorption, biotin-streptavidin interaction, and chemical conjugation1. Here, we present a new approach for surface-mediated gene delivery based on inclusion complex formation between the solid surface and delivery vehicles. Through this method, the delivery of transgenes can be spatially controlled by gene delivery through specific interactions between the delivery vehicles and the surface. Cyclodextrins (CD), cyclic oligomers of glucose, form inclusion complexes with small, hydrophobic compounds such as adamantane. In this work, nanoparticle composites of cyclodextrin polycations and nucleic acids were specifically immobilized on adamantane-coated surfaces. The interaction was characterized by surface plasmon resonance (SPR) spectroscopy and by atomic force microscopy (AFM).

Simple and versatile methods for the fabrication of arrays of live mammalian cells

Single-step methods for the generation of patterned surfaces on hydrogels are presented. Poly(vinyl alcohol) films covalently bonded on glass cover slips and commercially available hydrogel-coated polystyrene plates were used as cell-repellent surfaces. Cell-adhesive domains were created by spotting dilute solutions of sodium hypochlorite onto the surfaces. Alternatively, domains supporting cell attachment were created by exposure to UV light from a xenon excimer lamp, employing a contact mask. Rat skeletal myoblast cells, HEK 293 human embryonic kidney cells and Caco-2 colon carcinoma cells adhered and spread exclusively on modified areas. The surfaces are durable for weeks under cell culture conditions and re-usable after removal of the cells by trypsin treatment. Arrays of adhesive spots seeded with cells at a low density permitted dynamic monitoring of cell proliferation. Selected colonies can be harvested from the surfaces by means of local trypsination. Thus, these techniques may provide useful tools for the isolation of clonal cell populations. Additionally, we demonstrate the possibility of surface-mediated gene delivery from the micro patterns. We show that DNA, complexed with a lipid reagent, can be adsorbed on modified poly(vinyl alcohol) coatings, resulting in spatially controlled adhesion and reverse transfection of HEK 293 cells.