Gene-activated and cell-migration guiding PEG matrices based on three dimensional patterning of RGD peptides and DNA complexes
Introduction
Although biomaterials have generally been used to dictate the behaviour of cells transplanted with them, it has recently been highlighted that they can be designed to manipulate cells of the tissue in which they are implanted, also at a significant distance from the implant site. This manipulation can be achieved by either targeting the material to specific cells or anatomical locations, or controlling the movement of target cell populations [1]. Biomaterials may programme specific cell populations through their recruitment, subsequent activation, and final retention or release without transplantation. Those with such an ability, which act as selective bioreactors for use in the human body, look highly promising for the treatment of various diseases because of their potential to target disease sites, presently not clinically detectable. They have so far been used to generate anti-tumour responses by recruiting and programming immune cells in situ [2], although their potential has inspired encouraging studies aimed at producing several different biological responses, and overcoming the in vivo challenges to biomaterials. In particular, a combination of the abilities to envelope, spatially guide, reprogramme by gene transfer and release cells has the potential to enhance the in vivo challenges in the fields of tissue engineering, immunotherapy and gene therapy.
In order to contribute to the development of such biomaterials, a new approach, based on the combination of cell-controlled migration [3], [4] and gene transfer [5] methodologies, has been performed. In respect to the former methodology, it is worth noting that the design of a bioactive signals concentration gradient may represent a viable strategy to guide cells within the biomaterials [3], [4], [6], as the spatial distribution of bioactive signals within biomaterials strongly influences cell response [7], [8], [9]. In this context, researchers are seeking to identify techniques that permit the preparation of matrices with controlled gradients of biomacromolecular signals, able to guide cell migration into the matrices [9], [10], [11], [12], [13]. Microarchitectural features of the biomaterials also contribute to promoting and influencing cell behaviour and, in particular, to guiding cell colonization into the biomaterials [14]. Gene transfer is a powerful methodology for reprogramming cells and affecting their fate because genes naturally carry on specific instructions for cell function. This approach can be employed either to initiate or increase the production of specific proteins and antigens, or to block the expression of unwanted proteins. Additionally, gene transfer has the potential both to provide protein expression over a long period of time at effective concentration, and to target any cellular process by altering the expression of a specific protein [5]. Moreover, biomaterial-mediated gene transfer has the potential to maintain for a long period of time the effective DNA levels in a constrained area, extending the opportunity for cellular internalization and increasing the likelihood of gene transfer, and avoiding the possibility of dispersion [15], [16], [17], [18], [19], [20], [21]. In traditional gene delivery, gene vectors locate target cells, while in biomaterial-mediated gene transfer, cells locate vectors, following their migration into the matrix.
The potency of poly(ethylene glycol) (PEG) porous (to allow cell movement) gene activated matrix (GAM) to envelope, guide, transfect and release cells, and then to be used as a genetic bioreactor, has been investigated, based on the idea that, once enveloped, cells migrate through the matrix, in which they find pDNA complexes bound to the matrix, and become transfected before exiting. In order to continuously guide cells through the porous matrix, an appropriate stable gradient of the adhesive Gly-Arg-Gly-Asp-Ser (RGD) peptides has been realized. To locally transfect the cells, the matrix has also been functionalized by immobilizing poly(ethylenimine) (PEI)/DNA complexes with a pre-designed spatial distribution, through modification of PEI molecules with acrylated PEG. The multiple-bioactive porous matrix has been realized by combining, for the first time, particle templating [14] and fluidic-photopolymerization [22] technologies. The efficiency of the realized matrices has been evaluated in respect to their capability to englobe, guide (effect of RGD gradient on cell colonization), transfect (expression of green fluorescent protein (GFP)), and allow cells to migrate outwards.
Section snippets
Synthesis of acryloyl-PEG-RGD
The adhesive peptide sequence RGD was synthesized by the solid-phase method using standard 9-fluorenylmethoxycarboxyl (Fmoc) chemistry protocols on Rink-amide MBHA resin (scale of 2.0 mmol), by standard Fmoc protection for amino acid side-chains, on a scale of 2.0 mmol [23]. Peptide purity and integrity were confirmed by liquid chromatography–mass spectrometry (LC-MS) analysis using a LCQ DECA XP Ion Trap mass spectrometer (ThermoElectron, Milan, Italy) equipped with an OPTON ESI source,
PEG-PEI conjugate characterization
The modification of PEI with PEG was assessed by 1H-NMR spectroscopy in D2O. Results indicate that in the PEG-PEI conjugate produced, each PEI macromolecule was modified with five blocks of PEG, corresponding to a coupling efficiency of 0.9% [26] (Fig. 4).
DNA Complexes characterization
The size and zeta potential of PEG-PEI/DNA complexes (N/P = 6) were investigated by Zetasizer Nano-SZ. Results show that the formed complexes are characterized by a mean diameter of 106 ± 18 nm (Fig. 5), and a zeta potential of 0.9 ± 0.2 mV.
Microstructural properties of the biomaterials
The
Discussion
A combination of the abilities to envelope, spatially guide, influence by gene transfer, and then release cells has the potential to overcome the challenge to biomaterials in vivo in the field of tissue engineering, immunotherapy or gene therapy. With this aim, PEG matrices with modulated structural features and a defined spatial pattern of bioactive signals have been produced. In particular, an adhesive RGD peptide stable gradient, to direct and attract continuously NIH3T3 cells, and an
Conclusions
This study demonstrates the potential for newly designed and produced PEG multifunctional matrices to internalize, spatially guide, locally transfect, and release cells. The new matrices were realized by combining, for the first time, particle-templating and fluidic-photopolymerization technologies. Major challenges are a forced migration through the PEG porous matrix, achieved by imposing a stable RGD gradient, and a localized transfection obtained by immobilizing DNA complexes in the cellular
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Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage
2016, BiomaterialsCitation Excerpt :Unfortunately, this led to a low transfection efficiency of 2.8% GFP expressing cells but it is still encouraging for several tissue engineering applications to attract host cells through a gene-embedding biomaterial. Recruitment of cells inside the matrix could also be achieved thanks to a RGD peptide (promoting cell adhesion) [128] gradient. Several techniques of cells recruitment have been recently reviewed for cartilage repair applications [129].
MicroRNA-mediated immune modulation as a therapeutic strategy in host-implant integration
2015, Advanced Drug Delivery ReviewsCitation Excerpt :More recent applications of GAM are mainly in the areas of bone and cartilage regeneration. Modifications such as incorporation of PEIs have shown to enhance transfection efficiency and requiring much lower doses of plasmid DNA than earlier generation of GAMs [229-231]. For metal implants where titanium is used, the coating of the surface with hydroxyapatite nanocrystals allows PLGA particles to be electrostatically immobilized on them [232].
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2013, Current Opinion in BiotechnologyCitation Excerpt :Alginate hydrogels conjugated with various RGD densities for siRNA-mediated knockdown of eGFP demonstrated that increasing RGD density resulted in significantly higher knockdown of the targeted protein [46•]. Moreover, RGD gradients and presentation (homogeneous vs. clustered) in different scaffolds have been used to influence transfection [30••,47••]. Hydrogel stiffness can also be used to modulate migration and gene delivery rates; stiffer gels result in slower release rates of encapsulated polyplexes and decreased cell populations, spreading, and transfection [30••] (Figure 3b).
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