Elsevier

Acta Biomaterialia

Volume 8, Issue 9, September 2012, Pages 3228-3240
Acta Biomaterialia

Gene-activated and cell-migration guiding PEG matrices based on three dimensional patterning of RGD peptides and DNA complexes

https://doi.org/10.1016/j.actbio.2012.05.010Get rights and content

Abstract

Essential to the design of genetic bioreactors used in the human body is a consideration of how the properties of biomaterials can combine to envelope, spatially guide, reprogramme by gene transfer, and then release cells. In order to approach this goal, poly(ethylene glycol) (PEG) matrices with modulated structural features and defined spatial patterns of bioactive signals have been designed and produced. In particular, within such PEG matrices, both an adhesive RGD peptide gradient, to directionally attract NIH3T3 cells, and a designed spatial distribution of immobilized poly(ethylenimine) (PEI)/DNA complexes, to obtain a localized transfection, have been realized. These bioactive biomaterials have been designed bearing in mind that cells following an RGD gradient migrate through the matrix, in which they find the bound DNA and become transfected. Both cell migration and transfection have been monitored by fluorescence microscopy. Results show that this system is able to envelope cells, spatially guide them towards the immobilized gene complexes and locally transfect them. Therefore, the system, acting as a genetic bioreactor potentially useful for the regulation of biology at a distance, could be used to directly control cell trafficking and activation in the human body, and has many potential biomedical applications.

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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