The effects of an RGD-PAMAM dendrimer conjugate in 3D spheroid culture on cell proliferation, expression and aggregation

Abstract

By presenting biomolecular ligands on the surface in high density, ligand-decorated dendrimers are capable of binding to membrane receptors and cells with specificity and avidity. Despite the various uses, fundamental investigations on ligand-dendrimer conjugates have mainly focused on their binding behavior with cells, whereas their potential bioactivity and applications in multicellular systems, especially in three-dimensional (3D) culture systems, remains untapped. In this study, a typical adhesive peptide ligand – RGD – was modified to generation 4 polyamidoamine (PAMAM), and the bioactivity of suspended RGD-PAMAM conjugates was investigated on cells cultured as multicellular spheroids. Our results demonstrate that the RGD-PAMAM conjugates, after being incorporated into the 3D spheroids, were able to promote cellular proliferation and aggregation, and affect the mRNA expression of extracellular factors by NIH 3T3 cells. These bioactive functions were multivalency-dependent, as none of similar effects was observed for monovalent RGD ligand. Our study suggests that multivalent ligand-dendrimer conjugates may act as a unique type of artificial factors to mediate the cellular microenvironment in 3D culture, a property attributable to the spatial organization of the ligands and possible “cell-gluing” function of multivalent conjugates. This new finding opens the door for further exploring multivalent ligand-dendrimer conjugates for applications in 3D cell culture and tissue engineering.

Introduction

Dendrimers are a unique category of nanoscale materials that possess attractive structural features that include monodisperse molecular weight, tunable sizes and nanoscale starburst branches [1], [2], [3]. When decorated with peptide or carbohydrate ligands through the surface functional groups, the ligand-dendrimer conjugates can present bioactive ligands in high density with roughly spherical configuration [4], [5]. Such spatial characteristics of ligand arrangement has proven to be valuable to provide a basic model for probing fundamental ligand–receptor interactions, given that multivalency and clustering effects have been recognized as crucial mechanisms in membrane receptor-mediated signaling processes [6], [7]. Indeed, previous studies reported that ligand-dendrimer conjugates exhibited enhanced specificity and avidity in binding with cognate receptors, indicating the potential use of these materials where cell-material recognition is desired [8], [9], [10], [11]. For example, polyamidoamine (PAMAM) decorated with peptides containing adhesive arginine-glycine-aspartic acid (RGD) sequence, was found able to bind cells in a dosage- and cell type-dependent manner [10]. To date, ligand-decorated dendrimers have been investigated as smart probes in various biomedical applications from cell labeling, to diagnostic imaging and to drug delivery [12], [13], [14], [15].

In addition to investigation on the binding property of dendrimeric conjugates, some studies have been carried out regarding the possible toxic effects of the conjugates on the cellular and systemic levels. In the study by Yang et al, polyamidoamine (PAMAM) decorated with adhesive oligopeptide – RGD – was found to show toxic effects depending on the charged groups of PAMAM scaffolds [16]. In particular, anionic PAMAM conjugates did not exhibit effects on viability of cells in contrast to those containing cationic PAMAM scaffolds.

Despite the above studies, it is noted that our current understanding of dendrimeric conjugates have mainly focused on their binding capability and biocompatibility with cells/tissues. The bioactivity of conjugates bearing multivalent organization of ligands remains largely unknown. It is speculated that by virtue of clustered nanoscale arrangement, ligand-decorated dendrimers may enable discovery of new materials properties beyond their binding behavior with receptors. Indeed, receptor clustering is a fundamental process underlying many receptor-activated biological events. Through nanopatterning techniques on 2D surfaces, it is found that cell adhesion behavior and downstream intracellular signaling pathways can be affected by the spatial arrangement of adhesive ligands via the clustering mechanism of integrin receptors [17], [18].

To address the hypothesis on the bioactivity of dendrimeric materials, we here carry out studies on cell-material interactions via the multicellular spheroidal culture model. By promoting the cell–cell contact and recapitulating the multicellular condensation process occurring in natural tissue morphogenesis, multicellular spheroids provide a basic model for three-dimensional (3D) cellular studies [19], [20]. The spheroidal culture systems have been widely exploited in the maintenance, expansion and differentiation of cells in vitro [21], [22], [23], [24], [25]. It is hypothesized that within the constructs of multicellular spheroids, the dendrimeric conjugate molecules with nanospherical shapes would be subjected to real 3D cellular environment, and get exposed to multiple cells and function as a “gluing” material. In addition to the gluing effect, the conjugate material might as well affect the fundamental cell activities through multivalent mechanisms.

To investigate our hypothesis, a model adhesive peptide ligand, RGD, was modified to G4 polyamidoamine (PAMAM) and the resulting conjugate was supplemented to the culture of 3T3 fibroblast spheroids. Experiments were carried out to quantitatively examine the incorporation of suspended peptide-dendrimer conjugates into multicellular spheroids. To understand whether multivalent membrane-binding materials could usher in a new methodology to modulate cell activity in 3D culture, the effects of the RGD-PAMAM dendrimer conjugate on cell proliferation, gene expression and spheroidal sizes were analyzed and compared with those of the monovalent RGD ligand.