Engineering Patterned Aptamer-Functionalized Hydrogels for Controlling Vascularization

Introduction For controlling vascularization within engineered tissues, a high degree of spatiotemporal control on growth factors availability is needed.1 However, most of the current strategies for growth factors delivery often focuses on the immobilization or coupling of growth factors within the engineered matrices (hydrogel) via various linker proteins or peptides. These systems provide passive release rates and growth factor delivery on demand, but fail to adapt their release rates in accordance with the tissue development. To overcome this limitation, the present study employed nucleic acid based aptamers for spatiotemporally controlled growth factor delivery. Aptamers are an emerging class of affinity ligand that can be selected from DNA/RNA libraries to recognize proteins with high affinity and specificity.1 They are small-sized, stable structures with low immunogenicity.2 Aptamer based growth factor delivery systems are able to load/release multiple growth factors on demand with high specificity. In the present study, the authors have developed patterned aptamer-functionalized hydrogels to evaluate their potential for growth factor sequestering, controlled release and study their effect on vascular network formation. Methods The aptamer-functionalized hydrogels were prepared via photo-polymerization of gelatin methacryloyl (GelMA) and acrydite functionalized aptamers having sequence specific for binding to vascular endothelial growth factor (VEGF165). Visible light photoinitiator, tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate with sodium persulfate was used. For engineering patterned aptamer-functionalized hydrogels, different biofabrication techniques were employed such as 3D printing and photo patterning. The physicochemical properties of the patterned constructs were evaluated and compared with control samples. To study the programmable/triggered growth factor release efficiency, the complementary sequences (CSs) to VEGF specific aptamers were added into the system and evaluated using ELISA assay. For studying the effect of triggered growth factor release on vascular network formation, 3D culture of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) within aptamer-functionalized hydrogel matrices was performed. Results & Discussion The results obtained from physicochemical analysis of the aptamer-functionalized hydrogels confirmed the aptamer retention capacity of acrydite functionalized aptamers within the hydrogels, in comparison with the control aptamers for as long as 15 days at 37 °C. These results fit well with our hypothesis that the acrydite functionalized aptamers could covalently crosslink within the GelMA polymeric network whereas control aptamers (same DNA sequence with no chemical modification) tend to just physically entrapped within the hydrogel. The VEGF ELISA experiments confirmed triggered release of VEGF from the aptamer functionalized hydrogels in response to CS addition. Without CS addition, these hydrogels could sustain a controlled release for until 10 days. Furthermore, in co-culture experiments, the developed patterned aptamer-functionalized hydrogels showed high cellular viability and ability to control vascular network formation (by HUVECs and MSCs) over a span of 10 days within these hydrogels with triggered VEGF release on demand (VEGF release was triggered on day 5). These results further confirmed the bioactivity of the VEGF molecules after their loading within the aptamer functionalized hydrogels. Conclusions The present study shows the vasculogenic potential of patterned aptamer-functionalized hydrogels via spatiotemporally controlling VEGF availability within the hydrogel system. Acknowledgements: This work is supported by an ERC Consolidator Grant under grant agreement no 724469. References 1. B. Soontornworajit, et. al., Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides. Biomaterials 32 (2011) 6839-6849. 2. M.R. Battig, et. al., Programmable release of multiple protein drugs from aptamer-functionalized hydrogels via nucleic acid hybridization. J. Am. Chem. Soc. 134 (2012) 12410-12413.