Macroporous Hydrogels for Stable Sequestration and Sustained Release of Vascular Endothelial Growth Factor and Basic Fibroblast Growth Factor Using Nucleic Acid Aptamers

Abstract

Macroporous hydrogels have been widely studied for biological and biomedical applications such as drug delivery and tissue engineering. However, these hydrogels cannot stably sequester molecules of interest due to their high permeability. The purpose of this work was to study the feasibility of using two aptamers to sequester two protein drugs, quantify the apparent diffusivity of protein drugs in aptamer-functionalized macroporous hydrogels, and evaluate the function of aptamer-functionalized macroporous hydrogels in controlling protein release for angiogenesis. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) were used as model proteins. The data show that anti-VEGF and anti-bFGF aptamers could be uniformly incorporated into macroporous hydrogels for stable and specific sequestration of VEGF and bFGF. The aptamers could reduce the apparent diffusivity of VEGF and bFGF in the macroporous hydrogels by approximately three orders of magnitude. Moreover, as the aptamers could prolong the release of these growth factors, dual aptamer-functionalized macroporous hydrogels could stimulate synergistic angiogenesis. Therefore, this work has successfully demonstrated that aptamer-functionalized macroporous hydrogels hold great potential of stably sequestering multiple molecules of interest for various biological and biomedical applications.

Keywords: angiogenesis; aptamer; growth factor; macroporous hydrogel; protein delivery.

Fig. 1.

Synthesis and characterization of dual aptamer-functionalized macroporous hydrogels. A. Schematic illustration. B. Photographic image of the entire hydrogel. C. SEM images. Blank: hydrogel without aptamers; A-VEGF: anti-VEGF aptamer-functionalized hydrogel; A-bFGF: anti-bFGF aptamer-functionalized hydrogel; Dual: hydrogel functionalized with both anti-VEGF and anti-bFGF aptamers. D. Fluorescence (left) and confocal (right) microscopy images. The anti-VEGF and anti-bFGF aptamers were stained with FAM and Cy-5 labeled complementary sequences of the two aptamers, respectively.

Fig.2.

Growth factor sequestration in aptamer-functionalized macroporous hydrogel. A) Secondary structure of anti-VEGF (A-VEGF) and anti-bFGF (A-bFGF) aptamers. B) Schematic illustration of growth factor loading and binding in aptamer-functionalized macroporous hydrogel. C) VEGF retention in macroporous hydrogels. D) bFGF retention in macroporous hydrogels. Blank: macroporous hydrogel without aptamers. Dual: macroporous hydrogel functionalized with both A-VEGF and A-bFGF aptamers. (NS: Non-significant. ** p<0.01, *** p<0.001).

Fig. 3.

Examination of growth factors release from aptamer-functionalized macroporous hydrogels. A) Schematics showing the release of VEGF and bFGF from blank hydrogel versus from aptamer-functionalized hydrogels. B) Cumulative VEGF and bFGF release and data fitting based on Equation 2. C) Cumulative VEGF and bFGF release and data fitting based on Equation 3.

Fig. 4.

Comparison of apparent diffusivity. A) A summary of calculated diffusivity for bFGF and VEGF released from blank, single aptamer-functionalized and dual aptamer-functionalized macroporous hydrogels. B) Normalized apparent diffusivity. The calculated apparent diffusivity was divided by diffusivity of growth factors in aqueous solution (Do) for normalization.

Fig. 5.

Evaluation of dual aptamer-functionalized macroporous hydrogel in stimulating angiogenic response in vitro and in vivo. Hydrogels were incubated in the release medium for 3 days to mimic a potential in vivo release microenvironment before conducting the studies. A) Visualization of cell migration before and after treatment for all groups. The cells were stained with Calcein AM. B) Quantitative analysis of cell migration distance. C) Image of CAM at day 3 after implantation of hydrogels (Embryonic day 11). D) Quantitative analysis of new blood vessels formed after treatment. E) H&E staining of CAM treated with dual aptamer-functionalized hydrogels loaded with VEGF and bFGF (Red arrows point at small blood vessels. * hydrogel). Blank: hydrogel without aptamers (VEGF and bFGF loaded); A-VEGF: anti-VEGF aptamer-functionalized hydrogel (VEGF loaded); A-bFGF: anti-bFGF aptamer-functionalized hydrogel (bFGF loaded); Dual: hydrogel functionalized with both anti-VEGF and anti-bFGF aptamers (VEGF and bFGF loaded). (NS- Non-significant, *p<0.05, ** p<0.01, *** p<0.001).