Aptamer-functionalized Hydrogels Research Articles

An Aptamer-Based Hydrogel Sensing Platform Enabling Low Micromolar Detection of Small Molecules Using Low Sample Volumes.

Poor fluorescence recovery at low analyte dosages and slow ligand binding kinetics are critical challenges currently limiting the use of aptamer-functionalized hydrogels for sensing small molecules. In this paper, we report an adenosine-responsive hydrogel sensor that integrates FRET-signaling aptamer switches into in situ-gelling thin-film hydrogels. The hydrogel sensor is able to entrap a high proportion of the sensing probes (>70% following vigorous washing), delay nucleolytic degradation, stabilize weak aptamer complexes to improve hybridization affinity and suppress fluorescence background, and provide high sensitivity in biological fluids (i.e., undiluted human serum). Furthermore, the developed hydrogel sensors were able to achieve low limits of detection (5.3 μM in buffer and 8.8 μM in serum) within 4 min of exposure to the sample, with signal generation requiring only 20 μL/well of analyte sample. The physical nature of the aptamer encapsulation allows this approach to accommodate virtually any small-molecule aptamer, avoiding the need for covalent anchoring and the complex modification of nucleic acid sequences typically required for effective aptamer-based molecular recognition.

Aptamer-functionalized hydrogels promote bone healing by selectively recruiting endogenous bone marrow mesenchymal stem cells

Bone regeneration heavily relies on bone marrow mesenchymal stem cells (BMSCs). However, recruiting endogenous BMSCs for in situ bone regeneration remains challenging. In this study, we developed a novel BMSC-aptamer (BMSC-apt) functionalized hydrogel (BMSC-aptgel) and evaluated its functions in recruiting BMSCs and promoting bone regeneration. The functional hydrogels were synthesized between maleimide-terminated 4-arm polyethylene glycols (PEG) and thiol-flanked PEG crosslinker, allowing rapid in situ gel formation. The aldehyde group-modified BMSC-apt was covalently bonded to a thiol-flanked PEG crosslinker to produce high-density aptamer coverage on the hydrogel surface. In vitro and in vivo studies demonstrated that the BMSC-aptgel significantly increased BMSC recruitment, migration, osteogenic differentiation, and biocompatibility. In vivo fluorescence tomography imaging demonstrated that functionalized hydrogels effectively recruited DiR-labeled BMSCs at the fracture site. Consequently, a mouse femur fracture model significantly enhanced new bone formation and mineralization. The aggregated BMSCs stimulated bone regeneration by balancing osteogenic and osteoclastic activities and reduced the local inflammatory response via paracrine effects. This study's findings suggest that the BMSC-aptgel can be a promising and effective strategy for promoting in situ bone regeneration.

Development of a Biomimetic Extracellular Matrix with Functions of Protein Sequestration and Cell Attachment Using Dual Aptamer-Functionalized Hydrogels.

The extracellular matrix (ECM) has not only cell-binding sites for cell attachment but also protein-binding sites for molecular sequestration. Aptamers have high binding affinities and specificities against their target molecules. Thus, the purpose of this work was to develop dual aptamer-functionalized hydrogels for simultaneously recapitulating the two key features of the ECM in binding cells and sequestering proteins. We synthesized the hydrogels using free-radical polymerization in a freezing procedure. As the hydrogels were macroporous with pores of 40-50 μm, both cells and proteins could be loaded into the hydrogels after the synthesis. Importantly, the vascular endothelial growth factor (VEGF) aptamer improved VEGF sequestration and reduced the apparent diffusivity of VEGF by over 2 orders of magnitude, resultantly prolonging VEGF retention and release. The c-MET aptamer promoted the attachment of endothelial cells in the hydrogel network. When two aptamers were both incorporated into the hydrogel, they could produce synergistic effects on cell survival and growth. Thus, this work has successfully demonstrated the potential of developing biomimetic ECMs with two key functions of cell attachment and protein sequestration using dual aptamer-functionalized hydrogels.

Aptamer-functionalized hydrogels: An emerging class of biomaterials for protein delivery, cell capture, regenerative medicine, and molecular biosensing.

Molecular recognition is essential to the development of biomaterials. Aptamers are a unique class of synthetic ligands interacting with not only their target molecules with high affinities and specificities but also their complementary sequences with high fidelity. Thus, aptamers have recently attracted significant attention in the development of an emerging class of biomaterials, that is, aptamer-functionalized hydrogels. In this review, we introduce the methods of incorporating aptamers into hydrogels as pendant motifs or crosslinkers. We further introduce the functions of these hydrogels in recognizing proteins, cells, and analytes through four applications including protein delivery, cell capture, regenerative medicine, and molecular biosensing. Notably, as aptamer-functionalized hydrogels have the characteristics of both aptamers and hydrogels, their potential applications are broad and beyond the scope of this review. This article is categorized under: Biology-Inspired Nanomaterials > Nucleic Acid-Based Structures Implantable Materials and Surgical Technologies > Nanomaterials and Implants Therapeutic Approaches and Drug Discovery > Emerging Technologies.

Exogenous Signaling Molecules Released from Aptamer-Functionalized Hydrogels Promote the Survival of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cells (MSCs) have a very low survival rate after in vivo delivery, which limits their great promise for treating human diseases. Various strategies have been studied to overcome this challenge. However, an overlooked but important potential is to apply exogenous signaling molecules as biochemical cues to promote MSC survival, presumably because it is well-known that MSCs themselves can release a variety of potent signaling molecules. Thus, the purpose of this work was to examine and understand whether the release of exogenous signaling molecules from hydrogels can promote the survival of MSC spheroids. Our data show that more vascular endothelial growth factor (VEGF) but not platelet-derived growth factor BB (PDGF-BB) were released from MSC spheroids in comparison with 2D cultured MSCs. Aptamer-functionalized fibrin hydrogel (aFn) could release exogenous VEGF and PDGF-BB in a sustained manner. PDGF-BB-loaded aFn promoted MSC survival by ∼70% more than VEGF-loaded aFn under the hypoxic condition in vitro. Importantly, PDGF-BB-loaded aFn could double the survival rate of MSC spheroids in comparison with VEGF-loaded aFn during the one-week test in vivo. Therefore, this work demonstrated that defined exogenous signaling molecules (e.g., PDGF-BB) can function as biochemical cues for promoting the survival of MSC spheroids in vivo.

Enrichment of endogenous fractalkine and anti-inflammatory cells via aptamer-functionalized hydrogels

Early recruitment of non-classical monocytes and their macrophage derivatives is associated with augmented tissue repair and improved integration of biomaterial constructs. A promising therapeutic approach to recruit these subpopulations is by elevating local concentrations of chemoattractants such as fractalkine (FKN, CX3CL1). However, delivering recombinant or purified proteins is not ideal due to their short half-lives, suboptimal efficacy, immunogenic potential, batch variabilities, and cost. Here we report an approach to enrich endogenous FKN, obviating the need for delivery of exogenous proteins. In this study, modified FKN-binding-aptamers are integrated with poly(ethylene glycol) diacrylate to form aptamer-functionalized hydrogels ("aptagels") that localize, dramatically enrich and passively release FKN invitro for at least one week. Implantation in a mouse model of excisional skin injury demonstrates that aptagels enrich endogenous FKN and stimulate significant local increases in Ly6CloCX3CR1hi non-classical monocytes and CD206+ M2-like macrophages. The results demonstrate that orchestrators of inflammation can be manipulated without delivery of foreign proteins or cells and FKN-aptamer functionalized biomaterials may be a promising approach to recruit anti-inflammatory subpopulations to sites of injury. Aptagels are readily synthesized, highly customizable and could combine different aptamers to treat complex diseases in which regulation or enrichment of multiple proteins may be therapeutic.

Displacement and hybridization reactions in aptamer-functionalized hydrogels for biomimetic protein release and signal transduction.

A variety of hydrogels have been synthesized for controlling the release of signaling molecules in applications such as drug delivery and regenerative medicine. However, it remains challenging to synthesize hydrogels with the ability to control the release of signaling molecules sequentially or periodically under physiological conditions as living cells do in response to the variation of metabolism. The purpose of this work was to study a novel biomimetic hydrogel system with the ability of recapitulating the procedure of cellular signal transduction and controlling the sequential release of signaling molecules under physiological conditions. In the presence of a small chemical, the signaling molecule is regulated to change from a DNA-bound state to a free state and the freed signaling molecule is able to regulate intracellular signal transduction and cell migration. Moreover, periodic exposure of the hydrogel system to the small chemical leads to sequential protein release. Since signaling molecules are important for every activity of the cell, this hydrogel system holds potential as a metabolism-responsive platform for controlled release of signaling molecules and cell regulation in various applications.

Molecularly regulated release of growth factors from programmable hydrogels for angiogenesis

Event Abstract Back to Event Molecularly regulated release of growth factors from programmable hydrogels for angiogenesis Yong Wang1*, Nan Zhao1, Xiaolong Zhang1*, Li-Juan Duan2* and Guo-Hua Fong2* 1 Penn State University, Department of Biomedical Engineering, United States 2 UConn Health Center, Center for Vascular Biology, United States Introduction: Vascularization is important to treatment of ischemic diseases and survival of tissue-engineered constructs[1]. Delivery of growth factors is a promising strategy for vascularization. However, polymeric systems for growth factor delivery face several challenges including denaturation of growth factors, difficulty of controlling growth factor release kinetics, and/or lack of macroporous structures for cell growth[2]. The purpose of this work was to demonstrate that oligonucleotide aptamers and macroporous hydrogels could be used to develop a multifunctional biomaterial with great potential of overcoming all of these challenges. Materials and Methods: The multifunctional hydrogel was synthesized in a cylindrical mold with free radical polymerization and gas formation[3]. The key components in the pregel solution were oligonucleotide aptamers and acrylated gelatin that were incorporated into the hydrogel during the polymerization. The incorporation efficiency was determined by protein and oligonucleotide staining assays. The hydrogel was thoroughly washed to remove unreacted monomers and immersed in solution with VEGF and PDGF-BB for growth factor sequestration. The kinetics of growth factor release was examined using ELISA. The bioactivity of released or retained growth factors was measured using the standard HUVEC tube formation assay. To demonstrate directly the ability of this new hydrogel in promoting angiogenesis, the hydrogels with growth factors were subcutaneously implanted into a mouse model. The hydrogels were collected and analyzed after 4-week implantation. Endothelial cell surface biomarker CD31 was stained to examine populated vascular lumens in the hydrogels and tissues adjacent to the hydrogels. Results and Discussion: Macroporous hydrogels were successfully synthesized with free radical polymerization coupled with gas formation. The SEM images show that macroporous structures had an average pore diameter of 50 microns (Figure 1a). Both aptamers and gelatin were efficiently incorporated into the hydrogel network. Importantly, growth factors could be rapidly sequestered into the hydrogel network. When the superporous hydrogels were immersed into release media, aptamers effectively reduced the burst release effect and prolonged the growth factor release with desired kinetics. Moreover, the released and retained growth factors maintained high bioactivity (Figure 1b). For instance, at the end of two weeks, VEGF could maintain ~50% bioactivity that is much higher than the data reported in the literature. The animal studies showed that the multifunctional hydrogel stimulated angiogenesis much more effectively than the control hydrogels without aptamers (Figure 1c). Conclusions: Aptamer-functionalized hydrogels are a promising multifunctional biomaterial for controlled growth factor release. They hold great potential to stimulate vascularization for treatment of ischemic diseases or enhance survival of tissue-engineered constructs. U.S. NSF CAREER program under award number DMR-1332351; the National Heart, Lung, and Blood Institute of the NIH under award number R01HL122311

Aptamer-functionalized hydrogel as effective anti-cancer drugs delivery agents

An aptamer-functionalized hydrogel has been developed, which can be regulated by the AS1411 aptamer with the sol–gel conversion. Also the hydrogel can be further utilized for the controlled encapsulation and release of the cancer drugs. Specially, the AS1411 initiates the hybridization of acrydite-modified oligonucleotides to form the hydrogels and the presence of the target protein nucleolin leads the gel to dissolve as a result of reducing the cross-linking density by competitive target-aptamer binding. Based on the rheology of hydrogels, it is possible to utilize this material for storing and releasing molecules. In this research, the cancer drug doxorubicin is encapsulated inside the gel during the formation of the hydrogel and then released in the presence of nucleolin. Further experiments are carried out to prove the specific recognition of target matter. In vitro researches confirm that the aptamer-functionalized hydrogels can be used as drug carriers in targeted therapy and other biotechnological applications.

Endonuclease-responsive aptamer-functionalized hydrogel coating for sequential catch and release of cancer cells

Rare circulating tumor cells are a promising biomarker for the detection, diagnosis, and monitoring of cancer. However, it remains a challenge to develop biomedical devices for specific catch and nondestructive release of circulating tumor cells. The purpose of this study was to explore a unique system for cell catch and release by using aptamer-functionalized hydrogels and restriction endonucleases. The results show that the hydrogel coating was highly resistant to nonspecific cell binding with ∼5–15 cells/mm2 on the hydrogel surface. In contrast, under the same condition, the aptamer-functionalized hydrogel coating could catch target cancer cells with a density over 1000 cells/mm2. When the hydrogel coating was further treated with the restriction endonucleases, the bound cells were released from the hydrogel coating because of the endonuclease-mediated sequence-specific hydrolysis of the aptamer sequences. The release efficiency reached ∼99%. Importantly, ∼98% of the released cells maintained viability. Taken together, this study demonstrates that it is promising to apply endonuclease-responsive aptamer-functionalized hydrogels as a coating material to develop medical devices for specific catch and nondestructive release of rare circulating tumor cells.

Programmable Release of Multiple Protein Drugs from Aptamer-Functionalized Hydrogels via Nucleic Acid Hybridization

Polymeric delivery systems have been extensively studied to achieve localized and controlled release of protein drugs. However, it is still challenging to control the release of multiple protein drugs in distinct stages according to the progress of disease or treatment. This study successfully demonstrates that multiple protein drugs can be released from aptamer-functionalized hydrogels with adjustable release rates at predetermined time points using complementary sequences (CSs) as biomolecular triggers. Because both aptamer-protein interactions and aptamer-CS hybridization are sequence-specific, aptamer-functionalized hydrogels constitute a promising polymeric delivery system for the programmable release of multiple protein drugs to treat complex human diseases.

Aptamer-Functionalized Hydrogel Microparticles for Fast Visual Detection of Mercury(II) and Adenosine

With a low optical background, high loading capacity, and good biocompatibility, hydrogels are ideal materials for immobilization of biopolymers to develop optical biosensors. We recently immobilized mercury and lead binding DNAs within a monolithic gel and demonstrated ultrasensitive visual detection of these heavy metals. The high sensitivity was attributed to the enrichment of the analytes into the gels. The signaling kinetics was slow, however, taking about 1 h to obtain a stable optical signal because of a long diffusion distance. In this work, we aim to understand the analyte enrichment process and improve the signaling kinetics by preparing hydrogel microparticles. DNA-functionalized gel beads were synthesized using an emulsion polymerization technique and most of the beads were between 10 and 50 μm. Acrydite-modified DNA was incorporated by copolymerization. Visual detection of 10 nM Hg(2+) was still achieved and a stable signal was obtained in just 2 min. The gel beads could be spotted to form a microarray and dried for storage. A new visual sensor for adenosine was designed and immobilized within the gel beads. The adenosine aptamer binds its target about 1000-fold less tightly compared to the mercury binding DNA, allowing a comparison to be made on analyte enrichment by aptamer-functionalized hydrogels.

Cell adhesion on an artificial extracellular matrix using aptamer-functionalized PEG hydrogels

The development of an artificial extracellular matrix (ECM) is important to regenerative medicine because the ECM plays complex and dynamic roles in the regulation of cell behavior. In this study, nucleic acid aptamers were applied to functionalize hydrogels for mimicking the adhesion sites of the ECM. The results showed that nucleic acid aptamers could be incorporated into polyethylene glycol (PEG) hydrogels via free radical polymerization. The incorporation of the aptamers produced only a moderate effect on the mechanical properties of the PEG hydrogels. Importantly, the results also showed that the aptamers effectively induced cell type-specific adhesion to the PEG hydrogels without affecting cell viability. The cell adhesion was a function of the aptamer concentration, the spacer length and the cell seeding time. In addition, cell adhesion to the aptamer-functionalized hydrogel could be attenuated by means of aptamer inactivation in a physiological condition. Thus, aptamer-functionalized hydrogels are promising biomaterials for the development of artificial ECMs.

Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides

Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer–protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer–protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.