Elastic Macroporous Matrix-Supported In Situ Formation of Injectable Extracellular Matrix-Like Hydrogel for Carrying Growth Factors and Living Cells.

Hydrogels loaded with biologics hold great potential for various biomedical applications such as regenerative medicine. However, biologics may lose bioactivity during hydrogel preparation, shipping, and storage. While many injectable hydrogels do not have this issue, they face a dilemma between fast gelation causing the difficulty of injection and slow gelation causing the escape of solutions from an injection site. The purpose of this study is to develop an affinity hydrogel by integrating a pre-formed elastic macroporous matrix and an injectable hydrogel. The data shows that the macroporous hydrogel matrix can hold a large volume of solutions for the formation of in situ injectable hydrogels loaded with growth factors or living cells. The cells can proliferate in the composite hydrogels. The growth factors can be stably sequestered and sustainably released due to the presence of aptamers. When both living cells and growth factors are loaded together into the hydrogels, cells can proliferate under culture conditions with a reduced serum level. Therefore, a macroporous and elastic matrix-supported formation of aptamer-functionalized injectable hydrogels is a promising method for developing the carriers of biologics.

Biomimetic Bacterial Capsule for Enhanced Aptamer Display and Cell Recognition.

Great effort has been made to encapsulate or coat living mammalian cells for a variety of applications ranging from diabetes treatment to three-dimensional printing. However, no study has reported the synthesis of a biomimetic bacterial capsule to display high-affinity aptamers on the cell surface for enhanced cell recognition. Therefore, we synthesized an ultrathin alginate-polylysine coating to display aptamers on the surface of living cells with natural killer (NK) cells as a model. The results show that this coating-mediated aptamer display is more stable than direct cholesterol insertion into the lipid bilayer. The half-life of the aptamer on the cell surface can be increased from less than 1.5 to over 20 h. NK cells coated with the biomimetic bacterial capsule exhibit a high efficiency in recognizing and killing target cells. Therefore, this work has demonstrated a promising cell coating method for the display of aptamers for enhanced cell recognition.

Display of Polyvalent Hybrid Antibodies on the Cell Surface for Enhanced Cell Recognition.

Cell surface engineering with exogeneous receptors holds great promise for various applications. However, current biological methods face problems with safety, antigen escape, and receptor stoichiometry. The purpose of this study is to develop a biochemical method for displaying polyvalent antibodies (PAbs) on the cell surface. The PAbs are synthesized through the self-assembly of DNA-Ab conjugates under physiological conditions without the involvement of any factors harsh to cells. The data show that PAb-functionalized cells can recognize target cells much more effectively than monovalent controls. Moreover, dual Ab incorporation into the same PAb with a defined stoichiometric ratio leads to the formation of a polyvalent hybrid Ab (DPAb). DPAb-functionalized cells can effectively recognize target cell models with antigen escape, which cannot be achieved by PAbs with one type of Ab. Therefore, this work presents a novel biochemical method for Ab display on the cell surface for enhanced cell recognition.

High-affinity one-step aptamer selection using a non-fouling porous hydrogel.

Aptamers, commonly referred to as chemical antibodies, are used in a wide range of applications including drug delivery and biosensing. However, the process of aptamer selection poses a substantial challenge, as it requires numerous cycles of enrichment and involves issues with nonspecific binding. We present a simple, fast instrument-free method for aptamer enrichment and selection based on a diffusion-binding process in a three-dimensional non-fouling porous hydrogel with immobilized target proteins. Low-affinity aptamer candidates can be rapidly released from the hydrogel, whereas high-affinity candidates are restricted due to their strong binding to the immobilized protein targets. Consequently, a one-step enriched aptamer pool can strongly bind the protein targets. This enrichment is consistent across five proteins with isoelectric points in varying ranges. With thrombin as a representative model, the anti-thrombin aptamer identified from an enriched aptamer pool has been found to have a binding affinity that is comparable to those identified over ten cycles of selection using traditional methods.

Deoxyribonucleic Acid-Based Polyvalent Ligand-Receptor Binding for Engineering the Cell Surface with Nanoparticles.

Tethering nanoparticles (NPs) onto the cell surface is critical to cellular hitchhiking applications, such as targeted NP delivery and enhanced cell therapy. While numerous methods have been developed to achieve NP attachment onto the cell membrane, they often face limitations such as the use of complicated cell surface modifications or low-efficiency NP attachment. The purpose of this work was to explore a DNA-based synthetic ligand-receptor pair for NP attachment to the surface of live cells. Polyvalent ligand mimics were used to functionalize NPs, while the cell membrane was functionalized with DNA-based cell receptor mimics. Base pair-directed polyvalent hybridization allowed the NPs to bind to the cells quickly and efficiently. Notably, the process of attaching NPs to cells did not require sophisticated chemical conjugation on the cell membrane or involve any cytotoxic cationic polymers. Therefore, DNA-based polyvalent ligand-receptor binding is promising to various applications ranging from cell surface engineering to NP delivery.

Nanoparticle-Decorated Biomimetic Extracellular Matrix for Cell Nanoencapsulation and Regulation.

Cell encapsulation has been studied for various applications ranging from cell transplantation to biological production. However, current encapsulation technologies focus on cell protection rather than cell regulation that is essential to most if not all cell-based applications. Here we report a method for cell nanoencapsulation and regulation using an ultrathin biomimetic extracellular matrix as a cell nanocapsule to carry nanoparticles (CN2 ). This method allows high-capacity nanoparticle retention at the vicinity of cell surfaces. The encapsulated cells maintain high viability and normal metabolism. When gold nanoparticles (AuNPs) are used as a model to decorate the nanocapsule, light irradiation transiently increases the temperature, leading to the activation of the heat shock protein 70 (HSP70) promoter and the regulation of reporter gene expression. As the biomimetic nanocapsule can be decorated with any or multiple NPs, CN2 is a promising platform for advancing cell-based applications.

Thermoresponsive semi-interpenetrating gelatin-alginate networks for encapsulation and controlled release of scent molecules.

Plant-based meats, which are nutritious foods from non-animal sources, provide clues for addressing the negative externalities associated with conventional meat production. Interest in plant-based meat has increased and is driving the rapid growth of its market. Plant-based meat should be equipped with a temperature-dependent scent release system similar to the scent release mechanism of conventional meat, to deliver a desirable meat-like flavor to consumers and obtain higher market acceptance. In this study, we prepared thermoresponsive gelatin-alginate hybrid hydrogels to control the release of scent molecules. The polymer network of gelatin-alginate hydrogels was reinforced by a semi-interpenetrating network (sIPN). sIPN formation conferred resistance to external stimuli, such as shear force, swelling, and temperature, resulting in a sustained release of the meat scent. In addition, controlled size microcapsules fabricated from the same composition via an electrostatic extrusion process showed a sustained release pattern of the loaded scent at 70 °C, and the scent release rate was precisely controlled within an approximately 2-fold range by adjusting the alginate concentration. These observations suggest the potential use of edible biological macromolecules as food additives that can control the release of scent molecules from the plant-based meat during cooking.

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.

Synthetic DNA for Cell Surface Engineering: Experimental Comparison between Click Conjugation and Lipid Insertion in Terms of Cell Viability, Engineering Efficiency, and Displaying Stability.

The cell surface can be engineered with synthetic DNA for various applications ranging from cancer immunotherapy to tissue engineering. However, while elegant methods such as click conjugation and lipid insertion have been developed to engineer the cell surface with DNA, little effort has been made to systematically evaluate and compare these methods. Resultantly, it is often challenging to choose a right method for a certain application or to interpret data from different studies. In this study, we systematically evaluated click conjugation and lipid insertion in terms of cell viability, engineering efficiency, and displaying stability. Cells engineered with both methods can maintain high viability when the concentration of modified DNA is less than 25-50 µM. However, lipid insertion is faster and more efficient in displaying DNA on the cell surface than click conjugation. The efficiency of displaying DNA with lipid insertion is 10-40 times higher than that with click conjugation for a large range of DNA concentration. However, the half-life of physically inserted DNA on the cell surface is 3-4 times lower than that of covalently conjugated DNA, which depends on the working temperature. While the half-life of physically inserted DNA molecules on the cell surface is shorter than that of DNA molecules clicked onto the cell surface, lipid insertion is more effective than click conjugation in the promotion of cell-cell interactions under the two different experimental settings. The data acquired in this work are expected to act as a guideline for choosing an approximate method for engineering the cell surface with synthetic DNA or even other biomolecules.

Multifunctional DNA Nanogels for Aptamer-Based Targeted Delivery and Stimuli-Triggered Release of Cancer Therapeutics.

Targeted, stimulus-responsive DNA nanogels hold considerable promise for cancer therapeutics. To expand their functionality including thermoresponsiveness, here, multifunctional DNA nanogels are developed for potential application toward cancer-targeted delivery and stimuli-responsive release of cancer therapeutics. Three types of functionalized DNA nanobuilding units are formed into DNA nanogels of ≈200 nm via sequence-dependent self-assembly. The sequence-dependent assembly of nanobuilding units is precisely designed for controlled assembly and thermal disassembly at physiological temperatures. The supramolecular structure exhibits multifunctionalities including temperature-induced disassembly, aptamer-mediated cancer cell targeting, and light-triggered temperature increase. The nanogels support co-loading of cancer therapeutics including anti-sense oligonucleotides and doxorubicin along with stimuli-responsive release of loaded drugs through temperature-responsive structural disassembly and pH-responsive deintercalation. The nanogels exhibit efficient aptamer-mediated cancer-specific intracellular delivery and combinational anticancer effects upon light triggering. The developed DNA nanogels, thus, constitute potential noncationic nanovectors for targeted delivery of combinational cancer therapeutics.