Bio-Orthogonal Mediated Nucleic Acid Transfection of Cells via Cell Surface Engineering.

The efficient delivery of foreign nucleic acids (transfection) into cells is a critical tool for fundamental biomedical research and a pillar of several biotechnology industries. There are currently three main strategies for transfection including reagent, instrument, and viral based methods. Each technology has significantly advanced cell transfection; however, reagent based methods have captured the majority of the transfection market due to their relatively low cost and ease of use. This general method relies on the efficient packaging of a reagent with nucleic acids to form a stable complex that is subsequently associated and delivered to cells via nonspecific electrostatic targeting. Reagent transfection methods generally use various polyamine cationic type molecules to condense with negatively charged nucleic acids into a highly positively charged complex, which is subsequently delivered to negatively charged cells in culture for association, internalization, release, and expression. Although this appears to be a straightforward procedure, there are several major issues including toxicity, low efficiency, sorting of viable transfected from nontransfected cells, and limited scope of transfectable cell types. Herein, we report a new strategy (SnapFect) for nucleic acid transfection to cells that does not rely on electrostatic interactions but instead uses an integrated approach combining bio-orthogonal liposome fusion, click chemistry, and cell surface engineering. We show that a target cell population is rapidly and efficiently engineered to present a bio-orthogonal functional group on its cell surface through nanoparticle liposome delivery and fusion. A complementary bio-orthogonal nucleic acid complex is then formed and delivered to which chemoselective click chemistry induced transfection occurs to the primed cell. This new strategy requires minimal time, steps, and reagents and leads to superior transfection results for a broad range of cell types. Moreover the transfection is efficient with high cell viability and does not require a postsorting step to separate transfected from nontransfected cells in the cell population. We also show for the first time a precision transfection strategy where a single cell type in a coculture is target transfected via bio-orthogonal click chemistry.

General Dialdehyde Click Chemistry for Amine Bioconjugation.

The development of methods for conjugating a range of molecules to primary amine functional groups has revolutionized the fields of chemistry, biology, and material science. The primary amine is a key functional group and one of the most important nucleophiles and bases used in all of synthetic chemistry. Therefore, tremendous interest in the synthesis of molecules containing primary amines and strategies to devise chemical reactions to react with primary amines has been at the core of chemical research. In particular, primary amines are a ubiquitous functional group found in biological systems as free amino acids, as key side chain lysines in proteins, and in signaling molecules and metabolites and are also present in many natural product classes. Due to its abundance, the primary amine is the most convenient functional group handle in molecules for ligation to other molecules for a broad range of applications that impact all scientific fields. Because of the primary amine's central importance in synthetic chemistry, acid-base chemistry, redox chemistry, and biology, many methods have been developed to efficiently react with primary amines, including activated carboxylic acids, isothiocyanates, Michael addition type systems, and reaction with ketones or aldehydes followed by in situ reductive amination. Herein, we introduce a new traceless, high-yield, fast click-chemistry method based on the rapid and efficient trapping of amine groups via a functionalized dialdehyde group. The click reaction occurs in mild conditions in organic solvents or aqueous media and proceeds in high yield, and the starting dialdehyde reagent and resulting dialdehyde click conjugates are stable. Moreover, no catalyst or dialdehyde-activating group is required, and the only byproduct is water. The initial dialdehyde and the resulting conjugate are both straightforward to characterize, and the reaction proceeds with high atom economy. To demonstrate the broad scope of this new click-conjugation strategy, we designed a straightforward scheme to synthesize a suite of dialdehyde reagents. The dialdehyde molecules were used for applications in cell-surface engineering and for tailoring surfaces for material science applications. We anticipate the broad utility of the general dialdehyde click chemistry to primary amines in all areas of chemical research, ranging from polymers and bioconjugation to material science and nanoscience.

Scaffold Free Bio-orthogonal Assembly of 3-Dimensional Cardiac Tissue via Cell Surface Engineering.

There has been tremendous interest in constructing in vitro cardiac tissue for a range of fundamental studies of cardiac development and disease and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress towards studying 2-dimensional cardiac function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free 3-dimensional multiple cell type co-culture cardiac tissue models. Herein, we develop a programmed rapid self-assembly strategy to induce specific and stable cell-cell contacts among multiple cell types found in heart tissue to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a scaffold free and stable self assembled 3 cell line co-culture 3D cardiac tissue model by assembling cardiomyocytes, endothelial cells and cardiac fibroblast cells via a rapid inter-cell click ligation process. We compare and analyze the function of the 3D cardiac tissue chips with 2D co-culture monolayers by assessing cardiac specific markers, electromechanical cell coupling, beating rates and evaluating drug toxicity.

Generation of a Scaffold-Free Three-Dimensional Liver Tissue via a Rapid Cell-to-Cell Click Assembly Process.

There has been tremendous interest in constructing in vitro liver organ models for a range of fundamental studies of cell signaling, metabolism, and infectious diseases, and as a commercial system to evaluate therapeutic drug discovery prioritization and toxicity. Although there has been progress toward studying two-dimensional hepatic function in vitro, there remain challenging obstacles to generate rapid and efficient scaffold-free three-dimensional multiple cell line coculture tissue models of liver. Herein, we develop and employ a strategy to induce specific and stable cell-cell contacts among multiple hepatic cell lines to generate 3D tissues through cell-surface engineering based on liposome delivery and fusion to display bio-orthogonal functional groups from cell membranes. We generate, for the first time, a three cell line coculture 3D liver tissue model by assembling hepatocytes, hepatic endothelial cells, and hepatic stellate cells via a rapid intercell click ligation process. We compare and analyze the function of the superior 3D liver tissue chips with 2D coculture monolayer by assessing mitochondrial metabolic activity and evaluating drug toxicity.

Rewiring Gram-Negative Bacteria Cell Surfaces with Bio-Orthogonal Chemistry via Liposome Fusion.

The ability to tailor bacteria cell surfaces with non-native molecules is critical to advance the study of bacteria communication, cell behavior, and for next-generation therapeutics to improve livestock and human health. Such modifications would allow for novel control over cell behavior, cell-cell interactions, biofilm formation, adjuvant conjugation, and imaging. Current methods to engineer bacteria surfaces have made major advances but rely on complicated, slow, and often expensive molecular biology and metabolic manipulation methods with limited scope on the type of molecules installed onto the surface. In this report, we introduce a new straightforward method based on liposome fusion to engineer Gram-negative bacteria cells with bio-orthogonal groups that can subsequently be conjugated to a range of molecules (biomolecules, small molecules, probes, proteins, nucleic acids, ligands, and radiolabels) for further studies and programmed behavior of bacteria. This method is fast, efficient, inexpensive, and useful for installing a broad scope of ligands and biomolecules to Gram-negative bacteria surfaces.

Spheroid and Tissue Assembly via Click Chemistry in Microfluidic Flow.

Proper cell-cell contact and communication are essential for the correct development and survival of higher order organisms. In order to study complex cell interactions that occur in vivo, model systems that are able to recapitulate 3D cell-cell interactions in vitro are key to advancing new biotechnologies, therapeutics, and tissue engineering applications. Herein, we show a new strategy to rapidly and efficiently generate complex multiple cell line containing spheroids and tissues in microfluidic flow without the use of scaffolds, molecular biology, or metabolic biosynthesis. The method relies on the integration of microfluidics, liposome fusion, bio-orthogonal chemistry, and cell surface engineering to rapidly click coculture cell assemblies in flow. We demonstrate this strategy by assembling various combinations of cell types with an interfacial cell to cell click chemistry in microfluidic flow to generate a range of spheroid types and oriented tissue multilayers.

Photoreceptor cell death, proliferation and formation of hybrid rod/S-cone photoreceptors in the degenerating STK38L mutant retina.

A homozygous mutation in STK38L in dogs impairs the late phase of photoreceptor development, and is followed by photoreceptor cell death (TUNEL) and proliferation (PCNA, PHH3) events that occur independently in different cells between 7-14 weeks of age. During this period, the outer nuclear layer (ONL) cell number is unchanged. The dividing cells are of photoreceptor origin, have rod opsin labeling, and do not label with markers specific for macrophages/microglia (CD18) or Müller cells (glutamine synthetase, PAX6). Nestin labeling is absent from the ONL although it labels the peripheral retina and ciliary marginal zone equally in normals and mutants. Cell proliferation is associated with increased cyclin A1 and LATS1 mRNA expression, but CRX protein expression is unchanged. Coincident with photoreceptor proliferation is a change in the photoreceptor population. Prior to cell death the photoreceptor mosaic is composed of L/M- and S-cones, and rods. After proliferation, both cone types remain, but the majority of rods are now hybrid photoreceptors that express rod opsin and, to a lesser extent, cone S-opsin, and lack NR2E3 expression. The hybrid photoreceptors renew their outer segments diffusely, a characteristic of cones. The results indicate the capacity for terminally differentiated, albeit mutant, photoreceptors to divide with mutations in this novel retinal degeneration gene.