Metabolic glycan labeling and chemoselective functionalization of native biomaterials
Introduction
Tissue engineering aims to fabricate tissue constructs through the combination of cells and biomaterial scaffolds [1]. Because of their preserved biochemical, biomechanical and anatomical features, decellularized ECM biomaterials have been widely used in clinical applications to facilitate injury repair [[2], [3], [4]], and in organ bioengineering to facilitate cell engraftment and tissue maturation [[5], [6], [7], [8], [9], [10], [11], [12], [13], [14]]. ECM can be functionalized through the immobilization of bioactive molecules, such as growth factors and functional polysaccharides. These immobilized biomolecules endow the biomaterials with novel properties regarding their interactions with cells and host organisms. One of the common strategies for ECM functionalization utilizes amine-reactive chemical groups, such as N-hydroxysuccinimide esters, which specifically target the abundant primary amines within biological ECM [[15], [16], [17]]. However, the application of amine-reactive chemistry faces challenges when primary amines are also present in the biomolecules to be conjugated. Chemoselective chemistry utilizing bioinert chemical groups will be desired to enable efficient conjugation only between the two participating ligands with minimal side reactions. The copper-catalyzed click chemistry is one such chemoselective reaction that conjugates an azide with an alkyne, and has been widely used in bioconjugation [18,19]. Due to the biological and chemical inertness of azide and alkyne moieties in natural biological systems, the conjugation between azide- and alkyne-modified biomolecules is highly specific. However, the application of click chemistry to functionalizing decellularized ECM biomaterials has been hindered by the lack of methods for incorporating click-reactive ligands into native ECM in an efficient and biocompatible manner. In vivo metabolic labeling approaches have been developed to incorporate click-reactive ligands into amino acids, glycans, lipids and nucleic acids [[20], [21], [22], [23], [24], [25], [26], [27], [28], [29]]. By design, most studies have focused on the labeling of cellular components, and little attention has been paid to the feasibility, efficacy and stability of metabolic labeling of tissue and organ ECM.
Here we developed a metabolic glycan labeling approach that covalently incorporated click-reactive azide ligands into the ECM of a wide variety of rodent tissues and organs in vivo. We further demonstrated the feasibility of performing this metabolic ECM labeling procedure during the ex vivo culture of rat and porcine lungs, opening up the possibility of engineering the ECM of donor human organs. The azide ligands incorporated within the ECM remained stable after decellularization, and enabled chemoselective functionalization of ECM biomaterials with desired alkyne-modified biomolecules. Using azide-labeled lung ECM and heparin as an example, we demonstrated specific click-immobilization of alkyne-modified heparin, and its preserved bioactivity in immobilizing Antithrombin III and inhibiting Factor Xa activity.
Section snippets
In vivo metabolic labeling and organ/tissue decellularization
All animal experiments were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee and performed in compliance with the Animal Welfare Act. Male Sprague-Dawley rats (100–125 g, Charles River Laboratories) was administered with metabolic labeling reagents (Ac4GalNAz, Ac4GlcNAz or Ac4ManNAz) (prepared in 70% DMSO in PBS, 30 mg/day, Click Chemistry Tools) via intraperitoneal injection daily for three days. One day after the last administration of the metabolic
In vivo metabolic labeling of organ ECM in rat
To enable chemoselective modification of the ECM derived from non-genetically modified donor animals, we developed an approach to incorporate click-reactive azide into the native ECM of tissues and organs via in vivo metabolic glycan labeling. Specifically, we administered azide-modified monosaccharides (30 mg/day in 70% DMSO in PBS) to live rats (100–125 g) via intraperitoneal injection daily for three days [22], followed by organ harvest and perfusion decellularization as previously reported (
Discussion
In this study, we report a novel strategy to metabolically label native ECM biomaterials by covalent incorporation of azide ligands. This enables chemoselective functionalization of these biomaterials with desired features endowed by subsequent covalent immobilization of alkyne-modified bioactive molecules through click chemistry. We demonstrate that click-reactive azide ligands can be efficiently incorporated into the organs' ECM in vivo via physiologic metabolic pathways using Ac4GalNAz. We
Conclusion
In summary, by combining biologically selective azide incorporation into the ECM and subsequent chemoselective click ligation with desired alkyne-modified biomolecules, we provide an innovative solution to enable functionalization of native ECM biomaterials with high specificity and biocompatibility.
Acknowledgments
This study was supported by the United Therapeutics Corporation and the National Institutes of Health (NIH) Director's New Innovator Award (DP2-OD008749-01). The authors thank the Massachusetts General Hospital (MGH) Center for Skeletal Research Core (NIH P30 AR066261) for histological processing, and the MGH Knight Surgery Research Laboratory for assistance with swine surgeries.
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