Epitope‐Imprinted Nanoparticles as Transforming Growth Factor‐β3 Sequestering Ligands to Modulate Stem Cell Fate

Abstract

Growth factors (GFs) are biomolecules with potent biological effects but inherent limitations hinder their potential as therapeutic agents and cell culture supplements in tissue engineering and regenerative medicine (TERM). Biomaterials that sequester endogenous GFs by affinity binding might circumvent such limitations and thus are being increasingly investigated. Here, molecularly imprinted nanoparticles (MINPs) are proposed as specific abiotic ligands for GFs. As a proof of concept, a conformational epitope of transforming growth factor‐β3 (TGF‐β3) is designed and surface imprinted onto polyacrylamide‐based nanoparticles by inverse microemulsion polymerization. It is found that, depending on the polymerization mixture composition, MINPs can recognize and preferentially bind TGF‐β3, either in noncompetitive assays or from a complex human fluid (platelet lysate). Substrates functionalized with MINPs are then used for 2D culture of adipose‐derived stem cells. Remarkably, gene and protein expression profiles show a marked upregulation of SOX‐9, suggesting activation of TGF‐β3 signaling pathways without requiring supplementation with exogenous GF. Likewise, culturing these cells in pellets incorporating MINPs previously incubated with platelet lysate results in higher collagen II‐rich matrix deposition, compared to pellets incorporating non‐imprinted nanoparticles. In summary, results suggest MINPs can be used as cost‐effective, stable, and scalable alternative abiotic GF ligands to guide cell fate in TERM applications.

Epitope-imprinted polymers: Design principles of synthetic binding partners for natural biomacromolecules

“…Our laboratories have come up with novel methods for the production of materials capable of binding and recognizing specific proteins (44)(45)(46). To minimize the diffusive limitation of a material imprinted in the bulk, we have focused on surface adsorption (47)(48)(49). Correspondingly, our methods have been designed for the production of micro-or nanoparticles (NPs), to produce materials with large surfaceto-volume ratios.…”

“…To these ends, we have developed a surface imprinting technique whereby the template molecule is adsorbed at the interface between two distinct phases. Agitation during polymerization results in the formation of droplets, with subsequent polymerization yielding microparticles or NPs (48).…”

“…It is also crucial to assess the selectivity of the material, which is done by comparing its behavior when in contact with the target molecule versus other substances. This can be performed separately (one compound at a time) or in a competitive environment (mixture with multiple macromolecules) (47,48). From these experiments, it is possible to calculate another parameter, the selectivity coefficient (usually represented by the Greek letter ), relative to each competitor molecule, given by Eq.…”

“…The previous strategies have recently been combined by our groups to design a conformational epitope for transforming growth factor-3 (TGF-3) (48). The protein structure was scanned for surface-exposed domains that did not interfere in receptor binding using The US Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB) (126,127) and PyMOL software, followed by BLASTP to score their uniqueness.…”

Molecular Imprinting Strategies for Tissue Engineering Applications: A Review

“…The results showed that the nanoparticles could selectively bind the GF both in noncompetitive assays and in complex human fluid (platelet lysate). Human adipose stem cells, when incubated with platelet lysate and the imprinted nanoparticles, resulted in a higher collagen matrix deposition when compared to the control (non-imprinted nanoparticles) [ 111 ].…”