Biomaterial-Mediated Protein Expression Induced by Peptide-mRNA Nanoparticles Embedded in Lyophilized Collagen Scaffolds

Abstract

In our aging society, the number of patients suffering from poorly healing bone defects increases. Bone morphogenetic proteins (BMPs) are used in the clinic to promote bone regeneration. However, poor control of BMP delivery and thus activity necessitates high doses, resulting in adverse effects and increased costs. It has been demonstrated that messenger RNA (mRNA) provides a superior alternative to protein delivery due to local uptake and prolonged expression restricted to the site of action. Here, we present the development of porous collagen scaffolds incorporating peptide-mRNA nanoparticles (NPs). Nanoparticles were generated by simply mixing aqueous solutions of the cationic cell-penetrating peptide PepFect14 (PF14) and mRNA. Peptide-mRNA complexes were uniformly distributed throughout the scaffolds, and matrices fully preserved cell attachment and viability. There was a clear dependence of protein expression on the incorporated amount of mRNA. Importantly, after lyophilization, the mRNA formulation in the collagen scaffolds retained activity also at 4 °C over two weeks. Overall, our results demonstrate that collagen scaffolds incorporating peptide-mRNA complexes hold promise as off-the-shelf functional biomaterials for applications in regenerative medicine and constitute a viable alternative to lipid-based mRNA formulations.

Matrices Activated with Messenger RNA

“…This would make it feasible to reduce the dose required and, consequently, the side effects. This ability to retain expressed proteins has also been verified in collagen scaffolds [ 44 ].…”

“…Collagen is the major constituent of the ECM and one of the most used materials in the design of TAMs [ 9 , 20 , 23 , 31 , 33 , 37 , 43 , 44 , 45 , 46 , 47 ]. This material can be processed in diverse forms, such as sponges, membranes, and fibre matrices [ 31 , 43 , 47 , 48 ].…”

“…The main reason for this popularity is that they showed better transfection capacity than polymers [ 28 , 35 ]. Specifically, liposomes stand out from other structures because of their properties, such as high loading capacity [ 44 ]. However, when we are choosing a vector to integrate into TAMs, this feature may not be as advantageous as expected.…”

“…However, when we are choosing a vector to integrate into TAMs, this feature may not be as advantageous as expected. The reason lies in the fact that as they store a greater number of mRNA molecules inside, fewer nanoplexes are needed per matrix, and consequently, it is more difficult to achieve their homogeneous distribution in the system [ 44 ]. Another disadvantage of lipid systems is their complexity, as liposome formulation usually requires several components, such as structure-forming lipids, helper lipids, cholesterol, PEGylated lipids, and others [ 31 , 44 ].…”

“…Many TAMs integrate lipid-based commercial reagents such as Lipofectamine™, DreamFect™ Gold, or 3DfectIN™ [ 22 , 24 , 28 , 32 , 33 , 44 , 45 ]. The use of customized lipids has also been reported [ 9 , 21 , 31 , 34 , 43 , 66 ].…”