Mineral-Coated Microparticles Enhance mRNA-Based Transfection of Human Bone Marrow Cells

Abstract

The regenerative potential of bone marrow cells could be harnessed for tissue engineering applications. Bone marrow can be easily collected from patients, providing a valuable autologous source of therapeutic cells. However, years of delivery of bone marrow cells have highlighted the need for their genetic manipulation to overcome heterogeneity and to confer specificity to the regenerative process. In this study, we optimized the use of condensed mRNA as a non-viral alternative. As a proof of concept, we used mRNA encoding for reporter proteins such as EGFP or Firefly luciferase, which was condensed by complexing agents and delivered to human bone marrow cells using mineral-coated microparticles. We demonstrated that human bone marrow cells could be transfected with complexed mRNA, and that this approach was more efficient than the delivery of complexed plasmid DNA. In addition, human bone marrow cells were vulnerable to the toxicity of mRNA complexing agents, but these deleterious effects were mitigated by using mineral-coated microparticles as a carrier of complexed mRNA. Microparticle-mediated delivery of complexed mRNA also enabled higher cell metabolic activity and higher transfection in multiple in vitro culture conditions, including suspension culture and three-dimensional culture.

Sequential sequestrations increase the incorporation and retention of multiple growth factors in mineralized collagen scaffolds

“…In this manuscript, we described the use of modified simulated body fluid (mSBF) and sequential sequestrations to incorporate BMP2 and VEGF into mineralized collagen scaffolds. This work seeks to adapt promising results using mSBF to deposit a mineral layer that can sequester growth factors onto surfaces [58][59][60][61][62][63][64] to selectively incorporate growth factors used for bone repair applications into three-dimensional, porous biomaterials. Our primary goal is to improve growth factor incorporation and retention into mineralized collagen scaffolds via sequential sequestration strategies to enhance in vivo bone healing.…”

“…Modified simulated body fluid (mSBF) was made according to previous recipes [58][59][60][61][62][63][64] μm filter (Fisher Scientific, Hampton, New Hampshire USA). mSBF was stored at 4°C until use.…”

“…Our group has previously used strategies such as photopatterning 50,51 , covalent immobilization [52][53][54][55] , and sequestration modulated by glycosaminoglycan content 56 and small molecules 57 to add growth factors to non-mineralized and mineralized collagen scaffolds. Recently, the Murphy lab has described the use of modified simulated body fluid (mSBF) to create mineral coatings on material surfaces to deliver growth factors [58][59][60][61][62] , plasmid DNA lipoplexes 63 , and condensed mRNA 64 . We were particularly interested in the work by Clements et al in which they performed sequential sequestrations to layer Interleukin-1 Receptor Antagonist on nanoparticles to prolong in vivo activity 62 .…”

Calcium Phosphate Nanoparticles-Based Systems for RNAi Delivery: Applications in Bone Tissue Regeneration

“…Thus consideration of both the size and solubility of calcium phosphate nanoparticles is important in order to minimize cytotoxic effects on cells [121,126]. While HA remains the most frequently used calcium phosphate in gene delivery applications to date, amorphous calcium phosphate (ACP), beta tricalcium phosphate and dicalcium phosphate dihydrate (DCPD) have also shown promise [122,127]. There is significant scope for investigation of other calcium phosphate phases and the development of biphasic calcium phosphate-based materials that are specifically designed with tailored resorption kinetics for effective delivery of RNA [128].…”

Sequential sequestrations increase the incorporation and retention of multiple growth factors in mineralized collagen scaffolds

“…Recently, the Murphy lab has described the use of modied simulated body uid (mSBF) to create mineral coatings on material surfaces to deliver growth factors, [58][59][60][61][62] plasmid DNA lipoplexes, 63 and condensed mRNA. 64 We were particularly interested in the work by Clements et al in which they performed sequential sequestrations to layer interleukin-1 receptor antagonist on nanoparticles to prolong in vivo activity. 62 In this manuscript, we describe the use of mSBF and sequential sequestration to incorporate and retain BMP2 and VEGF in three-dimensional mineralized collagen scaffolds.…”