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.

Keywords: Jurkat; T cells; bone marrow; bone marrow aspirates; gene therapy; mRNA delivery; microparticles; mineral coating; non-viral vectors; transfection.

Graphical abstract

Figure 1

mRNA Delivery Can Overcome Current Limitations in Gene Therapy of Bone Marrow Cells (A) Schematic representation of the hBM processing. (B) hBM stained using acridine orange dye; the images show control cells and cells exposed to complexed mRNA, respectively. The cells in red have higher amounts of mRNA. (C and D) Jurkat cells (C) and hBM cells (D) incubated with pDNA or mRNA, respectively, encoding for EGFP and complexed with lipidic complexing agents. (E and F) Quantification of EGFP-positive Jurkat cells (E) and hBM (F) shows that the delivery of complexed mRNA is more effective than pDNA. Asterisk represents statistically significant differences using paired Student’s t test (n = 4), p < 0.05.

Figure 2

MCM as a Reservoir System for the Delivery of Complexed mRNA Schematic representation of the procedure used to manufacture MCM and their loading with mRNA complexes prior to exposure to hBM cells. The MCM facilitates the interaction between cells and complexed mRNA. The rate of release of complexed mRNA can be controlled by tuning the dissolution rate of the mineral coatings.

Figure 3

Optimization of mRNA Delivery via MCM (A and B) Contour plots displaying the results of the screening design performed on Jurkat cells. (A) Complexed mRNA encoding for Gaussia luciferase delivered via MCM. (B) Complexed mRNA encoding for Gaussia luciferase delivered via FMCM. (C) Direct comparison of complexed mRNA delivery to Jurkat cells using MCM or FMCM. Asterisk represents statistically significant differences using paired Student’s t test (n = 4), *p < 0.05. (D and E) Contour plots displaying the results of the screening design performed on hBM cells for the delivery of complexed mRNA encoding for Gaussia luciferase via MCM (D) or FMCM (E). (F) Direct comparison of complexed mRNA delivery to hBM cells using MCM or FMCM. Asterisks represent statistically significant differences using paired Student’s t test (n = 4), ****p < 0.05.

Figure 4

Transfection of hBM Is More Effective When mRNA Is Delivered via MCM (A and B) Transfection of Jurkat cells (A) and hBM cells (B) using complexed mRNA encoding for EGFP. (C and D) Exposure of Jurkat cells (C) or hBM cells (D) to MCM causes an increase of endosomal activity as shown by an increased uptake of labeled dextran. (E and F) Jurkat cells (E) or hBM cells (F) exposed to complexed mRNA encoding for Firefly luciferase. The extracellular environment had a considerable impact on transfection efficiency; when cells were cultured in whole bone marrow the transfection was significantly lower than cells cultured in media. The delivery of complexed mRNA via MCM enables significantly higher cell metabolic activity. Asterisk represents statistically significant differences using paired Student’s t test (n = 4), p < 0.05.

Figure 5

Cells Can Be Transfected Also in 3D Clot Mimic Hydrogels (A) Schematic representation of the bone marrow clots formation. Fresh bone marrow aspirates can be mixed with a solution of MCM at a 1:1 volume ratio and then let clot at 37°C. Alternatively, heparinized bone marrow can be concentrated by centrifugation, mixed with MCM solution and then enriched with clotting agents such as fibrinogen and thrombin, and let clot at 37°C. (B–D) Color-enhanced scanning electron micrographs showing, respectively, (B) a spontaneous clot formed using fresh bone marrow aspirates mixed with MCM and clots formed using heparinized bone marrow washed and then mixed with clotting agents, without MCM (C) or with MCM (D). (E) Delivery of complexed mRNA encoding for EGFP to 3D-clot mimic obtained by mixing hBM with clotting agents. mRNA complexes can transfect hBM cells also in 3D.

Figure 6

hBM Cells Can Be Enriched, Transfected, and Encapsulated within 30 min (A) Effect of incubation time on transfection of Jurkat cells. Complexed mRNA encoding for Firefly luciferase was incubated with Jurkat cells from a few seconds up to 24 h. The incubation time did not seem to have significative impact on transfection of Jurkat cells, but there was a trend indicating that longer incubation time could lead to lower transfection. (B) Incubation time had a significant impact on the transfection of hBM; the highest transfection was observed with the shortest incubation time. (C and D) Transfection of Jurkat cells (C) or hBM cells (D) encapsulated in 3D clot-mimic hydrogels. The delivery of mRNA complexes via MCM coincided with a higher preservation of metabolic activity. The extracellular environment had a significant effect on the transfection efficiency; in fact, higher transfection was observed when cells were cultured in media compared with culture in whole bone marrow. Asterisk represents statistically significant differences using paired Student’s t test (n = 4), p < 0.05.