"Introduction: The currently available treatments for inflammation often target the symptoms but not the causes, leading to an ineffective management of persistent inflammatory conditions.
Due to the central role of macrophages (Mφ) in the inflammatory response, and overall in healing, innovative strategies to fine-tune the states and functionalities of Mφ may unveil the pathophysiology of chronic inflammation with great promise for a wide range of human afflictions.
MicroRNAs (miRNAs) are powerful materials to program cell responses, offering immune-regulatory possibilities for resolution and prevention of uncontrolled inflammation (1). Nevertheless, efficient and precise delivery systems are still a challenge for translatable RNA-based technologies. A major obstacle is to find carriers that overcome the RNA instability and enhance intracellular release.
Magnetically-assisted strategies hold potential to modulate cell and tissue responses combining contactless control and tissue penetration for tracking, local retention, and real time monitoring (2). However, superparamagnetic iron oxide nanoparticles (SPIONs) have been scarcely explored for targeted delivery and cell programming. Therefore, we theorized that loading miRNAs onto previously functionalized SPIONs to transport them into cells may overcome the instability of miRNAs. Specifically, our aim is to magnetically deliver and study miRNA molecules in the modulation of Mφ responses by suppressing a miRNA sequence (miR-155-5p), known to be overexpressed in inflammatory states, and consequently increasing anti-inflammatory mediators.
Methodology: Commercially available SPIONs were conjugated with polyethylenimine and miRNA (miR-155-5p) to form magnetically-responsive complexes via electrostatic complexation (hereafter referred as magnetoplexes). The system was characterized according to dimension, shape, and charge as well as for miRNA binding efficiency. Stationary (SMF) and pulsed-electromagnetic field (PEMF) using MagnefectNano and MagnetoTherapy devices, respectively, were investigated for internalization and delivery of the magnetoplexes via magnetofection. THP1 cells were primed to an inflammatory state (Mφ1) with lipopolysaccharide and interferon-γ (100 and 20 ng/ml, respectively). Mφ1 viability and the expression of immune-modulatory molecules were assessed in the presence of the magnetoplexes. Two time-points (1 and 4 days) were studied together with different miRNA cargos (0.05 or 0.15 µg) to determine the impact of miRNA in inflammatory mediators. The outcomes were compared against non-treated Mφ1-primed THP1 (Ctrl).
Results: The magnetoplexes were successfully produced with 76±2nm, and 26.8±0.5mV. Iron as low as 40ng in magnetoplexes was effective for miRNA loading. An improved cell uptake was observed in SMF-stimulated cells comparing to PEMF. For that reason, a 20-minute SMF stimulus was used throughout the experiment. Additionally, intracellular magnetoplexes were detected by confocal microscopy. Four days after magnetoplexes treatment, anti-inflammatory molecules as ARG1 trended higher in Mφ1, independently of miRNA mass, comparing to Ctrl. These outcomes suggest that controlled delivery of the miRNA to Mφ1 via magnetoplexes enables precision functional pro-healing changes.
Conclusions: The work combines contactless with high precision control to reprogram Mφ1 profiles, whose outcomes will contribute to advanced targeted and guided macrophage communication favoring a pro-regenerative environment and contributing to improved healing outcomes.
Acknowledgements: NORTE-01-0145-FEDER-000021; ERC CoG MagTendon No.772817; EC Twinning project Achilles No.810850; FCT Doctoral Grant SFRD/BD/144816/2019.
References: 1.Peng, B, et al. Adv. Drug Deliv. Rev.,88, 108-122(2015);2.Gonçalves,A.I., et al. Biomed Mater(2018)."