Efficient mRNA Delivery In Vitro and In Vivo Using a Polycharged Biodegradable Nanomaterial

Abstract

As RNA rises as one of the most significant modalities for clinical applications and life science research, efficient tools for delivering and integrating RNA molecules into biological systems become essential. Herein, we report a formulation using a polycharged biodegradable nano-carrier, N1-501, which demonstrates superior efficiency and versatility in mRNA encapsulation and delivery in both cell and animal models. N1-501 is a polymeric material designed to function through a facile one-step formulation process suitable for various research settings. Its capability for mRNA transfection is investigated across a wide range of mRNA doses and in different biological models, including 18 tested cell lines and mouse models. This study also comprehensively analyzes N1-501's application for mRNA transfection by examining factors such as buffer composition and pH, incubation condition, and media type. Additionally, N1-501's superior in vivo mRNA transfection capability ensures its potential as an efficient and consistent tool for advancing mRNA-based therapies and genetic research.

Keywords: RNA research; RNA therapeutics; RNA translation; gene delivery; gene editing; mRNA transfection; protein translation; targeted delivery.

Figure 1

N1-501 effectively delivers mRNA across various cell lines as compared with Lipofectamine 3000. (A) The transfection efficiency was evaluated after 24 h of treatment. (B) Fluorescence microscopy images of 18 cell lines were captured 24 h after transfection to visualize eGFP expression levels. (Scale bar, 400 µm).

Figure 2

N1-501 demonstrated high spleen tropism during in vivo transfection. (A) In vivo bioluminescence (3, 6, and 12 h) and ex vivo bioluminescence (6 h) of major organs excised from Balb/c mice i.v. injected with N1-501/Fluc mRNA. (B) Quantification of total bioluminescence in various organs. (C) Quantification of total bioluminescence in mice at different time points.

Figure 3

Graphical depiction of operationally simple transfection protocols developed for N1-501.

Figure 4

The transfection performance and safety profiles of N1-501 at varied mRNA and reagent dose combinations. Black dots represent specific formulation conditions used in the study; the contour plot is filled with simulated data. (A) The contour plot for GFP expression, represented by MFI, was measured using a high-content screening system 24 h after transfection. (B) The contour plot showing the percentage of GFP-positive cells 24 h after transfection. (C) The contour plot showing HEK 293T cell viability 24 h after treatment with N1-501/eGFP mRNA complexes. Viability was evaluated using the CCK-8 assay.

Figure 5

Buffer and medium compatibility during formulation and transfection. N1-501 was diluted in 10 mM citrate buffer (pH = 4.0, 5.0, 6.0), 10 mM acetate buffer (pH = 4.0, 5.0, 6.0), 1X PBS buffer (pH = 7.4), 10 mM HEPES buffer (pH = 8.0), and water, respectively. After mixing with mRNA, the nanoparticle was diluted in Opti-MEM reduced-serum medium, serum-free DMEM [DMEM(−/−)], DMEM with 10% FBS [DMEM(10% FBS)], or DMEM containing 10% FBS and 1% penicillin/streptomycin [DMEM(+/+)] and applied to cells in the same media. The MFI (A) and percentage of GFP-positive cells (B) were assessed 24 h after transfection. Data are shown as the mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test (ns: no significance, **** p < 0.0001).

Figure 6

Effect of pre-incubation time on mRNA delivery by N1-501. N1-501 was formulated with eGFP mRNA and then held at 0 °C, 25 °C, and 37 °C for different lengths of time. (A) MFI and (B) percentage of GFP-positive cells were measured 24 h after transfection. Data are shown as the mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test (ns: no significance, ** p < 0.01).

Figure 7

N1-501’s shelf life, evaluated by transfection efficacy in vitro. (A) MFI and (B) percentage of GFP-positive cells were assessed 24 h after transfection. Data are shown as the mean ± SD. Statistical significance was determined using an unpaired two-tailed Student’s t test (ns: no significance, ** p < 0.01, *** p < 0.001, **** p < 0.0001).