Poly(aspartic acid)-Based Polymeric Nanoparticle for Local and Systemic mRNA Delivery

Abstract

Recently, therapeutics based on mRNA (mRNA) have attracted significant interest for vaccines, cancer immunotherapy, and gene editing. However, the lack of biocompatible vehicles capable of delivering mRNA to the target tissue and efficiently expressing the encoded proteins impedes the development of mRNA-based therapies for a variety of diseases. Herein, we report mRNA-loaded polymeric nanoparticles based on diethylenetriamine-substituted poly(aspartic acid) that induce protein expression in the lungs and muscles following intravenous and intramuscular injections, respectively. Animal studies revealed that the amount of polyethylene glycol (PEG) on the nanoparticle surface affects the translation of the delivered mRNA into the encoded protein in the target tissue. After systemic administration, only mRNA-loaded nanoparticles modified with PEG at a molar ratio of 1:1 (PEG/polymer) induce protein expression in the lungs. In contrast, protein expression was detected only following intramuscular injection of mRNA-loaded nanoparticles with a PEG/polymer ratio of 10:1. These findings suggest that the PEG density on the surface of poly(aspartic acid)-based nanoparticles should be optimized for different delivery routes depending on the purpose of the mRNA treatment.

Keywords: PEGylation; gene therapy; mRNA delivery; polymeric nanoparticle; systemic delivery.

Figure 1.

(a) Scheme for the synthesis of poly(β-benzyl-L-aspartate) (PBLA, 3) using ring-opening polymerization with β-benzyl L-aspartate N-carboxyanhydride (βLA-NCA, 1) initiated by n-butylamine(2). N-substituted polyaspartamide P[Asp(DET)] (5) is synthesized by the aminolysis reaction with diethylenetriamine (DET) (4). (b) Schematic illustration of the synthesis of the mRNA-loaded polymeric nanoparticles (PNP) and modification of the PNP by polyethylene glycol conjugation on the nanoparticle surface via amide bonds.

Figure 2.

Representative dynamic light scattering profiles of PNP, PEG(1x)-PNP, and PEG(10x)-PNP (N/P 32) and the summary of parameters for nanoparticles.

Figure 3.

(a) Representative fluorescence microscopy images of cells after 48 hours of incubation with PNP, PEG(1x) – PNP, and PEG(10x) – PNP. The blue pseudo color represents the fluorescence signal created by nuclei staining, the red pseudo color represents polymer, and the green pseudo color is generated by EGFP. Scale bars = 50 μm. (b) Background signals and mean fluorescence intensities in Huh7 human hepatocytes incubated with PNP, PEG(1x)-PNP, and PEG(10x)-PNP for 48 hours (EGFR mRNA 3 μg/mL) (n = 3). The fluorescence signal in the microscopy images was quantified using Image J software. (c) Cell viability of Huh7 cells treated for 48 hours with PNP, PEG(1x)-PNP, and PEG(10x)-PNP at EGFR mRNA concentrations of 0.5, 1.5, and 3 μg/mL (n = 3). The results of the experiment were presented as mean values ± standard deviation. The p-values, *P < 0.05, and *** P < 0.001, were used to designate statistical significance.

Figure 4.

Representative bioluminescence images of mice and excised organs 6 hours after intravenous injection of (a) PEG(1x)-PNP and (b) PEG(10x)-PNP at a dose of 0.44 mg Fluc mRNA kg⁻¹. Luciferase expressions of PEG(1x)-PNP are shown in the lungs. In mouse treated with PEG(1x)-PNP, the mean luminescence signals of the lungs in live mouse and excised lungs are 1.24 × 10⁴ and 5.16 × 10⁵, respectively.

Figure 5.

(a) Representative Cy7.5 fluorescence image of mouse organs after injection of Cy7.5 tagged PEG-PNP at 6 hours post-injection. (b) The mean fluorescence intensities of Cy7.5 of excised organs from mice given Cy7.5-tagged PEG-PNP intravenously.

Figure 6.

Representative luminescence image of mice following intramuscular injection of (a) PEG(1x)-PNP and (b) PEG(10x)-PNP at a dose of 0.22 mg mRNA kg⁻¹. For mice treated with PEG(10x)-PNP, the mean luminescence signal of the hind limb of treated mice is 4.47 × 10⁵.