Hybrid Biopolymer and Lipid Nanoparticles with Improved Transfection Efficacy for mRNA

Abstract

Hybrid nanoparticles from lipidic and polymeric components were assembled to serve as vehicles for the transfection of messenger RNA (mRNA) using different portions of the cationic lipid DOTAP (1,2-Dioleoyl-3-trimethylammonium-propane) and the cationic biopolymer protamine as model systems. Two different sequential assembly approaches in comparison with a direct single-step protocol were applied, and molecular organization in correlation with biological activity of the resulting nanoparticle systems was investigated. Differences in the structure of the nanoparticles were revealed by thorough physicochemical characterization including small angle neutron scattering (SANS), small angle X-ray scattering (SAXS), and cryogenic transmission electron microscopy (cryo-TEM). All hybrid systems, combining lipid and polymer, displayed significantly increased transfection in comparison to lipid/mRNA and polymer/mRNA particles alone. For the hybrid nanoparticles, characteristic differences regarding the internal organization, release characteristics, and activity were determined depending on the assembly route. The systems with the highest transfection efficacy were characterized by a heterogenous internal organization, accompanied by facilitated release. Such a system could be best obtained by the single step protocol, starting with a lipid and polymer mixture for nanoparticle formation.

Keywords: Covid-19; RNA; cancer immunotherapy; cationic lipid; cationic polymer; lipid-polymer hybrid nanoparticles; small angle scattering; vaccination.

Figure 1

Graphical representation of the concept of different assembly routes for the preparation of hybrid particles. Three different particle topologies with protamine as either the first added component (PC), the last added component (PS), or in a mixture with DOTAP before RNA addition (MP) were compared. The N/P ratio composition is given underneath the pie charts (protamine/DOTAP/mRNA).

Figure 2

Release of mRNA after the addition of heparin in H₂O, tested at an N/P ratio of 2 for protamine/mRNA particles (orange), DOTAP/mRNA particles (grey), MP_MID (blue), PC_MID (green), and PS_MID (red) particles. The curves are drawn to guide the eye.

Figure 3

(A) Double logarithmic plot of SAXS profiles of negatively charged PS particle intermediates (DOTAP/RNA, grey curves) of three different charge ratios of DOTAP (0.3, 0.7, 0.9) with a constant charge ratio of RNA (1.0) and for each curve below the corresponding curve after protamine addition to form the final PS particle (red curves) in HBG buffer (pH 7.2). Colour darkening indicates a higher proportion of protamine in the particle for the red curves and of DOTAP in the grey curves. The magnification shows the q-range around 0.11 Å⁻¹ where the peak is located. Curves were shifted vertically for clarity. (B) Area under the curve (AUC) for the three different N/P ratios. The blue arrow indicates the absolute reduction of the AUCs after protamine addition.

Figure 4

(A–C) Cryo-TEM pictures of the respective particles shown in the same colour as the corresponding curves. (D) Intensity scattering of PC_LOW particles in a q-range of 0.03–0.18 Å⁻¹ at different D₂O concentrations. The peak at 0.1 Å⁻¹ is present when the scattering length density of the buffer diverges from the scattering length density of the lipid. (E) Double logarithmic plot of SANS scattering of PC_LOW (N/P 0.3/1.7/1) and PS_HIGH (N/P 1.7/0.3/1) at 77% D₂O. Curves were shifted vertically for clarity. (F) Double logarithmic plot of the SAXS curve of MP_MID.

Figure 5

(A) HEK293 cell transfection of different protamine/DOTAP hybrid nanoparticles at different compositions in comparison to protamine/mRNA and DOTAP/mRNA particles. *** p < 0.001. Statistical significance was determined using two-way ANOVA combined with a Bonferroni comparison test. (B) Bioluminescence imaging of mice (n = 3 for particles, n = 2 for negative control) 6, 24, and 48 h after i.m. injection in the footpad of PC_MID, PS_MID, MP_MID, protamine/mRNA, DOTAP/mRNA or only PBS buffer (not shown). The average radiance is quantified as photons/sec/cm2/sr, meaning the amount of photons that are emitted per second per square centimeter tissue per square radian (solid angle). (C) Total flux kinetic analysis of the samples with respect to time. (D) Cumulative bioluminescence signal intensity at all time points indicating protein expression following injection of the respective samples. ** p < 0.01. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test.