The replacement of helper lipids with charged alternatives in lipid nanoparticles facilitates targeted mRNA delivery to the spleen and lungs

Abstract

The broad clinical application of mRNA therapeutics has been hampered by a lack of delivery vehicles that induce protein expression in extrahepatic organs and tissues. Recently, it was shown that mRNA delivery to the spleen or lungs is possible upon the addition of a charged lipid to a standard four-component lipid nanoparticle formulation. This approach, while effective, further complicates an already complex drug formulation and has the potential to slow regulatory approval and adversely impact manufacturing processes. We were thus motivated to maintain a four-component nanoparticle system while achieving shifts in tropism. To that end, we replaced the standard helper lipid in lipidoid nanoparticles, DOPE, with one of eight alternatives. These lipids included the neutral lipids, DOPC, sphingomyelin, and ceramide; the anionic lipids, phosphatidylserine (PS), phosphatidylglycerol, and phosphatidic acid; and the cationic lipids, DOTAP and ethyl phosphatidylcholine. While neutral helper lipids maintained protein expression in the liver, anionic and cationic lipids shifted protein expression to the spleen and lungs, respectively. For example, replacing DOPE with DOTAP increased positive LNP surface charge at pH 7 by 5-fold and altered the ratio of liver to lung protein expression from 36:1 to 1:56. Similarly, replacing DOPE with PS reduced positive charge by half and altered the ratio of liver to spleen protein expression from 8:1 to 1:3. Effects were consistent across ionizable lipidoid chemistries. Regarding mechanism, nanoparticles formulated with neutral and anionic helper lipids best transfected epithelial and immune cells, respectively. Further, the lung-tropic effect of DOTAP was linked to reduced immune cell infiltration of the lungs compared to neutral or anionic lipids. Together, these data show that intravenous non-hepatocellular mRNA delivery is readily achievable while maintaining a four-component formulation with modified helper lipid chemistry.

Keywords: Charge; Extrahepatic; Helper lipids; Lipid nanoparticles; Targeted delivery; mRNA delivery.

Figure 1:. Helper lipid charge influences the organ location of protein expression following mRNA delivery.

LNPs were formulated with one of three helper lipids: DOPE (net neutral charge), PS (net negative charge), or DOTAP (positive charge). Mice were injected intravenously with LNPs at a dose of 0.75 mg/kg of mRNA encoding Firefly luciferase. Luciferase signal was quantified three hours post-injection using an In Vivo Imaging System (IVIS). (A) Structures of the 3 helper lipids considered in this figure. (B) Luciferase expression occurred predominantly in the liver, regardless of helper lipid charge, for a standard LNP formulation (16 mol% helper lipid) incorporating the ionizable lipidoid 306O₁₀. The 3 columns in the images represent the 3 helper lipids, and the scale bar is on the right. (C)Luciferase expression shifted from the liver to the spleen or lungs when LNPs were formulated with 40 mol% PS or DOTAP instead of DOPE. (D) Helper lipid charge altered the organ location of protein expression irrespective of ionizable lipidoid identity for LNPs. Data for three ionizable lipidoids (200O_i10, 205O_6,10, and 306O₁₀) are shown here. Data represent mean values. Error bars represent standard deviation (n = 3).

Figure 2:. The effect of helper lipid charge on the location of protein expression holds is conserved across helper lipid chemistries.

LNPs were formulated with the ionizable lipidoid 306O₁₀ and 40 mol% helper lipid and IV injected into mice (0.75 mg/kg mRNA). Luciferase signal was measured three hours later. In addition to DOPE, neutral lipids 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), sphingomyelin (SM), and ceramide (Cer) induced protein expression primarily in the liver. Anionic lipids, including PS, phosphatidylglycerol (PG) and phosphatidic acid (PA) shifted expression to the spleen, and the cationic lipids DOTAP and ethyl phosphatidylcholine (EPC) shifted expression to the lungs. Luminescence values as a percentage of total luminescence of all organs (liver, lungs, spleen, kidneys, intestines, pancreas, and heart) are plotted above. Mean values shown. Error bars represent standard deviation (n = 3).

Figure 3:. In vivo lung and spleen specificity correlated with LNP physical properties.

The physical properties that correlated with lung specificity were (A) z-average size, (B) TNS fluorescence (a surrogate for LNP surface ionization) at pH 7 and (C) pH 5. The physical properties (D) zeta potential and (E) RNA entrapment correlated negatively with in vivo spleen specificity. (F) The correlation values of physical properties (z-average diameter, zeta potential, RNA entrapment and TNS ionization) were calculated for liver, spleen, and lung efficacy in vivo. Data shown represents mean. R-squared values are Pearson correlation coefficients. Boxes were shaded with colors indicating degree of correlation with <0.3 (low correlation) shaded red, 0.3–0.6 shaded yellow, and >0.6 (high correlation) shaded green. Error bars indicate s.d. (n = 6). R-squared values are shown on each plot.

Figure 4:. Adherent and immune cell lines were best transfected by neutral and anionic helper lipids, respectively.

Lipid nanoparticles formulated with 40 mol% helper lipid and mLuc were incubated with eight cell lines for 24 hours at 100 ng/well prior to measurement of luciferase signal. Cell lines included HeLa (human cervical), HepG2 (human hepatocytes), NIH 3T3 (mouse fibroblasts), A549 (human lung epithelial), Hulec-5a (human lung endothelial), RAW 264.7 (mouse macrophage), Jurkat (human T) and Raji B (human B). Data shown represent mean values. Error bars indicate standard deviation, 2-way ANOVA Tukey Test where *, **, *** and **** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001 and p ≤ 0.001 respectively.

Figure 5:. Organ specificity was linked to immune cell infiltration patterns.

LNPs were formulated with the lipidoid 306O₁₀ and 40 mol% DOPE, PS, or DOTAP. Three hours after LNP injection via tail vein at an mLuc dose of 0.75 mg/kg, cell populations in the liver, lungs, and spleen were analyzed via flow cytometry. Cell types are identified on the y-axes. Total immune cells were identified as CD45+, T cells as CD3+, B-cells as CD19+ with F4/80+ and CD11b+ labeled as such as further markers are needed for distinct cell definition. (A-E) LNP formulations did not alter the proportion of most cell types in the liver. (F-J) LNPs formulated with DOPE or PS increased the proportion of immune cells, and, specifically, myeloid cells in the lungs. DOTAP LNPs did not have an effect. (K-O) LNPs formulated with PS increased the proportion of myeloid cells in the spleen. Mean values are shown with error bars indicating s.d. (n = 3), 2-way ANOVA Tukey Test with * and ** representing p ≤ 0.05 and p ≤ 0.01, respectively.

Figure 6:. Helper lipid identity affected inflammatory state and cell-specific location of protein expression within organs.

LNPs were formulated with the lipidoid 306O₁₀ and at a helper lipid concentration of 40 mol%. (A) Three hours following intravenous LNP administration in mice, organs were harvested and prepared for hematoxylin & eosin staining. Hypercellularity was observed in the lungs of mice treated with DOTAP LNPs, suggesting the host response to DOTAP LNPs may contribute to the shift in efficacy to the lungs. (B) Tissues sections were also stained with antibodies against Firefly luciferase (green), phalloidin (red) and Hoescht (blue). Staining suggested that DOPE LNPs transfected the entirety of the liver emanating from liver sinusoidal endothelial cells. Transfection in the lungs appeared localized to the blood vessels. Splenic tissue sections suggested that transfection was throughout the spleen in DOPE and DOTAP LNP-treated mice, while PS LNP treated mice had more transfection in the white pulp marginal zones which contain adaptive immune cells such as B cells.