Lipid Nanoparticles With Fine-Tuned Composition Show Enhanced Colon Targeting as a Platform for mRNA Therapeutics

Abstract

Lipid Nanoparticles (LNPs) recently emerged as an invaluable RNA delivery platform. With many LNP-based therapeutics in the pre-clinical and clinical pipelines, there is extensive research dedicated to improving LNPs. These efforts focus mainly on the tolerability and transfectability of new ionizable lipids and RNAs, or modulating LNPs biodistribution with active targeting strategies. However, most formulations follow the well-established lipid proportions used in clinically approved products. Nevertheless, investigating the effects of LNPs composition on their biodistribution can expand the toolbox for particle design, leading to improved delivery strategies. Herein, a new LNPs (30-n-LNPs) formulation with increasing amounts of phospholipids is investigated as a possible mRNA delivery system for treating Inflammatory Bowel Diseases. Compared to LNPs with benchmark composition (b-LNPs), n-LNPs containing 30% distearoylphosphatidylcholine (DSPC) are well tolerated following intravenous administration and display natural targeting toward the inflamed colon in dextran sodium sulfate (DSS)-colitis bearing mice, while de-targeting clearing organs such as the liver and spleen. Using interleukin-10-encoding mRNA as therapeutic cargo, n-LNPs demonstrated a reduction of pathological burden in colitis-bearing mice. n-LNPs represent a starting point to further investigate the influence of LNPs composition on systemic biodistribution, ultimately opening new therapeutic modalities in different pathologies.

Keywords: colon; genetic medicines; inflammatory bowel disease; lipid nanoparticles; mRNA; targeting.

Figure 1

Physicochemical and structural characterization of LNPs. A) Schematic representation of the methods used for LNPs formulation and their characterization (AGE: agarose gel electrophoresis; Luc: in vivo Luc expression; FACS: Flow Cytometry; BW: body weight assessment; CL: colon length; Hist: histology). B) Summarized size and PDI of b‐LNPs and n‐LNPs measured by DLS (n = 9). C) LNPs size measured by NTA (n = 12). D) Measurement of particle diameter performed on TEM images (Data presented as Box‐Whiskers from minimum to maximum), exemplified in (E). F) zeta potential measurement (n = 6) and Ribogreen mRNA encapsulation assessment (n = 7, G). All data are presented as average ± SEM; Statistical analysis was performed using a one‐way ANOVA test (^* p < 0.05, ^** p < 0.01).

Figure 2

Biodistribution study in colitic mice. A) Representative pictures of the IVIS analysis performed on DSS mice 6 h post‐injection of mLuc‐b‐LNPs and mLuc‐n‐LNPs, and their relative radiance quantification for the colons (B), stomach and small intestines (C), Livers (D) and Spleens (E) (12 mice/group, data are presented as Box and Whiskers from minimum to maximum. Statistical analysis was performed using one‐way ANOVA; ^* p < 0.05, ^** p < 0.01, ^*** p,0.001).

Figure 3

Flow cytometry profiles of the tdTomato⁺ cells in the main cell populations were analyzed for the mice colon lamina propria, liver, spleen, and mesenteric lymph nodes (three mice were included in each experimental group. All data are presented as average ± SEM; Statistical analysis was performed using one‐way ANOVA; ^* p < 0.05, ^** p < 0.01).

Figure 4

Improved therapeutic outcome in colitic mice with IL‐10 mRNA delivered with n‐LNPs. Assessment of the mouse body weight (A) and colon length (B) during and after treatment with LNPs, respectively (n = 15 mice/group). C) representative hematoxylin/eosin histological sections of rolled mice colons. ELISA measurement of the colon levels of TNF‐α (D), IL‐6 (E), and IL‐12/IL‐23 p40 (F). 10 mice were included in each group; 5 mice were included for mIL‐10‐b‐LNPs. (All data are expressed as average ± SEM; Statistical analysis was performed using a one‐way ANOVA test ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001.)

Figure 5

Toxicity profile of LNPs. Assessment of the plasma levels of IL‐6 (A) and TNF‐α (B) 2 and 24 h after injection of mLuc‐b‐LNPs, mLuc‐n‐nLNPs, or LPS (n = 6 mice/group). Measurement of the blood levels of total bilirubin (C), albumin (D), creatinine (E), Urea (F), SGOT (G), and SGPT (H) (n = 5 or 6 mice/group). Min and Max values represent the normal range for each marker, as adapted from the literature.^{[ 51 ]} (I) representative images of Hematoxylin/Eosin‐stained mouse Lungs, Livers, Spleens, and Kidneys after treatment with LNPs. Scale bars equivalent to 500 µm. Data are presented as average ± SEM.