Lipid nanoparticles for lung endothelial targeting in experimental lung fibrosis

Introduction:: Dysfunction of the vasculature is a component in various lung-related pathophysiologies such as chronic obstructive pulmonary disease (COPD), pulmonary hypertension, acute lung injury (ALI), and Idiopathic Pulmonary Fibrosis (IPF) – a disease for which no curative therapeutics exist. In IPF, the lung vasculature adopts an aberrant phenotype characterized by vessel dilation, loss of microvasculature, and increased vascular permeability. Our group demonstrated a divergent response to fibrotic injury where young mice (8-week-old) spontaneously resolve post-injury fibrosis, accompanied by increased lung vascular density. Conversely, aged mice (18-month-old) lack the ability to resolve post-fibrotic injury and consistently display low vascular density¹. We demonstrated that matricellular protein, CCN3 whose gene expression follows the time course of resolution in young spontaneously resolving mice but not in aged non-resolving mice is involved in lung endothelial functions associated with tissue repair². Hence, lung vascular specific delivery of Ccn3 mRNA holds therapeutic potential for treatment of non-resolving lung fibrosis and vascular dysfunction. Lipid nanoparticle (LNP) strategies such as Selective Organ Targeting (SORT) demonstrate efficient mRNA encapsulation in a cationic lipid formulation with tropism to the healthy lung³. Leveraging the SORT strategy can facilitate mRNA delivery to the aged fibrotic lung vasculature in mice that are otherwise incapable of spontaneous resolution.


Materials and Methods:: SORT LNPs were prepared from a lipid solution of DPSC: Cholesterol: DLin-MC3-DMA: DMGPEG-2000: DOTAP and T7 NLS Cre mRNA. The aqueous phase (mRNA) and ethanol phase (lipids) were combined via microfluidic mixing with the NanoAssemblr Ignite platform. LNPs were then dialyzed using Pur-A-Lyzer Maxi 3500 Dialysis tubes against PBS to remove EtOH. To optimize desired LNP parameters, various conditions were modified such as: preparation via pipette mixing, flow rate ratio, N:P (ratio of total number of amines in lipid and total number of phosphates in mRNA), dialysis time and buffer, mRNA aqueous buffer composition and strength and incorporation of particle concentration methods such as Centrifugal filtration units. To validate formulation parameters, LNPs were characterized in terms of size, polydispersity index (PDI), and zeta potential (ξ) via Malvern Zetasizer and encapsulation efficiency was assessed via Quant-iT RiboGreen assay. To validate in vivo lung-selective delivery of the formulation, Cre mRNA encapsulated LNPs were administered intravenously to LSL-Luciferase: mTmG mice. Upon delivery of Cre mRNA, genetic recombination in these reporter mice results in deletion of the stop cassette and activation of luciferase and GFP expression in LNP-targeted cells. This allows for a bioluminescent and fluorescent readout of lipid nanoparticle uptake. After 48 hours, IVIS imaging was performed. To assess cell specific uptake of SORT LNPs, lungs were harvested and digested to a single cell suspension. To quantify cell specific transfection efficiency, the single cell suspension was incubated with antibodies recognizing endothelial, epithelial, and hematopoietic populations, and subjected to fluorescence activated cell sorting (FACS).


Results, Conclusions, and Discussions:: Results: SORT LNPs ranged in size from 100-170 nm with a polydispersity Index (PDI) of < 0.3, and a zeta potential (ξ) of -22 to +2 mV, along with mRNA encapsulation efficiency of 50-95%. We observed that optimal LNP parameters for lung specific delivery included a particle size close to 100 nm and a zeta potential (ξ) in the range of -1 to +1 mV. When LNP size exceeded 150 nm or particles were strongly anionic with a zeta potential (ξ) of -20 mV, spleen specific uptake was exclusively observed. IVIS imaging performed 48 hours after intravenous administration of optimized microfluidic prepared SORT LNPs encapsulating Cre mRNA in LSL-Luciferase: mTmG mice demonstrated lung selective delivery both in vivo and ex vivo with no off-target signal to the liver. Flow cytometry highlighted that approximately 81% of all transfected GFP⁺ cells in healthy mouse lungs were CD31⁺ (endothelial), whereas 4%, 10 %, and 5% were CD45⁺ (hematopoietic), CD326⁺ (epithelial), and lineage negative respectively.

Conclusions: Through microfluidic mixing, we produced LNPs with a high encapsulation efficiency (90-100%), small size (~100 nm), near neutral zeta potential (-1 to +1 mV), and nearly homodisperse particle distribution (< 0.3). We observed that maintaining LNP size close to 100 nm and zeta potential within the desired range was critical for lung-specific delivery. Findings also demonstrated that the choice of aqueous buffer composition and strength (citrate or acetate buffer), and dialysis buffer (PBS or Tris buffer) are fundamental to LNP parameters and transfection efficiency. Additionally, flow cytometry analysis revealed that GFP⁺CD31⁺ cells outnumbered the GFP⁺CD45⁺, GFP⁺CD326⁺, and GFP⁺ lineage negative cohorts.

Discussions: These findings allow us to implement the SORT strategy in the context of aging and disease models of experimental lung fibrosis to test delivery of therapeutic mRNA payloads such as Ccn3. By implementing this nano-delivery strategy for intervention at different phases within fibrotic development and characterizing any observed differences in transfection efficiency across lung cell types, we can ultimately identify a time point for intervention to the lung vasculature. This will better inform future studies that aim to target the lung vasculature in a pathologic context.


Acknowledgements (Optional): :

References (Optional): : 1 Caporarello, N. et al. Vascular dysfunction in aged mice contributes to persistent lung fibrosis. Aging Cell 19, e13196, doi:10.1111/acel.13196 (2020).

2 Betageri, K. R. et al. The matricellular protein CCN3 supports lung endothelial homeostasis and function. Am J Physiol Lung Cell Mol Physiol 324, L154-L168, doi:10.1152/ajplung.00248.2022 (2023).

3 Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol 15, 313-320, doi:10.1038/s41565-020-0669-6 (2020).