Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH
Abstract
:1. Introduction
2. Results
2.1. Characterization of Methionine Choline Deficient (MCD) Murine Model of NASH and RUNX1 Expression
2.2. Antibody Anchored RUNX1 siRNA-Engineered Stealth Immunonano-Lipocarriers
2.3. In Vitro Uptake, Cytotoxicity, and Efficacy of RUNX1 siRNA Immunonano-Lipocarriers in Liver Cells of MCD Mice
2.4. In Vivo Biodistribution of RUNX1 siRNA Immunonano-Lipocarriers
2.5. In Vivo Efficacy of RUNX1 siRNA Immunonano-Lipocarriers
2.6. RUNX1 siRNA Immunonano-Lipocarriers Decrease Expression of VCAM1 and ICAM1 in LSECs
2.7. RUNX1 siRNA Immunonano-Lipocarriers Decrease Percentage of T Cells and Monocytes/Kupffer Cells in Liver
2.8. RUNX1 siRNA Immunonano-Lipocarriers Reduce Liver Inflammation
3. Discussion
4. Materials and Methods
4.1. Materials
4.2. Animals
4.3. Methods
4.3.1. Isolation of Primary Hepatic Cells and Liver-Infiltrating Lymphocytes (LILs)
4.3.2. Characterization of LSECs and HSCs
4.3.3. Development and Characterization of RUNX1 siRNA-Engineered Stealth Immunonano-Lipocarriers
Preparation of RUNX1 siRNA Nano-Lipocarriers
Antibody Anchored RUNX1 siRNA-Engineered Stealth Immunonano-Lipocarriers
Particle Size, Polydispersity Index (PDI), and Zeta Potential
Cell Viability Assays with RUNX1 siRNA-Engineered Stealth Immunonano-Lipocarriers
4.3.4. In Vivo Biodistribution and In Vitro Uptake Studies
4.3.5. Real-Time qRT-PCR
4.3.6. Immunohistochemistry Analysis
4.3.7. Flow Cytometry Analysis
4.3.8. ELISA Assays for TNF-α and CCL2
4.3.9. Statistical Analysis
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Abbreviations
NAFLD | Non-alcoholic fatty liver disease |
NASH | Non-alcoholic steatohepatitis |
RUNX 1 | Runt-related transcription factor |
MCD | Methionine choline deficient |
LSECs | Liver sinusoidal endothelial cells |
NPCs | Non-parenchymal cells |
HSCs | Hepatic stellate cells |
LILs | Liver-infiltrating lymphocytes |
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Characteristics | Control | MCD |
---|---|---|
ALT (IU/L) | 56.7 ± 9.7 | 89.4 ± 8.9 |
Albumin (g/dL) | 3.6 ± 1.29 | 1.6 ± 0.12 |
Triglyceride (mmol/L) | 100 ± 4.9 | 102 ± 3.6 |
Cholesterol (mg/dL) | 67 ± 1.8 | 62 ± 2.3 |
Inflammatory grade | 0.2 ± 0.4 | 2.25 ± 0.5 |
Engineering Parameters | Desired Optimum Values/Features | Achievable Benefits |
---|---|---|
Size | <300 nm | Less size with a high surface area |
Shape | Spherical | Improved cellular delivery |
Matrix | Lipids (SC-shell) and phospholipids (SPC core) | Sustained RUNX1 siRNA release for improved biodistribution profile of engineered matrix deliverables |
Surface charge | Positive (after vegfr3 anchoring) | Improved physical and biological stability, avoid agglomeration |
Recognition by macrophages | Hydrated stealth (PEGylated) | Avoid recognition by other organ macrophages |
Surface ligand | Primary vegfr3 antibody | To trigger surface antigen–antibody interactions |
SC-shell | Solid and hydrophobic | To easily cross-cellular membrane |
SPC-core | Amphiphilic, vesicular, and soft matrix | Promote sustain delivery of RUNX1 siRNA |
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Tripathi, D.M.; Rohilla, S.; Kaur, I.; Siddiqui, H.; Rawal, P.; Juneja, P.; Kumar, V.; Kumari, A.; Naidu, V.G.M.; Ramakrishna, S.; et al. Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH. Int. J. Mol. Sci. 2021, 22, 8489. https://doi.org/10.3390/ijms22168489
Tripathi DM, Rohilla S, Kaur I, Siddiqui H, Rawal P, Juneja P, Kumar V, Kumari A, Naidu VGM, Ramakrishna S, et al. Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH. International Journal of Molecular Sciences. 2021; 22(16):8489. https://doi.org/10.3390/ijms22168489
Chicago/Turabian StyleTripathi, Dinesh Mani, Sumati Rohilla, Impreet Kaur, Hamda Siddiqui, Preety Rawal, Pinky Juneja, Vikash Kumar, Anupama Kumari, Vegi Ganga Modi Naidu, Seeram Ramakrishna, and et al. 2021. "Immunonano-Lipocarrier-Mediated Liver Sinusoidal Endothelial Cell-Specific RUNX1 Inhibition Impedes Immune Cell Infiltration and Hepatic Inflammation in Murine Model of NASH" International Journal of Molecular Sciences 22, no. 16: 8489. https://doi.org/10.3390/ijms22168489