Biomaterials Science (accepted manuscript; manuscript ID BM-ART-09-2020ResearchNon-viral Gene Delivery
Helper Lipid Structure Influences Protein Adsorption and Delivery of Lipid Nanoparticles to Spleen and Liver
Rui Zhang, Rakan El-Mayta, Timothy Murdoch, Claude Warzecha, Margaret Billingsley, Sarah Shepherd, Ningqiang Gong, Lili Wang, James Wilson, Daeyeon Lee, Michael J. Mitchell
Summary
LNP delivery to liver versus spleen is difficult to predict from formulation parameters, and the role of helper phospholipids in biodistribution and protein adsorption is incompletely understood. This study probed how helper lipid identity (DOPE vs DSPC) affects LNP accumulation in liver/spleen and interactions with apolipoprotein E (ApoE). DSPC-containing LNPs accumulated more in spleen than DOPE-containing LNPs (P = 0.0027); DOPE-containing LNPs accumulated more in liver (P < 0.0001). - QCM-D: DOPE LNP 42 showed stronger ApoE interaction than DSPC LNP.
Keywords
Lipid nanoparticleNanoparticlesmRNAsiRNADNABiodistributionTransfection
Purpose: LNP delivery to liver versus spleen is difficult to predict from formulation parameters, and the role of helper phospholipids in biodistribution and protein adsorption is incompletely understood. This study probed how helper lipid identity (DOPE vs DSPC) affects LNP accumulation in liver/spleen and interactions with apolipoprotein E (ApoE).
Hypothesis: Changing the helper lipid from DSPC to DOPE will alter LNP–ApoE binding and shift biodistribution: DOPE-containing LNPs will interact more strongly with ApoE and accumulate more in liver, while DSPC-containing LNPs will accumulate more in spleen.
Aims: Screen a 96-LNP library in vivo using DNA barcoding to identify helper-lipid-dependent biodistribution. - Measure LNP–ApoE binding using quartz crystal microbalance with dissipation monitoring (QCM-D). - Validate biodistribution differences using Cy3-siRNA LNPs and IVIS imaging of spleen. - Evaluate functional mRNA delivery and transfection in liver and spleen using luciferase mRNA LNPs and RT-qPCR/bioluminescence.
Delivery system: Platform: Lipid nanoparticles (LNPs) for nucleic acid delivery. - Ionizable lipid: C12-200 (screening); C14-4 used in additional QCM-D controls. - Helper lipids: DOPE vs DSPC. - Other components: Cholesterol; lipid-PEGs of varying molecular weight (C14-PEG1000, -2000, -3000, -5000). - Payloads: Barcoded DNA (b-DNA) for screening; Cy3-siRNA for imaging; firefly luciferase mRNA for functional delivery. - Formulation: Pipette mixing for b-DNA/Cy3-siRNA LNPs; microfluidic mixing for mRNA LNPs. - Targeting ligand: None; endogenous ApoE adsorption was studied.
Approach: In vivo screen: 96 LNPs pooled and injected IV into C57BL/6 mice (N = 4); tissues harvested 6 h post-injection; b-DNA quantified by deep sequencing and qPCR. - Validation: LNP 42 (DOPE) and LNP 90 (DSPC) were selected; identical except helper lipid. - siRNA imaging: Cy3-siRNA LNPs injected IV; spleens isolated 6 h post-injection; N = 3 mice/group. - mRNA delivery: Luciferase mRNA LNPs injected IV at 0.2 mg/kg; bioluminescence at 6 h; N = 4 mice/group; RT-qPCR for luciferase mRNA in liver/spleen. - Disease context: Healthy mice; no disease model.
Key methods: Dynamic light scattering (DLS) for LNP size and PDI. - Deep sequencing of b-DNA and qPCR normalization for tissue accumulation. - QCM-D for ApoE adsorption and LNP–ApoE interaction. - IVIS imaging for Cy3-siRNA spleen accumulation and luciferase bioluminescence. - RT-qPCR for luciferase mRNA abundance normalized to GAPDH.
Key results: DSPC-containing LNPs accumulated more in spleen than DOPE-containing LNPs (P = 0.0027); DOPE-containing LNPs accumulated more in liver (P < 0.0001). - QCM-D: DOPE LNP 42 showed stronger ApoE interaction than DSPC LNP 90; frequency shifts were about −350 Hz vs −200 Hz after PBS rinse. - Cy3-siRNA: DSPC LNP 90 showed about 2-fold higher spleen radiant efficiency than DOPE LNP 42 (P = 0.0019). - Luciferase mRNA: DOPE LNP 42 improved liver transfection about 3-fold (**P = 0.0001) and liver mRNA abundance about 2-fold (P = 0.0263). DSPC LNP 90 increased spleen mRNA abundance about 5-fold (***P = 0.0003), but spleen bioluminescence difference was not significant (P = 0.1493).
Interpretation: Helper lipid structure influences LNP–ApoE interactions and organ tropism: DOPE promotes ApoE binding and liver delivery, whereas DSPC favors spleen accumulation. Understanding helper-lipid-driven protein adsorption may improve rational design of LNPs for liver- or spleen-targeted nucleic acid therapeutics.
Limitations: Only healthy mice; no disease model or therapeutic efficacy endpoint. - Only two helper lipids compared (DOPE vs DSPC); other helper lipids not tested. - Spleen accumulation of DSPC LNPs did not translate into significantly higher functional mRNA transfection in spleen. - QCM-D is a simplified model of ApoE interaction; full protein corona composition was not characterized. - No toxicity, repeated-dose, or long-term safety evaluation. - No large-animal validation.
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