Role of Surface Chemistry on Serum Protein Corona-Mediated Cellular Delivery and Gene Silencing with Lipid Nanoparticles

Purpose: Protein corona formation in biological fluids can alter nanoparticle cellular uptake, transfection, and gene silencing, but the relationship between LNP surface chemistry and corona-mediated delivery is incompletely understood. This study examined how PEG lipid chain length and PEG molar ratio affect protein corona composition and siRNA delivery in hepatocellular carcinoma cells.

Hypothesis: Changing LNP surface chemistry—specifically PEG conjugated lipid chain length (C18 vs C14) and PEG molar ratio (6% vs 3%)—will change the serum protein corona composition, which in turn will alter cellular uptake, transfection, and gene silencing.

Aims: Formulate four EnCore LNPs (F1–F4) with similar composition but different PEG lipid chain lengths and molar ratios. - Characterize LNP size, surface charge, morphology, encapsulation, and surface PEG. - Analyze protein corona composition after incubation with fetal bovine serum (FBS). - Evaluate cellular uptake and CTNNB1 gene silencing in HepG2 cells with and without serum. - Test whether ApoA1 or ApoE affects LNP-mediated gene silencing.

Delivery system: Platform: EnCore lipid nanoparticles (LNPs). - Core: DL-048 with PEG-lipid (C18-PEG or C14-PEG). - Envelope: DSPC, cholesterol, DL-036 ionizable cationic lipid, with or without PEG-lipid. - Formulations: F1 = C18-C18 (6% PEG); F2 = C18-0 (3% PEG); F3 = C14-C14 (6% PEG); F4 = C14-0 (3% PEG). - Payload: CTNNB1 DsiRNA; Cy3-labeled siRNA; scrambled siRNA controls. - Targeting ligand: None. - Physicochemical properties: ~90 nm, PDI <0.1, encapsulation >90%, near-neutral zeta potential.

Approach: In vitro model: HepG2 human hepatocellular carcinoma cells. - Conditions: Serum-free vs. 5% FBS; LNP doses 10–400 nM for gene silencing. - Protein corona: LNPs incubated with FBS, separated by iodixanol ultracentrifugation, analyzed by SDS-PAGE and mass spectrometry. - Controls: Free siRNA, Lipofectamine RNAiMAX positive/negative controls, mock, untreated cells.

Key methods: Dynamic light scattering for size, PDI, zeta potential. - PicoGreen assay and UPLC for encapsulation and siRNA concentration. - TEM and cryo-EM for morphology. - XPS/ESCA and ¹H NMR for surface PEG characterization. - Fluorescence microscopy for cellular uptake. - qRT-PCR for CTNNB1 gene silencing. - SDS-PAGE, Bradford assay, LC-MS/MS for protein corona. - CellTiter-Blue for viability.

Key results: All four LNPs had similar size (~90 nm), PDI (<0.1), and encapsulation efficiency (>90%); zeta potential was near neutral. - Surface PEG molar ratio by ¹H NMR was approximately 1.9:1.0:2.0:1.0 for F1:F2:F3:F4, matching PEG input ratio. - FBS inhibited cellular uptake and transfection of F1 and F2 but facilitated uptake and transfection of F3 and F4. - Significant CTNNB1 knockdown was observed with F2 without FBS, and with F3 and F4 with FBS; F1 showed poor dose response. - ApoE addition facilitated gene silencing of F3 at 10 nM and F4 at all tested concentrations; ApoA1 addition did not change transfection. - Protein corona analysis: ApoA1 was most abundant in F2 and F4; ApoA2 was most abundant in F1; albumin was most abundant in F3. ApoE was present at low abundance in all formulations. - Surface PEG amount inversely correlated with total particle-bound protein. Serum-to-LNP ratio altered protein enrichment patterns, with ApoA1 enrichment increasing at higher serum ratios.

Interpretation: LNP surface chemistry determines protein corona composition, which in turn affects cellular delivery and gene silencing. ApoE is one corona protein that can enhance HepG2 uptake for C14-PEG LNPs. These findings suggest that rational LNP design can recruit beneficial endogenous proteins for targeted delivery and overcome PEG-related delivery barriers.

Limitations: In vitro only; no in vivo biodistribution, tumor delivery, or therapeutic efficacy. - Protein corona studied with FBS, not mouse or human serum/plasma. - Only HepG2 cells and one gene target (CTNNB1) were tested. - No direct receptor-blocking or knockout validation for ApoE-mediated uptake. - Protein corona was analyzed after static incubation; in vivo flow and immune interactions were not assessed. - Accepted manuscript; final peer-reviewed version may differ.