Role of Apolipoprotein- and Vitronectin-Enriched Protein Corona on Lipid Nanoparticles for In Vivo Targeted Delivery and Transfection of Oligonucleotides in Murine Tumor Models

Purpose: The protein corona that forms on lipid nanoparticles (LNPs) after systemic administration can alter their biological identity, but its impact on tumor delivery is not well understood. This study aimed to determine whether LNP formulation composition and surface charge can manipulate the protein corona and thereby affect in vitro transfection, in vivo biodistribution, and tumor-specific delivery of oligonucleotides.

Hypothesis: Specific binding of apolipoproteins from serum to LNPs can promote delivery to LDL receptor (LDLR)-expressing tumors. LNPs with different surface charges will bind apolipoproteins differently, and different protein corona profiles will lead to different in vivo biodistribution and tumor accumulation. PEG conjugated lipid chain length and PEG amount are also key factors for successful RNAi delivery to solid tumors.

Aims: Synthesize LNPs with variations in surface charge, PEG conjugated acyl chain length, and PEG amount. - Identify the protein corona composition of each LNP type. - Evaluate how protein corona affects in vitro cell transfection and gene silencing. - Assess in vivo biodistribution and tumor accumulation in a HepG2 hepatocellular carcinoma model. - Determine optimal PEG lipid length for tumor delivery and test CTNNB1 DsiRNA delivery and gene silencing.

Delivery system: Platform: Core-shell lipid nanoparticles (LNPs). - Core lipid: DL-048 with PEG2000-conjugated lipid. - Envelope lipids: DSPC or POPG (negative charge); cholesterol or DC-cholesterol (positive charge); DL-036 or DLin-MC3-DMA (MC3) ionizable cationic lipid; PEG-lipid C20, C18, C14, or C12; PEG amount 6% or 3%. - Payload: CTNNB1 DsiRNA; Cy5.5-labeled CTNNB1 DsiRNA for imaging. - Targeting ligand: None; endogenous targeting via protein corona. - Physicochemical properties: ~80–100 nm; PDI <0.1 for most; DsiRNA encapsulation ~90%; zeta potential negative except DC-CHOL LNPs positive.

Approach: In vitro: HepG2 cells transfected with CTNNB1 DsiRNA-loaded LNPs at 100 nM in serum-free or 5% nude mouse serum (NMS) conditions; qPCR for CTNNB1 expression. - Protein corona: LNPs incubated with NMS, separated by ultracentrifugation, analyzed by SDS-PAGE and LC-MS/MS. - In vivo: HepG2 xenograft tumors in nude mice; IVIS biodistribution of Cy5.5-labeled DsiRNA at 5 mg/kg; tumor/liver exposure and gene silencing after 3 mg/kg × 3 daily doses. - Groups/controls: Free DsiRNA, PBS, various LNP formulations; n = 2 for biodistribution, n = 5 for gene silencing.

Key methods: Dynamic light scattering and zeta potential for size, PDI, surface charge. - RiboGreen/PicoGreen and UPLC for encapsulation and DsiRNA concentration. - SDS-PAGE and LC-MS/MS for protein corona identification. - IVIS imaging for in vivo biodistribution. - Stem-loop PCR for DsiRNA tumor/liver exposure. - qPCR for CTNNB1 gene silencing. - CellTiter-Blue for cytotoxicity.

Key results: 20 LNPs were prepared with similar size (~80–100 nm), PDI, and ~90% encapsulation; DC-CHOL LNPs had positive zeta potential and were smaller (~70 nm). - Apolipoproteins (ApoE, ApoA4, ApoB) were major corona proteins for original, MC3, and POPG LNPs. DC-CHOL LNPs shifted the corona to vitronectin-rich regardless of PEG type/amount. - 3% PEG LNPs bound more ApoA4 than 6% PEG LNPs. - In vitro: serum enhanced transfection of C14-C14 and C14-0 LNPs; serum inhibited C18-0 and C20-0 LNPs; DC-CHOL LNPs were cytotoxic in serum-free conditions and transfection was inhibited by serum. - In vivo: C18-C18 and C18-0 LNPs had better tumor accumulation than C14 types. C18-C18 had the best tumor-to-liver and tumor-to-all-organs accumulation ratios. - C20-C20 and C18-C18 had the highest tumor accumulation, but C18-C18 had the best tumor selectivity. - DC-CHOL and POPG LNPs showed lower tumor accumulation and higher kidney accumulation. - C18-C18 and C18-0 LNPs encapsulating CTNNB1 DsiRNA showed significant gene silencing in both tumor and liver after systemic dosing.

Interpretation: The authors conclude that lipid composition and surface charge can predictably alter the LNP protein corona. Apolipoprotein-rich corona favors delivery to LDLR-expressing HepG2 tumors, whereas vitronectin-rich corona does not. LNPs with PEG-conjugated C18 lipids and apolipoprotein-rich corona were the best candidates for in vivo delivery to HepG2 tumors. Manipulating the protein corona and PEG components may help overcome limitations in targeted tumor delivery.

Limitations: Accepted manuscript; final version may differ. - In vivo biodistribution used small group sizes (n = 2); no survival or long-term therapeutic efficacy endpoint. - Only one tumor model (HepG2) and one oligonucleotide payload (CTNNB1 DsiRNA) were tested. - No active targeting ligand; tumor delivery relied on endogenous corona-mediated targeting. - Protein corona was studied only in nude mouse serum, not human serum. - Some formulations were not tested for serum stability due to the large number of LNPs. - Mechanistic role of specific corona proteins was inferred, not directly blocked or knocked down. - No large-animal validation.