Targeted Delivery of MicroRNA125a-5p by Engineered Lipid Nanoparticles for the Treatment of HER2 Positive Metastatic Breast Cancer

Purpose: miRNA-based therapeutics are limited by instability, poor cellular uptake, lack of targeting, and potential immune activation. No nonviral miRNA delivery strategy had been explored as a standalone treatment for HER2-positive metastatic breast cancer.

Hypothesis: Hyaluronic acid-coated lipid nanoparticles (HA-LNPs) will actively target CD44-overexpressing metastatic breast cancer cells, escape the endolysosomal pathway, deliver tumor suppressor miR125a-5p, knock down HER2 and downstream PI3K/AKT and MAPK signaling, suppress proliferation and migration, and outperform a commercial transfection reagent.

Aims: Engineer and characterize HA-coated LNPs for miRNA delivery. - Evaluate CD44-mediated targeting and intracellular delivery in breast cell lines. - Confirm endosomal escape and cytosolic localization. - Deliver miR125a-5p to patient-derived HER2+ metastatic breast cancer cells and assess HER2 knockdown, downstream signaling, proliferation, and migration. - Compare HA-LNP delivery with Lipofectamine 2000.

Delivery system: Platform: Lipid nanoparticles (LNPs) composed of L-α-phosphatidylcholine (PC), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), and cholesterol. - Surface modification: Hyaluronic acid (HA, ~1.65 MDa) conjugated via EDAC amide coupling to DPPE amines. - Payloads: FITC-dextran (10, 20, 70 kDa) as model cargo; human miR125a-5p mimic; scramble miRNA negative control. - Targeting ligand: Hyaluronic acid for CD44 receptor-mediated endocytosis. - Formulation process: Dry film hydration, extrusion to 80–100 nm, HA coating, lyophilization, rehydration with cargo. - Physicochemical properties: LNP ~93.6 nm, PDI 0.061, zeta −9.46 mV; HA-LNP ~157.2 nm, PDI 0.105, zeta −38.07 mV; HA-LNP-miR125a ~197.5 nm, PDI 0.182, zeta −43.24 mV; miRNA encapsulation 24.1 ± 4%. - Route/model: In vitro only; no in vivo administration.

Approach: Cell lines: MCF10A (normal breast), SKBR3 (HER2+ invasive breast cancer), and 21MT-1 (patient-derived HER2+ metastatic breast cancer from pleural effusion). - Targeting/uptake: Fluorescent microscopy, plate reader, live confocal microscopy, flow cytometry; HA competition assay. - Transfection: 21MT-1 cells treated with 50 pmol HA-LNP-miR125a-5p, 150 pmol HA-LNP-miR125a-5p, scramble miRNA, or Lipofectamine 2000 at 50/150 pmol. - Readouts: MTT proliferation, scratch migration assay, qRT-PCR for HER2, Western blot for HER2, PI3K, pAKT, total AKT, Ki67, pERK1/2, total ERK1/2, GAPDH/tubulin. - Replicates: Typically n = 3–4 biological replicates.

Key methods: Dynamic light scattering and zeta potential for particle characterization. - Transmission electron microscopy for morphology. - Fluorescence microscopy and plate reader for FITC-dextran uptake. - Live confocal microscopy for intracellular localization and lysosome colocalization. - Flow cytometry for HA-LNP uptake. - MTT assay for proliferation. - Scratch wound assay for migration. - qRT-PCR for HER2 mRNA. - Western blotting for HER2 pathway proteins.

Key results: HA-LNP uptake was significantly higher than naked FITC-dextran and highest in metastatic 21MT-1 cells, correlating with CD44 expression. - HA pre-treatment reduced HA-LNP uptake by 55%, supporting CD44-mediated targeting. - Flow cytometry showed ~4-fold higher HA-LNP uptake in 21MT-1 cells versus MCF10A cells. - HA-LNPs showed minimal lysosome colocalization and dispersed in the cytoplasm, indicating endosomal escape. - HER2 mRNA knockdown: 40% with 50 pmol HA-LNP-miR125a-5p and 60% with 150 pmol; Lipofectamine 2000 gave 0% at 50 pmol and 40% at 150 pmol. - HER2 protein reduction: 30% with HA-LNP versus 35% with high-dose Lipofectamine. - PI3K knockdown: 35% HA-LNP versus 40% Lipofectamine high dose. - pAKT knockdown: 40% with 50 pmol HA-LNP; Lipofectamine required 150 pmol for 60%. - Ki67 knockdown: ~40% HA-LNP versus 50% Lipofectamine high dose. - pERK1/2 reduction: 20% HA-LNP versus 30% Lipofectamine high dose. - Proliferation decreased by >15% at 72 h with HA-LNP-miR125a-5p; scramble had no effect. - Migration was reduced by >50% with HA-LNP-miR125a-5p and high-dose Lipofectamine; low-dose Lipofectamine gave 16% reduction; HA-LNP control had no effect.

Interpretation: HA-LNPs enable CD44-targeted, nonviral delivery of miR125a-5p to HER2+ metastatic breast cancer cells, producing HER2 knockdown and suppression of PI3K/AKT and MAPK pathways, proliferation, and migration. The platform performed comparably or better than Lipofectamine at lower miRNA doses, supporting further development for in vivo breast cancer gene therapy.

Limitations: In vitro only; no in vivo biodistribution, tumor efficacy, or safety data. - No animal tumor model or metastatic model validation. - Only one therapeutic miRNA and one patient-derived cell line were tested. - HA-LNP-miR125a-5p encapsulation efficiency was relatively low (24.1 ± 4%). - Serum stability, immune response, and repeat-dosing effects were not evaluated. - Mechanism of CD44 targeting was supported by competition and expression correlation, not receptor knockdown or blocking antibodies. - No long-term persistence, toxicity, or large-animal validation. - Clinical translation remains untested.