(274c) Award Submission: Targeted Delivery of Microrna By Engineered Lipid Nanoparticles for the Treatment of Metastatic Breast Cancer
Introduction:Human epidermal growth factor receptor-2 (HER2) positive breast cancer occurs in 20-30% of breast cancer patients and has been attributed to increased disease aggressiveness, reoccurrence, and overall worse prognosis (Engel & Kaklamani, 2007; Vrbic, Pejcic, Filipovic, Kocic, & Vrbic, 2013). Biological therapies such as monoclonal antibodies/tyrosine kinase inhibitors targeting HER2 have made substantial improvement in patient outcome, but resistance to these treatments over time has represented a real clinical challenge and highlighted a need to develop novel therapeutic approaches (Vrbic et al., 2013). MicroRNAs (miRNAs) are endogenous regulators of gene expression that play a pivotal role in biological processes spanning from global homeostasis to disease onset and progression. The ability to manipulate/ induce cellular re-equilibrium of deregulated miRNA expression profiles by inhibition of oncogenic miRNA or overexpression of tumor suppressor miRNA is a promising cancer strategy, but is currently hindered in application by the lack of nonviral delivery systems. Here, we developed a lipid nanoparticle (LNP) platform surface coated with Hyaluronic Acid (HA) for the targeted delivery of mature tumor suppressor MicroRNA125a-5p as a long term treatment option for breast cancer. We believe the successful implementation of this platform has major implications on future gene therapy regimens for breast cancer.
Methods and materials: LNPs were created via the traditional dry lipid film method, mechanically extruded down to the nanoscale, and covalently surface decorated with HA by crosslinking chemistry. Particle size, charge, and surface characteristics were measured by dynamic light scattering, zeta potential analysis, and transmission electron microscopy. High efficiency entrapment of MicroRNA inside the aqueous core of the HA-LNP was achieved by utilization of the scale up friendly lyophilization-rehydration active loading procedure. Three breast cell lines were employed to show the targeting potential of the nanoparticles: MCF10A (normal breast tissue, CD44 -), SKBR3 (invasive breast cancer, CD44 +), and 21MT-1 (metastatic breast cancer, CD44 ++). Western blot, qRT-PCR, MTT, and wound healing assay were performed to measure protein, mRNA, cellular proliferation, and cellular migration respectively following transfection. Live cell confocal microscopy was applied to validate cytosolic delivery of the nanoparticles, as well as validate the evasion of lyosomal degradation.
Results: The optimized HA-LNP platform was constructed from biocompatible lipids, cholesterol, and a surface coating of HA to achieve a nanocarrier system 160 nm in diameter, a polydispersity index of 0.105, a surface charge of -38 mV, and stability in solution for over 90 days. The HA-LNP delivery platform actively targets patient-derived metastatic breast cancer cells (21MT-1) isolated from the metastatic pleural effusion over normal breast tissue via an intrinsic HA-CD44 mediated endocytosis event, and has the ability to escape from the intracellular endolysosomal pathway for potent gene silencing. Knockdown of the HER2 proto-oncogene at the level of transcription and translation was achieved following HA-LNP mediated transfection with MicroRNA125a-5p. In addition, the PI3K/AKT and MAPK hyperactivated signaling pathways, cellular proliferation, and migration potential were also potently suppressed. Furthermore, the therapeutic efficacy of MicroRNA125a-5p by the HA-LNP platform was demonstrated to be significantly improved by over three fold as compared to the current gold standard commercial transfection reagent Lipofectamine.
Conclusions: Our HA-LNP platform was fabricated from a scale up friendly technique and achieved rapid and targeted intracellular delivery of MicroRNA via a HA-CD44 mediated endocytosis leading to potent oncogene silencing. This study demonstrates the use of a lipid based nonviral carrier to delivery MicroRNA as a promising strategy. In addition, we believe our HA-LNP platform can be used to study the mechanism of action for various MicroRNA and advance the field of MicroRNA therapeutics.