(M1330-01-03) Developing Lipid Nanoparticles (LNPs) for mRNA Delivery in Pancreatic Cancer (PC)

Purpose: Pancreatic cancer represents one of the most lethal malignancies, notorious for its dismal survival rates and limited treatment efficacy. Despite considerable advances in oncological therapies, including chemotherapy and molecular targeting agents, clinical outcomes for pancreatic cancer patients have not significantly improved, underscoring the urgency for novel therapeutic modalities. The application of lipid nanoparticle (LNP) technology for the administration of therapeutic mRNA molecules to pancreatic cancer cells heralds a new frontier in oncologic drug delivery systems. This technology facilitates the precise delivery of mRNA into the cancer cells, prompting intracellular RNA expression and potentially leading to a more potent therapeutic impact with a reduction in adverse systemic effects characteristic of conventional regimens. The focus of our research is to develop and optimize the LNP formulation parameters utilizing microfluidic assembly to enhance the intraperitoneal (IP) delivery of mRNA-based therapeutics. We are dedicated to the synthesis and characterization of LNPs fine-tuned for specific mRNA specifically for pancreatic cancer (PC) cells. Through this research, we aim to advance the development of mRNA-LNP as a potential therapeutic approach for PC.

Methods: We utilized microfluidic system to create a library of mRNA-LNP formulations. By changing different lipid types and lipid ratios in the lipid mixture, we aimed to fine-tune these formulations for the mRNA delivery to the pancreatic tumor. To evaluate our mRNA-LNP formulations, we looked at the physicochemical attributes such as size, homogeneity (PDI), zeta potential using dynamic light scattering (DLS) and encapsulation efficiency (EE%) using ribogreen assay. We checked the reproducibility and stability of these formulations. We also tested cytotoxicity of the formulations on KPC8060 cell line. The mRNA-LNP formulations that showed EE% more than 80% and were not toxic went through further studies. We looked at how well the LNP formulations can penetrate the in vitro tumor spheroids. To do this, we included a fluorescent marker, Rhodamine, to track the LNPs and eGFP to track the mRNA delivery. We imaged the spheroids under confocal microscope to see how deep they went into the spheroids. To investigate the in vivo biodistribution in tumor mice, we developed orthotopic pancreatic tumor mice model in c57BL/6 mice. For this study, we used luciferase mRNA (Fluc-mRNA) in our LNPs and tracked them using in vivo imaging system (IVIS).

Results: Changing different types of lipids and lipid ratio had a notable impact on the physicochemical properties of the mRNA-LNP formulations, particularly their size, PDI, zeta and EE% (Figure 1). The LNPs were found to effectively penetrate tumor spheroids and deliver mRNA inside cells during in vitro penetration assays (Figure 2D). In vivo biodistribution assays demonstrated a successful distribution of mRNA-LNPs and efficient delivery of the mRNA to pancreatic tumors. However, we observed significant difference the biodistribution for different LNP formulations (Figure 3).

Conclusion: The utilization of a microfluidic system for the development of mRNA-LNP formulations, coupled with the optimization of various process parameters, has yielded promising outcomes concerning their physicochemical attributes, cellular uptake, tumor penetration and in vivo biodistribution. As part of our future work, we aim to incorporate the targeting moiety conjugated lipid into the LNPs to enhance their specificity for pancreatic tumor delivery and reduce off target effect.


Figure 1: Physicochemical characterization of different LNP formulations.


Figure 2: Cell-based assays of LNP3 and LNP4 formulations conducted on pancreatic cancer cell line. Cytotoxicity assessment, as determined by the CTB assay on KPC8060 cell line, indicates the impact of LNP3 (Fig. 2A) and LNP4 (Fig. 2B) formulations compared to untreated (control) cells. The study involves a range of mRNA concentrations from 0.78 ng to 100 ng (C). Furthermore, the cellular uptake of LNP formulations is explored through luciferase assay, comparing it with lipofectamine-treated mRNA uptake and free mRNA uptake. (D) The in vitro tumor spheroid penetration capacity of LNP formulations is also evaluated, and the level of penetration is compared with lipofectamine-treated mRNA using post-treatment confocal imaging and z-stacking. N=3 biological replicates, scale: 100 µm.


Figure 3: Biodistribution assay for LNP formulations loaded with fLuc mRNA was conducted on male C57BL/6 tumor mice (n=5). Orthotopic tumor implantation in pancreas was done and ultrasound was used to detect tumor size. When the tumor reached to a specific size, the mice received treatment with 0.5 mg/kg fLuc mRNA LNP formulations. Following post-treatment incubation, the animals were euthanized, and their organs such as pancreas, spleen, colon, lung, liver and tumor were collected for imaging. The expression of mRNA in various organs was assessed using an in vivo imaging system to track the biodistribution of different LNP formulations.