(M1130-01-07) Map the Formulation Space of Lipid Nanoparticles (LNPs) Using Microfluidic Assembly in the Intraperitoneal (IP) Administration of RNA Therapeutics

Purpose: Pancreatic cancer (PC) is a devastating disease with a poor prognosis, and current treatment options are limited. Despite significant progress in the development of chemotherapy and targeted therapies, the treatment of pancreatic cancer remains a major challenge. One promising approach is the use of lipid nanoparticle (LNP) technology to deliver therapeutic RNA to pancreatic cancer cells. RNA-LNP technology allows for the specific delivery of RNA to cancer cells, leading to the expression of RNA within the cancer cells. This targeted delivery can be more effective and have fewer side effects compared to traditional therapies. The aim of this project is to map the formulation space of lipid nanoparticles (LNPs) using microfluidic assembly in the intraperitoneal (IP) administration of RNA therapeutics. Our goal is to synthesize and characterize LNPs optimized for a particular type of RNA, specifically for pancreatic cancer (PC) cells. Through this research, we aim to advance the development of RNA-LNP as a potential therapeutic approach for PC.

Methods: A microfluidic system was utilized in the development of the library of RNA-LNP formulations, where various parameters were modified to optimize the formulation. In order to characterize the formulations, their size, polydispersity index (PDI), encapsulation efficiency (EE%), and stability were assessed using different characterization techniques such as DLS, ribogreen assay, etc. Additionally, the in vitro cytotoxicity of the formulation was evaluated on pancreatic cell lines. RNA-LNP formulations with high EE% and no cytotoxicity were investigated for in vitro tumor spheroid penetration and in vivo biodistribution in mice. To track the biodistribution and level of penetration, a fluorescent dye (Rhodamine) was incorporated into the LNPs. Tumor penetration was observed under confocal microscopy. For biodistribution, c57BL/6 mice were given IP administration of RNA-LNP formulation and particles were tracked by in vivo imaging system. Luciferase mRNA was employed as a payload to determine the efficiency of mRNA delivery to cells and subsequent mRNA expression inside the cells.

Results: The modification of various process parameters had a notable impact on the physicochemical properties of the RNA-LNP formulations, particularly their size, PDI, and EE%. The LNPs were found to effectively penetrate tumor spheroids and deliver mRNA inside cells during in vitro penetration assays. In vivo biodistribution assays demonstrated a successful distribution of LNPs and efficient delivery of the payload to the pancreas.

Conclusion: In conclusion, the development of RNA-LNP formulations using a microfluidic system and optimization of various process parameters have shown promising results in delivering RNA therapeutics for pancreatic cancer treatment. The LNPs exhibited efficient penetration of tumor spheroids and successful delivery of the mRNA payload in vitro. Furthermore, in vivo biodistribution assays revealed the successful delivery of the LNP formulation to the pancreas. Future studies will focus on investigating the biodistribution of LNPs in pancreatic tumors. These findings suggest that RNA-LNP formulations have great potential as an effective delivery system for RNA therapeutics in pancreatic cancer treatment.

Figure 1. Physicochemical characterization such as size, PDI and encapsulation efficiency (EE%) of LNP3 and LNP4 for 15 days stored at 4 degrees. Size, PDI and EE% of LNP3 are stable for 15 days (A, B, C); Size, PDI and EE% of LNP4 are stable for 15 days (D, E, F). This study determines the stability of the formulations at 4 degrees.

Figure 2. Biodistribution analysis of LNP 3 in C57BL/6 mice measured by IVIS. 2(A) shows the untreated mice where no signal for luciferase or rhodamine was found; 2(B) shows C57BL/6 mice that were given IP administration of NP 3 formulation. After 24hr of treatment, the mice were imaged by IVIS (both whole-body imaging and organ imaging). Luciferase expression was found in the pancreas and colon and a rhodamine signal was found in the colon after 24hr.

Figure 3. In vitro KPC8060 pancreatic cancer tumor spheroid penetration assay. Spheroid penetration of NP 3 and NP 4 was investigated in comparison to spheroids without treatment and spheroids treated with GFP mRNA along with lipofectamine. Here, Rh (red) indicates the presence of LNPs and GFP (green) indicates protein expression. From this assay, it is found that both LNP 3 and NP 4 formulations can penetrate tumor spheroids and successful GFP mRNA expression into GFP protein.