DOI

https://doi.org/10.25772/PZJE-HF94

Author ORCID Identifier

0000-0003-3820-1986

Defense Date

2024

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmaceutical Sciences

First Advisor

Sandro da Rocha

Second Advisor

Douglas Sweet

Third Advisor

Xiang-Yang Wang

Abstract

Breast cancer remains the most prevalent cancer among women worldwide, with triple-negative breast cancer (TNBC) presenting distinct treatment hurdles due to its lack of hormone receptors and HER2 expression. In spite of recent advances including immune checkpoint inhibitors, there is still a significant unmet need in the treatment of TNBC. Innovative pharmacotherapies are crucial to support the treatment of TNBC. The tumor microenvironment (TME) is an important target for new therapies as it profoundly influences cancer progression and response to current treatments. Tumor-associated macrophages (TAMs) are the most abundant immune infiltrates in the TME and their phenotype correlates with treatment outcomes. While M1-like TAMs exhibit anti-tumor effects, M2-like TAMs promote tumor progression. Reprogramming M2-like TAMs into an M1-like phenotype thus represents a promising strategy. With recent developments and clinical translation of mRNA lipid nanoparticle (LNP) therapeutics (as in COVID-19 vaccines), there has been a significant focus on mRNA as an active pharmaceutical ingredient. However, efficient mRNA delivery to macrophages via LNP is hindered by challenges in cell uptake and intracellular delivery, thereby limiting cytosolic release and efficacy. Our study aims to optimize LNP formulations for enhanced mRNA delivery to macrophages, recognizing their essential role of TAMs in the TME, and the potential to improve treatment outcomes in TNBC.

We initiated our study by optimizing the formulation method and subsequently systematically evaluated various clinically relevant lipids to determine the optimal LNP composition for mRNA delivery to macrophages. We investigated the effect of the chemistry of the ionizable and helper lipids, as well as the nitrogen to phosphorus (N:P) ratio and particle size, on mRNA LNP internalization and reporter protein expression in murine macrophages (RAW 264.7), bone marrow derived macrophages (mBMDMs) and human peripheral monocyte-derived macrophages (hMDMs). The screening process comprised three phases. In the first phase, after the identification of optimum LNP preparation conditions based on desirable quality target product profile (QTPP), including high encapsulation efficiency (EE%), we assessed the effect of different types of ionizable lipids both in vitro and in vivo. Subsequently, in the second phase, for optimized ionizable lipid chemistry, we evaluated various phospholipids, followed by an evaluation of the impact of changing sterol lipids and their molar ratios in the third phase. Following the identification of the optimal formulation, we proceeded to investigate and validate its efficacy by evaluating the translation of reporter proteins in different macrophage models, including RAW264.7, mBMDMs, and hMDMs, and also in vivo. Additionally, we assessed the ability of the optimal formulation to deliver mRNA into macrophages of various phenotypes, including M0-, M1-, and M2-like macrophages. At this point, we also introduced an M2-targeting ligand into the formulation and assessed the potential of our formulations to target tumor promoting (M2-like) macrophages, which is the final step before the evaluation of the ability of mRNA LNP to efficiently shift the phenotype of TAMs in the TME of TNBC.

Our initial screenings across various ionizable lipids revealed that SM-102, CL1, and cKK-E12 showed superior efficiency in delivering the firefly luciferase (Fluc) reporter mRNA in macrophages in vitro, with SM-102 and ALC-0315 excelling in vivo translation of this reporter protein. Based on efficacy and cytotoxicity assessments, SM-102 was selected as the ionizable lipid for subsequent formulations. Further investigations into phospholipids indicated that DOPE-based LNPs enhanced the mRNA delivery in vitro, which is also supported by literature results. Additionally, exploring various sterols and their ratios pointed to β-sitosterol-based LNPs as the most effective sterol for this formulation and this type of cell (macrophage). Modifying the N:P ratio from 6 to 24 and increasing the LNP size significantly enhanced performance. The optimized formulation, containing SM-102, DOPE, β-sitosterol, and DMG-PEG2k (F6), outperformed Onpattro-like and Moderna-like LNPs in delivering reporter mRNA to primary and immortalized macrophages, which we tentatively attribute to an increased cellular uptake. However, the precise mechanisms, whether improved intracellular trafficking or endosomal escape also contributes to the enhanced efficacy of the designer formulation, remain to be clarified. Interestingly, LNPs with an N:P ratio of 24 did not exhibit increased cell uptake compared to those with a ratio of 6, suggesting the improvements may lie within intracellular processing pathways. Moreover, preliminary results indicated that targeting LNP composed of mannose modified F6 LNP enhanced eGFP expression in M2-like macrophages compared to those treated with non-targeting F6, suggesting its potential as a platform for targeting and repolarizing M2 towards M1-like macrophages. Finally, we also demonstrated that F6 facilitates mRNA translation within the tumor microenvironment in an in vivo TNBC model. Identification of expression in the various cell types in the TME will allow us to use such a model for further optimization of our formulation.

This investigation underscores the significant impact of lipid chemistry and quality attributes of the LNP on mRNA delivery efficiency in macrophages, laying the groundwork for further optimization efforts. The optimized formulation, F6, notably enhances mRNA delivery to macrophages, opening avenues for future research into repolarizing M2-like to M1-like macrophages with mRNA LNPs, representing a promising immunotherapeutic strategy for remodeling the TME and enhancing anti-tumor immunity.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

5-8-2024

Download

Available for download on Monday, May 07, 2029

Share

COinS