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Biomedical Engineering Education

Biomedical Engineering Education - Poster Session E

Poster E8 - Mannose-conjugated Poly(beta-Amino Ester) Nanoparticles for Specific and Enhanced mRNA Delivery to Antigen-Presenting Cells

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Macrophages, like dendritic cells, are bridges between the innate and adaptive immune system as antigen-presenting cells (APCs). As APCs, macrophages are integral to activating the adaptive immune system by processing and presenting antigens to T cells(1). Macrophages are highly plastic cells that can exhibit a range of phenotypes between the pro-inflammatory M1 and the anti-inflammatory M2 extremes. Focusing on their phenotypic plasticity allows us to enhance their beneficial functions as APCs, such as resolving inflammation or promoting tissue repair(2). M1 macrophages, known as “classically activated,” produce pro-inflammatory cytokines, phagocytose, and initiate immune response. In contrast, M2 macrophages, termed “alternatively activated,” are involved in tissue repair, wound healing, cell proliferation, and angiogenesis. The regulation and transition between these states are vital to the mechanisms of various diseases, including cancer(3). We use CD206/mannose receptor as our target, as it is primarily expressed on dendritic cells (DCs) and macrophages. In the context of cancer, tumor-associated macrophages (TAMs) often exhibit an M2-like phenotype. M2 macrophages upregulate the CD206 receptor, which enables selective delivery of nanoparticles to M2-like TAMs and facilitates the delivery of therapeutic agents specifically to these cells. This approach can enhance the effectiveness of cancer treatments by targeting the tumor microenvironment and modulating the behavior of TAMs to inhibit tumor progression. Thus, our goal is to engineer and optimize different formulations of polyethylene glycol (PEG) mannose functionalized nanoparticles (NPs) for improved nucleic acid delivery to APCs, which can be applied to a tumor microenvironment for enhancing cancer treatments.

Materials and

Methods:  We synthesized poly(beta-amino esters, PBAEs) as described previously(4). We utilized bone-marrow cells isolated from the tibias and femurs of C57BL/6 mice to differentiate into bone-marrow derived macrophages (BMDMs), which were polarized using lipopolysaccharide (LPS) and interleukin-4 (IL-4) into M1 and M2 macrophages, respectively. For NP formulation, lipid-polyethylene glycol (PEG) functionalized with mannose was conjugated to PBAE NPs, and green fluorescent protein (GFP) or Cy5 mRNA was encapsulated in the NPs to assess transfection or cellular uptake. Various polymer mass fractions of lipid-PEG-mannose , PEG, or a combination of both were formulated, with non-PEGylated NPs as a control. In vitro transfection efficacy and particle uptake were evaluated using measurements of the percentage of GFP-positive cells and mean fluorescence intensity of Cy5-labeled mRNA, respectively, via flow cytometry. To assess particle toxicity, we used a live/dead stain. Statistical comparisons were performed using one-way ANOVA with Dunnett’s test comparing viability to untreated cells (*, p < 0.05).

Results, Conclusions, and Discussions: Initially, panels A and B of Figure 1 highlight that high concentrations of lipid-PEG, whether alone or in combination with mannose, lead to reduced nanoparticle uptake in both M1 and M2 BMDMs. This phenomenon can be attributed to steric hindrance, where the bulky PEG molecules obstruct efficient interaction and internalization of nanoparticles by the cells. By contrast, the addition of mannose significantly enhances cellular uptake and transfection efficiency in M2 BMDMs, as evidenced by Figures 1B and 1C. This enhancement is explained by mannose-mediated endocytosis, utilizing mannose receptors on the macrophages to facilitate increased internalization and subsequent gene expression. Further analysis in Figure 1C specifically examines GFP expression, revealing that formulations incorporating PEG (without mannose) achieve similar GFP expression levels compared to those combined with mannose, which suggests that PEG on its own enhances cellular uptake and transfection efficiency. Importantly, this study identifies a differential response in M1 BMDMs, which are resistant to transfection, underscoring the complexity of macrophage biology and the necessity of delivery strategies tailored to different macrophage subtypes. These findings carry significant implications for advancing gene delivery systems targeted at macrophages, especially in therapeutic contexts where precise modulation of immune responses is critical. Studying how nanoparticle formulations impact uptake and transfection across different macrophage phenotypes can contribute to the design of more effective therapeutic strategies for more specific disease contexts and cellular targets.

Acknowledgements (Optional):  

References: [1] Hirayama et al., Int. J. Mol. Sci., 2017, 19(1), 92. [2] Martinez and Gordon, Front. Immunol., 2014, 5, 671. [3] Chávez-Galán, L.; Olleros, M. L.; Vesin, D.; Garcia, I., Front. Immunol., 2015, 6. [4] Yuan et al., AlChe J., 2017, 63(1470-1482).