Ionizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their... Show moreIonizable cationic lipids (ICLs) play an essential role in the effectiveness of lipid nanoparticles (LNPs) for delivery of mRNA therapeutics and vaccines; therefore, critical evaluations of their biological performance would extend the existing knowledge in the field. In the present study, we examined the effects of the three clinically-approved ICLs, Dlin-MC3-DMA, ALC-0315 and SM-102, as well as DODAP, on the in vitro and in vivo performance of LNPs for mRNA delivery and vaccine efficacy. mRNA-LNPs containing these lipids were successfully prepared, which were all found to be very similar in their physicochemical properties and mRNA encapsulation efficiencies. Furthermore, the results of the in vitro studies indicated that these mRNA-LNPs were efficiently taken up by immortalized and primary immune cells with comparable efficiency; however, SM-102-based LNPs were superior in inducing protein expression and antigen-specific T cell proliferation. In contrast, in vivo studies revealed that LNPs containing ALC-0315 and SM-102 yielded almost identical protein expression levels in zebrafish embryos, which were significantly higher than Dlin-MC3-DMA-based LNPs. Additionally, a mouse immunization study demonstrated that a single-dose subcutaneous administration of the mRNA-LNPs resulted in a high production of intracellular cytokines by antigen-specific T cells, but no significant differences among the three clinically approved ICLs were observed, suggesting a weak correlation between in vitro and in vivo outcomes. This study provides strong evidence that ICLs modulate the performance of mRNA-LNPs and that in vitro data does not adequately predict their behavior in vivo. Show less
Small interfering RNA (siRNA) therapeutics have shown tremendous potential for the treatment of a range of diseases, but there is still a need for novel siRNA delivery materials. Here, we introduce... Show moreSmall interfering RNA (siRNA) therapeutics have shown tremendous potential for the treatment of a range of diseases, but there is still a need for novel siRNA delivery materials. Here, we introduce M12L24 cages as siRNA delivery agents. We used four functionalized build-ing blocks to form M12L24 nanocages. Dynamic light scattering showed that well-defined 130 nm nanocage/siRNA assemblies formed with positive zeta potentials. Cell-specific siRNA-mediated green fluorescent protein (GFP) silencing, controlled by the metal used for nanocage formation, was obtained. A Pt12L24 nanocage was highly effective in delivering siRNA to U2OS cells but showed little efficiency for HeLa cells. The less stable Pd12L24 nanocage derived from the same building block displayed effective GFP silencing for HeLa cells but not for U2OS cells. The ease of prepara-tion and the ability to tune the binding strength, together with the specific siRNA delivery efficiency depending on the building blocks and metals, show potential for future siRNA delivery applications. Show less
Zeng, Y.; Shen, M.; Singhal, A.; Sevink, G.J.A.; Crone, N.S.A.; Boyle, A.L.; Kros, A. 2023
The induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as... Show moreThe induction of a potent T cell response is essential for successful tumor immunotherapy and protection against many infectious diseases. In the past few years, mRNA vaccines have emerged as potent immune activators and inducers of a robust T cell immune response. The recent approval of the Moderna and the Pfizer/BioNTech vaccines based on lipid nanoparticles (LNP) encapsulating antigen-encoding mRNA has revolutionized the field of vaccines. The advantages of LNPs are their ease of design and formulation resulting in potent, effective, and safe vaccines. However, there is still plenty of room for improvement with respect to LNP efficacy, for instance, by optimizing the lipid composition and tuning LNP for specific purposes. mRNA delivery is known to be strongly dependent on the lipid composition of LNPs and the efficiency is mainly determined by the ionizable lipids. Besides that, cholesterol and helper lipids also play important roles in mRNA transfection potency. Here, a panel of LNP formulations was studied by keeping the ionizable lipids constant, replacing cholesterol with β-sitosterol, and changing the fusogenic helper lipid DOPE content. We studied the ability of this LNP library to induce antigen presentation and T cell proliferation to identify superior LNP candidates eliciting potent T cell immune responses. We hypothesize that using β-sitosterol and increasing DOPE content would boost the mRNA transfection on immune cells and result in enhanced immune responses. Transfection of immortal immune cell lines and bone marrow dendritic cells (BMDCs) with LNPs was studied. Delivery of mRNA coding for the model antigen ovalbumin (OVA-mRNA) to BMDCs with a number of LNP formulations, resulted in a high level of activation, as evidenced by the upregulation of the co-stimulatory receptors (CD40 and CD86) and IL-12 in BMDCs. The enhancement of BMDC activation and T cell proliferation induced by the introduction of β-sitosterol and fusogenic DOPE lipids were cell dependent. Four LNP formulations (C12-200-cho-10%DOPE, C12-200-sito-10%DOPE, cKK-E12-cho-10%DOPE and cKK-E12-sito-30%DOPE) were identified that induced robust T cell proliferation and enhanced IFN-γ, TNF-α, IL-2 expression. These results demonstrate that T cell proliferation is strongly dependent on LNP composition and promising LNP-mRNA vaccine formulations were identified. Show less
This thesis focuses on the application of lipid-based nanomedicine in drug delivery, including small molecular antitumor drugs and biomacromolecules including mRNA, and evaluates their biological... Show moreThis thesis focuses on the application of lipid-based nanomedicine in drug delivery, including small molecular antitumor drugs and biomacromolecules including mRNA, and evaluates their biological performance. We have modified liposomes and LNPs with fusogenic coiled-coil peptides to enhance the drug/mRNA delivery efficiency (Chapter 2-4), and also investigated how the lipid composition of LNPs influences the immune response (Chapter 5). Show less
Zhou, X.Q.; Mytiliniou, M.; Hilgendorf, J.; Zeng, Y.; Papadopoulou, P.; Shao, Y.; ... ; Bonnet, S. 2021
Many drug delivery systems end up in the lysosome because they are built from covalent or kinetically inert supramolecular bonds. To reach other organelles, nanoparticles hence need to either be... Show moreMany drug delivery systems end up in the lysosome because they are built from covalent or kinetically inert supramolecular bonds. To reach other organelles, nanoparticles hence need to either be made from a kinetically labile interaction that allows re-assembly of the nanoparticles inside the cell following endocytic uptake, or, be taken up by a mechanism that short-circuits the classical endocytosis pathway. In this work, the intracellular fate of nanorods that self-assemble via the Pt horizontal ellipsis Pt interaction of cyclometalated platinum(II) compounds, is studied. These deep-red emissive nanostructures (638 nm excitation, approximate to 700 nm emission) are stabilized by proteins in cell medium. Once in contact with cancer cells, they cross the cell membrane via dynamin- and clathrin-dependent endocytosis. However, time-dependent confocal colocalization and cellular electron microscopy demonstrate that they directly move to mitochondria without passing by the lysosomes. Altogether, this study suggests that Pt horizontal ellipsis Pt interaction is strong enough to generate emissive, aggregated nanoparticles inside cells, but labile enough to allow these nanostructures to reach the mitochondria without being trapped in the lysosomes. These findings open new venues to the development of bioimaging nanoplatforms based on the Pt horizontal ellipsis Pt interaction. Show less
Magnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be... Show moreMagnetic-activated cell sorting (MACS) is an affinity-based technique used to separate cells according to the presence of specific markers. Current MACS systems generally require an antigen to be expressed at the cell surface; these antigen-presenting cells subsequently interact with antibody-labeled magnetic particles, facilitating separation. Here, we present an alternative MACS method based on coiled-coil peptide interactions. We demonstrate that HeLa, CHO, and NIH3T3 cells can either incorporate a lipid-modified coiled-coil-forming peptide into their membrane, or that the cells can be transfected with a plasmid containing a gene encoding a coiled-coil-forming peptide. Iron oxide particles are functionalized with the complementary peptide and, upon incubation with the cells, labeled cells are facilely separated from nonlabeled populations. In addition, the resulting cells and particles can be treated with trypsin to facilitate detachment of the cells from the particles. Therefore, our new MACS method promotes efficient cell sorting of different cell lines, without the need for antigen presentation, and enables simple detachment of the magnetic particles from cells after the sorting process. Such a system can be applied to rapidly developing, sensitive research areas, such as the separation of genetically modified cells from their unmodified counterparts. Show less
