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
There is an increasing interest in the application of metal-organic cages (MOCs) in a biomedicinal context, as they can offer non-classical distribution in organisms compared to molecular... Show moreThere is an increasing interest in the application of metal-organic cages (MOCs) in a biomedicinal context, as they can offer non-classical distribution in organisms compared to molecular substrates, while revealing novel cytotoxicity mechanisms. Unfortunately, many MOCs are not sufficiently stable under in vivo conditions, making it difficult to study their structure-activity relationships in living cells. As such, it is currently unclear whether MOC cytotoxicity stems from supramolecular features or their decomposition products. Herein, we describe the toxicity and photophysical properties of highly-stable rhodamine functionalized platinum-based Pt2L4 nanospheres as well as their building blocks under in vitro and in vivo conditions. We show that in both zebrafish and human cancer cell lines, the Pt2L4 nanospheres demonstrate reduced cytotoxicity and altered biodistribution within the body of zebrafish embryos compared to the building blocks. We anticipate that the composition-dependent biodistribution of Pt2L4 spheres together with their cytotoxic and photophysical properties provides the fundament for MOC application in cancer therapy. Show less
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
