Lipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for... Show moreLipid nanoparticles (LNPs) have gained great attention as carriers for mRNA-based therapeutics, finding applications in various indications, extending beyond their recent use in vaccines for infectious diseases. However, many aspects of LNP structure and their effects on efficacy are not well characterized. To further exploit the potential of mRNA therapeutics, better control of the relationship between LNP formulation composition with internal structure and transfection efficiency in vitro is necessary. We compared two well-established ionizable lipids, namely DODMA and MC3, in combination with two helper lipids, DOPE and DOPC, and two polymer-grafted lipids, either with polysarcosine (pSar) or polyethylene glycol (PEG). In addition to standard physicochemical characterization (size, zeta potential, RNA accessibility), small-angle X-ray scattering (SAXS) was used to analyze the structure of the LNPs. To assess biological activity, we performed transfection and cell-binding assays in human peripheral blood mononuclear cells (hPBMCs) using Thy1.1 reporter mRNA and Cy5-labeled mRNA, respectively. With the SAXS measurements, we were able to clearly reveal the effects of substituting the ionizable and helper lipid on the internal structure of the LNPs. In contrast, pSar as stealth moieties affected the LNPs in a different manner, by changing the surface morphology towards higher roughness. pSar LNPs were generally more active, where the highest transfection efficiency was achieved with the LNP formulation composition of MC3/DOPE/pSar. Our study highlights the utility of pSar for improved mRNA LNP products and the importance of pSar as a novel stealth moiety enhancing efficiency in future LNP formulation development. SAXS can provide valuable information for the rational development of such novel formulations by elucidating structural features in different LNP compositions. Show less
Nogueira, S.S.; Schlegel, A.; Maxeiner, K.; Weber, B.; Barz, M.; Schroer, M.A.; ... ; Haas, H. 2020
Polysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for... Show morePolysarcosine (pSar) is a polypeptoid based on the endogenous amino acid sarcosine (N-methylated glycine), which has previously shown potent stealth properties. Here, lipid nanoparticles (LNPs) for therapeutic application of messenger RNA were assembled using pSarcosinylated lipids as a tool for particle engineering. Using pSar lipids with different polymeric chain lengths and molar fractions enabled the control of the physicochemical characteristics of the LNPs, such as particle size, morphology, and internal structure. In combination with a suited ionizable lipid, LNPs were assembled, which displayed high RNA transfection potency with an improved safety profile after intravenous injection. Notably, a higher protein secretion with a reduced immunostimulatory response was observed when compared to systems based on polyethylene glycol (PEG) lipids. pSarcosinylated nanocarriers showed a lower proinflammatory cytokine secretion and reduced complement activation compared to PEGylated LNPs. In summary, the described pSar-based LNPs enable safe and potent delivery of mRNA, thus signifying an excellent basis for the development of PEG-free RNA therapeutics. Show less
Glycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation,... Show moreGlycans present on glycoproteins from the eggs of the parasite Schistosoma mansoni are mediators of various immune responses of the human host, including T-cell modulation and granuloma formation, and they are the target of glycan-specific antibodies. Here we have analyzed the glycosylation of kappa-5, a major glycoprotein antigen from S. mansoni eggs using a targeted approach of lectin purification followed by mass spectrometry of glycopeptides as well as released glycans. We demonstrate that kappa-5 has four fully occupied N-glycosylation sites carrying unique triantennary glycans composed of a difucosylated and xylosylated core region, and immunogenic GalNAc beta 1-4GlcNAc (LDN) termini. Furthermore, we show that the kappa-5 specific IgE antibodies in sera of S. mansoni-infected individuals are directed against the core region of the kappa-5 glycans. Whereas two previously analyzed immunomodulatory egg glycoproteins, IPSE/alpha-1 and omega-1, both express diantennary N-glycans with a difucosylated core and one or two Gal beta 1-4(Fuc alpha 1-3)GlcNAc (Lewis X) antennae, the kappa-5 glycosylation appears unique among the major soluble egg antigens of S. mansoni. The distinct structural and antigenic properties of kappa-5 glycans suggest a specific role for kappa-5 in schistosome egg immunogenicity. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.005710, 1-14, 2011. Show less
Soluble egg antigens (SEA) of the human parasite Schistosoma mansoni are among the strongest natural stimuli of Th2 responses. Omega-1, a major glycoprotein in SEA, initiates these characteristic... Show moreSoluble egg antigens (SEA) of the human parasite Schistosoma mansoni are among the strongest natural stimuli of Th2 responses. Omega-1, a major glycoprotein in SEA, initiates these characteristic Th2 responses through conditioning of dendritic cells (DCs). In view of the reported immunomodulatory potential of SEA glycans, we have investigated omega-1 glycosylation, using an approach combining mass spectrometric techniques and enzyme treatments at the glycopeptide level. We demonstrate that omega-1 has two fully occupied N-glycosylation sites, each mainly carrying core-difucosylated diantennary glycans with one or more Lewis X motifs in the antennae. Using a specific approach of nanoscale LC MS(/MS) and MALDI-TOF(/TOF) MS in combination with exoglycosidase treatments of tryptic glycopeptides, we were able to provide a detailed, site-specific glycosylation analysis of a single, native S. mansoni glycoprotein. The obtained knowledge of the glycans present on omega-1 contributes to a full understanding of the mode of action of this immunomodulatory glycoprotein. Show less
