Preparation, Characterization, and Surface Modification of Trifluoroethyl Ester-Terminated Silicon Nanoparticles

Platinum-catalyzed hydrosilylation of hydrogen-terminated silicon nanoparticles (Si NPs) with 2,2,2-trifluoroethyl 4-pentenoate gave well-defined nanoparticles (NPs) with surface groups that are reactive toward amines. The particles showed a diameter of 1.4 ± 0.2 nm as revealed by transmission electron microscopy (TEM) measurements. Characterization with ¹H, ¹³C and heteronuclear NMR techniques revealed that the trifluoroethyl pentenoate group is attached to the Si NP surface via the terminal carbon atom. The trifluoroethyl ester is reactive toward primary amines, allowing for additional surface functionalization. Modification of the Si NPs was performed with benzylamine, 1,2-diaminoethane, and propargylamine. The modification gave a complete substitution of the trifluoroethyl group to amide groups. The modified Si NPs were characterized in detail by a series of one-dimensional (1-D) and two-dimensional (2-D) NMR techniques and by FT-IR. The propargylamide-terminated Si NPs were further functionalized with an azide-terminated fluorescent dye (Azide-Fluor 585 sulphorhodamine) using a copper-catalyzed azide-alkyne cycloaddition reaction (CuAAC). Gel permeation chromatography and time-resolved fluorescence anisotropy spectroscopy of the dye reveal a significant increase in the hydrodynamic radius upon clicking of the dye. Additionally, NMR spectroscopy reveals the presence of a 1,2,3-triazole ring in the product, which confirms that the increase in the hydrodynamic radius is caused by the attachment of the dye to the Si NP surface via the CuAAC reaction

Immunopeptidome of hepatocytes isolated from patients with HBV infection and hepatocellular carcinoma

Background & Aims: Antigen-specific immunotherapy is a promising strategy to treat HBV infection and hepatocellular carcinoma (HCC). To facilitate killing of malignant and/or infected hepatocytes, it is vital to know which T cell targets are presented by human leucocyte antigen (HLA)-I complexes on patient-derived hepatocytes. Here, we aimed to reveal the hepatocyte-specific HLA-I peptidome with emphasis on peptides derived from HBV proteins and tumour-associated antigens (TAA) to guide development of antigen-specific immunotherapy. Methods: Primary human hepatocytes were isolated with high purity from (HBV-infected) non-tumour and HCC tissues using a newly designed perfusion-free procedure. Hepatocyte-derived HLA-bound peptides were identified by unbiased mass spectrometry (MS), after which source proteins were subjected to Gene Ontology and pathway analysis. HBV antigen and TAA-derived HLA peptides were searched for using targeted MS, and a selection of peptides was tested for immunogenicity. Results: Using unbiased data-dependent acquisition (DDA), we acquired a high-quality HLA-I peptidome of 2 × 105 peptides that contained 8 HBV-derived peptides and 14 peptides from 8 known HCC-associated TAA that were exclusive to tumours. Of these, 3 HBV- and 12 TAA-derived HLA peptides were detected by targeted MS in the sample they were originally identified in by DDA. Moreover, 2 HBV- and 2 TAA-derived HLA peptides were detected in samples in which no identification was made using unbiased MS. Finally, immunogenicity was demonstrated for 5 HBV-derived and 3 TAA-derived peptides. Conclusions: We present a first HLA-I immunopeptidome of isolated primary human hepatocytes, devoid of immune cells. Identified HBV-derived and TAA-derived peptides directly aid development of antigen-specific immunotherapy for chronic HBV infection and HCC. The described methodology can also be applied to personalise immunotherapeutic treatment of liver diseases in general. Lay summary: Immunotherapy that aims to induce immune responses against a virus or tumour is a promising novel treatment option to treat chronic HBV infection and liver cancer. For the design of successful therapy, it is essential to know which fragments (i.e. peptides) of virus-derived and tumour-specific proteins are presented to the T cells of the immune system by diseased liver cells and are thus good targets for immunotherapy. Here, we have isolated liver cells from patients who have chronic HBV infection and/or liver cancer, analysed what peptides are presented by these cells, and assessed which peptides are able to drive immune responses