Summary of main results:
The protocols for the synthesis of fluorescent molecularly imprinted polymers (MIPs) of 100-200 nm size against the human sugar structure sialic acid (SA), SA-MIPs, have been developed, optimized and evaluated. The flow cytometry method show that SA-MIPs can target and bind to different cancer cells to a various degree, by detecting a fluorescent signal on the cancer cell. With fluorescence microscopy, the binding of the SA-MIPs to cancer cells was visualized. Specificity studies were performed by using pentavalent SA conjugates.
Flow cytometry and fluorescence microscopy also show that 20 nm red carbon nanodots (CNDs) and silica-coated red CNDs (R-CSNs) against the more cancer specific glycan biomarker sialylated Tn (STn) bind and target cancer cells. In addition, protocols for the synthesis of fluorescent 100 nm MIPs against human Tn, Tn-MIPs, were evaluated.
Digital holographic microscopy for measurements of cellular morphology in 3D, with or without MIPs, have been used analyze cancer cells. For that purpose, fluorescent or non-fluorescent SA-MIPs were evaluated. The SA-MIPs ability to affect cell motility and survival was investigated. The SA-MIPs seems to be non-inflammatory and non-toxic for phagocytosing cells in vitro. To determine the possibility to reduce possible unspecific uptake of SA-MIPs by phagocytosing cells, PEGylation of the SA-MIP particles was performed and tested. Indeed, the uptake of PEGylated MIPs was decreased.
Cell libraries were generated of Tn and STn O-glycoforms, as well as of different mucins by using genetically engineered HEK293 CRISPR/Cas9 isogenic cell lines. Thereafter, secreted mucin constructs were prepared for the use to identify and validate the glyco-structures in the library by mass spectrometry. Even more complex glycan structures on the mucins MUC1, MUC2 and MUC7 have been analysed. However, more work needs to be performed for using MIPs in binding assays with whole mucin constructs, since the specificity of the MIPs could not be verified with the current protocols. O-GalNAc truncation in cell lines such as MCF-7, MDAMB231 and PC3 cells was investigated for differences in cell motility and migration. Moreover, the energy metabolism profiles of O-GalNAc truncated PC3 cells were compared with PC3 WT cells.
Investigation of the ability of the glycosaminoglycan heparin to reduce cellular infection with SARS-CoV-2 was performed.
Preparations have been done in animal models to be able to study in vivo MIP-targeting to cancer cells in mice, using SA-MIPs with different fluorophores. More work needs to be performed to select the most optimal fluorophore for MIPs in future work.
A patent “Fluorescent particles with molecularly imprinted fluorescent polymer shells for cell staining applications in cytometry and microscopy” was filed in Germany including ESRs from GlycoImaging. Moreover, two additional patents were filed in 2020, not yet published or granted, both involving the work of ESRs in GlycoImaging.
GlycoImaging ESRs participated in fourteen different conferences presenting posters during 2019-2021. The conferences from April 2020 have been virtual. Seventeen publications with connection to GlycoImaging have been published between 2018-2021. Three doctoral theses´ were defended during 2021. The exhibition “Fighting cancer with plastic bullets” 2019-2020 at the Malmö University main library was a joint outreach activity together with EU-ITN Biocapture. The Graduate student symposium on Molecular Imprinting and a Summer School was co-organized with MU-ITN Biocapture in Berlin in August 2019 (BAM was the host). ESRs at BAM in Berlin participated in the public scientific event “Long night of Science” 2019.
