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  • br The fact that albumin masking was largely eliminated from


    The fact that albumin ‘masking’ was largely eliminated from the in vivo liposomal corona (Fig. 1D) prompted us to investigate whether intravenously injected liposomes can form coronas with Dalbavancin pro-teins that cannot be directly detected by proteomic analysis of plasma control samples. Plasma control samples were prepared from blood collected from non-injected healthy and melanoma-bearing mice and without any further processing they were subjected to mass spectro-metry analysis. To offer a comprehensive identification of proteins in plasma and corona samples LC-MS/MS was performed and data ana-lysis was performed using two software tools, Scaffold and Progenesis. Scaffold software was initially used as a bioinformatics tool to identify and compare proteins in plasma and corona samples using relative protein abundance quantification, based on spectral counting. Proces-sing of the raw data generated from LC-MS/MS with Progenesis dis-covery software was then carried out to more thoroughly, quantita-tively and statistically, compare the levels of identified proteins in samples derived from healthy and tumor-bearing mice in to order dis-cover potential biomarker proteins.
    The Venn diagrams in Fig. 2A show the number of common and unique proteins in healthy and melanoma-bearing C57 mice, as iden-tified by mass spectrometry analysis of plasma control and corona samples. A significantly higher total number of proteins was detected in the corona samples (n = 932 in healthy mice; n = 897 in melanoma mice) in comparison with the number of proteins identified by plasma analysis (n = 263 in healthy mice; n = 225 in melanoma mice). In addition, the most abundant plasma proteins were not the predominant corona proteins as depicted in Fig. 2B and Table S3. These findings demonstrate the ability of liposomes to capture and amplify low abundant blood proteins that cannot be directly detected by conven-tional plasma proteomic analysis.
    Of special interest for biomarker discovery is the low-abundance plasma proteome, secreted from the tumor microenvironment into the blood circulation, likely to contain the most clinically relevant and previously undiscovered markers. Only low MW intact proteins or
    Fig. 1. Protein Corona formation in tumor-bearing mice: (A) Representative bioluminescence images of melanoma-bearing C57 mice 2 weeks post subcutaneous injection of B16f10-luc cells and of lung carcinoma-bearing SCID mice 4 weeks post intravenous injection of A549-luc cells using the IVIS Lumina II camera; (B) Negative stain TEM of liposomes before and after their I.V injection and recovery from C57 and SCID mice. All scale bars are 100 nm; (C) The total amount of proteins adsorbed in vivo onto liposomes recovered from the blood circulation of healthy and tumor-bearing mice. Pb values (μg of protein/μM lipid) represent the average and standard deviation from 3 biological replicates, each using 3 mice. *indicates p < 0.05; (D) Imperial stained SDS-PAGE gel of plasma proteins control and corona proteins associated with liposomes.
    (caption on next page)
    Fig. 2. Comprehensive identification corona proteins associated with PEGylated liposomes in the blood circulation of healthy and melanoma-bearing C57 mice by LC-MS/MS: (A) Venn diagrams report the number of unique and common proteins between proteins identified in healthy and melanoma-bearing mice, in plasma control and corona samples. Peptide identifications were accepted if they could be established at greater than 50.0% probability and protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides. Proteins shown in the Venn diagrams were identified in at least one of the three biological replicates; (B) Heatmap of Relative Protein Abundance (RPA %) values for plasma and corona proteins. Only proteins with RPA > 1% in at least one of the samples are shown. Protein-rows are sorted according to the RPA% values (from highest to lowest) of the first sample (plasma healthy). The list of illustrated plasma and corona proteins and their respective accession numbers and RPA values are shown in Table S3; (C) Classification of plasma and corona proteins identified according to their molecular mass. The RPA% values for each molecular weight group represents the average of 3 biological replicates (n = 3 mice/replicate); (D) Most abundant proteins (top-20) identified in the protein corona of liposomes intravenously injected in healthy and melanoma-bearing mice. RPA% values represent the average and standard deviation from 3 biological replicates (n = 3 mice/replicate).