Udvikling af et Nyt Peptid Baseret Probe til Billeddannelse af Tumorer

Development of a Novel Peptide Based Probe for Tumour Imaging

Bennike, Tue

4. term

Nanotechnology, Master

2011

2011-06-07

92

Radioaktive stoffer og receptor-målrettede peptider bruges ofte til kræftdiagnostik og -behandling. Den epidermale vækstfaktor receptor (EGFR) er ikke tumorspecifik, men er ofte overudtrykt i mange kræfttyper og er forbundet med dårlig prognose. Derfor er EGFR et relevant mål. Den naturlige ligand EGF har stærk mitogen aktivitet og er derfor uegnet som målretningsmolekyle. I stedet blev peptidet GE11 (en EGF-peptidanalog) valgt som et muligt EGFR-målrettet peptid. GE11 blev syntetiseret ved fast fase peptidsyntese og mærket med den fluorescerende farve 5(6)-carboxyfluorescein. Massespektrometri bekræftede korrekt vægt og aminosyresekvens, men oprensning var vanskelig og tegn på nedbrydning blev observeret. Protokollen blev derefter optimeret ved at indsætte lysin i N-terminalen (lysin-GE11), hvilket gav højere udbytte, og dette peptid blev også fluorescensmærket. Det fluorescerende peptid blev testet in vitro på NR6-cellelinjen (murine fibroblaster) uden endogen EGFR, samt på varianter stabilt transfekteret til at udtrykke enten fysiologiske eller høje niveauer af human EGFR, eller den afkortede, konstitutivt aktive og kræftspecifikke variant EGFRvIII. Receptorudtryk blev delvist bekræftet med immunfluorescens (to antistoffer) og analyseret med fluorescensmikroskopi og konfokal laserskannende mikroskopi. Ved inkubation med det fluorescensmærkede peptid var affiniteten for EGFR lav. Mikroskopi viste højere signal i linjer med EGFRvIII og fysiologisk EGFR, mens flowcytometri bekræftede lav affinitet og desuden viste lavest signal i EGFRvIII-linjen, i modsætning til mikroskopien. For at muliggøre in vivo PET/CT blev en DOTA-mærket version (DOTA-lysin-GE11) syntetiseret og oprenset. Peptidet dannede komplekser med stabile gallium- og fluorescerende europium-ioner, men på grund af lav affinitet og begrænset projekttid blev in vivo-test ikke gennemført. Det konkluderes, at den lave affinitet for EGFR sandsynligvis skyldes den ekstra lysinrest. Fremtidigt arbejde kan undersøge GE11 uden lysin eller andre peptider med affinitet for EGFRvIII for at udvikle bedre receptor-målrettede midler til kræftdiagnostik og -behandling.

Radioactive compounds and receptor-targeted peptides are commonly used in cancer diagnosis and therapy. The epidermal growth factor receptor (EGFR) is not tumor-specific, but it is frequently overexpressed in many cancers and associated with poor outcomes, making it a relevant target. The natural ligand EGF has strong mitogenic activity and is therefore unsuitable as a targeting molecule. Instead, the peptide GE11 (an EGF peptide analog) was selected as a potential EGFR-targeting peptide. GE11 was synthesized by solid-phase peptide synthesis and labeled with the fluorescent dye 5(6)-carboxyfluorescein. Mass spectrometry confirmed the correct mass and amino acid sequence, but purification was challenging and signs of degradation were observed. The protocol was then optimized by inserting lysine at the N-terminus (lysine-GE11), which improved yield, and this peptide was also fluorescence-labeled. The fluorescent peptide was tested in vitro on the NR6 cell line (murine fibroblasts) lacking endogenous EGFR, as well as on variants stably transfected to express either physiological or high levels of human EGFR, or the truncated, constitutively active, cancer-specific variant EGFRvIII. Receptor expression was partially confirmed by immunofluorescence (two antibodies) and analyzed using fluorescence microscopy and confocal laser scanning microscopy. Upon incubation with the fluorescent peptide, affinity for EGFR was low. Microscopy showed higher signal in lines expressing EGFRvIII and physiological EGFR, while flow cytometry confirmed low affinity and additionally showed the lowest signal in the EGFRvIII line, in contrast to microscopy. To enable in vivo PET/CT, a DOTA-labeled version (DOTA-lysine-GE11) was synthesized and purified. The peptide formed complexes with stable gallium and fluorescent europium ions, but due to low affinity and limited project time, in vivo testing was not performed. It is concluded that the low EGFR affinity is likely due to the extra lysine residue. Future work could examine GE11 without lysine or other peptides with affinity for EGFRvIII to advance receptor-targeted agents for cancer diagnosis and treatment.

[This abstract was generated with the help of AI]

Development ; Novel ; Labelled ; affinity ; Peptide ; Tumour ; Tumor ; Imaging ; macromolecular ; tracer ; probe ; cancer ; master ; thesis ; Aalborg ; university ; Tue ; Bennike ; petct ; spect ; cell ; synthesis ; spps ; carboxyfluorescein ; fluorescence ; DOTA ; chelator ; chelating ; europium ; gallium ; confocal ; microscopy ; flow ; cytometry ; internalisation ; nano ; nanotechnology ; nanobiotechnology

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