Lipid based sensor for the detection of tumor biomarkers

Research project

Description

Lipid-based sensors are inspired to the sensor functions of biological membranes. Ligand-receptor interactions mediated by lipid bilayers are in fact involved in physiological activities of biological membranes such as signal transduction and information processing. The reorganization and response to physicochemical stimuli by the membrane lipid bilayer greatly facilitates signal transduction.
Artificial lipid membranes can embed compounds such as peptides, enzymes, antibodies, receptors, ionophores, and redox species that can specifically detect counterparts such as substrates, antigens, hormones, ions and electron donors or acceptors.
This project proposes a synergic experimental and computational approach to develop novel lipid-based sensors for dosing the activity of three specific enzymes that are biomarker of many solid tumours, namely thymidylate synthase, thymidine phosphorylase and dihydropyrimidine dehydrogenase (DPD). These enzymes are involved in the metabolism of pyrimidines and are the target of a potent chemotherapic agent, 5-fluorouracil (5-FU), widely employed in the treatment of some of the most frequently occurring malignant tumours (breast, colon, and skin cancer). Their presence or their absence in biological fluids is indeed related to a specific state of health and their easy and fast detection is a problem of major concern. In fact, for patients affected by DPD deficiency (around 8% of patients) 5-FU can even be fatal at the very first dose. Moreover, the drug can have severe toxic effects if patients are overdosed whereas it will have a reduced therapeutic efficacy if patients are underdosed. Thus it is clear the need for a rapid and accurate detection of the activity of these enzymes before and during 5-FU treatment to individuate patients who cannot be treated and the optimal doses of treatment. At present simple, fast, reliable and cheap screening methods to dose the activity of the three enzymes before and during the treatment with 5-FU does not exist. Currently used methods based on liquid chromatography and mass spectroscopy only enable to measure the 5-FU content in the blood and require the use of expensive and not widely available equipment, thus hindering the widespread diffusion of personalised dose management. The current gold standard approach for calibrating 5-FU drug therapy is based on body surface area estimation. However, literature reports show variations up to 100 fold in the levels of 5-FU in plasma between different subjects with the same body surface area, thus resulting in the ineffectiveness of the therapy and in harmful side effects for the patients.
This project proposes the development of functionalized lipid vesicles (liposomes) designed and formulated in such a way that the interaction of the lipid embedded substrate with the target enzymes will induce lipid reorganization that will trigger an optical response. To this end, mechanisms of induction of optical signals such as formation of excimers, fluorescence resonance energy transfer, and fluorescence dequenching by chromophores in the lipid bilayer and/or in the internal aqueous phase of liposomes will be exploited. Liposomes will be formulated with both commercial and ad hoc designed lipids. Spectroscopic properties of liposomes either in aggregative conditions or not will be studied and spectral variations induced by the interaction of liposomes with the target enzymes will be investigated. Lipid composition, fluorescent probe concentration, liposome concentration, effect of medium and overall concentration conditions will be investigated for observing a proper and highly sensitive optical response. Besides conventional fluorescence measurements, non-conventional equipment (i.e. a computer screen or a mobile phone as light source and a webcam as detector) will be used to perform spectroscopic measurements to develop a cheap and alternative approach to be used in the clinical laboratories. Besides the induction of an optical response, the induction of a gravimetrical signal or an increase of bilayer permeability upon the interaction of the enzymes with their substrate anchored to lipid mono- and bi-layers immobilized on surface will be investigated by quartz crystal microbalance and by cyclic voltammetry and impedance spectroscopy measurements, respectively.
The design of lipids and the overall experimental investigations will be guided and supported by molecular dynamic simulations on model systems suitable to mimic the response of the lipid bilayers to the interaction with the enzyme and by the theoretical description of the molecular mechanisms regulating the interactions between the enzymes and their counterpart.
The success of this methodology will produce a proof of principle that could be extended to the detection of other biomarkers thus constituting a very general approach to the fast and easy detection of biological markers.
StatusActive
Effective start/end date1/1/12 → …

Funding

  • MIUR - FIRB

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Artificial Organs
Edema Disease of Swine
Buccal Administration
Contraception Behavior
Biomarkers
Substrates
Signal transduction
British Columbia
Acetone
Catalysis
Fluorescence
Immunologic Cytotoxicity
Inborn Errors Amino Acid Metabolism
Feline Sarcoma Viruses
African horse sickness virus
Carnitine Acyltransferases
Cytoplasmic Streaming
Callosities
Association Learning
Pica