Development of milk exosome-based probes for biomedical imaging

dc.contributor.advisorSalinas Rodríguez, Beatriz
dc.contributor.advisorDesco Menéndez, Manuel
dc.contributor.authorGonzález Gutiérrez, María Isabel
dc.contributor.departamentoUC3M. Departamento de Bioingenieríaes
dc.contributor.funderComunidad de Madrides
dc.contributor.tutorDesco Menéndez, Manuel
dc.description.abstractExosomes are extracellular vesicles naturally secreted by living cells, intended for the exchange of information between them. Their physicochemical characteristics, featured by nanometric size and lipid bilayer structure, make them similar to the synthetic liposomes designed to be applied in the field of biomedicine. In addition, exosomes have associated an intrinsic biological role, which confers them natural tropism to certain tissues and pathological processes. Taken together, these characteristics suggest the potential use of exosomes as natural platforms to design new nanoprobes for molecular imaging, overcoming the limitations presented by nanoparticles traditionally used in this research field, generally associated with their toxicity and in vivo stability. As source of exosomes, milk stands out due to its high content of these nanovesicles, with proven functionality as carriers of molecules and drugs. The main goal of this thesis was to develop milk exosome-based probes for the detection of several pathologies by molecular imaging. To reach this aim, the project was divided in four specific objectives, as follows: i) establishing an optimal protocol for the isolation of exosomes from, in this case, commercial goat milk, according to the milk characteristics and the resources available in the research laboratory, ii) exploring alternative strategies for the radioactive and fluorescent labeling of exosomes, overcoming the limitations of current approaches, iii) determining the in vivo pharmacokinetics and biodistribution of the exosome-based probes, to elucidate their natural behavior in healthy mice and iv) evaluating the capacity of the exosome-based probes to target inflammatory processes, as potential nanoplatforms for diagnosis. Thus, first contribution of this thesis is to describe for first time an isolation protocol based on the combination of physical and biological approaches to obtain pure and homogeneous milk exosome samples. By means of physicochemical, proteomic and biological characterization, the efficacy of the biophysical protocol was demonstrated, as well as the exosomal nature of the isolated vesicles and their non-toxicity in healthy mice. The second contribution of this thesis is based on the development of new chemical strategies for the labeling of exosomes with both radioisotopes and fluorophores. In the case of the radiochemical labeling, exosomes were labeled with Technetium-99, by the passive incorporation of this isotope into the nanovesicle structure. The main advantages of this straightforward approach is that it does not require a chelator for the coordination of the radiometal and employs mild conditions that avoid the chemical modification or degradation of the exosomes. The second chemical approach presented in this thesis is the fluorescent labeling of exosomes. Current methodologies for the fluorescent labeling of exosomes involve advanced knowledge of genetic engineering or result in unstable fluorescent probes that can lead to false positives. This thesis proposes a labeling strategy based on the covalent binding of commercial fluorophores to the functional groups available on the exosome surface. This strong chemical bond ensures the stability of the nanoprobe, preventing the release of the fluorophore. In both cases, physicochemical characterization of the resulting molecular probes showed that the original properties of the exosomes are not altered after the corresponding labeling. The third contribution of this work is the in vivo evaluation by nuclear and optical techniques of the natural behavior of labeled goat milk exosomes after exogenous administration in healthy mice. By means of SPECT/CT imaging and ex vivo evaluation, it was determined that the way of administering the exosome-based probe significantly alters their pharmacokinetic and biodistribution parameters. On the other hand, both optical and nuclear imaging revealed the high accumulation of the nanoprobes in liver after intravenous injection, due to its high uptake by hepatocytes and Kupffer cells. This finding could endorse their future used in the field of hepatic diseases. Last contribution of this thesis deals with the evaluation of the exosome-based probes for diagnostic of inflammatory pathologies. Based on the literature, goat milk exosomes are involved in the immune response and inflammation. For this reason, a fluorescent exosome-based probe developed in this thesis was evaluated in vitro against cells related to the inflammatory process and in vivo in a peritonitis mouse model, by different optical imaging techniques. Fluorescent exosomes demonstrated their ability to detect the inflammatory process underlying the disease, presenting particular uptake by the proinflammatory population of macrophages. In conclusion, this thesis provides novel methodologies to the labeling and characterization of milk exosomes for use as natural nanoprobes for molecular imaging. These approaches improve the current labeling protocols, enabling the development of stable molecular probes that do not alter the original properties of the exosomes. Pharmacokinetic and biodistribution evaluation of the developed nanoprobes highlight the importance of choosing the appropriate route of administration of exosomes according to their purpose, as well as their potential applicability in liver pathologies. Finally, studies performed in the in vivo model of peritonitis support their use as imaging probes for the detection of inflammatory response, enabling the evaluation of the process at cellular level as well as the in vivo localization of the inflammatory focus.en
dc.description.degreePrograma de Doctorado en Ciencia y Tecnología Biomédica por la Universidad Carlos III de Madrides
dc.description.responsabilityPresidente: Carlos Pérez Medina.- Secretario: Benito Jorge Rubio Retama.- Vocal: Francisca Mulero Aniortees
dc.description.sponsorshipThe work developed in this PhD thesis was framed within several national and international research projects, supported by the following funding: - Instituto de Salud Carlos III, through the project “PI16/02037”, co-funded by European Regional Development Fund (ERDF), “A way to make Europe” - Comunidad de Madrid, project “Y2018/NMT-4949 (NanoLiver-CM)” and “S2017/BMD-3867 (RENIM-CM)”, co-funded by European Structural and Investment Fund. During part of the pre-doctoral period (2022 – 2023), the author of this thesis has been also funded by Instituto de Investigación Sanitaria Gregorio Marañón, Intramural Programme for the Promotion of R&D&I 2021, Sub-programme "Pre-doctoral training contract".en
dc.relation.projectIDComunidad de Madrid. Y2018/NMT-4949/NanoLiveres
dc.relation.projectIDComunidad de Madrid. S2017/BMD-3867/RENIMes
dc.rightsAtribución-NoComercial-SinDerivadas 3.0 España*
dc.rights.accessRightsopen accessen
dc.subject.ecienciaBiología y Biomedicinaes
dc.subject.otherBiomedical imagingen
dc.subject.otherMilk exosomeen
dc.titleDevelopment of milk exosome-based probes for biomedical imagingen
dc.typedoctoral thesis*
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