Development of milk exosome-based probes for biomedical imaging

Resumen

The term nanotechnology encompasses scientific fields such as physics, chemistry, biology and technology with the aim of developing new materials and devices of nanometric size (10-9 m). Renewable energies or electronics are some of the applications of this scientific area, but it is in the biomedical field where the development of new drug transport systems and imaging agents has been a true revolution. However, despite their promising applications, several disadvantages associated with these synthetic devices, such as poor in vivo stability and biocompatibility, potential toxic profile and low scalability and cost-effective production, limit their translation to the clinical field. In this context, exosomes are emerging as a promising alternative to synthetic nanoparticles; exosomes are nanometer-sized (20-200 nm) extracellular vesicles that cells naturally release into the biological medium for intercellular communication purposes. These nanovesicles present cup-shape morphology under electron microscope and negative surface charge in buffer, but one of their most attractive characteristics is phospholipid bilayer structure, which enables hydrophilic, hydrophobic and lipophilic compounds to be loaded inside. In addition to their physical properties, another interesting feature of exosomes is their natural targeting for certain biomolecular pathways and pathologies. It has been described that exosomes are involved in the development of neurodegenerative pathologies such as Alzheimer's or Parkinson's diseases, in the spread of viral and bacterial infections, and in oncological-associated processes such as angiogenesis or macrophage immunomodulation. Altogether, their physicochemical characteristics, similar to those of synthetic lipid-based nanoparticles, and their biological role in several pathologies, postulate exosomes as novel natural nanoparticles for biomedical applications. Since all living cells of the organism produce exosomes with specific characteristics depending on the cell of origin, the choice of the exosome source on which to design the new nanoplatform should meet the following requirements: i) resistance to conditions other than physiological, to withstand the chemical treatments required for the probe/tracer design, ii) non-associated toxicity or immunogenicity and iii) high availability and cost-effective isolation. These requirements can be found in the exosomes contained in food sources, where milk stands out as a raw material for the collection of exosomes that present high resistance to pH and temperature changes, cross-species tolerance and a biological role mainly associated to the regulation of the inflammation response and the infant immune system maturation. Among the different milk sources that are part of the human diet, goat milk exosomes have not been extensively evaluated, despite the recent literature which demonstrates their natural immunoregulatory effects and the effective drug encapsulation compared to other milk-derived exosomes. Imaging techniques are a useful tool for the non-invasive assessment of the biological behavior of novel therapeutic systems, by the diagnostic of the stage and severity of a disease or evaluating the response to drug treatment. Among the different modalities available, molecular (also called functional) imaging enables the detection and visualization of biological processes in living organisms at the cellular and molecular level. In this category, optical and nuclear imaging stand out as those with better preclinical application and translation to the clinical field. Optical imaging is a technique based on the detection of light for signaling molecular and cellular processes. This methodology is commonly employed in preclinical research laboratories due to the low cost of the technique, its safety and easy use, including bioluminescence and fluorescence modalities. The detection of the light signal comes from the result of a substrate-enzyme reaction in the first case or after irradiating a commercial fluorophore with a laser in the second one. Particularly, near-infrared fluorescence is gaining relevance in clinical applications due to the possibility of improving the imaging resolution and depths, as well as reducing the background signal from endogenous molecules and tissues, presenting promising applicability in image-guided surgery. However, with a view to clinical translation, nuclear imaging still stands out as a powerful tool for diagnostic imaging in biomedicine. This technique includes Positron Emission Tomography (PET) and Single-Photon Emission Computed Tomography (SPECT) and is based on the detection of the radiation emitted by a radioisotope. Two- or three-dimensional images can be registered using these modalities, with high sensitivity and limitless depth of tissue penetration. In addition, PET or SPECT imaging are normally combined with X-Ray Computed Tomography (CT) to correlate the functional data from nuclear images and the anatomical characteristics of the event under evaluation. Both optical and nuclear imaging can support the further translation of exosomes as natural nanoparticles into the diagnostics and therapeutic field. These imaging techniques can provide a more in-depth study of their pharmacokinetic properties and enable the evaluation of their promising use as diagnostic agents of inflammation-related pathologies, in a non-invasive manner. To date, different methodologies have been explored for both radioactive and fluorescent labeling of exosomes, considering various cellular sources. However, current protocols present several disadvantages; fluorescent exosome labeling techniques involve genetic engineering expertise to incorporate the fluorescent agent into the exosome structure. Alternatively, other studies have evaluated the integration of commercial fluorophores into the membrane of these nanovesicles or the encapsulation within them, but both approaches result in the uncontrolled labeling, the modification of the exosome surface or the unintended release of the fluorophore, and can lead to false positives. In the case of nuclear imaging, the exosome labeling using Technetium-99 is one of the most employed. This isotope can be anchored to the exosome using a surface complex, or can be intraluminally entrapped after crossing the exosomal lipid membrane. However, both approaches involve sensitive chemical conditions and lead to unstable tracers or the surface modification of the exosomes. Thus, this thesis work explores the development of milk exosome-based probes as diagnostic agents for molecular imaging, taking advantage of its exceptional physicochemical properties (nanometric size, lipid bilayer, richness in functional groups) and its biological role in several pathologies. For this purpose, the thesis is divided into four specific objectives: i) to define an optimal protocol for the isolation of exosomes from goat milk, as well as to present a complete characterization of these nanovesicles at physicochemical and biological terms. ii) to evaluate alternative radioactive and fluorescent labeling strategies to develop exosome-based nanoprobes, resolving the drawbacks associated with currently use approaches. iii) to determine the in vivo pharmacokinetics and biodistribution properties of the exosome-based probes in healthy mice, for establishing their natural behavior. iv) to investigate the ability of the exosome-based probes for detecting inflammatory processes underlying pathologies, as potential diagnostic tools. In general terms, this thesis offers labeling alternatives to improve the exosome research as novel nanoplatforms for biomedical imaging but also to upgrade the knowledge on their pharmacokinetics and cell interaction, mainly related to inflammation-related diseases. First, this thesis proposes a novel methodology for the isolation of exosomes from goat milk. There are no standardized protocols for this aim and the efficiency of the current ones varies according to the inherent characteristics of this biological fluid and the equipment available in the research laboratory. Differential centrifugation is one of the most employed techniques for the collection of exosomes from milk, due to the possibility of working with large volumes of raw material, as well as its potential scalability. However, the co-precipitation of compounds with nanovesicle-like characteristics reduces the effectiveness of this methodology. Thus, differential centrifugation should be complemented by additional techniques to improve the purity of the isolated nanovesicles. Among the different approaches, size exclusion chromatography has proven to be effective in the separation of exosomes from other residual milk components. However, other techniques focused on the removal of non-exosomal compounds, such as acidification by acetic or hydrochloric acid, can affect the composition of exosomes. The novel procedure developed in this thesis combines differential ultracentrifugation and size exclusion chromatography, both as well-known physical approaches, with the biological treatment of milk with microbial rennet, enabling the isolation of pure and homogeneous samples of exosomes. Because goat milk has a high casein content, its treatment with this biological agent enables the coagulation and precipitation of this milk component, increasing the purity of the isolated nanovesicles. Other residual milk elements, such as fat, cell debris or large extracellular vesicles, are removed during differential centrifugation, varying the speed values at each step, until the final precipitation of the exosome pellet. Lastly, co-isolated nanovesicle-like components are excluded by the size exclusion chromatography, and exosomes are re-suspended in phosphate buffer. The efficacy of this biophysical approach, as well as the exosomal nature and non-toxic effects of the isolate vesicles, were evaluated by complete physicochemical and biological characterization. Transmission Electron Microscopy showed the typical “cup-shape” morphology of these nanovesicles, and Dynamic Light Scattering and Nanoparticle Tracking Analysis confirmed the homogenous size distribution of the isolated vesicles, in the nanometric range. The latter technique, together with the quantification of the protein content by Bradford-Coomassie assay, demonstrated the exosome enrichment of the collected samples. Regarding the nature of the isolated vesicles, Western Blot and proteomic evaluation revealed the presence of common exosomal surface biomarkers, as well as its endosomal origin and its implication in immunological biological pathways. On the other hand, the plasma biochemical analysis of healthy mice treated with the isolated milk exosomes exhibited no changes at basic, hepatic and inflammatory levels, suggesting the non-toxic profile of these nanovesicles. Once exosomes were successfully isolated, this thesis work presents new chemical strategies for the labeling of exosomes with either radioisotopes or fluorophores. On the one hand, exosomes were labeled with Technetium-99, by the passive incorporation of this isotope into the nanovesicle structure under physiological conditions. This straightforward methodology avoids the use of chelators and prevents the chemical modification or degradation of the exosomes, which are some of the main limitations of current labeling techniques. First, Technetium-99, 99mTc, was reduced into its reactive form (Tc (IV)) by using SnCl2. Then, the isotope was incorporate to the exosome structure by incubation at physiological conditions. The chelation with the radiometal is established on phosphonate groups of the exosomal membrane, as reported previously on synthetic liposomes studies. The chemical labeling conditions were optimized from the point of view of the amount of exosomes and reducing agent, and the radiochemical yield of the synthesis as well as the purity of the final tracer were evaluated by means of Thin Layer Chromatography and High Performance Liquid Chromatography, respectively. Transmission Electron Microscopy and Dynamic Light Scattering confirmed that 99mTc-exosomes maintained the original physicochemical properties of the starting isolated nanovesicles. Goat milk exosomes were also fluorescently labeled with two different commercial fluorophores through their covalent binding to the functional groups available on the surface of the exosomes. This strong chemical bond ensures the stability of the nanoprobe and overcomes the limitations associated to current methodologies, mainly related to the unspecific labeling and the uncontrolled release of the fluorophore. This approach was not only validated on goat milk exosomes but also on exosomes derived from tumor cell lines, demonstrating in both cases a high labeling yield, measured by flow cytometry. As in the case of radioactively labeled exosomes, the physicochemical characterization of the nanoprobes showed that the natural properties of the exosomes were not altered after the covalent fluorescent labeling. The high stability of the nanoprobe along time was demonstrated by High Performance Liquid Chromatography. The biodistribution properties of goat milk exosomes were assessed by nuclear and optical techniques after the exogenous administration of the exosome-based nanoprobes in healthy mice. Significant changes were observed in the in vivo pharmacokinetics of 99mTc-exosomes depending on the administration route (intravenous, intraperitoneal or intranasal), based on combined SPECT/CT imaging. Ex vivo biodistribution by gamma counter, blood half-life assay and autoradiography images confirmed these findings, supporting the importance of choosing an appropriate route of administration depending on the intended use of the exosomes. As observed in nuclear imaging of 99mTc-exosomes, optical techniques revealed the significant liver uptake of fluorescently labeled exosomas after intravenous injection. Results achieved by in vitro and ex vivo confocal imaging exhibited the internalization of the exosomes in hepatocytes and Kupffer cells, supporting the potential used of these nanovesicles in hepatic therapy. Last step of this thesis work has been the in vitro and in vivo assessment of these novel exosome-based optical probes as diagnostic tools for inflammation-related diseases. The non-toxic effects of both unlabeled and fluorescently labeled exosomes was first tested against macrophages, at different times and concentrations of the nanovesicles. Once demonstrated the harmlessness of the molecular nanoprobes in this cell line, in vitro flow cytometry and confocal microscopy studies showed that the uptake of exosomes in macrophages differs depending on their polarization status, highlighting the internalization of the fluorescent exosomes in the proinflammatory macrophage population, compared to the antiinflammatory phenotype and the non-activated macrophages. Finally, to confirm the ability of the exosome-based nanoprobe for signaling inflammatory focus, a peritonitis model was induced by the intraperitoneal injection of thioglycolate in mice, three hours before the exosome administration. In vivo optical imaging revealed a significant fluorescent signal in the peritonitis model mice compared to healthy animals, located in the region where the inflammatory process was generated. The in vivo uptake of the fluorescent exosomes by the myeloid population (in particular, macrophages and neutrophils) was assessed by the isolation of the peritoneal exudate from the untreated peritonitis model mice and the nanoprobe-treated peritonitis model animals. Flow cytometry and confocal imaging analysis of the exudates confirmed the in vivo internalization of the fluorescent exosomes in both cell lines, with statistical significance compared to the untreated controls. As future lines of research, this work would benefit from a complete evaluation of the immunogenicity, immunoreactivity and possible adverse effects associated to the administration of milk exosomes, considering their potential clinical transference. In addition, the involvement of these nanovesicles in certain pathologies or biological pathways, such as tumor development, should also be ruled out. The functionality of goat milk exosomes as drug delivery platforms could also be explored, taking advantage of their structural characteristics, as well as the optimization of the developed exosome-based nanoprobes by bioengineering to enhance their accumulation in specific biological sites, enriching their surface with specific target-relevant vectors. In summary, this thesis work provides new tools for the evaluation of milk exosomes as natural nanoplatforms with potential use in the field of molecular imaging. The proposed biophysical protocol for the isolation of exosomes from goat milk enables the collection a pure and homogeneous sample of nanovesicles with typical exosome-like features and no observed toxic effects. The efficacy of this methodology in relation to the elimination of co-isolated residual components could be evaluated in other milk sources in future lines of research. On the other hand, the optimized approaches for the development of both nuclear and fluorescent exosome-based probes keep “exosome-friendly” conditions, resulting in highly pure and stable products while maintaining the original physicochemical characteristics of the initial nanovesicles. These strategies also overcome common limitations associated to currently used protocols, mainly related to unspecific and/or unstable labeling. The development of these exosome-based probes enabled to gather more information about the natural behavior of exosomes in healthy organism. Thus, the relevance of the route of administration chosen for the dosage of exosomes have been confirmed, concluding that it alters drastically the pharmacokinetics and biodistribution properties of these nanovesicles. The high liver accumulation after intravenous injection, observed by both nuclear and optical imaging, also stands out the potential applicability of milk exosomes for the diagnosis and treatment of hepatic pathologies. Lastly, this thesis presents the ability of a fluorescent exosome-based nanoprobe to detect inflammatory processes underlying pathologies, demonstrating its in vivo internalization in myeloid cells associated to the inflammation focus and highlighting the uptake of goat milk exosome by the proinflammatory phenotype of macrophages. This thesis project has been framed in several national and international research projects. From the results achieved on this work, three research articles have been published in high impact scientific journals, and two others are currently under preparation. In addition, the findings achieved in this thesis have been presented in fifteen scientific congresses and have enabled the establishment of collaborations with relevant research centers such as Technische Universität München (München, Germany) and Centro Nacional de Investigaciones Oncológicas (CNIO) Carlos III (Madrid, Spain).