Multitasking Fe3O4@Cu@Au and Fe3O4@SiO2 nanoparticles for biomedical applications

Tra i nanomateriali utili in ambito biomedico, le nanoparticelle di magnetite offrono grandi vantaggi sia in campo diagnostico che terapeutico, permettendo di combinare le loro peculiari proprietà magnetiche con le note proprietà chimico fisiche che caratterizzano le nanoparticelle. Realizzando una struttura core-shell e funzionalizzando la superficie della nanoparticella con sostanze biologicamente o farmacologicamente attive, è possibile, da un lato, conservare le proprietà superparamagnetiche del nucleo e, dall'altro, ottenere nanosistemi multifunzionali più selettivi e/o efficaci nei confronti dei target biologici prescelti. Lo scopo di questa tesi ha riguardato, quindi, la progettazione e la realizzazione di nanosistemi basati su nanoparticelle core-shell costituite da Fe3O4@Cu@Au o Fe3O4@SiO2 utilizzabili per diverse applicazioni biomediche. La sintesi delle nanoparticelle è stata effettuata utilizzando reagenti biocompatibili mentre una approfondita caratterizzazione chimico-fisica dei nanosistemi è stata ottenuta tramite l'analisi con differenti tecniche microscopiche e spettroscopiche. Attraverso esperimenti biologici in vitro, è stata valutata l' efficacia terapeutica e/o l' internalizzazione in cellule immunitarie di nanosistemi opportunamente funzionalizzati con farmaci chemioterapici, macromolecole biologiche o metaboliti secondari con attivià antibiotica. Infine, nanoparticelle funzionalizzate con specifici anticorpi sono state utilizzate per lo sviluppo di strumenti diagnostici per la rivelazione di neurotrasmettitori in vitro

Analytical tools for the multiplex rapid detection of SARS-CoV-2

There is a high demand for analytical tools that can readily be applied to effectively diagnose the Covid-19 but also to carry out screening and surveillance detection with enough frequency to get the transmission rates under control and thus help to timely tackling the disease. On the one hand, high throughput analytical benchtop approaches are still highly demanding for accelerating diagnostics. Such platforms are required to show multiplexed capabilities while in turn reducing the turnaround times of currently applied techniques such as the RT-PCR gold standard. On the other hand, massive screening and surveillance protocols still require for effective tools at the point of need that could reliably detect the virus in individuals after being exposed or the likelihood of being immunized after suffering from the disease. The rapid detection of coronavirus biomarkers, including RNA as well as spike and nucleocapsid proteins in nasopharyngeal and oropharyngeal samples, together with host biomarkers such as immunoglobulins and cytokines in serum has been addressed in this work. We aim to produce tools that provided with a global response to the diagnosis, prognosis and follow-up of the disease (Figure 1). All the biocomponents and corresponding bioassay protocols required for measuring these biomarkers have been tailored made and implemented in three different platforms. A calorimetric device based on a lateral flow assay format [1, 2] and a multiplex electrochemical platform comprising an electrochemical transducer array and a paper microfluidic component [3] have been adapted to produce tools to be used at the point of care. Likewise, a fluorescence microarray platform has been set up with the potential for high-throughput screening by recording molecular signatures thanks to the its multiplexing and miniaturization capabilities. REFERENCES [1] E. Polo et al. PCT, ES2013/070549 [2] E. Polo et al. Chem. Commun., 49 (2013) 3676 [3] C. Fernández-Sánchez et al. Application no. EP20382721.

SELF-ASSEMBLED PEG-SN38 NANOSYSTEMS

The development of biocompatible and selective drug delivery systems still nowadays represents a crucial challenge for poorly soluble drugs used in cancer treatment [1]. For advanced colorectal cancer irinotecan (CPT-11), a soluble 7-ethyl-10-camptothecin (SN-38) derivative, is currently used in clinics. Since CPT-11 is only partially converted in its active metabolite, the development of other potential pro-drugs or drug delivery systems that release SN-38 in higher extension can generate more effective treatments with lower doses [2]. The covalent conjugation of SN-38 to a polymeric carrier, such as the biocompatible polyethylene glycol (PEG), generates a water-soluble conjugate with improved drug pharmacokinetic profile, protecting it from rapid degradation and stabilizing its bioactive closed-lactone form, allowing its systemic administration [3]. In this context, we have designed and synthesized diverse SN38-conjugates based on homo-bivalent and hetero-bivalent PEG-based platforms, where SN38 was conjugated through an ester bond at the C20 position. The use of a labile bond, sensitive to pH and esterases, can allow the stimuli-selective release of the intact drug. These systems have amphiphilic character due to the presence of a hydrophilic moiety of monodispersed PEG27 bound to a nitrilotriacetic acid core (NTA) [4] and a hydrophobic part containing SN38 and lipids (cholesterol or fatty acids). By adjusting the hydrophilic/hydrophobic ratio we obtain compounds able to self-assembly at ¿M concentration in aqueous media forming regular and nanostructures (10-15 nm or 40-60 nm) with different tridimensional shape, spherical or cylindrical depending on the chemical feature of the system. The preliminary results suggest that those compounds could be versatile candidates as SN38-delivery systems. References [1] Senapati S., Kumar A., Kumar S., et al., Sig. Transduct. Target Ther., 2018, 3. [2] Palakurthi S., Expert Opin Drug Deliv., 2015, 12, 1911-1921. [3] Zhao H., Rubio B., Sapra P., et al., Bioconj. Chem. 2008, 19, 849-859. [4] Pulido D., Albericio F., Royo M., Org. Letters. 2014, 16, 1318-1321

Multivalent Self-Assembled PEG-SN38 Nanosystems for Advanced Colorectal Cancer Therapy

Introduction: The improvement of solubility and stability of poorly water-soluble chemotherapeutic drugs still remains a big challenge in cancer therapy.1 For advanced colorectal cancer, Irinotecan, a soluble 7-ethyl-10-camptothecin (SN38) derivative, is currently used in clinics, although it is only partially converted in its active metabolite. The development of drug delivery systems which release SN38 in higher extension can lead to more effective treatments using lower doses.2 The pharmacokinetic profile and solubility of the drug can be improved through covalent conjugation to a polymeric carrier, such as polyethylene glycol (PEG). The presence of the polymer can also allow the stabilization of SN38 bioactive closed-lactone form, protecting it from rapid degradation.3 Objective: The development of SN38-PEG conjugates, with high biocompatibility and water solubility, able to assemble in nanovesicles that can efficiently and selectively deliver and release the drug into tumors in its active form. Material and Methods: Organic compounds were synthesized by optimizing known procedures, purified and characterized by HPLC-MS, HPLC-PDA and NMR. The self-assembly properties of the systems were evaluated by CMC and DLS experiments while the morphological characterization was carried out by means of TEM and cryo-TEM. The drug release studies were performed by HPLC-PDA. Results: New SN38-conjugates based on multivalent PEG platforms4 were designed and synthesized. The SN38 conjugation by ester bond at the C20 position can allow the selective release of the intact drug, triggered by chemical or biological stimuli. The simultaneous presence of hydrophilic moiety of PEG bound to nitrilotriacetic acid core and hydrophobic part containing SN38 and/or lipids gives the system amphiphilic character. By tuning the hydrophilic/hydrophobic ratio, SN38-nanosystems are able to self-assembly into homogeneous structures at ¿M concentration in aqueous media. Depending on the chemical features of the systems, spherical or cylindrical shapes were obtained. Kinetic studies of SN38 release at physiological condition and acid pH demonstrated the high stability of these nanosystems. Conclusions: Multivalent amphiphilic SN38-PEG platforms able to self-assembly into regular nanostructures in aqueous media were obtained. According to preliminary results on stability, water solubility and drug release profile, these compounds can be promising candidates as SN38-delivery systems for the treatment of advanced colorectal cancer. 1 Senapati S., et al. Sig. Transduct. Target Ther., 2018, 3:7. 2 Palakurthi S., Expert Opin Drug Deliv., 2015, 12, 1911-1921. 3 Zhao H., et al., Bioconj. Chem., 2008, 19, 849-859. 4 Pulido D., et al., J. Med. Chem. Org. Letters., 2018, 61, 9335-9346

Immunochemical diagnostic and therapy strategies based on Quorum Sensing

Worldwide infectious diseases are one of the major concerns entailing a dramatic impact on society. Particularly, respiratory infections, caused by the pathogenic bacteria Pseudomonas aeruginosa and Staphylococcus aureus, are responsible of more than 3 millions deaths per year1. In order to better monitor and manage these diseases, there is still the need of reliable diagnostic tools able to early detect the presence of these bacteria in biological samples and innovative therapeutic strategies to avoid the development of bacterial resistance processes. A promising approach consists on interrupting or attenuating bacterial virulence pathways (antivirulence therapies). This can be achieved by affecting the bacterial communication system Quorum Sensing (QS), a regulation mechanism that coordinates the expression of a myriad of genes in response to the presence of small signal molecules known as autoinducers2. Moreover, the detection and quantification of these molecules can provide valuable information on disease state and the subsequent treatment. Currently, immunochemical tools targeting different QS components for both diagnostic and therapeutic purposes are being developed, exploiting the high binding affinity of an antibody towards its hapten