Cancer is estimated to impact more than 20 million people globally in the next years. To control this global burden, more selective and effective treatments are required, relying on combinatorial therapies targeting multiple components of the disease, minimizing compensatory mechanisms that could result in relapse and resistance to treatment. Nanotechnology, and Nanoparticles (NPs) in particular, are expected to provide a range of devices for cancer diagnosis and treatment (theranostics) as their sizes are well matched in size to biologic molecules and cell structures. Gold NPs (AuNPs) have been extensively investigated and applied in conjunction with biomolecules due to their ease of synthesis and functionalization derived from their large surface area to volume ratio. This way, several strategies have been proposed based on AuNPs simultaneously functionalized with therapeutic moieties (drugs, compounds, and RNAi molecules) and targeting molecules (peptides, antibodies, etc) for the delivery of the effector molecules to specific cells, which may be combined with other therapeutic modalities, such as for photo-triggered hyperthermia.

We have been using AuNPs for combined delivery of anticancer nanotherapeutics in vitro/in vivo via precise active targeting to cancer cells, capable of gene silencing of crucial pathways involved in cancer development. We have also demonstrated the potential for the combined delivery of gene silencing with chemotherapeutics-loaded nanoparticles to downregulate proliferation pathways and angiogenesis. More recently, we have used AuNPs for photothermal potentiation of chemotherpaeutics. We shall discuss the potential of these combinatory approaches towards the development of effective nanomedicines.

Achieving to determine the local temperature of nano-objects would suppose a major breakthrough in many technological and scientific fields. Magnetic and photo-induced hyperthermia therapies employ nanoparticles as heating sources for efficient therapeutic purposes in the fight of oncological diseases. However, these therapeutic effects have been also observed by localized nanoparticle heating without a detectable macroscopic temperature rise [1]. Local heating effects produced at the nanoparticle´s surface are a key issue to evaluate the onset of thermal doses and quantify possible side effects derived from achieving high local temperatures (i.e. destroying healthy cells in the tissue, degradation of proteins and enzymes in the extracellular medium, etc). In this work, we investigate the heating efficiency of nanoparticles subjected to magneto- and photo-thermal effects in combination with other therapeutic approaches methods [2, 3]. Moreover, we also present the use of the extended X-ray absorption fine structure (EXAFS) analysis as a direct and in situ probe to determine the local temperature at the nanoscale of gold-based nanoparticles upon laser photo-excitation, revealing significant nanothermal gradients [4].

[1]        E. Cazares-Cortes et al., ACS Appl. Mater. Inter. 9, 25775 (2017).

[2]        A. Espinosa et al., Adv. Funct. Mater. 28, 1803660 (2018).

[3]        A. Espinosa et al., Small 16, 1904960 (2020).

[4]        A. Espinosa et al., Nano Letters 21, 769 (2021).

Optical hyperthermia mediated by plasmonic materials is a non-invasive technique that allows controlled temperature increase of biological tissues. Many factors, conditions, and parameters influence the therapeutic effects of heat, including intensity and irradiation time and number of pulses. Increasing evidence suggests heat-based therapies not only for cancer treatment but also in regenerative medicine, to enhance wound healing and tissue regeneration. Recently, photothermal agents such as gold nanoparticles (AuNPs) have been used as nano-hotspot to selectively generate heat in a spatiotemporal fashion, which is known as photothermal therapy [1]. Cnidarians have a long history as experimental models for regeneration due to their spectacular ability to rebuild any missing body part from tiny pieces of tissue, representing unique models for regenerative medicine. The effects induced by heat or light irradiation on the freshwater polyp Hydra vulgaris have been recently investigated [2-3], opening an interesting scenario on the possibility of developing new optothermal actuators to enhance their regenerative potential. Gold nanoparticles due to plasmonic features can release precise doses of heat under near-infrared (NIR) irradiation and in Hydra they have been shown able to induce diverse responses [4], ranging from cell ablation to programmed cell death or thermo tolerance by simply tuning the NP shape, size and in turn their thermal properties. By tuning the NIR irradiation and the AuNPs dose, the capability of treated polyps to regenerate the missing heads under photostimulation has been dissected at whole animal, cellular and molecular levels and compared to exposure to external macroscopic heat sources, suggesting an innovative application of mild hyperthermia mediated by AuNPs to enhance tissue regeneration. The results reveal the action of heat on animal physiology and open new perspectives for the development of technologies based on hyperthermia for tissue regeneration.

The CRISPR/Cas9 has revolutionized not only the process of gene editing but also  the way of understanding research. Now, we have the tools to theoretically modify the genetics of potentially any organism for industrial, biotechnological, and medical applications. Regarding the latter one, novel therapies are being developed aiming to directly intervene to the genome and treat previously incurable diseases. Despite this scenario is moving faster and getting closer towards a reality, it might not be true yet due to the unresolved issues related to delivery and safety. To help with this, nanomedicine and synthetic biology are emerging with cutting-edge solutions and new perspectives to overcome the above mentioned limitations, as for instance, controlled delivery and remote spatio-temporal activation of gene editing. Here, we propose a nano-formulation of Cas9 consisting of the Cas9 enzyme conjugated to a gold nanoparticle (AuNP-Cas9). Our results showed high cleavage and gene editing efficiency in vitro, and zebrafish embryos, respectively, and the ability to spontaneously localize in the nucleus of human melanoma cells. Thanks to the plasmonic properties of gold nanoparticles, this nano-formulation serves as a nano-source of heat validated in zebrafish embryos and could consequently combine efficient gene editing with targeted photothermal therapy in cells.

Colloidal Nanoparticles (NPs) have great potential in many applications including biological applications. The biomedical application efficacy of the NPs is determined and limited by different parameters, such as the formation of protein corona upon the interaction of the NPs with the biological milieu. Which in some cases negatively influences their final fate and reduces their circulation time. There are already different strategies aiming to suppress or at least minimize the effect of the protein corona. In the current work, we have developed a universal surface modification method based on amphiphilic polymers to modify the surface charge of the NPs from positive, negative, and zwitterionic charges, while keeping the same surface chemistry. In addition to that, studying their protein and cell interactions versus different surface charges. The physicochemical characterization of the NPs has been studied using different methods such as Dynamic light scattering (DLS), Zeta potential (ζ), UV-Vis spectroscopy, and Drop shape analyzer. The protein-NPs adsorption has been studied using Fluorescence Correlation Spectroscopy (FCS) to evaluate the change in the overall hydrodynamic size of the NPs upon interaction with different protein concentrations. Furthermore, their cellular interactions, in terms of biocompatibility and cellular uptake, have been studied using Flow cytometer and ICP-MS techniques. We have found that the protein adsorption is charge-dependent as well as the cellular uptake, regardless of the size and type of the core. Moreover, the zwitterionic structure showed significant suppression of the protein corona formation, and our results suggest the orientation and nature of the zwitterionic ligand play a major role in such suppression effect. 

Anti-cancer drugs are usually associated with lack of specificity and high systemic toxicity. Thus, new strategies are needed for reducing these side effects. Among them, enzyme prodrug therapy is one of the latest. It is based on the conversion of a low toxicity molecule into a cytotoxic agent only in presence of a specific enzyme.
Here, we propose an enzyme-nanoparticles hybrid that synergistically integrate the specific recognition, selectivity, and unique catalytic properties of enzymes with the size-dependent unique features of nanomaterials. In this sense, we have co-entrapped magnetic nanoparticles (MNPs) with a therapeutic enzyme (Horseradish Peroxidase, HRP) in a biomimetic silica matrix in mild and biocompatible conditions [1] to exploit the HRP activity on an innocuous pro-drugthat induces oxidative stress over tumour cells and the possibility of MNPs to generate heat under an alternative magnetic field.
The nanohybrids (nHs) display good co-entrapment efficiencies in terms of HRP immobilization (62 ± 6 %), expressed activity (79 ± 15 %) and entrapped iron (80 ± 6 %) and good catalytic properties. Besides, we assure a time-controlled activation of the enzyme, and thus of the cytotoxic effect, by a remote enzymatic nanoactuation. Indeed, in vitro results with nHs show that the system is not cytotoxic per se and displays enhanced cytotoxic activity only in the presence of the prodrug and the AMF application towards a pancreatic cancer cell line.

Nanotechnology allows the creation of a myriad of materials by producing composite self-assembled architectures, e.g., inorganic and/or organic nanoparticles, biomolecules, therapeutics, etc. The fine tuning over their properties provides powerful solutions for the development of smart nanotherapeutics. However, despite the multiple advantages that nanoformulations provides, very limited success has been achieved in complex models or in vivo.

Design smart nanoformulations enabling effectively targeting and dose control and thereby, avoiding off-target effects as clearance by liver and spleen, remains among the most critical challenges in the field of the nanomedicine. New synthetic approaches to design more efficient and selective nanocarriers are therefore needed.

The properties of engineered nanomaterials in terms of size, composition, shape, degradability, stimulus-responsive behaviour, elasticity, etc., can be finely modulated by several synthetic approaches. Here we will discuss some approximations developed by our group, combining stimulus-responsiveness and surface engineering, aiming to develop efficient and smart nanocarriers for biological applications. One of these approaches is based on developing biomimetic nanocarriers with a surface that mimics different cellular compositions (tumoral cells, platelets, immune cells, etc.). These nanocarriers can be programmed to perform specific biological tasks such as evade immune cells, prolong systemic circulation time, homotypic targeting, and high efficiency of intracellular drug release. Moreover, enabling effectively dose control can be achieved by developing stimuli-responsive controlled delivery systems triggered by stimuli such as NIR light.

Advancements in the use of nanoparticles for biomedical applications have clearly shown their potential for the preparation of improved imaging and drug-delivery systems. However, only a few successfully translate into clinical practice, because, a common “barrier” preventing nanoparticles from delivering efficiently their payload to the target site after administration, is related to the nanoparticle uptake by macrophages. We have recently reported disulfide-bridged organosilica nanoparticles with cage-like morphology, and assessed in detail their bioaccumulation in vivo. [1] The fate of intravenously injected 20 nm nanocages was investigated in both healthy and tumor bearing mice. Interestingly, the nanoparticles exclusively co-localize with hepatic sinusoidal endothelial cells (LSECs), while avoiding Kupffer-cell uptake (less than 6%), in both physiological and pathological condition. Our findings suggest that organosilica nanocages hold the potential to be used as nanotools for LSECs modulation, potentially impacting key biological processes such as tumor cell extravasation and hepatic immunity to invading metastatic cells or a tolerogenic state in intrahepatic immune cells in autoimmune diseases. 

Such nanocages are also able to stabilize out of equilibrium species and transport them inside cells were they can be released and evolve towards the equilibrium state. [2]

Finally  the combination of nanoparticles with injectable hydrogels  can be used for tumor resection or tissue regeneration. [3-4]. Indeed, injectable nanocomposite hydrogel able to form in situ a tissue, are an innovative material that can be employed for the treatment of esophageal fistulas. [4]

The material, easily injectable with an endoscopic needle, is formed in a time compatible with the surgical procedure and has final mechanical properties suitable for cell proliferation. The in vivo experiments (porcine model) on esophageal-cutaneous fistulas, showed improved healing in the animals treated with the hydrogel compared with the control group.

Most studies about the interaction of nanoparticles (NPs) with cells are focused on how the physicochemical properties of NPs will influence their uptake by cells. However, much less is known about their potential excretion from cells. In order to control and manipulate the number of NPs in a cell however both, cellular uptake and excretion need to be studied quantitatively. Monitoring the intracellular and extracellular amount of NPs over time (after residual non-internalized NPs have been removed), enables to disentangle the influence of cell proliferation and exocytosis, which are the major pathways for the reduction of NPs per cell. Proliferation depends on the type of cells, and exocytosis depends in addition to the type of cells also on the properties of the NPs, such as their size. Examples are given on the role of these two different processes for different cells and NPs.

Metal-based nanomaterials (NM) find applications today in the biomedical field for imaging, sensing, and therapy. Moreover, due to the possibility to functionalize their surface, they offer scope for theranostics applications, in which imaging properties, targeting agents, and drugs are carried by a single nano-platform. Therefore, it is crucial to assess the stability of these engineered nanomaterials in in vitro biological models, in order to contribute to the design of biocompatible functional NM with intact optical properties and ideally no toxicity.

Synchrotron techniques as X-ray Fluorescence (XRF) micro/nano-imaging and X-ray Absorption Spectroscopy (XAS) are unique experimental techniques that can reveal the fate of metallic NM in cells and tissues. XRF micro/nano-imaging provides the biodistribution, down to the sub-cellular level, of native and exogenous elements, allowing for the visualization of the trafficking pathways of the NM and of the target cellular compartments. XAS interrogates a selected metal and provides information about its chemical state. Therefore, it is an invaluable tool to disclose the physicochemical transformations of metallic NM in vivo/in cellulo, as it detects not only intact NM but also their degradation products, which are most often optically silent.

We will present two studies making use of synchrotron techniques to unravel the biotransformations of two distinct NM formulations. The first focuses on Ag nanoparticles (NP), which are extensively used as antibacterial agents in medical devices such as catheters, wound dressing, and implants. We investigated the transformations of Ag NP, as a function of the coating, in a hepatic cell line, including in 3D cultures mimicking liver functions. This approach allowed us to follow the in cellulo dissolution and the distribution of Ag species until biliary excretion. [1, 2]

The second example concerns indium phosphide (InP) –based quantum dots (QD): these nanocrystals exhibit a narrow and tunable photoluminescence peak in the UV/Visible range, which makes them excellent candidates for biosensing and theranostics. We probed their transformations in the polyp Hydra vulgaris, an invertebrate animal model. Although InP QDs proved stable in vitro, even after 24 h at acidic pH, we highlighted their fast and almost complete degradation in less than 3 h in vivo. [3]  

In summary, we propose a panel of experimental tools that enable the investigation of the in cellulo and in vivo stability of metal-based nanomaterials for nanomedicine.

In the last decades, inorganic nanoparticles have been steadily gaining more attention from scientists from a wide variety of fields such as material science, engineering, physics, or chemistry. The very different properties compared to that of the respective bulk, and thus intriguing characteristics of materials in the nanometre scale, have driven nanoscience to be the centre of many basic and applied research topics. Moreover, a wide variety of recently developed methodologies for their surface functionalization provide these materials with very specific properties such as drug delivery and circulating cancer biomarkers detection. In this talk we describe the synthesis and functionalization of magnetic and gold nanoparticles as therapeutic and diagnosis tools against cancer.

Gold nanoprisms (NPRs) have been functionalized with PEG, glucose, cell penetrating peptides, antibodies and/or fluorescent dyes, aiming to enhance NPRs stability, cellular uptake, and imaging capabilities, respectively. Cellular uptake and impact were assayed by a multiparametric investigation on the impact of surface modified NPRs on mice and human primary and transform cell lines. Under NIR illumination, these nanoprobes can cause apoptosis. Moreover, these nanoparticles have also been used for optoacoustic imaging, as well as for tumoral marker detection using a novel type of thermal ELISA and LFIA nanobiosensor using a thermosensitive support.

The recent cutting-edge advances on nanomaterials is anticipated to overcome some of the therapeutic window and clinical applicability of many drug/peptide molecules and can also act as innovative theranostic platforms and tool for the clinic in the future [1-4].

In the last decade, research on cancer and cardiovascular diseases resulted in a new set of potential treatments with promising results in the clinics [5-9]. Amongst the different experimental treatments, active cancer immunotherapy and targeted to the injured heart hold great promise for the future treatment of these diseases. In this work, prominent nanosystems, such as biohybrid nanocomposites made of different nanoparticles (porous silicon and oncolytic virus) and cancer cell-based membrane materials are presented and discussed as potential platforms for the individualization of medical intervention and biomedical applications.

Examples on how these biohybrid nanomaterials can be prepared and scaled-up, as well as how they can be used to enhance the drug’s targetability, intracellular drug delivery for both cancer chemo- and immune-therapy applications as well as other applications, will be highlighted and discussed.

Overall, our results suggest that biohybrid nanomaterials are a versatile and advanced platform for treatment of different diseases with an interesting potential for present and future clinical impact given its easy tailorability to each patient.

[1] Zhang, [..], Santos*, M. Hai*, D. A. Weitz*, Proc. Natl. Acad. Sci. U.S.A. 2019, 116, 7744.

[2] Liu*, [...], Santos*, Adv. Mater. 2018, 30, 1703393.

[3] Ferreira*, [...], Santos*, Adv. Funct. Mater. 2018, 28, 1705134.

[4] Ferreira*, [...], Santos*, Small 2017, 13, 1701276.

[5] Fontana*, [...], Santos*, Adv. Mater. 2017, 29, 1603239.

[6] Fontana, [...], Cerullo*, Santos*, ACS Nano 2019, 13, 6477.

[7] Yong, [...], Santos*, Yang*, Nature Commun. 2019, 10, 3838.

[8] Fusciello, [...], Santos*, Cerullo*, Nature Commun. 2019, 10, 5747.

[9] Zhang, [..] Fang*, Santos*, Zhang*, Sci. Adv. 2022, 8, eabj8207.

Herein, the synthesis and application of nanostructured materials will be described with a special focus on the properties they could offer for medical applications. Examples will be given in which (i) multilayered magnetic nanobeads are explored for the delivery of peptides molecules triggered by intracellular proteases,^[1] (ii) extracellular matrix-like magnetic nanofibers are realized as manipulative hyperthermia material and switchable drug release platforms (Figure 1).^[2]

Figure 1. Magnetically hybrid nanostructured systems.

[1] Quarta, A.; Rodio, M.; Cassani, M.; Gigli, G.; Pellegrino, T.; del Mercato, L.L. Multilayered Magnetic Nanobeads for the Delivery of Peptides Molecules Triggered by Intracellular Proteases. ACS Applied Materials and Interfaces, 2017, 9, 35095-35104.

[2] Serio, F.; Silvestri, N.; Kumar Avugadda, S.; Nucci, G.; Nitte, S.; D’Amone, E.; Onesto, V.; Gigli, G.; del Mercato, L.L.; Pellegrino, T. Co-loading of Doxorubicin and Iron Oxide Nanocubes in Polycaprolactone Fibers for Combining Magneto-Thermal and Chemotherapeutic Effects on Cancer Cells. Journal of Colloid And Interface Science, 2022, 607, 34-44.

Micro-engineered cell culture models, termed Organs-on-Chips, have emerged as a new tool to recapitulate human physiology and drug responses. Multiple studies and research programs have shown that Organs-on-Chips can capture the multicellular architectures, vascular-parenchymal tissue interfaces, chemical gradients, mechanical cues, and vascular perfusion of the body. Accordingly, these models can reproduce tissue and organ functionality and mimic human disease states to an extent thus far unattainable with conventional 2D or 3D culture systems. In this talk, we will present two approaches of using this technology. The first, will demonstrate how drug can be tested by linking of 8 human-Organ-on-a-Chip and showing results that are comparable to clinical data. Furthermore, we demonstrate how to exploit the micro-engineering technology in a novel system-level approach to decompose the integrated functions of the neurovascular unit into individual cellular compartments, while retaining their paracellular metabolic coupling. Using individual, fluidically-connected chip units, we have created a system that models influx and efflux functions of the brain vasculature and the metabolic interaction with the brain parenchyma. This model reveals a previously unknown role of the brain endothelium in neural cell metabolism: In addition to its well-established functions in metabolic transport, the brain endothelium secretes metabolites that are directly utilized by neurons. This discovery would have been impossible to achieve using conventional in vitro or in vivo measurements.

The tumor microenvironment (TME) comprises non-cancerous stromal components such as the extracellular matrix (ECM), blood vessels, infiltrating immune cells, and various associated tissue-specific cells [1]. This unique environment, which emerges during tumor progression via complex interactions between host and tumorous tissue, has been proposed as a target for anti-tumor therapies [2]. Understanding the unique nature of the TME and implementing rational design by engineering biodegradable, multivalent polymeric nanocarriers (such as polypeptides [3]) may foster the development of more efficient anticancer nanotherapeutics.

Our studies demonstrated that polyglutamate-based polymers (PGA) represent excellent candidates for TME targeting due to their architectural versatility, biodegradability, and multivalency that allows the rational design of polymer-based combination therapies and the implementation of targeting strategies. We obtained PGAs through N-carboxyanhydride ring-opening polymerization (NCA-ROP) and introduced various functionalities through post-polymerization techniques to yield a set of orthogonal reactive attachment sites [4]. Following a bottom-up strategy, we obtained self-assembling star-based polypeptide architectures that formed supramolecular nanostructures with interesting properties [5], including a lymphotropic character highly suitable for nanovaccine development. This strategy, combined with adequate bioresponsive polymer-drug linker design [6] and drug selection and tumor-associated antigens or targeting moieties [7], allowed us to achieve proof-of-concept for metastatic breast cancer [6,7], melanoma, pancreatic cancer and castration-resistant prostate cancer treatment.

The rational design of polypeptide-based therapeutics, incorporating bioresponsive elements and targeting moieties for the TME, could significantly enhance their anticancer therapeutic efficiency. Adequate tropism and appropriate drug release kinetics represent crucial parameters for achieving an adequate safety:efficacy ratio and securing an adequate therapeutic window for future treatments.

Conductive polymers are very attractive for biomedical applications. The potential to respond to light and electrical stimuli finds application in a wide range of nanomedical strategies, such as nerve and tissue regeneration (i.e. polypyrrole), enhanced neuronal growth (i.e. by polypyrrole functionalized with laminin), drug delivery and phototherapy. Often the effectiveness of new approaches is impaired by low biocompatibility, reduced biodegradability and the inefficient recognition of endogenous components or specific targets. In this direction, an effective strategy is the engineering of physiological processes in situ, in a non-invasive way, leading to the addition of non-native properties and functionality to endogenous components and biological systems. The fluorophore dithienothiophene-S,S-dioxide (DTTO) is able to spontaneously enter human and mouse cell lines and become incorporated into protein structures giving rise to fluorescent and conducting fibrils, without causing adverse effects on cell viability and proliferation. Moreover, by using the freshwater polyp Hydra vulgaris, we demonstrated in vivo the stable incorporation of the dye into supramolecular protein-dye co-assembled microfibers without signs of toxicity. Electric force microscopy showed electrical conductivity and circular dichroism analysis confirmed the presence of proteins within the fibers. In order to understand the molecular mechanism underlaying the fiber biogenesis and to improve their use for new biomedical approaches, here we demonstrated the possibility to induce DTTO-fibers production in different cell lines and in vivo models (such as Nematostella vectensis) and to modulate the production process using diverse treatment conditions. This approach could open the path to directly engineering a living organism, allowing to manipulate and to control physiological processes

Cancer metastasis is a complex process in which cells break out the primary tumor mass by invading and colonizing a new site. When patients are diagnosed with metastatic cancer, such as colorectal cancer (CRC), metastasis has already spread to another part of the body and conventional therapies fail to target cancer cells. The transforming growth factor-β inhibitor Galunisertib has been recently approved for the treatment of metastatic CRC due to its ability to revert the metastatic process and induce cell epithelization. However, the oral administration of Galunisertib causes systemic toxicity and side effects. The controlled delivery of Galunisertib to CRC through nanoparticle-based approaches can be a valid weapon to increase the drug concentration at the tumor site and improve the therapy outcomes. To this aim, porous diatomite nanoparticles (DNPs) were loaded with Galunisertib and capped with gelatin to provide the nanocarrier with pH-responsive properties. In this study, the tumor acidic microenvironment triggered the release of Galunisertib from DNPs in a sustained and selective manner. The gelatin-capped platform showed a pH and time-dependent release profile provided by the presence of gelatin. Indeed, gelatin remains folded in physiological conditions, hampering the release of Galunisertib from the nanosystem, whereas the acidic environment promoted the drug release. Here, for the first time, the drug release was monitored in living CRC cells via Surface-Enhanced Raman Scattering. The surface of DNPs was decorated with gold nanoparticles that enhanced the Raman signal of the loaded drug and enabled to trace the Galunisertib delivery from the nanocarrier to cells down to femtogram scales. Finally, in vitro studies performed on the CRC cell line confirmed that the controlled delivery of Galunisertib from modified-DNPs induced the cell epithelization more efficiently than the free drug.

Surgery, chemotherapy and radiotherapy are currently the most used strategies in the treatment of cancer, a disease whose worldwide incidence and mortality is increasing considerably over projections being expected 27.5 million new cases of cancer each year by 2040 (61.7% increase from 2018). Recent investigations on cancer treatment gravitate towards the
use of alternative therapies that overcome the problem of classical ones viz. insufficient concentration of the drug at the
tumor site, limited biodistribution, systemic toxicity, lack of selectivity or differentiation between cancer and normal
cells, and development of cancer therapy resistance.
In this sense, the use of nanobased therapeutic agents has been shown to be advantageous for exploiting the well-known
enhanced permeability and retention (EPR) effect that provokes the accumulation of particulates (bigger than 40 kDa) at
the tumor site. This is due to a combination of fenestration in the vasculature and poor lymphatic drainage from tumors.
Nevertheless, nanoparticles cannot avoid non-selective distribution even though being functionalized with targeting
molecules to induce an improvement of their therapeutic efficacy by increasing their specific uptake by tumoral cells.
To overcome the limitations of passive and active targeting in achieving consistent delivery to a varied clinical target,
more inclusive targeting strategies are being studied that do not relay on the fixed, passive accumulation capacity inherent
to a given tumor. These strategies aim to improve the delivery of nanomedicines across many solid tumor phenotypes,
thereby maximizing their clinical applicability by exploiting the use of remote stimuli responsive nanocarriers to
complement the EPR effect. In this sense, the ability of magnetic nanoparticles (MNPs) to respond to the application of
alternating magnetic fields (AMF) by locally converting magnetic field energy into thermal energy is being used as
external stimulus to gain control both in space and time of cancer therapy. The use of AMF to trigger temperature gradients
at a length scale of few tens of nanometers from the surface of MNPs has advantages compared to the use of light due
to its higher tissue penetration into soft tissues (generally >1m at field frequencies <1kHz for AMF versus few centimetres
for NIR light). In this regard, during this talk we would focus on describing complementary strategies triggered by
magnetic heatin that we have developed to gain spatio-temporal control over i) direct killing of cancer cells, ii) modulation
of the tumour stroma, and iii) drug delivery.

I will present different approaches using inorganic nanoparticles or extracellular vesicles activable by an external field to target and modulate the tumor microenvironment by thermal phototherapy or photodynamic therapy in models of cholangiocarcinoma and peritoneal metastasis. The rigidity and mechanical heterogeneity of tumors being an important factor in their progression and resistance to treatment, we will show how acting on the extracellular matrix and softening the tumor can improve the migration of T lymphocytes in different preclinical models and promote the response to immunotherapies. Measurement of tumor stiffness by elastography could be a physical biomarker, accessible by imaging, to stratify patients who may benefit from antistromal therapy to increase their response to immunotherapy

Promoting tissue repair and regeneration by manipulating intracellular pathways is currently a hot topic in biomedicine research. In living cells, external mechanical stimuli are converted into biochemical signals by the mechanotransduction processes. Cadherins are crucial cell membrane proteins that promote cell-cell adhesion through their calcium-dependent homophilic interactions. In addition, cadherins are important mechanosensors which induce intracellular cascades in order to activate collective epithelial remodeling during tissue repair. Thus, cadherins are an attractive target to manipulate intracellular pathways implicated in these processes.

Our main objective is to selectively bind magnetic microparticles (MMPs) functionalized with oriented E-cadherin fragments to the membrane of E-cadherin-expressing cells, and under the application of an external magnetic field, the pulling force on the MMPs will be used to trigger mechanotransduction processes and intracellular signallings.

To do so, we have generated different engineered molecules of E-cadherin, composed of the first two extracellular domains, which are enough to establish stable homophilic interactions with the cadherins present on the cellular membrane. We have used the wild type E-cadherin recombinant fragment, and two E-cadherin mutants generated by site directed mutagenesis, in order to control the binding affinity. The cadherin fragments have been modified with a histidine tag (His-Tag) at the C-terminus to allow their oriented attachment to NTA-Cobalt-MMPs via metal-chelate affinity.

We have optimized the functionalization of the MMPs with the E-cadherin fragments, and the amount of attached molecules has been measured by flow cytometry using an anti-E-cadherin antibody that recognize specifically these proteins present in MMPs. To test if the cadherin fragments were functional and could recognize other cadherin fragments we incubated the MMPs in presence or absence of Ca2+. Only in the presence of Ca2+ homophilic interaction between cadherins occurs and, therefore, aggregates of MMPs are formed.

Finally, we have inmmobilized the E-cadherin-MMPs on membrane of living cells that express E-cadherin, visualizing this attachment using fluorescence microscopy and SEM. MMPs have been incubated with the cells at different concentrations and times, to establish the optimal conditions of membrane labelling without premature MMP internalization. With this fine-tuning of the MMPs-cell interaction assays, the next step will be the activation of mechanotransduction processes using magnetic actuators.

Iron oxide nanoparticles (IONP) are the eternal promise to change the molecular imaging field. This promise has not become a reality due to the unfavourable imaging features of traditional iron oxide nanoparticles for many diseases, i.e. the negative contrast in magnetic resonance imaging (MRI). Our group has focused on making IONP really useful for molecular imaging. For that, we developed extremely small 68Ga core-doped iron oxide nano-radiomaterials. These nanotracers provide simultaneous positive contrast in MRI and PET signal allowing for an unambiguous diagnosis of several diseases in animal models. Recently, these nano-radiomaterials have been used for the diagnosis of cancer,[1,2] inflammatory diseases,[3] atherosclerosis[4,5] and thrombosis.[6]

Here, I’ll show our latest results in the use of these nano-radiomaterials, particularly for the diagnosis of vascular diseases and how the combined use of these nanotracers with bioorthogonal chemistry may change the way molecular imaging is carried out.