How to obtain an integrated picture of the molecular networks involved in adaptation to microgravity in different biological systems?

Periodically, the European Space Agency (ESA) updates scientific roadmaps in consultation with the scientific community. The ESA SciSpacE Science Community White Paper (SSCWP) 9, "Biology in Space and Analogue Environments", focusses in 5 main topic areas, aiming to address key community-identified knowledge gaps in Space Biology. Here we present one of the identified topic areas, which is also an unanswered question of life science research in Space: "How to Obtain an Integrated Picture of the Molecular Networks Involved in Adaptation to Microgravity in Different Biological Systems?" The manuscript reports the main gaps of knowledge which have been identified by the community in the above topic area as well as the approach the community indicates to address the gaps not yet bridged. Moreover, the relevance that these research activities might have for the space exploration programs and also for application in industrial and technological fields on Earth is briefly discussed.

Development and characterization of lipid nanocapsules loaded with iron oxide nanoparticles for magnetic targeting to the blood-brain barrier.

Brain drug delivery is severely hindered by the presence of the blood-brain barrier (BBB). Its functionality relies on the interactions of the brain endothelial cells with additional cellular constituents, including pericytes, astrocytes, neurons, or microglia. To boost brain drug delivery, nanomedicines have been designed to exploit distinct delivery strategies, including magnetically driven nanocarriers as a form of external physical targeting to the BBB. Herein, a lipid-based magnetic nanocarrier prepared by a low-energy method is first described. Magnetic nanocapsules with a hydrodynamic diameter of 256.7 ± 8.5 nm (polydispersity index: 0.089 ± 0.034) and a ξ-potential of -30.4 ± 0.3 mV were obtained. Transmission electron microscopy-energy dispersive X-ray spectroscopy analysis revealed efficient encapsulation of iron oxide nanoparticles within the oily core of the nanocapsules. Both thermogravimetric analysis and phenanthroline-based colorimetric assay showed that the iron oxide percentage in the final formulation was 12 wt.%, in agreement with vibrating sample magnetometry analysis, as the specific saturation magnetization of the magnetic nanocapsules was 12% that of the bare iron oxide nanoparticles. Magnetic nanocapsules were non-toxic in the range of 50-300 µg/mL over 72 h against both the human cerebral endothelial hCMEC/D3 and Human Brain Vascular Pericytes cell lines. Interestingly, higher uptake of magnetic nanocapsules in both cell types was evidenced in the presence of an external magnetic field than in the absence of it after 24 h. This increase in nanocapsules uptake was also evidenced in pericytes after only 3 h. Altogether, these results highlight the potential for magnetic targeting to the BBB of our formulation.

Piezoelectric nanomaterials: latest applications in biomedicine and challenges in clinical translation.

Tweetable abstract Nano-sized piezoelectric materials allow for precise interaction with living systems to local deliver electrical cues. Recent innovations enhance their potential in tissue engineering and regenerative medicine.

Ultrasound-Activated Piezoelectric Nanoparticles Trigger Microglia Activity Against Glioblastoma Cells.

Glioblastoma multiforme (GBM) is the most aggressive brain cancer, characterized by a rapid and drug-resistant progression. GBM "builds" around its primary core a genetically heterogeneous tumor-microenvironment (TME), recruiting surrounding healthy brain cells by releasing various intercellular signals. Glioma-associated microglia (GAM) represent the largest population of collaborating cells, which, in the TME, usually exhibit the anti-inflammatory M2 phenotype, thus promoting an immunosuppressing environment that helps tumor growth. Conversely, "classically activated" M1 microglia could provide proinflammatory and antitumorigenic activity, expected to exert a beneficial effect in defeating glioblastoma. In this work, an immunotherapy approach based on proinflammatory modulation of the GAM phenotype is proposed, through a controlled and localized electrical stimulation. The developed strategy relies on the wireless ultrasonic excitation of polymeric piezoelectric nanoparticles coated with GBM cell membrane extracts, to exploit homotypic targeting in antiglioma applications. Such camouflaged nanotransducers locally generate electrical cues on GAM membranes, activating their M1 phenotype and ultimately triggering a promising anticancer activity. Collected findings open new perspectives in the modulation of immune cell activities through "smart" nanomaterials and, more specifically, provide an innovative auspicious tool in glioma immunotherapy.

Drug-Loaded Polydopamine Nanoparticles for Chemo/Photothermal Therapy against Colorectal Cancer Cells.

Colorectal cancer (CRC) is a common and deadly malignancy, ranking second in terms of mortality and third in terms of incidence on a global scale. The survival rates for CRC patients are unsatisfactory primarily because of the absence of highly effective clinical strategies. The efficacy of existing CRC treatments, such as chemotherapy (CT), is constrained by issues such as drug resistance and damage to healthy tissues. Alternative approaches such as photothermal therapy (PTT), while offering advantages over traditional therapies, suffer instead from a low efficiency in killing tumor cells when used alone. In this context, nanostructures can efficiently contribute to a selective and targeted treatment. Here, we combined CT and PTT by developing a nanoplatform based on polydopamine nanoparticles (PDNPs), selected for their biocompatibility, drug-carrying capabilities, and ability to produce heat upon exposure to near-infrared (NIR) irradiation. As a chemotherapy drug, sorafenib has been selected, a multikinase inhibitor already approved for clinical use. By encapsulating sorafenib in polydopamine nanoparticles (Sor-PDNPs), we were able to successfully improve the drug stability in physiological media and the consequent uptake by CRC cells, thereby increasing its therapeutic effects. Upon NIR stimulus, Sor-PDNPs can induce a temperature increment of about 10 °C, encompassing both PTT and triggering a localized and massive drug release. Sor-PDNPs were tested on healthy colon cells, showing minimal adverse outcomes; conversely, they demonstrated excellent efficacy against CRC cells, with a strong capability to hinder cancer cell proliferation and induce apoptosis. Obtained findings pave the way to new synergistic chemo-photothermal approaches, maximizing the therapeutic outcomes against CRC while minimizing side effects on healthy cells.

Establishment of an orthotopic glioblastoma mouse model for preclinical studies.

Glioblastoma accounts almost 50% of all brain cancers, being the most common and lethal brain tumor in adults. Despite the current standard gold treatment based on surgery, chemotherapy, and radiotherapy, other treatment strategies are needed. Different in vitro models are currently used, including commercial cell lines, patient-derived cell lines, organoids, as well as in vivo models, being orthotopic xenografts the most used ones. In this chapter, we describe a standard protocol for the intracranial inoculation of glioblastoma cells in immunodeficient mice, and how to follow up the tumor progression and analyze the data.

How are cell and tissue structure and function influenced by gravity and what are the gravity perception mechanisms?

Progress in mechanobiology allowed us to better understand the important role of mechanical forces in the regulation of biological processes. Space research in the field of life sciences clearly showed that gravity plays a crucial role in biological processes. The space environment offers the unique opportunity to carry out experiments without gravity, helping us not only to understand the effects of gravitational alterations on biological systems but also the mechanisms underlying mechanoperception and cell/tissue response to mechanical and gravitational stresses. Despite the progress made so far, for future space exploration programs it is necessary to increase our knowledge on the mechanotransduction processes as well as on the molecular mechanisms underlying microgravity-induced cell and tissue alterations. This white paper reports the suggestions and recommendations of the SciSpacE Science Community for the elaboration of the section of the European Space Agency roadmap "Biology in Space and Analogue Environments" focusing on "How are cells and tissues influenced by gravity and what are the gravity perception mechanisms?" The knowledge gaps that prevent the Science Community from fully answering this question and the activities proposed to fill them are discussed.

Nanomaterials as Microglia Modulators in the Treatment of Central Nervous System Disorders.

Microglia play a pivotal role in the central nervous system (CNS) homeostasis, acting as housekeepers and defenders of the surrounding environment. These cells can elicit their functions by shifting into two main phenotypes: pro-inflammatory classical phenotype, M1, and anti-inflammatory alternative phenotype, M2. Despite their pivotal role in CNS homeostasis, microglia phenotypes can influence the development and progression of several CNS disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, ischemic stroke, traumatic brain injuries, and even brain cancer. It is thus clear that the possibility of modulating microglia activation has gained attention as a therapeutic tool against many CNS pathologies. Nanomaterials are an unprecedented tool for manipulating microglia responses, in particular, to specifically target microglia and elicit an in situ immunomodulation activity. This review focuses the discussion on two main aspects: analyzing the possibility of using nanomaterials to stimulate a pro-inflammatory response of microglia against brain cancer and introducing nanostructures able to foster an anti-inflammatory response for treating neurodegenerative disorders. The final aim is to stimulate the analysis of the development of new microglia nano-immunomodulators, paving the way for innovative and effective therapeutic approaches for the treatment of CNS disorders.

Monitoring Cell Cytoskeleton Variations upon Piezoelectric Stimulation: Implications for the Immune System.

Piezoelectric stimulation can have a significant impact on different cellular functions with possible applications in several fields, such as regenerative medicine, cancer therapy, and immunoregulation. For example, piezoelectric stimulation has been shown to modulate cytoskeleton variations: the implications of this effect range from the regulation of migration and invasion of cancer cells to the activation of pro- or anti-inflammatory phenotypes in immune cells. In this chapter, we will present different methodologies to evaluate cytoskeleton variations, focusing on modifications on f-/g-actin ratio and on the migration and invasion ability of tumor cells.

Microglia Polarization and Antiglioma Effects Fostered by Dual Cell Membrane-Coated Doxorubicin-Loaded Hexagonal Boron Nitride Nanoflakes.

Microglial cells play a critical role in glioblastoma multiforme (GBM) progression, which is considered a highly malignant brain cancer. The activation of microglia can either promote or inhibit GBM growth depending on the stage of the tumor development and on the microenvironment conditions. The current treatments for GBM have limited efficacy; therefore, there is an urgent need to develop novel and efficient strategies for drug delivery and targeting: in this context, a promising strategy consists of using nanoplatforms. This study investigates the microglial response and the therapeutic efficacy of dual-cell membrane-coated and doxorubicin-loaded hexagonal boron nitride nanoflakes tested on human microglia and GBM cells. Obtained results show promising therapeutic effects on glioma cells and an M2 microglia polarization, which refers to a specific phenotype or activation state that is associated with anti-inflammatory and tissue repair functions, highlighted through proteomic analysis.

Sensorization of microfluidic brain-on-a-chip devices: Towards a new generation of integrated drug screening systems.

Brain-on-a-chip (BoC) devices show typical characteristics of brain complexity, including the presence of different cell types, separation in different compartments, tissue-like three-dimensionality, and inclusion of the extracellular matrix components. Moreover, the incorporation of a vascular system mimicking the blood-brain barrier (BBB) makes BoC particularly attractive, since they can be exploited to test the brain delivery of different drugs and nanoformulations. In this review, we introduce the main innovations in BoC and BBB-on-a-chip models, especially focusing sensorization: electrical, electrochemical, and optical biosensors permit the real-time monitoring of different biological phenomena and markers, such as the release of growth factors, the expression of specific receptors/biomarkers, the activation of immune cells, cell viability, cell-cell interactions, and BBB crossing of drugs and nanoparticles. The recent improvements in signal amplification, miniaturization, and multiplication of the sensors are discussed in an effort to highlight their benefits versus limitations and delineate future challenges in this field.

How do gravity alterations affect animal and human systems at a cellular/tissue level?

The present white paper concerns the indications and recommendations of the SciSpacE Science Community to make progress in filling the gaps of knowledge that prevent us from answering the question: "How Do Gravity Alterations Affect Animal and Human Systems at a Cellular/Tissue Level?" This is one of the five major scientific issues of the ESA roadmap "Biology in Space and Analogue Environments". Despite the many studies conducted so far on spaceflight adaptation mechanisms and related pathophysiological alterations observed in astronauts, we are not yet able to elaborate a synthetic integrated model of the many changes occurring at different system and functional levels. Consequently, it is difficult to develop credible models for predicting long-term consequences of human adaptation to the space environment, as well as to implement medical support plans for long-term missions and a strategy for preventing the possible health risks due to prolonged exposure to spaceflight beyond the low Earth orbit (LEO). The research activities suggested by the scientific community have the aim to overcome these problems by striving to connect biological and physiological aspects in a more holistic view of space adaptation effects.

Drug-Loaded Lipid Magnetic Nanoparticles for Combined Local Hyperthermia and Chemotherapy against Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is a devastating tumor of the central nervous system, currently missing an effective treatment. The therapeutic gold standard consists of surgical resection followed by chemotherapy (usually with temozolomide, TMZ) and/or radiotherapy. TMZ does not, however, provide significant survival benefit after completion of treatment because of development of chemoresistance and of heavy side effects of systemic administration. Improvement of conventional treatments and complementary therapies are urgently needed to increase patient survival and quality of life. Stimuli-responsive lipid-based drug delivery systems offer promising prospects to overcome the limitations of the current treatments. In this work, multifunctional lipid-based magnetic nanovectors functionalized with the peptide angiopep-2 and loaded with TMZ (Ang-TMZ-LMNVs) were tested to enhance specific GBM therapy on an in vivo model. Exposure to alternating magnetic fields (AMFs) enabled magnetic hyperthermia to be performed, that works in synergy with the chemotherapeutic agent. Studies on orthotopic human U-87 MG-Luc2 tumors in nude mice have shown that Ang-TMZ-LMNVs can accumulate and remain in the tumor after local administration without crossing over into healthy tissue, effectively suppressing tumor invasion and proliferation and significantly prolonging the median survival time when combined with the AMF stimulation. This powerful synergistic approach has proven to be a robust and versatile nanoplatform for an effective GBM treatment.

Magnetic self-assembly of 3D multicellular microscaffolds: A biomimetic brain tumor-on-a-chip for drug delivery and selectivity testing.

In recent years, the need for highly predictive brain cancer models to test new anticancer compounds and experimental therapeutic approaches has significantly increased. Realistic in vitro brain tumor-on-a-chip platforms would allow a more accurate selection of valid candidate drugs and nanomedicines, therefore alleviating the economic and ethical issues of unsuccessful studies in vivo. Here, we present a multi-functional self-assembled brain tumor-on-a-chip model characterized by 3D glioma cultures interfaced both to nonmalignant brain cells of the peritumoral niche and to a 3D-real-scale blood-brain barrier (BBB) microfluidic system. This platform allowed us to screen multiple features, such as BBB crossing capabilities, apoptotic efficacy against GBM cells, and side effects on nonmalignant brain cells of a promising anticancer drug, nutlin-3a, which is fundamental for the treatment of brain cancer.

Cell-Membrane-Coated and Cell-Penetrating Peptide-Conjugated Trimagnetic Nanoparticles for Targeted Magnetic Hyperthermia of Prostate Cancer Cells.

Prostate malignancy represents the second leading cause of cancer-specific death among the male population worldwide. Herein, enhanced intracellular magnetic fluid hyperthermia is applied in vitro to treat prostate cancer (PCa) cells with minimum invasiveness and toxicity and highly specific targeting. We designed and optimized novel shape-anisotropic magnetic core-shell-shell nanoparticles (i.e., trimagnetic nanoparticles - TMNPs) with significant magnetothermal conversion following an exchange coupling effect to an external alternating magnetic field (AMF). The functional properties of the best candidate in terms of heating efficiency (i.e., Fe3O4@Mn0.5Zn0.5Fe2O4@CoFe2O4) were exploited following surface decoration with PCa cell membranes (CM) and/or LN1 cell-penetrating peptide (CPP). We demonstrated that the combination of biomimetic dual CM-CPP targeting and AMF responsiveness significantly induces caspase 9-mediated apoptosis of PCa cells. Furthermore, a downregulation of the cell cycle progression markers and a decrease of the migration rate in surviving cells were observed in response to the TMNP-assisted magnetic hyperthermia, suggesting a reduction in cancer cell aggressiveness.

A Novel Patient-Personalized Nanovector Based on Homotypic Recognition and Magnetic Hyperthermia for an Efficient Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the deadliest brain tumor, characterized by an extreme genotypic and phenotypic variability, besides a high infiltrative nature in healthy tissues. Apart from very invasive surgical procedures, to date, there are no effective treatments, and life expectancy is very limited. In this work, an innovative therapeutic approach based on lipid-based magnetic nanovectors is proposed, owning a dual therapeutic function: chemotherapy, thanks to an antineoplastic drug (regorafenib) loaded in the core, and localized magnetic hyperthermia, thanks to the presence of iron oxide nanoparticles, remotely activated by an alternating magnetic field. The drug is selected based on ad hoc patient-specific screenings; moreover, the nanovector is decorated with cell membranes derived from patients' cells, aiming at increasing homotypic and personalized targeting. It is demonstrated that this functionalization not only enhances the selectivity of the nanovectors toward patient-derived GBM cells, but also their blood-brain barrier in vitro crossing ability. The localized magnetic hyperthermia induces both thermal and oxidative intracellular stress that lead to lysosomal membrane permeabilization and to the release of proteolytic enzymes into the cytosol. Collected results show that hyperthermia and chemotherapy work in synergy to reduce GBM cell invasion properties, to induce intracellular damage and, eventually, to prompt cellular death.

Self-assembled three-dimensional hydrogels based on graphene derivatives and cerium oxide nanoparticles: scaffolds for co-culture of oligodendrocytes and neurons derived from neural stem cells.

Stem cell-based therapies have shown promising results for the regeneration of the nervous system. However, the survival and integration of the stem cells in the neural circuitry is suboptimal and might compromise the therapeutic outcomes of this approach. The development of functional scaffolds capable of actively interacting with stem cells may overcome the current limitations of stem cell-based therapies. In this study, three-dimensional hydrogels based on graphene derivatives and cerium oxide (CeO2) nanoparticles are presented as prospective supports allowing neural stem cell adhesion, migration and differentiation. The morphological, mechanical and electrical properties of the resulting hydrogels can be finely tuned by controlling several parameters of the self-assembly of graphene oxide sheets, namely the amount of incorporated reducing agent (ascorbic acid) and CeO2 nanoparticles. The intrinsic properties of the hydrogels, as well as the presence of CeO2 nanoparticles, clearly influence the cell fate. Thus, stiffer adhesion substrates promote differentiation to glial cell lineages, while softer substrates enhance mature neuronal differentiation. Remarkably, CeO2 nanoparticle-containing hydrogels support the differentiation of neural stem cells to neuronal, astroglial and oligodendroglial lineage cells, promoting the in vitro generation of nerve tissue grafts that might be employed in neuroregenerative cell therapies.

Sumac (Rhus coriaria) Extract-Loaded Polymeric Nanosheets Efficiently Protect Human Dermal Fibroblasts from Oxidative Stress.

Under healthy physiological conditions, living organisms possess a variety of antioxidant mechanisms to scavenge overproduced reactive oxygen species (ROS). However, under pathological circumstances, endogenous antioxidant systems may not be adequate to eliminate the excessive amount of oxidants, and thus, a continuous exogenous antioxidant income is required. In this regard, sumac (Rhus coriaria) extract is a good candidate for therapeutic applications, because of its high content of antioxidant polyphenolic compounds. In this work, sumac extract-loaded nanosheets (sumac-nanosheet) have been exploited for loading and controlled release of sumac extract, envisioning topical drug delivery applications. Sumac extract has been obtained through the solvent extraction method, and polymeric nanosheets have been thereafter prepared through the spin coating-assisted layer-by-layer deposition of polycaprolactone (PCL), sumac extract, and poly(d,l-lactic acid) (PDLLA). The collected data show a rich content of the sumac extract in terms of polyphenolic compounds, as well as its strong antioxidant properties. Moreover, for the first time in the literature, we demonstrated the possibility of efficiently loading such extract in polymeric nanosheets and the suitability of this nanoplatform as a reactive oxygen species scavenger in human dermal fibroblasts treated with a pro-oxidant insult.

Combining confocal microscopy, dSTORM, and mass spectroscopy to unveil the evolution of the protein corona associated with nanostructured lipid carriers during blood-brain barrier crossing.

Upon coming into contact with the biological environment, nanostructures are immediately covered by biomolecules, particularly by proteins forming the so-called "protein corona" (PC). The phenomenon of PC formation has gained great attention in recent years due to its implication in the use of nanostructures in biomedicine. In fact, it has been shown that the formation of the PC can impact the performance of nanostructures by reducing their stability, causing aggregation, increasing their toxicity, and providing unexpected and undesired nanostructure-cell interactions. In this work, we decided to study for the first time the formation and the evolution of PC on the surface of nanostructured lipid carriers loaded with superparamagnetic iron oxide nanoparticles, before and after the crossing of an in vitro model of the blood-brain barrier (BBB). Combining confocal microscopy, direct STochastic Optical Reconstruction Microscopy (dSTORM), and proteomic analysis, we were able to carry out a complete analysis of the PC formation and evolution. In particular, we highlighted that PC formation is a fast process, being formed around particles even after just 1 min of exposure to fetal bovine serum. Moreover, PC formed around particles is extremely heterogeneous: while some particles have no associated PC at all, others are completely covered by proteins. Lastly, the interaction with an in vitro BBB model strongly affects the PC composition: in particular, a large amount of the proteins forming the initial PC is lost after the BBB passage and they are partially replaced by new proteins derived from both the brain endothelial cells and the cell culture medium. Altogether, the obtained data could potentially provide new insights into the design and fabrication of lipid nanostructures for the treatment of central nervous system disorders.