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.

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.

Current advances in temozolomide encapsulation for the enhancement of glioblastoma treatment.

Glioblastoma is the most common and lethal brain tumor in adults. The incorporation of temozolomide (TMZ) into the standard treatment has increased the overall survival rate of glioblastoma patients. Since then, significant advances have been made in understanding the benefits and limitations of TMZ. Among the latter, the unspecific toxicity of TMZ, poor solubility, and hydrolyzation are intrinsic characteristics, whereas the presence of the blood-brain barrier and some tumor properties, such as molecular and cellular heterogeneity and therapy resistance, have limited the therapeutic effects of TMZ in treating glioblastoma. Several reports have revealed that different strategies for TMZ encapsulation in nanocarriers overcome those limitations and have shown that they increase TMZ stability, half-life, biodistribution, and efficacy, offering the promise for future nanomedicine therapies in handling glioblastoma. In this review, we analyze the different nanomaterials used for the encapsulation of TMZ to improve its stability, blood half-life and efficacy, paying special attention to polymer- and lipid-based nanosystems. To improve TMZ drug resistance, present in up to 50% of patients, we detail TMZ combined therapeutic with i) other chemotherapies, ii) inhibitors, iii) nucleic acids, iv) photosensitizers and other nanomaterials for photodynamic therapy, photothermal therapy, and magnetic hyperthermia, v) immunotherapy, and vi) other less explored molecules. Moreover, we describe targeting strategies, such as passive targeting, active targeting to BBB endothelial cells, glioma cells, and glioma cancer stem cells, and local delivery, where TMZ has demonstrated an improved outcome. To finish our study, we include possible future research directions that could help decrease the time needed to move from bench to bedside.

Ultrasound-responsive nutlin-loaded nanoparticles for combined chemotherapy and piezoelectric treatment of glioblastoma cells.

Glioblastoma multiforme (GBM), also known as grade IV astrocytoma, represents the most aggressive primary brain tumor. The complex genetic heterogeneity, the acquired drug resistance, and the presence of the blood-brain barrier (BBB) limit the efficacy of the current therapies, with effectiveness demonstrated only in a small subset of patients. To overcome these issues, here we propose an anticancer approach based on ultrasound-responsive drug-loaded organic piezoelectric nanoparticles. This anticancer nanoplatform consists of nutlin-3a-loaded ApoE-functionalized P(VDF-TrFE) nanoparticles, that can be remotely activated with ultrasound-based mechanical stimulations to induce drug release and to locally deliver anticancer electric cues. The combination of chemotherapy treatment with chronic piezoelectric stimulation resulted in activation of cell apoptosis and anti-proliferation pathways, induction of cell necrosis, inhibition of cancer migration, and reduction of cell invasiveness in drug-resistant GBM cells. Obtained results pave the way for the use of innovative multifunctional nanomaterials in less invasive and more focused anticancer treatments, able to reduce drug resistance in GBM. STATEMENT OF SIGNIFICANCE: Piezoelectric hybrid lipid-polymeric nanoparticles, efficiently encapsulating a non-genotoxic drug (nutlin-3a) and functionalized with a peptide (ApoE) that enhances their passage through the BBB, are proposed. Upon ultrasound stimulation, nanovectors resulted able to reduce cell migration, actin polymerization, and invasion ability of glioma cells, while fostering apoptotic and necrotic events. This wireless activation of anticancer action paves the way to a less invasive, more focused and efficient therapeutic strategy.

Discrimination of glioma patient-derived cells from healthy astrocytes by exploiting Raman spectroscopy.

Glioblastoma multiforme (GBM) is one of the most common and aggressive brain tumors. It presents a very bad prognosis with a patients' overall survival of 12-15 months; treatment failure is mainly ascribable to tumor recurrence. The development of new tools, that could help the precise detection of the tumor border, is thus an urgent need. During the last decades, different vibrational spectroscopy techniques have been developed to distinguish cancer tissue from heathy tissue; in the present work, we compared GBM cells deriving from four patients with healthy human astrocytes using Raman spectroscopy. We have shown that the region between 1000 and 1300 cm-1 is enough informative for this discrimination, indeed highlighting that peaks related to DNA/RNA and cytochrome c are increased in cancer cells. Finally, our model has been able to discriminate cancer cells from healthy cells with an average accuracy of 92.5%. We believe that this study might help to further understand which are the essential Raman peaks exploitable in the detection of cancer cells, with important perspectives under a diagnostic point of view.

The unpredictable carbon nanotube biocorona and a functionalization method to prevent protein biofouling.

BACKGROUND: The intrinsic physicochemical properties of carbon nanotubes (CNTs) make them unique tools in nanotechnology. Their elemental composition, resilience, thermal properties, and surface reactivity make CNTs also of undisputed interest in biotechnology. In particular, their extraordinary ability to capture biomolecules on their surface makes them essential in this field. The proteins adsorbed on the CNTs create a biological coating that endows them the ability to interact with some cell receptors, penetrate membranes or interfere with cell biomechanics, thus behaving as an active bio-camouflage. But some of these proteins unfold, triggering an immune response that unpredictably changes the biological activity of CNTs. For this reason, the control of the biocorona is fundamental in the nanobiotechnology of CNTs. RESULTS: Using TEM and AFM here we demonstrate a significant increase in CNTs diameter after protein functionalization. A quantitative analysis using TGA revealed that between 20 and 60% of the mass of functionalized nanotubes corresponds to protein, with single-walled CNTs capturing the highest amounts. To qualitatively/quantitatively characterize these biocoatings, we studied the biochemical "landscape" of the proteins captured by the different nanotubes after functionalization under various conditions. This study revealed a significant variability of the proteins in the corona as a function of the type of nanotube, the functionalization temperature, or the time after exposure to serum. Remarkably, the functionalization of a single type of CNT with sera from various human donors also resulted in different protein landscapes. Given the unpredictable assortment of proteins captured by the corona and the biological implications of this biocoating, we finally designed a method to genetically engineer and produce proteins to functionalize nanotubes in a controlled and customizable way. CONCLUSIONS: We demonstrate the high unpredictability of the spontaneous protein corona on CNTs and propose a versatile functionalization technique that prevents the binding of nonspecific proteins to the nanotube to improve the use of CNTs in biomedical applications.

Lipid-Based Nanocarriers for The Treatment of Glioblastoma.

Glioblastoma multiforme (GBM) is the most common and malignant neoplasia having origin in the brain. The current treatments involve surgery, radiotherapy, and chemotherapy, being complete surgical resection the best option for the patient survival chances. However, in those cases where a complete removal is not possible, radiation and chemotherapy are applied. Herein, the main challenges of chemotherapy, and how they can be overcome with the help of nanomedicine, are approached. Natural pathways to cross the blood-brain barrier (BBB) are detailed, and different in vivo studies where these pathways are mimicked functionalizing the nanomaterial surface are shown. Later, lipid-based nanocarriers, such as liposomes, solid lipid nanoparticles, and nanostructured lipid carriers, are presented. To finish, recent studies that have used lipid-based nanosystems carrying not only therapeutic agents, yet also magnetic nanoparticles, are described. Although the advantages of using these types of nanosystems are explained, including their biocompatibility, the possibility of modifying their surface to enhance the cell targeting, and their intrinsic ability of BBB crossing, it is important to mention that research in this field is still at its early stage, and extensive preclinical and clinical investigations are mandatory in the close future.

Design of Polymeric and Biocompatible Delivery Systems by Dissolving Mesoporous Silica Templates.

There are many nanoencapsulation systems available today. Among all these, mesoporous silica particles (MSPs) have received great attention in the last few years. Their large surface-to-volume ratio, biocompatibility, and versatility allow the encapsulation of a wide variety of drugs inside their pores. However, their chemical instability in biological fluids is a handicap to program the precise release of the therapeutic compounds. Taking advantage of the dissolving capacity of silica, in this study, we generate hollow capsules using MSPs as transitory sacrificial templates. We show how, upon MSP coating with different polyelectrolytes or proteins, fully customized hollow shells can be produced. These capsules are biocompatible, flexible, and biodegradable, and can be decorated with nanoparticles or carbon nanotubes to endow the systems with supplementary intrinsic properties. We also fill the capsules with a fluorescent dye to demonstrate intracellular compound release. Finally, we document how fluorescent polymeric capsules are engulfed by cells, releasing their encapsulated agent during the first 96 h. In summary, here, we describe how to assemble a highly versatile encapsulation structure based on silica mesoporous cores that are completely removed from the final polymeric capsule system. These drug encapsulation systems are highly customizable and have great versatility as they can be made using silica cores of different sizes and multiple coatings. This provides capsules with unique programmable attributes that are fully customizable according to the specific needs of each disease or target tissue for the development of nanocarriers in personalized medicine.

A custom-made functionalization method to control the biological identity of nanomaterials.

Here we propose a one-step strategy to endow nanomaterials with a custom-designed bio-identity. This study designs a universal 'nanomaterial binding domain' that can be genetically attached to any protein ensuring precise and spontaneous protein orientation. We demonstrate how, despite the simplicity of the method, the bioconjugation achieved: (i) is highly efficient, even in the presence of competing proteins, (ii) is stable at extreme physiological conditions (pH ranges 5.2-9.0; NaCl concentrations 0-1 M); (iii) prevents unwanted protein biofouling days after incubation in biologically-relevant conditions; and finally, (iv) avoids nanoparticle interaction with promiscuous unspecific receptors. In summary, this protein biocoating technique, applicable to a wide array of nano-designs, integrates material science and molecular biology procedures to create hybrid nanodevices with well-defined surfaces and predictable biological behaviors, opening a chapter in precision nanodiagnostics, nanosensing or nanotherapeutic applications.

Engineering Sub-Cellular Targeting Strategies to Enhance Safe Cytosolic Silica Particle Dissolution in Cells.

Mesoporous silica particles (MSP) are major candidates for drug delivery systems due to their versatile, safe, and controllable nature. Understanding their intracellular route and biodegradation process is a challenge, especially when considering their use in neuronal repair. Here, we characterize the spatiotemporal intracellular destination and degradation pathways of MSP upon endocytosis by HeLa cells and NSC-34 motor neurons using confocal and electron microscopy imaging together with inductively-coupled plasma optical emission spectroscopy analysis. We demonstrate how MSP are captured by receptor-mediated endocytosis and are temporarily stored in endo-lysosomes before being finally exocytosed. We also illustrate how particles are often re-endocytosed after undergoing surface erosion extracellularly. On the other hand, silica particles engineered to target the cytosol with a carbon nanotube coating, are safely dissolved intracellularly in a time scale of hours. These studies provide fundamental clues for programming the sub-cellular fate of MSP and reveal critical aspects to improve delivery strategies and to favor MSP safe elimination. We also demonstrate how the cytosol is significantly more corrosive than lysosomes for MSP and show how their biodegradation is fully biocompatible, thus, validating their use as nanocarriers for nervous system cells, including motor neurons.

Controlled drug delivery systems for cancer based on mesoporous silica nanoparticles.

The implementation of nanotechnology in medicine has opened new research horizons particularly in the field of therapeutic delivery. Mesoporous silica particles have emerged as biocompatible drug delivery systems with an enormous potential in the treatment of cancer among many other pathologies. In this review, we focus on the unique properties of these particles as chemotherapy delivery carriers. Here, we summarize the general characteristics of these nanomaterials - including their physicochemical properties and customizable surfaces - different stimuli that can be used to trigger targeted drug release, biocompatibility and finally, the drawbacks of these types of nanomaterials, highlighting some of the most important features of mesoporous silica nanoparticles in drug delivery.

A Biomimetic Escape Strategy for Cytoplasm Invasion by Synthetic Particles.

The translocation of nanomaterials or complex delivery systems into the cytosol is a major challenge in nanobiotechnology. After receptor-mediated endocytosis, most nanomaterials are sequestered and undergo degradation, therapy inactivation, or exocytosis. Herein we explore a novel surface particle coating made of adsorbed carbon nanotubes that provides coated materials with new properties that reproduce the viral cell-invasive mechanisms, namely, receptor-mediated endocytosis, endolysosomal escape, and cytosolic particle release preserving cell viability. This novel biomimetic coating design will enable the intracytoplasmic delivery of many different functional materials endowed with therapeutic, magnetic, optical, or catalytic functionalities, thus opening the door to a wide array of chemical and physical processes within the cytosolic or nuclear domains, and supporting new developments in the biotechnological, pharmaceutical, and biomedical industries.

Carbon nanotubes gathered onto silica particles lose their biomimetic properties with the cytoskeleton becoming biocompatible.

Carbon nanotubes (CNTs) are likely to transform the therapeutic and diagnostic fields in biomedicine during the coming years. However, the fragmented vision of their side effects and toxicity in humans has proscribed their use as nanomedicines. Most studies agree that biocompatibility depends on the state of aggregation/dispersion of CNTs under physiological conditions, but conclusions are confusing so far. This study designs an experimental setup to investigate the cytotoxic effect of individualized multiwalled CNTs compared to that of identical nanotubes assembled on submicrometric structures. Our results demonstrate how CNT cytotoxicity is directly dependent on the nanotube dispersion at a given dosage. When CNTs are gathered onto silica templates, they do not interfere with cell proliferation or survival becoming highly compatible. These results support the hypothesis that CNT cytotoxicity is due to the biomimetics of these nanomaterials with the intracellular nanofilaments. These findings provide major clues for the development of innocuous CNT-containing nanodevices and nanomedicines.