Today, bio-medical efforts are entering the subcellular level, which is witnessed with the fast-developing fields of nanomedicine, nanodiagnostics and nanotherapy in conjunction with the implementation of nanoparticles for disease prevention, diagnosis, therapy and follow-up. Nanoparticles or nanocontainers offer advantages including high sensitivity, lower toxicity and improved safety—characteristics that are especially valued in the oncology field. Cancer cells develop and proliferate in complex microenvironments leading to heterogeneous diseases, often with a fatal outcome for the patient. Although antibody-based therapy is widely used in the clinical care of patients with solid tumours, its efficiency definitely needs improvement. Limitations of antibodies result mainly from their big size and poor penetration in solid tissues. Nanobodies are a novel and unique class of antigen-binding fragments, derived from naturally occurring heavy-chain-only antibodies present in the serum of camelids. Their superior properties such as small size, high stability, strong antigen-binding affinity, water solubility and natural origin make them suitable for development into next-generation biodrugs. Less than 30 years after the discovery of functional heavy-chain-only antibodies, the nanobody derivatives are already extensively used by the biotechnology research community. Moreover, a number of nanobodies are under clinical investigation for a wide spectrum of human diseases including inflammation, breast cancer, brain tumours, lung diseases and infectious diseases. Recently, caplacizumab, a bivalent nanobody, received approval from the European Medicines Agency (EMA) and the US Food and Drug Administration (FDA) for treatment of patients with thrombotic thrombocytopenic purpura.

Cancer is a heterogeneous disease, which provides a broad field for investigation, while simultaneously reducing the chances for a universal treatment. Malignant gliomas are the most common type of primary brain tumors. The heterogeneity of gliomas regarding clinical presentation, pathology and response to treatment makes this type of tumor a challenging area of research. As the clinical symptoms may be unspecific (e.g., seizures and headaches) it is often difficult to diagnose a patient in the early stages of the disease. Thus far, there are no known genetic patterns of inheritance of this disease. Currently, the treatment of glioblastoma involves surgery, whenever possible, followed by radiation and chemotherapy. Experimental procedures, such as passive and active immunotherapy, use of angiogenesis inhibitors in combination with chemotherapeutics and gene/antibody therapy, are additional treatment options. However, as the brain is difficult to access due to the presence of the blood‑brain barrier (BBB), none of the above‑mentioned therapies have been successful in curing this disease. The lack of knowledge regarding the mechanisms underlying the development and progression of gliomas further adds to the difficulties. Currently, investigations are focused on the development of novel methods for improving the outcome of this disease. however, despite the extensive investigations, 88% of all glioblastoma multiforme (GBM) patients succumb to the disease within 3 years. GBM remains one of the most challenging malignancies worldwide.

Advances in technology of the past decades led to development of new nanometer scale diagnosis and treatment approaches in cancer medicine leading to establishment of nanooncology. Inorganic and organic nanomaterials have been shown to improve bioimaging techniques and targeted drug delivery systems. Their favorable physico-chemical characteristics, like small sizes, large surface area compared to volume, specific structural characteristics, and possibility to attach different molecules on their surface transform them into excellent transport vehicles able to cross cell and/or tissue barriers, including the blood–brain barrier. The latter is one of the greatest challenges in diagnosis and treatment of brain cancers. Application of nanomaterials can prolong the circulation time of the drugs and contrasting agents in the brain, posing an excellent opportunity for advancing the treatment of the most aggressive form of the brain cancer—glioblastomas. However, possible unwanted side-effects and toxicity issues must be considered before final clinical translation of nanoparticles.

Differentially expressed proteins in glioblastoma multiforme identified with a nanobody-based anti-proteome approach and confirmed by OncoFinder as possible tumor-class predictive biomarker candidates Differentially expressed proteins in glioblastoma multiforme identified with a nanobody-based anti-proteome approach and confirmed by OncoFinder as possible tumor-class predictive biomarker candidates

Glioblastoma (GBM), the most common primary brain tumor, is a complex and extremely aggressive disease. Despite recent advances in molecular biology, there is a lack of biomarkers, which would improve GBM’s diagnosis, prognosis, and therapy. Here, we analyzed by qPCR the expression levels of a set of miRNAs in GBM and lower-grade glioma human tissue samples and performed a survival analysis in silico. We then determined the expression of same miRNAs and their selected target mRNAs in small extracellular vesicles (sEVs) of GBM cell lines. We showed that the expression of miR-21-5p was significantly increased in GBM tissue compared to lower-grade glioma and reference brain tissue, while miR-124-3p and miR-138-5p were overexpressed in reference brain tissue compared to GBM. We also demonstrated that miR-9-5p and miR-124-3p were overexpressed in the sEVs of GBM stem cell lines (NCH421k or NCH644, respectively) compared to the sEVs of all other GBM cell lines and astrocytes. VIM mRNA, a target of miR-124-3p and miR-138-5p, was overexpressed in the sEVs of U251 and U87 GBM cell lines compared to the sEVs of GBM stem cell line and also astrocytes. Our results suggest VIM mRNA, miR-9-5p miRNA, and miR-124-3p miRNA could serve as biomarkers of the sEVs of GBM cells.

Owing to the advancement of technology combined with our deeper knowledge of human nature and diseases, we are able to move towards precision medicine, where patients are treated at the individual level in concordance with their genetic profiles. Lately, the integration of nanoparticles in biotechnology and their applications in medicine has allowed us to diagnose and treat disease better and more precisely. As a model disease, we used a grade IV malignant brain tumor (glioblastoma). Significant improvements in diagnosis were achieved with the application of fluorescent nanoparticles for intraoperative magnetic resonance imaging (MRI), allowing for improved tumor cell visibility and increasing the extent of the surgical resection, leading to better patient response. Fluorescent probes can be engineered to be activated through different molecular pathways, which will open the path to individualized glioblastoma diagnosis, monitoring, and treatment. Nanoparticles are also extensively studied as nanovehicles for targeted delivery and more controlled medication release, and some nanomedicines are already in early phases of clinical trials. Moreover, sampling biological fluids will give new insights into glioblastoma pathogenesis due to the presence of extracellular vesicles, circulating tumor cells, and circulating tumor DNA. As current glioblastoma therapy does not provide good quality of life for patients, other approaches such as immunotherapy are explored. To conclude, we reason that development of personalized therapies based on a patient’s genetic signature combined with pharmacogenomics and immunogenomic information will significantly change the outcome of glioblastoma patients.