Electrochemistry: A basic and powerful tool for micro- and nanomotor fabrication and characterization

Electrochemistry, although an ancient field of knowledge, has become of paramount importance in the synthesis of materials at the nanoscale, with great interest not only for fundamental research but also for practical applications. One of the promising fields in which electrochemistry meets nanoscience and nanotechnology is micro/nanoscale motors. Micro/nano motors, which are devices able to perform complex tasks at the nanoscale, are commonly multifunctional nanostructures of different materials - metals, polymers, oxides- and shapes -spheres, wires, helices- with the ability to be propelled in fluids. Here, we first introduce the topic of micro/nanomotors and make a concise review of the field up to day. We have analyzed the field from different points of view (e.g. materials science and nanotechnology, physics, chemistry, engineering, biology or environmental science) to have a broader view of how the different disciplines have contributed to such exciting and impactful topic. After that, we focus our attention on describing what electrochemical technology is and how it can be successfully used to fabricate and characterize micro/nanostructures composed of different materials and showing complex shapes. Finally, we will review the micro and nanomotors fabricated using electrochemical techniques with applications in biomedicine and environmental remediation, the two main applications investigated so far in this field. Thus, different strategies have thus been shown capable of producing core-shell nanomaterials combining the properties of different materials, multisegmented nanostructures made of, for example, alternating metal and polymer segments to confer them with flexibility or helicoidal systems to favor propulsion. Moreover, further functionalization and interaction with other materials to form hybrid and more complex objects is also shown

Evaluation of nanobodies against the selected protein biomarkers of glioblastoma and attempt of their delivery with exosomes

Glioblastom (GBM) je najpogostejši primarni možganski tumor, ki se pojavlja s pogostnostjo 3,2 primera na 100 000 prebivalcev. Kljub uveljavljenem zdravljenju, ki obsega kirurško odstranitev tumorja, kemoterapijo s temozolomidom in radioterapijo, večina bolnikov ne preživi več kot 18 mesecev po postavljeni diagnozi. Eden izmed sodobnih možnih načinov zdravljenja GBM je uporaba nanoteles, antigen-prepoznavnih delov težkoverižnih protiteles, ki jih proizvajajo le nekatere živali, npr. lame. Nanotelesa imajo namreč v primerjavi s klasičnimi protiteles precej prednosti, kot so visoka stabilnost, možnost proizvodnje z bakterijo E. coli in hitrejše prehajanje v tumor. V okviru doktorskega dela smo proučili vpliv nanoteles na preživetje celic, proti osmim možnim označevalcem glioblastoma, njihovo migracijo in tvorjenje kolonij. Najprej smo z uporabo imunohistokemije ugotovili, da na osnovi navzočnosti biooznačevalec oziroma razlik v izražanju, vimentin lahko razlikuje med glioblastomom, gliomi nižje stopnje in normalno možganovino, medtem ko biooznačevalci TUFM, DPYSL1 in CRMP1 razlikujejo med glioblastomom in normalno možganovino. Rezultati proučevanja citotoksičnega delovanja nanoteles kažejo, da nanotelesa Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) in Nb314 (anti-DPYSL2) delujejo citotoksično na glioblastomske celice. Posebno velik vpliv na citotoksičnost glioblastomskih matičnih celic ima nanotelo anti-TUFM (Nb225). Na migracijo glioblastomskih celic pa najbolj vpliva nanotelo anti-vimentin (Nb79), ki je popolnoma inhibiralo migracijo celic glioblastomske celične linije U87MG. V drugem delu raziskave smo razvili dostavni sistem, ki temelji na zunajceličnih veziklih eksosomih, v katere smo zapakirali nanotelesa, da bi izboljšali njihovo dostavo in povečali učinkovitost. Eksosomi so najmanjši zunajcelični vezikli, ki jih izločajo celice, in naj bi, v primerjavi s primerljivimi dostavljalci, liposomi, hitreje prehajali v tarčne celice, obenem pa naj bi tudi imeli daljši razpolovni čas. Eksosome smo izolirali iz glioblastomske celične linije U251MG in jih opredelili z uporabo prenosa western za detekcijo eksosomalnih označevalcev, z uporabo metode sledenja nanodelcem za določitev števila in velikosti eksosomov, ter z elektronsko mikroskopijo za ugotavljanje njihove oblike. V eksosome smo nanotelesa uspešno zapakirali z metodama inkubacije z 0,4 % saponinom in sonikacije, ki sta bili približno enako učinkoviti, medtem ko posredno pakiranje nanoteles v eksosome preko inkubacije celic z nanotelesi ni bilo uspešno. Eksosomi so poleg tega, da so potencialni dostavljalci oz. komponente sistemov za dostavo zdravil do tkiv in celic, tudi možen vir biooznačevalcev. V naši študiji smo z metodo qPCR analizirali izražanje izbranih mRNA, miRNA in proteinov v eksosomih celic glioblastomskih celičnih linij. Ugotovili smo, da so miR-9-5p, miR-124-3p, mRNA TUFM in mRNA CRMP1 možni označevalci eksosomov glioblastomskih matičnih celic, mRNA VIM pa primeren označevalec eksosomov diferenciranih glioblastomskih celic. Za razliko od omenjenih molekul RNA so bili proteini v eksosomih proučevanih celic slabše zastopani, v njih smo lahko detektirali samo proteinska biooznačevalca ALYREF in DPYSL2. V doktorski nalogi smo pokazali, da so nanotelesa primerno sredstvo za doseganje citotoksičnega učinka in zmanjševanje migracije glioblastomskih celic. Uspešno smo razvili dostavno sredstvo, eksosome, ki vsebujejo nanotelesa, in te bi lahko v prihodnosti uporabili za povečanje učinkovitosti samih nanoteles. Naši rezultati kažejo, da so citotoksična nanotelesa in eksosomi, kot dostavni sistem, obetavna učinkovita in specifična oblika terapije za zdravljenje GBM. Vzporedno smo v naši študiji tudi določili možne eksosomalne označevalce glioblastomskih celic, za katere predlagamo ovrednotenje pri nadaljnjem proučevanju eksosomov, izoliranih iz telesnih tekočin bolnikov. Ti bi v prihodosti lahko služili kot potencialni biomarkerji GBM iz krvi ali cerebrospinalne tekočine bolnikov z GBM.Glioblastoma is the most common primary brain tumor, occurring with a frequency of 3.2 cases per 100 000 population. Despite established treatment, which includes surgical removal of the tumor, chemotherapy with temozolomide, and radiotherapy, most patients do not survive more than 18 months after diagnosis. One of the modern possible ways of treating GBM is the use of nanobodies, antigen-recognizing parts of heavy chain-only antibodies produced by only some animals, e.g. llamas. Namely, nanobodies have many advantages over classical antibodies, such as high stability, possibility of production with E. coli and faster transition and penetration to the tumor. In the doctoral thesis, we studied the influence of nanobodies against eight possible biomarkers of glioblastoma on cell survival, migration and colony formation. First, using immunohistochemistry, we found that, based on differences in expression or presence, the biomarker vimentin could distinguish between glioblastoma, lower-grade gliomas, and normal brain, while biomarkers TUFM, DPYSL1, and CRMP1 distinguished between glioblastoma and normal brain. The results of the study of the cytotoxic action of nanobodies showed that nanobodies Nb79 (anti-vimentin), Nb179 (anti-NAP1L1), Nb225 (anti-TUFM) and Nb314 (anti-DPYSL2) have a cytotoxic effect on glioblastoma cells. The anti-TUFM nanobody (Nb225) has a particularly large effect on the cytotoxicity of glioblastoma stem cells. The migration of glioblastoma cells is mostly affected by the anti-vimentin nanobody (Nb79), which completely inhibited the migration of cells of the glioblastoma cell line U87MG. In the second part of the study, we developed a delivery system based on extracellular vesicles, exosomes, into which we packaged nanobodies to improve their delivery and increase efficiency. Exosomes are the smallest extracellular vesicles secreted by cells and are thought to pass more rapidly into target cells than comparable delivery systems, while also having a longer half-life. Exosomes were isolated from the U251MG glioblastoma cell line cells and characterized by detection of exosomal markers using Western blot, a nanoparticle tracking analysis method to determine their number and size, and by electron microscopy to determine their shape. Nanobodies were successfully packaged into exosomes by 0.4% saponin incubation and sonication methods, which were approximately equally effective, while indirect packaging by incubating cells with nanobodies was not successful. In addition to being carriers of a potential delivery system, exosomes are also a possible source of biomarkers. In our study, we analyzed the expression of selected mRNAs, miRNAs, and proteins in the exosomes of glioblastoma cell lines using the qPCR method. We found that miR-9-5p, miR-124-3p, TUFM mRNA, and CRMP1 mRNA are possible markers of glioblastoma stem cell exosomes, and VIM mRNA is a suitable marker of differentiated glioblastoma cell exosomes. In contrast to the mentioned RNA molecules, proteins were less represented in the exosomes of the studied cells, in which only the protein biomarkers ALYREF and DPYSL2 could be detected. In our doctoral dissertation, we showed that nanobodies are a suitable means of achieving a cytotoxic effect and reducing the migration of glioblastoma cells. We have successfully developed a delivery vehicle, exosomes that contain nanobodies, and these could be used in the future to increase the efficiency of the nanobodies themselves. Our results suggest that cytotoxic nanobodies and exosomes, as a delivery system, are a promising form of efficient and specific therapy for the treatment of GBM. In parallel, in this study we also identified possible exosomal markers of glioblastoma cells, for which we propose evaluation in the further study of exosomes isolated from patients’ body fluids. These could in the future serve as potential biomarkers of GBM from the blood or cerebrospinal fluid of patients with GBM

Nanotechnology meets oncology

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

Nanotechnology Meets Oncology: Nanomaterials in Brain Cancer Research, Diagnosis and Therapy

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

Coding of glioblastoma progression and therapy resistance through long noncoding RNAs

Glioblastoma is the most aggressive and lethal primary brain malignancy, with an average patient survival from diagnosis of 14 months. Glioblastoma also usually progresses as a more invasive phenotype after initial treatment. A major step forward in our understanding of the nature of glioblastoma was achieved with large-scale expression analysis. However, due to genomic complexity and heterogeneity, transcriptomics alone is not enough to define the glioblastoma "fingerprint", so epigenetic mechanisms are being examined, including the noncoding genome. On the basis of their tissue specificity, long noncoding RNAs (lncRNAs) are being explored as new diagnostic and therapeutic targets. In addition, growing evidence indicates that lncRNAs have various roles in resistance to glioblastoma therapies (e.g., MALAT1, H19) and in glioblastoma progression (e.g., CRNDE, HOTAIRM1, ASLNC22381, ASLNC20819). Investigations have also focused on the prognostic value of lncRNAs, as well as the definition of the molecular signatures of glioma, to provide more precise tumor classification. This review discusses the potential that lncRNAs hold for the development of novel diagnostic and, hopefully, therapeutic targets that can contribute to prolonged survival and improved quality of life for patients with glioblastoma

Non-animal glioblastoma models for personalized treatment

Glioblastoma is an extremely lethal cancer characterized by great heterogeneity at different molecular and cellular levels. As a result, treatment options have moved far from systemic and universal therapies toward targeted treatments and personalized medicine. However, for successful translation from preclinical studies to clinical trials, experiments must be performed on reliable disease models. Numerous experimental models have been developed for glioblastoma, ranging from simple 2D cell cultures to study the nature of the disease to complex 3D models such as neurospheres, organoids, tissue-slice cultures, bioprinted models, and tumor on chip, as perfect prototypes to evaluate the therapeutic potential of different drugs. The presence of multiple research models is consistent with the complexity and molecular diversity of glioblastoma. The advantage of such models is the recapitulation of the tumor environment, and in some cases the preservation of immune system components as well as the creation of simple vessels. There are also two case studies translating in vitro studies on glioblastoma organoids to patients as well as four ongoing clinical trials using glioblastoma models, indicating high clinical potential of glioblastoma models

Nanomedicine and immunotherapy

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

Algorithmically deduced FREM2 molecular pathway is a potent grade and survival biomarker of human gliomas

Gliomas are the most common malignant brain tumors with high mortality rates. Recently we showed that the FREM2 gene has a role in glioblastoma progression. Here we reconstructed the FREM2 molecular pathway using the human interactome model. We assessed the biomarker capacity of FREM2 expression and its pathway as the overall survival (OS) and progression-free survival (PFS) biomarkers. To this end, we used three literature and one experimental RNA sequencing datasets collectively covering 566 glioblastomas (GBM) and 1097 low-grade gliomas (LGG). The activation level of deduced FREM2 pathway showed strong biomarker characteristics and significantly outperformed the FREM2 expression level itself. For all relevant datasets, it could robustly discriminate GBM and LGG (p 0.74). High FREM2 pathway activation level was associated with poor OS in LGG (p < 0.001), and low PFS in LGG (p < 0.001) and GBM (p < 0.05). FREM2 pathway activation level was poor prognosis biomarker for OS (p < 0.05) and PFS (p < 0.05) in LGG with IDH mutation, for PFS in LGG with wild type IDH (p < 0.001) and mutant IDH with 1p/19q codeletion (p < 0.05), in GBM with unmethylated MGMT (p < 0.05), and in GBM with wild type IDH (p < 0.05). Thus, we conclude that the activation level of the FREM2 pathway is a potent new-generation diagnostic and prognostic biomarker for multiple molecular subtypes of GBM and LGG

Cytoskeletal proteins as glioblastoma biomarkers and targets for therapy

Glioblastoma, the most common primary brain malignancy, is an exceptionally fatal cancer. Lack of suitable biomarkers and efficient treatment largely contribute to the therapy failure. Cytoskeletal proteins are crucial proteins in glioblastoma pathogenesis and can potentially serve as biomarkers and therapeutic targets. Among them, GFAP, has gained most attention as potential diagnostic biomarker, while vimentin and microtubules are considered as prospective therapeutic targets. Microtubules represent one of the best anti-cancer targets due to their critical role in cell proliferation. Despite testing in clinical trials, the efficiency of taxanes, epothilones, vinca-domain binding drugs, colchicine-domain binding drugs and γ-tubulin binding drugs remains to be confirmed. Moreover, tumor treating field that disrupts microtubules draw attention because of its high efficiency and is called “the fourth cancer treatment modality”. Thereby, because of the involvement of cytoskeleton in key physiological and pathological processes, its therapeutic potential in glioblastoma is currently extensively investigated

Sonication is a suitable method for loading nanobody into glioblastoma small extracellular vesicles

Glioblastoma is one of the deadliest cancers, therefore novel efficient therapeutic approaches are urgently required. One of such are nanobodies, prospective nano-sized bio-drugs with advantageous characteristics. Nanobodies can target intracellular proteins, but to increase their efficiency, the delivery system should be applied. Here, we examined small extracellular vesicles as a delivery system for anti-vimentin nanobody Nb79. Nb79 was loaded in small extracellular vesicles either by incubation with glioblastoma cells, by passive loading into isolated small extracellular vesicles or by sonication of isolated small extracellular vesicles. Small extracellular vesicles secreted by glioblastoma cells were isolated by ultracentrifugation on sucrose cushion. The size distribution and average size of sonicated and non-sonicated small extracellular vesicles were determined by nanoparticle tracking analysis method. The loading of Nb79 into small extracellular vesicles by incubation with cells, passive loading or sonication was confirmed by Western blot and electron microscopy. The effect of small extracellular vesicles on cell survival was determined by WST-1 reagent. Loading of small extracellular vesicles by incubation of cells with Nb79 was unsuccessful and resulted in substantial cell death. On the other hand, as confirmed by Western blot and electron microscopy, sonication is a successful method for obtaining Nb79-loaded small extracellular vesicles. Small extracellular vesicles also had an effect on cell viability. Small extracellular vesicles without Nb79 increased survival of U251 and NCH644 cells for 20–25%, while the Nb79-loaded small extracellular vesicles decreased survival of NCH421k by 11%. We demonstrated that sonication is a suitable method to load nanobodies into exosome, and these small extracellular vesicles could in turn reduce cell survival. The method could be translated also to other applications, such as targeted delivery system of other protein-based drugs