Technological and biomedical applications of magnetic nanostructures

The battle against cancer is not an easy task, due to the complexity and great heterogeneity of this disease. However, with the contribution of nanotechnology that has been increasing in recent years, it is possible to effectively deal with it. Magnetically activated treatments, which are already at the stage of clinical trials or medical practice, may compete with or complement the decades-long established treatments, such as chemotherapy and radiation therapy, with the aim of increasing the therapeutic efficiency and at the same time minimizing the side effects of these invasive treatments. The combination of magnetic fields and magnetic nanoparticles (MNPs) is already used in diagnosis (MRI contrast agents), therapy (drug deposition, magnetic hyperthermia) and manipulation of cancerous tissues (biolabeling, bioseparation). The magnetic fields used penetrate biological tissues and are safe for humans. The same situation prevails in the case of MNPs, which easily penetrate many kinds of biological pathways and are also, safe for humans. Thus, magnetic nanoparticle hyperthermia (MPH) is one of the most interesting treatment methods that involves the local heating of tumors and cancer cells, in the temperature range of 41-45 °C, through MNPs that release heat, after their exposure to an alternating magnetic field.In recent years, in the fight against cancer, much attention is also shifting to the magneto-mechanical action of nanoparticles, that induce static or time-varying magnetic fields, in the absence of heat. More specifically, mechanical forces generated through magnetic fields "manipulate" magnetic nanoparticles (MNPs), within variable environments. Electromagnetic energy is converted into mechanical energy, by combining various types of fields, (such as static, pulsed, alternating, rotating) of very low frequencies, with MNPs, allowing them to function and bind specifically to cell membranes (malignant and non-malignant), destroying, only the cancerous tissues. Magnetic stress through MNPs and the induced apoptotic death of cancer cells has already been studied in specially designed setups, where permanent magnets or electromagnets are used to produce controlled field gradients applied to MNPs incubated in cancer cell cultures.In this thesis, the objective is the thermal and mechanical activation of magnetic nanoparticles (MNPs), as cancer treatment strategies, under the influence of magnetic fields. However, side effects such as Eddy currents occur during MPH, which cause discomfort to patients and energy losses, an important factor, limiting the performance of MPH, in clinical trials. Therefore, the primary objective of this thesis is to minimize the unwanted heating of healthy tissues, without sparing the therapeutic ability of MNPs to effectively treat the tumor area. To achieve this goal, two alternative magnetic hyperthermia protocols are proposed, on the one hand using an intermittently applied alternating magnetic field instead of a continuous alternating magnetic field and on the other hand with the relative movement of the field toward the sample, through a motion-table. These two alternative ways of conducting MPH are examined not only separately, but also in combination, as well, in order to accomplish an even greater reduction of these healthy tissues side effects.To simulate the cancerous and healthy tissues, agarose phantoms were synthesized in the presence and absence of MNPs, respectively. The MNPs used are made of iron oxides (Fe3O4), as they are widely used, due to their biocompatibility, functionality and specific magnetic properties both in the treatment and in the diagnosis of various diseases. These MNPs were developed in the laboratory by the aqueous co-precipitation method and structural, morphological and magnetic characterization was carried out. For a more complete picture and a deeper understanding of the response of a real animal tissue, the same protocol was implemented in ex vivo measurements.In a subsequent stage, the magneto-mechanical stress of cancerous and healthy breast cells is studied, applying various types of field modes, such as static, pulsed and rotating, at very low frequencies, intending to reduce the viability, only in cancerous cells. With the help of fluorescent dyes and confocal microscopy, the alterations suffered by the cytoskeleton and the nucleus of the cells after a magneto-mechanical treatment, are recorded. The MNPs used in this research are starch coated iron oxides, commercial nanoparticles. Therefore, the final stage of this PhD thesis and the main research question it poses, concerns the combination of thermal and mechanical therapy, in order to investigate the cumulative effect of these two approaches on the destruction of breast cancer cells, through MNPs stimulation by magnetic fields.Η μάχη κατά τον καρκίνο δεν είναι εύκολη υπόθεση, λόγω της πολυπλοκότητας και της μεγάλης ετερογένειας που αυτή η νόσος παρουσιάζει. Ωστόσο, με την συμβολή της νανοτεχνολογίας που ολοένα και αυξάνεται τα τελευταία χρόνια είναι δυνατή η αποτελεσματική αντιμετώπιση του. Μαγνητικά ενεργοποιημένες θεραπείες, που βρίσκονται ήδη στο στάδιο των κλινικών δοκιμών ή της ιατρικής πράξης, είναι δυνατόν να ανταγωνιστούν τις εδώ και δεκαετίες καθιερωμένες θεραπείες, τη χημειοθεραπεία και την ακτινοθεραπεία ή να δράσουν συμπληρωματικά προς αυτές, με στόχο την αύξηση της θεραπευτικής απόδοσης και την ταυτόχρονη μείωση των παρενεργειών, αυτών των επεμβατικών θεραπειών. Ο συνδυασμός μαγνητικών πεδίων και μαγνητικών νανοσωματιδίων (ΜΝΣ) χρησιμοποιείται ήδη στη διάγνωση (σκιαγραφικοί παράγοντες MRI), στη θεραπεία (απόθεση φαρμάκων, μαγνητική υπερθερμία) και στον χειρισμό καρκινικών ιστών (βιοσήμανση, βιοδιαχωρισμός). Τα μαγνητικά πεδία που χρησιμοποιούνται διαπερνούν βιολογικούς ιστούς και είναι ασφαλή για τον άνθρωπο. Το ίδιο συμβαίνει και με τα ΜΝΣ, τα οποία διαπερνούν εύκολα πολλών ειδών βιολογικές διαδρομές και είναι επίσης, ασφαλή για τον άνθρωπο. Έτσι, η μαγνητική υπερθερμία νανοσωματιδίων (ΜΥΝ) αποτελεί μια από τις πιο ενδιαφέρουσες μεθόδους θεραπείας, η οποία στοχεύει στη τοπική θέρμανση των όγκων και των καρκινικών κυττάρων, στο θερμοκρασιακό εύρος 41-45 οC, μέσω ΜΝΣ που εκλύουν θερμότητα, όταν αυτά εκτεθούν σε εναλλασσόμενο μαγνητικό πεδίο.Τα τελευταία χρόνια, στη μάχη κατά του καρκίνου, ιδιαίτερη προσοχή δίνεται και στη μαγνητο-μηχανική δράση νανοσωματιδίων που προκαλούν στατικά ή χρονικά μεταβαλλόμενα μαγνητικά πεδία, απουσία θερμότητας. Πιο συγκεκριμένα, μηχανικές δυνάμεις που δημιουργούνται μέσω μαγνητικών πεδίων "χειραγωγούν" τα μαγνητικά νανοσωματίδια (ΜΝΣ), μέσα σε μεταβλητά περιβάλλοντα. Η ηλεκτρομαγνητική ενέργεια μετατρέπεται σε μηχανική ενέργεια, συνδυάζοντας διάφορους τύπους πεδίων, (όπως στατικό, παλμικό, εναλλασσόμενο, περιστρεφόμενο), πολύ χαμηλών συχνοτήτων, με ΜΝΣ, επιτρέποντάς τα να λειτουργούν και να συνδέονται ειδικά με κυτταρικές μεμβράνες (κακοήθεις και μη), καταστρέφοντας μόνο τους καρκινικούς ιστούς. Η μαγνητική καταπόνηση μέσω ΜΝΣ και ο επαγόμενος αποπτωτικός θάνατος των καρκινικών κυττάρων έχει ήδη μελετηθεί σε ειδικά σχεδιασμένες διατάξεις, όπου μόνιμοι μαγνήτες ή ηλεκτρομαγνήτες χρησιμοποιούνται για την παραγωγή ελεγχόμενων βαθμίδων πεδίου που εφαρμόζονται σε ΜΝΣ επωασμένα σε καρκινικές κυτταρικές καλλιέργειες.Στη παρούσα διδακτορική διατριβή λοιπόν, στόχος είναι η θερμική και μηχανική ενεργοποίηση μαγνητικών νανοσωματιδίων (ΜΝΣ) ως στρατηγικές αντιμετώπισης του καρκίνου, υπό την επίδραση μαγνητικών πεδίων. Ωστόσο, κατά τη ΜΥΝ εμφανίζονται ανεπιθύμητα ρεύματα Eddy, τα οποία προκαλούν δυσφορία στους ασθενείς και απώλειες ισχύος αποτελώντας σημαντικό παράγοντα που περιορίζει την απόδοση της ΜΥΝ, σε κλινικές δοκιμές. Επομένως, πρωταρχικός στόχος της συγκεκριμένης διατριβής είναι η ελαχιστοποίηση της ανεπιθύμητης θέρμανσης των υγιών ιστών, χωρίς να περιορίζεται η θεραπευτική ικανότητα των ΜΝΣ, για αποτελεσματική θεραπεία της προβληματικής περιοχής. Για την επίτευξη αυτού του στόχου προτείνονται δύο εναλλακτικά πρωτόκολλα μαγνητικής υπερθερμίας, αφενός με τη χρήση διακοπτόμενα εφαρμοζόμενου εναλλασσόμενου μαγνητικού πεδίου αντί συνεχόμενου εναλλασσόμενου μαγνητικού πεδίου και αφ’ ετέρου με την σχετική κίνηση του πεδίου ως προς το δείγμα, μέσω μιας τράπεζας - κίνησης. Οι δύο αυτοί εναλλακτικοί τρόποι διεξαγωγής της ΜΥΝ, εξετάζονται και χωριστά, αλλά και συνδυαστικά με σκοπό την ακόμη μεγαλύτερη μείωση των παρενεργειών αυτών στους υγιείς ιστούς.Για τη προσομοίωση των καρκινικών και υγιών ιστών συντέθηκαν ομοιώματα αγαρόζης παρουσία και μη ΜΝΣ, αντίστοιχα. Τα ΜΝΣ που χρησιμοποιούνται είναι από οξείδια του σιδήρου (Fe3O4), καθώς χρησιμοποιούνται ευρέως, λόγω της βιοσυμβατότητας, της λειτουργικότητας και των ιδιαίτερων μαγνητικών τους ιδιοτήτων, τόσο στη θεραπεία, όσο και στη διάγνωση διαφόρων ασθενειών. Τα ΜΝΣ αυτά, αναπτύχθηκαν στο εργαστήριο με τη μέθοδο της υδατικής συγκαταβύθισης και πραγματοποιήθηκε δομικός, μορφολογικός και μαγνητικός χαρακτηρισμός. Για μια πιο ολοκληρωμένη εικόνα και βαθύτερη κατανόηση της ανταπόκρισης ενός πραγματικού ζωικού ιστού, το ίδιο πρωτόκολλο υλοποιήθηκε και σε ex vivo μετρήσεις.Σε επόμενο στάδιο, μελετάται η μαγνητο-μηχανική καταπόνηση καρκινικών και υγιών κυττάρων του μαστού, εφαρμόζοντας διάφορους τύπους πεδίου, όπως στατικό, παλμικό και περιστρεφόμενο, σε πολύ χαμηλές συχνότητες με στόχο τη μείωση της βιωσιμότητας, μόνο των καρκινικών κυττάρων. Με τη βοήθεια φθορίζουσων χρωστικών και της συνεστιακής μικροσκοπίας, αποτυπώνονται οι αλλοιώσεις που έχουν υποστεί ο κυτταροσκελετός και ο πυρήνας των κυττάρων, ύστερα από μία μαγνητο-μηχανική θεραπεία. Τα ΜΝΣ που χρησιμοποιούνται και σε αυτήν την έρευνα είναι από οξείδια του σιδήρου, εμπορικά και επικαλυμμένα με άμυλο. Επομένως, το τελευταίο στάδιο αυτής της διδακτορικής διατριβής και το κύριο ερευνητικό ερώτημα που θέτει, αφορά το συνδυασμό της θερμικής και της μηχανικής θεραπείας, ώστε να διερευνηθεί η αθροιστική επίδραση των δύο αυτών προσεγγίσεων στην καταστροφή των καρκινικών κυττάρων του μαστού, μέσω της διέγερσης ΜΝΣ από μαγνητικά πεδία.

Numerical Simulation of Temperature Variations during the Application of Safety Protocols in Magnetic Particle Hyperthermia

Unavoidably, magnetic particle hyperthermia is limited by the unwanted heating of the neighboring healthy tissues, due to the generation of eddy currents. Eddy currents naturally occur, due to the applied alternating magnetic field, which is used to excite the nanoparticles in the tumor and, therefore, restrict treatment efficiency in clinical application. In this work, we present two simply applicable methods for reducing the heating of healthy tissues by simultaneously keeping the heating of cancer tissue, due to magnetic nanoparticles, at an adequate level. The first method involves moving the induction coil relative to the phantom tissue during the exposure. More specifically, the coil is moving symmetrically—left and right relative to the specimen—in a bidirectional fashion. In this case, the impact of the maximum distance (2–8 cm) between the coil and the phantom is investigated. In the second method, the magnetic field is applied intermittently (in an ON/OFF pulsed mode), instead of the continuous field mode usually employed. The parameters of the intermittent field mode, such as the time intervals (ON time and OFF time) and field amplitude, are optimized based on the numerical assessment of temperature increase in healthy tissue and cancer tissue phantoms. Different ON and OFF times were tested in the range of 25–100 s and 50–200 s, respectively, and under variable field amplitudes (45–70 mT). In all the protocols studied here, the main goal is to generate inside the cancer tissue phantom the maximum temperature increase, possible (preferably within the magnetic hyperthermia window of 4–8 °C), while restricting the temperature increase in the healthy tissue phantom to below 4 °C, signifying eddy current mitigation

Synergistic Effect of Combined Treatment with Magnetic Hyperthermia and Magneto-Mechanical Stress of Breast Cancer Cells

With the development of nanotechnology, the emergence of new anti-tumor techniques using nanoparticles such as magnetic hyperthermia and magneto-mechanical activation have been the subject of much attention and study in recent years, as anticancer tools. Therefore, the purpose of the current in vitro study was to investigate the cumulative effect of a combination of these two techniques, using magnetic nanoparticles against breast cancer cells. After 24 h of incubation, human breast cancer (MCF-7) and non-cancerous (MCF-10A) cells with and without MNPs were treated (a) for 15 min with magnetic hyperthermia, (b) for 30 min with magneto-mechanical activation, and (c) by a successive treatment consisting of a 15-min magnetic hyperthermia cycle and 30 min of magneto-mechanical activation. The influence of treatments on cell survival and morphology was studied by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay and light microscopy. When applied, separately, magneto-mechanical and thermal (hyperthermia) treatment did not demonstrate strong reduction in cell viability. No morphological changes were observed in non-cancerous cells after treatments. On the other hand, the combination of magneto-mechanical and thermal treatment in the presence of MNPs had a synergistic effect on decreased cell viability, and apoptosis was demonstrated in the cancer cell line. Synergism is most evident in the cancer cell line, incubated for 120 h, while in the non-cancerous line after 120 h, an increase in proliferation is clearly observed. MCF-7 cells showed more rounded cell morphology, especially after 120 h of combined treatment

Cell Behavioral Changes after the Application of Magneto-Mechanical Activation to Normal and Cancer Cells

In vitro cell exposure to nanoparticles, depending on the applied concentration, can help in the development of theranostic tools to better detect and treat human diseases. Recent studies have attempted to understand and exploit the impact of magnetic field-actuated internalized magnetic nanoparticles (MNPs) on the behavior of cancer cells. In this work, the viability rate of MNP’s-manipulated cancerous (MCF-7, MDA-MB-231) and non-cancerous (MCF-10A) cells was investigated in three different types of low-frequency magnetic fields: static, pulsed, and rotating field mode. In the non-cancerous cell line, the cell viability decreased mostly in cells with internalized MNPs and those treated with the pulsed field mode. In both cancer cell lines, the pulsed field mode was again the optimum magnetic field, which together with internalized MNPs caused a large decrease in cells’ viability (50–55% and 70% in MCF-7 and MDA-MB-231, respectively) while the static and rotating field modes maintained the viability at high levels. Finally, F-actin staining was used to observe the changes in the cytoskeleton and DAPI staining was performed to reveal the apoptotic alterations in cells’ nuclei before and after magneto-mechanical activation. Subsequently, reduced cell viability led to a loss of actin stress fibers and apoptotic nuclear changes in cancer cells subjected to MNPs triggered by a pulsed magnetic field

Biomimetic and biodegradable cellulose acetate scaffolds loaded with dexamethasone for bone implants

There is, as a matter of fact, an ever increasing number of patients requiring total hip replacement (Pabinger, C.; Geissler, A. Osteoarthritis Cartilage 2014, 22, 734–741). Implant-associated acute inflammations after an invasive orthopedic surgery are one of the major causes of implant failure. In addition, there are instability, aseptic loosening, infection, metallosis and fracture (Melvin, J. S.; Karthikeyan, T.; Cope, R.; Fehring, T. K. J. Arthroplasty 2014, 29, 1285–1288). In this work, a drug-delivery nanoplatform system consisting of polymeric celluloce acetate (CA) scaffolds loaded with dexamethasone was fabricated through electrospinning. Atomic force microscopy (AFM) and scanning electron microscopy (SEM) indicated the successful fabrication of these structures. Cytotoxicity studies were performed by using MTT assay, methylene-blue staining and SEM fixation and showed very good cell adhesion and proliferation, indicating the cytocompatibility of these fibrous scaffolds. Drug-release kinetics was measured for the evaluation of a controllable and sustained release of anti-inflammatory drug onto the engineered implants and degradation study was conducted in order to assess the mass loss of polymers. This drug-delivery nanoplatform as coating on titanium implants may be a promising approach not only to alleviate but also to prevent implant-associated acute inflammations along with a simultaneous controlled release of the drug

Synergistic Effect of Combined Treatment with Magnetic Hyperthermia and Magneto-Mechanical Stress of Breast Cancer Cells

With the development of nanotechnology, the emergence of new anti-tumor techniques using nanoparticles such as magnetic hyperthermia and magneto-mechanical activation have been the subject of much attention and study in recent years, as anticancer tools. Therefore, the purpose of the current in vitro study was to investigate the cumulative effect of a combination of these two techniques, using magnetic nanoparticles against breast cancer cells. After 24 h of incubation, human breast cancer (MCF-7) and non-cancerous (MCF-10A) cells with and without MNPs were treated (a) for 15 min with magnetic hyperthermia, (b) for 30 min with magneto-mechanical activation, and (c) by a successive treatment consisting of a 15-min magnetic hyperthermia cycle and 30 min of magneto-mechanical activation. The influence of treatments on cell survival and morphology was studied by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay and light microscopy. When applied, separately, magneto-mechanical and thermal (hyperthermia) treatment did not demonstrate strong reduction in cell viability. No morphological changes were observed in non-cancerous cells after treatments. On the other hand, the combination of magneto-mechanical and thermal treatment in the presence of MNPs had a synergistic effect on decreased cell viability, and apoptosis was demonstrated in the cancer cell line. Synergism is most evident in the cancer cell line, incubated for 120 h, while in the non-cancerous line after 120 h, an increase in proliferation is clearly observed. MCF-7 cells showed more rounded cell morphology, especially after 120 h of combined treatment