Computational and Experimental Evaluation of the Immune Response of Neoantigens for Personalized Vaccine Design.

In the last few years, the importance of neoantigens in the development of personalized antitumor vaccines has increased remarkably. In order to study whether bioinformatic tools are effective in detecting neoantigens that generate an immune response, DNA samples from patients with cutaneous melanoma in different stages were obtained, resulting in a total of 6048 potential neoantigens gathered. Thereafter, the immunological responses generated by some of those neoantigens ex vivo were tested, using a vaccine designed by a new optimization approach and encapsulated in nanoparticles. Our bioinformatic analysis indicated that no differences were found between the number of neoantigens and that of non-mutated sequences detected as potential binders by IEDB tools. However, those tools were able to highlight neoantigens over non-mutated peptides in HLA-II recognition (p-value 0.03). However, neither HLA-I binding affinity (p-value 0.08) nor Class I immunogenicity values (p-value 0.96) indicated significant differences for the latter parameters. Subsequently, the new vaccine, using aggregative functions and combinatorial optimization, was designed. The six best neoantigens were selected and formulated into two nanoparticles, with which the immune response ex vivo was evaluated, demonstrating a specific activation of the immune response. This study reinforces the use of bioinformatic tools in vaccine development, as their usefulness is proven both in silico and ex vivo.

A UHPLC-Mass Spectrometry View of Human Melanocytic Cells Uncovers Potential Lipid Biomarkers of Melanoma.

Melanoma is the deadliest form of skin cancer due to its ability to colonize distant sites and initiate metastasis. Although these processes largely depend on the lipid-based cell membrane scaffold, our understanding of the melanoma lipid phenotype lags behind most other aspects of this tumor cell. Here, we examined a panel of normal human epidermal and nevus melanocytes and primary and metastatic melanoma cell lines to determine whether distinctive cell-intrinsic lipidomes can discern non-neoplastic from neoplastic melanocytes and define their metastatic potential. Lipidome profiles were obtained by UHPLC-ESI mass-spectrometry, and differences in the signatures were analyzed by multivariate statistical analyses. Significant and highly specific changes in more than 30 lipid species were annotated in the initiation of melanoma, whereas less numerous changes were associated with melanoma progression and the non-malignant transformation of nevus melanocytes. Notably, the "malignancy lipid signature" features marked drops in pivotal membrane lipids, like sphingomyelins, and aberrant elevation of ether-type lipids and phosphatidylglycerol and phosphatidylinositol variants, suggesting a previously undefined remodeling of sphingolipid and glycerophospholipid metabolism. Besides broadening the molecular definition of this neoplasm, the different lipid profiles identified may help improve the clinical diagnosis/prognosis and facilitate therapeutic interventions for cutaneous melanoma.

PLGA-PEI nanoparticle covered with poly(I:C) for personalised cancer immunotherapy.

Melanoma is the main cause of death among skin cancers and its incidence worldwide has been experiencing an appalling increase. However, traditional treatments lack effectiveness in advanced or metastatic patients. Immunotherapy, meanwhile, has been shown to be an effective treatment option, but the rate of cancers responding remains far from ideal. Here we have developed a personalized neoantigen peptide-based cancer vaccine by encapsulating patient derived melanoma neoantigens in polyethylenimine (PEI)-functionalised poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and coating them with polyinosinic:polycytidylic acid (poly(I:C)). We found that PLGA NPs can be effectively modified to be coated with the immunoadjuvant poly(I:C), as well as to encapsulate neoantigens. In addition, we found that both dendritic cells (DCs) and lymphocytes were effectively stimulated. Moreover, the developed NP was found to have a better immune activation profile than NP without poly(I:C) or without antigen. Our results demonstrate that the developed vaccine has a high capacity to activate the immune system, efficiently maturing DCs to present the antigen of choice and promoting the activity of lymphocytes to exert their cytotoxic function. Therefore, the immune response generated is optimal and specific for the elimination of melanoma tumour cells.

Small extracellular vesicle-based human melanocyte and melanoma signature.

Intercellular communication is a cell-type and stimulus-dependent event driven not only by soluble factors but also by extracellular vesicles (EVs). EVs include vesicles of different size and origin that contain a myriad of molecules. Among them, small EVs (sEV; <200 nm) have been shown to modulate not just regional cell responses but also distant organ behavior. In cancer, distant organ modulation by sEVs has been associated to disease dissemination, which is one of the main concerns in melanoma. Description of broadly conserved alterations in sEV-contained molecules represents a strategy to identify key modifications in cellular communication as well as new disease biomarkers. Here, we characterize proteomes of cutaneous melanocyte and melanoma-derived sEVs to deepen on the landscape of normal and disease-related cell communication. Results reveal the presence of unique protein signatures for melanocytes and melanoma cells that reflect cellular transformation-related profound modifications. Melanocyte-derived sEVs are enriched in oxidative metabolism (e.g., aconitase 2, ACO2) or pigmentation (e.g., tyrosinase, TYR) related proteins while melanoma-derived sEVs reflect a generalized decrease in mature melanocytic markers (e.g., melanoma antigen recognized by T-cells 1, MART-1, also known as MLANA) and an increase in epithelial to mesenchymal transition (EMT)-related adhesion molecules such as tenascin C (TNC).

A Universal Antigen-Ranking Method to Design Personalized Vaccines Targeting Neoantigens against Melanoma.

Background: The main purpose of this article is to introduce a universal mathematics-aided vaccine design method against malignant melanoma based on neoantigens. The universal method can be adapted to the mutanome of each patient so that a specific candidate vaccine can be tailored for the corresponding patient. Methods: We extracted the 1134 most frequent mutations in melanoma, and we associated each of them to a vector with 10 components estimated with different bioinformatics tools, for which we found an aggregated value according to a set of weights, and then we ordered them in decreasing order of the scores. Results: We prepared a universal table of the most frequent mutations in melanoma ordered in decreasing order of viability to be used as candidate vaccines, so that the selection of a set of appropriate peptides for each particular patient can be easily and quickly implemented according to their specific mutanome and transcription profile. Conclusions: We have shown that the techniques that are commonly used for the design of personalized anti-tumor vaccines against malignant melanoma can be adapted for the design of universal rankings of neoantigens that originate personalized vaccines when the mutanome and transcription profile of specific patients is considered, with the consequent savings in time and money, shortening the design and production time.

Melanoma Clinical Decision Support System: An Artificial Intelligence-Based Tool to Diagnose and Predict Disease Outcome in Early-Stage Melanoma Patients.

This study set out to assess the performance of an artificial intelligence (AI) algorithm based on clinical data and dermatoscopic imaging for the early diagnosis of melanoma, and its capacity to define the metastatic progression of melanoma through serological and histopathological biomarkers, enabling dermatologists to make more informed decisions about patient management. Integrated analysis of demographic data, images of the skin lesions, and serum and histopathological markers were analyzed in a group of 196 patients with melanoma. The interleukins (ILs) IL-4, IL-6, IL-10, and IL-17A as well as IFNγ (interferon), GM-CSF (granulocyte and macrophage colony-stimulating factor), TGFß (transforming growth factor), and the protein DCD (dermcidin) were quantified in the serum of melanoma patients at the time of diagnosis, and the expression of the RKIP, PIRIN, BCL2, BCL3, MITF, and ANXA5 proteins was detected by immunohistochemistry (IHC) in melanoma biopsies. An AI algorithm was used to improve the early diagnosis of melanoma and to predict the risk of metastasis and of disease-free survival. Two models were obtained to predict metastasis (including "all patients" or only patients "at early stages of melanoma"), and a series of attributes were seen to predict the progression of metastasis: Breslow thickness, infiltrating BCL-2 expressing lymphocytes, and IL-4 and IL-6 serum levels. Importantly, a decrease in serum GM-CSF seems to be a marker of poor prognosis in patients with early-stage melanomas.

Dihydroceramide accumulation and reactive oxygen species are distinct and nonessential events in 4-HPR-mediated leukemia cell death.

4-(Hydroxyphenyl)retinamide (4-HPR) is a synthetic retinoid with a strong apoptotic effect towards different cancer cell lines in vitro, and it is currently tested in clinical trials. Increases of reactive oxygen species (ROS) and modulation of endogenous sphingolipid levels are well-described events observed upon 4-HPR treatment, but there is still a lack of understanding of their relationship and their contribution to cell death. LC-MS analysis of sphingolipids revealed that in human leukemia CCRF-CEM and Jurkat cells, 4-HPR induced dihydroceramide but not ceramide accumulation even at sublethal concentrations. Myriocin prevented the 4-HPR-induced dihydroceramide accumulation, but it did not prevent the loss of viability and increase of intracellular ROS production. On the other hand, ascorbic acid, Trolox, and vitamin E reversed 4-HPR effects on cell death but not dihydroceramide accumulation. NDGA, described as a lipoxygenase inhibitor, exerted a significantly higher antioxidant activity than vitamin E and abrogated 4-HPR-mediated ROS. It did not however rescue cellular viability. Taken together, this study demonstrates that early changes observed upon 4-HPR treatment, i.e., sphingolipid modulation and ROS production, are mechanistically independent events. Furthermore, the results indicate that 4-HPR-driven cell death may occur even in the absence of dihydroceramide or ROS accumulation. These observations should be taken into account for an improved design of drug combinations.

Upregulated phospholipase D2 expression and activity is related to the metastatic properties of melanoma.

The incidence rates of melanoma have increased steadily in recent decades and nearly 25% of the patients diagnosed with early-stage melanoma will eventually develop metastasis, for which there is currently no fully effective treatment. The link between phospholipases and tumors has been studied extensively, particularly in breast and colon cancers. With the aim of finding new biomarkers and therapeutic options for melanoma, the expression of different phospholipases was assessed in 17 distinct cell lines in the present study, demonstrating that phospholipase D2 (PLD2) is upregulated in metastatic melanoma as compared to normal skin melanocytes. These results were corroborated by immunofluorescence and lipase activity assays. Upregulation of PLD2 expression and increased lipase activity were observed in metastatic melanoma relative to normal skin melanocytes. So far, the implication of PLD2 activity in melanoma malignancies has remained elusive. To the best of our knowledge, the present study was the first to demonstrate that the overexpression of PLD2 enhances lipase activity, and its effect to increase the proliferation, migration and invasion capacity of melanoma cells was assessed with XTT and Transwell assays. In addition, silencing of PLD2 in melanoma cells reduced the metastatic potential of these cells. The present study provided evidence that PLD2 is involved in melanoma malignancy and in particular, in its metastatic potential, and established a basis for future studies evaluating PLD2 blockade as a therapeutic strategy to manage this condition.

MGRN1 as a Phenotypic Determinant of Human Melanoma Cells and a Potential Biomarker.

Mahogunin Ring Finger 1 (MGRN1), a ubiquitin ligase expressed in melanocytes, interacts with the α melanocyte-stimulating hormone receptor, a well-known melanoma susceptibility gene. Previous studies showed that MGRN1 modulates the phenotype of mouse melanocytes and melanoma cells, with effects on pigmentation, shape, and motility. Moreover, MGRN1 knockdown augmented the burden of DNA breaks in mouse cells, indicating that loss of MGRN1 promoted genomic instability. However, data concerning the roles of MGRN1 in human melanoma cells remain scarce. We analyzed MGRN1 knockdown in human melanoma cells. Transient MGRN1 depletion with siRNA or permanent knockdown in human melanoma cells by CRISPR/Cas9 caused an apparently MITF-independent switch to a more dendritic phenotype. Lack of MGRN1 also increased the fraction of human cells in the S phase of the cell cycle and the burden of DNA breaks but did not significantly impair proliferation. Moreover, in silico analysis of publicly available melanoma datasets and estimation of MGRN1 in a cohort of clinical specimens provided preliminary evidence that MGRN1 expression is higher in human melanomas than in normal skin or nevi and pointed to an inverse correlation of MGRN1 expression in human melanoma with patient survival, thus suggesting potential use of MGRN1 as a melanoma biomarker.

Evaluation of bioactive sphingolipids in 4-HPR-resistant leukemia cells.

BACKGROUND: N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a synthetic retinoid with potent pro-apoptotic activity against several types of cancer, but little is known regarding mechanisms leading to chemoresistance. Ceramide and, more recently, other sphingolipid species (e.g., dihydroceramide and dihydrosphingosine) have been implicated in 4-HPR-mediated tumor cell death. Because sphingolipid metabolism has been reported to be altered in drug-resistant tumor cells, we studied the implication of sphingolipids in acquired resistance to 4-HPR based on an acute lymphoblastic leukemia model. METHODS: CCRF-CEM cell lines resistant to 4-HPR were obtained by gradual selection. Endogenous sphingolipid profiles and in situ enzymatic activities were determined by LC/MS, and resistance to 4-HPR or to alternative treatments was measured using the XTT viability assay and annexin V-FITC/propidium iodide labeling. RESULTS: No major crossresistance was observed against other antitumoral compounds (i.e. paclitaxel, cisplatin, doxorubicin hydrochloride) or agents (i.e. ultra violet C, hydrogen peroxide) also described as sphingolipid modulators. CCRF-CEM cell lines resistant to 4-HPR exhibited a distinctive endogenous sphingolipid profile that correlated with inhibition of dihydroceramide desaturase. Cells maintained acquired resistance to 4-HPR after the removal of 4-HPR though the sphingolipid profile returned to control levels. On the other hand, combined treatment with sphingosine kinase inhibitors (unnatural (dihydro)sphingosines ((dh)Sph)) and glucosylceramide synthase inhibitor (PPMP) in the presence or absence of 4-HPR increased cellular (dh)Sph (but not ceramide) levels and were highly toxic for both parental and resistant cells. CONCLUSIONS: In the leukemia model, acquired resistance to 4-HPR is selective and persists in the absence of sphingolipid profile alteration. Therapeutically, the data demonstrate that alternative sphingolipid-modulating antitumoral strategies are suitable for both 4-HPR-resistant and sensitive leukemia cells. Thus, whereas sphingolipids may not be critical for maintaining resistance to 4-HPR, manipulation of cytotoxic sphingolipids should be considered a viable approach for overcoming resistance.

The diversity profile of TP53 is influenced by positive selection on the immediately upstream locus WDR79.

BACKGROUND/AIM: TP53 is an efficient central node in a signal transduction network that responds to minimize cancer. However, over 50% of tumors show some mutation in TP53. Thus, one might argue that this single central node network lacks robustness. Therefore, we wanted to investigate if natural selection has played a role in shaping the genomic region containing TP53. METHODS: We have analyzed the HapMap data for evidence of selection using F(ST) pairwise comparisons and the extended haplotype homozygosity test on a 200-kb region encompassing TP53. We have also resequenced 4 kb upstream TP53 in Europeans (including melanoma patients), Asians, Australian Aborigines and Africans. RESULTS: Genetic hitchhiking by a linked, positively selected allele at the nearby gene WDR79 may be partly responsible for the sequence diversity profile of TP53. It can help explain why the TP53 Arg72 allele is the major allele in Europeans even when the alternative allele, 72Pro, has been reported to offer an increased longevity after disease. CONCLUSIONS: Despite the important role of TP53, a complex interplay with other evolutionary forces, which are extrinsic to TP53 function, may have driven the genetic diversity pattern of this locus, and, as a consequence, its structure and function.

Inhibition of the Human Hsc70 System by Small Ligands as a Potential Anticancer Approach.

Heat shock protein (Hsp) synthesis is upregulated in a wide range of cancers to provide the appropriate environment for tumor progression. The Hsp110 and Hsp70 families have been associated to cancer cell survival and resistance to chemotherapy. In this study, we explore the strategy of drug repurposing to find new Hsp70 and Hsp110 inhibitors that display toxicity against melanoma cancer cells. We found that the hits discovered using Apg2, a human representative of the Hsp110 family, as the initial target bind also to structural regions present in members of the Hsp70 family, and therefore inhibit the remodeling activity of the Hsp70 system. One of these compounds, the spasmolytic agent pinaverium bromide used for functional gastrointestinal disorders, inhibits the intracellular chaperone activity of the Hsp70 system and elicits its cytotoxic activity specifically in two melanoma cell lines by activating apoptosis. Docking and molecular dynamics simulations indicate that this compound interacts with regions located in the nucleotide-binding domain and the linker of the chaperones, modulating their ATPase activity. Thus, repurposing of pinaverium bromide for cancer treatment appears as a promising novel therapeutic approach.

The multifunctional role of SPANX-A/D protein subfamily in the promotion of pro-tumoural processes in human melanoma.

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes encode a protein family (SPANX-A, -B, -C and -D), whose expression is limited to the testis and spermatozoa in normal tissues and various tumour cells. SPANX-A/D proteins have been detected in metastatic melanoma cells, but their contribution to cancer development and the underlying molecular mechanisms of skin tumourigenesis remain unknown. Combining functional and proteomic approaches, the present work describes the presence of SPANX-A/D in primary and metastatic human melanoma cells and how it promotes pro-tumoural processes such as cell proliferation, motility and migration. We provide insights into the molecular features of skin tumourigenesis, describing for the first time a multifunctional role of the SPANX-A/D protein family in nuclear function, energy metabolism and cell survival, considered key hallmarks of cancer. A better comprehension of the SPANX-A/D protein subfamily and its molecular mechanisms will help to describe new aspects of tumour cell biology and develop new therapeutic targets and tumour-directed pharmacological drugs for skin tumours.

Plasma membrane and nuclear envelope integrity during the blebbing stage of apoptosis: a time-lapse study.

BACKGROUND INFORMATION: The execution phase of apoptosis is characterized by extensive blebbing of the plasma membrane, which usually results in secondary lysis in vitro. To analyse the permeability of cellular membranes during this process, we induced apoptosis in human melanoma A375 cells that had been transfected with fluorescently tagged proteins which were targeted to different subcellular locations. RESULTS: The dual treatment of resveratrol and butyrate produced a synergistic induction of apoptosis by blocking different phases of the cell cycle. Changes in the plasma membrane, nuclear envelope and nucleoli were monitored by time-lapse confocal microscopy. Fluorescently labelled proteins were not mis-localized from their original locations in any of the cells undergoing blebbing for several hours. Thus the maintenance of karyophilic and nucleolar proteins within the nucleus during the blebbing stage and the accessibility of vital selective chromatin dyes confirmed a functional preservation of the nuclear compartment until the final necrotic blister. The translocation of phosphatidylserine to the outer leaflet of the plasma membrane was not detected during the blebbing period. CONCLUSION: These results show that the functional integrity of the nuclear envelope and plasma membrane may be conserved until the end of the execution phase of apoptosis.

BRAF V600E mutational load as a prognosis biomarker in malignant melanoma.

Analyzing the mutational load of driver mutations in melanoma could provide valuable information regarding its progression. We aimed at analyzing the heterogeneity of mutational load of BRAF V600E in biopsies of melanoma patients of different stages, and investigating its potential as a prognosis factor. Mutational load of BRAF V600E was analyzed by digital PCR in 78 biopsies of melanoma patients of different stages and 10 nevi. The BRAF V600E load was compared among biopsies of different stages. Results showed a great variability in the load of V600E (0%-81%). Interestingly, we observed a significant difference in the load of V600E between the early and late melanoma stages, in the sense of an inverse correlation between BRAF V600E mutational load and melanoma progression. In addition, a machine learning approach showed that the mutational load of BRAF V600E could be a good predictor of metastasis in stage II patients. Our results suggest that BRAF V600E is a promising biomarker of prognosis in stage II patients.

Serum markers improve current prediction of metastasis development in early-stage melanoma patients: a machine learning-based study.

Metastasis development represents an important threat for melanoma patients, even when diagnosed at early stages and upon removal of the primary tumor. In this scenario, determination of prognostic biomarkers would be of great interest. Serum contains information about the general status of the organism and therefore represents a valuable source for biomarkers. Thus, we aimed to define serological biomarkers that could be used along with clinical and histopathological features of the disease to predict metastatic events on the early-stage population of patients. We previously demonstrated that in stage II melanoma patients, serum levels of dermcidin (DCD) were associated with metastatic progression. Based on the relevance of the immune response on the cancer progression and the recent association of DCD with local and systemic immune response against cancer cells, serum DCD was analyzed in a new cohort of patients along with interleukin 4 (IL-4), IL-6, IL-10, IL-17A, interferon γ (IFN-γ), transforming growth factor-ß (TGF- ß), and granulocyte-macrophage colony-stimulating factor (GM-CSF). We initially recruited 448 melanoma patients, 323 of whom were diagnosed as stages I-II according to AJCC. Levels of selected cytokines were determined by ELISA and Luminex, and obtained data were analyzed employing machine learning and Kaplan-Meier techniques to define an algorithm capable of accurately classifying early-stage melanoma patients with a high and low risk of developing metastasis. The results show that in early-stage melanoma patients, serum levels of the cytokines IL-4, GM-CSF, and DCD together with the Breslow thickness are those that best predict melanoma metastasis. Moreover, resulting algorithm represents a new tool to discriminate subjects with good prognosis from those with high risk for a future metastasis.

The nucleus does not significantly affect the migratory trajectories of amoeba in two-dimensional environments.

For a wide range of cells, from bacteria to mammals, locomotion movements are a crucial systemic behavior for cellular life. Despite its importance in a plethora of fundamental physiological processes and human pathologies, how unicellular organisms efficiently regulate their locomotion system is an unresolved question. Here, to understand the dynamic characteristics of the locomotion movements and to quantitatively study the role of the nucleus in the migration of Amoeba proteus we have analyzed the movement trajectories of enucleated and non-enucleated amoebas on flat two-dimensional (2D) surfaces using advanced non-linear physical-mathematical tools and computational methods. Our analysis shows that both non-enucleated and enucleated amoebas display the same kind of dynamic migration structure characterized by highly organized data sequences, super-diffusion, non-trivial long-range positive correlations, persistent dynamics with trend-reinforcing behavior, and move-step fluctuations with scale invariant properties. Our results suggest that the presence of the nucleus does not significantly affect the locomotion of amoeba in 2D environments.

Evidence of conditioned behavior in amoebae.

Associative memory is the main type of learning by which complex organisms endowed with evolved nervous systems respond efficiently to certain environmental stimuli. It has been found in different multicellular species, from cephalopods to humans, but never in individual cells. Here we describe a motility pattern consistent with associative conditioned behavior in the microorganism Amoeba proteus. We use a controlled direct-current electric field as the conditioned stimulus, and a specific chemotactic peptide as the unconditioned stimulus. The amoebae are capable of linking two independent past events, generating persistent locomotion movements that can prevail for 44 min on average. We confirm a similar behavior in a related species, Metamoeba leningradensis. Thus, our results indicate that unicellular organisms can modify their behavior during migration by associative conditioning.

Complex signatures of selection for the melanogenic loci TYR, TYRP1 and DCT in humans.

BACKGROUND: The observed correlation between ultraviolet light incidence and skin color, together with the geographical apportionment of skin reflectance among human populations, suggests an adaptive value for the pigmentation of the human skin. We have used Affymetrix U133a v2.0 gene expression microarrays to investigate the expression profiles of a total of 9 melanocyte cell lines (5 from lightly pigmented donors and 4 from darkly pigmented donors) plus their respective unirradiated controls. In order to reveal signatures of selection in loci with a bearing on skin pigmentation in humans, we have resequenced between 4 to 5 kb of the proximal regulatory regions of three of the most differently expressed genes, in the expectation that variation at regulatory regions might account for intraespecific morphological diversity, as suggested elsewhere. RESULTS: Contrary to our expectations, expression profiles did not cluster the cells into unirradiated versus irradiated melanocytes, or into lightly pigmented versus darkly pigmented melanocytes. Instead, expression profiles correlated with the presence of Bovine Pituitary Extract (known to contain alpha-MSH) in the media. This allowed us to differentiate between melanocytes that are synthesizing melanin and those that are not. TYR, TYRP1 and DCT were among the five most differently expressed genes between these two groups. Population genetic analyses of sequence haplotypes of the proximal regulatory flanking-regions included Tajima's D, HEW and DHEW neutrality tests analysis. These were complemented with EHH tests (among others) in which the significance was obtained by a novel approach using extensive simulations under the coalescent model with recombination. We observe strong evidence for positive selection for TYRP1 alleles in Africans and for DCT and TYRP1 in Asians. However, the overall picture reflects a complex pattern of selection, which might include overdominance for DCT in Europeans. CONCLUSION: Diversity patterns clearly evidence adaptive selection in pigmentation genes in Africans and Asians. In Europeans, the evidence is more complex, and both directional and balancing selection may be involved in light skin. As a result, different non-African populations may have acquired light skin by alternative ways, and so light skin, and perhaps dark skin too, may be the result of convergent evolution.

Cost-Effective Design of High-Magnetic Moment Nanostructures for Biotechnological Applications.

Disk-shaped magnetic nanostructures present distinctive features for novel biomedical applications. Fine tuning of geometry and dimensions is demanded to evaluate efficiency and capability of such applications. This work addresses a cost-effective, versatile, and maskless design of biocompatible high-magnetic moment elements at the sub-micrometer scale. Advantages and disadvantages of two high throughput fabrication routes using interference lithography were evaluated. Detrimental steps such as the release process of nanodisks into aqueous solution were optimized to fully preserve the magnetic properties of the material. Then, cell viability of the nanostructures was assessed in primary melanoma cultures. No toxicity effects were observed, validating the potential of these nanostructures in biotechnological applications. The present methodology will allow the fabrication of magnetic nanoelements at the sub-micrometer scale with unique spin configurations, such as vortex state, synthetic antiferromagnets, or exchange-coupled heterostructures, and their use in biomedical techniques that require a remote actuation or a magneto-electric response.