Detection of the Uveal Melanoma-Associated Mutation GNAQ Q209P from Liquid Biopsy Using CRISPR/Cas12a Technology.

Uveal melanoma (UM) is a rare ocular tumor characterized by high metastasis risk and poor prognosis. The in-depth characterization of UM's molecular profile is critical for better disease classification and prognosis. Furthermore, the development of detection tools to monitor UM evolution upon treatment is of great interest for designing optimal therapeutic strategies. However, commonly used techniques, such as ddPCR or NGS, are costly, and they involve sophisticated equipment and complex experimental design. The development of alternative sensing methods that are fast, simple, and inexpensive would be of great benefit to improve UM's diagnosis and management, especially when combined with liquid biopsy. Samples from liquid biopsy can be obtained with minimal invasiveness, and the detection of circulating tumor DNA (ctDNA) in UM patients' plasma has proven useful for the diagnosis of metastasis, prognosis prediction, and disease monitoring. In this context, CRISPR/Cas12a-derived molecular sensors, thanks to their high specificity and sensitivity and their potential for point of care diagnosis, are particularly interesting. Here, we developed a CRISPR/Cas12a-based approach for the specific detection of the UM-related mutation GNAQ Q209P that relies on the design of highly specific crRNAs. Coupled with allele-specific PCR, it constitutes a sensitive platform for liquid biopsy detection, capable of sensing GNAQ Q209P in plasma samples with a low ctDNA concentration and fractional abundance. Finally, our method was validated using plasma samples from metastatic UM patients.

Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures for sensitive and selective SARS-CoV-2 sensing.

The development of DNA-sensing platforms based on new synthetized Methylene Blue functionalized carbon nanodots combined with different shape gold nanostructures (AuNs), as a new pathway to develop a selective and sensitive methodology for SARS-CoV-2 detection is presented. A mixture of gold nanoparticles and gold nanotriangles have been synthetized to modify disposable electrodes that act as an enhanced nanostructured electrochemical surface for DNA probe immobilization. On the other hand, modified carbon nanodots prepared a la carte to contain Methylene Blue (MB-CDs) are used as electrochemical indicators of the hybridization event. These MB-CDs, due to their structure, are able to interact differently with double and single-stranded DNA molecules. Based on this strategy, target sequences of the SARS-CoV-2 virus have been detected in a straightforward way and rapidly with a detection limit of 2.00 aM. Moreover, this platform allows the detection of the SARS-CoV-2 sequence in the presence of other viruses, and also a single nucleotide polymorphism (SNPs). The developed approach has been tested directly on RNA obtained from nasopharyngeal samples from COVID-19 patients, avoiding any amplification process. The results agree well with those obtained by RT-qPCR or reverse transcription quantitative polymerase chain reaction technique.

Amplification-free detection of SARS-CoV-2 using gold nanotriangles functionalized with oligonucleotides.

Gold nanotriangles (AuNTs) functionalized with dithiolated oligonucleotides have been employed to develop an amplification-free electrochemical biosensor for SARS-CoV-2 in patient samples. Gold nanotriangles, prepared through a seed-mediated growth method and exhaustively characterized by different techniques, serve as an improved electrochemical platform and for DNA probe immobilization. Azure A is used as an electrochemical indicator of the hybridization event. The biosensor detects either single stranded DNA or RNA sequences of SARS-CoV-2 of different lengths, with a low detection limit of 22.2 fM. In addition, it allows to detect point mutations in SARS-CoV-2 genome with the aim to detect more infective SARS-CoV-2 variants such as Alpha, Beta, Gamma, Delta, and Omicron. Results obtained with the biosensor in nasopharyngeal swab samples from COVID-19 patients show the possibility to clearly discriminate between non-infected and infected patient samples as well as patient samples with different viral load. Furthermore, the results correlate well with those obtained by the gold standard technique RT-qPCR, with the advantage of avoiding the amplification process and the need of sophisticated equipment.

Albumin-based nanostructures for uveal melanoma treatment.

Uveal melanoma (UM) is an intraocular tumor which is almost lethal at the metastatic stage due to the lack of effective treatments. In this regard, we have developed an albumin-based nanostructure (ABN) containing AZD8055 (ABN-AZD), which is a potent mTOR kinase inhibitor, for its efficient delivery to the tumors. The drug has been conjugated to ABN using tailored linkers that have a disulfide moiety, allowing its release selectively and effectively in the presence of an elevated concentration of glutathione, such as inside the tumoral cells. Our therapeutic approach induced significant cellular toxicity in uveal melanoma cells, but not in non-tumoral keratinocytes, highlighting the excellent selectivity of the system. In addition, these nanostructures showed excellent activity in vivo, decreasing the tumor surface compared to the free AZD8055 in mice models. Remarkably, the results obtained were achieved employing a dose 23 times lower than those used in previous reports.

Targeting autophagy using metallic nanoparticles: a promising strategy for cancer treatment.

Despite the extensive genetic and phenotypic variations present in the different tumors, they frequently share common metabolic alterations, such as autophagy. Autophagy is a self-degradative process in response to stresses by which damaged macromolecules and organelles are targeted by autophagic vesicles to lysosomes and then eliminated. It is known that autophagy dysfunctions can promote tumorigenesis and cancer development, but, interestingly, its overstimulation by cytotoxic drugs may also induce cell death and chemosensitivity. For this reason, the possibility to modulate autophagy may represent a valid therapeutic approach to treat different types of cancers and a variety of clinical trials, using autophagy modulators, are currently employed. On the other hand, recent progress in nanotechnology offers plenty of tools to fight cancer with innovative and efficient therapeutic agents by overcoming obstacles usually encountered with traditional drugs. Interestingly, nanomaterials can modulate autophagy and have been exploited as therapeutic agents against cancer. In this article, we summarize the most recent advances in the application of metallic nanostructures as potent modulators of autophagy process through multiple mechanisms, stressing their therapeutic implications in cancer diseases. For this reason, we believe that autophagy modulation with nanoparticle-based strategies would acquire clinical relevance in the near future, as a complementary therapy for the treatment of cancers and other diseases.

MicroRNA sensors based on gold nanoparticles.

MicroRNAs (miRNAs) are small regulatory RNAs, the dysregulation of which has been associated with the progression of several human diseases, including cancer. Interestingly, these molecules can be used as biomarkers for early disease diagnosis and can be found in a variety of body fluids and tissue samples. However, their specific properties and very low concentrations make their detection rather challenging. In this regard, current detection methods are complex, cost-ineffective, and of limited application in point-of-care settings or resource-limited facilities. Recently, nanotechnology-based approaches have emerged as promising alternatives to conventional miRNA detection methods and paved the way for research towards sensitive, fast, and low-cost detection systems. In particular, due to their exceptional properties, the use of gold nanoparticles (AuNPs) has significantly improved the performance of miRNA biosensors. This review discusses the application of AuNPs in different miRNA sensor modalities, commenting on recently reported examples. A practical overview of each modality is provided, highlighting their future use in clinical diagnosis. Graphical abstract ᅟ.

The phenotype of target pancreatic cancer cells influences cell death by magnetic hyperthermia with nanoparticles carrying gemicitabine and the pseudo-peptide NucAnt.

In this paper we show that conjugation of magnetic nanoparticles (MNPs) with Gemcitabine and/or NucAnt (N6L) fostered their internalization into pancreatic tumor cells and that the coupling procedure did not alter the cytotoxic potential of the drugs. By treating tumor cells (BxPC3 and PANC-1) with the conjugated MNPs and magnetic hyperthermia (43 °C, 60 min), cell death was observed. The two pancreatic tumor cell lines showed different reactions against the combined therapy according to their intrinsic sensitivity against Gemcitabine (cell death, ROS production, ability to activate ERK 1/2 and JNK). Finally, tumors (e.g. 3 mL) could be effectively treated by using almost 4.2 × 105 times lower Gemcitabine doses compared to conventional therapies. Our data show that this combinatorial therapy might well play an important role in certain cell phenotypes with low readiness of ROS production. This would be of great significance in distinctly optimizing local pancreatic tumor treatments.

Recent advances in uveal melanoma treatment.

Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Recent advances in the understanding of molecular characteristics helped to determine which tumors are most likely to progress. About 50% of patients carrying genetic alterations such as chromosomal aberrations and mutations are at significant risk for metastatic disease of which the majority will succumb to UM within few months. Currently, there is no effective treatment for metastatic uveal melanoma, and we hope this review will encourage researchers and clinicians to work to find a better standard of care. In this article we provide a comprehensive overview of the molecular framework of UM, highlighting the most common mutations involved in this kind of cancer. It also covers the most recent treatments from basic research to clinical trials, including small molecules, nucleic acids or immunotherapy, among others. It is intended to serve as a key reference for clinicians and researchers working in this field.

Copper(II)-Thymine Coordination Polymer Nanoribbons as Potential Oligonucleotide Nanocarriers.

The direct reaction between copper nitrate, thymine-1-acetic acid, and 4,4'-bipyridine in water leads to the formation of a blue colloid comprising uniform crystalline nanoribbons (length >1 µm; width ca. 150-185 nm; diameter ca. 15-60 nm) of a coordination polymer. The polymer displays a thymine-based structure freely available for supramolecular interactions. These nanostructures show significant selective interaction with single-stranded oligonucleotides based on adenine. Remarkably, they present low cell toxicity in three cell lines-despite the copper(II) content-and can be used as nanocarriers of oligonucleotides. These results suggest the potential of these types of nanostructures in several biological applications.

Multifunctionalized iron oxide nanoparticles for selective drug delivery to CD44-positive cancer cells.

Nanomedicine nowadays offers novel solutions in cancer therapy and diagnosis by introducing multimodal treatments and imaging tools in one single formulation. Nanoparticles acting as nanocarriers change the solubility, biodistribution and efficiency of therapeutic molecules, reducing their side effects. In order to successfully  apply these novel therapeutic approaches, efforts are focused on the biological functionalization of the nanoparticles to improve the selectivity towards cancer cells. In this work, we present the synthesis and characterization of novel multifunctionalized iron oxide magnetic nanoparticles (MNPs) with antiCD44 antibody and gemcitabine derivatives, and their application for the selective treatment of CD44-positive cancer cells. The lymphocyte homing receptor CD44 is overexpressed in a large variety of cancer cells, but also in cancer stem cells (CSCs) and circulating tumor cells (CTCs). Therefore, targeting CD44-overexpressing cells is a challenging and promising anticancer strategy. Firstly, we demonstrate the targeting of antiCD44 functionalized MNPs to different CD44-positive cancer cell lines using a CD44-negative non-tumorigenic cell line as a control, and verify the specificity by ultrastructural characterization and downregulation of CD44 expression. Finally, we show the selective drug delivery potential of the MNPs by the killing of CD44-positive cancer cells using a CD44-negative non-tumorigenic cell line as a control. In conclusion, the proposed multifunctionalized MNPs represent an excellent biocompatible nanoplatform for selective CD44-positive cancer therapy in vitro.

Modified RNAs in CRISPR/Cas9: An Old Trick Works Again.

Old tricks, new dog: CRISPR/Cas9 is a powerful tool for gene editing that requires an endonuclease (Cas9) and RNA strands. It has been shown that chemical modification of the RNA structures, an approach that has been used to improve the efficiency of RNA interference, can also be applied to enhance the activity of CRISPR/Cas9 and reduce its off-target effects.

Efficient treatment of breast cancer xenografts with multifunctionalized iron oxide nanoparticles combining magnetic hyperthermia and anti-cancer drug delivery.

INTRODUCTION: Tumor cells can effectively be killed by heat, e.g. by using magnetic hyperthermia. The main challenge in the field, however, is the generation of therapeutic temperatures selectively in the whole tumor region. We aimed to improve magnetic hyperthermia of breast cancer by using innovative nanoparticles which display a high heating potential and are functionalized with a cell internalization and a chemotherapeutic agent to increase cell death. METHODS: The superparamagnetic iron oxide nanoparticles (MF66) were electrostatically functionalized with either Nucant multivalent pseudopeptide (N6L; MF66-N6L), doxorubicin (DOX; MF66-DOX) or both (MF66-N6LDOX). Their cytotoxic potential was assessed in a breast adenocarcinoma cell line MDA-MB-231. Therapeutic efficacy was analyzed on subcutaneous MDA-MB-231 tumor bearing female athymic nude mice. RESULTS: All nanoparticle variants showed an excellent heating potential around 500 W/g Fe in the alternating magnetic field (AMF, conditions: H=15.4 kA/m, f=435 kHz). We could show a gradual inter- and intracellular release of the ligands, and nanoparticle uptake in cells was increased by the N6L functionalization. MF66-DOX and MF66-N6LDOX in combination with hyperthermia were more cytotoxic to breast cancer cells than the respective free ligands. We observed a substantial tumor growth inhibition (to 40% of the initial tumor volume, complete tumor regression in many cases) after intratumoral injection of the nanoparticles in vivo. The proliferative activity of the remaining tumor tissue was distinctly reduced. CONCLUSION: The therapeutic effects of breast cancer magnetic hyperthermia could be strongly enhanced by the combination of MF66 functionalized with N6L and DOX and magnetic hyperthermia. Our approach combines two ways of tumor cell killing (magnetic hyperthermia and chemotherapy) and represents a straightforward strategy for translation into the clinical practice when injecting nanoparticles intratumorally.

Detection of GNAQ mutations and reduction of cell viability in uveal melanoma cells with functionalized gold nanoparticles.

BACKGROUND: Uveal melanoma (UM) is the most common primary intraocular malignancy in adults. Early treatment may improve any chances of preventing metastatic disease, but diagnosis of small UM is challenging. Up to 95 % of all UMs carry somatic mutations in the G-coupled proteins GNAQ and GNA11 promoting anchorage-independent growth and proliferation. About 50 % of UMs are fatal. Once metastatic, patients have limited options for successful therapy. METHODS: We have developed functionalized gold nanoparticles (AuNPs) to visualize transcripts of mutant GNAQ mRNA in living cells. In addition to their suitability as a specific tool for GNAQ mutation detection, we have developed a novel linker that enables conjugation of siRNAs to AuNPs allowing for greater and more rapid intracellular release of siRNAs compared to previously described approaches. RESULTS: Binding of modified AuNPs to matching target mRNA leads to conformational changes, resulting in a detectable fluorescent signal that can be used for mutation detection in living cells. Knockdown of GNAQ with siRNA-AuNPs effectively reduced downstream signals and decreased cell viability in GNAQ mutant uveal melanoma cells. CONCLUSION: AuNPs may in future be developed to serve as sensors for mutations of vital importance. The new release system for siRNA-AuNP improves previous systems, which conceivably will be useful for future therapeutic gene regulatory approaches.

Designed Modular Proteins as Scaffolds To Stabilize Fluorescent Nanoclusters.

Proteins have been used as templates to stabilize fluorescent metal nanoclusters thus obtaining stable fluorescent structures, and their fluorescent properties being modulated by the type of protein employed. Designed consensus tetratricopeptide repeat (CTPR) proteins are suited candidates as templates for the stabilization of metal nanoclusters due to their modular structural and functional properties. Here, we have studied the ability of CTPR proteins to stabilize fluorescent gold nanoclusters giving rise to designed functional hybrid nanostructures. First, we have investigated the influence of the number of CTPR units, as well as the presence of cysteine residues in the CTPR protein, on the fluorescent properties of the protein-stabilized gold nanoclusters. Synthetic protocols to retain the protein structure and function have been developed, since the structural and functional integrity of the protein template is critical for further applications. Finally, as a proof-of-concept, a CTPR module with specific binding capabilities has been used to stabilize gold nanoclusters with positive results. Remarkably, the protein-stabilized gold nanocluster obtained combines both the fluorescence properties of the nanoclusters and the functional properties of the protein. The fluorescence changes in nanoclusters fluorescence have been successfully used as a sensor to detect when the specific ligand was recognized by the CTPR module.

Gold-Coated Superparamagnetic Nanoparticles for Single Methyl Discrimination in DNA Aptamers.

Au- and iron-based magnetic nanoparticles (NPs) are promising NPs for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core puts together the benefits from adding the magnetic properties to the robust chemistry provided by the thiol functionalization of gold. Here, the use of Au-coated magnetic NPs for molecular detection of a single methylation in DNA aptamer is described. Binding of α-thrombin to two aptamers conjugated to these NPs causes aggregation, a phenomenon that can be observed by UV, DLS and MRI. These techniques discriminate a single methylation in one of the aptamers, preventing aggregation due to the inability of α-thrombin to recognize it. A parallel study with gold and ferromagnetic NPs is detailed, concluding that the Au coating of FexOy NP does not affect their performance and that they are suitable as complex biosensors. These results prove the high detection potency of Au-coated SPIONs for biomedical applications especially for DNA repair detection.

Multicore iron oxide nanoparticles for magnetic hyperthermia and combination therapy against cancer cells.

HYPOTHESIS: Multicore flower-like iron oxide nanoparticles (IONPs) are among the best candidates for magnetic hyperthermia applications against cancers. However, they are rarely investigated in physiological environments and their efficacy against cancer cells has been even less studied. The combination of magnetic hyperthermia, using multicore IONPs, with selected bioactive molecules should lead to an enhanced activity against cancer cells. EXPERIMENTS: Multicore IONPs were synthesized by a seeded-growth thermal decomposition approach. Then, the cytotoxicity, cell uptake, and efficacy of the magnetic hyperthermia approach were studied with six cancer cell lines: PANC1 (pancreatic carcinoma), Mel202 (uveal melanoma), MCF7 (breast adenocarcinoma), MB231 (triple-negative breast cancer line), A549 (lung cancer), and HCT116 (colon cancer). Finally, IONPs were modified with a chemotherapeutic drug (SN38) and tumor suppressor microRNAs (miR-34a, miR-182, let-7b, and miR-137), to study their activity against cancer cells with and without combination with magnetic hyperthermia. FINDINGS: Two types of multicore IONPs with very good heating abilities under magnetic stimulation have been prepared. Their concentration-dependent cytotoxicity and internalization have been established, showing a strong dependence on the cell line and the nanoparticle type. Magnetic hyperthermia causes significant cell death that is dramatically enhanced in combination with the bioactive molecules.

Free PCR virus detection via few-layer bismuthene and tetrahedral DNA nanostructured assemblies.

In this work we describe a highly sensitive method based on a biocatalyzed electrochemiluminescence approach. The system combines, for the first time, the use of few-layer bismuthene (FLB) as a platform for the oriented immobilization of tetrahedral DNA nanostructures (TDNs) specifically designed and synthetized to detect a specific SARS-CoV-2 gene sequence. In one of its vertices, these TDNs contain a DNA capture probe of the open reading frame 1 ab (ORF1ab) of the virus, available for the biorecognition of the target DNA/RNA. At the other three vertices, there are thiol groups that enable the stable anchoring/binding to the FLB surface. This novel geometry/approach enables not only the binding of the TDNs to surfaces, but also the orientation of the capture probe in a direction normal to the bismuthine surface so that it is readily accessible for binding/recognition of the specific SARS-CoV-2 sequence. The analytical signal is based on the anodic electrochemiluminescence (ECL) intensity of luminol which, in turn, arises as a result of the reaction with H2O2, generated by the enzymatic reaction of glucose oxidation, catalyzed by the biocatalytic label avidin-glucose oxidase conjugate (Av-GOx), which acts as co-reactant in the electrochemiluminescent reaction. The method exhibits a limit of detection (LOD) of 4.31 aM and a wide linear range from 14.4 aM to 1.00 µM, and its applicability was confirmed by detecting SARS-CoV-2 in nasopharyngeal samples from COVID-19 patients without the need of any amplification process.

Emerging clinically tested detection methods for COVID-19.

At the time of writing, there were 486 761 597 global cases of COVID-19 with 6 142 735 confirmed deaths (World Health Organization, 4 April 2022). According to the scarcity of information about estimation of cases with mild or no symptoms, it is suggested that they could represent 25-80% of all infections. The majority of these cases remain untested, although they are infective. The molecular diagnosis of COVID-19 is based mainly on quantitative reverse transcription PCR. However, this approach faces several challenges related to the shortage of resources and people who are adequately trained to run the tests. Alternative testing methods, targeting effectively several viral compounds at different stages of the infection, have quickly emerged. However, universal systems that are specific, sensitive, affordable, easy, portable and scalable are still warranted. In this review, a comprehensive compilation of the methods available is provided.

Multifunctional magnetic nanoparticles elicit anti-tumor immunity in a mouse melanoma model.

Immunotherapy has emerged as a promising strategy to eradicate cancer cells. Particularly, the development of cancer vaccines to induce a potent and sustained antigen-specific T cell response has become a center of attention. Herein, we describe a novel immunotherapy based on magnetic nanoparticles (MNP) covalently modified with the OVA254-267 antigen and a CpG oligonucleotide via disulfide bonds. The MNP-CpG-COVA significantly enhances dendritic cell activation and CD8+ T cell antitumoral response against B16-OVA melanoma cells in vitro. Notably, the immune response induced by the covalently modified MNP is more potent and sustained over time than that triggered by the free components, highlighting the advantage of nanoformulations in immunotherapies. What is more, the nanoparticles are stable in the blood after in vivo administration and induce potent levels of systemic tumor-specific effector CD8 + T cells. Overall, our findings highlight the potential of covalently functionalized MNP to induce robust immune responses against mouse melanoma.

Nanomedical research and development in Spain: improving the treatment of diseases from the nanoscale.

The new and unique possibilities that nanomaterials offer have greatly impacted biomedicine, from the treatment and diagnosis of diseases, to the specific and optimized delivery of therapeutic agents. Technological advances in the synthesis, characterization, standardization, and therapeutic performance of nanoparticles have enabled the approval of several nanomedicines and novel applications. Discoveries continue to rise exponentially in all disease areas, from cancer to neurodegenerative diseases. In Spain, there is a substantial net of researchers involved in the development of nanodiagnostics and nanomedicines. In this review, we summarize the state of the art of nanotechnology, focusing on nanoparticles, for the treatment of diseases in Spain (2017-2022), and give a perspective on the future trends and direction that nanomedicine research is taking.