Application of magnetically induced hyperthermia on the model protozoan Crithidia fasciculata as a potential therapy against parasitic infections

Magnetic hyperthermia is currently an EU-approved clinical therapy against tumor cells that uses magnetic nanoparticles under a time varying magnetic field (TVMF). The same basic principle seems promising against trypanosomatids causing Chagas disease and sleeping sickness, since therapeutic drugs available display severe side effects and drug-resistant strains. However, no applications of this strategy against protozoan-induced diseases have been reported so far. In the present study, Crithidia fasciculata, a widely used model for therapeutic strategies against pathogenic trypanosomatids, was targeted with Fe_{3}O_{4} magnetic nanoparticles (MNPs) in order to remotely provoke cell death using TVMFs. The MNPs with average sizes of d approx. 30 nm were synthesized using a precipitation of FeSO_{4}4 in basic medium. The MNPs were added to Crithidia fasciculata choanomastigotes in exponential phase and incubated overnight. The amount of uploaded MNPs per cell was determined by magnetic measurements. Cell viability using the MTT colorimetric assay and flow cytometry showed that the MNPs were incorporated by the cells with no noticeable cell-toxicity effects. When a TVMF (f = 249 kHz, H = 13 kA/m) was applied to MNP-bearing cells, massive cell death was induced via a non-apoptotic mechanism. No effects were observed by applying a TVMF on control (without loaded MNPs) cells. No macroscopic rise in temperature was observed in the extracellular medium during the experiments. Scanning Electron Microscopy showed morphological changes after TVMF experiments. These data indicate (as a proof of principle) that intracellular hyperthermia is a suitable technology to induce the specific death of protozoan parasites bearing MNPs. These findings expand the possibilities for new therapeutic strategies that combat parasitic infections.Comment: 9 pages, four supplementary video file

Design of stable magnetic hybrid nanoparticles of Si-entrapped HRP

Hybrid and composite nanoparticles represent an attractive material for enzyme integration due to possible synergic advantages of the structural builders in the properties of the nanobiocatalyst. In this study, we report the synthesis of a new stable hybrid nanobiocatalyst formed by biomimetic silica (Si) nanoparticles entrapping both Horseradish Peroxidase (HRP) (EC 1.11.1.7) and magnetic nanoparticles (MNPs). We have demonstrated that tailoring of the synthetic reagents and post immobilization treatments greatly impacted physical and biocatalytic properties such as an unprecedented ~280 times increase in the half-life time in thermal stability experiments. The optimized nanohybrid biocatalyst that showed superparamagnetic behaviour, was effective in the batch conversion of indole-3-acetic acid, a prodrug used in Direct Enzyme Prodrug Therapy (DEPT). Our system, that was not cytotoxic per se, showed enhanced cytotoxic activity in the presence of the prodrug towards HCT-116, a colorectal cancer cell line. The strategy developed proved to be effective in obtaining a stabilized nanobiocatalyst combining three different organic/inorganic materials with potential in DEPT and other biotechnological applications

Triggering antitumoural drug release and gene expression by magnetic hyperthermia

Magnetic nanoparticles (MNPs) are promising tools for a wide array of biomedical applications. One of their most outstanding properties is the ability to generate heat when exposed to alternating magnetic fields, usually exploited in magnetic hyperthermia therapy of cancer. In this contribution, we provide a critical review of the use of MNPs and magnetic hyperthermia as drug release and gene expression triggers for cancer therapy. Several strategies for the release of chemotherapeutic drugs from thermo-responsive matrices are discussed, providing representative examples of their application at different levels (from proof of concept to in vivo applications). The potential of magnetic hyperthermia to promote in situ expression of therapeutic genes using vectors that contain heat-responsive promoters is also reviewed in the context of cancer gene therapy

Remote activation of enzyme nanohybrids for cancer prodrug therapy controlled by magnetic heating

Herein, we have developed nanohybrids (nHs) to remotely activate a therapeutic enzyme for its use in Directed Enzyme Prodrug Therapy (DEPT). The coencapsulation of magnetic nanoparticles (MNPs) with horseradish peroxidase (HRP) using biomimetic silica as an entrapment matrix was optimized to obtain nanosized hybrids (∼150 nm) for remote activation of the therapeutic enzyme. HRP converts indole-3-acetic acid (3IAA) into peroxylated radicals, whereas MNPs respond to alternating magnetic fields (AMFs) becoming local hotspots. The AMF application triggered an increase in the bioconversion rate of HRP matching the activity displayed at the optimal temperature of the nHs (Topt = 50 °C) without altering the temperature of the reaction media. This showed that enzyme nanoactuation is possible with MNPs even if they are not covalently bound. After an extensive physicochemical/magnetic characterization, the spatial location of each component of the nH was deciphered, and an insulating role of the silica matrix was suggested as critical for introducing remote control over HRP. In vitro assays, using a human pancreatic cancer cell line (MIA PaCa-2), showed that only upon exposure to AMF and in the presence of the prodrug, the enzyme-loaded nHs triggered cell death. Moreover, in vivo experiments showed higher reductions in the tumor volume growth in those animals treated with nHs in the presence of 3IAA when exposed to AMF. Thus, this work demonstrates the feasibility of developing a spatiotemporally controlled DEPT strategy to overcome unwanted off-target effects

Dependence of intracellular magnetic nanoparticles amount in the apoptotic death pathway triggered by magnetic hyperthermia treatment

Trabajo presentado a la 4th Spanish Conference on Biomedical Applications of Nanomaterials, celebrada online del 2 al 4 de junio de 2021.Peer reviewe

Magnetic Nanoparticles for Cancer Therapy

Abstract: Today, technologies based on magnetic nanoparticles (MNPs) are routinely applied to biological systems with diagnostic or therapeutic purposes. The paradigmatic example is the magnetic resonance imaging (MRI), a technique that uses the magnetic moments of MNPs as a disturbance of the proton resonance to obtain images. Similarly, magnetic fluid hyperthermia (MFH) uses MNPs as heat generators to induce localized cell death. The physical basis of these techniques relies on the interaction with external magnetic fields, and therefore the magnetic moment of the particles has to be maximized for these applications. Targeted drug-delivery based on 'smart' nanoparticles is the next step towards more efficient oncologic therapies, by delivering a minimal dose of drug only to the vicinity of the target. Current improvements in this fields relay on a) particle functionalization with specific ligands for targeting cell membrane receptors and b) loading MNPs onto cells (e.g., dendritic cells, T-cells, macrophages) having an active role in tumor grow. Here we review the current state of research on applications of magnetic carriers for cancer therapy, discussing the advances and drawbacks of both passive and targeted delivery of MNPs. The most promising strategies for targeted delivery of MNPs are analyzed, evaluating the expected impact on clinical MRI and MFH protocols

Designing novel nano-immunoassays: antibody orientation versus sensitivity

International audienceThere is a growing interest in the use of magnetic nanoparticles (MNPs) for their application in quantitative and highly-sensitive biosensors. The use of them as labels of biological recognition events and their detection by means of some magnetic method constitutes a very promising strategy for quantitative high-sensitive lateral-flow assays. In the present article, we report the importance of nanoparticle functionalization for the improvement of sensitivity for a lateral flow immunoassay. More precisely, we have found that immobilization of IgG anti-hCG through its polysaccharide moieties on magnetic nanoparticles allows more successful recognition of the hCG hormone. Although we used the detection of hCG as a model in this work, the strategy of binding antibodies to MNPs through its sugar chains reported here is applicable to other antibodies. Its potential is huge as it will be very useful for the development of quantitative and high-sensitive lateral-flow assays for its use on human and veterinary, medicine, food and beverage manufacturing, pharmaceutical, medical biologics and personal care product production, environmental remediation, etc

Heterofunctional supports for the one-step purification, immobilization and stabilization of large multimeric enzymes: Amino-glyoxyl versus amino-epoxy supports

For the immobilization-stabilization of multimeric enzymes, we propose a novel heterofunctional support containing a very low concentration of ionized amino groups and a very high concentration of very poorly reactive glyoxyl (aldehyde) groups. A large tetrameric enzyme, β-galactosidase from Thermus sp., was purified and dramatically stabilized with this novel support. The enzyme was first immobilized by physical adsorption via selective multipoint anionic exchange involving the largest region of the enzyme containing all enzyme subunits. Then, an additional long incubation of the immobilized derivative under alkaline conditions was performed in order to promote an intense intramolecular multipoint covalent attachment between amino groups of the adsorbed enzyme and the very stable glyoxyl groups on the support. This novel β-galactosidase derivative is the first one in which the four subunits of this enzyme become attached to a pre-existing support. Additionally, the novel amino-glyoxyl supports were much more suitable than amino-epoxy supports for intramolecular multipoint covalent immobilization of the adsorbed enzyme onto the support. In fact, at pH 7.0, the new supports covalently immobilize the physically adsorbed protein 24-fold more rapidly than epoxy supports. Furthermore, derivatives prepared on amino-glyoxyl supports preserved 85% of catalytic activity and were 5-fold more stable than derivatives prepared on amino-epoxy supports and more than 1000-fold more stable than soluble enzyme. © 2010 Elsevier Ltd.Peer Reviewe