Membrane-localized magnetic hyperthermia promotes intracellular delivery of cell-impermeant probes.

In this work, we report the disruptive use of membrane-localized magnetic hyperthermia to promote the internalization of cell-impermeant probes. Under an alternating magnetic field, magnetic nanoparticles (MNPs) immobilized on the cell membrane via bioorthogonal click chemistry act as nanoheaters and lead to the thermal disruption of the plasma membrane, which can be used for internalization of different types of molecules, such as small fluorescent probes and nucleic acids. Noteworthily, no cell death, oxidative stress and alterations of the cell cycle are detected after the thermal stimulus, although cells are able to sense and respond to the thermal stimulus through the expression of different types of heat shock proteins (HSPs). Finally, we demonstrate the utility of this approach for the transfection of cells with a small interference RNA (siRNA), revealing a similar efficacy to a standard transfection method based on the use of cationic lipid-based reagents (such as Lipofectamine), but with lower cell toxicity. These results open the possibility of developing new procedures for "opening and closing" cellular membranes with minimal disturbance of cellular integrity. This on-demand modification of cell membrane permeability could allow the direct intracellular delivery of biologically relevant (bio)molecules, drugs and nanomaterials, thus overcoming traditional endocytosis pathways and avoiding endosomal entrapment.

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.

Iron Speciation in Animal Tissues Using AC Magnetic Susceptibility Measurements: Quantification of Magnetic Nanoparticles, Ferritin, and Other Iron-Containing Species.

The simultaneous detection and quantification of several iron-containing species in biological matrices is a challenging issue. Especially in the frame of studies using magnetic nanoparticles for biomedical applications, no gold-standard technique has been described yet and combinations of different techniques are generally used. In this work, AC magnetic susceptibility measurements are used to analyze different organs from an animal model that received a single intratumor administration of magnetic nanoparticles. The protocol used for the quantification of iron associated with the magnetic nanoparticles is carefully described, including the description of the preparation of several calibration standard samples of nanoparticle suspensions with different degrees of dipolar interactions. The details for the quantitative analysis of other endogenous iron-containing species such as ferritin or hemoglobin are also described. Among the advantages of this technique are that tissue sample preparation is minimal and that large amounts of tissue can be characterized each time (up to hundreds of milligrams). In addition, the very high specificity of the magnetic measurements allows for tracking of the nanoparticle transformations. Furthermore, the high sensitivity of the instrumentation results in very low limits of detection for some of the iron-containing species. Therefore, the presented technique is an extremely valuable tool to track iron oxide magnetic nanoparticles in samples of biological origin.

Unveiling the role of surface, size, shape and defects of iron oxide nanoparticles for theranostic applications.

Iron oxide nanoparticles (IONPs) are well-known contrast agents for MRI for a wide range of sizes and shapes. Their use as theranostic agents requires a better understanding of their magnetic hyperthermia properties and also the design of a biocompatible coating ensuring their stealth and a good biodistribution to allow targeting of specific diseases. Here, biocompatible IONPs of two different shapes (spherical and octopod) were designed and tested in vitro and in vivo to evaluate their abilities as high-end theranostic agents. IONPs featured a dendron coating that was shown to provide anti-fouling properties and a small hydrodynamic size favoring an in vivo circulation of the dendronized IONPs. While dendronized nanospheres of about 22 nm size revealed good combined theranostic properties (r2 = 303 mM s-1, SAR = 395 W gFe-1), octopods with a mean size of 18 nm displayed unprecedented characteristics to simultaneously act as MRI contrast agents and magnetic hyperthermia agents (r2 = 405 mM s-1, SAR = 950 W gFe-1). The extensive structural and magnetic characterization of the two dendronized IONPs reveals clear shape, surface and defect effects explaining their high performance. The octopods seem to induce unusual surface effects evidenced by different characterization techniques while the nanospheres show high internal defects favoring Néel relaxation for magnetic hyperthermia. The study of octopods with different sizes showed that Néel relaxation dominates at sizes below 20 nm while the Brownian one occurs at higher sizes. In vitro experiments demonstrated that the magnetic heating capability of octopods occurs especially at low frequencies. The coupling of a small amount of glucose on dendronized octopods succeeded in internalizing them and showing an effect of MH on tumor growth. All measurements evidenced a particular signature of octopods, which is attributed to higher anisotropy, surface effects and/or magnetic field inhomogeneity induced by tips. This approach aiming at an analysis of the structure-property relationships is important to design efficient theranostic nanoparticles.

Nano-Second Laser Interference Photoembossed Microstructures for Enhanced Cell Alignment.

Photoembossing is a powerful photolithographic technique to prepare surface relief structures relying on polymerization-induced diffusion in a solventless development step. Conveniently, surface patterns are formed by two or more interfering laser beams without the need for a lithographic mask. The use of nanosecond pulsed light-based interference lithography strengthens the pattern resolution through the absence of vibrational line pattern distortions. Typically, a conventional photoembossing protocol consists of an exposure step at room temperature that is followed by a thermal development step at high temperature. In this work, we explore the possibility to perform the pulsed holographic exposure directly at the development temperature. The surface relief structures generated using this modified photoembossing protocol are compared with those generated using the conventional one. Importantly, the enhancement of surface relief height has been observed by exposing the samples directly at the development temperature, reaching approximately double relief heights when compared to samples obtained using the conventional protocol. Advantageously, the light dose needed to reach the optimum height and the amount of photoinitiator can be substantially reduced in this modified protocol, demonstrating it to be a more efficient process for surface relief generation in photopolymers. Kidney epithelial cell alignment studies on substrates with relief-height optimized structures generated using the two described protocols demonstrate improved cell alignment in samples generated with exposure directly at the development temperature, highlighting the relevance of the height enhancement reached by this method. Although cell alignment is well-known to be enhanced by increasing the relief height of the polymeric grating, our work demonstrates nano-second laser interference photoembossing as a powerful tool to easily prepare polymeric gratings with tunable topography in the range of interest for fundamental cell alignment studies.

Critical Parameters to Improve Pancreatic Cancer Treatment Using Magnetic Hyperthermia: Field Conditions, Immune Response, and Particle Biodistribution.

Magnetic hyperthermia (MH) was used to treat a murine model of pancreatic cancer. This type of cancer is generally characterized by the presence of dense stroma that acts as a barrier for chemotherapeutic treatments. Several alternating magnetic field (AMF) conditions were evaluated using three-dimensional (3D) cell culture models loaded with magnetic nanoparticles (MNPs) to determine which conditions were producing a strong effect on the cell viability. Once the optimal AMF conditions were selected, in vivo experiments were carried out using similar frequency and field amplitude parameters. A marker of the immune response activation, calreticulin (CALR), was evaluated in cells from a xenograft tumor model after the MH treatment. Moreover, the distribution of nanoparticles within the tumor tissue was assessed by histological analysis of tumor sections, observing that the exposure to the alternating magnetic field resulted in the migration of particles toward the inner parts of the tumor. Finally, a relationship between an inadequate body biodistribution of the particles after their intratumoral injection and a significant decrease in the effectiveness of the MH treatment was found. Animals in which most of the particles remained in the tumor area after injection showed higher reductions in the tumor volume growth in comparison with those animals in which part of the particles were found also in the liver and spleen. Therefore, our results point out several factors that should be considered to improve the treatment effectiveness of pancreatic cancer by magnetic hyperthermia.

The Intracellular Number of Magnetic Nanoparticles Modulates the Apoptotic Death Pathway after Magnetic Hyperthermia Treatment.

Magnetic hyperthermia is a cancer treatment based on the exposure of magnetic nanoparticles to an alternating magnetic field in order to generate local heat. In this work, 3D cell culture models were prepared to observe the effect that a different number of internalized particles had on the mechanisms of cell death triggered upon the magnetic hyperthermia treatment. Macrophages were selected by their high capacity to uptake nanoparticles. Intracellular nanoparticle concentrations up to 7.5 pg Fe/cell were measured both by elemental analysis and magnetic characterization techniques. Cell viability after the magnetic hyperthermia treatment was decreased to <25% for intracellular iron contents above 1 pg per cell. Theoretical calculations of the intracellular thermal effects that occurred during the alternating magnetic field application indicated a very low increase in the global cell temperature. Different apoptotic routes were triggered depending on the number of internalized particles. At low intracellular magnetic nanoparticle amounts (below 1 pg Fe/cell), the intrinsic route was the main mechanism to induce apoptosis, as observed by the high Bax/Bcl-2 mRNA ratio and low caspase-8 activity. In contrast, at higher concentrations of internalized magnetic nanoparticles (1-7.5 pg Fe/cell), the extrinsic route was observed through the increased activity of caspase-8. Nevertheless, both mechanisms may coexist at intermediate iron concentrations. Knowledge on the different mechanisms of cell death triggered after the magnetic hyperthermia treatment is fundamental to understand the biological events activated by this procedure and their role in its effectiveness.

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.

Dual Role of Magnetic Nanoparticles as Intracellular Hotspots and Extracellular Matrix Disruptors Triggered by Magnetic Hyperthermia in 3D Cell Culture Models.

Magnetic hyperthermia is a promising therapy for the localized treatment of cancer based on the exposure of magnetic nanoparticles to an external alternating magnetic field. In order to evaluate some of the mechanisms involved in the cellular damage caused by this treatment, two different 3D cell culture models were prepared using collagen, which is the most abundant protein of the extracellular matrix. The same amount of nanoparticles was added to cells either before or after their incorporation into the 3D structure. Therefore, in one model, particles were located only inside cells (In model), while the other one had particles both inside and outside cells (In&Out model). In the In&Out model, the hyperthermia treatment facilitated the migration of the particles from the outer areas of the 3D structure to the inner parts, achieving a faster homogeneous distribution throughout the whole structure and allowing the particles to gain access to the inner cells. The cell death mechanism activated by the magnetic hyperthermia treatment was different in both models. Necrosis was observed in the In model and apoptosis in the In&Out model 24 h after the hyperthermia application. This was clearly correlated with the amount of nanoparticles located inside the cells. Thus, the combination of both 3D models allowed us to demonstrate two different roles of the magnetic particles during the hyperthermia treatment: (i) The modulation of the cell death mechanism depending on the amount of intracellular particles and (ii) the disruption of the collagen matrix caused by the extracellular nanoparticles.

Beyond Traditional Hyperthermia: In Vivo Cancer Treatment with Magnetic-Responsive Mesoporous Silica Nanocarriers.

In this study, we present an innovation in the tumor treatment in vivo mediated by magnetic mesoporous silica nanoparticles. This device was built with iron oxide magnetic nanoparticles embedded in a mesoporous silica matrix and coated with an engineered thermoresponsive polymer. The magnetic nanoparticles act as internal heating sources under an alternating magnetic field (AMF) that increase the temperature of the surroundings, provoking the polymer transition and consequently the release of a drug trapped inside the silica pores. By a synergic effect between the intracellular hyperthermia and chemotherapy triggered by AMF application, significant tumor growth inhibition was achieved in 48 h after treatment. Furthermore, the small magnetic loading used in the experiments indicates that the treatment is carried out without a global temperature rise of the tissue, which avoids the problem of the necessity to employ large amounts of magnetic cores, as is common in current magnetic hyperthermia.

Formation Mechanism of Maghemite Nanoflowers Synthesized by a Polyol-Mediated Process.

Magnetic nanoparticles are being developed as structural and functional materials for use in diverse areas, including biomedical applications. Here, we report the synthesis of maghemite (γ-Fe2O3) nanoparticles with distinct morphologies: single-core and multicore, including hollow spheres and nanoflowers, prepared by the polyol process. We have used sodium acetate to control the nucleation and assembly process to obtain the different particle morphologies. Moreover, from samples obtained at different time steps during the synthesis, we have elucidated the formation mechanism of the nanoflowers: the initial phases of the reaction present a lepidocrocite (γ-FeOOH) structure, which suffers a fast dehydroxylation, transforming to an intermediate "undescribed" phase, possibly a partly dehydroxylated lepidocrocite, which after some incubation time evolves to maghemite nanoflowers. Once the nanoflowers have been formed, a crystallization process takes place, where the γ-Fe2O3 crystallites within the nanoflowers grow in size (from ∼11 to 23 nm), but the particle size of the flower remains essentially unchanged (∼60 nm). Samples with different morphologies were coated with citric acid and their heating capacity in an alternating magnetic field was evaluated. We observe that nanoflowers with large cores (23 nm, controlled by annealing) densely packed (tuned by low NaAc concentration) offer 5 times enhanced heating capacity compared to that of the nanoflowers with smaller core sizes (15 nm), 4 times enhanced heating effect compared to that of the hollow spheres, and 1.5 times enhanced heating effect compared to that of single-core nanoparticles (36 nm) used in this work.

Cell death induced by AC magnetic fields and magnetic nanoparticles: current state and perspectives.

This review analyses the advances in the field of magnetically induced cell death using intracellular magnetic nanoparticles (MNPs). Emphasis has been given to in vitro research results, discussing the action of radiofrequency (RF) waves on biological systems as well as those results of thermally induced cell death in terms of MNP cell interactions. Our main goal has been to provide a unified depiction of many recent experiments and theoretical models relevant to the effect of applied electromagnetic fields on MNPs after cellular uptake and the cytotoxicity assessment of MNPs. We have addressed the effects of RF waves used for in vitro magnetic hyperthermia on eukaryotic cells regarding physical modifications of the cellular local environment and cell viability.

Prognostic significance and diagnostic value of protein S-100 and tyrosinase in patients with malignant melanoma.

OBJECTIVES: The utility of many molecules as tumor markers in melanoma has been investigated with different results. The aims of this study was to compare the value of tyrosinase mRNA by reverse transcription polymerase chain reaction (RT-PCR) in peripheral blood and of serum S-100 protein in patients with melanoma at different stages of disease. METHODS: We have studied 90 peripheral blood samples corresponding to 90 patients that had been diagnosed with melanoma. The clinical staging at the time of blood sampling was performed according to the American Join Committee on Cancer guidelines. S-100 protein in serum was measured by enzyme-linked immunosorbent assay (normal range: 0-0.150 microg) and the presence of tyrosinase mRNA was assessed by RT-PCR. RESULTS: Median progression-free survival was 281 days for tyrosinase positive patients and it has not been reached for tyrosinase negative patients (P = 0.03). Median progression free survival was 213 days for patients with elevated serum S-100 and it has not been reached for patients with normal level of serum S-100 (P < 0.001). Median overall survival (OS) was 396 days for tyrosinase positive patients and it has not been reached for negative patients (P = 0.0096). Median OS was 282 days for patients with elevated serum S-100 and it has not been reached for patients with normal level of serum S-100 (P < 0.001). In a multivariate analysis, both markers have significant prognostic value for time to progression and for survival (chi(2) test). CONCLUSIONS: RT-PCR for tyrosinase mRNA and S-100 are significant prognostic factors for progression-free survival and OS in melanoma. S-100 has higher sensitivity and specificity than tyrosinase.

A variant associated with nicotine dependence, lung cancer and peripheral arterial disease.

Smoking is a leading cause of preventable death, causing about 5 million premature deaths worldwide each year. Evidence for genetic influence on smoking behaviour and nicotine dependence (ND) has prompted a search for susceptibility genes. Furthermore, assessing the impact of sequence variants on smoking-related diseases is important to public health. Smoking is the major risk factor for lung cancer (LC) and is one of the main risk factors for peripheral arterial disease (PAD). Here we identify a common variant in the nicotinic acetylcholine receptor gene cluster on chromosome 15q24 with an effect on smoking quantity, ND and the risk of two smoking-related diseases in populations of European descent. The variant has an effect on the number of cigarettes smoked per day in our sample of smokers. The same variant was associated with ND in a previous genome-wide association study that used low-quantity smokers as controls, and with a similar approach we observe a highly significant association with ND. A comparison of cases of LC and PAD with population controls each showed that the variant confers risk of LC and PAD. The findings provide a case study of a gene-environment interaction, highlighting the role of nicotine addiction in the pathology of other serious diseases.

Two variants on chromosome 17 confer prostate cancer risk, and the one in TCF2 protects against type 2 diabetes.

We performed a genome-wide association scan to search for sequence variants conferring risk of prostate cancer using 1,501 Icelandic men with prostate cancer and 11,290 controls. Follow-up studies involving three additional case-control groups replicated an association of two variants on chromosome 17 with the disease. These two variants, 33 Mb apart, fall within a region previously implicated by family-based linkage studies on prostate cancer. The risks conferred by these variants are moderate individually (allele odds ratio of about 1.20), but because they are common, their joint population attributable risk is substantial. One of the variants is in TCF2 (HNF1beta), a gene known to be mutated in individuals with maturity-onset diabetes of the young type 5. Results from eight case-control groups, including one West African and one Chinese, demonstrate that this variant confers protection against type 2 diabetes.

Results of a pilot trial of immunotherapy with dendritic cells pulsed with autologous tumor lysates in patients with advanced cancer.

AIMS AND BACKGROUND: The purpose of the study was to test the immunological and clinical effects of infusions of dendritic cells pulsed with autologous tumor lysate in patients with advanced cancer. PATIENTS AND METHODS: Peripheral blood mononuclear cells from 15 patients with metastatic cancer (melanoma in 10, lung cancer in 2, renal cell carcinoma in 1, sarcoma in 1, breast cancer in 1) were harvested by leukapheresis after mobilization with GM-CSF (5 microg/kg/day s.c. for 4 days). Mononuclear cells were separated and cultured in GM-CSF (1000 U/ml) and interleukin-4 (1000 U/ml) for 7 days. Phenotype was assessed by 2-color flow cytometry and immunocytochemistry. On day 6, dendritic cells were pulsed with 1 g of fresh autologous tumor lysate for 24 h and infused intravenously. Interleukin-2 (6 million IU), interferon a (4 million IU) and GM-CSF (400 microg) were injected s.c. daily for 10 days beginning on the day of dendritic cell infusion. Treatment was repeated every 21 days for 3 courses. RESULTS: The morphology, immunocytochemistry and phenotype of cultured cells was consistent with dendritic cells: intense positivity for HLA-DR and CD86, with negativity for markers of other lineages, including CD3, CD4, CD8 and CD14. More than 5 x 10(7) dendritic cells were injected in all patients. Nine patients developed >5 mm delayed type cutaneous hypersensitivity reactions to tumor lysate+/-GM-CSF after the first immunization (larger than GM-CSF in all cases). Median delayed type cutaneous hypersensitivity to lysate +/- GM-CSF was 3 cm after the third immunization. One melanoma patient with skin, liver, lung and bone metastases had a partial response lasting 8 months (followed by progression in the brain). Seven patients had stable disease for >3 months, and 7 had progression. CONCLUSIONS: Infusion of tumor lysate-pulsed dendritic cells induces a strong cell-mediated antitumor immune reaction in patients with advanced cancer and has some clinical activity.