Magneto-mechanical destruction of cancer-associated fibroblasts using ultra-small iron oxide nanoparticles and low frequency rotating magnetic fields.

The destruction of cells using the mechanical activation of magnetic nanoparticles with low-frequency magnetic fields constitutes a recent and interesting approach in cancer therapy. Here, we showed that superparamagnetic iron oxide nanoparticles as small as 6 nm were able to induce the death of pancreatic cancer-associated fibroblasts, chosen as a model. An exhaustive screening of the amplitude, frequency, and type (alternating vs. rotating) of magnetic field demonstrated that the best efficacy was obtained for a rotating low-amplitude low-frequency magnetic field (1 Hz and 40 mT), reaching a 34% ratio in cell death induction; interestingly, the cell death was not maximized for the largest amplitudes of the magnetic field. State-of-the-art kinetic Monte-Carlo simulations able to calculate the torque undergone by assemblies of magnetic nanoparticles explained these features and were in agreement with cell death experiments. Simulations showed that the force generated by the nanoparticles once internalized inside the lysosome was around 3 pN, which is in principle not large enough to induce direct membrane disruption. Other biological mechanisms were explored to explain cell death: the mechanical activation of magnetic nanoparticles induced lysosome membrane permeabilization and the release of the lysosome content and cell death was mediated through a lysosomal pathway depending on cathepsin-B activity. Finally, we showed that repeated rotating magnetic field exposure halted drastically the cell proliferation. This study established a proof-of-concept that ultra-small nanoparticles can disrupt the tumor microenvironment through mechanical forces generated by mechanical activation of magnetic nanoparticles upon low-frequency rotating magnetic field exposure, opening new opportunities for cancer therapy.

A Preclinical Validation of Iron Oxide Nanoparticles for Treatment of Perianal Fistulizing Crohn's Disease.

Fistulizing anoperineal lesions are severe complications of Crohn's disease (CD) that affect quality of life with a long-term risk of anal sphincter destruction, incontinence, permanent stoma, and anal cancer. Despite several surgical procedures, they relapse in about two-thirds of patients, mandating innovative treatments. Ultrasmall particles of iron oxide (USPIO) have been described to achieve in vivo rapid healing of deep wounds in the skin and liver of rats thanks to their nanobridging capability that could be adapted to fistula treatment. Our main purpose was to highlight preclinical data with USPIO for the treatment of perianal fistulizing CD. Twenty male Sprague Dawley rats with severe 2,4,6-trinitrobenzenesulfonic acid solution (TNBS)-induced proctitis were operated to generate two perianal fistulas per rat. At day 35, two inflammatory fistulas were obtained per rat and perineal magnetic resonance imaging (MRI) was performed. After a baseline MRI, a fistula tract was randomly drawn and topically treated either with saline or with USPIO for 1 min (n = 17 for each). The rats underwent a perineal MRI on postoperative days (POD) 1, 4, and 7 and were sacrificed for pathological examination. The primary outcome was the filling or closure of the fistula tract, including the external or internal openings. USPIO treatment allowed the closure and/or filling of all the treated fistulas from its application until POD 7 in comparison with the control fistulas (23%). The treatment with USPIO was safe, permanently closed the fistula along its entire length, including internal and external orifices, and paved new avenues for the treatment of perianal fistulizing Crohn's disease.

Iron Oxide Nanoparticles Functionalized with Fucoidan: a Potential Theranostic Nanotool for Hepatocellular Carcinoma.

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 µM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.

Assessment of the Morphological, Optical, and Photoluminescence Properties of HfO2 Nanoparticles Synthesized by a Sol-Gel Method Assisted by Microwave Irradiation.

In this work, we optimized the synthesis of HfO2 nanoparticles (NPs) with a nonaqueous sol-gel method assisted by microwave heating, with a direct surfactant-free extraction and stabilization in water. To tune the structural, morphological, and photophysical properties, we explored the influence of reaction time, heating temperature, and type and concentration of a salt precursor. The controlled size, shape, crystallinity associated with high stability, a good yield of production, and stabilization in water without any surfactant modification of these HfO2 NPs open possibilities for future optoelectronic and biomedical applications. The investigation of their optical properties, revealed a high absorption in the UV range and the presence of a large band gap, originating in transparency at visible wavelengths. Under UV excitation, photoluminescence (PL) shows three emission bands centered at 305, 381, and 522 nm and are assigned to the vibronic transition of an excited OH•* radical or to a self-trapped exciton, to threefold oxygen vacancies VO3 with recombination to the valence band, and to defect level, respectively. The presence of oxygen vacancies associated with PL properties is particularly attractive for optoelectronic, photocatalysis, scintillator, and UV photosensor applications. Finally, by changing the nature of the hafnium precursor salt, using hafnium ethoxide or hafnium acetylacetonate, low-crystallized and aggregated NPs were obtained, which requires further investigation.

Real-Time Observation and Analysis of Magnetomechanical Actuation of Magnetic Nanoparticles in Cells.

The origin of cell death in the magnetomechanical actuation of cells induced by magnetic nanoparticle motion under low-frequency magnetic fields is still elusive. Here, a miniaturized electromagnet fitted under a confocal microscope is used to observe in real time cells specifically targeted by superparamagnetic nanoparticles and exposed to a low-frequency rotating magnetic field. Our analysis reveals that the lysosome membrane is permeabilized in only a few minutes after the start of magnetic field application, concomitant with lysosome movements toward the nucleus. Those events are associated with disorganization of the tubulin microtubule network and a change in cell morphology. This miniaturized electromagnet will allow a deeper insight into the physical, molecular, and biological process occurring during the magnetomechanical actuation of magnetic nanoparticles.

Gold, Silver, and Iron Oxide Nanoparticle Incorporation into Silk Hydrogels for Biomedical Applications: Elaboration, Structure, and Properties.

Silk fibroin (SF) is a versatile material with biodegradable and biocompatible properties, which make it fit for broad biomedical applications. In this context, the incorporation of nanosized objects into SF allows the development of a variety of bionanocomposites with tailored properties and functions. Herein, we report a thorough investigation on the design, characterization, and biological evaluation of SF hydrogels incorporating gold, silver, or iron oxide nanoparticles. The latter are synthesized in aqueous media using a biocompatible ligand allowing their utilization in various biomedical applications. This ligand seems to play a pivotal role in nanoparticle dispersion within the hydrogel. Results show that the incorporation of nanoparticles does not greatly influence the mechanism of SF gelation and has a minor impact on the mechanical properties of the so-obtained bionanocomposites. By contrast, significant changes are observed in the swelling behavior of these materials, depending on the nanoparticle used. Interestingly, the main characteristics of these bionanocomposites, related to their potential use for biomedical purposes, show the successful input of nanoparticles, including antibacterial properties for gold and silver nanoparticles and magnetic properties for iron oxide ones.

Covalent Organic Framework Embedded with Magnetic Nanoparticles for MRI and Chemo-Thermotherapy.

Nanoscale imine-linked covalent organic frameworks (nCOFs) were first loaded with the anticancer drug Doxorubicin (Dox), coated with magnetic iron oxide nanoparticles (γ-Fe2O3 NPs), and stabilized with a shell of poly(l-lysine) cationic polymer (PLL) for simultaneous synergistic thermo-chemotherapy treatment and MRI imaging. The pH responsivity of the resulting nanoagents (γ-SD/PLL) allowed the release of the drug selectively within the acidic microenvironment of late endosomes and lysosomes of cancer cells (pH 5.4) and not in physiological conditions (pH 7.4). γ-SD/PLL could efficiently generate high heat (48 °C) upon exposure to an alternating magnetic field due to the nCOF porous structure that facilitates the heat conduction, making γ-SD/PLL excellent heat mediators in an aqueous solution. The drug-loaded magnetic nCOF composites were cytotoxic due to the synergistic toxicity of Dox and the effects of hyperthermia in vitro on glioblastoma U251-MG cells and in vivo on zebrafish embryos, but they were not significantly toxic to noncancerous cells (HEK293). To the best of our knowledge, this is the first report of multimodal MRI probe and chemo-thermotherapeutic magnetic nCOF composites.

USPIO-PEG nanoparticles functionalized with a highly specific collagen-binding peptide: a step towards MRI diagnosis of fibrosis.

Fibrosis is characterized by a pathologic deposition of collagen I, leading to impaired function of organs. Tissue biopsy is the gold standard method for the diagnosis of fibrosis but this is an invasive procedure, subject to sampling errors. Several non-invasive techniques such as magnetic resonance imaging (MRI) using non-specific probes have been developed but they are not fully satisfying as they allow diagnosis at a late stage. In this study, collagelin, a collagen-binding peptide has been covalently linked using click chemistry to pegylated Ultra Small Super Paramagnetic Iron Oxide Nanoparticles (USPIO-PO-PEG-collagelin NPs) with the aim of diagnosing fibrosis at an early stage by MRI. USPIO-PO-PEG-collagelin NPs showed a high affinity for collagen I, two times higher than that of free collagelin whereas not peptide labeled USPIO NPs (USPIO-PO-PEG-yne) did not present any affinity. NPs were not toxic for macrophages and fibroblasts. Diffusion through collagen hydrogels concentrated at 3 and 10 mg mL-1 revealed a large accumulation of USPIO-PO-PEG-collagelin NPs within the collagen network after 72 hours, ca. 3 times larger than that of unlabeled USPIO, thereby evidencing the specific targeting of collagen I. Moreover, the quantity of USPIO-PO-PEG-collagelin NPs accumulated within hydrogels was proportional to the collagen concentration. Subsequently, the NPs diffusion through collagen hydrogels was monitored by MRI. The MRI T2 time relaxation decreased much more significantly with depth for USPIO-PO-PEG-collagelin NPs compared to unlabeled ones. Taken together, these results show that USPIO-PEG-collagelin NPs are promising as effective MRI nanotracers for molecular imaging of fibrosis at an early stage.

Endosomal Confinement of Gold Nanospheres, Nanorods, and Nanoraspberries Governs Their Photothermal Identity and Is Beneficial for Cancer Cell Therapy.

Gold nanoparticles can act as photothermal agents to generate local tumor heating and subsequent depletion upon laser exposure. Herein, photothermal heating of four gold nanoparticles and the resulting induced cancer cell death are systematically assessed, within extra- or intracellular localizations. Two state-of-the-art gold nanorods are compared with small nanospheres (single-core) and nanoraspberries (multicore). Heat generation is measured in water dispersion and in cancer cells, using lasers at wavelengths of 680, 808, and 1064 nm, covering the entire range used in photothermal therapy, defined as near infrared first (NIR-I) and second (NIR-II) windows, with NIR-II offering more tissue penetration. When dispersed in water, gold nanospheres provide no significant heating, gold nanorods are efficient in NIR-I, and only gold nanoraspberries are still heating in NIR-II. However, in cells, due to endosomal confinement, all nanoparticles present an absorption red-shift translating visible and NIR-I absorbing nanoparticles into effective NIR-I and NIR-II nanoheaters, respectively. The gold nanorods then become competitive with the multicore nanoparticles (nanoraspberries) in NIR-II. Similarly, once in cells, gold nanospheres can be envisaged for NIR-I heating. Remarkably, nanoraspberries are efficient nanoheaters, whatever the laser applied, and the extra- versus intra-cellular localization demonstrates treatment versatility.

Synthesis and activation of an iron oxide immobilized drug-mimicking reporter under conventional and pulsed X-ray irradiation conditions.

An efficient nano-sized delivery system is presented here allowing the immobilized, picolinium-tethered organic ligand to be released by X-ray irradiation. A marked difference was observed in the fragmentation efficiency by using conventional Cs-137 vs. pulsed sources.

Impact of magnetic nanoparticle surface coating on their long-term intracellular biodegradation in stem cells.

Magnetic nanoparticles (MNPs) internalized within stem cells have paved the way for remote magnetic cell manipulation and imaging in regenerative medicine. A full understanding of their interactions with stem cells and of their fate in the intracellular environment is then required, in particular with respect to their surface coatings. Here, we investigated the biological interactions of MNPs composed of an identical magnetic core but coated with different molecules: phosphonoacetic acid, polyethylene glycol phosphonic carboxylic acid, caffeic acid, citric acid, and polyacrylic acid. These coatings vary in the nature of the chelating function, the number of binding sites, and the presence or absence of a polymer. The nanoparticle magnetism was systematically used as an indicator of their internalization within human stem cells and of their structural long-term biodegradation in a 3D stem cell spheroid model. Overall, we evidence that the coating impacts the aggregation status of the nanoparticles and subsequently their uptake within stem cells, but it has little effect on their intracellular degradation. Only a high number of chelating functions (polyacrylic acid) had a significant protective effect. Interestingly, when the nanoparticles aggregated prior to cellular internalization, less degradation was also observed. Finally, for all coatings, a robust dose-dependent intracellular degradation rate was demonstrated, with higher doses of internalized nanoparticles leading to a lower degradation extent.

Raspberry-like small multicore gold nanostructures for efficient photothermal conversion in the first and second near-infrared windows.

Gold nanoraspberries were synthesized by a seed-mediated synthesis with polyethylene glycol-functionalized bisphosphonates. The original structure shifted the optical absorption to infrared, revealing very efficient photothermal properties within the 2nd biological transparency window and leading to cancer cell necrosis at moderate intracellular doses and low (safe) laser power.

Bimodal Fucoidan-Coated Zinc Oxide/Iron Oxide-Based Nanoparticles for the Imaging of Atherothrombosis.

A polyol method was used to obtain ultrasmall ZnO nanoparticles (NPs) doped with iron ions and coated with a low molecular weight fucoidan in order to perform in vivo MR and ex vivo fluorescence imaging of athrothrombosis. During the synthesis, the early elimination of water by azeotropic distillation with toluene allowed us to produce NPs which size, determined by XRD and TEM, decreased from 7 nm to 4 nm with the increase of iron/zinc ratios from 0.05 to 0.50 respectively. For the highest iron content (NP-0.50) NPs were evidenced as a mixture of nanocrystals made of wurtzite and cubic phase with a molar ratio of 2.57:1, although it was not possible to distinguish one from the other by TEM. NP-0.50 were superparamagnetic and exhibited a large emission spectrum at 470 nm when excited at 370 nm. After surface functionalization of NP-0.50 with fucoidan (fuco-0.50), the hydrodynamic size in the physiological medium was 162.0 ± 0.4 nm, with a corresponding negative zeta potential of -48.7 ± 0.4 mV, respectively. The coating was evidenced by FT-IR spectra and thermogravimetric analysis. Aqueous suspensions of fuco-0.50 revealed high transverse proton relaxivities (T2) with an r2 value of 173.5 mM-1 s-1 (300 K, 7.0 T) and remained stable for more than 3 months in water or in phosphate buffer saline without evolution of the hydrodynamic size and size distribution. No cytotoxic effect was observed on human endothelial cells up to 48 h with these NPs at a dose of 0.1 mg/mL. After injection into a rat model of atherothrombosis, MR imaging allowed the localization of diseased areas and the subsequent fluorescence imaging of thrombus on tissue slices.

Hybrid Au@alendronate nanoparticles as dual chemo-photothermal agent for combined cancer treatment.

A gold therapeutic nanoplatform with the same molecule used as reductant, coating and therapeutic agent has been developed in a one-pot, one-phase process using alendronate, a drug from the bisphosphonate family known for its antitumor effects. In addition, the core made of gold nanoparticles (NPs) brings thermal functionalities under irradiation within the first biological window (650-900 nm). The Au@alendronate nanoplatform thus provided a combined antitumor activity through drug delivery and photothermal therapy. Au@alendronate NPs inhibited in vitro the proliferation of prostate cancer cells (PC3) in a dose-dependent manner, with an IC50 value of 100 µM. Under NIR irradiation a temperature increase was observed leading to a reduction of the IC50 value to 1 µM, with total tumor cell death at 100 µM.

Tolerogenic Iron Oxide Nanoparticles in Type 1 Diabetes: Biodistribution and Pharmacokinetics Studies in Nonobese Diabetic Mice.

Nanoparticle (NP) administration is among the most attractive approaches to exploit the synergy of different copackaged molecules for the same target. In this work, iron oxide NPs are surface-engineered for the copackaging of the autoantigen proinsulin, a major target of adaptive immunity in type 1 diabetes (T1D), and 2-(1'H-indole-3'-carbonyl)-thiazole-4-carboxylic acid methylester (ITE), a small drug conditioning a tolerogenic environment. Magnetic resonance imaging (MRI) combined with magnetic quantification are used to investigate NP biokinetics in nonobese diabetic (NOD) mice and control mice in different organs. Different NP biodistribution, with in particular enhanced kidney elimination and a stronger accumulation in the pancreas for prediabetic NOD mice, is observed. This is related to preferential NP accumulation in the pancreatic inflammatory zone and to enhancement of renal elimination by diabetic nephropathy. For both mouse strains, an MRI T2 contrast enhancement at 72 h in the liver, pancreas, and kidneys, and indicating recirculating NPs, is also found. This unexpected result is confirmed by magnetic quantification at different time points as well as by histological evaluation. Besides, such NPs are potential MRI contrast agents for early diagnosis of T1D.

Nanohybrids with Magnetic and Persistent Luminescence Properties for Cell Labeling, Tracking, In Vivo Real-Time Imaging, and Magnetic Vectorization.

Once injected into a living organism, cells diffuse or migrate around the initial injection point and become impossible to be visualized and tracked in vivo. The present work concerns the development of a new technique for therapeutic cell labeling and subsequent in vivo visualization and magnetic retention. It is hypothesized and subsequently demonstrated that nanohybrids made of persistent luminescence nanoparticles and ultrasmall superparamagnetic iron oxide nanoparticles incorporated into a silica matrix can be used as an effective nanoplatform to label therapeutic cells in a nontoxic way in order to dynamically track them in real-time in vitro and in living mice. As a proof-of-concept, it is shown that once injected, these labeled cells can be visualized and attracted in vivo using a magnet. This first step suggests that these nanohybrids represent efficient multifunctional nanoprobes for further imaging guided cell therapies development.

Palladium-Loaded Cucurbit[7]uril-Modified Iron Oxide Nanoparticles for C-C Cross-Coupling Reactions.

Cucurbit[7]uril modified iron oxide nanoparticles (CB[7]NPs) were loaded with palladium to form nano-catalysts (Pd@CB[7]NPs) that, with microwave heating, catalysed Suzuki-Miyaura, Sonogashira, and Mizoroki-Heck cross-coupling reactions. Reactions were run in environmentally benign 1:1 ethanol/water solvent under convenient aerobic conditions. In a preliminary screening, conversions and yields were uniformly high with turn over frequencies (TOF) ranging from 64 to 7360 h-1 . The nano-catalysts could be recovered with a magnet and reused several times (6 times for Suzuki-Miayura reaction) without loss of activity.

Synthetic melanin bound to subunit vaccine antigens significantly enhances CD8+ T-cell responses.

Cytotoxic T-lymphocytes (CTLs) play a key role in immunity against cancer; however, the induction of CTL responses with currently available vaccines remains difficult. Because several reports have suggested that pigmentation and immunity might be functionally linked, we investigated whether melanin can act as an adjuvant in vaccines. Short synthetic peptides (8-35 amino acids long) containing T-cell epitopes were mixed with a solution of L-Dopa, a precursor of melanin. The mixture was then oxidized to generate nanoparticles of melanin-bound peptides. Immunization with melanin-bound peptides efficiently triggered CTL responses in mice, even against self-antigens and at a very low dose of peptides (microgram range). Immunization against a tumor antigen inhibited the growth of established tumors in mice, an effect that was abrogated by the depletion of CD8+ lymphocytes. These results demonstrate the efficacy of melanin as a vaccine adjuvant.

Synthesis of metastable hard-magnetic ε-Fe2O3 nanoparticles from silica-coated akaganeite nanorods.

We present a simple preparation route to obtain a nanoscale metastable hard-magnetic ε-Fe2O3 phase, using silica coated ß-FeOOH nanorods as a precursor and an annealing process. The synthesized ε-Fe2O3 nanoparticles exhibit large coercivity (HC ∼ 20 kOe at 300 K and HC ∼ 1.6 kOe at 400 K), confirming their high potential for practical applications.

Effects of coating spherical iron oxide nanoparticles.

We investigate the effect of several coatings applied in biomedical applications to iron oxide nanoparticles on the size, structure and composition of the particles. The four structural techniques employed - TEM, DLS, VSM, SAXS and EXAFS - show no significant effects of the coatings on the spherical shape of the bare nanoparticles, the average sizes or the local order around the Fe atoms. The NPs coated with hydroxylmethylene bisphosphonate or catechol have a lower proportion of magnetite than the bare and citrated ones, raising the question whether the former are responsible for increasing the valence state of the oxide on the NP surfaces and lowering the overall proportion of magnetite in the particles. VSM measurements show that these two coatings lead to a slightly higher saturation magnetization than the citrate. This article is part of a Special Issue entitled "Science for Life" Guest Editor: Dr. Austen Angell, Dr. Salvatore Magazù and Dr. Federica Migliardo.