Comprehensive and systematic characterization of multi-functionalized cisplatin nano-conjugate: from the chemistry and proteomic biocompatibility to the animal model.

BACKGROUND: Nowadays, nanoparticles (NPs) have evolved as multifunctional systems combining different custom anchorages which opens a wide range of applications in biomedical research. Thus, their pharmacological involvements require more comprehensive analysis and novel nanodrugs should be characterized by both chemically and biological point of view. Within the wide variety of biocompatible nanosystems, iron oxide nanoparticles (IONPs) present mostly of the required features which make them suitable for multifunctional NPs with many biopharmaceutical applications. RESULTS: Cisplatin-IONPs and different functionalization stages have been broadly evaluated. The potential application of these nanodrugs in onco-therapies has been assessed by studying in vitro biocompatibility (interactions with environment) by proteomics characterization the determination of protein corona in different proximal fluids (human plasma, rabbit plasma and fetal bovine serum),. Moreover, protein labeling and LC-MS/MS analysis provided more than 4000 proteins de novo synthetized as consequence of the nanodrugs presence defending cell signaling in different tumor cell types (data available via ProteomeXchanges with identified PXD026615). Further in vivo studies have provided a more integrative view of the biopharmaceutical perspectives of IONPs. CONCLUSIONS: Pharmacological proteomic profile different behavior between species and different affinity of protein coating layers (soft and hard corona). Also, intracellular signaling exposed differences between tumor cell lines studied. First approaches in animal model reveal the potential of theses NPs as drug delivery vehicles and confirm cisplatin compounds as strengthened antitumoral agents.

Real-Time Intracellular Temperature Imaging Using Lanthanide-Bearing Polymeric Micelles.

Measurement of thermogenesis in individual cells is a remarkable challenge due to the complexity of the biochemical environment (such as pH and ionic strength) and to the rapid and yet not well-understood heat transfer mechanisms throughout the cell. Here, we present a unique system for intracellular temperature mapping in a fluorescence microscope (uncertainty of 0.2 K) using rationally designed luminescent Ln3+-bearing polymeric micellar probes (Ln = Sm, Eu) incubated in breast cancer MDA-MB468 cells. Two-dimensional (2D) thermal images recorded increasing the temperature of the cells culture medium between 296 and 304 K shows inhomogeneous intracellular temperature progressions up to ∼20 degrees and subcellular gradients of ∼5 degrees between the nucleolus and the rest of the cell, illustrating the thermogenic activity of the different organelles and highlighting the potential of this tool to study intracellular processes.

Identifying early pathogenic events during vascular calcification in uremic rats.

Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.

The antidiabetic effect of superparamagnetic iron oxide nanoparticles highlights the role of WNT/AMPK/mTOR/FOXO1/mitochondrial DNA in muscle and kidney.

Aim: To explore the antidiabetic effect of superparamagnetic iron oxide nanoparticles (SPIONs)-PEG-550 and its related metabolic pathways in muscles and kidney. Materials & methods: Diabetes was induced in 5-day neonatal rats; after confirming diabetes, treatment with SPIONs-PEG-550 started at different doses for 4 weeks. Routine analysis of glucose, insulin, adipocytokines, urea and creatinine was performed. The expression of several genes involved in metabolic pathways and the corresponding protein levels were examined. Results & conclusion: SPIONs-PEG-550 normalized the disturbed glucose homeostasis, reversed insulin resistance, adjusted the serum level of adipocytokines, and improved several disturbed downstream effectors of the insulin signaling and WNT pathway in both tissues. Histological examination of the muscle and pancreas has shown almost normal functional characteristics without remarkable adverse effects on the kidney.

Enhancement of Tumor Cell Immunogenicity and Antitumor Properties Derived from Platinum-Conjugated Iron Nanoparticles.

From chemistry design to clinical application, several approaches have been developed to overcome platinum drawbacks in antitumoral therapies. An in-depth understanding of intracellular signaling may hold the key to the relationship of both conventional drugs and nanoparticles. Within these strategies, first, nanotechnology has become an essential tool in oncotherapy, improving biopharmaceutical properties and providing new immunomodulatory profiles to conventional drugs mediated by activation of endoplasmic reticulum (ER) stress. Secondly, functional proteomics techniques based on microarrays have proven to be a successful method for high throughput screening of proteins and profiling of biomolecule mechanisms of action. Here, we conducted a systematic characterization of the antitumor profile of a platinum compound conjugated with iron oxide nanoparticles (IONPs). As a result of the nano-conjugation, cytotoxic and proteomics profiles revealed a significant improvement in the antitumor properties of the starting material, providing selectivity in certain tumor cell lines tested. Moreover, cell death patterns associated with immunogenic cell death (ICD) response have also been identified when ER signaling pathways have been triggered. The evaluation in several tumor cell lines and the analysis by functional proteomics techniques have shown novel perspectives on the design of new cisplatin-derived conjugates, the high value of IONPs as drug delivery systems and ICD as a rewarding approach for targeted oncotherapy and onco-immunotherapies.

Local Temperature Increments and Induced Cell Death in Intracellular Magnetic Hyperthermia.

The generation of temperature gradients on nanoparticles heated externally by a magnetic field is crucially important in magnetic hyperthermia therapy. But the intrinsic low heating power of magnetic nanoparticles, at the conditions allowed for human use, is a limitation that restricts the general implementation of the technique. A promising alternative is local intracellular hyperthermia, whereby cell death (by apoptosis, necroptosis, or other mechanisms) is attained by small amounts of heat generated at thermosensitive intracellular sites. However, the few experiments conducted on the temperature determination of magnetic nanoparticles have found temperature increments that are much higher than the theoretical predictions, thus supporting the local hyperthermia hypothesis. Reliable intracellular temperature measurements are needed to get an accurate picture and resolve the discrepancy. In this paper, we report the real-time variation of the local temperature on γ-Fe2O3 magnetic nanoheaters using a Sm3+/Eu3+ ratiometric luminescent thermometer located on its surface during exposure to an external alternating magnetic field. We measure maximum temperature increments of 8 °C on the surface of the nanoheaters without any appreciable temperature increase on the cell membrane. Even with magnetic fields whose frequency and intensity are still well within health safety limits, these local temperature increments are sufficient to produce a small but noticeable cell death, which is enhanced considerably as the magnetic field intensity is increased to the maximum level tolerated for human use, consequently demonstrating the feasibility of local hyperthermia.

Metal-free highly luminescent silica nanoparticles.

Stable, cost-effective, brightly luminescent, and metal-free organosilica nanoparticles (NPs) were prepared using the Stöber method without any thermal treatment above 318 K. The white-light photoluminescence results from a convolution of the emission originated in the NH(2) groups of the organosilane and oxygen defects in the silica network. The time-resolved emission spectra are red-shifted, relative to those acquired in the steady-state regime, pointing out that the NPs emission is governed by donor-acceptor (D-A) recombination mechanisms. Moreover, the increase of the corresponding lifetime values with the monitored wavelength further supports that the emission is governed by a recombination mechanism typical of a D-A pair attributed to an exceptionally broad inhomogeneous distribution of the emitting centers peculiar to silica-based NPs. These NPs exhibit the highest emission quantum yield value (0.15 ± 0.02) reported so far for organosilica biolabels without activator metals. Moreover, the emission spectra and the quantum yield values are quite stable over time showing no significant aging effects after exposure to the ambient environment for more than 1 year, stressing the potential of these NPs as metal-free biolabels.

The Anti-Obesity Potential of Superparamagnetic Iron Oxide Nanoparticles against High-Fat Diet-Induced Obesity in Rats: Possible Involvement of Mitochondrial Biogenesis in the Adipose Tissues.

BACKGROUND: Obesity is a pandemic disease that is rapidly growing into a serious health problem and has economic impact on healthcare systems. This bleak image has elicited creative responses, and nanotechnology is a promising approach in obesity treatment. This study aimed to investigate the anti-obesity effect of superparamagnetic iron oxide nanoparticles (SPIONs) on a high-fat-diet rat model of obesity and compared their effect to a traditional anti-obesity drug (orlistat). METHODS: The obese rats were treated daily with orlistat and/or SPIONs once per week for 8 weeks. At the end of the experiment, blood samples were collected for biochemical assays. Then, the animals were sacrificed to obtain white adipose tissues (WAT) and brown adipose tissues (BAT) for assessment of the expression of thermogenic genes and mitochondrial DNA copy number (mtDNA-CN). RESULTS: For the first time, we reported promising ameliorating effects of SPIONs treatments against weight gain, hyperglycemia, adiponectin, leptin, and dyslipidemia in obese rats. At the molecular level, surprisingly, SPIONs treatments markedly corrected the disturbed expression and protein content of inflammatory markers and parameters controlling mitochondrial biogenesis and functions in BAT and WAT. CONCLUSIONS: SPIONs have a powerful anti-obesity effect by acting as an inducer of WAT browning and activator of BAT functions.

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification.

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca²(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM² but not with 2 × 1 or 1 × 4 Ca × P(i) mM². Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.

Magnetic and relaxation properties of multifunctional polymer-based nanostructured bioferrofluids as MRI contrast agents.

A series of maghemite/polymer composite ferrofluids with variable magnetic core size, which show a good efficiency as MRI contrast agents, are presented. These ferrofluids are biocompatible and can be proposed as possible platforms for multifunctional biomedical applications, as they contain anchoring groups for biofunctionalization, can incorporate fluorescent dyes, and have shown low cellular toxicity. The magnetic properties of the ferrofluids have been determined by means of magnetization and ac susceptibility measurements as a function of temperature and frequency. The NMR dispersion profiles show that the low frequency behavior of the longitudinal relaxivity r(1) is well described by the heuristic model of (1)H nuclear relaxation induced by superparamagnetic nanoparticles proposed by Roch and co-workers. The contrast efficiency parameter, i.e., the nuclear transverse relaxivity r(2), for samples with d > 10 nm assumes values comparable with or better than the ones of commercial samples, the best results obtained in particles with the biggest magnetic core, d = 15 nm. The contrast efficiency results are confirmed by in vitro MRI experiments at ν = 8.5 MHz, thus allowing us to propose a set of optimal microstructural parameters for multifunctional ferrofluids to be used in MRI medical diagnosis.

The Thermodynamics of Medial Vascular Calcification.

Medial vascular calcification (MVC) is a degenerative process that involves the deposition of calcium in the arteries, with a high prevalence in chronic kidney disease (CKD), diabetes, and aging. Calcification is the process of precipitation largely of calcium phosphate, governed by the laws of thermodynamics that should be acknowledged in studies of this disease. Amorphous calcium phosphate (ACP) is the key constituent of early calcifications, mainly composed of Ca2+ and PO4 3- ions, which over time transform into hydroxyapatite (HAP) crystals. The supersaturation of ACP related to Ca2+ and PO4 3- activities establishes the risk of MVC, which can be modulated by the presence of promoter and inhibitor biomolecules. According to the thermodynamic parameters, the process of MVC implies: (i) an increase in Ca2+ and PO4 3- activities (rather than concentrations) exceeding the solubility product at the precipitating sites in the media; (ii) focally impaired equilibrium between promoter and inhibitor biomolecules; and (iii) the progression of HAP crystallization associated with nominal irreversibility of the process, even when the levels of Ca2+ and PO4 3- ions return to normal. Thus, physical-chemical processes in the media are fundamental to understanding MVC and represent the most critical factor for treatments' considerations. Any pathogenetical proposal must therefore comply with the laws of thermodynamics and their expression within the medial layer.

Efficient Brownian oscillators and nanoheaters based on gallium-doped ε-Fe2O3.

Wireless actuation at the nanoscale is vital in many contexts, and magnetic fields acting on nanoparticles (NPs) are among the most effective tools when actuation concerns linear forces. However, effective tools to apply torques at the nanoscale are still missing, because NPs where the magnetic moment is strongly coupled to the lattice agglomerate due to their high magnetic moment. Here, we show that gallium-doped ε-iron oxide NPs have small interparticle magnetic interactions and huge lattice-coupling for efficiently applying torques at the nanoscale. In this view, they are expected to be useful tools to efficiently apply mechanical forces to induce cellular apoptosis and to discern between mechanical and thermal contributions to cellular apoptosis currently under debate.

Medial Arterial Calcification: JACC State-of-the-Art Review.

Medial arterial calcification (MAC) is a chronic systemic vascular disorder distinct from atherosclerosis that is frequently but not always associated with diabetes mellitus, chronic kidney disease, and aging. MAC is also a part of more complex phenotypes in numerous less common diseases. The hallmarks of MAC include disseminated and progressive precipitation of calcium phosphate within the medial layer, a prolonged and clinically silent course, and compromise of hemodynamics associated with chronic limb-threatening ischemia. MAC increases the risk of complications during vascular interventions and mitigates their outcomes. With the exception of rare monogenetic defects affecting adenosine triphosphate metabolism, MAC pathogenesis remains unknown, and causal therapy is not available. Implementation of genetics and omics-based approaches in research recognizing the critical importance of calcium phosphate thermodynamics holds promise to unravel MAC molecular pathogenesis and to provide guidance for therapy. The current state of knowledge concerning MAC is reviewed, and future perspectives are outlined.

Whither Magnetic Hyperthermia? A Tentative Roadmap.

The scientific community has made great efforts in advancing magnetic hyperthermia for the last two decades after going through a sizeable research lapse from its establishment. All the progress made in various topics ranging from nanoparticle synthesis to biocompatibilization and in vivo testing have been seeking to push the forefront towards some new clinical trials. As many, they did not go at the expected pace. Today, fruitful international cooperation and the wisdom gain after a careful analysis of the lessons learned from seminal clinical trials allow us to have a future with better guarantees for a more definitive takeoff of this genuine nanotherapy against cancer. Deliberately giving prominence to a number of critical aspects, this opinion review offers a blend of state-of-the-art hints and glimpses into the future of the therapy, considering the expected evolution of science and technology behind magnetic hyperthermia.

Effect of superparamagnetic iron oxide nanoparticles on glucose homeostasis on type 2 diabetes experimental model.

AIMS: Evaluation of the anti-diabetic effect of superparamagnetic iron oxide nanoparticles (SPIONs) on Type 2 diabetic rats and compared their effect to metformin treatment. MAIN METHODS: Diabetic rats were treated with different doses of nanoparticles one time per week for 4 weeks. Fasting blood glucose level was determined for studied groups during the experimental period (30 days). At the end of the experiment, oral glucose tolerance test was carried out, serum samples were collected for biochemical assays. Then animals were sacrificed to obtain tissues for assessment of glucose transporters, insulin receptors and insulin signaling proteins. KEY FINDING: SPIONs treatment normalized fasting blood glucose and lowering insulin level in diabetic rats compared to untreated diabetic rats. SPIONs significantly ameliorate the glucose sensing and the active components of insulin signaling pathway. The anti-diabetic effects of SPIONs may be mediated through its effect on (i) hepatic peroxisome proliferator-activated receptor gamma coactivator 1-alpha content, which induced by SPIONs treatment in a dose-dependent manner, (ii) adipocytokines as SPIONs treated diabetic rats showed significantly higher levels of adiponectin and lower retinol binding protein 4 compared to untreated diabetic rats, (iii) lipid profile as SPIONs treatment significantly corrected the lipid profile in a dose-dependent manner and to a similar extent as metformin or even better. SIGNIFICANCE: To our knowledge, this is the first study that explores the anti-diabetic effects of SPIONs on diabetic model.

Magnetic hyperthermia with ε-Fe2O3 nanoparticles.

Biocompatibility restrictions have limited the use of magnetic nanoparticles for magnetic hyperthermia therapy to iron oxides, namely magnetite (Fe3O4) and maghemite (γ-Fe2O3). However, there is yet another magnetic iron oxide phase that has not been considered so far, in spite of its unique magnetic properties: ε-Fe2O3. Indeed, whereas Fe3O4 and γ-Fe2O3 have a relatively low magnetic coercivity, ε-Fe2O3 exhibits a giant coercivity. In this report, the heating power of ε-Fe2O3 nanoparticles in comparison with γ-Fe2O3 nanoparticles of similar size (∼20 nm) was measured in a wide range of field frequencies and amplitudes, in uncoated and polymer-coated samples. It was found that ε-Fe2O3 nanoparticles primarily heat in the low-frequency regime (20-100 kHz) in media whose viscosity is similar to that of cell cytoplasm. In contrast, γ-Fe2O3 nanoparticles heat more effectively in the high frequency range (400-900 kHz). Cell culture experiments exhibited no toxicity in a wide range of nanoparticle concentrations and a high internalization rate. In conclusion, the performance of ε-Fe2O3 nanoparticles is slightly inferior to that of γ-Fe2O3 nanoparticles in human magnetic hyperthermia applications. However, these ε-Fe2O3 nanoparticles open the way for switchable magnetic heating owing to their distinct response to frequency.

Polymer-coated superparamagnetic iron oxide nanoparticles as T2 contrast agent for MRI and their uptake in liver.

AIM: To study the efficiency of multifunctional polymer-based superparamagnetic iron oxide nanoparticles (bioferrofluids) as a T2 magnetic resonance contrast agent and their uptake and toxicity in liver. MATERIALS & METHODS: Mice were intravenously injected with bioferrofluids and Endorem®. The magnetic resonance efficiency, uptake and in vivo toxicity were investigated by means of magnetic resonance imaging (MRI) and histological techniques. RESULTS: Bioferrofluids are a good T2 contrast agent with a higher r2/r1 ratio than Endorem. Bioferrofluids have a shorter blood circulation time and persist in liver for longer time period compared with Endorem. Both bioferrofluids and Endorem do not generate any noticeable histological lesions in liver over a period of 60 days post-injection. CONCLUSION: Our bioferrofluids are powerful diagnostic tool without any observed toxicity over a period of 60 days post-injection.

PEG-copolymer-coated iron oxide nanoparticles that avoid the reticuloendothelial system and act as kidney MRI contrast agents.

In vitro experiments have shown the great potential of magnetic nanocarriers for multimodal imaging diagnosis and non-invasive therapies. However, their extensive clinical application is still jeopardized by a fast retention in the reticuloendothelial system (RES). The other issue that restrains their potential performance is slow degradation and excretion, which increases their risks of toxicity. We report a promising case in which multicore iron oxide nanoparticles coated with a poly(4-vinylpyridine) polyethylene glycol copolymer show low RES retention and high urinary excretion, as confirmed by single photon emission computerized tomography (SPECT), gamma counting, magnetic resonance imaging (MRI) and electron microscopy (EM) biodistribution studies. These iron oxide-copolymer nanoparticles have a high PEG density in their coating which may be responsible for this effect. Moreover, they show a clear negative contrast in the MR imaging of the kidneys. These nanoparticles with an average hydrodynamic diameter of approximately 20 nm were nevertheless able to cross the glomerulus wall which has an effective pore size of approximately 6 nm. A transmission electron microscopy inspection of kidney tissue revealed the presence of iron containing nanoparticle clusters in proximal tubule cells. This therefore makes them exceptionally useful as magnetic nanocarriers and as new MRI contrast agents for the kidneys.

MRI Study of the Influence of Surface Coating Aging on the In Vivo Biodistribution of Iron Oxide Nanoparticles.

Medical imaging is an active field of research that fosters the necessity for novel multimodal imaging probes. In this line, nanoparticle-based contrast agents are of special interest, since those can host functional entities either within their interior, reducing potential toxic effects of the imaging tracers, or on their surface, providing high payloads of probes, due to their large surface-to-volume ratio. The long-term stability of the particles in solution is an aspect usually under-tackled during probe design in research laboratories, since their performance is generally tested briefly after synthesis. This may jeopardize a later translation into practical medical devices, due to stability reasons. To dig into the effects of nanoparticle aging in solution, with respect to their behavior in vivo, iron oxide stealth nanoparticles were used at two stages (3 weeks vs. 9 months in solution), analyzing their biodistribution in mice. Both sets of nanoprobes showed similar sizes, zeta potentials, and morphology, as observed by dynamic light scattering (DLS) and transmission electronic microscopy (TEM), but fresh nanoparticles accumulated in the kidneys after systemic administration, while aged ones accumulated in liver and spleen, confirming an enormous effect of particle aging on their in vivo behavior, despite barely noticeable changes perceived on a simple inspection of their structural integrity.