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Massive Intracellular Biodegradation of Iron Oxide Nanoparticles Evidenced Magnetically at Single Endosome and Tissue Levels
Abstract
Quantitative studies of the long-term fate of iron oxide nanoparticles inside cells, a prerequisite for regenerative medicine applications, are hampered by the lack of suitable biological tissue models and analytical methods. Here we propose stem cell spheroids as a tissue model to track intracellular magnetic nanoparticles transformations during long-term tissue maturation. We show that global spheroid magnetism can serve as a fingerprint of the degradation process and we evidence a near-complete nanoparticle degradation over a month of tissue maturation, as confirmed by electron microscopy. Remarkably, the same massive degradation was measured at the endosome level by single-endosome nano-magnetophoretic tracking in cell-free endosomal extract. Interestingly, this spectacular nanoparticles breakdown barely affected iron homeostasis: only the genes coding for ferritin light chain (iron loading) and ferroportin (iron export) were upregulated two-fold by the degradation process. Besides, these unique magnetic and tissular tools allow screening the bio-stability of magnetic nanomaterials, as demonstrated here with iron oxide nanocubes and nanodimers. Hence we demonstrate that stem cells spheroids and purified endosomes are suitable models needed to monitor nanoparticle degradation in conjunction with magnetic, chemical and biological characterizations at the cellular scale, quantitatively, in the long term, in situ and in real time.
Supplementary resource (1)
... However, particle degradation dynamics differ between spherical and cube-shaped particles, thus it is not prudent to extrapolate these results to the present nanoparticles. Previous reports have shown the edges of cube-shaped nanoparticles are prone to enhanced biodegradation kinetics, particularly in lysosomal compartments of cells with the edges of the cubes more vulnerable than the faces [85]. Nanocubes experienced partial degradation leaving smaller, spherical particles, mostly at the endosome edges after 27 days of cellular exposure [85]. ...
... Previous reports have shown the edges of cube-shaped nanoparticles are prone to enhanced biodegradation kinetics, particularly in lysosomal compartments of cells with the edges of the cubes more vulnerable than the faces [85]. Nanocubes experienced partial degradation leaving smaller, spherical particles, mostly at the endosome edges after 27 days of cellular exposure [85]. On the other hand, spherical nanoparticles coated with DMSA [83] and other coatings [85,86] have been shown to degrade particle by particle over time, with some particles remaining intact longer than others. ...
... Nanocubes experienced partial degradation leaving smaller, spherical particles, mostly at the endosome edges after 27 days of cellular exposure [85]. On the other hand, spherical nanoparticles coated with DMSA [83] and other coatings [85,86] have been shown to degrade particle by particle over time, with some particles remaining intact longer than others. Therefore, the next steps prior to translation of these nanoparticles to clinical settings include determining their stability and retention in an in vivo model. ...
Background: Superparamagnetic iron core iron oxide shell nanocubes have previously shown superior performance in magnetic resonance imaging T2 contrast enhancement compared with spherical nanoparticles. Methods: Iron core iron oxide shell nanocubes were synthesized, stabilized with dimercaptosuccinic acid (DMSA-NC) and physicochemically characterized. Magnetic resonance imaging (MRI) contrast enhancement and biocompatibility were assessed in vitro. Results: DMSA-NC showed a transverse relaxivity of 122.59 mM ⁻¹ ·s ⁻¹ Fe. Treatment with DMSA-NC did not induce cytotoxicity or oxidative stress in U-251 cells, and electron microscopy demonstrated DMSA-NC localization within endosomes and lysosomes in cells following internalization. Global proteomics revealed dysregulation of iron storage, transport, transcription and mRNA processing proteins. Conclusion: DMSA-NC is a promising T2 MRI contrast agent which, in this preliminary investigation, demonstrates favorable biocompatibility with an astrocyte cell model.
... This knowledge is extremely useful for the design of efficient delivery systems. TEM has demonstrated that endocytosis is the most common uptake mode; nanocarriers have been observed making contact with the plasma membrane occurring in plasma membrane invaginations and entering the cell enclosed in endosomes [59,[61][62][63][64][65][66][67][68][69][70]. Endocytosis may take place for single nanoparticles and also for small nanoparticle groups whereas single large nanoconstructs or large clusters of nanoparticulates enter the cells by means of phagocytosis or macropinocytosis, respectively [63,[71][72][73][74]. ...
... Endosomes/phagosomes are destined to fuse with primary lysosomes with the consequent degradation of the contained nanoparticles by the action of lytic enzymes [83,84]. Therefore, the nanoconstructs internalized via endocytosis/phagocytosis will generally be trapped in vacuolar structures without making contact with any organelle; only the degradation products that can cross the lysosomal membrane will spread to the cytosol whereas nanoparticle remnants will remain inside secondary lysosomes and residual bodies [61][62][63][64][65][66][67][68]72,73,[85][86][87]. ...
... Endosomal-escaped nanoparticles were observed to undergo exocytosis [108][109][110] but they can also re-enter the lytic pathway by the autophagic route [72,87,96,101,111]. TEM has allowed the observation of non-degradable nanoparticle remnants persisting inside vacuolar structures [61,62,[64][65][66][67][68]72,73,86,87,112], thus providing unique information on their biodegradability. It is worth noting that fluorescent microscopy does not allow entire nanoparticles to be distinguished from their remnants, provided that they keep their binding with the marker. ...
Nanomedical research necessarily involves the study of the interactions between nanoparticulates and the biological environment. Transmission electron microscopy has proven to be a powerful tool in providing information about nanoparticle uptake, biodistribution and relationships with cell and tissue components, thanks to its high resolution. This article aims to overview the transmission electron microscopy techniques used to explore the impact of nanoconstructs on biological systems, highlighting the functional value of ultrastructural morphology, histochemistry and microanalysis as well as their fundamental contribution to the advancement of nanomedicine.
... [11], while their intercellular trafficking can be extremely regulated [12,13], as evidenced in the case of Abraxane [2]. General concerns regarding degradation of non-biological nanocarriers exist because of the safety issues that may hamper the clinical translation of these technologies, in particular when dealing with regenerative medicine applications [14]. It is commonly accepted that if the degradation products of these carriers are water-soluble, they can be excreted via renal or hepatobiliary clearance. ...
... It is commonly accepted that if the degradation products of these carriers are water-soluble, they can be excreted via renal or hepatobiliary clearance. Mazuel et al. [14,15] investigated the degradation and clearance of iron nanocarriers in cellular spheroids, by developing an analytic tool that could measure carrier degradation as a function of their magnetic properties. The analysis was so sensitive that allowed following a carrier digestion in a single endosome, representing a milestone in the nanocarrier degradation detection. ...
... 29 However, IONPs are ionized very slowly in vivo according to the previous studies. 30,31 Thus, the iron ion concentration following IONP injection would be low and would not cause any oxidative stress or toxicity. Moreover, Ferumoxytol, which is approved by the U.S. FDA as a therapeutic for patients with iron deficiency anemia, was used as an IONP in this study, implying the safety of IONP and raising the clinical potential of MSC-IONPs. ...
... IONPs slowly ionized and were maintained for at least 10 days, suggesting that the effect of magnetic guidance would maintain for such a long time. According to a previous study, 31 the low pH in the endosome induces a slow ionization of IONP in the endosome of MSCs into iron ions. Also, we have now demonstrated that MSC-IONPs can be still attracted by magnetic force 10 days after IONP internalization into MSCs as shown in the following ( Figure S4). ...
Alzheimer's disease (AD) is a neurodegenerative disease with multifactorial pathogenesis. However, most current therapeutic approaches for AD target a single pathophysiological mechanism, generally resulting in unsatisfactory therapeutic outcomes. Recently, mesenchymal stem cell (MSC) therapy, which targets multiple pathological mechanisms of AD, has been explored as a novel treatment. However, the low brain retention efficiency of administered MSCs limits their therapeutic efficacy. In addition, autologous MSCs from AD patients may have poor therapeutic abilities. Here, we overcome these limitations by developing iron oxide nanoparticle (IONP)-incorporated human Wharton's jelly-derived MSCs (MSC-IONPs). IONPs promote therapeutic molecule expression in MSCs. Following intracerebroventricular injection, MSC-IONPs showed a higher brain retention efficiency under magnetic guidance. This potentiates the therapeutic efficacy of MSCs in murine models of AD. Furthermore, human Wharton's jelly-derived allogeneic MSCs may exhibit higher therapeutic abilities than those of autologous MSCs in aged AD patients. This strategy may pave the way for developing MSC therapies for AD.
... [63]. MNPs' degradation could affect their magnetic properties [64] and, thus, their ability to apply the magnetomechanical force partly responsible for the observed growth inhibition effects in +MNPs+Field cells. ...
... Indeed, it was found that the acidic pH of the lysosomes affects the magnetic properties of the MNPs, an observation confirming previously published results [64]. The lack of correlation between the degree of MNPs' and lysosomes' co-localization and the degree of the loss of the pH gradient in +MNPs+Field cells is not completely clear, but could be attributed to magnetization loss. ...
The application of magnetomechanical stress in cells using internalized magnetic nanoparticles (MNPs) actuated by low-frequency magnetic fields has been attracting considerable interest in the field of cancer research. Recent developments prove that magnetomechanical stress can inhibit cancer cells’ growth. However, the MNPs’ type and the magnetic field’s characteristics are crucial parameters. Their variability allows multiple combinations, which induce specific biological effects. We previously reported the antiproliferative effects induced in HT29 colon cancer cells by static-magnetic-field (200 mT)-actuated spherical MNPs (100 nm). Herein, we show that similar growth inhibitory effects are induced in other colon cancer cell lines. The effect of magnetomechanical stress was also examined in the growth rate of tumor spheroids. Moreover, we examined the biological mechanisms involved in the observed cell growth inhibition. Under the experimental conditions employed, no cell death was detected by PI (propidium iodide) staining analysis. Flow cytometry and Western blotting revealed that G2/M cell cycle arrest might mediate the antiproliferative effects. Furthermore, MNPs were found to locate in the lysosomes, and a decreased number of lysosomes was detected in cells that had undergone magnetomechanical stress, implying that the mechanical activation of the internalized MNPs could induce lysosome membrane disruption. Of note, the lysosomal acidic conditions were proven to affect the MNPs’ magnetic properties, evidenced by vibrating sample magnetometry (VSM) analysis. Further research on the combination of the described magnetomechanical stress with lysosome-targeting chemotherapeutic drugs could lay the groundwork for the development of novel anticancer combination treatment schemes.
... The co-precipitation is the first synthesis method of nanoparticles developed by R. Massard in the 1980s [20], and still currently the most used [14,21,[39][40][41][42][43][44][45][46][47][48][49] by researchers working on magnetic iron oxide nanoparticles. It is based on joint precipitation in aqueous solution of ferric and ferrous ions by addition of a basis such as ammoniac or sodium hydroxide. ...
... Citrates are part of the first molecules for the stabilization of iron oxide nanoparticles [48,49,91,102]. Citric acid is a tricarboxylic acid α-hydroxylated with chemical formula C 6 H 8 O 7 (M = 192 g·mol −1 ) and showing no toxicity to mammals [103]. Commonly used as an acidifier in the food industry, it also produced in all cells of all aerobic organisms during the Krebs cycle by condensation of oxaloacetate and acetyl-coenzyme A. Citric acid is a weak triacid which therefore has three separate pKa: there also are four different predominant forms in function of pH ( Figure 8). ...
Nanoparticles have experienced increasing interest over the past three decades owing to the development of new synthesis methods and the adaptation of analysis tools with spatial resolutions below one micrometer. Among the different types of nanoparticles developed in recent years (metals, metal oxides, silica, polymers, etc.), significant scientific interest has developed around iron oxide nanoparticles. This review will focus on these magnetic iron oxide nanoparticles. We will first discuss the magnetic properties of iron oxide nanoparticles, then the different methods of synthesis and washing. Finally, we will discuss some functionalization strategies of iron oxide nanoparticles which are developed within our research team.
... MNPs, however, enable the non-destructive evaluation of cellular uptake by high-sensitivity Superconducting Quantum Interference Device (SQUID) magnetometry and even facilitate monitoring of the nanoparticles fate through finer details of their magnetic behaviour using both static (DC) [27][28][29][30][31] and dynamic (AC) measurements [32][33][34]. Nevertheless, the relatively small amplitudes available in AC magnetometers (now commonly used to characterize the absorption rate for magnetic hyperthermia) are often unable to saturate the magnetic response and, thus, are unable to measure the mass of internalized MNPs [35]. ...
Nanoparticle uptake by cells is a key parameter in their performance in biomedical applications. However, the use of quantitative, non-destructive techniques to obtain the amount of nanoparticles internalized by cells is still uncommon. We have studied the cellular uptake and the toxicity of core-shell maghemite-silica magnetic nanoparticles (MNPs), with a core diameter of 9 nm and a shell thickness of 3 nm. The internalization of the nanoparticles by mouse neuroblastoma 2a cells was evaluated by sensitive and non-destructive Superconducting Quantum Interference Device (SQUID) magnetometry and corroborated by graphite furnace atomic absorption spectroscopy. We were thus able to study the toxicity of the nanoparticles for well-quantified MNP uptake in terms of nanoparticle density within the cell. No significant variation in cell viability or growth rate was detected for any tested exposure. Yet, an increase in both the amount of mitochondrial superoxide and in the lysosomal activity was detected for the highest concentration (100 μg ml⁻¹) and incubation time (24 h), suggesting the onset of a disruption in ROS homeostasis, which may lead to an impairment in antioxidant responses. Our results validate SQUID magnetometry as a sensitive technique to quantify MNP uptake and demonstrate the non-toxic nature of these core-shell MNPs under our culture conditions.
... It is The copyright holder for this preprint this version posted May 15, 2024. ; https://doi.org/10.1101/2024.05.15.594430 doi: bioRxiv preprint product of the former 55 . To further support the production of INPs by RJ-1, unstained sections were examined under an HAADF-STEM mode. ...
The supportive role of extracellular matrix in tumor growth and the drug resistance of tumor cells stand the major challenges in treating cancer. Here, we report the discovery of an oncolytic Clostridium strain, named RJ-1, which simultaneously expresses collagenases and produces iron-containing particles through intrabacterial mineralization. The secreted collagenases degrade pre-existing collagen mesh and show a collagen concentration-dependent degradation feature. Iron-containing particles released from RJ-1 by autolysis generate ferrous ions in the lysosomal acid environment following cell uptake and subsequently induce ferroptosis via the Fenton reaction. Due to selective germination in tumor sites, systemic administration of RJ-1 spores synchronously destructs dense tumor extracellular matrix and causes non-apoptotic tumor cell death. In collagen-dense tumor-bearing mice, a single intravenous injection achieves a favorable tolerance and rapid oncolysis of giant tumors with a size of ∼1000 mm ³ . This work finds a dual collagenase-expressing and iron-concentrating bacterium, proposing an alternative strategy to combat tumors.
... The surface smoothness of the NC membranes may be altered by the incorporation of GO into bio-NCs. The biofouling properties of the polymer membranes in biological applications can be overcome by incorporating GO due to its hydrophilic nature and electrostatic repulsion characteristics, which induce biodegradation or oppose bioadsorption and (Mazuel et al., 2016) thus can be used in biosensors (Qiu et al., 2020). Nafion exhibits high conductivity for proton and excellent chemical and physical stability. ...
The Au partially embedded nanostructure (PEN) is synthesized by ion irradiation on an Au thin film deposited on a glass substrate using a 50 keV Ar ion. Scanning electron microscopy results show ion beam‐induced restructuring from irregularly shaped nanostructures (NSs) to spherical Au NSs, and further ion irradiation leads to the formation of well‐separated spherical nanoparticles. Higuchi's algorithm of surface analysis is utilized to find the evolution of surface morphology with ion irradiation in terms of the Hurst exponent and fractal dimension. The Au PEN is evidenced by Rutherford backscattering spectrometry and optical studies. Also, the depth of the mechanism behind synthesized PEN is explained on the basis of theoretical simulations, namely, a unified thermal spike and a Monte Carlo simulation consisting of dynamic compositional changes (TRIDYN). Another set of plasmonic NSs was formed on the surface by thermal annealing of the Au film on the substrate. Glucose sensing has been studied on the two types of plasmonic layers: nanoparticles on the surface and PEN. The results reveal the sensing responses of both types of plasmonic layers. However, PEN retains its plasmonic behavior as the NSs are still present after washing with water, which demonstrates the potential for reusability.
Research Highlights
Synthesis of PENs by ion irradiation
Utilization of Higuchi's algorithm to explore the surface morphology.
Unified thermal spike and TRIDYN simulations being used to explain the results.
Glucose is only used as a test case for reusability of substrate.
... With regard to OVERCOME, vector replication in the carrier macrophages would be induced via small molecule, heat, or even magneto-mechanical actuation [54] once they have reached the tumor(s). Excess iron oxide nanoparticle deposition in the body from carrier macrophage lysis may not be ideal, but they should be degraded eventually [55]. ...
Recently, I described a novel approach for cancer therapy in an article entitled “Promoting Oncolytic Vector Replication with Switches that Detect Ubiquitous Mutations”. However, there are a few more design details that should potentially be taken into consideration. Also, multiregion, multisample tumor sequencing may be simplified by analyzing cell-free DNA in the blood of patients.
... 44 Aer being introduced into the endosome, IONPs are broken down and discharge iron ions into the cytoplasm. 45 Degradation of IONPs is notable in the endosomal medium, which has a pH of 4.7 and contains citrate. Acidic pH is unable to degrade IONPs without citrate. ...
Air pollution is a major risk factor for neurological disorders. Both indoor and outdoor dusts comprise different types of iron oxides in the nano-scale range. Due to their small size and unique physico-chemical properties, iron oxide nanoparticles (IONPs) adopt the intracellular path to agglomerate inside the cell cytoplasm. Moreover, they can cross the blood–brain barrier to invade cortical tissues in the brain and impair neuronal functions. Hence, analysis of the effects of IONPs on the Central Nervous System (CNS) structure and functions is indispensable from medical perspective. A literature search was performed using three scientific databases: ScienceDirect, PubMed, and Google Scholar. Articles published till December, 2023 were screened for their relevancy. Analyses of the appropriate literature have revealed that IONPs are being employed in drug delivery systems and diagnosis of CNS-related ailments that favor neuroprotection. However, the inhalation of IONPs from air and other sources can lead to excessive accumulation of iron in the neuronal tissues, leading to a disturbance in neuronal signaling and augmenting the onset of neurodegenerative disorders. Therefore, it is essential to monitor and control the abundance of IONPs in the environment to combat adverse impacts on the human nervous system.
... In addition, according to Fig. S2, the MNPs exist in the endosomal structures of the cells instead of being dispersed in cytosols. Assuming through the distribution pattern of the intracellularly delivered MNPs, degradation of the MNPs under low-pH conditions in endosomes could be another factor of the MNPs dilution [87]. Further, the detailed mechanisms related to the synergic effect of the MNPs on neural induction should be revealed, and thus, the exact role of the MNPs on neural induction of the hESCs has to be established. ...
Background: To improve the efficiency of neural development from human embryonic stem cells, human embryoid body (hEB) generation is vital through 3-dimensional formation. However, conventional approaches still have limitations: long-term cultivation and laborious steps for lineage determination. Methods: In this study, we controlled the size of hEBs for ectodermal lineage specification using cell-penetrating magnetic nanoparticles (MNPs), which resulted in reduced time required for initial neural induction. The magnetized cells were applied to concentrated magnetic force for magnet-derived multicellular organization. The uniformly sized hEBs were differentiated in neural induction medium (NIM) and suspended condition. This neurally induced MNP-hEBs were compared with other groups. Results: As a result, the uniformly sized MNP-hEBs in NIM showed significantly improved neural inductivity through morphological analysis and expression of neural markers. Signaling pathways of the accelerated neural induction were detected via expression of representative proteins; Wnt signaling, dopaminergic neuronal pathway, intercellular communications, and mechanotransduction. Consequently, we could shorten the time necessary for early neurogenesis, thereby enhancing the neural induction efficiency. Conclusion: Overall, this study suggests not only the importance of size regulation of hEBs at initial differentiation stage but also the efficacy of MNP-based neural induction method and stimulations for enhanced neural tissue regeneration.
... With regard to OVERCOME, vector replication in the carrier macrophages would be induced via small molecule, heat, or even magneto-mechanical actuation [52] once they have reached the tumor(s). Excess iron oxide nanoparticle deposition in the body from carrier macrophage lysis may not be ideal, but they should be degraded eventually [53]. ...
Recently, I described a novel approach for cancer therapy in an article entitled “Promoting Oncolytic Vector Replication with Switches that Detect Ubiquitous Mutations”. However, there are a few more design details that should potentially be taken into consideration. Also, multiregion, multisample tumor sequencing may be simplified by analyzing cell-free DNA in the blood of patients.
... However, we noticed that exposure to vigorous shear forces resulted in noticeable particle detachment, particularly problematic at sites with significant shear forces, such as the portal vein. On the other hand, IONPs tend to be rapidly internalized by cells, potentially leading to premature degradation in the acidic environment of endosomes [20], posing challenges for precise labeling. To address these challenges, we encapsulated IONPs within PLGA particles ranging from 1 to 5 μm to enhance the retention. ...
Post-transplantation tracking of pancreatic islets is a prerequisite for advancing cell therapy to treat type 1 diabetes. Magnetic resonance imaging (MRI) has emerged as a safe and non-invasive technique for visualizing cells in clinical applications. In this study, we proposed a novel MRI contrast agent formulation by encapsulating iron oxide nanoparticles (IONPs) in poly(lactic-co-glycolic acid) (PLGA) particles functionalized with a tissue adhesive polydopamine (PD) layer (IONP-PLGA-PD MS). Intriguingly, our particles facilitated efficient and robust labeling through a one-step process, allowing for the incorporation of a substantial amount of IONPs without detrimental impacts on the viability and functionality of pancreatic islets. The MRI signals emanating from islets labeled using our particles were found to be stable over 30 days in vitro and 60 days when transplanted under kidney capsules of diabetic mice. These results suggest that our approach provides a potential platform for monitoring the fate of pancreatic islets after transplantation.
... With regard to OVERCOME, vector replication in the carrier macrophages would be induced via small molecule, heat, or even magneto-mechanical actuation [43] once they have reached the tumor(s). Excess iron oxide nanoparticle deposition in the body from carrier macrophage lysis may not be ideal, but they should be degraded eventually [44]. ...
Some tumors occur in anatomical regions that are hard to biopsy with a needle. Such regions include the brain, spinal cord, liver, and lungs. For the latter two, magnetic nanoparticle-loaded macrophages could be intravenously infused and driven via an MRI machine into the tumor or tumors. Once there, they can be induced to phagocytose whole tumor cells. They would keep their target in a non-digested form by inhibiting phagosome maturation - and be directed via magnetotaxis or chemotaxis to an extraction point in the body where they can be more easily collected via needle.
... With regard to OVERCOME, vector replication in the carrier macrophages would be induced via small molecule, heat, or even magneto-mechanical actuation [ 29 ] once they have reached the tumor(s). Excess iron oxide nanoparticle deposition in the body from carrier macrophage lysis may not be ideal, but they should be degraded eventually [ 30 ]. ...
Some tumors occur in anatomical regions that are hard to biopsy with a needle. Such regions include the brain, spinal cord, liver, and lungs. For the latter two, magnetic nanoparticle-loaded macrophages could be intravenously infused and driven via an MRI machine into the tumor or tumors. Once there, they can be induced to phagocytose whole tumor cells. They would keep their target in a non-digested form by inhibiting phagosome maturation - and be directed via magnetotaxis or chemotaxis to an extraction point in the body where they can be more easily collected via needle.
... The mechanism of NP ion release to facilitate stem cell therapy involves using the NPs to transport therapeutic molecules to the cells. 138,139 The NPs are coated with a material, allowing the therapeutic molecules to pass into the cells without degradation or elimination. Once inside the cells, the therapeutic molecules can stimulate the stem cells to differentiate and proliferate, enhancing the therapeutic benefits. ...
Engineered nanomaterials (ENMs) with different topographies provide effective nano−bio interfaces for controlling the differentiation of stem cells. The interaction of stem cells with nanoscale topographies and chemical cues in their microenvironment at the nano−bio interface can guide their fate. The use of nanotopographical cues, in particular nanorods, nanopillars, nanogrooves, nanofibers, and nanopits, as well as biochemical forces mediated factors, including growth factors, cytokines, and extracellular matrix proteins, can significantly impact stem cell differentiation. These factors were seen as very effective in determining the proliferation and spreading of stem cells. The specific outgrowth of stem cells can be decided with size variation of topographic nanomaterial along with variation in matrix stiffness and surface structure like a special arrangement. The precision chemistry enabled controlled design, synthesis, and chemical composition of ENMs can regulate stem cell behaviors. The parameters of size such as aspect ratio, diameter, and pore size of nanotopographic structures are the main factors for specific termination of stem cells. Protein corona nanoparticles (NPs) have shown a powerful facet in stem cell therapy, where combining specific proteins could facilitate a certain stem cell differentiation and cellular proliferation. Nano−bio reactions implicate the interaction between biological entities and nanoparticles, which can be used to tailor the stem cells' culmination. The ion release can also be a parameter to enhance cellular proliferation and to commit the early differentiation of stem cells. Further research is needed to fully understand the mechanisms underlying the interactions between engineered nano−bio interfaces and stem cells and to develop optimized regenerative medicine and tissue engineering designs.
... [10][11][12] Mazuel et al. evidenced a near-complete intracellular degradation of Fe 3 O 4 NPs by using stem cell spheroids as a tissue model and global spheroid magnetism as a ngerprint of the degradation process. 13 However, because of the relatively low drug loading and iron leakage, Fe 3 O 4 NPs presents the weak antitumor efficacy when used as drug carriers and iron source for CDT. 14 In addition, the CDT efficacy and clinical translation are restricted by the low conversion efficiency from Fe 2+ to Fe 3+ , consumption of hydroxyl radicals by glutathione (GSH), 15 the limited endogenous supply of H 2 O 2 . ...
Cascade catalytic therapy has been recognized as a promising cancer treatment strategy, which is due in part to the induced tumor apoptosis when converting intratumoral hydrogen peroxide (H2O2) into highly toxic hydroxyl radicals (˙OH) based on the Fenton or Fenton-like reactions. Moreover this is driven by the efficient catalysis of glucose oxidization associated with starving therapy. The natural glucose oxidase (GO x ), recognized as a "star" enzyme catalyst involved in cancer treatment, can specially and efficiently catalyze the glucose oxidization into gluconic acid and H2O2. Herein, pH-responsive biodegradable cascade therapeutic nanocomposites (Fe3O4/GO x -PLGA) with dual enzymatic catalytic features were designed to respond to the tumor microenvironment (TME) and to catalyze the cascade reaction (glucose oxidation and Fenton-like reaction) for inducing oxidase stress. The GO x -motivated oxidation reaction could effectively consume intratumoral glucose to produce H2O2 for starvation therapy and the enriched H2O2 was subsequently converted into highly toxic ˙OH by a Fe3O4-mediated Fenton-like reaction for chemodynamic therapy (CDT). In addition, the acidity amplification owing to the generation of gluconic acid will in turn accelerate the degradation of the nanocomposite and initiate the Fe3O4-H2O2 reaction for enhancing CDT. The resultant cooperative cancer therapy was proven to provide highly efficient tumor inhibition on HeLa cells with minimal systemic toxicity. This cascade catalytic Fenton nanocomposite might provide a promising strategy for efficient cancer therapy.
... MNP degradation first occurs at the level of the PC associated with the MNP surface after coming into contact with blood or other biological fluids [40]. The coating or functionalization of the MNPs then degrades and finally, the metallic core disintegrates [78]. Each of these processes is influenced by the nature of the MNPs, the type of cell in which degradation occurs and the cell's metabolic state [79]. ...
Background: The surface coating of iron oxide magnetic nanoparticle (MNPs) drives their intracellular trafficking and degradation in endolysosomes, as well as dictating other cellular outcomes. As such, we assessed whether MNP coatings might influence their biodistribution, their accumulation in certain organs and their turnover therein, processes that must be understood in vivo to optimize the design of nanoformulations for specific therapeutic/diagnostic needs.
Results: In this study, three different MNP coatings were analyzed, each conferring the identical 12 nm iron oxide cores with different physicochemical characteristics: 3-aminopropyl-triethoxysilane (APS), dextran (DEX), and dimercaptosuccinic acid (DMSA). When the biodistribution of these MNPs was analyzed in C57BL/6 mice, they all mainly accumulated in the spleen and liver one week after administration. The coating influenced the proportion of the MNPs in each organ, with more APS-MNPs accumulating in the spleen and more DMSA-MNPs accumulating in the liver, remaining there until they were fully degraded. The changes in the physicochemical properties of the MNPs (core size and magnetic properties) was also assessed during their intracellular degradation when internalized by two murine macrophage cell lines. The decrease in the size of the MNPs iron core was influenced by their coating and the
organ in which they accumulated. Finally, MNP degradation was analyzed in the liver and spleen of C57BL/6 mice from 7 days to 15 months after the last intravenous MNP administration.
Conclusions: The MNPs degraded at different rates depending on the organ and their coating, the former representing the feature that was fundamental in determining the time they persisted. In the liver, the rate of degradation was similar for all three coatings, and it was faster than in the spleen. This information regarding the influence of coatings
... MNP degradation first occurs at the level of the PC associated with the MNP surface after coming into contact with blood or other biological fluids [40]. The coating or functionalization of the MNPs then degrades and finally, the metallic core disintegrates [78]. Each of these processes is influenced by the nature of the MNPs, the type of cell in which degradation occurs and the cell's metabolic state [79]. ...
Background
The surface coating of iron oxide magnetic nanoparticle (MNPs) drives their intracellular trafficking and degradation in endolysosomes, as well as dictating other cellular outcomes. As such, we assessed whether MNP coatings might influence their biodistribution, their accumulation in certain organs and their turnover therein, processes that must be understood in vivo to optimize the design of nanoformulations for specific therapeutic/diagnostic needs.
Results
In this study, three different MNP coatings were analyzed, each conferring the identical 12 nm iron oxide cores with different physicochemical characteristics: 3-aminopropyl-triethoxysilane (APS), dextran (DEX), and dimercaptosuccinic acid (DMSA). When the biodistribution of these MNPs was analyzed in C57BL/6 mice, they all mainly accumulated in the spleen and liver one week after administration. The coating influenced the proportion of the MNPs in each organ, with more APS-MNPs accumulating in the spleen and more DMSA-MNPs accumulating in the liver, remaining there until they were fully degraded. The changes in the physicochemical properties of the MNPs (core size and magnetic properties) was also assessed during their intracellular degradation when internalized by two murine macrophage cell lines. The decrease in the size of the MNPs iron core was influenced by their coating and the organ in which they accumulated. Finally, MNP degradation was analyzed in the liver and spleen of C57BL/6 mice from 7 days to 15 months after the last intravenous MNP administration.
Conclusions
The MNPs degraded at different rates depending on the organ and their coating, the former representing the feature that was fundamental in determining the time they persisted. In the liver, the rate of degradation was similar for all three coatings, and it was faster than in the spleen. This information regarding the influence of coatings on the in vivo degradation of MNPs will help to choose the best coating for each biomedical application depending on the specific clinical requirements.
Graphical Abstract
... Indeed, each single spheroid's magnetism is a fingerprint of nanoparticles integrity, and therefore of potential degradation. Remarkably, it was found that magnetic nanoparticles can be more than 90% purged inside the stem cells spheroids in the first ten days of tissue maturation [102]. This near-total nanoparticle degradation yet barely affected cellular iron homeostasis, which bodes well for their safety in regenerative medicine applications. ...
An attractive approach in cell therapies and medically oriented nanotechnologies is to interface magnetic nanoparticles with cells. This will supply the cells with sufficient magnetization for theranostic applications and for external magnetic field manipulation.
In tissue engineering, one challenge is to produce tissue analogues that are large, precisely organized, and responsive to stimuli, preferably without the need for an artificial supporting scaffold. One powerful tool for such biofabrication is certainly the bioprinting technology.
In magnetic tissue engineering, it appears possible to use magnetic forces to manipulate cells, both individually and within aggregates, and thereby to produce three-dimensional artificial tissues with inherent capacities for further physical stimulation, a possibility that bioprinting does not offer yet.
We here introduce the feasibility of using magnetic forces created by external (micro)magnets to form 3D tissue-like scaffold-free structures. Because stem cells are essential in tissue engineering, such magnetic technologies were developed with magnetized stem cells, and applied for instance to vascular or cartilage tissue engineering. One precondition to this approach, which lies in the magnetization of (stem) cells endowed through internalization of iron oxide magnetic nanoparticles, is to ensure the safety of magnetic nanoparticles with respect to cellular functions, which is initially discussed.
Finally, we introduce a magnetic tissue stretcher which, in a single step, allows to create a tissue composed of any type of component cell, then to mature it, stimulate it by compression or stretching at any desired frequency, e.g. cyclically, opening new possibilities in the cardiac muscle tissue engineering field.
... The main issue with using nano-and microparticles as mini-scaffolds is their potential cytotoxicity. From one point of view, they biodegrade to ferritin, which is a natural component of human blood, that can be removed with urea [51]. From another point of view, undesirable accumulation of magnetic nanoparticles in human organs, such as the spleen and liver, with the potential development of hemosiderosis has been reported. ...
The concept of “lockyballs” or interlockable mini-scaffolds fabricated by two-photon polymerization from biodegradable polymers for the encagement of tissue spheroids and their delivery into the desired location in the human body has been recently introduced. In order to improve control of delivery, positioning, and assembly of mini-scaffolds with tissue spheroids inside, they must be functionalized. This review describes the design, fabrication, and functionalization of mini-scaffolds as well as perspectives on their application in tissue engineering for precisely controlled cell and mini-tissue delivery and patterning. The development of functionalized mini-scaffolds advances the original concept of “lockyballs” and opens exciting new prospectives for mini-scaffolds’ applications in tissue engineering and regenerative medicine and their eventual clinical translation.
... Our presented studies showed that IONP is an excellent contrast agent candidate for intraoperative PG imaging. The biodegradation mechanism of IONPs has previously been elucidated that cellular internalized IONPs are metabolized into Fe 3+ ions in the acidic and enzyme-rich environment of lysosomes, where ferritin and transferrin proteins bind Fe 3+ ions and subsequently facilitate systematic iron absorption and recycle for many important biological processes including erythropoiesis, liver and heart functions (30)(31)(32). Owing to their outstanding human safety and biodegradability, intravenous administration of IONPs has already been clinically approved by FDA and NMPA for treating acute iron deficiency anemia (23)(24)(25). Our in vivo results further validated that most IONP10s can be biodegraded within the thyroid microenvironment and are not detectable by eye after 90 days in a head-to-head comparison with CNPs ( Figure 6). ...
Parathyroid gland (PG) injury is the most common complication of thyroidectomy owing to the lack of approaches for surgeons to effectively distinguish PGs from surrounding thyroid glands (TGs) in the operation room. Herein, we report the development of biodegradable iron oxide nanoparticles (IONPs) as a promising contrast agent candidate for intraoperative PG visualization. We elucidated that locally administrated dark-colored IONPs readily diffuse in TGs but cannot infiltrate tissue-dense PGs, yielding a distinguishable contrast enhancement between PGs and TGs by naked eye observation. We performed unbiased and quantitative in vivo screenings to optimize particle size and concentration of IONPs for PG/TG contrast enhancement. Moreover, in vivo applications of IONPs via the local administration route demonstrate no adverse toxicities and can be biodegraded in the thyroid microenvironment within 3 months. To our knowledge, these promising findings provide the first in vivo evidence that IONPs can serve as a safe, biodegradable, and effective contrast agent candidate for improving PG visualization in thyroidectomy.
... Interestingly, nanoparticle breakdown barely affected iron homeostasis: only the genes coding for ferritin light chain (iron loading) and ferroportin (iron export) were up-regulated from day 3 on up to twofold on day 25 by the degradation process, whereas genes for ferritin heavy chain and the divalent metal transporter DMT1 were not upregulated. [55] In short term observations up to a few days, magnetic nanoparticle internalization impacted iron homeostasis slightly by up-regulation of iron-storage and/or iron-export proteins. [56][57][58] The transferrin receptor mRNA and protein levels (TFR1) were transiently down regulated. ...
In regenerative medicine, noncontact manipulation of cells enables new possibilities for tissue engineering. Due to their physicochemical properties, superparamagnetic iron oxide nanoparticles (SPIONs) are used in biomedicine for various applications, e.g., as drug transporters, contrast agents or to make cells maneuverable by magnetic forces. SPIONs attached to and/or taken up by cells enable their magnetic targeting for adoptive immune therapies or tissue engineering. Remote control of different “magnetized” cell types can be used to construct multilayered tissues without the need for a scaffold structure. Here, the suitability of SPIONs with various coatings, such as polyacrylic acid‐co‐maleic acid (PAM), lauric acid (LA), lauric acid‐human serum albumin (HSA), and citrate to magnetize cells is compared with the commercially available NanoShuttle‐PL, designed for use in magnetic 3D cell cultures. Depending on the amount of cellular labeling, magnetic control is more or less effective. In particular, PAM‐ and citrate‐coated SPIONs achieve good cellular loading and provide magnetic controllability of cells. In 2D cell culture, the magnetic cargo allows the patterned culture of cells. In 3D, SPIONs enable and accelerate spheroid formation as well as micropatterning using unloaded and loaded cells in parallel.
... Nanoparticles enter cells by different endocytic pathways and are transported into endosomes via endosomal-lysosomal pathway. In this low pH condition, degradation of the nanoparticles occurs with subsequent iron ions release [51], leading to an increase in the iron labile pool and lipid peroxidation accumulation via Fenton reaction. In this context, iron chelators like DFO and CPX can prevent iron-dependent lipid ROS production and ferroptosis by sequestering iron ions. ...
The use of nanomaterials rationally engineered to treat cancer is a burgeoning field that has reported great medical achievements. Iron-based polymeric nano-formulations with precisely tuned physicochemical properties are an expanding and versatile therapeutic strategy for tumor treatment. Recently, a peculiar type of regulated necrosis named ferroptosis has gained increased attention as a target for cancer therapy. Here, we show for the first time that novel iron oxide nanoparticles coated with gallic acid and polyacrylic acid (IONP–GA/PAA) possess intrinsic cytotoxic activity on various cancer cell lines. Indeed, IONP–GA/PAA treatment efficiently induces ferroptosis in glioblastoma, neuroblastoma, and fibrosarcoma cells. IONP–GA/PAA-induced ferroptosis was blocked by the canonical ferroptosis inhibitors, including deferoxamine and ciclopirox olamine (iron chelators), and ferrostatin-1, the lipophilic radical trap. These ferroptosis inhibitors also prevented the lipid hydroperoxide generation promoted by the nanoparticles. Altogether, we report on novel ferroptosis-inducing iron encapsulated nanoparticles with potent anti-cancer properties, which has promising potential for further in vivo validation.
... Indeed, they have been used as T2 contrast agents for MRI, [8][9][10] they are known to have a limited toxicity and to be degraded within cells, being then less toxic to the organism as compared to other magnetic nanomaterials. 6,11,12 For instance, these external fields-responsive IO NPs show great promise as new theranostics for localized and remotely activated magnetic hyperthermia therapy [13][14][15][16] combined with MRI. Upon alternative magnetic field application, they produce a local heat which is used for tumor treatment by magnetic field induced hyperthermia. ...
The chemical design of smart nanocarriers, providing in one nanoformulation combined anticancer therapies, still remains a challenge in the field of nanomedicine. Among nanomaterials, iron oxide-based core-shell nanostructures have been already studied for their intrinsic magnetic hyperthermia features that may be coupled with drug delivery. However, despite the great interest today for photo-induced hyperthermia, very few studies investigated the potential of such nanocarriers to combine photothermia and drug delivery. In this work, our aim was to design functional iron [email protected] mesoporous silica nanoparticles (denoted [email protected] NPs) loaded with a drug and able to combine in a same formulation near infrared (NIR) light induced photothermia with antitumor drug release. Herein, the NIR photothermal properties (SAR, specific absorption rates) of such nanomaterials were quantified for the first time as a function of the laser power and the NP amount. Aside the response to NIR light, the conditions to obtain very high drug loading (drug payloads up to 91 wt%) of the model antitumor drug doxorubicin (DOX) were optimized by varying different parameters, such as the NP surface chemistry (BARE (Si-OH), aminopropylsiloxane (APTES) and isobutyramide (IBAM)) and the pH of the drug impregnation aqueous solution. The drug release study of these core-shell systems in the presence or absence of NIR light demonstrated that the DOX release efficiency is mainly influenced by two parameters: surface chemistry (BARE ≥ IBAM ≥ APTES) and pH (pH 5.5 ≥ pH 6.5 ≥ pH 7.5). Furthermore, the temperature profiles under NIR light are found similar and independent from the pH range, the surface chemistry and the cycle number. Hence, the combination of local photothermia with lysosomal-like pH induced drug delivery (up to 40% release of the loaded drug) with these nanostructures could open the way towards new drug delivery nanoplatforms for nanomedecine applications.
... However, the intracellular fate of MNPs has only recently been explored in an attempt to understand how MNP degradation might affect their short-and long-term toxicity. In 3D mesenchymal stem cell spheroids, the extensive intracellular biodegradation of different MNPs leads to putative ferritin-rich spots appearing in or close to endolysosomes, in association with a loss of magnetization [35]. Likewise, magnetosomes (cubic MNPs) also undergo significant intracellular degradation in human mesenchymal stem cells, shifting the magnetite phase state to a ferrihydrite phase [36]. ...
Magnetic nanoparticles (MNPs) are potential theranostic tools that are biodegraded through different endocytic pathways. However, little is known about the endolysosomal network through which MNPs transit and the influence of the surface coating in this process. Here, we studied the intracellular transit of two MNPs with a similar iron oxide core size but with two distinct coatings: 3-aminopropyl-trietoxysilane (APS) and dimercaptosuccinic acid (DMSA). Using endolysosomal markers and a high throughput analysis of the associated proteome, we tracked the MNPs intracellularly in two different mouse cell lines, RAW264.7 (macrophages) and Pan02 (tumor cells). We did not detect differences in the MNP trafficking kinetics nor in the MNP-containing endolysosome phenotype in Pan02 cells. Nonetheless, DMSA-MNPs transited at slower rate than APS-MNPs in macrophages as measured by MNP accumulation in Rab7⁺ endolysosomes. Macrophage DMSA-MNP-containing endolysosomes had a higher percentage of lytic enzymes and catalytic proteins than their APS-MNP counterparts, concomitantly with a V-type ATPase enrichment, suggesting an acidic nature. Consequently, more autophagic vesicles are induced by DMSA-MNPs in macrophages, enhancing the expression of iron metabolism-related genes and proteins. Therefore, unlike Pan02 cells, the MNP coating appears to influence the intracellular trafficking rate and the endolysosome nature in macrophages. These results highlight how the MNP coating can determine the nanoparticle intracellular fate and biodegradation in a cell-type bias.
... 30 Amino acids are inexpensive, non-toxic, and biocompatible. [31][32][33][34] Amino acids play important roles in the body. They can reduce tumor cells. ...
Introduction: Due to the side effects of drugs, the development of nanoscale drug delivery systems has led to a significant improvement in medicinal therapies due to drug pharmacokinetics changes, decreased toxicity, and increased half-life of the drug. This study aimed to synthesize tamoxifen (TMX)-loaded L-lysine coated magnetic iron oxide nanoparticles as a nano-carrier to investigate its cytotoxic effects and anti-cancer properties against MCF-7 cancer cells.
Methods: Magnetic Fe3O4 nanoparticles were synthesized and coated with L-lysine (F-Lys NPs). Then, TMX was loaded onto these NPs. The characteristics of synthesized nanoparticles (F-Lys-TMX NPs) were evaluated by X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), differential scanning calorimetry (DSC), vibrating sample magnetometer (VSM), and thermogravimetric analysis (TGA). The drug release was analyzed at pH 5.8 and pH 7.4. The MCF-7 cells were exposed to F-Lys-TMX NPs, F-Lys NPs, and TMX for 24, 48, and 72 hours. To evaluate the cytotoxic potential of designed nanoparticles, MTT and apoptosis assays, real-time PCR, and cell cycle analysis was carried out.
Results: The F-Lys-TMX NPs had spherical morphology with a size ranging from 9 to 30 nm. By increasing the nanoparticles concentration and treatment time, more cell proliferation inhibition and apoptosis induction were observed in F-Lys-TMX NPs-treated cells compared to the TMX. The expression levels of ERBB2, cyclin D1, and cyclin E genes were down-regulated and expression levels of the caspase-3 and caspase-9 genes were up-regulated. Studies on the drug release revealed a slow and controlled pH-dependent release of the nanoparticles. Cell cycle analysis indicated that F-Lys-TMX NPs could arrest the cells at the G0/G1 phase.
Conclusion: The findings suggest that F-Lys-TMX NPs are more effective and have the potential for cell proliferation inhibition and apoptosis induction compared to the TMX. Hence, F-Lys-TMX NPs can be considered as an anti-cancer agent against MCF-7 breast cancer cells.
... Aside from the mechanism by which normal cells are not damaged by INPs treatments for cancer, and that cancer cells' behavior differs extremely from normal cells, here we can confirm that MNPs do not damage the surrounding host's environment as previously described [59] . That is, the excess intracellular breakdown products of these INPs are sequestered in intracellular ferritin stores, hence limiting exaggerated toxic oxidative response. ...
In this review, we provide an overview of methods for synthesizing magnetic nanoparticles (MNPs) with potential applications to biomedical research. We explore how the structure and properties of these particles are related to their diverse uses in medical diagnostics and bioanalysis. Special emphasis is placed on MNPs containing noble metals, which serve as biomarkers or active agents. Specifically, we focus on the application of direct and combined methods of atomic spectroscopy (ETAAS, AES/ICP–MS) to biomedical research. Experimental approaches to studying the behavior and transformations of MNPs in vitro and in vivo are considered. The importance of proper sample preparation in simulating the behavior of nanoparticles in biological media is highlighted. We also examine the significance of preparation techniques for the accurate determination of dissolved and nanosized forms in biological samples. Lastly, we assess the potential for the comprehensive studies of MNP behavior within complex biological systems, pointing toward future directions in this dynamic and promising field of research.
The field of nanomedicine is rapidly evolving, with new materials and formulations being reported almost daily. In this respect, inorganic and inorganic-organic composite nanomaterials have gained significant attention. However, the...
With their distinctive physicochemical features, nanoparticles have gained recognition as effective multifunctional tools for biomedical applications, with designs and compositions tailored for specific uses. Notably, magnetic nanoparticles stand out as first-in-class examples of multiple modalities provided by the iron-based composition. They have long been exploited as contrast agents for magnetic resonance imaging (MRI) or as anti-cancer agents generating therapeutic hyperthermia through high-frequency magnetic field application, known as magnetic hyperthermia (MHT). This review focuses on two more recent applications in oncology using iron-based nanomaterials: photothermal therapy (PTT) and ferroptosis. In PTT, the iron oxide core responds to a near-infrared (NIR) excitation and generates heat in its surrounding area, rivaling the efficiency of plasmonic gold-standard nanoparticles. This opens up the possibility of a dual MHT + PTT approach using a single nanomaterial. Moreover, the iron composition of magnetic nanoparticles can be harnessed as a chemotherapeutic asset. Degradation in the intracellular environment triggers the release of iron ions, which can stimulate the production of reactive oxygen species (ROS) and induce cancer cell death through ferroptosis. Consequently, this review emphasizes these emerging physical and chemical approaches for anti-cancer therapy facilitated by magnetic nanoparticles, combining all-in-one functionalities.
The uptake and the fate of Zr-based metal–organic-framework nanoparticles labeled with organic fluorophores in HeLa cells has been monitored with fluorescence detection and elemental analysis. The nanoparticles have been selected as a model system of carrier nanoparticles (here Zr-based metal–organic-framework nanoparticles) with integrated cargo molecules (here organic fluorophores), with aze that does not allow for efficient exocytosis, a material which only partly degrades under acidic conditions as present in endosomes/lysosomes, and with limited colloidal stability. Data show that, for Zr-based metal–organic-framework nanoparticles of 40 nm size as investigated here, the number of nanoparticles per cells decreases faster due to particle redistribution upon proliferation than due to nanoparticle exocytosis and that, thus, also for this system, exocytosis is not an efficient pathway for clearance of the nanoparticles from the cells.
The field of nanomedicine is rapidly evolving, with new materials and formulations being reported almost daily. In this respect, inorganic and inorganic-organic composite nanomaterials have gained significant attention. However, the use of new materials in clinical trials and their final approval as drugs has been hampered by several challenges, one of which is the complex and difficult to control nanomaterial chemistry that takes place within the body. Several reviews have summarized investigations on inorganic nanomaterial stability in model body fluids, cell cultures, and organisms, focusing on their degradation as well as the influence of corona formation. However, in addition to these aspects, various chemical reactions of nanomaterials, including phase transformation and/or the formation of new/secondary nanomaterials, have been reported. In this review, we discuss recent advances in our understanding of biochemical transformations of medically relevant inorganic (composite) nanomaterials in environments related to their applications. We provide a refined terminology for the primary reaction mechanisms involved to bridge the gaps between different disciplines involved in this research. Furthermore, we highlight suitable analytical techniques that can be harnessed to explore the described reactions. Finally, we highlight opportunities to utilize them for diagnostic and therapeutic purposes and discuss current challenges and research priorities.
Regenerative medicine has the potential to revolutionize healthcare by providing transplant options for patients suffering from tissue disease or organ failure. Cryopreservation offers a promising solution for long-term tissue and...
An attractive approach in cell therapies and medically oriented nanotechnologies is to interface magnetic nanoparticles with cells. This will endow the cells with sufficient magnetization for theranostic applications and for external magnetic field manipulation.
In tissue engineering, one challenge is to produce tissue analogues that are large, precisely organized and responsive to stimuli, preferably without the need for a supporting scaffold. One powerful tool for such biofabrication is certainly the bioprinting technology.
In magnetic tissue engineering, it is possible to use magnetic forces to manipulate cells, both individually and within aggregates, and thereby to produce three-dimensional artificial tissues with inherent capacities for further physical stimulation, a possibility that bioprinting does not offer yet.
Here, we first introduce the feasibility of using magnetic forces created by external (micro)magnets to form 3D tissue-like, scaffold-free structures. Because stem cells are essential in tissue engineering, such magnetic technologies were developed with magnetized stem cells and applied for instance to vascular or cartilage tissue engineering. One precondition to this approach, which lies in the magnetization of cells through the internalization of iron oxide magnetic nanoparticles, is to ensure the safety of magnetic nanoparticles with respect to cellular functions, which is initially discussed.
Then, we describe a magnetic tissue stretcher which, in a single step, allows to create a tissue composed of any type of cell, then to mature it and stimulate it by compression or stretching at any desired frequency, e.g. cyclically, offering new possibilities in the cardiac muscle tissue engineering field.
Visualizing nanoparticles made of organic material (e.g., polysaccharides, proteins, non-osmiophilic lipids) inside cells and tissues at transmission electron microscopyTransmission electron microscopy (TEM) is a difficult task due to the intrinsic weak electron density of these nanoconstructs, which makes them hardly distinguishable in the biological environment. We describe here a simple protocol to apply photooxidation to fluorescently labeled nanoparticles administered to cultured cells in vitro. The conversion of the fluorescent signal into a granular electron-dense reaction product through light irradiation in the presence of diaminobenzidine makes the nanoparticles clearly visible at the ultrastructural level. Our procedure proved to be reliable with various fluorophores and may be applied to any cell type.
Superparamagnetic iron oxide (SPIO)‐labeling of cells has been applied for magnetic resonance imaging (MRI) cell tracking for over 30 years, having resulted in a dozen or so clinical trials. SPIO nanoparticles are biodegradable and can be broken down into elemental iron, and hence the tolerance of cells to magnetic labeling has been overall high. Over the years, however, single reports have accumulated demonstrating that the proliferation, migration, adhesion, and differentiation of magnetically labeled cells may differ from unlabeled cells, with inhibition of chondrocytic differentiation of labeled human mesenchymal stem cells (hMSCs) as a notable example. This historical perspective provides an overview of some of the drawbacks that can be encountered with magnetic labeling. Now that magnetic particle imaging (MPI) cell tracking is emerging as a new in vivo cellular imaging modality, there has been a renaissance in the formulation of SPIO nanoparticles this time optimized for MPI. Lessons learned from the occasional past pitfalls encountered with SPIO‐labeling of cells for MRI may expedite the possible future clinical translation of (combined) MRI/MPI cell tracking.
The diverse applications of porphyrin-based nano-sized metal-organic frameworks (NMOFs) lead to great exposure risks to human and environment. Understanding the cellular biological effects (such as toxicity, distribution, and localization) of porphyrinic NMOFs is a prerequisite to the assessment of their health risk. However, the characteristics of distribution, localization, and immune response induced by porphyrinic NMOFs have not been studied yet. Here, we report the size-dependent biological effects of porphyrinic NMOFs under sublethal dose. Various sizes of PCN-224 (30, 90, and 180 nm) were taken as model porphyrinic NMOFs. We found that 30 nm PCN-224 gave the highest uptake content, followed by 90 and 180 nm PCN-224. The mechanism for uptake was clathrin-mediated for 30 and 90 nm PCN-224, but clathrin- and glycosylphosphatidylinositol-mediated for 180 nm PCN-224. All PCN-224 were localized in lysosome with size-dependent velocity of colocalization transport. 30 nm PCN-224 induced the highest released cytokines than 90 and 180 nm PCN-224 accompanied with the activation of NF-κB pathway. This work reveals the mechanisms for the endocytosis of PCN-224 and the release of cytokine induced by PCN-224, which is helpful for the health risk assessment of NMOFs.
Ultrasmall superparamagnetic iron oxide nanoparticles (uSPIOs) are attractive platforms for the development of smart contrast agents for magnetic resonance imaging (MRI). Oleic acid-capped uSPIOs are commercially available yet hydrophobic, hindering in vivo applications. A hydrophilic ligand with high affinity toward uSPIO surfaces can render uSPIOs water-soluble, biocompatible, and highly stable under physiological conditions. A small overall hydrodynamic diameter ensures optimal pharmacokinetics, tumor delivery profiles, and, of particular interest, enhanced T1 MR contrasts. In this study, for the first time, we synthesized a ligand that not only fulfills the as-proposed properties but also provides multiple reactive groups for further modifications. The synthesis delivers a facile approach using commercially available reactants, with resultant uSPIO-ligand constructs assembled through a single-step ligand exchange process. Structural and molecular size analyses confirmed size uniformity and small hydrodynamic diameter of the constructs. On average, 43 reactive amine groups were present per uSPIO nanoparticle. Its r1 relaxivity has been tested on a 7 Tesla MR instrument and is comparable to that of the clinically available T1 gadolinium-based contrast agent GBCA (1 vs 3 mM-1 s-1, respectively). A significant decrease in tumor T1 (15%) within 1 h of injection and complete signal recovery after 2 h were detected with a dose of 7 μg Fe/g mouse. The agent also has high r2 relaxivity and can be used for T2 contrast-enhanced MRI. Taken together, good relaxation and delivery properties and the presence of multiple surface reactive groups can facilitate its application as a universal MRI-compatible nanocarrier platform.
Toxoplasma gondii is a protozoan parasite capable of infecting a wide range of living beings, including felines that are the definitive hosts of the disease, toxoplasmosis, and livestock, birds and fish. In humans, the parasite can also be present in a latent or cystic form, the latter being able to become chronic, leading to lodging in brain, retina or muscles. Infection occurs upon consuming water or food contaminated with oocysts. The tachyzoites of RH strain have fast replication and relative difficulty of maintanence exclusively in vitro, often requiring stages of in vivo cultivation in experimental animals. Three-dimensional nanoestrutucured cell cultures can be helpful to build new forms of in vitro production with potential gains in practicality and yield. This work aimed to demonstrate the feasibility of use of three-dimensional culture of murine fibroblasts aggregated to nanoparticles as substrate for T. gondii tachyzoites with the intention of facilitating the management and in vitro replication of the parasite. Magnetic aggregation was used to produce cell spheroids, which were infected with tachyzoites of RH strain and maintained in culture. After infection spheroids were evaluated by transmission electron microscopy and fluorescence microscopy with 3D rendering of image stacks. The presence of the parasite was confirmed by PCR and the number of free parasites in culture was evaluated by flow cytometry. The three-dimensional culture model used showed sustainable production of tachyzoites within 24 hours after inoculum, showing itself as a potential surrogate for the use of animals for the maintenance of the parasite.
Les nanoparticules magnétiques (NPM) sont une classe prometteuse de nanomatériaux fonctionnels dans le domaine biomédical pour diverses applications telles que la thérapie anticancéreuse par hyperthermie ou la manipulation magnétique intracellulaire. Cependant, une limitation majeure de ces approches est leur piégeage endosomal après internalisation par la voie d'endocytose, conduisant à une chute de leur pouvoir chauffant et réduisant les possibilités de ciblage intracellulaire. L’objectif de ce projet était d'améliorer l'accès des NPM au cytosol en les fonctionnalisant avec des peptides cationiques. Arg9 est un peptide vecteur (cell-penetrating peptides, CPP) classique, capable de pénétrer dans les cellules par translocation directe, une perturbation transitoire de la membrane plasmique permettant d'atteindre le cytosol. D'autre part, il a été démontré que des séquences riches en résidus histidine favorisent l'échappement endosomal, probablement par effet d'éponge à protons. Les NPM utilisées sont des nanoparticules d’oxydes de fer enrobées de silice γ-Fe2O3@SiO2 permettant de fonctionnaliser leur surface par les peptides cationiques en utilisant la chimie click. Dans un premier temps, la synthèse et caractérisation des peptides et des nanoparticules cœur-coquilles ont été réalisées, puis la fonctionnalisation de ces nanoparticules par les peptides. L’internalisation des nanoparticules dans les cellules a ensuite été étudiée, par microscopie confocale combinée à des expériences de fuite de calcéine, et par microscopie électronique à transmission. Il a ainsi pu être montré que le greffage sur les nanoparticules de peptides riches en histidine permet de favoriser leur accès au cytosol.
Nanotechnology provides enormous momentum for the growth of various fields and contributes a huge number of nanoscale substances either as products or by-products to human society and the environment. However, exposure to various nanomaterials has been reported to elicit toxic effects on diverse organisms, which provokes worries about nanosafety issues and yields the nanotoxicology field. Here, we provide an informative review on this field aiming to further advance the toxicological research and safety evaluation of nanomaterials. Firstly, the toxicokinetics (i.e., absorption, distribution, metabolism, and excretion) of nanomaterials in terms of common exposure routes are introduced. Secondly, we discuss nanomaterial toxicity and the underlying mechanisms on the levels of organs, tissues, cells, and molecules. Because physicochemical properties play a significant role in nanomaterial toxicity, we briefly present how various properties impact nanotoxicity. Following, the major toxicological topics and their corresponding analytical approaches are provided to facilitate the advancement of the nanotoxicology field. Last but not the least, we stress the challenges facing and the possible solutions to fulfill the reliable and efficient safety evaluation of nanomaterials.
In this paper, we have employed K-d tree algorithmic based multiscale entropy analysis (MSE) to distinguish alcoholic subjects from non-alcoholic ones. Traditional MSE techniques have been used in many applications to quantify the dynamics of physiological time series at multiple temporal scales. However, this algorithm requires O(N²), i.e. exponential time and space complexity which is inefficient for long-term correlations and online application purposes. In the current study, we have employed a recently developed K-d tree approach to compute the entropy at multiple temporal scales. The probability function in the entropy term was converted into an orthogonal range. This study aims to quantify the dynamics of the electroencephalogram (EEG) signals to distinguish the alcoholic subjects from control subjects, by inspecting various coarse grained sequences formed at different time scales, using traditional MSE and comparing the results with fast MSE (fMSE). The performance was also measured in terms of specificity, sensitivity, total accuracy and receiver operating characteristics (ROC). Our findings show that fMSE, with a K-d tree algorithmic approach, improves the reliability of the entropy estimation in comparison with the traditional MSE. Moreover, this new technique is more promising to characterize the physiological changes having an affect at multiple time scales.
Magnetic labeling of stem cells enables their non-invasive detection by magnetic resonance imaging (MRI). Practically, most MRI studies have been limited to visualization of local engraftment as other sources of endogenous hypointense contrast complicate the interpretation of systemic (whole body) cell distribution. In addition, MRI cell tracking is inherently non-quantitative in nature. We report here on the potential of magnetic particle imaging (MPI) as a novel tomographic technique for non-invasive hot spot imaging and quantification of stem cells using superparamagnetic iron oxide (SPIO) tracers. Neural and mesenchymal stem cells, representing small and larger cell bodies, were labeled with three different SPIO tracer formulations, including two preparations that have previously been used in clinical MRI cell tracking studies (Feridex® and Resovist®). Magnetic particle spectroscopy (MPS) measurements demonstrated a linear correlation between MPI signal and iron content, for both homogeneous solutions of free particles in solution and for internalized and aggregated particles in labeled cells over a wide range of concentrations. The overall MP signal ranged from 1×10(-3) - 3×10(-4) Am(2)/g Fe, which was equivalent to 2×10(-14) - 1×10(-15) Am(2) per cell, indicating that cell numbers can be quantified with MPI analogous to the use of radiotracers in nuclear medicine or fluorine tracers in (19)F MRI. When SPIO-labeled cells were transplanted in mouse brain, they could be readily detected by MPI at a detection threshold of about 5×10(4) cells, with MPI/MRI overlays showing an excellent agreement between the hypointense MRI areas and MPI hot spots. The calculated tissue MPI signal ratio for 100,000 vs. 50,000 implanted cells was 2.08. Hence, MPI has potential to be further developed for quantitative and easy-to-interpret, tracer-based non-invasive imaging of cells, preferably with MRI as an adjunct anatomical imaging modality.
Long-term in vivo studies in murine models have shown that DMSA-coated nanoparticles accumulate in spleen, liver and lung tissues during extended periods of time (at least up to 3 months) without any significant signs of toxicity detected. During that time, nanoparticles undergo a process of biotransformation either by reducing the size or the particle aggregation or both. Using a rat model, we have evaluated the transformations of magnetic nanoparticles injected at low doses. Particles with two different coatings, dimercaptosuccinic acid (NP-DMSA) and polyethylene glycol (NP-PEG-(NH2)2) have been administered to animals, to evaluate the role of coating in the degradation of the particles. We have found that low doses of magnetic nanoparticles are quickly metabolized by the animals. In fact, using a nanoparticle dose four times lower than in previous experiments, NP-DMSA were not observed 24 h after the administration either in the liver or in the lungs. Interestingly, an increased amount of ferritin, the iron storage protein, was observed in liver tissues from rats that were treated with the low dose of NP-DMSA in comparison with the control ones, suggesting a rapid metabolization of the particles into ferritin iron. On the other side we have found that, NP-PEG-(NH2)2 are still detectable in several organs 24 h after their administration at low doses. Probably, due to the longer circulation times of the NP-PEG-(NH2)2, there is a delay in the arrival of the particles to the tissue and this is the reason why we are able to see the particles 24 h post-administration. PEG coating could also be protecting the nanoparticles from rapid degradation of the reticuloendothelial system. Knowledge on the biodistribution, circulation time and degradation processes is required to gain a better understanding of the safety evaluation of this kind of nanomaterial for biomedical applications.
A deeper knowledge on the effects of the degradation of magnetic nanoparticles on their magnetic properties is required to develop tools for the identification and quantification of magnetic nanoparticles in biological media by magnetic means.
Citric acid and phosphonoacetic acid-coated magnetic nanoparticles have been degraded in a medium that mimics lysosomal conditions. Magnetic measurements and transmission electron microscopy have been used to follow up the degradation process.
Particle size is reduced significantly in 24 h at pH 4.5 and body temperature. These transformations affect the magnetic properties of the compounds. A reduction of the interparticle interactions is observed just 4 h after the beginning of the degradation process. A strong paramagnetic contribution coming from the degradation products appears with time.
A model for the in vivo degradation of magnetic nanoparticles has been followed to gain insight on the changes of the magnetic properties of iron oxides during their degradation. The degradation kinetics is affected by the particle coating, in our case being the phosphonoacetic acid-coated particles degraded faster than the citric acid-coated ones.
Cellular aggregates (spheroids) are widely used in biophysics and tissue engineering as model systems for biological tissues. In this Letter we propose novel methods for molding stem-cell spheroids, deforming them, and measuring their interfacial and elastic properties with a single method based on cell tagging with magnetic nanoparticles and application of a magnetic field gradient. Magnetic molding yields spheroids of unprecedented sizes (up to a few mm in diameter) and preserves tissue integrity. On subjecting these spheroids to magnetic flattening (over 150g), we observed a size-dependent elastocapillary transition with two modes of deformation: liquid-drop-like behavior for small spheroids, and elastic-sphere-like behavior for larger spheroids, followed by relaxation to a liquidlike drop.
A current challenge for tissue engineering while restoring the function of diseased or damaged tissue is to customize the tissue according to the target area. Scaffold-free approaches usually yield spheroid shapes with the risk of necrosis at the center due to poor nutrient and oxygen diffusion. Here, we used magnetic forces developed at the cellular scale by miniaturized magnets to create rod-shaped aggregates of stem cells that subsequently matured into a tissue-like structure. However, during the maturation process, the tissue-rods spontaneously bent and coiled into sphere-like structures, triggered by the increasing cell-cell adhesion within the initially non-homogeneous tissue. Optimisation of the intra-tissular magnetic forces successfully hindered the transition, in order to produce stable rod-shaped stem cells aggregates.
In regenerative medicine, clinical imaging is indispensable for characterizing damaged tissue and for measuring the safety and efficacy of therapy. However, the ability to track the fate and function of transplanted cells with current technologies is limited. Exogenous contrast labels such as nanoparticles give a strong signal in the short term but are unreliable long term. Genetically encoded labels are good both short- and long-term in animals, but in the human setting they raise regulatory issues related to the safety of genomic integration and potential immunogenicity of reporter proteins. Imaging studies in brain, heart and islets share a common set of challenges, including developing novel labeling approaches to improve detection thresholds and early delineation of toxicity and function. Key areas for future research include addressing safety concerns associated with genetic labels and developing methods to follow cell survival, differentiation and integration with host tissue. Imaging may bridge the gap between cell therapies and health outcomes by elucidating mechanisms of action through longitudinal monitoring.
We report a highly reproducible route to synthesize iron oxide nanoparticles (IONPs) with control over size and shape and with size dispersions around 10%. By tuning the relative ratio of squalane to dibenzyl ether, which were used as solvents in the synthesis, the size of the particles could be varied from 14 to around 100 nm, while their shape evolved from cubic (for size ranges up to 35 nm) to truncated octahedra and octahedra (for sizes from 40 nm up to 100 nm). Fine tuning of the size within each of these ranges could be achieved by varying the heating ramp and the iron precursor to decanoic acid ratio. We also demonstrate direct water transfer of the as-synthesized IONPs via in situ ligand exchange with gallol polyethylene glycol molecules, the latter simply added to the crude nanocrystal mixture at 70 °C. The specific absorption rate (SAR) values measured on the water transferred IONPs, at frequencies and applied magnetic fields that are considered safe for patients, confirmed their high heating performance. Finally, this method allows the transfer of 35 nm nanocubes as individually coated and stable particles to the water phase. For the first time, the heating performance of such large IONPs has been studied. This work uncovers the possibility of using large IONPs for magnetic hyperthermia in tumor therapy.
Stem cell therapies offer great potentials in the treatment for a wide range of diseases and conditions. With so many stem cell replacement therapies going through clinical trials currently, there is a great need to understand the mechanisms behind a successful therapy, and one of the critical points of discovering them is to track stem cell migration, proliferation and differentiation in vivo. To be of most use tracking methods should ideally be non-invasive, high resolution and allow tracking in three dimensions. Magnetic resonance imaging (MRI) is one of the ideal methods, but requires a suitable contrast agent to be loaded to the cells to be tracked, and one of the most wide-spread in stem cell tracking is a group of agents known as magnetic nanoparticles. This review will explore the current use of magnetic nanoparticles in developing and performing stem cell therapies, and will investigate their potential limitations and the future directions magnetic nanoparticle tracking is heading in.
Despite remarkable efforts, metastatic melanoma (MM) still presents with significant mortality. Recently, mono-chemotherapies are increasingly replenished by more cancer-specific combination therapies involving death ligands and drugs interfering with cell signaling. Still, MM remains a fatal disease because tumors rapidly develop resistance to novel therapies thereby regaining tumorigenic capacity. Although genetically engineered mouse models for MM have been developed, at present no model is available that reliably mimics the human disease and is suitable for studying mechanisms of therapeutic obstacles including cell death resistance. To improve the increasing requests on new therapeutic alternatives, reliable human screening models are demanded that translate the findings from basic cellular research into clinical applications. By developing an organotypic full skin equivalent, harboring melanoma tumor spheroids of defined sizes we have invented a cell-based model that recapitulates both the 3D organization and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention. By extending our previous findings on melanoma cell sensitization toward TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) by co-application of sublethal doses of ultraviolet-B radiation (UVB) or cisplatin, we show significant differences in the therapeutical outcome to exist between regular two-dimensional (2D) and complex in vivo-like 3D models. Of note, while both treatment combinations killed the same cancer cell lines in 2D culture, skin equivalent-embedded melanoma spheroids are potently killed by TRAIL+cisplatin treatment but remain almost unaffected by the TRAIL+UVB combination. Consequently, we have established an organotypic human skin-melanoma model that will facilitate efforts to improve therapeutic outcomes for malignant melanoma by providing a platform for the investigation of cytotoxic treatments and tailored therapies in a more physiological setting.
The unique magnetic properties of iron oxide nanoparticles have paved the way for various biomedical applications, such as magnetic resonance cellular imaging or magnetically induced therapeutic hyperthermia. Living cells interact with nanoparticles by internalizing them within intracellular acidic compartments. Although no acute toxicity of iron oxide nanoparticles has been reported up to now, the mechanisms of nanoparticle degradation by the cellular environment are still unknown. In the organism, the long term integrity and physical state of iron-based nanoparticles are challenged by iron homeostasis. In this study, we monitored the degradation of 7 nm sized maghemite nanoparticles in a medium mimicking the intracellular environment. Magnetic nanoparticles with three distinct surface coatings, currently evaluated as MRI contrast agents, were shown to exhibit different kinetics of dissolution at an acidic pH in the presence of a citrate chelating agent. Our assessment of the physical state of the nanoparticles during degradation revealed that the magnetic properties, size distribution and structure of the remaining nanocrystals were identical to those of the initial suspension. This result suggests a model for nanoparticle degradation with rapidly dissolved nanocrystals and a reservoir of intact nanoparticles.
Aim:
Most of the research efforts in magnetic targeting have been focused on the development of magnetic nanovectors, while the investigation of methods for tracking their magnetic targeting efficiency remains inappropriately addressed. We propose herein a miniaturized approach for appraising magnetophoretic mobility at the nanoscale.
Materials & methods:
A simple and easy-to-use chamber including a microtip as a magnetic attractor was developed to perform magnetophoretic measurement at the size scale of nano-objects, and under bright field or fluorescence microscopy. Different sets of magnetic nanocontainers were produced and their magnetophoretic mobility was investigated. Real-time observations of the Brownian motion of the nanocontainers were also carried out for simultaneous size determination.
Results:
Attraction of the nanocontainers at the microtip is demonstrated as a qualitative method that immediately distinguishes magnetically responsive nano-objects. The combination of the analysis of Brownian motion, together with the magnetophoretic mobility, inferred both the size, the magnetophoretic velocity and the magnetic content of the nanocontainers. Additionally, nanomagnetophoresis experiments under fluorescence microscopy provided information on the constitutive core/shell integrity of the nanocontainers and the co-internalization of a fluorescent cargo.
Conclusion:
This nanomagnetophoresis method represents a promising tool to estimate the feasibility of magnetic targeting in laboratory routine.
This work demonstrated that ultrasmall gold nanoparticles (AuNPs) smaller than 10 nm display unique advantages over nanoparticles larger than 10 nm in terms of localization to, and penetration of, breast cancer cells, multicellular tumor spheroids, and tumors in mice. Au@tiopronin nanoparticles that have tunable sizes from 2 to 15 nm with identical surface coatings of tiopronin and charge were successfully prepared. For monolayer cells, the smaller the Au@tiopronin NPs, the more AuNPs found in each cell. In addition, the accumulation of Au NPs in the ex vivo tumor model was size-dependent: smaller AuNPs were able to penetrate deeply into tumor spheroids, whereas 15 nm nanoparticles were not. Owing to their ultrasmall nanostructure, 2 and 6 nm nanoparticles showed high levels of accumulation in tumor tissue in mice after a single intravenous injection. Surprisingly, both 2 and 6 nm Au@tiopronin nanoparticles were distributed throughout the cytoplasm and nucleus of cancer cells in vitro and in vivo, whereas 15 nm Au@tiopronin nanoparticles were found only in the cytoplasm, where they formed aggregates. The ex vivo multicellular spheroid proved to be a good model to simulate in vivo tumor tissue and evaluate nanoparticle penetration behavior. This work gives important insights into the design and functionalization of nanoparticles to achieve high levels of accumulation in tumors.
Aims: Therapeutic intracavitary stem cell infusion currently suffers from poor myocardial homing. We examined whether cardiac cell retention could be enhanced by magnetic targeting of endothelial progenitor cells (EPCs) loaded with iron oxide nanoparticles.Methods and Results: EPCs were magnetically labeled with citrate-coated iron oxide nanoparticles. Cell proliferation, migration and CXCR4 chemokine receptor expression were assessed in different labeling conditions and no adverse effects of the magnetic label were observed. The magnetophoretic mobility of labeled EPCs was determined in vitro, with the same magnet as that subsequently used in vivo. Coronary artery occlusion was induced for 30 minutes in 36 rats (31 survivors), followed by 20 minutes of reperfusion. The rats were randomized to receive, during brief aortic cross-clamping, direct intraventricular injection of culture medium (n=7) or magnetically labeled EPCs (n=24), with (n=14) or without (n=10) subcutaneous insertion of a magnet over the chest cavity (n=14). The hearts were explanted 24 h later and engrafted cells were visualized by magnetic resonance imaging (MRI) of the heart at 1.5 T. Their abundance in the myocardium was also analyzed semi-quantitatively by immunofluorescence, and quantitatively by real time polymerase chain reaction (RT-PCR). Although differences in cell retention between groups failed to be statistically significant using RT-PCR quantification, due to the variability of the animal model, immunostaining showed that the average number of engrafted EPCs was significantly ten times higher with than without magnetic targeting. There was thus a consistent trend favoring the magnet-treated hearts, thereby suggesting magnetic targeting as a potentially new mean of enhancing myocardial homing of intravascularly delivered stem cells.Conclusion: Magnetic targeting has the potential to enhance myocardial retention of intravascularly delivered endothelial progenitor cells.
The potential applications of superparamagnetic iron oxide nanoparticles (SPIONs) in several nanomedical fields have attracted intense interest based on the cell-nano interaction. However, the mechanisms underlying cell uptake, the intracellular trail, final fate and the biological effects of SPIONs have not yet been clearly elucidated. Here, we showed that multiple endocytic pathways were involved in the internalization process of SPIONs in the RAW264.7 macrophage. The internalized SPIONs were biocompatible and used three different metabolic pathways: The SPIONs were distributed to daughter cells during mitosis; they were degraded in the lysosome and free iron was released into the intracellular iron metabolic pool; and, the intact SPIONs were potentially exocytosed out of the cells. The internalized SPIONs did not induce cell damage but affected iron metabolism, inducing the upregulation of ferritin light chain at both the mRNA and protein levels and ferroportin 1 at the mRNA level. These results may contribute to the development of nanobiology and to the safe use of SPIONs in medicine when administered as a contrast medium or a drug delivery tool.
This paper presents the outcomes from a workshop of the European Network on the Health and Environmental Impact of Nanomaterials (NanoImpactNet). During the workshop, 45 experts in the field of safety assessment of engineered nanomaterials addressed the need to systematically study sets of engineered nanomaterials with specific metrics to generate a data set which would allow the establishment of dose-response relations. The group concluded that international cooperation and worldwide standardization of terminology, reference materials and protocols are needed to make progress in establishing lists of essential metrics. High quality data necessitates the development of harmonized study approaches and adequate reporting of data. Priority metrics can only be based on well-characterized dose-response relations derived from the systematic study of the bio-kinetics and bio-interactions of nanomaterials at both organism and (sub)-cellular levels. In addition, increased effort is needed to develop and validate analytical methods to determine these metrics in a complex matrix.
Superparamagnetic iron oxide nanoparticles (SPION) are increasingly used to label human bone marrow stromal cells (BMSCs, also called "mesenchymal stem cells") to monitor their fate by in vivo MRI, and by histology after Prussian blue (PB) staining. SPION-labeling appears to be safe as assessed by in vitro differentiation of BMSCs, however, we chose to resolve the question of the effect of labeling on maintaining the "stemness" of cells within the BMSC population in vivo. Assays performed include colony forming efficiency, CD146 expression, gene expression profiling, and the "gold standard" of evaluating bone and myelosupportive stroma formation in vivo in immuncompromised recipients. SPION-labeling did not alter these assays. Comparable abundant bone with adjoining host hematopoietic cells were seen in cohorts of mice that were implanted with SPION-labeled or unlabeled BMSCs. PB+ adipocytes were noted, demonstrating their donor origin, as well as PB+ pericytes, indicative of self-renewal of the stem cell in the BMSC population. This study confirms that SPION labeling does not alter the differentiation potential of the subset of stem cells within BMSCs.
Cell culture is an essential tool in drug discovery, tissue engineering and stem cell research. Conventional tissue culture produces two-dimensional cell growth with gene expression, signalling and morphology that can be different from those found in vivo, and this compromises its clinical relevance. Here, we report a three-dimensional tissue culture based on magnetic levitation of cells in the presence of a hydrogel consisting of gold, magnetic iron oxide nanoparticles and filamentous bacteriophage. By spatially controlling the magnetic field, the geometry of the cell mass can be manipulated, and multicellular clustering of different cell types in co-culture can be achieved. Magnetically levitated human glioblastoma cells showed similar protein expression profiles to those observed in human tumour xenografts. Taken together, these results indicate that levitated three-dimensional culture with magnetized phage-based hydrogels more closely recapitulates in vivo protein expression and may be more feasible for long-term multicellular studies.
What happens to inorganic nanoparticles (NPs), such as plasmonic gold or silver, superparamagnetic iron oxide, or fluorescent quantum dot NPs after they have been administrated to a living being? This review discusses the integrity, biodistribution, and fate of NPs after in vivo administration. The hybrid nature of the NPs is described, conceptually divided into the inorganic core, the engineered surface coating comprising of the ligand shell and optionally also bio-conjugates, and the corona of adsorbed biological molecules. Empirical evidence shows that all of these three compounds may degrade individually in vivo and can drastically modify the life cycle and biodistribution of the whole heterostructure. Thus, the NPs may be decomposed into different parts, whose biodistribution and fate would need to be analyzed individually. Multiple labeling and quantification strategies for such a purpose will be discussed. All reviewed data indicate that NPs in vivo should no longer be considered as homogeneous entities, but should be seen as inorganic/organic/biological nano-hybrids with complex and intricately linked distribution and degradation pathways.
Understanding transport of carbon nanotubes (CNTs) within tissues is essential for biomedical imaging and drug delivery using these carriers. Compared to traditional cell cultures in animal studies, three-dimensional tissue replicas approach the complexity of the actual organs and enable high temporal and spatial resolution of the CNT permeation. We investigated diffusional transport of CNTs in highly uniform spheroids of hepatocellular carcinoma and found that apparent diffusion coefficients of CNTs in these tissue replicas are comparable to diffusion rates of similarly charged molecules with molecular weight 10,000× lower. Moreover, diffusivity of CNTs in tissues is enhanced after functionalization with transforming growth factor β1. The anomalous trend is attributed to the planar diffusion of CNTs along cellular membranes reducing effective dimensionality of diffusional space. These findings indicate that nanotubes and potentially similar nanostructures are capable of fast and deep permeation into the tissue, which is often difficult to realize with anticancer agents.
Safe implementation of nanotechnology and nanomedicine requires an in-depth understanding of the life cycle of nanoparticles in the body. Here we investigate the long term fate of gold/iron oxide heterostructures after intravenous injection in mice. We show these heterostructures degrade in vivo and that the magnetic and optical properties change during the degradation process. These particles eventually eliminate from the body. The comparison of two different coating shells for heterostructures - amphiphilic polymer or polyethylene glycol - reveals the long lasting impact of initial surface properties on the nanocrystal degradability and on the kinetics of elimination of magnetic iron and gold from liver and spleen. Modulate nanoparticles reactivity to the biological environment by the choice of materials and surface functionalization may provide new directions in the design of multifunctional nanomedicines with predictable fate.
Inorganic nanoparticles are frequently engineered with an organic surface coating to improve their physicochemical properties, and it is well known that their colloidal properties may change upon internalization by cells. While the stability of such nanoparticles is typically assayed in simple in vitro tests, their stability in a mammalian organism remains unknown. Here, we show that firmly grafted polymer shells around gold nanoparticles may degrade when injected into rats. We synthesized monodisperse radioactively labelled gold nanoparticles ((198)Au) and engineered an (111)In-labelled polymer shell around them. Upon intravenous injection into rats, quantitative biodistribution analyses performed independently for (198)Au and (111)In showed partial removal of the polymer shell in vivo. While (198)Au accumulates mostly in the liver, part of the (111)In shows a non-particulate biodistribution similar to intravenous injection of chelated (111)In. Further in vitro studies suggest that degradation of the polymer shell is caused by proteolytic enzymes in the liver. Our results show that even nanoparticles with high colloidal stability can change their physicochemical properties in vivo.
An overview of the current understanding of the behavior of iron oxide nanoparticles (IONPs), Q-Dots, silver and ZnO NPs, and Au NPs as a result of highly varying conditions to which they will be exposed when used in biomedical research or when distributed in the environment. It was shown that depending on the nature of the core material degradation of the NPs can occur, and its extent depends on the microenvironment to which they are exposed. In this connection, distinction should be made between degradation of the inorganic core, which highly depends on the material of the core, and degradation of the organic surface coating, which in particular affects colloidal stability and thus biodistribution of the NPs. For degrading NPs, the process of degradation brings along substantial difficulties in understanding their toxicological profile as not only the NP but also its released ions and the combination of the two can all have different biodistributions and toxic effects, which must be studied in detail. Other focus points are NP shape and size, where the ratio of surface area over volume should be kept as low as possible as degradation will occur at the surface of the NPs. Furthermore, the composition of the chemical core can be adjusted by the addition of other metal ions and hereby altering the matrix of the inorganic cores, which can result in enhanced chemical stability against pH-dependent degradation.
Magnetization relaxation mechanisms strongly influence how magnetic nanoparticles respond to high-frequency fields in applications such as magnetic hyperthermia. The dominant mechanism depends on the mobility of the particles, which will be affected in turn by their microenvironment. In this study AC susceptometry was used to follow the in situ magnetic response of model systems of blocked and superparamagnetic nanoparticles, following their cellular internalization and subsequent release by freeze-thaw lysis. The AC susceptibility signal from internalized particles in live cells showed only Néel relaxation, consistent with measurements of immobilized nanoparticle suspensions. However, Brownian relaxation was restored after cell lysis, indicating that the immobilization effect was reversible and that nanoparticle integrity was maintained in the cells. The results presented demonstrate that cellular internalization can disable Brownian relaxation, which has significant implications for designing suitable nanoparticles for intracellular hyperthermia applications. Further to this, the results highlight the possibility that particles could be released in reusable form from degrading cells following hyperthermia treatment, and subsequently reabsorbed by viable cells.
Quantitatively tracking engraftment of intracerebrally or intravenously transplanted stem cells and evaluating their concomitant therapeutic efficacy for stroke has been a challenge in the field of stem cell therapy. In this study, first, an MRI/SPECT/fluorescent tri-modal probe (125I-fSiO4@SPIOs) is synthesized for quantitatively tracking mesenchymal stem cells (MSCs) transplanted intracerebrally or intravenously into stroke rats, and then the therapeutic efficacy of MSCs delivered by both routes and the possible mechanism of the therapy are evaluated. It is demonstrated that (125)I-fSiO4@SPIOs have high efficiency for labeling MSCs without affecting their viability, differentiation, and proliferation capacity , and found that 35% of intracerebrally injected MSCs migrate along the corpus callosum to the lesion area, while 90% of intravenously injected MSCs remain trapped in the lung at 14 days after MSC transplantation. However, neurobehavioral outcomes are significantly improved in both transplantation groups, which are accompanied by increases of vascular endothelial growth factor, basic fibroblast growth factor, and tissue inhibitor of metalloproteinases-3 in blood, lung, and brain tissue (p < 0.05). The study demonstrates that 125I-fSiO4@SPIOs are robust probe for long-term tracking of MSCs in the treatment of ischemic brain and MSCs delivered via both routes improve neurobehavioral outcomes in ischemic rats.
Cell aggregates, or spheroids, have been used as building blocks to fabricate scaffold-free tissues that can closely mimic the native three-dimensional in vivo environment for broad applications including regenerative medicine and high throughput testing of drugs. The incorporation of magnetic nanoparticles (MNPs) into spheroids permits the manipulation of spheroids into desired shapes, patterns, and tissues using magnetic forces. Current strategies incorporating MNPs often involve cellular uptake, and should therefore be avoided because it induces adverse effects on cell activity, viability, and phenotype. Here, we report a Janus structure of magnetic cellular spheroids (JMCS) with spatial control of MNPs to form two distinct domains: cells and extracellular MNPs. This separation of cells and MNPs within magnetic cellular spheroids was successfully incorporated into cellular spheroids with various cellular and extracellular compositions and contents. The amount of cells that internalized MNPs was quantified and showed that JMCSs resulted in significantly lower internalization (35%) compared to uptake spheroids (83%, p < 0.05). Furthermore, the addition of MNPs to cellular spheroids using the Janus method has no adverse effects on cellular viability up to seven weeks, with spheroids maintaining at least 82% viability over 7 weeks when compared to control spheroids without MNPs. By safely incorporating MNPs into cellular spheroids, results demonstrated that JMCSs were capable of magnetic manipulation, and that magnetic forces used during magnetic force assembly mediate fusion into controlled patterns and complex tissues. Finally, JMCSs were assembled and fused into a vascular tissue construct 5 mm in diameter using magnetic force assembly.
Recent advances in cell therapy and tissue engineering open new windows for regenerative medicine, but still necessitate innovative non invasive imaging technologies. We demonstrate that high resolution Magnetic Resonance Imaging (MRI) allows combining cellular-scale resolution with the ability to detect several cell types simultaneously at any tissue depth. Two contrast agents, based on iron oxide and gadolinium oxide rigid nano-platforms, were used to "tattoo" endothelial cells and stem cells, respectively, with no impact on cells functions, including their capacity for differentiation. The labeled cells contrast properties were optimized for simultaneous MRI detection: endothelial cells and stem cells seeded together in a polysaccharide-based scaffold material for tissue engineering appeared respectively in black and white, and could be tracked, at the cellular level, both in vitro and in vivo. In addition, endothelial cells labeled with iron oxide nanoparticles could be remotely manipulated by applying a magnetic field, allowing the creation of vessel substitutes with in depth detection of individual cellular components.
Although iron oxide magnetic nanoparticles (MNP) have been proposed for numerous biomedical applications, little is known about their biotransformation and long-term toxicity in the body. Dimercaptosuccinic acid (DMSA)-coated magnetic nanoparticles have been proven efficient for in vivo drug delivery, but these results must nonetheless be sustained by comprehensive studies of long-term distribution, degradation and toxicity. We studied DMSA-coated magnetic nanoparticles effects in vitro on NCTC 1469 non-parenchymal hepatocytes, and analyzed their biodistribution and biotransformation in vivo in C57BL/6 mice. Our results indicate that DMSA-coated magnetic nanoparticles have little effect on cell viability, oxidative stress, cell cycle or apoptosis on NCTC 1469 cells in vitro. In vivo distribution and transformation was studied by alternating current magnetic susceptibility measurements, a technique that permits distinction of MNP from other iron species. Our results show that DMSA-coated MNP accumulate in spleen, liver and lung tissues for extended periods of time, in which nanoparticles undergo a process of conversion from superparamagnetic iron oxide nanoparticles to other non-superparamagnetic iron forms, with no significant signs of toxicity. This work provides first evidence of DMSA-coated magnetite nanoparticles biotransformation in vivo.
The long term fate of nanomaterials in biological environment represents a critical matter, which determines environmental effects and potential risks for human health. Predicting these risks requires understanding of nanoparticle transformations, persistence and degradation, some issues somehow ignored so far. Safe by design, inorganic nanostructures are being envisioned for therapy, yet fundamental principles of their processing in biological systems, change in physical properties and in situ degradability have not been thoroughly assessed. Here we report the longitudinal visualization of iron oxide nanocube transformations inflicted by intracellular-like environment. Structural degradation of individual nanocubes with two different surface coatings (amphiphilic polymer shell and polyethylene-glycol ligand molecules) was monitored at the atomic scale with aberration-corrected high-resolution transmission electron microscopy. Our results suggest that the polymer coating controls surface reactivity and that availability and access of chelating agents to the crystal surface govern the degradation rate. This in situ study of single nanocube degradation was compared to intracellular transformations observed in mice over fourteen days after intravenous injection, revealing the role of nanoparticle clustering, intracellular sorting within degradation compartments and iron transfer and recycling into ferritin storage proteins. Our approach reduces the gap between in situ nanoscale observations in mimicking biological environments and in vivo real tracking of nanoparticle fate.
Magnetic forces induce cell condensation necessary for stem cell differentiation into cartilage and elicit the formation of a tissue-like structure: Magnetically driven fusion of aggregates assembled by micromagnets results in the formation of a continuous tissue layer containing abundant cartilage matrix.
The specific targeting of cells to sites of tissue damage in vivo is a major challenge precluding the success of stem cell-based therapies. Magnetic particle-based targeting may provide a solution. Our aim was to provide a model system to study the trapping and potential targeting of human mesenchymal stem cells (MSCs) during in vitro fluid flow, which ultimately will inform cell targeting in vivo. In this system magnet arrays were used to trap superparamagnetic iron oxide particle-doped MSCs. The in vitro experiments demonstrated successful cell trapping, where the volume of cells trapped increased with magnetic particle concentration and decreased with increasing flow rate. Analysis of gene expression revealed significant increases in COL1A2 and SOX9. Using principles established in vitro, a proof-of-concept in vivo experiment demonstrated that magnetic particle-doped, luciferase-expressing MSCs were trapped by an implanted magnet in a subcutaneous wound model in nude mice. Our results demonstrate the effectiveness of using an in vitro model for testing superparamagnetic iron oxide particles to develop successful MSC targeting strategies during fluid flow, which ultimately can be translated to in vivo targeted delivery of cells via the circulation in a variety of tissue-repair models. Copyright © 2012 John Wiley & Sons, Ltd.
The labeling of stem cells with iron oxide nanoparticles is increasingly used to enable MRI cell tracking and magnetic cell manipulation, stimulating the fields of tissue engineering and cell therapy. However, the impact of magnetic labeling on stem-cell differentiation is still controversial. One compromising factor for successful differentiation may arise from early interactions of nanoparticles with cells during the labeling procedure. It is hypothesized that the lack of control over nanoparticle colloidal stability in biological media may lead to undesirable nanoparticle localization, overestimation of cellular uptake, misleading MRI cell tracking, and further impairment of differentiation. Herein a method is described for labeling mesenchymal stem cells (MSC), in which the physical state of citrate-coated nanoparticles (dispersed versus aggregated) can be kinetically tuned through electrostatic and magnetic triggers, as monitored by diffusion light scattering in the extracellular medium and by optical and electronic microscopy in cells. A set of statistical cell-by-cell measurements (flow cytometry, single-cell magnetophoresis, and high-resolution MRI cellular detection) is used to independently quantify the nanoparticle cell uptake and the effects of nanoparticle aggregation. Such aggregation confounds MRI cell detection as well as global iron quantification and has adverse effects on chondrogenetic differentiation. Magnetic labeling conditions with perfectly stable nanoparticles-suitable for obtaining differentiation-capable magnetic stem cells for use in cell therapy-are subsequently identified.
A simple, fast, efficient, and widely applicable method to radiolabel the cores of monodisperse superparamagnetic iron oxide nanoparticles (SPIOs) with (59)Fe was developed. These cores can be used as precursors for a variety of functionalized nanodevices. A quality control using filtration techniques, size-exclusion chromatography, chemical degradation methods, transmission electron microscopy, and magnetic resonance imaging showed that the nanoparticles were stably labeled with (59)Fe. Furthermore, the particle structure and the magnetic properties of the SPIOs were unchanged. In a second approach, monodisperse SPIOs stabilized with (14)C-oleic acid were synthesized, and the stability of this shell labeling was studied. In proof of principle experiments, the (59)Fe-SPIOs coated with different shells to make them water-soluble were used to evaluate and compare in vivo pharmacokinetic parameters such as blood half-life. It could also be shown that our radiolabeled SPIOs embedded in recombinant lipoproteins can be used to quantify physiological processes in closer detail than hitherto possible. In vitro and in vivo experiments showed that the (59)Fe label is stable enough to be applied in vivo, whereas the (14)C label is rapidly removed from the iron core and is not adequate for in vivo studies. To obtain meaningful results in in vivo experiments, only (59)Fe-labeled SPIOs should be used.
To investigate the cellular consequences of a prolonged cellular presence of large amounts of iron oxide nanoparticles (IONPs) as well as the fate of such particles in brain cells, cultured primary astrocytes were loaded for 4h with dimercaptosuccinate-coated IONPs. Subsequently, the IONP-treated cells were incubated for up to 7 days in IONP-free medium and the cell viability, metabolic parameters and iron metabolism of the cells were investigated. Despite an up to 100-fold elevated specific cellular iron content, IONP-loaded cells remained viable throughout the 7 day main incubation and did not show any substantial alteration in glucose and glutathione metabolism. During the incubation, the high cellular iron content of IONP-loaded astrocytes remained almost constant. Electron microscopy revealed that after 7 days of incubation most of the cellular iron was still present in IONP-filled vesicles. However, the transient appearance of reactive oxygen species (ROS) as well as a strong increase in cellular levels of the iron storage protein ferritin suggest that at least some low-molecular-weight iron was liberated from the accumulated IONPs. These results demonstrate that even the prolonged presence of large amounts of accumulated IONPs does not harm astrocytes and that these cells store IONP-derived iron in ferritin.
Superparamagnetic iron oxide (SPIO) nanoparticles have been widely used for stem cell labeling and tracking. Surface modification has been known to improve biocompatibility, biodistribution, and labeling efficiency of SPIO nanoparticles. However, the effects of amine (NH 3+)-surface-modified SPIO nanoparticles on proliferation and differentiation of human mesenchymal stem cells (hMSCs) remain unclear. The purpose of this study is to investigate how amine-surface-modified SPIO nanoparticles affected hMSCs. In this study, intracellular uptake and the contiguous presence of amine-surface-modified SPIO nanoparticles in hMSCs were demonstrated by Prussian blue staining, transmission electron microscopy and magnetic resonance imaging. Moreover, accelerated cell proliferation was found to be associated with cellular internalization of amine-surface-modified SPIO nanoparticles. The osteogenic and chondrogenic differentiation potentials of hMSCs were impaired after treating with SPIO, while adipogenic potential was relatively unaffected. Altered cytokine production profile in hMSCs caused by amine-surface-modified SPIO nanoparticles may account for the increased proliferation and impaired differentiation potentials; concentrations of the growth factors in the SPIO-labeled condition medium including amphiregulin, glial cell-derived neurotrophic factor, heparin-binding EGF-like growth factor and vascular endothelial growth factor, as well as soluble form of macrophage colony-stimulating factor receptor and SCF receptor, were higher than in the unlabeled-condition medium. In summary, although amine-surface-modified SPIO labeling is effective for cell tracking, properties of hMSCs may alter as a consequence and this needs to be taken into account when evaluating therapeutic efficacies of SPIO-labeled stem cells in vivo.
Iron oxide nanoparticles are a useful diagnostic contrast agent and have great potential for therapeutic applications. Multiple emerging diagnostic and therapeutic applications and the numerous versatile parameters of the nanoparticle platform require a robust biological model for characterization and assessment. Here we investigate the use of iron oxide nanoparticles that target tumor vasculature, via the tumstatin peptide, in a novel three-dimensional tissue culture model. The developed tissue culture model more closely mimics the in vivo environment with a leaky endothelium coating around a glioma tumor mass. Tumstatin-iron oxide nanoparticles showed penetration and selective targeting to endothelial cell coating on the tumor in the three-dimensional model, and had approximately 2 times greater uptake in vitro and 2.7 times tumor neo-vascularization inhibition. Tumstatin provides targeting and therapeutic capabilities to the iron oxide nanoparticle diagnostic contrast agent platform. And the novel endothelial cell-coated tumor model provides an in vitro microtissue environment to evaluate nanoparticles without moving into costly and time-consuming animal models.
A general strategy is described which allows for transferring hydrophobically capped nanocrystals from organic to aqueous solution by wrapping an amphiphilic polymer around the particles. In particular, high quality CoPt3, Au, CdSe/ZnS, and Fe2O3 nanocrystals have been water-solubilized in this way. Analysis with transmission electron microscopy, gel electrophoresis, and fluorescence correlation spectroscopy demonstrates that monodispersity of the particles is conserved upon phase transfer to aqueous solution.
Magnetic iron oxide nanoparticles (IONPs) have been used for a variety of neurobiological applications, although little is yet known as to the fate of such particles in brain cells. To address these questions, we have exposed oligodendroglial OLN-93 cells to dimercaptosuccinate-coated IONPs. Treatment of the cells strongly increased the specific cellular iron content proportional to the IONP concentrations applied (0-1000 μM total iron as IONPs) up to 300-fold, but did not cause any acute cytotoxicity or induce oxidative stress. To investigate the potential of OLN-93 cells to liberate iron from the accumulated IONPs, we have studied the upregulation of the iron storage protein ferritin and the cell proliferation as cellular processes that depend on the availability of low-molecular-weight iron. The presence of IONPs caused a concentration-dependent increase in the amount of cellular ferritin and partially bypassed the inhibition of cell proliferation by the iron chelator deferoxamine. These data demonstrate that viable OLN-93 cells efficiently take up IONPs and suggest that these cells are able to use iron liberated from accumulated IONPs for their metabolism.
Superparamagnetic iron oxide nanoparticles are used in various medical applications including magnetic resonance imaging, magnetic hyperthermia, and targeted drug and gene delivery. When used in vivo, these nanoparticles interact with endothelial cells lining all blood vessels, therefore it is crucial to understand endothelial cell functional changes and toxicity upon nanoparticle exposure. We incubated porcine aortic endothelial cells with varying concentrations of bare iron oxide nanoparticles (20-40 nm), and measured cellular reactive oxygen species (ROS) formation, morphology and cytoskeletal organization, death, and elastic modulus. Intracellular ROS increased more than 800% after 3 h of nanoparticle exposure (0.5 mg mL(-1)). Endothelial cells elongated to more than twice their initial length by 12 h, and actin stress fibers formed within the cells. This change in the actin cytoskeleton increased cell elastic modulus by 50%. When ROS formation was blocked using scavengers, initial cell morphology and the actin cytoskeleton remained intact, and cell viability increased. These studies suggest that iron oxide nanoparticles induce ROS formation, which disrupts the actin cytoskeleton and alters endothelial cell morphology and mechanics. If ROS formation is decreased using ROS inhibitors, either as a component of the nanoparticle coating or by systemic administration, higher nanoparticle concentrations might be used with greater efficacy and diminished side effects.
Iron oxide nanoparticles (NPs) are frequently employed in biomedical research as magnetic resonance (MR) contrast agents where high intracellular levels are required to clearly depict signal alterations. To date, the toxicity and applicability of these particles have not been completely unraveled. Here, we show that endosomal localization of different iron oxide particles results in their degradation and in reduced MR contrast, the rate of which is governed mainly by the stability of the coating. The release of ferric iron generates reactive species, which greatly affect cell functionality. Lipid-coated NPs display the highest stability and furthermore exhibit intracellular clustering, which significantly enhances their MR properties and intracellular persistence. These findings are of considerable importance because, depending on the nature of the coating, particles can be rapidly degraded, thus completely annihilating their MR contrast to levels not detectable when compared to controls and greatly impeding cell functionality, thereby hindering their application in functional in vivo studies.
Skeletal muscle tissue engineering is currently applied in a variety of research fields, including regenerative medicine, drug screening, and bioactuator development, all of which require the fabrication of biomimic and functional skeletal muscle tissues. In the present study, magnetite cationic liposomes were used to magnetically label C2C12 myoblast cells for the construction of three-dimensional artificial skeletal muscle tissues by an applied magnetic force. Skeletal muscle functions, such as biochemical and contractile properties, were evaluated for the artificial tissue constructs. Histological studies revealed that elongated and multinucleated myotubes were observed within the tissue. Expression of muscle-specific markers, such as myogenin, myosin heavy chain and tropomyosin, were detected in the tissue constructs by western blot analysis. Further, creatine kinase activity increased during differentiation. In response to electric pulses, the artificial tissue constructs contracted to generate a physical force (the maximum twitch force, 33.2 μN [1.06 mN/mm2]). Rheobase and chronaxie of the tissue were determined as 4.45 V and 0.72 ms, respectively. These results indicate that the artificial skeletal muscle tissue constructs fabricated in this study were physiologically functional and the data obtained for the evaluation of their functional properties may provide useful information for future skeletal muscle tissue engineering studies.
The success of cardiac stem cell therapies is limited by low cell retention, due at least in part to washout via coronary veins.
We sought to counter the efflux of transplanted cells by rendering them magnetically responsive and imposing an external magnetic field on the heart during and immediately after injection.
Cardiosphere-derived cells (CDCs) were labeled with superparamagnetic microspheres (SPMs). In vitro studies revealed that cell viability and function were minimally affected by SPM labeling. SPM-labeled rat CDCs were injected intramyocardially, with and without a superimposed magnet. With magnetic targeting, cells were visibly attracted toward the magnet and accumulated around the ischemic zone. In contrast, the majority of nontargeted cells washed out immediately after injection. Fluorescence imaging revealed more retention of transplanted cells in the heart, and less migration into other organs, in the magnetically targeted group. Quantitative PCR confirmed that magnetic targeting enhanced cell retention (at 24 hours) and engraftment (at 3 weeks) in the recipient hearts by approximately 3-fold compared to nontargeted cells. Morphometric analysis revealed maximal attenuation of left ventricular remodeling, and echocardiography showed the greatest functional improvement, in the magnetic targeting group. Histologically, more engrafted cells were evident with magnetic targeting, but there was no incremental inflammation.
Magnetic targeting enhances cell retention, engraftment and functional benefit. This novel method to improve cell therapy outcomes offers the potential for rapid translation into clinical applications.
Most models for ferritin iron release are based on reduction and chelation of iron. However, newer models showing direct Fe(III) chelation from ferritin have been proposed. Fe(III) chelation reactions are facilitated by gated pores that regulate the opening and closing of the channels.
Results suggest that iron core reduction releases hydroxide and phosphate ions that exit the ferritin interior to compensate for the negative charge of the incoming electrons. Additionally, chloride ions are pumped into ferritin during the reduction process as part of a charge balance reaction. The mechanism of anion import or export is not known but is a natural process because phosphate is a native component of the iron mineral core and non-native anions have been incorporated into ferritin in vitro. Anion transfer across the ferritin protein shell conflicts with spin probe studies showing that anions are not easily incorporated into ferritin. To accommodate both of these observations, ferritin must possess a mechanism that selects specific anions for transport into or out of ferritin. Recently, a gated pore mechanism to open the 3-fold channels was proposed and might explain how anions and chelators can penetrate the protein shell for binding or for direct chelation of iron.
These proposed mechanisms are used to evaluate three in vivo iron release models based on (1) equilibrium between ferritin iron and cytosolic iron, (2) iron release by degradation of ferritin in the lysosome, and (3) metallo-chaperone mediated iron release from ferritin.
Iron oxide nanoparticle internalization exerts detrimental effects on cell physiology for a variety of particles, but little is known about the mechanism involved. The effects of high intracellular levels of four types of iron oxide particles (Resovist, Endorem, very small organic particles, and magnetoliposomes (MLs)) on the viability and physiology of murine C17.2 neural progenitor cells and human blood outgrowth endothelial cells are reported. The particles diminish cellular proliferation and affect the actin cytoskeleton and microtubule network architectures as well as focal adhesion formation and maturation. The extent of the effects correlates with the intracellular concentration (= iron mass) of the particles, with the biggest effects for Resovist and MLs at the highest concentration (1000 microg Fe mL(-1)). Similarly, the expression of focal adhesion kinase (FAK) and the amount of activated kinase (pY397-FAK) are affected. The data suggest that high levels of perinuclear localized iron oxide nanoparticles diminish the efficiency of protein expression and sterically hinder the mature actin fibers, and could have detrimental effects on cell migration and differentiation.