Biosynthesis of magnetic nanoparticles from nano-degradation products revealed in human stem cells.

While magnetic nanoparticles offer exciting possibilities for stem cell imaging or tissue bioengineering, their long-term intracellular fate remains to be fully documented. Besides, it appears that magnetic nanoparticles can occur naturally in human cells, but their origin and potentially endogenous synthesis still need further understanding. In an effort to explore the life cycle of magnetic nanoparticles, we investigated their transformations upon internalization in mesenchymal stem cells and as a function of the cells' differentiation status (undifferentiated, or undergoing adipogenesis, osteogenesis, and chondrogenesis). Using magnetism as a fingerprint of the transformation process, we evidenced an important degradation of the nanoparticles during chondrogenesis. For the other pathways, stem cells were remarkably "remagnetized" after degradation of nanoparticles. This remagnetization phenomenon is the direct demonstration of a possible neosynthesis of magnetic nanoparticles in cellulo and could lay some foundation to understand the presence of magnetic crystals in human cells. The neosynthesis was shown to take place within the endosomes and to involve the H-subunit of ferritin. Moreover, it appeared to be the key process to avoid long-term cytotoxicity (impact on differentiation) related to high doses of magnetic nanoparticles within stem cells.

Ever-Evolving Identity of Magnetic Nanoparticles within Human Cells: The Interplay of Endosomal Confinement, Degradation, Storage, and Neocrystallization.

Considerable knowledge has been acquired in inorganic nanoparticles' synthesis and nanoparticles' potential use in biomedical applications. Among different materials, iron oxide nanoparticles remain unrivaled for several reasons. Not only do they respond to multiple physical stimuli (e.g., magnetism, light) and exert multifunctional therapeutic and diagnostic actions but also they are biocompatible and integrate endogenous iron-related metabolic pathways. With the aim to optimize the use of (magnetic) iron oxide nanoparticles in biomedicine, different biophysical phenomena have been recently identified and studied. Among them, the concept of a "nanoparticle's identity" is of particular importance. Nanoparticles' identities evolve in distinct biological environments and over different periods of time. In this Account, we focus on the remodeling of magnetic nanoparticles' identities following their journey inside cells. For instance, nanoparticles' functions, such as heat generation or magnetic resonance imaging, can be highly impacted by endosomal confinement. Structural degradation of nanoparticles was also evidenced and quantified in cellulo and correlates with the loss of magnetic nanoparticle properties. Remarkably, in human stem cells, the nonmagnetic products of nanoparticles' degradation could be subsequently reassembled into neosynthesized, endogenous magnetic nanoparticles. This stunning occurrence might account for the natural presence of magnetic particles in human organs, especially the brain. However, mechanistic details and the implication of such phenomena in homeostasis and disease have yet to be completely unraveled.This Account aims to assess the short- and long-term transformations of magnetic iron oxide nanoparticles in living cells, particularly focusing on human stem cells. Precisely, we herein overview the multiple and ever-evolving chemical, physical, and biological magnetic nanoparticles' identities and emphasize the remarkable intracellular fate of these nanoparticles.

Janus Magnetic-Plasmonic Nanoparticles for Magnetically Guided and Thermally Activated Cancer Therapy.

Progress of thermal tumor therapies and their translation into clinical practice are limited by insufficient nanoparticle concentration to release therapeutic heating at the tumor site after systemic administration. Herein, the use of Janus magneto-plasmonic nanoparticles, made of gold nanostars and iron oxide nanospheres, as efficient therapeutic nanoheaters whose on-site delivery can be improved by magnetic targeting, is proposed. Single and combined magneto- and photo-thermal heating properties of Janus nanoparticles render them as compelling heating elements, depending on the nanoparticle dose, magnetic lobe size, and milieu conditions. In cancer cells, a much more effective effect is observed for photothermia compared to magnetic hyperthermia, while combination of the two modalities into a magneto-photothermal treatment results in a synergistic cytotoxic effect in vitro. The high potential of the Janus nanoparticles for magnetic guiding confirms them to be excellent nanostructures for in vivo magnetically enhanced photothermal therapy, leading to efficient tumor growth inhibition.

Photothermia at the nanoscale induces ferroptosis via nanoparticle degradation.

The Fe(II)-induced ferroptotic cell death pathway is an asset in cancer therapy, yet it calls into question the biocompatibility of magnetic nanoparticles. In the latter, Fe(II) is sequestered within the crystal structure and is released only upon nanoparticle degradation, a transition that is not well understood. Here, we dissect the chemical environment necessary for nanoparticle degradation and subsequent Fe(II) release. Importantly, temperature acts as an accelerator of the process and can be triggered remotely by laser-mediated photothermal conversion, as evidenced by the loss of the nanoparticles' magnetic fingerprint. Remarkably, the local hot-spot temperature generated at the nanoscale can be measured in operando, in the vicinity of each nanoparticle, by comparing the photothermal-induced nanoparticle degradation patterns with those of global heating. Further, remote photothermal irradiation accelerates degradation inside cancer cells in a tumor spheroid model, with efficiency correlating with the endocytosis progression state of the nanoparticles. High-throughput imaging quantification of Fe2+ release, ROS generation, lipid peroxidation and cell death at the spheroid level confirm the synergistic thermo-ferroptotic therapy due to the photothermal degradation at the nanoparticle level.

Emergence of magnetic nanoparticles in photothermal and ferroptotic therapies.

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.

Biomineralization of magnetic nanoparticles in stem cells.

Iron is one of the most common metals in the human body, with an intrinsic metabolism including proteins involved in its transport, storage, and redox mechanisms. A less explored singularity is the presence of magnetic iron in the organism, especially in the brain. The capacity of human stem cells to biosynthesize magnetic nanoparticles was recently demonstrated, using iron released by the degradation of synthetic magnetic nanoparticles. To evidence a magnetic biomineralization in mammalian cells, it is required to address the biosynthesis of magnetic nanoparticles in cells supplied exclusively with non-magnetic iron salt precursors. Herein, mouse and human mesenchymal stem cells were incubated with ferric quinate for up to 36 days. By optimizing the concentration and culture time, and by measuring both total intracellular iron content and cellular magnetic signals, the biosynthesis of magnetic nanoparticles was found to occur from 14 days of continuous iron incubation and was correlated with important doses of intracellular iron. The local electronic structure and chemical environment of intracellular iron were further characterized by XAS spectroscopy at the Fe K-edge, showing a total conversion of Fe2+ to Fe3+ when using ferrous salts (ascorbate and sulfate), and a transformation towards ferrihydrite as well as a small proportion of a magnetic phase.

The role of tumor model in magnetic targeting of magnetosomes and ultramagnetic liposomes.

The combined passive and active targeting of tumoral tissue remains an active and relevant cancer research field. Here, we exploit the properties of two highly magnetic nanomaterials, magnetosomes and ultramagnetic liposomes, in order to magnetically target prostate adenocarcinoma tumors, implanted orthotopically or subcutaneously, to take into account the role of tumor vascularization in the targeting efficiency. Analysis of organ biodistribution in vivo revealed that, for all conditions, both nanomaterials accumulate mostly in the liver and spleen, with an overall low tumor retention. However, both nanomaterials were more readily identified in orthotopic tumors, reflecting their higher tumor vascularization. Additionally, a 2- and 3-fold increase in nanomaterial accumulation was achieved with magnetic targeting. In summary, ultramagnetic nanomaterials show promise mostly in the targeting of highly-vascularized orthotopic murine tumor models.

In Vivo Assimilation of CuS, Iron Oxide and Iron Oxide@CuS Nanoparticles in Mice: A 6-Month Follow-Up Study.

Nanoparticles (NPs) are at the leading edge of nanomedicine, and determining their biosafety remains a mandatory precondition for biomedical applications. Herein, we explore the bioassimilation of copper sulfide NPs reported as powerful photo-responsive anticancer therapeutic agents. The nanoparticles investigated present a hollow shell morphology, that can be left empty (CuS NPs) or be filled with an iron oxide flower-like core (iron oxide@CuS NPs), and are compared with the iron oxide nanoparticles only (iron oxide NPs). CuS, iron oxide@CuS and iron oxide NPs were injected in 6-week-old mice, at doses coherent with an antitumoral treatment. Cu and Fe were quantified in the liver, spleen, kidneys, and lungs over 6 months, including the control animals, thus providing endogenous Cu and Fe levels in the first months after animal birth. After intravenous NPs administration, 77.0 ± 3.9% of the mass of Cu injected, and 78.6 ± 3.8% of the mass of Fe, were detected in the liver. In the spleen, we found 3.3 ± 0.6% of the injected Cu and 3.8 ± 0.6% for the Fe. No negative impact was observed on organ weight, nor on Cu or Fe homeostasis in the long term. The mass of the two metals returned to the control values within three months, a result that was confirmed by transmission electron microscopy and histology images. This bioassimilation with no negative impact comforts the possible translation of these nanomaterials into clinical practice.

Flow with variable pulse frequencies accelerates vascular recellularization and remodeling of a human bioscaffold.

Despite significant advances in vascular tissue engineering, the ideal graft has not yet been developed and autologous vessels remain the gold standard substitutes for small diameter bypass procedures. Here, we explore the use of a flow field with variable pulse frequencies over the regeneration of an ex vivo-derived human scaffold as vascular graft. Briefly, human umbilical veins were decellularized and used as scaffold for cellular repopulation with human smooth muscle cells (SMC) and endothelial cells (EC). Over graft development, the variable flow, which mimics the real-time cardiac output of an individual performing daily activities (e.g., resting vs. exercising), was implemented and compared to the commonly used constant pulse frequency. Results show marked differences on SMC and EC function, with changes at the molecular level reflecting on tissue scales. First, variable frequencies significantly increased SMC proliferation rate and glycosaminoglycan production. These results can be tied with the SMC gene expression that indicates a synthetic phenotype, with a significant downregulation of myosin heavy chain. Additionally and quite remarkably, the variable flow frequencies motivated the re-endothelialization of the grafts, with a quiescent-like structure observed after 10 days of conditioning, contrasting with the low surface coverage and unaligned EC observed under constant frequency (CF). Besides, the overall biomechanics of the generated grafts (conditioned with both pulsed and CFs) evidence a significant remodeling after 55 days of culture, depicted by high burst pressure and Young's modulus. These last results demonstrate the positive recellularization and remodeling of a human-derived scaffold toward an arterial vessel.

Using Magnetometry to Monitor Cellular Incorporation and Subsequent Biodegradation of Chemically Synthetized Iron Oxide Nanoparticles.

Magnetic nanoparticles, made of iron oxide, present a peculiar interest for a wide range of biomedical applications for which they are often internalized in cells and then left within. One challenge is to assess their fate in the intracellular environment with reliable and precise methodologies. Herein, we introduce the use of the vibrating sample magnetometer (VSM) to precisely quantify the integrity of magnetic nanoparticles within cells by measuring their magnetic moment. Stem cells are first labeled with two types of magnetic nanoparticles; the nanoparticles have the same core produced via a fast and efficient microwave-based nonaqueous sol gel synthesis and differ in their coating: the commonly used citric acid molecule is compared to polyacrylic acid. The formation of 3D cell-spheroids is then achieved via centrifugation and the magnetic moment of these spheroids is measured at different times with the VSM. The obtained moment is a direct fingerprint of the nanoparticles' integrity, with decreasing values indicative of a nanoparticle degradation. For both nanoparticles, the magnetic moment decreases over culture time revealing their biodegradation. A protective effect of the polyacrylic acid coating is also shown, when compared to citric acid.

Sequential adaptation of perfusion and transport conditions significantly improves vascular construct recellularization and biomechanics.

Recellularization of ex vivo-derived scaffolds remains a significant hurdle primarily due to the scaffolds subcellular pore size that restricts initial cell seeding to the scaffolds periphery and inhibits migration over time. With the aim to improve cell migration, repopulation, and graft mechanics, the effects of a four-step culture approach were assessed. Using an ex vivo-derived vein as a model scaffold, human smooth muscle cells were first seeded onto its ablumen (Step 1: 3 hr) and an aggressive 0-100% nutrient gradient (lumenal flow under hypotensive pressure) was created to initiate cell migration across the scaffold (Step 2: Day 0 to 19). The effects of a prolonged aggressive nutrient gradient created by this single lumenal flow was then compared with a dual flow (lumenal and ablumenal) in Step 3 (Day 20 to 30). Analyses showed that a single lumenal flow maintained for 30 days resulted in a higher proportion of cells migrating across the scaffold toward the vessel lumen (nutrient source), with improved distribution. In Step 4 (Day 31 to 45), the transition from hypotensive pressure (12/8 mmHg) to normotensive (arterial-like) pressure (120/80 mmHg) was assessed. It demonstrated that recellularized scaffolds exposed to arterial pressures have increased glycosaminoglycan deposition, physiological modulus, and Young's modulus. By using this stepwise conditioning, the challenging recellularization of a vein-based scaffold and its positive remodeling toward arterial biomechanics were obtained.

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

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

Transformation Cycle of Magnetosomes in Human Stem Cells: From Degradation to Biosynthesis of Magnetic Nanoparticles Anew.

The nanoparticles produced by magnetotactic bacteria, called magnetosomes, are made of a magnetite core with high levels of crystallinity surrounded by a lipid bilayer. This organized structure has been developed during the course of evolution of these organisms to adapt to their specific habitat and is assumed to resist degradation and to be able to withstand the demanding biological environment. Herein, we investigated magnetosomes' structural fate upon internalization in human stem cells using magnetic and photothermal measurements, electron microscopy, and X-ray absorption spectroscopy. All measurements first converge to the demonstration that intracellular magnetosomes can experience an important biodegradation, with up to 70% of their initial content degraded, which is associated with the progressive storage of the released iron in the ferritin protein. It correlates with an extensive magnetite to ferrihydrite phase transition. The ionic species delivered by this degradation could then be used by the cells to biosynthesize magnetic nanoparticles anew. In this case, cell magnetism first decreased with magnetosomes being dissolved, but then cells remagnetized entirely, evidencing the neo-synthesis of biogenic magnetic nanoparticles. Bacteria-made biogenic magnetosomes can thus be totally remodeled by human stem cells, into human cells-made magnetic nanoparticles.

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

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

Role of growth factors and oxygen to limit hypertrophy and impact of high magnetic nanoparticles dose during stem cell chondrogenesis.

Due to an unmet clinical need of curative treatments for osteoarthritic patients, tissue engineering strategies that propose the development of cartilage tissue replacements from stem cells have emerged. Some of these strategies are based on the internalization of magnetic nanoparticles into stem cells to then initiate the chondrogenesis via magnetic compaction. A major difficulty is to drive the chondrogenic differentiation of the cells such as they produce an extracellular matrix free of hypertrophic collagen. An additional difficulty has to be overcome when nanoparticles are used, knowing that a high dose of nanoparticles can limit the chondrogenesis. We here propose a gene-based analysis of the effects of chemical factors (growth factors, hypoxia) on the chondrogenic differentiation of human mesenchymal stem cells both with and without nanoparticles. We focus on the synthesis of two of the most important constituents present in the cartilaginous extracellular matrix (Collagen II and Aggrecan) and on the expression of collagen X, the signature of hypertrophic cartilage, in order to provide a quantitative index of the type of cartilage produced (i.e. hyaline, hypertrophic). We demonstrate that by applying specific environmental conditions, gene expression can be directed toward the production of hyaline cartilage, with limited hypertrophy. Besides, a combination of the growth factors IGF-1, TGF-ß3, with a hypoxic conditioning remarkably reduced the impact of high nanoparticles concentration.

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Human Perinatal-Derived Biomaterials.

Human perinatal tissues have been used for over a century as allogeneic biomaterials. Due to their advantageous properties including angiogenecity, anti-inflammation, anti-microbial, and immune privilege, these tissues are being utilized for novel applications across wide-ranging medical disciplines. Given continued clinical success, increased adoption of perinatal tissues as a disruptive technology platform has allowed for significant penetration into the multi-billion dollar biologics market. Here, we review current progress and future applications of perinatal biomaterials, as well as associated regulatory issues.

The consequence of biologic graft processing on blood interface biocompatibility and mechanics.

Processing ex vivo derived tissues to reduce immunogenicity is an effective approach to create biologically complex materials for vascular reconstruction. Due to the sensitivity of small diameter vascular grafts to occlusive events, the effect of graft processing on critical parameters for graft patency, such as peripheral cell adhesion and wall mechanics, requires detailed analysis. Isolated human umbilical vein sections were used as model allogenic vascular scaffolds that were processed with either: 1. sodium dodecyl sulfate (SDS), 2. ethanol/acetone (EtAc), or 3. glutaraldehyde (Glu). Changes in material mechanics were assessed via uniaxial tensile testing. Peripheral cell adhesion to the opaque grafting material was evaluated using an innovative flow chamber that allows direct observation of the blood-graft interface under physiological shear conditions. All treatments modified the grafts tensile strain and stiffness properties, with physiological modulus values decreasing from Glu 240±12 kPa to SDS 210±6 kPa and EtAc 140±3 kPa, P<.001. Relative to glutaraldehyde treatments, neutrophil adhesion to the decellularized grafts increased, with no statistical difference observed between SDS or EtAc treatments. Early platelet adhesion (% surface coverage) showed no statistical difference between the three treatments; however, quantification of platelet aggregates was significantly higher on SDS scaffolds compared to EtAc or Glu. Tissue processing strategies applied to the umbilical vein scaffold were shown to modify structural mechanics and cell adhesion properties, with the EtAc treatment reducing thrombotic events relative to SDS treated samples. This approach allows time and cost effective prescreening of clinically relevant grafting materials to assess initial cell reactivity.