Nickel as a modifier of calcium oxalate: an in situ liquid cell TEM investigation of nucleation and growth.

Despite extensive research on the nucleation and growth of calcium oxalate (CaOx) crystals, there are still several challenges and unknowns that remain. In particular, the role of trace metal elements in the promotion or inhibition of CaOx crystals is not well understood. In the present study, in situ graphene liquid cell transmission electron microscopy (in situ GLC TEM) was used to observe real-time, nanoscale transformations of CaOx crystals in the presence of nickel ions (Ni2+). The results showed that Ni2+ form Ni-water complexes, acting as a shape-directing species, generating a unique morphology and altering growth kinetics. Transient adsorption of Ni-water complexes resulted in a metastable phase formation of calcium oxalate trihydrate. Atomistic molecular dynamics simulations confirmed that Ni2+ acts as a weak inhibitor which slows down the CaOx crystallization, elucidating that Ni2+ impacts small-sized CaOx clusters by bringing more water into the clusters. This work highlighted the intricacies behind the effect of Ni2+ on CaOx biomineralization that were made possible to discern using in situ GLC TEM.

Collagen membrane functionalized with magnesium oxide via room-temperature atomic layer deposition promotes osteopromotive and antimicrobial properties.

Artificial bone grafting materials such as collagen are gaining interest due to the ease of production and implantation. However, collagen must be supplemented with additional coating materials for improved osteointegration. Here, we report room-temperature atomic layer deposition (ALD) of MgO, a novel method to coat collagen membranes with MgO. Characterization techniques such as X-ray photoelectron spectroscopy, Raman spectroscopy, and electron beam dispersion mapping confirm the chemical nature of the film. Scanning electron and atomic force microscopies show the surface topography and morphology of the collagen fibers were not altered during the ALD of MgO. Slow release of magnesium ions promotes bone growth, and we show the deposited MgO film leaches trace amounts of Mg when incubated in phosphate-buffered saline at 37 °C. The coated collagen membrane had a superhydrophilic surface immediately after the deposition of MgO. The film was not toxic to human cells and demonstrated antibacterial properties against bacterial biofilms. Furthermore, in vivo studies performed on calvaria rats showed MgO-coated membranes (200 and 500 ALD) elicit a higher inflammatory response, leading to an increase in angiogenesis and a greater bone formation, mainly for Col-MgO500, compared to uncoated collagen. Based on the characterization of the MgO film and in vitro and in vivo data, the MgO-coated collagen membranes are excellent candidates for guided bone regeneration.

Real-time TEM observations of ice formation in graphene liquid cell.

The study of ice nucleation and growth at the nanoscale is of utmost importance in geological and atmospheric sciences. However, existing transmission electron microscopy (TEM) approaches have been unsuccessful in imaging ice formation directly. Herein, we demonstrate how radical scavengers - such as TiO2 - encased with water in graphene liquid cells (GLCs) facilitate the observation of ice nucleation phenomena at low temperatures. Atomic-resolution imaging reveals the nucleation and growth of cubic ice-phase crystals at close proximity to TiO2-water nanointerfaces at low temperatures. Interestingly, both heterogeneously and homogeneously nucleated ice crystals exhibited this cubic phase. Ice crystal nuclei were observed to be more stable at the TiO2-water nanointerface, as compared with crystals in the bulk liquid (homogeneous nucleation), suggesting the radical scavenging efficacy of TiO2 nanoparticles mitigating the electron beam by-products. The present work demonstrates that the use of radical scavengers in GLC TEM shows great promise towards unveiling the nanoscale pathways for ice nucleation and growth dynamic events.

Enhanced Bacterial Growth by Polyelemental Glycerolate Particles.

While polyelemental alloys are shown to be promising for healthcare applications, their effectiveness in promoting bacterial growth remains unexplored. In the present work, we evaluated the interaction of polyelemental glycerolate particles (PGPs) with Escherichia coli (E. coli) bacteria. PGPs were synthesized using the solvothermal route, and nanoscale random distribution of metal cations in the glycerol matrix of PGPs was confirmed. We observed 7-fold growth of E. coli bacteria upon 4 h of interaction with quinary glycerolate (NiZnMnMgSr-Gly) particles in comparison to control E. coli bacteria. Nanoscale microscopic studies on bacteria interactions with PGPs showed the release of metal cations in the bacterium cytoplasm from PGPs. The electron microscopy imaging and chemical mapping indicated bacterial biofilm formation on PGPs without causing significant cell membrane damage. The data showed that the presence of glycerol in PGPs is effective in controlling the release of metal cations, thus preventing bacterial toxicity. The presence of multiple metal cations is expected to provide synergistic effects of nutrients needed for bacterial growth. The present work provides key microscopic insights of mechanisms by which PGPs enhance biofilm growth. This study opens the door for future applications of PGPs in areas where bacterial growth is essential including healthcare, clean energy, and the food industry.

In Situ Microscopic Studies on the Interaction of Multi-Principal Element Nanoparticles and Bacteria.

Multi-principal element nanoparticles are an emerging class of materials with potential applications in medicine and biology. However, it is not known how such nanoparticles interact with bacteria at nanoscale. In the present work, we evaluated the interaction of multi-principal elemental alloy (FeNiCu) nanoparticles with Escherichia coli (E. coli) bacteria using the in situ graphene liquid cell (GLC) scanning transmission electron microscopy (STEM) approach. The imaging revealed the details of bacteria wall damage in the vicinity of nanoparticles. The chemical mappings of S, P, O, N, C, and Cl elements confirmed the cytoplasmic leakage of the bacteria. Our results show that there is selective release of metal ions from the nanoparticles. The release of copper ions was much higher than that for nickel while the iron release was the lowest. In addition, the binding affinity of bacterial cell membrane protein functional groups with Cu, Ni, and Fe cations is found to be the driving force behind the selective metal cations' release from the multi-principal element nanoparticles. The protein functional groups driven dissolution of multielement nanoparticles was evaluated using the density functional theory (DFT) computational method, which confirmed that the energy required to remove Cu atoms from the nanoparticle surface was the least in comparison with those for Ni and Fe atoms. The DFT results support the experimental data, indicating that the energy to dissolve metal atoms exposed to oxidation and/or the to presence of oxygen atoms at the surface of the nanoparticle catalyzes metal removal from the multielement nanoparticle. The study shows the potential of compositional design of multi-principal element nanoparticles for the controlled release of metal ions to develop antibacterial strategies. In addition, GLC-STEM is a promising approach for understanding the nanoscale interaction of metallic nanoparticles with biological structures.

Nanoscale chemical and structural investigation of solid solution polyelemental transition metal oxide nanoparticles.

Although it has been shown that configurational entropy can improve the structural stability in transition metal oxides (TMOs), little is known about the oxidation state of transition metals under random mixing of alloys. Such information is essential in understanding the chemical reactivity and properties of TMOs stabilized by configurational entropy. Herein, utilizing electron energy loss spectroscopy (EELS) technique in an aberration-corrected scanning transmission electron microscope (STEM), we systematically studied the oxidation state of binary (Mn, Fe)3O4, ternary (Mn, Fe, Ni)3O4, and quinary (Mn, Fe, Ni, Cu, Zn)3O4 solid solution polyelemental transition metal oxides (SSP-TMOs) nanoparticles. Our findings show that the random mixing of multiple elements in the form of solid solution phase not only promotes the entropy stabilization but also results in stable oxidation state in transition metals spanning from binary to quinary transition metal oxide nanoparticles.

A Light-Curable and Tunable Extracellular Matrix Hydrogel for In Situ Suture-Free Corneal Repair.

Corneal injuries are a major cause of blindness worldwide. To restore corneal integrity and clarity, there is a need for regenerative bio-integrating materials for in-situ repair and replacement of corneal tissue. Here, we introduce Light-curable COrnea Matrix (LC-COMatrix), a tunable material derived from decellularized porcine cornea extracellular matrix containing un-denatured collagen and sulfated glycosaminoglycans. It is a functionalized hydrogel with proper swelling behavior, biodegradation, and viscosity that can be cross-linked in situ with visible light, providing significantly enhanced biomechanical strength, stability, and adhesiveness. Cross-linked LC-COMatrix strongly adheres to human corneas ex vivo and effectively closes full-thickness corneal perforations with tissue loss. Likewise, in vivo, LC-COMatrix seals large corneal perforations, replaces partial-corneal stromal defects and bio-integrates into the tissue in rabbit models. LC-COMatrix is a natural ready-to-apply bio-integrating adhesive that is representative of native corneal matrix with potential applications in corneal and ocular surgeries.

Optimization of the Mechanical Properties and the Cytocompatibility for the PMMA Nanocomposites Reinforced with the Hydroxyapatite Nanofibers and the Magnesium Phosphate Nanosheets.

Commercial poly methyl methacrylate (PMMA)-based cement is currently used in the field of orthopedics. However, it suffers from lack of bioactivity, mechanical weakness, and monomer toxicity. In this study, a PMMA-based cement nanocomposite reinforced with hydroxyapatite (HA) nanofibers and two-dimensional (2D) magnesium phosphate MgP nanosheets was synthesized and optimized in terms of mechanical property and cytocompatibility. The HA nanofibers and the MgP nanosheets were synthesized using a hydrothermal homogeneous precipitation method and tuning the crystallization of the sodium-magnesium-phosphate ternary system, respectively. Compressive strength and MTT assay tests were conducted to evaluate the mechanical property and the cytocompatibility of the PMMA-HA-MgP nanocomposites prepared at different ratios of HA and MgP. To optimize the developed nanocomposites, the standard response surface methodology (RSM) design known as the central composite design (CCD) was employed. Two regression models generated by CCD were analyzed and compared with the experimental results, and good agreement was observed. Statistical analysis revealed the significance of both factors, namely, the HA nanofibers and the MgP nanosheets, in improving the compressive strength and cell viability of the PMMA-MgP-HA nanocomposite. Finally, it was demonstrated that the HA nanofibers of 7.5% wt and the MgP nanosheets of 6.12% wt result in the PMMA-HA-MgP nanocomposite with the optimum compressive strength and cell viability.

Ultrafast Synthesis of High Entropy Oxide Nanoparticles by Flame Spray Pyrolysis.

The synthesis of high entropy oxide (HEO) nanoparticles (NPs) possesses many challenges in terms of process complexity and cost, scalability, tailoring nanoparticle morphology, and rapid synthesis. Herein, we report the synthesis of novel single-phase solid solution (Mn, Fe, Ni, Cu, Zn)3(O)4 quinary HEO NPs produced by a flame spray pyrolysis route. The aberration-corrected scanning transmission electron microscopy (STEM) technique is utilized to investigate the spinel crystal structure of synthesized HEO NPs, and energy-dispersive X-ray spectroscopy analysis confirmed the high entropy configuration of five metal elements in their oxide form within a single HEO nanoparticle. Selected area electron diffraction, X-ray diffraction, and Raman spectroscopy analysis results are in accordance with STEM results, providing the key attributes of a spinel crystal structure of HEO NPs. X-ray photoelectron spectroscopy results provide the insightful understanding of chemical oxidation states of individual elements and their possible cation occupancy sites in the spinel-structured HEO NPs.

In-situ porcine corneal matrix hydrogel as ocular surface bandage.

PURPOSE: Bioactive substrates can be used therapeutically to enhance wound healing. Here, we evaluated the effect of an in-situ thermoresponsive hydrogel from decellularized porcine cornea ECM, COMatrix (COrnea Matrix), for application as an ocular surface bandage for corneal epithelial defects. METHODS: COMatrix hydrogel was fabricated from decellularized porcine corneas. The effects of COMatrix hydrogel on attachment and proliferation of human corneal epithelial cells (HCECs) were evaluated in vitro. The effect of COMatrix on the expressions of the inflammatory genes, IL-1ß, TNF-α, and IL-6 was assessed by RT-PCR. The in-situ application and also repairing effects of COMatrix hydrogel as an ocular bandage was studied in a murine model of corneal epithelial wound. The eyes were examined by optical coherence tomography (OCT) and slit-lamp microscopy in vivo and by histology and immunofluorescence post-mortem. RESULTS: In vitro, COMatrix hydrogel significantly enhanced the attachment and proliferation of HCECs relative to control. HCECs exposed to COMatrix had less induced expression of TNF-α (P < 0.05). In vivo, COMatrix formed a uniform hydrogel that adhered to the murine ocular surface after in-situ curing. Corneal epithelial wound closure was significantly accelerated by COMatrix hydrogel compared to control (P < 0.01). There was significant increase in the expression of proliferation marker Ki-67 in wounded corneal epithelium by COMatrix hydrogel compared to control (P < 0.05). CONCLUSIONS: COMatrix hydrogel is a naturally derived bioactive material with potential application as an ocular surface bandage to enhance epithelial wound healing.

Fabrication, Rheological, and Compositional Characterization of Thermoresponsive Hydrogel from Cornea.

Fabricating thermoresponsive hydrogels from decellularized tissues is a trending and promising approach to develop novel biomaterials for tissue engineering and therapeutic purposes. There are differences in the characteristics of the produced hydrogels related to the source tissue as well as the decellularization and solubilization protocols used. Detailed characterization of the hydrogels will support the efforts to optimize their application as biomaterials for tissue engineering and therapeutics. Here, we describe an optimized method for fabricating an in situ thermoresponsive hydrogel from decellularized porcine cornea extracellular matrix (COMatrix), and provide a detailed characterization of its structure, thermoresponsive rheological behavior (heat-induced sol-gel transition), as well as exploring its protein composition using proteomics. COMatrix forms a transparent gel (10-min time to gelation) after in situ curing with heat, characterized by alteration in light absorbance and rheological indexes. The rheological characterization of heat-formed COMatrix gel shows similar behavior to common biomaterials utilized in tissue engineering. The fibrillar structure of COMatrix gel was observed by scanning electron microscopy showing that the density of fibers attenuates in lower concentrations. Mass spectrometry-based proteomic analysis revealed that COMatrix hydrogel is rich in proteins with known regenerative properties such as lumican, keratocan, and laminins in addition to structural collagen proteins (Data is available via ProteomeXchange with identifier PXD020606). COMatrix hydrogel is a naturally driven biomaterial with favorable biomechanical properties and protein content with potential application as a therapeutic biomaterial in ocular regeneration and tissue engineering. Impact statement Fabrication and application of decellularized porcine corneal extracellular matrix is an emerging approach for corneal tissue engineering and regeneration. There are several protocols for decellularization of porcine cornea with various efficiencies. Here, we are presenting an optimized protocol for decellularization of porcine cornea followed by fabrication of a thermoresponsive hydrogel from the decellularized cornea matrix. Moreover, the fabricated hydrogel was rheologically and compositionally characterized as crucial features to be employed for further application of this hydrogel in corneal tissue engineering and regeneration.

Targeted sonodynamic destruction of glioblastoma cells using antibody-titanium dioxide nanoparticle conjugates.

Aim: We present data on sonodynamic therapy (SDT) against glioblastoma cells utilizing titanium dioxide (TiO2) nanoparticles conjugated to anti-EGFR antibody. Materials & methods: TiO2 nanoparticles were bound to anti-EGFR antibody to form antibody-nanoparticle conjugates (ANCs), then characterized by x-ray photoelectron spectroscopy and transmission electron microscopy. Cells underwent ultrasound and assessment on viability, reactive oxygen species and apoptosis were performed. Results: X-ray photoelectron spectroscopy analysis revealed the formation of an ANC. Transmission electron microscopy showed internalization of the ANCs by glioblastoma cells. With SDT, cell viabilities were reduced in the presence of ANCs, reactive oxygen species production was formed, but minimal effect on apoptosis was seen. Conclusion: For the first time, an ANC can be used with SDT to kill glioblastoma cells.

Etoposide-Bound Magnetic Nanoparticles Designed for Remote Targeting of Cancer Cells Disseminated Within Cerebrospinal Fluid Pathways.

Magnetic nanoparticles (MNPs) have potential for enhancing drug delivery in selected cancer patients, including those which have cells that have disseminated within cerebrospinal fluid (CSF) pathways. Here, we present data related to the creation and in vitro use of new two-part MNPs consisting of magnetic gold-iron alloy cores which have streptavidin binding sites, and are coated with biotinylated etoposide. Etoposide was chosen due to its previous use in the CSF and ease of biotinylation. Etoposide magnetic nanoparticles ("Etop-MNPs") were characterized by several different methods, and moved at a distance by surface-walking of MNP clusters, which occurs in response to a rotating permanent magnet. Human cell lines including D283 (medulloblastoma), U138 (glioblastoma), and H2122 (lung adenocarcinoma) were treated with direct application of Etop-MNPs (and control particles), and after remote particle movement. Cell viability was determined by MTT assay and trypan blue exclusion. Results indicated that the biotinylated etoposide was successfully bound to the base MNPs, with the hybrid particle attaining a maximum velocity of 0.13 ± 0.018 cm/sec. Etop-MNPs killed cancer cells in a dose-dependent fashion, with 50 ± 6.8% cell killing of D283 cells (for example) with 24 h of treatment after remote targeting. U138 and H2122 cells were found to be even more susceptible to the killing effect of Etop-MNPs than D283 cells. These findings indicate that the novel Etop-MNPs have a cytotoxic effect, and can be moved relatively rapidly at physiologic distances, using a rotating magnet. While further testing is needed, intrathecal administration of Etop-MNPs holds promise for magnetically-enhanced eradication of cancer cells distributed within CSF pathways, particularly if given early in the course of the disease.

Revealing nanoscale mineralization pathways of hydroxyapatite using in situ liquid cell transmission electron microscopy.

To treat impairments in hard tissues or overcome pathological calcification in soft tissues, a detailed understanding of mineralization pathways of calcium phosphate materials is needed. Here, we report a detailed mechanistic study of hydroxyapatite (HA) mineralization pathways in an artificial saliva solution via in situ liquid cell transmission electron microscopy (TEM). It is found that the mineralization of HA starts by forming ion-rich and ion-poor solutions in the saliva solution, followed by coexistence of the classical and nonclassical nucleation processes. For the nonclassical path, amorphous calcium phosphate (ACP) functions as the substrate for HA nucleation on the ACP surface, while the classical path features direct HA nucleation from the solution. The growth of HA crystals on the surface of ACP is accompanied by the ACP dissolution process. The discoveries reported in this work are important to understand the physiological and pathological formation of HA minerals, as well as to engineer the biomineralization process for bone healing and hard tissue repairs.

Combination targeting of 'platelets + fibrin' enhances clot anchorage efficiency of nanoparticles for vascular drug delivery.

Occlusive thrombosis is a central pathological event in heart attack, stroke, thromboembolism, etc. Therefore, pharmacological thrombolysis or anticoagulation is used for treating these diseases. However, systemic administration of such drugs causes hemorrhagic side-effects. Therefore, there is significant clinical interest in strategies for enhanced drug delivery to clots while minimizing systemic effects. One such strategy is by using drug-carrying nanoparticles surface-decorated with clot-binding ligands. Efforts in this area have focused on binding to singular targets in clots, e.g. platelets, fibrin, collagen, vWF or endothelium. Targeting vWF, collagen or endothelium maybe sub-optimal since in vivo these entities will be rapidly covered by platelets and leukocytes, and thus inaccessible for sufficient nanoparticle binding. In contrast, activated platelets and fibrin are majorly accessible for particle-binding, but their relative distribution in clots is highly heterogeneous. We hypothesized that combination-targeting of 'platelets + fibrin' will render higher clot-binding efficacy of nanoparticles, compared to targeting platelets or fibrin singularly. To test this, we utilized liposomes as model nanoparticles, decorated their surface with platelet-binding peptides (PBP) or fibrin-binding peptides (FBP) or combination (PBP + FBP) at controlled compositions, and evaluated their binding to human blood clots in vitro and in a mouse thrombosis model in vivo. In parallel, we developed a computational model of nanoparticle binding to single versus combination entities in clots. Our studies indicate that combination targeting of 'platelets + fibrin' enhances the clot-anchorage efficacy of nanoparticles while utilizing lower ligand densities, compared to targeting platelets or fibrin only. These findings provide important insights for vascular nanomedicine design.

TEM Studies on Antibacterial Mechanisms of Black Phosphorous Nanosheets.

PURPOSE: Recently, two-dimensional (2D) nanomaterials are gaining tremendous attention as novel antibacterial platforms to combat against continuously evolving antimicrobial resistance levels. Among the family of 2D nanomaterials, black phosphorus (BP) nanosheets have demonstrated promising potential for biomedical applications. However, there is a need to gain nanoscale insights of the antibacterial activity of BP nanosheets which lies at the center of technical challenges. METHODS: Ultra-large BP nanosheets were synthesized by liquid-exfoliation method in the eco-friendly deoxygenated water. Synthesized BP nanosheets were characterized by TEM, AFM, and Raman spectroscopy techniques and their chemical stability was evaluated by EDS and EELS elemental analysis. The antibacterial activity of BP nanosheets was evaluated at nanoscale by the ultramicrotome TEM technique. Further, HAADF-STEM image and EDS elemental line map of the damaged bacterium were utilized to analyze the presence of diagnostic ions. Supportive SEM and ATR-FTIR studies were carried out to confirm the bacterial cell wall damage. In vitro colony counting method was utilized to evaluate the antibacterial performance of ultra-large BP nanosheets. RESULTS: Elemental EELS and EDS analysis of BP nanosheets stored in deoxygenated water confirmed the absence of oxygen peak. TEM studies indicate the various events of bacterial cell damage with the lost cellular metabolism and structural integrity. Colony counting test results show that as-synthesized BP nanosheets (100 µg/mL) can kill ~95% bacteria within 12 hours. CONCLUSION: TEM studies demonstrate the various events of E. coli membrane damage and the loss of structural integrity. These events include the BP nanosheets interaction with the bacterial cell wall, cytoplasmic leakage, detachment of cytoplasm from the cell membrane, reduced density of lipid bilayer and agglomerated DNA structure. The EDS elemental line mapping of the damaged bacterium confirms the disrupted cell membrane permeability and the lost cellular metabolism. SEM micrographs and ATR-FTIR supportive results confirm the bacterial cell wall damage.

Novel PMMA bone cement nanocomposites containing magnesium phosphate nanosheets and hydroxyapatite nanofibers.

Lack of bioactivity and monomer toxicity are limiting factors of polymethyl methacrylate (PMMA) bone cement in orthopedic applications. Herein, we address these shortcomings by proposing two-dimensional magnesium phosphate (MgP) nanosheets and hydroxyapatite (HA) nanofibers as novel fillers in PMMA bone cement nanocomposites. Two-dimensional MgP nanosheets and one-dimensional HA nanofibers were synthesized by tuning the crystallization of the sodium-magnesium-phosphate ternary system and hydrothermal homogeneous precipitation, respectively. We show that MgP nanosheets exhibit antibacterial properties against Escherichia coli (E. coli). In addition, HA nanofibers with high level of bioactivity are the proper choice to induce cell viability in the nanocomposite. Results indicate that the combination of both fillers can act as deformation locks enhancing the compressive strength of the nanocomposites. The synthesized nanocomposite possesses excellent bioactivity, mechanical properties, and cytocompatibility potentially opening new paradigm in the design of next generation bone cement composites.

Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology.

BACKGROUND: Nanoscale surface roughness has been suggested to have antibacterial and antifouling properties. Several existing models have attempted to explain the antibacterial mechanism of nanoscale rough surfaces without direct observation. Here, conventional and liquid-cell TEM are implemented to observe nanoscale bacteria/surface roughness interaction. The visualization of such interactions enables the inference of possible antibacterial mechanisms. METHODS AND RESULTS: Nanotextures are synthesized on biocompatible polymer microparticles (MPs) via plasma etching. Both conventional and liquid-phase transmission electron microscopy observations suggest that these MPs may cause cell lysis via bacterial binding to a single protrusion of the nanotexture. The bacterium/protrusion interaction locally compromises the cell wall, thus causing bacterial death. This study suggests that local mechanical damage and leakage of the cytosol kill the bacteria first, with subsequent degradation of the cell envelope. CONCLUSION: Nanoscale surface roughness may act via a penetrative bactericidal mechanism. This insight suggests that future research may focus on optimizing bacterial binding to individual nanoscale projections in addition to stretching bacteria between nanopillars. Further, antibacterial nanotextures may find use in novel applications employing particles in addition to nanotextures on fibers or films.

In Situ Visualization of Ferritin Biomineralization via Graphene Liquid Cell-Transmission Electron Microscopy.

Ferritin biomineralization is essential to regulate the toxic Fe2+ iron ions in the human body. Unravelling the mechanism of biomineralization in ferritin facilitates our understanding of the causes underlying many iron disorder-related diseases. Until now, no report of in situ visualization of ferritin biomineralization events at nanoscale exists due to the requirement for high-resolution imaging of nanometer-sized ferritin proteins in their hydrated states. Herein, for the first time, we show that the biomineralization processes within individual ferritin proteins can be visualized by means of graphene liquid cell-transmission electron microscopy (GLC-TEM). The increase in the ratio of Fe3+/Fe2+ ions over time monitored via electron energy loss spectroscopy (EELS) reveals the change in oxidation state of iron oxide phases with time. This study lays a foundation for future investigations on iron regulation mechanisms in healthy and dysfunctional ferritins.

On the structure and chemistry of iron oxide cores in human heart and human spleen ferritins using graphene liquid cell electron microscopy.

Ferritin is a protein that regulates the iron ions in humans by storing them in the form of iron oxides. Despite extensive efforts to understand the ferritin iron oxide structures, it is still not clear how ferritin proteins with a distinct light (L) and heavy (H) chain subunit ratio impact the biomineralization process. In situ graphene liquid cell-transmission electron microscopy (GLC-TEM) provides an indispensable platform to study the atomic structure of ferritin mineral cores in their native liquid environment. In this study, we report differences in the iron oxide formation in human spleen ferritins (HSFs) and human heart ferritins (HHFs) using in situ GLC-TEM. Scanning transmission electron microscopy (STEM) along with selected area electron diffraction (SAED) of the mineral core and electron energy loss spectroscopy (EELS) analyses enabled the visualization of morphologies, crystal structures and the chemistry of iron oxide cores in HSFs and HHFs. Our study revealed the presence of metastable ferrihydrite (5Fe2O3·9H2O) as a dominant phase in hydrated HSFs and HHFs, while a stable hematite (α-Fe2O3) phase predominated in non-hydrated HSFs and HHFs. In addition, a higher Fe3+/Fe2+ ratio was found in HHFs in comparison with HSFs. This study provides new understanding on iron-oxide phases that exist in hydrated ferritin proteins from different human organs. Such new insights are needed to map ferritin biomineralization pathways and possible correlations with various iron-related disorders in humans.