The Protein Corona around Nanoparticles Facilitates Stem Cell Labeling for Clinical MR Imaging.

Purpose To evaluate if the formation of a protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for in vivo tracking with magnetic resonance (MR) imaging. Materials and Methods Ferumoxytol was incubated in media containing human serum (group 1), fetal bovine serum (group 2), StemPro medium (group 3), protamine (group 4), and protamine plus heparin (group 5). Formation of a protein corona was characterized by means of dynamic light scattering, ζ potential, and liquid chromatography-mass spectrometry. Iron uptake was evaluated with 3,3'-diaminobenzidine-Prussian blue staining, lysosomal staining, and inductively coupled plasma spectrometry. To evaluate the effect of a protein corona on stem cell labeling, human mesenchymal stem cells (hMSCs) were labeled with the above formulations, implanted into pig knee specimens, and investigated with T2-weighted fast spin-echo and multiecho spin-echo sequences on a 3.0-T MR imaging unit. Data in different groups were compared by using a Kruskal-Wallis test. Results Compared with bare nanoparticles, all experimental groups showed significantly increased negative ζ values (from -37 to less than -10; P = .008). Nanoparticles in groups 1-3 showed an increased size because of the formation of a protein corona. hMSCs labeled with group 1-5 media showed significantly shortened T2 relaxation times compared with unlabeled control cells (P = .0012). hMSCs labeled with group 3 and 5 media had the highest iron uptake after cells labeled with group 1 medium. After implantation into pig knees, hMSCs labeled with group 1 medium showed significantly shorter T2 relaxation times than hMSCs labeled with group 2-5 media (P = .0022). Conclusion The protein corona around ferumoxytol nanoparticles can facilitate stem cell labeling for clinical cell tracking with MR imaging. © RSNA, 2017 Online supplemental material is available for this article.

Synthesis and Characterization of Graphite-Encapsulated Iron Nanoparticles from Ball Milling-Assisted Low-Pressure Chemical Vapor Deposition.

Graphite-encapsulated Fe nanoparticles were synthesized using a combined method of high-energy ball milling and low-pressure chemical vapor deposition (LPCVD). Fe2O3 and graphite powders were milled to increase their surface areas and obtain a more homogeneous distribution. LPCVD was performed at a pressure of ~0.57 Torr in a tube furnace under a CH4/H2 atmosphere at 1050°C for 1 and 3 h. As-synthesized samples were purified in a 2 M HF solution. Characterization was performed using X-ray diffractometry (XRD), scanning and transmission electron microscopy (SEM and TEM) and alternating gradient magnetometry (AGM). XRD revealed the presence of body centered cubic (BCC) and face centered cubic (FCC) Fe phases without residual iron oxides. SEM confirmed the powders were better mixed and smaller after ball milling compared to mortar and pestle milled powders. High resolution TEM showed all nanoparticles had at least four and on average 16 graphitic layers, around an Fe core ranging from 20-300 nm. Magnetic measurements indicated that nanoparticles exhibit soft ferromagnetic behavior with low saturation magnetization (17-21 emu/g) and coercivity (110 Oe). A chemical stability test performed in a 2 M HCl solution showed that graphitic shells did not degrade, nor was there evidence of core dissolution or shell discontinuity.

Remotely controlled near-infrared-triggered photothermal treatment of brain tumours in freely behaving mice using gold nanostars.

Current clinical brain tumour therapy practices are based on tumour resection and post-operative chemotherapy or X-ray radiation. Resection requires technically challenging open-skull surgeries that can lead to major neurological deficits and, in some cases, death. Treatments with X-ray and chemotherapy, on the other hand, cause major side-effects such as damage to surrounding normal brain tissues and other organs. Here we report the development of an integrated nanomedicine-bioelectronics brain-machine interface that enables continuous and on-demand treatment of brain tumours, without open-skull surgery and toxicological side-effects on other organs. Near-infrared surface plasmon characteristics of our gold nanostars enabled the precise treatment of deep brain tumours in freely behaving mice. Moreover, the nanostars' surface coating enabled their selective diffusion in tumour tissues after intratumoral administration, leading to the exclusive heating of tumours for treatment. This versatile remotely controlled and wireless method allows the adjustment of nanoparticles' photothermal strength, as well as power and wavelength of the therapeutic light, to target tumours in different anatomical locations within the brain.

Exploring valence states of abnormal mineral deposits in biological tissues using correlative microscopy and spectroscopy techniques: A case study on ferritin and iron deposits from Alzheimer's disease patients.

Abnormal accumulation of inorganic trace elements in a human brain, such as iron, zinc and aluminum, oftentimes manifested as deposits and accompanied by a chemical valence change, is pathologically relevant to various neurodegenerative diseases. In particular, Fe2+ has been hypothesized to produce free radicals that induce oxidative damage and eventually cause Alzheimer's disease (AD). However, traditional biomedical techniques, e.g. histology staining, are limited in studying the chemical composition and valence states of these inorganic deposits. We apply commonly used physical (phys-) science methods such as X-ray energy dispersive spectroscopy (EDS), focused-ion beam (FIB) and electron energy loss spectroscopy (EELS) in transmission electron microscopy in conjunction with magnetic resonance imaging (MRI), histology and optical microscopy (OM) to study the valence states of iron deposits in AD patients. Ferrous ions are found in all deposits in brain tissues from three AD patients, constituting 0.22-0.50 of the whole iron content in each specimen. Such phys-techniques are rarely used in medical science and have great potential to provide unique insight into biomedical problems.

Comparative electron and photon excitation of localized surface plasmon resonance in lithographic gold arrays for enhanced Raman scattering.

The ability to tune the localized surface plasmon resonance (LSPR) of nanostructures is desirable for surface enhanced Raman spectroscopy (SERS), plasmon-assisted chemistry and other nanophotonic applications. Although historically the LSPR is mainly studied by optical techniques, with the recent advancement in electron monochromators and correctors, it has attracted considerable attention in transmission electron microscopy (TEM). Here, we use electron energy loss spectroscopy (EELS) in a scanning TEM to study individual gold nanodiscs and bowties in lithographic arrays with variable LSPRs by adjusting the size, interspacing, shape and dielectric environment during the nanofabrication process. We observe the strongest Raman signal enhancement when the LSPR frequency is close to the incident laser frequency in Raman spectroscopy. A simplified harmonic oscillator model is used to estimate SERS enhancement factor (EF) from EELS, bridging the connection between electron and photon excitation of plasmonic arrays. This work demonstrates that STEM-EELS shows promise for revealing the contributions of specific LSPR modes to SERS EF. Our results provide guidelines to fine-tune nanoparticle parameters to deliver the maximum signal enhancement in biosensing applications, such as early cancer detection.

Correlative Microscopy to Localize and Characterize Iron Deposition in Alzheimer's Disease.

BACKGROUND: Recent evidence suggests that the accumulation of iron, specifically ferrous Fe2+, may play a role in the development and progression of neurodegeneration in Alzheimer's disease (AD) through the production of oxidative stress. OBJECTIVE: To localize and characterize iron deposition and oxidation state in AD, we analyzed human hippocampal autopsy samples from four subjects with advanced AD that have been previously characterized with correlative MRI-histology. METHODS: We perform scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), and electron energy loss spectroscopy (EELS) in the higher resolution transmission electron microscope on the surface and cross-sections of specific iron-rich regions of interest. RESULTS: Specific previously analyzed regions were visualized using SEM and confirmed to be iron-rich deposits using EDS. Subsequent analysis using focused ion beam cross-sectioning and SEM characterized the iron deposition throughout the 3-D volumes, confirming the presence of iron throughout the deposits, and in two out of four specimens demonstrating colocalization with zinc. Analysis of traditional histology slides showed the analyzed deposits overlapped both with amyloid and tau deposition. Following higher resolution analysis of a single iron deposit using scanning transmission electron microscope (STEM), we demonstrated the potential of monochromated STEM-EELS to discern the relative oxidation state of iron within a deposit. CONCLUSION: These findings suggest that iron is present in the AD hippocampus and can be visualized and characterized using combined MRI and EM techniques. An altered relative oxidation state may suggest a direct link between iron and oxidative stress in AD. These methods thus could potentially measure an altered relative oxidation state that could suggest a direct link between iron and oxidative stress in AD. Furthermore, we have demonstrated the ability to analyze metal deposition alongside commonly used histological markers of AD pathology, paving the way for future insights into the molecular interactions between Aß, tau, iron, and other putative metals, such as zinc.

Nanomedicine for Spontaneous Brain Tumors: A Companion Clinical Trial.

Nanoparticles' enhanced permeation and retention (EPR) variations due to tumor heterogeneity in naturally occurring brain tumors are commonly neglected in preclinical nanomedicine studies. Recent pathological studies have shown striking similarities between brain tumors in humans and dogs, indicating that canine brain tumors may be a valuable model to evaluate nanoparticles' EPR in this context. We recruited canine clinical cases with spontaneous brain tumors to investigate nanoparticles' EPR in different brain tumor pathologies using surface-enhanced Raman spectroscopy (SERS). We used gold nanoparticles due to their surface plasmon effect that enables their sensitive and microscopic resolution detection using the SERS technique. Raman microscopy of the resected tumors showed heterogeneous EPR of nanoparticles into oligodendrogliomas and meningiomas of different grades, without any detectable traces in necrotic parts of the tumors or normal brain. Raman observations were confirmed by scanning electron microscopy (SEM) and X-ray elemental analyses, which enabled localization of individual nanoparticles embedded in tumor tissues. Our results demonstrate nanoparticles' EPR and its variations in clinically relevant, spontaneous brain tumors. Such heterogeneities should be considered alongside routine preoperative imaging and histopathological analyses in order to accelerate clinical management of brain tumors using nanomedicine approaches.

Observing Plasmon Damping Due to Adhesion Layers in Gold Nanostructures Using Electron Energy Loss Spectroscopy.

Gold plasmonic nanostructures with several different adhesion layers have been studied with monochromated electron energy loss spectroscopy in the scanning transmission electron microscope (STEM-EELS) and with surface enhanced Raman spectroscopy (SERS). Compared to samples with no adhesion layer, those with 2nm of Cr or Ti show broadened, lower intensity plasmon peaks as measured with EELS. This broadening is observed in both optically active ("bright") and inactive ("dark") plasmon modes. When the former are probed with SERS, the signal enhancement factor is lower for samples with Cr or Ti, another indication of reduced plasmon resonance. This work illustrates the capability of STEM-EELS to provide direct near-field measurement of changes in plasmon excitation probability with nano-scale spatial resolution. Additionally, it demonstrates that applications which require high SERS enhancement, such as biomarker detection and cancer diagnostics, can be improved by avoiding the use of a metallic adhesion layer.

The Exosome Total Isolation Chip.

Circulating tumor-derived extracellular vesicles (EVs) have emerged as a promising source for identifying cancer biomarkers for early cancer detection. However, the clinical utility of EVs has thus far been limited by the fact that most EV isolation methods are tedious, nonstandardized, and require bulky instrumentation such as ultracentrifugation (UC). Here, we report a size-based EV isolation tool called ExoTIC (exosome total isolation chip), which is simple, easy-to-use, modular, and facilitates high-yield and high-purity EV isolation from biofluids. ExoTIC achieves an EV yield ∼4-1000-fold higher than that with UC, and EV-derived protein and microRNA levels are well-correlated between the two methods. Moreover, we demonstrate that ExoTIC is a modular platform that can sort a heterogeneous population of cancer cell line EVs based on size. Further, we utilize ExoTIC to isolate EVs from cancer patient clinical samples, including plasma, urine, and lavage, demonstrating the device's broad applicability to cancers and other diseases. Finally, the ability of ExoTIC to efficiently isolate EVs from small sample volumes opens up avenues for preclinical studies in small animal tumor models and for point-of-care EV-based clinical testing from fingerprick quantities (10-100 µL) of blood.