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.

Rotating Magnetic Nanoparticle Clusters as Microdevices for Drug Delivery.

BACKGROUND: Magnetic nanoparticles (MNPs) hold promise for enhancing delivery of therapeutic agents, either through direct binding or by functioning as miniature propellers. Fluid-filled conduits and reservoirs within the body offer avenues for MNP-enhanced drug delivery. MNP clusters can be rotated and moved across surfaces at clinically relevant distances in response to a rotating magnet. Limited data are available regarding issues affecting MNP delivery by this mechanism, such as adhesion to a cellular wall. Research reported here was initiated to better understand the fundamental principles important for successful implementation of rotational magnetic drug targeting (rMDT). METHODS: Translational movements of four different iron oxide MNPs were tested, in response to rotation (3 Hz) of a neodymium-boron-iron permanent magnet. MNP clusters moved along biomimetic channels of a custom-made acrylic tray, by surface walking. The effects of different distances and cellular coatings on MNP velocity were analyzed using videography. Dyes (as drug surrogates) and the drug etoposide were transported by rotating MNPs along channels over a 10 cm distance. RESULTS: MNP translational velocities could be predicted from magnetic separation times. Changes in distance or orientation from the magnet produced alterations in MNP velocities. Mean velocities of the fastest MNPs over HeLa, U251, U87, and E297 cells were 0.24 ± 0.02, 0.26 ± 0.02, 0.28 ± 0.01, and 0.18 ± 0.03 cm/sec, respectively. U138 cells showed marked MNP adherence and an 87.1% velocity reduction at 5.5 cm along the channel. Dye delivery helped visualize the effects of MNPs as microdevices for drug delivery. Dye delivery by MNP clusters was 21.7 times faster than by diffusion. MNPs successfully accelerated etoposide delivery, with retention of chemotherapeutic effect. CONCLUSION: The in vitro system described here facilitates side-by-side comparisons of drug delivery by rotating MNP clusters, on a human scale. Such microdevices have the potential for augmenting drug delivery in a variety of clinical settings, as proposed.

Nanoscale imaging using differential expansion microscopy.

Expensive and time-consuming approaches of immunoelectron microscopy of biopsy tissues continues to serve as the gold-standard for diagnostic pathology. The recent development of the new approach of expansion microscopy (ExM) capable of fourfold lateral expansion of biological specimens for their morphological examination at approximately 70 nm lateral resolution using ordinary diffraction limited optical microscopy, is a major advancement in cellular imaging. Here we report (1) an optimized fixation protocol for retention of cellular morphology while obtaining optimal expansion, (2) an ExM procedure for up to eightfold lateral and over 500-fold volumetric expansion, (3) demonstrate that ExM is anisotropic or differential between tissues, cellular organelles and domains within organelles themselves, and (4) apply image analysis and machine learning (ML) approaches to precisely assess differentially expanded cellular structures. We refer to this enhanced ExM approach combined with ML as differential expansion microscopy (DiExM), applicable to profiling biological specimens at the nanometer scale. DiExM holds great promise for the precise, rapid and inexpensive diagnosis of disease from pathological specimen slides.

An in vitro Model System for Evaluating Remote Magnetic Nanoparticle Movement and Fibrinolysis.

BACKGROUND: Thrombotic events continue to be a major cause of morbidity and mortality worldwide. Tissue plasminogen activator (tPA) is used for the treatment of acute ischemic stroke and other thrombotic disorders. Use of tPA is limited by its narrow therapeutic time window, hemorrhagic complications, and insufficient delivery to the location of the thrombus. Magnetic nanoparticles (MNPs) have been proposed for targeting tPA delivery. It would be advantageous to develop an improved in vitro model of clot formation, to screen thrombolytic therapies that could be enhanced by addition of MNPs, and to test magnetic drug targeting at human-sized distances. METHODS: We utilized commercially available blood and endothelial cells to construct 1/8th inch (and larger) biomimetic vascular channels in acrylic trays. MNP clusters were moved at a distance by a rotating permanent magnet and moved along the channels by surface walking. The effect of different transport media on MNP velocity was studied using video photography. MNPs with and without tPA were analyzed to determine their velocities in the channels, and their fibrinolytic effect in wells and the trays. RESULTS: MNP clusters could be moved through fluids including blood, at human-sized distances, down straight or branched channels, using the rotating permanent magnet. The greatest MNP velocity was closest to the magnet: 0.76 ± 0.03 cm/sec. In serum, the average MNP velocity was 0.10 ± 0.02 cm/sec. MNPs were found to enhance tPA delivery, and cause fibrinolysis in both static and dynamic studies. Fibrinolysis was observed to occur in 85% of the dynamic MNP + tPA experiments. CONCLUSION: MNPs hold great promise for use in augmenting delivery of tPA for the treatment of stroke and other thrombotic conditions. This model system facilitates side by side comparisons of MNP-facilitated drug delivery, at a human scale.

Human Skeletal Muscle Cells on Engineered 3D Platform Express Key Growth and Developmental Proteins.

Current approaches in regenerative medicine to develop human skeletal muscle replicating native tissue for engrafts and high-throughput drug screening and gene therapy are still in their infancy and have not proven to recapitulate the behavior and regulatory processes present in endogenous skeletal muscle tissue. This stems at least in part from the lack of a comprehensive understanding of the emergent properties of in vitro skeletal muscle growth and development. To address this gap in our current knowledge, we have developed a stretchable micropatterned 3D human skeletal muscle platform that recapitulates organized and parallel growth of muscle cells and fibers as opposed to the randomly oriented cells growth on a 2D glass surface. Mass spectrometry of the muscle cells growing on the 3D platform express key myogenic proteins such as myoferlin for myoblast fusion required in the formation of muscle tissue, and proteins involved in mitochondrial health and biogenesis, in contrast to cells growing on 2D glass surface. These results demonstrate that the engineered human muscle cells grown on the 3D platform holds great promise to further establish the emergent properties of in vitro skeletal muscle growth and development for a wide range of biomedical applications.

Human skeletal muscle cell atlas: Unraveling cellular secrets utilizing 'muscle-on-a-chip', differential expansion microscopy, mass spectrometry, nanothermometry and machine learning.

The 'Human Cell Atlas' project has been launched to obtain a comprehensive understanding of all cell types, the fundamental living units that constitute the human body. This is a global partnership and effort involving experts from many disciplines, from computer science, engineering to medicine, and is supported by several private and public organizations, among them, the Chan Zuckerberg Foundation, the National Institutes of Health, and Google, that will greatly benefit humanity. Nearly 37 trillion cells of various shapes, sizes, and composition, are precisely organized to constitute the human body. Humans, like all other living organisms, are dynamic, and therefore a comprehensive understanding of different cells in their various dynamic states is required to provide a reference map for the early diagnosis and various preventive approach to disease, and in the development of precision therapeutics. Skeletal muscles being the most abundant tissue and the largest locomotor and metabolic organ in the human body, requires a global understanding of its structure, composition, and function. The objective of creating a 'Human Skeletal Muscle Cell Atlas', necessitates therefore a comprehensive understanding of the emergent properties of skeletal muscle cell growth, development, structure, function and chemistry, under conditions of activity and inactivity. To achieve this objective would require a very precise yet rapid and cost-effective approach of combined multimodal imaging, including our new and novel 'Differential Expansion Microscopy', our 'Nanoscale Thermometry', combined with 'Mass Spectrometry', 'Motor Protein Motility Assay' and 'Machine Learning' tools.

Hydroxyapatite as a Vehicle for the Selective Effect of Superparamagnetic Iron Oxide Nanoparticles against Human Glioblastoma Cells.

Despite the early promises of magnetic hyperthermia (MH) as a method for treating cancer, it has been stagnating in the past decade. Some of the reasons for the low effectiveness of superparamagnetic nanoparticles (SPIONs) in MH treatments include (a) low uptake in cancer cells; (b) generation of reactive oxygen species that cause harm to the healthy cells; (c) undeveloped targeting potential; and (d) lack of temperature sensitivity between cancer cells and healthy cells. Here we show that healthy cells, including human mesenchymal stem cells (MSCs) and primary mouse kidney and lung fibroblasts, display an unfavorably increased uptake of SPIONs compared to human brain cancer cells (E297 and U87) and mouse osteosarcomas cells (K7M2). Hydroxyapatite (HAP), the mineral component of our bones, may offer a solution to this unfavorably selective SPION delivery. HAP nanoparticles are commended not only for their exceptional biocompatibility but also for the convenience of their use as an intracellular delivery agent. Here we demonstrate that dispersing SPIONs in HAP using a wet synthesis method could increase the uptake in cancer cells and minimize the risk to healthy cells. Specifically, HAP/SPION nanocomposites retain the superparamagnetic nature of SPIONs, increase the uptake ratio between U87 human brain cancer cells and human MSCs versus their SPION counterparts, reduce migration in a primary brain cancer spheroid model compared to the control, reduce brain cancer cell viability compared to the treatment with SPIONs alone, and retain the viability of healthy human MSCs. A functional synergy between the two components of the nanocomposites was established; as a result, the cancer versus healthy cell (U87/MSC) selectivity in terms of both the uptake and the toxicity was higher for the composite than for SPIONs or HAP alone, allowing it to be damaging to cancer cells and harmless to the healthy ones. The analysis of actin cytoskeleton order at the microscale revealed that healthy MSCs and primary cancer cells after the uptake of SPIONs display reduced and increased anisotropy in their cytoskeletal arrangement, respectively. In contrast, the uptake of SPION/HAP nanocomposites increased the cytoskeletal anisotropy of both the healthy MSCs and the primary cancer cells. In spite of the moderate specific magnetization of HAP/SPION nanohybrids, reaching 15 emu/g for the 28.6 wt % SPION-containing composite, the cancer cell treatment in an alternating magnetic field resulted in an intense hyperthermia effect that increased the temperature by ca. 1 °C per minute of exposure and reduced the cell population treated for 30 min by more than 50%, while leaving the control populations unharmed. These findings on nanocomposites of HAP and SPIONs may open a new avenue for cancer therapies that utilize MH.

Earthicle: The Design of a Conceptually New Type of Particle.

The conception and the steps made in the design of a conceptually new type of composite particle, so-called "earthicle", are being described. This particle is meant to roughly mimic the layered structure of the Earth, having zerovalent iron core, silicate mantle, and a thin carbonaceous crust resembling the biosphere and its geological remnants. Particles are made in a stable colloidal form in an aqueous medium, involving chemical precipitation and pyrolysis of citric acid in the solution. The effects of various synthesis parameters were studied, including borohydride and oleate concentrations, APTES/TEOS molar ratio, chemical nature of the carbon precursors, and others. XRD analysis confirmed the predominantly zerovalent iron composition of the core, amorphous silica and crystalline iron silicate/silicide composition of the mesolayer, and the carbonaceous, amorphous graphitic composition of the surface coating. The atomically thin carbon shell was also detected as a distinct shoulder on the broad n-π* absorption resonance and the peak at ∼300 nm, a signature of sp2 hybridized electronic orbitals and the result of the interband π-π* transition characteristic of graphitic structures. The irregularity of the shape of generally round Fe0 particles has caused the uniformity of the silica shell to be directly proportional to the particle size. The size of the earthicles ranged from 60 to 500 nm depending on the ionic concentration of the precursors and additives. Silica layer effectively prevented the aggregation of the iron core and increased the biocompatibility of the particles. The point of zero charge first increased from the acidic to the neutral range after coating Fe0 core with the APTES-functionalized, aminated silica shell and then restored its low value after depositing the carboxylated carbonic crust in a charge-reversal process designed to facilitate the formation of core-multishell structures. Tested on K7M2 osteosarcoma cell line and primary kidney and lung fibroblasts, cytotoxicity was cell-line dependent; however, the trend assessed in both planar and 3D cell culture with respect to the three types of particles, Fe0, Fe/SiO2, and Fe/SiO2/C, was general and independent of the cell line. Thus, the pronounced toxicity of Fe0 alone became neutralized after the silica layer was coated around Fe0. The further addition of the carbonic layer reduced the viability as compared to Fe/SiO2, albeit in a statistically significant manner only for K7M2 cell line when compared against the untreated control. Cell response also varied depending on the formulation: while some formulations exhibited lethal effects on kidney fibroblasts, were harmless to lung fibroblasts, and boosted the proliferation of K7M2 osteosarcoma cells, other formulations exhibited the opposite behavior despite being similar in terms of their core/double-shell structure. Compared across three different cancerous cell lines, K7M2 osteosarcoma and U87 and E297 glioblastoma, a similar cell-line dependency in response was observed, yet the viability reduction was consistent for all Fe/SiO2/C particles, ranging from 80% to 85% of the untreated control. Carbon surface layer, albeit of graphitic structural nature, was of a markedly more viable character than that of nanosized graphene oxide. The viability of lung fibroblasts incubated with Fe/SiO2/C particles was reduced in the presence of the alternating magnetic field of 312.75 A/m and 1 MHz, while the viability reduction caused by Fe/SiO2/C particles in kidney fibroblasts and K7M2 cells was converted from statistically insignificant to significant, suggesting that the composite particles could be used for hyperthermia treatments, although their properties should be optimized for a more intense effect. A single-cell immunofluorescent analysis of the interaction of primary kidney fibroblasts and K7M2 osteosarcoma cells with Fe/SiO2/C particles demonstrated that the cell uptake and perinuclear localization may be responsible for the necrotic effects. This analysis also showed that composite Fe/SiO2/C particles may have the ability to cause the rupture of the cancer cell nucleus while having a harmless effect on the primary cells. Such a promising and selective anticancer activity will be investigated in more detail in future studies.

Self-Setting Calcium Phosphate Cements with Tunable Antibiotic Release Rates for Advanced Antimicrobial Applications.

Osteomyelitis, an infectious disease predominantly tied to poor sanitary conditions in underdeveloped regions of the world, is in need of inexpensive, easily in situ synthesizable and administrable materials for its treatment. The results of this study stem from the attempt to create one such affordable and minimally invasive therapeutic platform in the form of a self-setting, injectable cement with a tunable drug release profile, composed of only nanoparticulate hydroxyapatite, the synthetic version of the bone mineral. Cements comprised two separately synthesized hydroxyapatite powders, one of which, HAP2, was precipitated abruptly, retaining the amorphous nature longer, and the other one of which, HAP1, was precipitated at a slower rate, more rapidly transitioning to the crystalline structure. Cements were made with four different weight ratios of the two hydroxyapatite components: 100/0, 85/15, 50/50, and 0/100 with respect to HAP1 and HAP2. Both the setting and the release rates measured on two different antibiotics, vancomycin and ciprofloxacin, were controlled using the weight ratio of the two hydroxyapatite components. Various inorganic powder properties were formerly used to control drug release, but here we demonstrate for the first time that the kinetics of the mechanism of formation of a solid compound can be controlled to produce tunable drug release profiles. Specifically, it was found that the longer the precursor calcium phosphate component of the cement retains the amorphous nature of the primary precipitate, the more active it was in terms of speeding up the diffusional release of the adsorbed drug. The setting rate was, in contrast, inversely proportional to the release rate and to the content of this active hydroxyapatite component, HAP2. The empirical release profiles were fitted to a set of equations that could be used to tune the release rate to the therapeutic occasion. All of the cements loaded with vancomycin or ciprofloxacin inhibited the growth of Gram-positive Staphylococcus aureus and Gram-negative Escherichia coli and Pseudomonas aeruginosa in both agar diffusion assays and broth dilution tests with intensities either comparable to the antibiotic per se, as in the case of ciprofloxacin, or even larger than the antibiotic alone, as in the case of vancomycin. Interestingly, even the pure cements exhibited an antibacterial effect ranging from moderate to strong, while demonstrating high levels of biocompatibility with osteoclastic RAW264.7 cells and only slightly affecting the viability of the osteoblastic MC3T3-E1 cells, in direct proportion with the amount of the more active hydroxyapatite component in the cements. This antibacterial effect was especially noticeable against Gram-negative bacteria, where the growth inhibition by the cements was comparable to or even stronger than that of the pure antibiotics. The antibiofilm assay against P. aeruginosa biofilms reiterated the antibiotic effectiveness of pure, antibiotic-free cements. That the carrier per se, composed of a nontoxic, easily prepared, bone mineral composite, can exhibit a strong antibacterial effect even in the absence of an antibiotic drug is an insight highly relevant in view of the rising resistance of an array of pathogens to traditional antibiotic therapies and the demands for the timely development of suitable alternatives.