DNA origami scaffold promoting nerve guidance and regeneration.

Self-assembly of biological elements into biomimetic cargo carriers for targeting and delivery is a promising approach. However, it still holds practical challenges. We developed a functionalization approach of DNA origami (DO) nanostructures with neuronal growth factor (NGF) for manipulating neuronal systems. NGF bioactivity and its interactions with the neuronal system were demonstrated in vitro and in vivo models. The DO elements fabricated by molecular self-assembly have manipulated the surrounding environment through static spatially and temporally controlled presentation of ligands to the cell surface receptors. Our data showed effective bioactivity in differentiating PC12 cells in vitro. Furthermore, the DNA origami NGF (DON) affected the growth directionality and spatial capabilities of dorsal root ganglion neurons in culture by introducing a chemotaxis effect along a gradient of functionalized DO structures. Finally, we showed that these elements provide enhanced axonal regeneration in a rat sciatic nerve injury model in vivo. This study is a proof of principle for the functionality of DO in neuronal manipulation and regeneration. The approach proposed here, of an engineered platform formed out of programmable nanoscale elements constructed of DO, could be extended beyond the nervous system and revolutionize the fields of regenerative medicine, tissue engineering, and cell biology.

A streptavidin-biotin system combined with magnetic actuators for remote neuronal guidance.

The ability to control neuronal mobility and organization is of great importance in developing neuronal interfaces and novel therapeutic approaches. An emerging promising method is the manipulation of neuronal cells from afar via magnetic forces. Nevertheless, using magnetic iron oxide nanoparticles as internal actuators may lead to biotoxicity, adverse influence on intracellular processes, and thus requires prerequisite considerations for therapeutic approaches. Magnetizing the cells via the incorporation of magnetic particles that can be applied extracellularly is advantageous. Herein, we have developed a magnetic system based on streptavidin-biotin interaction to decorate cellular membrane with magnetic elements. In this model, superparamagnetic microparticles, coated with streptavidin, were specifically bound to biotinylated PC12 cells. We demonstrated that cell movement can be directed remotely by the forces produced by pre-designed magnetic fields. First, using time lapse imaging, we analyzed the kinetics of cell migration towards the higher flux zone. Next, to form organized networks of cells we designed and fabricated micro-patterned magnetic devices. The fabricated devices were composed of a variety of ferromagnetic shapes, sputter-deposited onto glass substrates. Cells that were conjugated to the magnetic particles were plated atop the micro-patterned substrates, attracted to the magnetic actuators and became fixed onto the magnetic patterns. In all, our study presents a novel system based on a well-known molecular technology combined with nanotechnology that may well lead to the expansion of implantable magnetic actuators to organize and direct cellular growth.

Controlled synthesis of multifunctional dome-shaped micro- and nano-structures via a robust physical route for biological applications.

Micro- and nano-particles are elemental for many current and developing technologies. Specifically, these particles are being used extensively in biological studies and applications, which include imaging, drug delivery and therapeutics. Recent advances have led to the development of multifunctional particles, which have the potential to further enhance their effectiveness, enabling novel applications. Therefore, many efforts have been devoted to producing well-defined particles for specific needs. However, the conventional fabrication methodologies used to develop particles are time consuming, making it extremely challenging to fine-tune the properties of the particles for multifunctional applications. Herein, we present a simple and facile method to fabricate dome-shaped micron- and nano-sized particles via a robust physical route. The presented method enables particles to be designed using a vast range of materials, with different sizes and compositions. The versatility of this method enables the engineering of multifunctional particles with pre-defined properties that can be adjusted to a specific biological application. We demonstrate the fabrication of dome-shaped particles using physical vapor deposition (PVD) and a polystyrene-bead-monolayer-based mechanical mask. We show domes from several materials and coatings; in particular, we demonstrate the development process for biocompatible magnetic iron oxide domes. We find that our magnetic domes exhibit an Fe3O4 structure with a high magnetization saturation. In addition, we examine the biocompatibility of the magnetic domes by performing viability tests and morphological analysis. The ability to design and fabricate micro- and nano-particles upon request in a simple and relatively high-throughput manner opens possibilities for the development of new smart multifunctional particles for both therapeutic and diagnostic applications.

Remote photonic sensing of action potential in mammalian nerve cells via histogram-based analysis of temporal spatial acoustic vibrations.

Label free and remote action potential detection in neurons can be of great importance in the neuroscience research field. This paper presents a novel label free imaging modality based on the detection of temporal vibrations of speckle patterns illuminating the sample. We demonstrated the feasibility of detecting action potentials originating from spontaneous and stimulated activity in cortical cell culture. The spatiotemporal vibrations of isolated cortical cells were extracted by illuminating the culture with a laser beam while the vibrations of the random back scattered secondary speckle patterns are captured by a camera. The postulated action potentials were estimated following correlation-based analysis on the captured vibrations, where the variance deviation of the signal from a Gaussian distribution is directly associated with the action potential events. The technique was validated in a series of experiments in which the optical signals were acquired concurrently with microelectrode array (MEA) recordings. Our results demonstrate the ability of detecting action potential events in mammalian cells remotely via extraction of acoustic vibrations.

An Engineered Nanocomplex with Photodynamic and Photothermal Synergistic Properties for Cancer Treatment.

Photodynamic therapy (PDT) and photothermal therapy (PTT) are promising therapeutic methods for cancer treatment; however, as single modality therapies, either PDT or PTT is still limited in its success rate. A dual application of both PDT and PTT, in a combined protocol, has gained immense interest. In this study, gold nanoparticles (AuNPs) were conjugated with a PDT agent, meso-tetrahydroxyphenylchlorin (mTHPC) photosensitizer, designed as nanotherapeutic agents that can activate a dual photodynamic/photothermal therapy in SH-SY5Y human neuroblastoma cells. The AuNP-mTHPC complex is biocompatible, soluble, and photostable. PDT efficiency is high because of immediate reactive oxygen species (ROS) production upon mTHPC activation by the 650-nm laser, which decreased mitochondrial membrane potential (∆ψm). Likewise, the AuNP-mTHPC complex is used as a photoabsorbing (PTA) agent for PTT, due to efficient plasmon absorption and excellent photothermal conversion characteristics of AuNPs under laser irradiation at 532 nm. Under the laser irradiation of a PDT/PTT combination, a twofold phototoxicity outcome follows, compared to PDT-only or PTT-only treatment. This indicates that PDT and PTT have synergistic effects together as a combined therapeutic method. Our study aimed at applying the AuNP-mTHPC approach as a potential treatment of cancer in the biomedical field.

A predictive model for personalization of nanotechnology-based phototherapy in cancer treatment.

A major challenge in radiation oncology is the prediction and optimization of clinical responses in a personalized manner. Recently, nanotechnology-based cancer treatments are being combined with photodynamic therapy (PDT) and photothermal therapy (PTT). Predictive models based on machine learning techniques can be used to optimize the clinical setup configuration, including such parameters as laser radiation intensity, treatment duration, and nanoparticle features. In this article we demonstrate a methodology that can be used to identify the optimal treatment parameters for PDT and PTT by collecting data from in vitro cytotoxicity assay of PDT/PTT-induced cell death using a single nanocomplex. We construct three machine learning prediction models, employing regression, interpolation, and low- degree analytical function fitting, to predict the laser radiation intensity and duration settings that maximize the treatment efficiency. To examine the accuracy of these prediction models, we construct a dedicated dataset for PDT, PTT, and a combined treatment; this dataset is based on cell death measurements after light radiation treatment and is divided into training and test sets. The preliminary results show that the performance of all three models is sufficient, with death rate errors of 0.09, 0.15, and 0.12 for the regression, interpolation, and analytical function fitting approaches, respectively. Nevertheless, due to its simple form, the analytical function method has an advantage in clinical application and can be used for further analysis of the sensitivity of performance to the treatment parameters. Overall, the results of this study form a baseline for a future personalized prediction model based on machine learning in the domain of combined nanotechnology- and phototherapy-based cancer treatment.

Fabrication of Magnetic Platforms for Micron-Scale Organization of Interconnected Neurons.

The ability to direct neurons into organized neural networks has great implications for regenerative medicine, tissue engineering, and bio-interfacing. Many studies have aimed at directing neurons using chemical and topographical cues. However, reports of organizational control on a micron-scale over large areas are scarce. Here, an effective method has been described for placing neurons in preset sites and guiding neuronal outgrowth with micron-scale resolution, using magnetic platforms embedded with micro-patterned, magnetic elements. It has been demonstrated that loading neurons with magnetic nanoparticles (MNPs) converts them into sensitive magnetic units that can be influenced by magnetic gradients. Following this approach, a unique magnetic platform has been fabricated on which PC12 cells, a common neuron-like model, were plated and loaded with superparamagnetic nanoparticles. Thin films of ferromagnetic (FM) multilayers with stable perpendicular magnetization were deposited to provide effective attraction forces toward the magnetic patterns. These MNP-loaded PC12 cells, plated and differentiated atop the magnetic platforms, were preferentially attached to the magnetic patterns, and the neurite outgrowth was well aligned with the pattern shape, forming oriented networks. Quantitative characterization methods of the magnetic properties, cellular MNP uptake, cell viability, and statistical analysis of the results are presented. This approach enables the control of neural network formation and improves neuron-to-electrode interface through the manipulation of magnetic forces, which can be an effective tool for in vitro studies of networks and may offer novel therapeutic biointerfacing directions.

3D Printing-Enabled Nanoparticle Alignment: A Review of Mechanisms and Applications.

3D printing (additive manufacturing (AM)) has enormous potential for rapid tooling and mass production due to its design flexibility and significant reduction of the timeline from design to manufacturing. The current state-of-the-art in 3D printing focuses on material manufacturability and engineering applications. However, there still exists the bottleneck of low printing resolution and processing rates, especially when nanomaterials need tailorable orders at different scales. An interesting phenomenon is the preferential alignment of nanoparticles that enhance material properties. Therefore, this review emphasizes the landscape of nanoparticle alignment in the context of 3D printing. Herein, a brief overview of 3D printing is provided, followed by a comprehensive summary of the 3D printing-enabled nanoparticle alignment in well-established and in-house customized 3D printing mechanisms that can lead to selective deposition and preferential orientation of nanoparticles. Subsequently, it is listed that typical applications that utilized the properties of ordered nanoparticles (e.g., structural composites, heat conductors, chemo-resistive sensors, engineered surfaces, tissue scaffolds, and actuators based on structural and functional property improvement). This review's emphasis is on the particle alignment methodology and the performance of composites incorporating aligned nanoparticles. In the end, significant limitations of current 3D printing techniques are identified together with future perspectives.

Patterning of Particles and Live Cells at Single Cell Resolution.

The ability to manipulate and selectively position cells into patterns or distinct microenvironments is an important component of many single cell experimental methods and biological engineering applications. Although a variety of particles and cell patterning methods have been demonstrated, most of them deal with the patterning of cell populations, and are either not suitable or difficult to implement for the patterning of single cells. Here, we describe a bottom-up strategy for the micropatterning of cells and cell-sized particles. We have configured a micromanipulator system, in which a pneumatic microinjector is coupled to a holding pipette capable of physically isolating single particles and cells from different types, and positioning them with high accuracy in a predefined position, with a resolution smaller than 10 µm. Complementary DNA sequences were used to stabilize and hold the patterns together. The system is accurate, flexible, and easy-to-use, and can be automated for larger-scale tasks. Importantly, it maintains the viability of live cells. We provide quantitative measurements of the process and offer a file format for such assemblies.

Brief Electrical Stimulation Triggers an Effective Regeneration of Leech CNS.

The search for therapeutic strategies to promote neuronal regeneration following injuries toward functional recovery is of great importance. Brief low-frequency electrical stimulation (ES) has been reported as a useful method to improve neuronal regeneration in different animal models; however, the effect of ES on single neuron behavior has not been shown. Here, we study the effect of brief ES on neuronal regeneration of the CNS of adult medicinal leeches. Studying the regeneration of selected sets of identified neurons allow us to quantify the ES effect per cell type at the single-cell level. Chains of the CNS that were subjected to cut injury were observed for 3 d, and the spontaneous regeneration was compared with the electrically stimulated injured chains. We show that the ES improves the efficiency of regeneration of Retzius cells, as larger masses of the total branching tree traverse the injury site with better directed growth with no effect on the average branching tree length. No antero-posterior polarity was found along regeneration within the leech CNS. Moreover, the microglial cell distribution was examined revealing more microglial cells in proximity to the stimulation site compared with non-stimulated. Our results lay a foundation for future ES-based neuroregenerative therapies.

Axonal Tree Morphology and Signal Propagation Dynamics Improve Interneuron Classification.

Neurons are diverse and can be differentiated by their morphological, electrophysiological, and molecular properties. Current morphology-based classification approaches largely rely on the dendritic tree structure or on the overall axonal projection layout. Here, we use data from public databases of neuronal reconstructions and membrane properties to study the characteristics of the axonal and dendritic trees for interneuron classification. We show that combining signal propagation patterns observed by biophysical simulations of the activity along ramified axonal trees with morphological parameters of the axonal and dendritic trees, significantly improve classification results compared to previous approaches. The classification schemes introduced here can be utilized for robust neuronal classification. Our work paves the way for understanding and utilizing form-function principles in realistic neuronal reconstructions.

Large-scale acoustic-driven neuronal patterning and directed outgrowth.

Acoustic manipulation is an emerging non-invasive method enabling precise spatial control of cells in their native environment. Applying this method for organizing neurons is invaluable for neural tissue engineering applications. Here, we used surface and bulk standing acoustic waves for large-scale patterning of Dorsal Root Ganglia neurons and PC12 cells forming neuronal cluster networks, organized biomimetically. We showed that by changing parameters such as voltage intensity or cell concentration we were able to affect cluster properties. We examined the effects of acoustic arrangement on cells atop 3D hydrogels for up to 6 days and showed that assembled cells spontaneously grew branches in a directed manner towards adjacent clusters, infiltrating the matrix. These findings have great relevance for tissue engineering applications as well as for mimicking architectures and properties of native tissues.

Fluorescent Mantle Carbon Coated Core-Shell SPIONs for Neuroengineering Applications.

Superparamagnetic iron oxide nanoparticles (SPIONs) have been used for a variety of biomedical applications, from multimodal imaging to the mechanical activity of cells and tissues. Herein, we present fluorescently mantled carbon coated core-shell superparamagnetic iron oxide nanoparticles (FC-SPIONs) as an excellent material to promote the neuronal differentiation and neuronal network outgrowth in neural tissue engineering applications. Morphological, structural, and functional group characterizations were systematically investigated. FC-SPIONs showed superior magnetic and inherent fluorescence characteristic properties. Furthermore, FC-SPIONs interactions against neuronal PC12 cells showed promising results and deliberate their potential for significant applications in neuroengineering. Interestingly, FC-SPIONs were assessed as biocompatible and promoted the neuronal PC12 cell differentiation process. Accompanied by these results, network outgrowth and branching patterns of neuronal processes can be regulated using FC-SPIONs. Importantly, FC-SPIONs are promising due to their biocompatibility and selective affinity toward neuronal cells, paving the way for neuronal differentiation and outgrowth and neuronal therapeutics in neuroengineering applications.

Magnetic Targeting of mTHPC To Improve the Selectivity and Efficiency of Photodynamic Therapy.

Photodynamic therapy (PDT) is a promising recognized treatment for cancer. To date, PDT drugs are injected systemically, and the tumor area is irradiated to induce cell death. Current clinical protocols have several drawbacks, including limited accessibility to solid tumors and insufficient selectivity of drugs. Herein, we propose an alternative approach to improve PDT effectiveness by magnetic targeting of responsive carriers conjugated to the PDT drug. We coordinatively attached a meso-tetrahydroxyphenylchlorin (mTHPC) photosensitizer to Ce-doped-γ-Fe2O3 maghemite nanoparticles (MNPs). These MNPs are superparamagnetic and biocompatible, and the resulting mTHPC-MNPs nanocomposites are stable in aqueous suspensions. MDA-MB231 (human breast cancer) cells incubated with the mTHPC-MNPs showed high uptake and high death rates in cell population after PDT. The exposure to external magnetic forces during the incubation period directed the nanocomposites to selected sites enhancing drug accumulation that was double that of cells with no magnetic exposure. Next, breast cancer tumors were induced subcutaneously in mice and treated magnetically. In vivo results showed accelerated drug accumulation in tumors of mice injected with mTHPC-MNP nanocomposites, compared to the free drug. PDT irradiation led to a decrease in tumor size of both groups, whereas treatment with the focused magnetic nanocomposites led to significant tumor regression. Our results demonstrate a method to improve the current PDT treatments by applying magnetic forces to effectively direct the drug to cancerous tissue. This approach leads to a highly localized and effective PDT process, opening new directions for clinical PDT protocols.

Neuroprotective Effect of Nerve Growth Factor Loaded in Porous Silicon Nanostructures in an Alzheimer's Disease Model and Potential Delivery to the Brain.

Nerve growth factor (NGF) plays a vital role in reducing the loss of cholinergic neurons in Alzheimer's disease (AD). However, its delivery to the brain remains a challenge. Herein, NGF is loaded into degradable oxidized porous silicon (PSiO2 ) carriers, which are designed to carry and continuously release the protein over a 1 month period. The released NGF exhibits a substantial neuroprotective effect in differentiated rat pheochromocytoma PC12 cells against amyloid-beta (Aß)-induced cytotoxicity, which is associated with Alzheimer's disease. Next, two potential localized administration routes of the porous carriers into murine brain are investigated: implantation of PSiO2 chips above the dura mater, and biolistic bombardment of PSiO2 microparticles through an opening in the skull using a pneumatic gene gun. The PSiO2 -implanted mice are monitored for a period of 8 weeks and no inflammation or adverse effects are observed. Subsequently, a successful biolistic delivery of these highly porous microparticles into a live-mouse brain is demonstrated for the first time. The bombarded microparticles are observed to penetrate the brain and reach a depth of 150 µm. These results pave the way for using degradable PSiO2 carriers as potential localized delivery systems for NGF to the brain.

Comparing Transcriptome Profiles of Neurons Interfacing Adjacent Cells and Nanopatterned Substrates Reveals Fundamental Neuronal Interactions.

Developing neuronal axons are directed by chemical and physical signals toward a myriad of target cells. According to current dogma, the resulting network architecture is critically shaped by electrical interconnections, the synapses; however, key mechanisms translating neuronal interactions into neuronal growth behavior during network formation are still unresolved. To elucidate these mechanisms, we examined neurons interfacing nanopatterned substrates and compared them to natural interneuron interactions. We grew similar neuronal populations under three connectivity conditions, (1) the neurons are isolated, (2) the neurons are interconnected, and (3) the neurons are connected only to artificial substrates, then quantitatively compared both the cell morphologies and the transcriptome-expression profiles. Our analysis shows that whereas axon-guidance signaling pathways in isolated neurons are predominant, in isolated neurons interfacing nanotopography, these pathways are downregulated, similar to the interconnected neurons. Moreover, in nanotopography, interfacing neuron genes related to synaptogenesis and synaptic regulation are highly expressed, that is, again resembling the behavior of interconnected neurons. These molecular findings demonstrate that interactions with nanotopographies, although not leading to electrical coupling, play a comparable functional role in two major routes, neuronal guidance and network formation, with high relevance to the design of regenerative interfaces.

Designing Porous Silicon Films as Carriers of Nerve Growth Factor.

Despite the great potential of NGF for treating neurodegenerative diseases, its therapeutic administration represents a significant challenge as the protein does not cross the blood-brain barrier, owing to its chemical properties, and thus requires long-term delivery to the brain to have a biological effect. This work describes fabrication of nanostructured PSi films as degradable carriers of NGF for sustained delivery of this sensitive protein. The PSi carriers are specifically tailored to obtain high loading efficacy and continuous release of NGF for a period of four weeks, while preserving its biological activity. The behavior of the NGF-PSi carriers as a NGF delivery system is investigated in vitro by examining their capability to induce neuronal differentiation and outgrowth of PC12 cells and dissociated DRG neurons. Cell viability in the presence of neat and NGF-loaded PSi carriers is evaluated. The bioactivity of NGF released from the PSi carriers is compared to the conventional treatment of repetitive free NGF administrations. PC12 cell differentiation is analyzed and characterized by the measurement of three different morphological parameters of differentiated cells; (i) the number of neurites extracting from the soma (ii) the total neurites' length and (iii) the number of branching points. PC12 cells treated with the NGF-PSi carriers demonstrate a profound differentiation throughout the release period. Furthermore, DRG neuronal cells cultured with the NGF-PSi carriers show an extensive neurite initiation, similar to neurons treated with repetitive free NGF administrations. The studied tunable carriers demonstrate the long-term implants for NGF release with a therapeutic potential for neurodegenerative diseases.

Fluorescent metal-doped carbon dots for neuronal manipulations.

There is a growing need for biocompatible nanocomposites that may efficiently interact with biological tissues through multiple modalities. Carbon dots (CDs) could serve as biocompatible fluorescence nanomaterials for targeted tissue/cell imaging. Important goals toward this end are to enhance the fluorescence quantum yields of the CDs and to increase their targetability to cells. Here, sonochemistry was used to develop a one-pot synthesis of CDs, including metal-doped CDs (M@CDs), demonstrating how various experimental parameters, such as sonication time, temperature, and power of sonication affect the size of the CDs (2-10 nm) and their fluorescence properties. The highest measured quantum yield of emission was ∼16%. Similarly, we synthesized CDs doped with different metals (M@CDs) including Ga, Sn, Zn, Ag, and Au. The interaction of M@CDs with neuron-like cells was examined and showed efficient uptake and low cytotoxicity. Moreover, the influence of the M@CDs on the improvement of neurites during initiation and elongation growth phases were compared with pristine CDs. Our research demonstrates the use of M@CDs for imaging and for neuronal interactions. The M@CD nanocomposites are promising due to their biocompatibility, photo-stability and potential selective affinity, paving the way for multifunctional biomedical applications.

Magnetic Targeting of Growth Factors Using Iron Oxide Nanoparticles.

Growth factors play an important role in nerve regeneration and repair. An attractive drug delivery strategy, termed "magnetic targeting", aims to enhance therapeutic efficiency by directing magnetic drug carriers specifically to selected cell populations that are suitable for the nervous tissues. Here, we covalently conjugated nerve growth factor to iron oxide nanoparticles (NGF-MNPs) and used controlled magnetic fields to deliver the NGF⁻MNP complexes to target sites. In order to actuate the magnetic fields a modular magnetic device was designed and fabricated. PC12 cells that were plated homogenously in culture were differentiated selectively only in targeted sites out of the entire dish, restricted to areas above the magnetic "hot spots". To examine the ability to guide the NGF-MNPs towards specific targets in vivo, we examined two model systems. First, we injected and directed magnetic carriers within the sciatic nerve. Second, we injected the MNPs intravenously and showed a significant accumulation of MNPs in mouse retina while using an external magnet that was placed next to one of the eyes. We propose a novel approach to deliver drugs selectively to injured sites, thus, to promote an effective repair with minimal systemic side effects, overcoming current challenges in regenerative therapeutics.

Ultrafine Highly Magnetic Fluorescent γ-Fe2O3/NCD Nanocomposites for Neuronal Manipulations.

In this work, we describe a low-cost, two-step synthesis of composites of nitrogen-doped carbon quantum dots (NCDs) with γ-Fe2O3 (NCDs/γ-Fe2O3), which is based on a hydrothermal cum co-precipitation method. The product is a fine powder of particles having an average diameter of 9 ± 3 nm. The physical and chemical properties of NCDs/γ-Fe2O3 were studied, as well as the superconducting quantum interference device and Mossbauer analysis of the magnetic properties of these nanocomposites. The interaction of NCDs/γ-Fe2O3 nanocomposites with neuron-like cells was examined, showing efficient uptake and low toxicity. Our research demonstrates the use of the nanocomposites for imaging and for controlling the cellular motility. The NCDs/γ-Fe2O3 nanocomposites are promising because of their biocompatibility, photostability, and potential selective affinity, paving the way for multifunctional biomedical applications.