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New strategies to promote neuronal regeneration should aim to increase the speed of axonal elongation. Biochemical signaling is a key factor in axon growth, but recent discoveries have shown that mechanical force, through a process referred to as stretch growth, can significantly influence the elongation rate. Here, we develop a method to apply forces to primary hippocampal neurons from mice using magnetic microposts that actuate in response to an external magnetic field. Neurons are cultured onto these microposts and subjected to an average displacement of 0.2 μm at a frequency of 5 Hz. We find that the mechanical stimulation promotes an increase in the length of the axons compared to control conditions. In addition, there is an increase in the density of microtubules and in the amount of cisternae of the endoplasmic reticulum, providing evidence that stretch growth is accompanied by a mass addition to the neurite. Together, these results indicate that magnetically-actuated microposts can accelerate the rate of axon growth, paving the way for future applications in neuronal regeneration. VIDEO ABSTRACT., (Copyright © 2021 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

Neurons are mechanosensitive cells. The role of mechanical force in the process of neurite initiation, elongation and sprouting; nerve fasciculation; and neuron maturation continues to attract considerable interest among scientists. Force is an endogenous signal that stimulates all these processes in vivo. The axon is able to sense force, generate force and, ultimately, transduce the force in a signal for growth. This opens up fascinating scenarios. How are forces generated and sensed in vivo? Which molecular mechanisms are responsible for this mechanotransduction signal? Can we exploit exogenously applied forces to mimic and control this process? How can these extremely low forces be generated in vivo in a non-invasive manner? Can these methodologies for force generation be used in regenerative therapies? This review addresses these questions, providing a general overview of current knowledge on the applications of exogenous forces to manipulate axonal outgrowth, with a special focus on forces whose magnitude is similar to those generated in vivo. We also review the principal methodologies for applying these forces, providing new inspiration and insights into the potential of this approach for future regenerative therapies.

There is a growing body of evidence indicating the importance of physical stimuli for neuronal growth and development. Specifically, results from published experimental studies indicate that forces, when carefully controlled, can modulate neuronal regeneration. Here, we validate a non-invasive approach for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles (MNPs) and magnetic fields (Ms). The concept is that the application of a tensile force to a neuronal cell can stimulate neurite initiation or axon elongation in the desired direction, the MNPs being used to generate this tensile force under the effect of a static external magnetic field providing the required directional orientation. In a neuron-like cell line, we have confirmed that MNPs direct the neurite outgrowth preferentially along the direction imposed by an external magnetic field, by inducing a net angle displacement (about 30°) of neurite direction. From the clinical editor: This study validates that non-invasive approaches for physical guidance of nerve regeneration based on the synergic use of magnetic nanoparticles and magnetic fields are possible. The hypothesis was confirmed by observing preferential neurite outgrowth in a cell culture system along the direction imposed by an external magnetic field., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Purpose: It has been proposed in the literature that Fe(3)O(4) magnetic nanoparticles (MNPs) could be exploited to enhance or accelerate nerve regeneration and to provide guidance for regenerating axons. MNPs could create mechanical tension that stimulates the growth and elongation of axons. Particles suitable for this purpose should possess (1) high saturation magnetization, (2) a negligible cytotoxic profile, and (3) a high capacity to magnetize mammalian cells. Unfortunately, the materials currently available on the market do not satisfy these criteria; therefore, this work attempts to overcome these deficiencies., Methods: Magnetite particles were synthesized by an oxidative hydrolysis method and characterized based on their external morphology and size distribution (high-resolution transmission electron microscopy [HR-TEM]) as well as their colloidal (Z potential) and magnetic properties (Superconducting QUantum Interference Devices [SQUID]). Cell viability was assessed via Trypan blue dye exclusion assay, cell doubling time, and MTT cell proliferation assay and reactive oxygen species production. Particle uptake was monitored via Prussian blue staining, intracellular iron content quantification via a ferrozine-based assay, and direct visualization by dual-beam (focused ion beam/scanning electron microscopy [FIB/SEM]) analysis. Experiments were performed on human neuroblastoma SH-SY5Y cell line and primary Schwann cell cultures of the peripheral nervous system., Results: This paper reports on the synthesis and characterization of polymer-coated magnetic Fe(3)O(4) nanoparticles with an average diameter of 73 ± 6 nm that are designed as magnetic actuators for neural guidance. The cells were able to incorporate quantities of iron up to 2 pg/cell. The intracellular distribution of MNPs obtained by optical and electronic microscopy showed large structures of MNPs crossing the cell membrane into the cytoplasm, thus rendering them suitable for magnetic manipulation by external magnetic fields. Specifically, migration experiments under external magnetic fields confirmed that these MNPs can effectively actuate the cells, thus inducing measurable migration towards predefined directions more effectively than commercial nanoparticles (fluidMAG-ARA supplied by Chemicell). There were no observable toxic effects from MNPs on cell viability for working concentrations of 10 μg/mL (EC(25) of 20.8 μg/mL, compared to 12 μg/mL in fluidMAG-ARA). Cell proliferation assays performed with primary cell cultures of the peripheral nervous system confirmed moderate cytotoxicity (EC(25) of 10.35 μg/mL)., Conclusion: These results indicate that loading neural cells with the proposed MNPs is likely to be an effective strategy for promoting non-invasive neural regeneration through cell magnetic actuation.

We investigated the ability of cationic liposomes composed of 1,5-dihexadecyl N-arginyl-L-glutamate (Arg-Glu2C(16)) to carry nucleic acids into neuronal cells. Such liposomes have been shown to have a remarkable capacity for transfecting immortalized cell lines. Lipoplexes between the Arg-Glu2C(16) liposomes and plasmid DNA encoding green fluorescent protein (GFP) were analyzed in terms of lipoplex formation, intracellular DNA trafficking, transfection efficiency, and cytotoxicity in neuronal SH-SY5Y cells. A maximum number of cells expressing GFP was obtained with lipoplexes at a lipid-to-DNA ratio of 15. With these lipoplexes, 16% of the cells were GFP-positive, which was approximately fourfold higher than the level obtained with a commercially available transfection reagent, Lipofectamine 2000. Furthermore, as a result of the low cytotoxicity of the Arg-Glu2C(16) lipoplexes, the proportion of GFP-positive cells could be increased to 25% by increasing the concentration of lipoplexes that was applied to the cells. We have demonstrated that Arg-Glu2C(16), as a model cationic amino acid-based lipid, has a high capability as a gene carrier, even for neuronal transfection., From the Clinical Editor: In this study, specific cationic liposomes were characterized as nucleic acid transfection agents for neuronal cells. A fourfold higher transfection rate with low cytotoxicity was reported compared to Lipofectamine 2000, a commercial reagent. The authors conclude that the studied cationic liposomes have a high capability as a gene carrier for neuronal transfection. This may become clinically significant in future gene therapy efforts of neuronal diseases., (Copyright 2010 Elsevier Inc. All rights reserved.)

The explosive growth of nanotechnology in the last years has led to dramatic innovations in pharmacology, and it is revolutioning the development of biologically active compounds. Carbon nanotubes (CNTs) are widely explored for biomedical applications such as intracellular transporters for (bio)molecules, and represent promising future tools for efficient and safe cell therapy. Due to their nanoscale dimensions, the ability to interact with cells, and their easy functionalization, CNTs are close-to-ideal vectors for an efficient and safe cell therapy, obviating the risks associated with the use of viral vectors. Notwithstanding, conflicting data concerning the biocompatibility of CNTs have been reported in the literature; while some studies point toward very low toxicity of CNTs both in vitro and in vivo, others reveal various toxic effects such as oxidative stress, DNA damage, and cell apoptosis. Thus, standardized methods and independent test systems are urgently needed to verify cytotoxicity data in this research field. In this chapter, we summarize the used methods and the achieved main results in our laboratories concerning multiwalled carbon nanotubes (MWCNTs) biocompatibility studies. The in vitro response of human neuroblastoma cell line and primary mouse neurons was investigated following the exposure to different samples of MWCNTs in order to evaluate their effects on cell viability, oxidative stress, and apoptosis. Moreover, in vivo neurocompatibility tests were carried out through injections in mouse brains.

The use of polymeric carriers containing dispersed magnetic nanocrystalline particles for targeted delivery of drugs in clinical practice has attracted the interest of the scientific community. In this paper a system comprised of alginate microparticles with a core of magnetite and carrying nerve growth factor (NGF) is described. The magnetic properties of these microspheres, typical of superparamagnetic materials, allow precise and controlled delivery to the intended tissue environment. Experiments carried out on PC12 cells with magnetic alginate microspheres loaded with NGF have confirmed the induction of cell differentiation which is strongly dependent on the distance from the microsphere cluster. In addition, finite element modelling (FEM) of the release profile from the microspheres in culture, indicated the possibility of creating defined and predictable NGF gradients from the loaded microspheres. These observations on the carriage and release of growth factors by the proposed microparticles open new therapeutic options for both neuronal regeneration and of the development of effective neuronal interfaces.

Carbon nanotubes (CNTs) are nanodevices with important potential applications in biomedicine such as drug and gene delivery. Brain diseases with no current therapy could be candidates for CNT-based therapies. Little is known about toxicity of CNTs and of their dispersion factors in the brain. Here we show that multiwall CNTs (MWCNTs) coated with Pluronic F127 (PF127) surfactant can be injected in the mouse cerebral cortex without causing degeneration of the neurons surrounding the site of injection. We also show that, contrary to previous reports on lack of PF127 toxicity on cultured cell lines, concentrations of PF127 as low as 0.01% can induce apoptosis of mouse primary cortical neurons in vitro within 24 hours. However, the presence of MWCNTs can avoid PF127-induced apoptosis. These results suggest that PF127-coated MWCNTs do not induce apoptosis of cortical neurons. Moreover, the presence of MWCNTs can reduce PF127 toxicity.

Boron nitride nanotubes (BNNTs) have unique physical properties, which can be exploited in the biomedical field. Hence, the surprising lack of reported studies on their biocompatibility and interactions with living cells, addressed by the present paper which deals the results of such an investigation based on 72 h culture of human neuroblastoma cell line (SH-SY5Y) in the presence of an aqueous suspension of polyethyleneimine (PEI)-coated BNNTs. BNNTs conjugated with fluorescent markers (quantum dots) are employed to enable tracking of their uptake by living cells. The results demonstrate good cytocompatibility together with unequivocal BNNT cellular uptake by an energy-dependent endocytic process.

"Neurocompatibility" is a broad definition which comprises aspects of biocompatibility, chemical and physical surface properties, and biostability of an artificial substrate interfaced with a neural tissue. The main issue coming from the analysis of the state of art of neuroprosthesis and neuron/electrode interfaces is the strong influence of electrode surface morphology on neurocompatibility. Enhanced functions of neurons have been observed on nano-structured materials. This paper proposes the use of focused ion beam (FIB) technology as high precision machining technique to modify the surface morphology of an interface material. By controlling the ion milling in three dimensions, the fabrication of a surface with any predefined morphology becomes possible with nanometric precision. In vitro tests on PC12 cells cultured on surfaces with different morphologies show that the surface morphology influences the cell adhesion. Experimental results suggest an enhancement of the interaction between cells and artificial surfaces at a specific scale (tens of nanometres) which is the typical scale of cellular interaction in the extra-cellular matrix (ECM) of living organisms.

This paper presents a drug delivery system based on alginate microspheres. The biocompatibility, the flexibility in size and shape, the ability to entrap biomolecules as well as cells make alginate based systems ideal for in vivo drug delivery. Specifically, considering the target application of neural regeneration and the issue of neuroprotection for the development of innovative neuroprostheses, the authors describe a system for controlled release of Netrin-1, an axonal guidance protein. Microspheres dimensioning (based on specifications of drug release time and release modality), microspheres realization, and mass transport tests are described. The release efficiency is finally assessed by in vitro experiments of axonal guidance performed on embryonic neuronal cells. Preliminary results show that neuronal axons grow approaching the Netrin-1 source, thus indicating an efficient entrapment and release of the protein in the microspheres, in agreement with the microsphere modelling described before.

Abstract: Carbon nanotubes (CNTs) are nanodevices with important potential applications in biomedicine such as drug and gene delivery. Brain diseases with no current therapy could be candidates for CNT-based therapies. Little is known about toxicity of CNTs and of their dispersion factors in the brain. Here we show that multiwall CNTs (MWCNTs) coated with Pluronic F127 (PF127) surfactant can be injected in the mouse cerebral cortex without causing degeneration of the neurons surrounding the site of injection. We also show that, contrary to previous reports on lack of PF127 toxicity on cultured cell lines, concentrations of PF127 as low as 0.01% can induce apoptosis of mouse primary cortical neurons in vitro within 24 hours. However, the presence of MWCNTs can avoid PF127-induced apoptosis. These results suggest that PF127-coated MWCNTs do not induce apoptosis of cortical neurons. Moreover, the presence of MWCNTs can reduce PF127 toxicity. [Copyright &y& Elsevier]