Engineered extracellular vesicles derived from primary M2 macrophages with anti-inflammatory and neuroprotective properties for the treatment of spinal cord injury.

BACKGROUND: Uncontrollable inflammation and nerve cell apoptosis are the most destructive pathological response after spinal cord injury (SCI). So, inflammation suppression combined with neuroprotection is one of the most promising strategies to treat SCI. Engineered extracellular vesicles with anti-inflammatory and neuroprotective properties are promising candidates for implementing these strategies for the treatment of SCI. RESULTS: By combining nerve growth factor (NGF) and curcumin (Cur), we prepared stable engineered extracellular vesicles of approximately 120 nm from primary M2 macrophages with anti-inflammatory and neuroprotective properties (Cur@EVs-cl-NGF). Notably, NGF was coupled with EVs by matrix metalloproteinase 9 (MMP9)-a cleavable linker to release at the injured site accurately. Through targeted experiments, we found that these extracellular vesicles could actively and effectively accumulate at the injured site of SCI mice, which greatly improved the bioavailability of the drugs. Subsequently, Cur@EVs-cl-NGF reached the injured site and could effectively inhibit the uncontrollable inflammatory response to protect the spinal cord from secondary damage; in addition, Cur@EVs-cl-NGF could release NGF into the microenvironment in time to exert a neuroprotective effect against nerve cell damage. CONCLUSIONS: A series of in vivo and in vitro experiments showed that the engineered extracellular vesicles significantly improved the microenvironment after injury and promoted the recovery of motor function after SCI. We provide a new method for inflammation suppression combined with neuroprotective strategies to treat SCI.

Epigallocatechin-3-Gallate-Loaded Liposomes Favor Anti-Inflammation of Microglia Cells and Promote Neuroprotection.

Microglia-mediated neuroinflammation is recognized to mainly contribute to the progression of neurodegenerative diseases. Epigallocatechin-3-gallate (EGCG), known as a natural antioxidant in green tea, can inhibit microglia-mediated inflammation and protect neurons but has disadvantages such as high instability and low bioavailability. We developed an EGCG liposomal formulation to improve its bioavailability and evaluated the neuroprotective activity in in vitro and in vivo neuroinflammation models. EGCG-loaded liposomes have been prepared from phosphatidylcholine (PC) or phosphatidylserine (PS) coated with or without vitamin E (VE) by hydration and membrane extrusion method. The anti-inflammatory effect has been evaluated against lipopolysaccharide (LPS)-induced BV-2 microglial cells activation and the inflammation in the substantia nigra of Sprague Dawley rats. In the cellular inflammation model, murine BV-2 microglial cells changed their morphology from normal spheroid to activated spindle shape after 24 h of induction of LPS. In the in vitro free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay, EGCG scavenged 80% of DPPH within 3 min. EGCG-loaded liposomes could be phagocytized by BV-2 cells after 1 h of cell culture from cell uptake experiments. EGCG-loaded liposomes improved the production of BV-2 microglia-derived nitric oxide and TNF-α following LPS. In the in vivo Parkinsonian syndrome rat model, simultaneous intra-nigral injection of EGCG-loaded liposomes attenuated LPS-induced pro-inflammatory cytokines and restored motor impairment. We demonstrated that EGCG-loaded liposomes exert a neuroprotective effect by modulating microglia activation. EGCG extracted from green tea and loaded liposomes could be a valuable candidate for disease-modifying therapy for Parkinson's disease (PD).

Neural crest stem cells protect spinal cord neurons from excitotoxic damage and inhibit glial activation by secretion of brain-derived neurotrophic factor.

The acute phase of spinal cord injury is characterized by excitotoxic and inflammatory events that mediate extensive neuronal loss in the gray matter. Neural crest stem cells (NCSCs) can exert neuroprotective and anti-inflammatory effects that may be mediated by soluble factors. We therefore hypothesize that transplantation of NCSCs to acutely injured spinal cord slice cultures (SCSCs) can prevent neuronal loss after excitotoxic injury. NCSCs were applied onto SCSCs previously subjected to N-methyl-D-aspartate (NMDA)-induced injury. Immunohistochemistry and TUNEL staining were used to quantitatively study cell populations and apoptosis. Concentrations of neurotrophic factors were measured by ELISA. Migration and differentiation properties of NCSCs on SCSCs, laminin, or hyaluronic acid hydrogel were separately studied. NCSCs counteracted the loss of NeuN-positive neurons that was otherwise observed after NMDA-induced excitotoxicity, partly by inhibiting neuronal apoptosis. They also reduced activation of both microglial cells and astrocytes. The concentration of brain-derived neurotrophic factor (BDNF) was increased in supernatants from SCSCs cultured with NCSCs compared to SCSCs alone and BDNF alone mimicked the effects of NCSC application on SCSCs. NCSCs migrated superficially across the surface of SCSCs and showed no signs of neuronal or glial differentiation but preserved their expression of SOX2 and Krox20. In conclusion, NCSCs exert neuroprotective, anti-apoptotic and glia-inhibitory effects on excitotoxically injured spinal cord tissue, some of these effects mediated by secretion of BDNF. However, the investigated NCSCs seem not to undergo neuronal or glial differentiation in the short term since markers indicative of an undifferentiated state were expressed during the entire observation period.

Borneol and lactoferrin dual-modified crocetin-loaded nanoliposomes enhance neuroprotection in HT22 cells and brain targeting in mice.

Crocetin (CCT), a natural bioactive compound extracted and purified from the traditional Chinese medicinal herb saffron, has been shown to play a role in neurodegenerative diseases, particularly depression. However, due to challenges with solubility, targeting, and bioavailability, formulation development and clinical use of CCT are severely limited. In this study, we used the emulsification-reverse volatilization method to prepare CCT-loaded nanoliposomes (CN). We further developed a borneol (Bor) and lactoferrin (Lf) dual-modified CCT-loaded nanoliposome (BLCN) for brain-targeted delivery of CCT. The results of transmission electron microscope (TEM) and particle size analysis indicated that the size of BLCN (∼140 nm) was suitable for transcellular transport across olfactory axons (∼200 nm), potentially paving a direct path to the brain. Studies on lipid solubility, micropolarity, and hydrophobicity showed that BLCN had a relatively high Lf grafting rate (81.11 ± 1.33 %) and CCT entrapment efficiency (83.60 ± 1.04 %) compared to other liposomes, likely due to Bor improving the lipid solubility of Lf, and the combination promoting the orderly arrangement of liposome membrane molecules. Microplate reader and fluorescence microscopy analysis showed that BLCN efficiently promoted the endocytosis of fluorescent coumarin 6 into HT22 cells with a maximal fluorescence intensity of (13.48 ± 0.80 %), which was significantly higher than that of CCT (5.73 ± 1.17 %) and CN (12.13 ± 1.01 %). BLCN also exhibited sustained function, remaining effective for more than 12 h after reaching a peak at 1 h in cells, while CN showed a significant decrease after 4 h. The uptake mechanisms of BLCN in HT22 cells mainly involve energy-dependent, caveolae-mediated, and microtubule-mediated endocytosis, as well as micropinocytosis. Furthermore, BLCN displayed a significant neuroprotective effect on HT22 cells in glutamate-, corticosterone-, and H2O2-induced models. Tissue fluorescence image analysis of mice showed that BLCN exhibited substantial retention of fluorescent DiR in the brain after nasal administration for 12 h. These findings suggest that CCT has the potential for cellular uptake, neuroprotection, and targeted delivery to the brain following intranasal administration when encapsulated in Bor and Lf dual-modified nanoliposomes.

Highlighting the usage of polymeric nanoparticles for the treatment of traumatic brain injury: A review study.

There are very limited options for treating traumatic brain injury (TBI). Nanoparticles offer the potential of targeting specific cell types, and, potentially, crossing the BBB under the right conditions making them an area of active research for treating TBI. This review focuses on polymeric nanoparticles and the impact of their chemistry, size, and surface groups on their interactions with the vasculature and cells of the brain following injury. The vast majority of the work in the field focuses on acute injury, and when the work is looked at closely, it suggests that nanoparticles rely on interactions with vascular and immune cells to alter the environment of the brain. Nonetheless, there are promising results from a number of approaches that lead to behavioral improvements coupled with neuroprotection that offer promise for therapeutic outcomes. The majority of approaches have been tested immediately following injury. It is not entirely clear what impact these approaches will have in chronic TBI, but being able to modulate inflammation specifically may have a role both during and after the acute phase of injury.

Thymoquinone Lipid Nanoparticles Cut the Gordian Knots of Depression via Neuroprotective BDNF and Downregulation of Neuro-inflammatory NF-κB, IL-6, and TNF-α in LPS Treated Rats.

BACKGROUND: In over 300 million clinical cases, antidepressant drugs seem to provide only symptomatic relief and limited protection in life-threatening depressive events. OBJECTIVES: To compare neuronal-signaling mechanism and neuroprotective roles of Thymoquinone (TQ) suspension and its SLN (TQSLN) against standard antidepressant drug fluoxetine. METHODS: This research investigated in-silico docking at NF-KB p50 active site, CLSM based gut permeation, screening of antidepressant activities and neurosignaling pathways involved. RESULTS: As compared to fluoxetine, TQ reporteda significantly better docking score (-6.83 v/s -6.22) and a better lower free binding energy of (-34.715 Kcal/mol v/s -28.537 Kcal/mol). While poorly oral bioavailable and P-gp substrate TQ reported approximately 250% higher gut permeation if delivered as TQSLN formulation. In locomotor studies, as compared to TQS, TQSLN favored more prominent (p< 0.010) elevation in average time, horizontalactivity, average-velocity, and total-movement with reduced rest time LPS treated groups. However, in the tail suspension test, TQSLN significantly reduced immobility time (p<0.010). Similarly, In the modified force swimming test, TQSLN also significantly reduced immobility time (p<0.010), but swimming time (p<0.010) and climbing time (p<0.050) were significantly elevated. Subsequently, TQSLN reported significantly elevated neuroprotective BDNF (p<0.010) as well as hippocampal 5HT/TRP; accompanied with reduced levels of hippocampal inflammatory markers TNF-α (p<0.001) and IL-6 (p<0.010) as well as lower kynurenine and tryptophan ratio (KYN/TRP). Similarly, the hippocampal CA1 region further revealed TQSL more predominantly attenuated NF-kB nuclear translocation in the brain. CONCLUSION: Despite the poor bioavailability of TQ, TQSLN potentially attenuates neuroinflammatory transmitters and favors BDNF to modulate depressive neurobehavioral states.

Xenon Nanobubbles for the Image-Guided Preemptive Treatment of Acute Ischemic Stroke via Neuroprotection and Microcirculatory Restoration.

Early lesion site diagnosis and neuroprotection are crucial to the theranostics of acute ischemic stroke. Xenon (Xe), as a nontoxic gaseous neuroprotectant, holds great promise for ischemic stroke therapy. In this study, Xe-encapsulated lipid nanobubbles (Xe-NBs) have been prepared for the real-time ultrasound image-guided preemptive treatment of the early stroke. The lipids are self-assembled at the interface of free Xe bubbles, and the mean diameter of Xe-NBs is 225 ± 11 nm with a Xe content of 73 ± 2 µL/mL. The in vitro results show that Xe-NBs can protect oxygen/glucose-deprived PC12 cells against apoptosis and oxidative stress. Based on the ischemic stroke mice model, the biodistribution, timely ultrasound imaging, and the therapeutic effects of Xe-NBs for stroke lesions were investigated in vivo. The accumulation of Xe-NBs to the ischemic lesion endows ultrasound contrast imaging with the lesion area. The cerebral blood flow measurement indicates that the administration of Xe-NBs can improve microcirculatory restoration, resulting in reduced acute microvascular injury in the lesion area. Furthermore, local delivery of therapeutic Xe can significantly reduce the volume of cerebral infarction and restore the neurological function with reduced neuron injury against apoptosis. Therefore, Xe-NBs provide a novel nanosystem for the safe and rapid theranostics of acute ischemic stroke, which is promising to translate into the clinical management of stroke.

Polydopamine Nanoparticles as an Organic and Biodegradable Multitasking Tool for Neuroprotection and Remote Neuronal Stimulation.

Oxidative stress represents a common issue in most neurological diseases, causing severe impairments of neuronal cell physiological activity that ultimately lead to neuron loss of function and cellular death. In this work, lipid-coated polydopamine nanoparticles (L-PDNPs) are proposed both as antioxidant and neuroprotective agents, and as a photothermal conversion platform able to stimulate neuronal activity. L-PDNPs showed the ability to counteract reactive oxygen species (ROS) accumulation in differentiated SH-SY5Y, prevented mitochondrial ROS-induced dysfunctions and stimulated neurite outgrowth. Moreover, for the first time in the literature, the photothermal conversion capacity of L-PDNPs was used to increase the intracellular temperature of neuron-like cells through near-infrared (NIR) laser stimulation, and this phenomenon was thoroughly investigated using a fluorescent temperature-sensitive dye and modeled from a mathematical point of view. It was also demonstrated that the increment in temperature caused by the NIR stimulation of L-PDNPs was able to produce a Ca2+ influx in differentiated SH-SY5Y, being, to the best of our knowledge, the first example of organic nanostructures used in such an approach. This work could pave the way to new and exciting applications of polydopamine-based and of other NIR-responsive antioxidant nanomaterials in neuronal research.

Composite nanofibers incorporating alpha lipoic acid and atorvastatin provide neuroprotection after peripheral nerve injury in rats.

Despite the new treatment strategies within the last 30 years, peripheral nerve injury (PNI) is still a worldwide clinical problem. The incidence rate of PNIs is 1 in 1000 individuals per year. In this study, we designed a composite nanoplatform for dual therapy in peripheral nerve injury and investigated the in-vivo efficacy in rat sciatic nerve crush injury model. Alpha-lipoic acid (ALA) was loaded into poly lactic-co-glycolic acid (PLGA) electrospun nanofibers which would release the drug in a faster manner and atorvastatin (ATR) loaded chitosan (CH) nanoparticles were embedded into PLGA nanofibers to provide sustained release. Sciatic nerve crush was generated via Yasargil aneurism clip with a holding force of 50 g/cm2. Nanofiber formulations were administered to the injured nerve immediately after trauma. Functional recovery of operated rat hind limb was evaluated using the sciatic functional index (SFI), extensor postural thrust (EPT), withdrawal reflex latency (WRL) and Basso, Beattie, and Bresnahan (BBB) test up to one month in the post-operative period at different time intervals. In addition to functional recovery assessments, ultrastructural and biochemical analyses were carried out on regenerated nerve fibers. L-929 mouse fibroblast cell line and B35 neuroblastoma cell line were used to investigate the cytotoxicity of nanofibers before in-vivo experiments. The neuroprotection potential of these novel nanocomposite fiber formulations has been demonstrated after local implantation of composite nanofiber sheets incorporating ALA and ATR, which contributed to the recovery of the motor and sensory function and nerve regeneration in a rat sciatic nerve crush injury model.

Astrocyte-Like Cells Differentiated from Dental Pulp Stem Cells Protect Dopaminergic Neurons Against 6-Hydroxydopamine Toxicity.

Dental pulp stem cells (DPSCs) are promising for use in neurodegenerative-diseases because of their neural crest origin. While neuronal differentiation of DPSCs has been shown, their plasticity towards astrocyte-like cells remains to be studied. We aimed to examine differentiation potential of DPSCs to astrocytes and their consequent neuroprotective role towards dopaminergic (DA) neurons under 6-hydroxydopamine (6-OHDA) toxicity. Induction of DPSCs to astrocytes with differentiation factors showed definitive increase in astrocyte-specific markers glial fibrillary acidic protein (GFAP), and excitatory amino acid transporter 2 along with glial calcium-binding protein S100ß through FACS and immunofluorescence assays. RT-PCR and ELISA showed significant increase in BDNF and GDNF expression and secretion in astrocyte-differentiated DPSCs over naïve DPSCs. Neuroprotective role of these cells on DA neurons under 6-OHDA stress was evaluated by both contact and non-contact methods. FACS analysis of PKH26-stained SH-SY5Y homogenous cells in contact method and of TH immunopositive cells in primary midbrain culture in non-contact method both indicated higher survival of DA neurons in astrocyte-differentiated DPSCs over naïve DPSCs. Recovery of ß-tubulin III and TH immunopositive cells was reduced in the presence of TrkB inhibitor, suggesting a key neuroprotective role of BDNF secretion by DPSCs. When nitric oxide (NO) release was inhibited by L-NAME in primary midbrain culture, BDNF release in co-culture under 6-OHDA stress reduced further in naïve DPSCs than in astrocyte-differentiated DPSCs, suggesting that BDNF release in naïve DPSCs is primarily regulated by paracrine signaling while for differentiated DPSCs, it is equally through autocrine and paracrine signaling with NO being the mediator. In conclusion, we suggest that DPSCs exposed to glial commitment cues exhibit substantial differentiation towards astrocyte-like cells with better neuroprotective activity against 6-OHDA toxicity than naïve DPSCs.

Thermosensitive heparin-poloxamer hydrogels enhance the effects of GDNF on neuronal circuit remodeling and neuroprotection after spinal cord injury.

Traumatic spinal cord injury (SCI) results in paraplegia or quadriplegia, and currently, therapeutic interventions for axonal regeneration after SCI are not clinically available. Animal studies have revealed that glial cell-derived neurotrophic factor (GDNF) plays multiple beneficial roles in neuroprotection, glial scarring remodeling, axon regeneration and remyelination in SCI. However, the poor physicochemical stability of GDNF, as well as its limited ability to cross the blood-spinal cord barrier, hampers the development of GDNF as an effective therapeutic intervention in clinical practice. In this study, a novel temperature-sensitive heparin-poloxamer (HP) hydrogel with high GDNF-binding affinity was developed. HP hydrogels showed a supporting scaffold for GDNF when it was injected into the lesion epicenter after SCI. GDNF-HP by orthotopic injection on lesioned spinal cord promoted the beneficial effects of GDNF on neural stem cell proliferation, reactive astrogliosis inhibition, axonal regeneration or plasticity, neuroprotection against cell apoptosis, and body functional recovery. Most interestingly, GDNF demonstrated a bidirectional regulation of autophagy, which inhibited cell apoptosis at different stages of SCI. Furthermore, the HP hydrogel promoted the inhibition of autophagy-induced apoptosis by GDNF in SCI. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2816-2829, 2017.

Neuroprotection against cerebral ischemia/reperfusion injury by intravenous administration of liposomal fasudil.

Fasudil, a Rho-kinase inhibitor, is a promising neuroprotectant against ischemic stroke; however, its low bioavailability is an obstacle to be overcome. Our previous study revealed that the liposomal drug delivery system is a hopeful strategy to increase the therapeutic efficacy of neuroprotectants. In the present study, the usefulness of intravenously administered liposomal fasudil for cerebral ischemia/reperfusion (I/R) injury treatment was examined in transient middle cerebral artery occlusion (t-MCAO) rats. The results showed that PEGylated liposomes of approximately 100nm in diameter accumulated more extensively in the I/R region compared with those of over 200nm. Confocal images showed that fluorescence-labeled liposomal fasudil was widely distributed in the I/R region, and was not noticeably taken up by microglia, which are well-known resident macrophages in the brain, and neuronal cells. These data indicated that liposomal fasudil mainly exerted its pharmacological activity by releasing fasudil from the liposomes in the I/R region. Moreover, liposomal fasudil effectively suppressed neutrophil invasion and brain cell damage in the t-MCAO rats, resulting in amelioration of their motor function deficits. These findings demonstrated both the importance of particle size for neuroprotectant delivery and the effectiveness of liposomal fasudil for the treatment of cerebral I/R injury.

A dual brain-targeting curcumin-loaded polymersomes ameliorated cognitive dysfunction in intrahippocampal amyloid-ß1-42-injected mice.

Due to the impermeability of the blood-brain barrier and the nonselective distribution of drugs in the brain, the therapeutic access to intractable neurological disorders is challenging. In this study, dual brain-targeting polymersomes (POs) functionalized by transferrin and Tet-1 peptide (Tf/Tet-1-POs) promoted the transportation of curcumin into the brain and provided neuroprotection. The modification of the ligands that bind to the surface of POs was revealed by X-ray photoelectron spectroscopy analysis. The cell uptake of a coculture model of mouse brain capillary endothelial cells with neurons showed that the Tf/Tet-1-POs had significant transportation properties and possessed affinity for neurons. The pharmacokinetic analysis showed that the blood-brain barrier permeability-surface efficiency of the Tf/Tet-1-POs was 0.28 mL/h/g and that the brain tissue uptake rate (% ID/g) was 0.08, which were significant compared with the controls (P<0.05). The curcumin-encapsulated Tf/Tet-1-POs provided neuroprotection and ameliorated cognitive dysfunction in intrahippocampal amyloid-ß1-42-injected mice. These results suggest that the dual brain-targeting POs are more capable of drug delivery to the brain that can be exploited as a multiple noninvasive vehicle for targeting therapeutics.