Internalization of rabies virus glycoprotein differs between pathogenic and attenuated virus strains.

The zoonotic rabies virus (RABV) is a non-segmented negative-sense RNA virus classified within the family Rhabdoviridae, and is the most common aetiological agent responsible for fatal rabies disease. The RABV glycoprotein (G) forms trimeric spikes that protrude from RABV virions and mediate virus attachment, entry and spread, and is a major determinant of RABV pathogenesis. A range of RABV strains exist that are highly pathogenic in part due to their ability to evade host immune detection. However, some strains are disease-attenuated and can be cleared by host defences. A detailed molecular understanding of how strain variation relates to pathogenesis is currently lacking. Here, we reveal key differences in the trafficking profiles of RABV-G proteins from the challenge virus standard strain (CVS-11) and a highly attenuated vaccine strain SAD-B19 (SAD). We show that CVS-G traffics to the cell surface and undergoes rapid internalization through both clathrin- and cholesterol-dependent endocytic pathways. In contrast, SAD-G remains resident at the plasma membrane and internalizes at a significantly slower rate. Through engineering hybrids of CVS-G and SAD-G, we show that the cytoplasmic tail of CVS-G is the key determinant of these different internalization profiles. Alanine scanning further revealed that mutation of Y497 in CVS-G (H497 in SAD-G) could reduce the rate of internalization to SAD-G levels. Together, these data reveal new phenotypic differences between CVS-G and SAD-G proteins that may contribute to altered in vivo pathogenicity.

sEVsRVG selectively delivers antiviral siRNA to fetus brain, inhibits ZIKV infection and mitigates ZIKV-induced microcephaly in mouse model.

Zika virus (ZIKV), a flavivirus associated with neurological disorders, constitutes a global health threat. During pregnancy, ZIKV traverses the placenta and causes congenital disease such as microcephaly and Guillain-Barré syndrome in newborns. To develop a specific antiviral therapy against ZIKV-induced microcephaly that could cross placental and blood-brain barriers, we designed targeted small extracellular vesicles (sEVs) encapsulating antiviral siRNA (small interfering RNA) to inhibit ZIKV. The neuro-specific targeting was achieved by engineering EVs membrane protein lamp2b fused with a neuron-specific rabies virus glycoprotein derived peptide (RVG). Intravenous administration of the RVG-engineered sEVs loaded with siRNA (ZIKV-specific siRNA) protected pregnant AG6 mice against vertical transmission of ZIKV. Particularly, sEVsRVG-siRNA traversed placental and blood-brain barriers and suppressed ZIKV infection in fetal brains. Moreover, sEVsRVG-siRNA alleviated the neuroinflammation and neurological damage caused by ZIKV in the fetal mouse model. In general, we developed a sEVs-based targeted system of antiviral therapy for brain and fetal brain infections.

Migration of gastric cancer is suppressed by recombinant Newcastle disease virus (rL-RVG) via regulating α7-nicotinic acetylcholine receptors/ERK- EMT.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) have been reported to be overexpressed in malignancies in humans and is associated with tumorigenesis and cell migration. In previous studies of gastric cancer, alpha7 nicotinic acetylcholine receptor (α7-nAChR) overexpression leads to epithelial-mesenchymal transition (EMT) and promotes the migration of gastric cancer cells. Recombinant avirulent LaSota strain of Newcastle disease virus (NDV) expressing the rabies virus glycoprotein (rL-RVG) may promote apoptosis of gastric cancer cells and reduces the migration of lung cancer metastasis. However, whether rL-RVG inhibits migration of gastric cancer cells and what the underlying functional mechanism is remains unknown. METHODS: The gastric cancer cell lines BGC and SGC were randomly divided into 3 groups: rL-RVG, NDV and Phosphate Buffered Solution (PBS) control groups. Furthermore,we adopted ACB and MLA,α7nAChR-siRNA for the overexpression and silencing of α7-nAChR.Corynoxenine was used for inhibiting the MEK-ERK pathway. Western blot, Immunofluoresce,cell proliferation assays,cell migration analyses through wound-healing assays and Transwell assays were used to explore the underlying mechanisms. A mouse xenograft model was used to investigate the effects of rL-RVG,NDV on tumor growth. RESULTS: In this study, our findings demonstrate that rL-RVG suppressed the migration of gastric cancer cells and reduced EMT via α7-nAChR in vitro. Furthermore rL-RVG decreased the phosphorylation levels of the MEK/ERK signaling pathway such as down-regulating the expression of P-MEK and P-ERK. Additionally, rL-RVG also reduced the expression level of mesenchymal markers N-cadherin and Vimentin and enhanced the expression of the epithelial marker E-cadherin. Lastly, rL-RVG inhibited nicotinic acetylcholine receptors (nAChRs) to suppress cell migration and epithelial to mesenchymal transition (EMT) in gastric cell. We also found that rL-RVG suppresses the growth of gastric cancer subcutaneous tumor cells in vivo. CONCLUSION: rL-RVG inhibits α7-nAChR-MEK/ERK-EMT to suppress migration of gastric cancer cells.

Nanoparticle encapsulation increases the brain penetrance and duration of action of intranasal oxytocin.

The blood-brain barrier (BBB) limits the therapeutic use of large molecules as it prevents them from passively entering the brain following administration by conventional routes. It also limits the capacity of researchers to study the role of large molecules in behavior, as it often necessitates intracerebroventricular administration. Oxytocin is a large-molecule neuropeptide with pro-social behavioral effects and therapeutic promise for social-deficit disorders. Although preclinical and clinical studies are using intranasal delivery of oxytocin to improve brain bioavailability, it remains of interest to further improve the brain penetrance and duration of action of oxytocin, even with intranasal administration. In this study, we evaluated a nanoparticle drug-delivery system for oxytocin, designed to increase its brain bioavailability through active transport and increase its duration of action through encapsulation and sustained release. We first evaluated transport of oxytocin-like large molecules in a cell-culture model of the BBB. We then determined in vivo brain transport using bioimaging and cerebrospinal fluid analysis in mice. Finally, we determined the pro-social effects of oxytocin (50 µg, intranasal) in two different brain targeting and sustained-release formulations. We found that nanoparticle formulation increased BBB transport both in vitro and in vivo. Moreover, nanoparticle-encapsulated oxytocin administered intranasally exhibited greater pro-social effects both acutely and 3 days after administration, in comparison to oxytocin alone, in mouse social-interaction experiments. These multimodal data validate this brain targeting and sustained-release formulation of oxytocin, which can now be used in animal models of social-deficit disorders as well as to enhance the brain delivery of other neuropeptides.

Achieving scientific and regulatory success in implementing non-animal approaches to human and veterinary rabies vaccine testing: A NICEATM and IABS workshop report.

This two-day workshop, co-sponsored by NICEATM and IABS-NA, brought together over 60 international scientists from government, academia, and industry to advance alternative methods for human and veterinary Rabies Virus Vaccine (RVV) potency testing. On day one, workshop presentations focused on regulatory perspectives related to in vitro potency testing, including recent additions to the European Pharmacopoeia (5.2.14) that provide a scientific rationale for why in vivo methods may be less suitable for vaccine quality control than appropriately designed in vitro methods. Further presentations reviewed the role of the consistency approach to manufacturing and vaccine batch comparison to provide supportive data for the substitution of existing animal-based methods with in vitro assays. In addition, updates from research programs evaluating and validating RVV glycoprotein (G) quantitation by ELISA as an in vitro potency test were presented. On the second day, RVV stakeholders participated in separate human and veterinary vaccine discussion groups focused on identifying potential obstacles or additional requirements for successful implementation of non-animal alternatives to the in vivo potency test. Workshop outcomes and proposed follow up activities are discussed herein.

DROSOPHILA S2 cell culture in a WAVE Bioreactor: potential for scaling up the production of the recombinant rabies virus glycoprotein.

The transmembrane rabies virus glycoprotein (RVGP) is the main antigen of vaccine formulations used around the world to prevent rabies, the most lethal preventable infectious disease known. The objective of this work was to evaluate the potential of a bioreactor using wave-induced agitation in the initial steps of scaling up the rRVGP production process by a Drosophila melanogaster S2 cell line to produce rRVGP in sufficient quantities for immunization and characterization studies. Taking advantage of some remarkable features recognized in Drosophila S2 cells for scaling the culture process, a robust recombinant lineage (S2MtRVGPH-His) engineered by our group for the expression of rRVGP using a copper-inducible promoter was used in the bioreactor cultures. The WAVE Bioreactor was chosen because it represents an innovative approach to the cultivation of animal cells using single-use technology. For that purpose, we firstly established a procedure for culturing the S2MtRVGPH-His lineage in 100 mL Schott flasks. Using an inoculum of 5 × 105 cells/mL in culture medium (Sf900-III) induced with solution of CuSO4 (0.7 mM) and a convenient pH range (6.2-7.0), optimal parameter values such as time of induction (72 h) and temperature (28 °C) to increase rRVGP production could be defined. This procedure was reproduced in culture experiments conducted in a WAVE Bioreactor™ 2/10 using a 2 L Cellbag. The results in Schott flasks and in WAVE Bioreactor™ were very similar, yielding a maximum titer of rRVGP above of 1 mg.L-1. The immunization study showed that the rRVGP produced in the bioreactor was of high immunogenic quality.

Recombinant Newcastle disease virus rL-RVG enhances the apoptosis and inhibits the migration of A549 lung adenocarcinoma cells via regulating alpha 7 nicotinic acetylcholine receptors in vitro.

BACKGROUND: The aim of this study were to investigate the possible pro-apoptotic mechanisms of the recombinant Newcastle disease virus (NDV) strain rL-RVG, which expresses the rabies virus glycoprotein, in A549 lung adenocarcinoma cells via the regulation of alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) and to analyze the relationships between α7 nAChR expression in lung cancer and the clinical pathological features. METHODS: α7 nAChR expression in A549, LΑ795, and small-cell lung carcinoma (SCLC) cells, among others, was detected using reverse transcription polymerase chain reaction (RT-PCR). The optimal α7 nAChR antagonist and agonist concentrations for affecting A549 lung adenocarcinoma cells were detected using MTT assays. The α7 nAChR expression in A549 cells after various treatments was assessed by Western blot, immunofluorescence and RT-PCR analyses. Apoptosis in the various groups was also monitored by Western blot and TUNEL assays, followed by the detection of cell migration via transwell and scratch tests. Furthermore, α7 nAChR expression was examined by immunohistochemistry in lung cancer tissue samples from 130 patients and 40 pericancerous tissue samples, and the apoptotis in lung adenocarcinoma tissue was detected by Tunel assay, Then, the expression levels and clinicopathological characteristics were analyzed. RESULTS: Of the A549, LΑ795, SCLC and U251 cell lines, the A549 cells exhibited the highest α7 nAChR expression. The cells infected with rL-RVG exhibited high RVG gene and protein expression. The rL-RVG group exhibited weaker α7 nAChR expression compared with the methyllycaconitine citrate hydrate (MLA, an α7 nAChR antagonist) and NDV groups. At the same time, the MLA and rL-RVG treatments significantly inhibited proliferation and migration and promoted apoptosis in the lung cancer cells (P < 0.05). The expression of α7 nAChR was upregulated in lung cancer tissue compared with pericancerous tissue (P = 0.000) and was significantly related to smoking, clinical tumor-node-metastases stage, and histological differentiation (P < 0.05). The AI in lung adenocarcinoma tissue in high-medium differentiation group was lower than that in low differentiation group (p < 0.01). CONCLUSIONS: An antagonist of α7 nAChR may be used as a molecular target for lung adenocarcinoma therapy. Recombinant NDV rL-RVG enhances the apoptosis and inhibits the migration of A549 lung adenocarcinoma cells by regulating α7 nAChR signaling pathways.

Expression of rabies virus glycoprotein in the methylotrophic yeast Pichia pastoris.

Rabies is a fatal disease that can be prevented by vaccination. Different approaches were investigated to develop novel human rabies vaccines with improved features compared to the current available vaccines, among them is the use of heterologous gene expression technology. Here, we describe the expression of the surface rabies virus glycoprotein (RABV-G), which is the major antigen responsible for the induction of protective immunity, in Pichia pastoris. Six transformants were selected according to their gene copy number as determined by real time qPCR. Upon induction by methanol, low level of RABV-G was secreted into the culture medium, around 60 ng/mL. To understand the effect of foreign gene dosage on cellular physiology of P. pastoris, transcriptional analysis of key genes involved in unfolded protein response (UPR) and endoplasmic reticulum associated degradation (ERAD) pathway was performed. Results showed that these pathways were highly activated; misfolded RABV-G was degraded in the cytosol via the ERAD mechanism. To study the functionality of the secreted RABV-G, in vitro competitive neutralizing assay was conducted. Data showed the secreted recombinant RABV-G had enabled a reduction of the neutralizing activity of human immune rabies serum, indicating that the secreted recombinant protein had reached its correct conformational form.

Targeting DNA vaccines to myeloid cells using a small peptide.

Targeting DNA vaccines to dendritic cells (DCs) greatly enhances immunity. Although several approaches have been used to target protein Ags to DCs, currently there is no method that targets DNA vaccines directly to DCs. Here, we show that a small peptide derived from the rabies virus glycoprotein fused to protamine residues (RVG-P) can target DNA to myeloid cells, including DCs, which results in enhanced humoral and T-cell responses. DCs targeted with a DNA vaccine encoding the immunodominant vaccinia B8R gene via RVG-P were able to restimulate vaccinia-specific memory T cells in vitro. Importantly, a single i.v. injection of B8R gene bound to RVG-P was able to prime a vaccinia-specific T-cell response that was able to rapidly clear a subsequent vaccinia challenge in mice. Moreover, delivery of DNA in DCs was enough to induce DC maturation and efficient Ag presentation without the need for adjuvants. Finally, immunization of mice with a DNA-vaccine encoding West Nile virus (WNV) prM and E proteins via RVG-P elicited high titers of WNV-neutralizing Abs that protected mice from lethal WNV challenge. Thus, RVG-P provides a reagent to target DNA vaccines to myeloid cells and elicit robust T-cell and humoral immune responses.

Optical Imaging and Gene Therapy with Neuroblastoma-Targeting Polymeric Nanoparticles for Potential Theranostic Applications.

Recently, targeted delivery systems based on functionalized polymeric nanoparticles have attracted a great deal of attention in cancer diagnosis and therapy. Specifically, as neuroblastoma occurs in infancy and childhood, targeted delivery may be critical to reduce the side effects that can occur with conventional approaches, as well as to achieve precise diagnosis and efficient therapy. Thus, biocompatible poly(d,l-lactide-co-glycolide) (PLG) nanoparticles containing an imaging probe and therapeutic gene are prepared, followed by modification with rabies virus glycoprotein (RVG) peptide for neuroblastoma-targeting delivery. RVG peptide is a well-known neuronal targeting ligand and is chemically conjugated to PLG nanoparticles without changing their size or shape. RVG-modified nanoparticles are effective in specifically targeting neuroblastoma both in vitro and in vivo. RVG-modified nanoparticles loaded with a fluorescent probe are useful to detect the tumor site in a neuroblastoma-bearing mouse model, and those encapsulating a therapeutic gene cocktail (siMyc, siBcl-2, and siVEGF) significantly suppressed tumor growth in the mouse model. This approach to designing and tailoring of polymeric nanoparticles for targeted delivery may be useful in the development of multimodality systems for theranostic approaches.

A rabies mRNA vaccine with H270P mutation in its glycoprotein induces strong cellular and humoral immunity.

Rabies is a lethal zoonotic disease that kills approximately 60,000 people each year. As the sole virion-surface protein, the rabies virus glycoprotein (RABV-G) mediates its host-cell entry. RABV-G's pre-fusion conformation displays major known neutralizing antibody epitopes, which can be used as immunogen for prophylaxis. H270P targeted mutation can stabilize RABV-G in the pre-fusion conformation. Herein, we report the development of a highly promising rabies mRNA vaccine composed of H270P targeted mutation packaged in lipid nanoparticle (LNP), named LNP-mRNA-G-H270P. Humoral and cellular immunity of this vaccine were assessed in mice comparing to the unmodified LNP-mRNA-G and a commercially available inactivated vaccine using one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. The results show the titer of RABV-G-specific IgG and virus-neutralization antibody titers (VNTs) in LNP-mRNA-G-H270P group were significant higher than those in LNP-mRNA-G and inactivated vaccine groups. Likewise, IFN-γ-secreting splenocytes, level of IL-2 in the supernatant of spleen cells, as well as IFN-γ-producing CD4+ T cells in LNP-mRNA-G-H270P group were significant higher than those in the other two vaccine groups. Hence, these results demonstrated that targeting the H270P mutation in RABV-G through an mRNA-LNP vaccine platform represents a promising strategy for developing a more efficacious rabies vaccine.

Engineered exosomes mediated targeted delivery of neuroprotective peptide NR2B9c for the treatment of traumatic brain injury.

Neuroprotection is one of the core treatment strategies for brain injuries including traumatic brain injury (TBI). NR2B9c is a promising neuroprotective peptide but its clinical translation is limited because of poor brain penetrability. Exosomes are naturally occurring nanovesicles having therapeutic potential for TBI as well as an efficient drug delivery carrier to the brain. Here, we engineered exosomes with neuron targeting peptide rabies virus glycoprotein (RVG29) via bio-orthogonal click chemistry technique and loaded it with NR2B9c, developing RVG-ExoNR2B9c. RVG29 conjugated exosome had higher neuron targeting efficiency compared to naïve exosomes both in vivo and in vitro. RVG-ExoNR2B9c had great cytoprotective effect against oxygen glucose deprived Neuro2a cells. Intravenous administration of RVG-ExoNR2B9c significantly improved behavioral outcomes and reduced the lesion volume after TBI injury in a mice controlled cortical impact model. Due to their multifunctionality and significant efficacy, we anticipate that RVG-ExoNR2B9c have the potential to be translated both as therapeutic agent as well as cargo delivery system to the brain for the treatment of TBI.

The human alpha7 nicotinic acetylcholine receptor is a host target for the rabies virus glycoprotein.

The rabies virus enters the nervous system by interacting with several molecular targets on host cells to modify behavior and trigger receptor-mediated endocytosis of the virion by poorly understood mechanisms. The rabies virus glycoprotein (RVG) interacts with the muscle acetylcholine receptor and the neuronal α4ß2 subtype of the nicotinic acetylcholine receptor (nAChR) family by the putative neurotoxin-like motif. Given that the neurotoxin-like motif is highly homologous to the α7 nAChR subtype selective snake toxin α-bungarotoxin (αBTX), other nAChR subtypes are likely involved. The purpose of this study is to determine the activity of the RVG neurotoxin-like motif on nAChR subtypes that are expressed in brain regions involved in rabid animal behavior. nAChRs were expressed in Xenopus laevis oocytes, and two-electrode voltage clamp electrophysiology was used to collect concentration-response data to measure the functional effects. The RVG peptide preferentially and completely inhibits α7 nAChR ACh-induced currents by a competitive antagonist mechanism. Tested heteromeric nAChRs are also inhibited, but to a lesser extent than the α7 subtype. Residues of the RVG peptide with high sequence homology to αBTX and other neurotoxins were substituted with alanine. Altered RVG neurotoxin-like peptides showed that residues phenylalanine 192, arginine 196, and arginine 199 are important determinants of RVG peptide apparent potency on α7 nAChRs, while serine 195 is not. The evaluation of the rabies ectodomain reaffirmed the observations made with the RVG peptide, illustrating a significant inhibitory impact on α7 nAChR with potency in the nanomolar range. In a mammalian cell culture model of neurons, we confirm that the RVG peptide binds preferentially to cells expressing the α7 nAChR. Defining the activity of the RVG peptide on nAChRs expands our understanding of basic mechanisms in host-pathogen interactions that result in neurological disorders.

Corrigendum: A single vaccination of nucleoside-modified Rabies mRNA vaccine induces prolonged highly protective immune responses in mice.

[This corrects the article DOI: 10.3389/fimmu.2022.1099991.].

Selective neurodegeneration generated by intravenous α-synuclein pre-formed fibril administration is not associated with endogenous α-synuclein levels in the rat brain.

Selective loss of discrete neuronal populations is a prominent feature of many neurodegenerative conditions, but the molecular basis of this is poorly understood. A central role of α-synuclein in the selective neurodegeneration of Parkinson's disease has been speculated, as its level of expression critically determines the propensity of this protein to misfold. To investigate whether the propensity of neuronal cell loss is associated with the level of endogenous α-synuclein expression, non-transgenic rats were given a single intravenous administration of α-synuclein pre-formed fibrils (PFFs) reversibly complexed with the rabies virus glycoprotein peptide (RVG9R). The number of surviving cells in different neuronal populations was systematically quantified using unbiased stereology. Our data demonstrated that a non-selective, transvascular delivery of α-synuclein PFFs led to a time-dependent loss of specific populations of midbrain (but not olfactory) dopaminergic neurons, medullary (but not pontine) cholinergic neurons, and brainstem serotonergic neurons. Contrary to the central role of endogenous α-synuclein expression in determining the seeding and aggregation propensity of pathological α-synuclein, we did not observe an association between the levels of α-synuclein expression in different regions of the rodent brain (although did not ascertain this at the individual cell level) and neurodegenerative propensity. The results from our study highlight the complexity of the neurodegenerative process generated by α-synuclein seeding. Further investigations are therefore required to elucidate the molecular basis of neurodegeneration driven by exogenous pathogenic α-synuclein spread.

Optimized lipopolymers with curcumin to enhance AZD5582 and GDC0152 activity and downregulate inhibitors of apoptosis proteins in glioblastoma multiforme.

Inhibition to glioblastoma multiforme (GBM) propagation is a critical challenge in clinical practice because binding of inhibitors of apoptosis proteins (IAPs) to caspase prevents cancer cells from death. In this study, folic acid (FA), lactoferrin (Lf) and rabies virus glycoprotein (RVG) were grafted on lipopolymers (LPs) composed of poly(ε-caprolactone) and Compritol 888 ATO to encapsulate AZD5582 (AZD), GDC0152 (GDC) and curcumin (CURC). The standard deviations of initial particle diameter and particle diameter after storage for 30 days were involved in LP composition optimization. The functionalized LPs were used to permeate the blood-brain barrier (BBB) and constrain IAP quantity in GBM cells. Experimental results revealed that an increase in Span 20 (emulsifier) concentration enlarged the size of LPs, and enhanced the entrapment and releasing efficiency of AZD, DGC and CURC. 1H nuclear magnetic resonance spectra showed that the hydrogen bonds between the LPs and drugs supported the sustained release of AZD, DGC and CURC from the LPs. The LPs modified with the three targeting biomolecules facilitated the penetration of AZD, GDC and CURC across the BBB, and could recognize U87MG cells and human brain cancer stem cells. Immunofluorescence staining, flow cytometry and western blot demonstrated that CURC-incorporated LPs enhanced AZD and GDC activity in suppressing cellular IAP 1 (cIAP1) and X-linked IAP (XIAP) levels, and raising caspase-3 level in GBM. Surface FA, Lf and RVG also promoted the ability of the drug-loaded LPs to avoid carcinoma growth. The current FA-, Lf- and RVG-crosslinked LPs carrying AZD, DGC and CURC can be promising in hindering IAP expressions for GBM management.

Bio-clickable, small extracellular vesicles-COCKTAIL therapy for ischemic stroke.

Delivering large therapeutic molecules via the blood-brain barrier to treat ischemic stroke remains challenging. NR2B9c is a potent neuroprotective peptide but it's safe and targeted delivery to the brain requires an efficient, natural, and non-immunogenic delivery technique. Small extracellular vesicles (sEVs) have shown great potential as a non-immunogenic, natural cargo delivery system; however, tailoring of its inefficient brain targeting is desired. Here, we coupled rabies virus glycoprotein 29 with sEVs surface via bio-orthogonal click chemistry reactions, followed by loading of NR2B9c, ultimately generating stroke-specific therapeutic COCKTAIL (sEVs-COCKTAIL). Primary neurons and Neuro-2a cells were cultured for in vitro and transient middle cerebral artery occlusion model was used for in vivo studies to evaluate neuron targeting and anti-ischemic stroke potential of the sEVs-COCKTAIL. Bio-clickable sEVs were selectively taken up by neurons but not glial cells. In the in vitro ischemic stroke model of oxygen-glucose deprivation, the sEVs-COCKTAIL exhibited remarkable potential against reactive oxygen species and cellular apoptosis. In vivo studies further demonstrated the brain targeting and increased half-life of bio-clickable sEVs, delivering NR2B9c to the ischemic brain and reducing stroke injury. Treatment with the sEVs-COCKTAIL significantly increased behavioral recovery and reduced neuronal apoptosis after transient middle cerebral artery occlusion. NR2B9c was delivered to neurons binding to post-synaptic density protein-95, inhibiting N-methyl-d-Aspartate receptor-mediated over production of oxidative stress and mitigating protein B-cell lymphoma 2 and P38 proteins expression. Our results provide an efficient and biocompatible approach to a targeted delivery system, which is a promising modality for stroke therapy.

Exosomes derived from human umbilical cord mesenchymal stem cells loaded with RVG-Lamp2b and Netrin-1 promotes Schwann cell invasion and migration.

BACKGROUND: Netrin-1 has a neuroprotective effect by regulating angiogenesis, autophagy, apoptosis, and neuroinflammation. This study investigated the effects of netrin-1 delivery to mouse Schwann cells and vascular endothelial cells using exosomes modified with rabies virus glycoprotein (RVG) peptides. MATERIALS AND METHODS: RVG-Lamp2b and/or Netrin-1 were overexpressed in human umbilical cord mesenchymal stem cells to obtain exosomes modified with RVG-Lamp2b and/or loaded with Netrin-1. Then, exosomes were labeled with carboxyfluorescein diacetate succinimidyl ester and co-cultured with mouse Schwann cells and endothelial cells. Netrin-1 expression in Schwann cells and endothelial cells was measured using quantitative polymerase chain reaction and immunoblotting. Moreover, methyl thiazolyl tetrazolium assays and Transwell assays were used to detect proliferation, migration, and invasion of Schwann cells and endothelial cells. RESULTS: Exosomes with RVG-Lamp2b entered Schwann cells more readily compared with the exosomes without RVG-Lamp2b. Meanwhile, this was not the case in endothelial cells. Netrin-1-loaded exosomes significantly promoted Netrin-1 expression, cell proliferation, migration, invasion, and epithelial-mesenchymal transition in Schwann cells and endothelial cells. These effects were further enhanced by Netrin-1-loaded exosomes modified with RVG-Lamp2b in Schwann cells, but not in endothelial cells. CONCLUSION: HucMSC-derived exosomes loaded with RVG-Lamp2b and Netrin-1 promote proliferation, migration, and invasion of Schwann cells.

Preparation of baicalin-loaded ligand-modified nanoparticles for nose-to-brain delivery for neuroprotection in cerebral ischemia.

Neuroprotection in cerebral ischemia (CI) has received increasing attention. However, efficient delivery of therapeutic agents to the brain remains a major challenge due to the complex environment of the brain. Nose-to-brain-based delivery is a promising approach. Here, we optimized a nanocarrier formulation of neuroprotective agents that can be used for nose-to-brain delivery by obtaining RVG29 peptide-modified polyethylene glycol-polylactic acid-co-glycolic acid nanoparticles (PEG-PLGA RNPs) that have physicochemical properties that lead to stable and sustained drug release and thereby improve the bioavailability of neuroprotective agents. The brain-targeting ability of PEG-PLGA RNPs administered through nasal inhalation was verified in a rat model of CI. It was found that delivery to the whole brain can be achieved with little delivery to the peripheral circulation. Baicalin (BA) was selected as the neuroprotective agent for delivery. After intranasal administration of BA-PEG-PLGA RNPs, the neurological dysfunction of rats with ischemic brain injury was significantly alleviated, the cerebral infarction area was reduced, and nerve trauma and swelling were relieved. Furthermore, it was demonstrated that the neuroprotective effects of BA in a rat model of CI may be mediated by inhibition of inflammation and alleviation of oxidative stress. The immunohistochemical results obtained after treatment with nanoparticles loaded with BA showed that Nrf2/HO-1 was activated in the area in which ischemic brain damage had occurred and that its expression was significantly higher in the group treated with BA-PEG-PLGA RNPs than in the other groups. The ELISA results showed that the levels of IL-1ß, IL-6, and TNF-α were abnormally increased in the serum of rats with cerebral ischemia. After treatment with BA-loaded nanoparticles, IL-1ß, IL-6, and TNF-α levels decreased significantly. Oxidative stress was alleviated; the levels of glutathione and superoxide dismutase increased; and the levels of reactive oxygen species and malondialdehyde decreased, in animals to which BA-PEG-PLGA RNPs were delivered by intranasal inhalation. In conclusion, BA-PEG-PLGA RNPs can effectively deliver BA to rats and thereby exert neuroprotective effects against CI.

Rabies Virus Glycoprotein-Mediated Transportation and T Cell Infiltration to Brain Tumor by Magnetoelectric Gold Yarnballs.

T lymphocyte infiltration with immunotherapy potentially suppresses most devastating brain tumors. However, local immune privilege and tumor heterogeneity usually limit the penetration of immune cells and therapeutic agents into brain tumors, leading to tumor recurrence after treatment. Here, a rabies virus glycoprotein (RVG)-camouflaged gold yarnball (RVG@GY) that can boost the targeting efficiency at a brain tumor via dual hierarchy- and RVG-mediated spinal cord transportation, facilitating the decrease of tumor heterogeneity for T cell infiltration, is developed. Upon magnetoelectric irradiation, the electron current generated on the GYs activates the electrolytic penetration of palbociclib-loaded dendrimer (Den[Pb]) deep into tumors. In addition, the high-density GYs at brain tumors also induces the disruption of cell-cell interactions and T cell infiltration. The integration of the electrolytic effects and T cell infiltration promoted by drug-loaded RVG@GYs deep in the brain tumor elicits sufficient T cell numbers and effectively prolongs the survival rate of mice with orthotopic brain tumors.