Novel brain-targeted nanomicelles for anti-glioma therapy mediated by the ApoE-enriched protein corona in vivo.

BACKGROUND: The interactions between nanoparticles (NPs) and plasma proteins form a protein corona around NPs after entering the biological environment, which provides new biological properties to NPs and mediates their interactions with cells and biological barriers. Given the inevitable interactions, we regard nanoparticle‒protein interactions as a tool for designing protein corona-mediated drug delivery systems. Herein, we demonstrate the successful application of protein corona-mediated brain-targeted nanomicelles in the treatment of glioma, loading them with paclitaxel (PTX), and decorating them with amyloid ß-protein (Aß)-CN peptide (PTX/Aß-CN-PMs). Aß-CN peptide, like the Aß1-42 peptide, specifically binds to the lipid-binding domain of apolipoprotein E (ApoE) in vivo to form the ApoE-enriched protein corona surrounding Aß-CN-PMs (ApoE/PTX/Aß-CN-PMs). The receptor-binding domain of the ApoE then combines with low-density lipoprotein receptor (LDLr) and LDLr-related protein 1 receptor (LRP1r) expressed in the blood-brain barrier and glioma, effectively mediating brain-targeted delivery. METHODS: PTX/Aß-CN-PMs were prepared using a film hydration method with sonication, which was simple and feasible. The specific formation of the ApoE-enriched protein corona around nanoparticles was characterized by Western blotting analysis and LC-MS/MS. The in vitro physicochemical properties and in vivo anti-glioma effects of PTX/Aß-CN-PMs were also well studied. RESULTS: The average size and zeta potential of PTX/Aß-CN-PMs and ApoE/PTX/Aß-CN-PMs were 103.1 nm, 172.3 nm, 7.23 mV, and 0.715 mV, respectively. PTX was efficiently loaded into PTX/Aß-CN-PMs, and the PTX release from rhApoE/PTX/Aß-CN-PMs exhibited a sustained-release pattern in vitro. The formation of the ApoE-enriched protein corona significantly improved the cellular uptake of Aß-CN-PMs on C6 cells and human umbilical vein endothelial cells (HUVECs) and enhanced permeability to the blood-brain tumor barrier in vitro. Meanwhile, PTX/Aß-CN-PMs with ApoE-enriched protein corona had a greater ability to inhibit cell proliferation and induce cell apoptosis than taxol. Importantly, PTX/Aß-CN-PMs exhibited better anti-glioma effects and tissue distribution profile with rapid accumulation in glioma tissues in vivo and prolonged median survival of glioma-bearing mice compared to those associated with PMs without the ApoE protein corona. CONCLUSIONS: The designed PTX/Aß-CN-PMs exhibited significantly enhanced anti-glioma efficacy. Importantly, this study provided a strategy for the rational design of a protein corona-based brain-targeted drug delivery system. More crucially, we utilized the unfavorable side of the protein corona and converted it into an advantage to achieve brain-targeted drug delivery.

Efficient Anti-Glioma Therapy Through the Brain-Targeted RVG15-Modified Liposomes Loading Paclitaxel-Cholesterol Complex.

BACKGROUND: Glioma is the most common primary malignant brain tumor with a dreadful overall survival and high mortality. One of the most difficult challenges in clinical treatment is that most drugs hardly pass through the blood-brain barrier (BBB) and achieve efficient accumulation at tumor sites. Thus, to circumvent this hurdle, developing an effectively traversing BBB drug delivery nanovehicle is of significant clinical importance. Rabies virus glycoprotein (RVG) is a derivative peptide that can specifically bind to nicotinic acetylcholine receptor (nAChR) widely overexpressed on BBB and glioma cells for the invasion of rabies virus into the brain. Inspired by this, RVG has been demonstrated to potentiate drugs across the BBB, promote the permeability, and further enhance drug tumor-specific selectivity and penetration. METHODS: Here, we used the RVG15, rescreened from the well-known RVG29, to develop a brain-targeted liposome (RVG15-Lipo) for enhanced BBB permeability and tumor-specific delivery of paclitaxel (PTX). The paclitaxel-cholesterol complex (PTX-CHO) was prepared and then actively loaded into liposomes to acquire high entrapment efficiency (EE) and fine stability. Meanwhile, physicochemical properties, in vitro and in vivo delivery efficiency and therapeutic effect were investigated thoroughly. RESULTS: The particle size and zeta potential of PTX-CHO-RVG15-Lipo were 128.15 ± 1.63 nm and -15.55 ± 0.78 mV, respectively. Compared with free PTX, PTX-CHO-RVG15-Lipo exhibited excellent targeting efficiency and safety in HBMEC and C6 cells, and better transport efficiency across the BBB in vitro model. Furthermore, PTX-CHO-RVG15-Lipo could noticeably improve the accumulation of PTX in the brain, and then promote the chemotherapeutic drugs penetration in C6luc orthotopic glioma based on in vivo imaging assays. The in vivo antitumor results indicated that PTX-CHO-RVG15-Lipo significantly inhibited glioma growth and metabasis, therefore improved survival rate of tumor-bearing mice with little adverse effect. CONCLUSION: Our study demonstrated that the RVG15 was a promising brain-targeted specific ligands owing to the superior BBB penetration and tumor targeting ability. Based on the outstanding therapeutic effect both in vitro and in vivo, PTX-CHO-RVG15-Lipo was proved to be a potential delivery system for PTX to treat glioma in clinic.

Aggravated chronic brain injury after focal cerebral ischemia in aquaporin-4-deficient mice.

The water channel aquaporin-4 (AQP4) is important in brain water homeostasis, and is also involved in astrocyte growth and glial scar formation. It has been reported that AQP4 deficiency attenuates acute ischemic brain injury as a result of reducing cytotoxic edema. Here, we determined whether AQP4 deficiency influences chronic brain injury after focal cerebral ischemia induced by 30 min of middle cerebral artery occlusion (MCAO). AQP4(-/-) mice exhibited a lower survival rate and less body weight gain than wild-type mice, but their neurological deficits were similar to wild-type mice during 35 days after MCAO. At 35 days after MCAO, AQP4(-/-) mice showed more severe brain atrophy and cavity formation in the ischemic hemisphere as well as more neuronal loss in the hippocampus. Furthermore, astrocyte proliferation and glial scar formation were impaired in AQP4(-/-) mice. Therefore, AQP4 deficiency complicated by astrocyte dysfunction aggravates chronic brain injury after focal cerebral ischemia, suggesting that AQP4 may be important in the chronic phase of the post-ischemic recovery process.

CysLT2 receptor-mediated AQP4 up-regulation is involved in ischemic-like injury through activation of ERK and p38 MAPK in rat astrocytes.

AIMS: We previously reported that cysteinyl leukotriene receptor 2 (CysLT(2)) mediates ischemic astrocyte injury, and leukotriene D(4)-activated CysLT(2) receptor up-regulates the water channel aquaporin 4 (AQP4). Here we investigated the mechanism underlying CysLT(2) receptor-mediated ischemic astrocyte injury induced by 4-h oxygen-glucose deprivation and 24-h recovery (OGD/R). MAIN METHODS: Primary cultures of rat astrocytes were treated by OGD/R to construct the cell injury model. AQP4 expression was inhibited by small interfering RNA (siRNA). The expressions of AQP4 and CysLTs receptors, and the MAPK signaling pathway were determined. KEY FINDINGS: OGD/R induced astrocyte injury, and increased expression of the CysLT(2) (but not CysLT(1)) receptor and AQP4. OGD/R-induced cell injury and AQP4 up-regulation were inhibited by a CysLT(2) receptor antagonist (Bay cysLT2) and a non-selective CysLT receptor antagonist (Bay u9773), but not by a CysLT(1) receptor antagonist (montelukast). Knockdown of AQP4 by siRNA attenuated OGD/R injury. Furthermore, OGD/R increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the cell injury and AQP4 up-regulation. SIGNIFICANCE: The CysLT(2) receptor mediates AQP4 up-regulation in astrocytes, and up-regulated AQP4 leads to OGD/R-induced injury, which results from activation of the ERK1/2 and p38 MAPK pathways.

Antibacterial activity and cytotoxicity of two novel cross-linking antibacterial monomers on oral pathogens.

OBJECTIVES: The antibacterial activity and cytotoxicity of two novel cross-linking antibacterial monomers, 2-methacryloxylethyl dodecyl methyl ammonium bromide (MAE-DB) and 2-methacryloxylethyl hexadecyl methyl ammonium bromide (MAE-HB) were tested in this study. DESIGN: The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of unpolymerized MAE-DB and MAE-HB against eight strains of oral bacteria were tested using a broth dilution test. Time-kill determinations were performed to examine the kinetics of unpolymerized MAE-DB and MAE-HB against Streptococcus mutans UA159 and Streptococcus sanguinis ATCC6715. Bacterial morphology was observed using a field emission scanning electron microscope (Fe-SEM). The cytotoxicity of unpolymerized two new monomers and Bis-GMA on the human gingival fibroblast cell line H2620 was assessed using a methyl thiazolyl tetrazolium assay. RESULTS: Unpolymerized MAE-DB and MAE-HB showed strong bactericidal activity against oral bacteria. The MBC value of MAE-DB ranged from 12.2 to 24.4µg/ml and the MBC value of MAE-HB ranged from 6.2 to 48.8µg/ml. Time-kill determinations indicated that unpolymerized MAE-DB and MAE-HB had rapid killing effects against S. mutans UA159 and S. sanguinis ATCC6715 at the concentration of 4× MBC. The Fe-SEM observation showed that MAE-DB and MAE-HB could disturb the integrity of bacteria and cause lysis of bacterial cells. The median lethal concentration values on human gingival fibroblast for both monomers were between 10 and 20µg/ml, and greater than that of Bis-GMA. CONCLUSIONS: Unpolymerized MAE-DB and MAE-HB monomers had strong bactericidal activity against eight strains of oral bacteria. Their cytotoxicities were less than that of Bis-GMA.

Baicalin attenuates oxygen-glucose deprivation-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase activation in PC12 cells.

AIM: To determine whether the flavonoid baicalin attenuates oxygen-glucose deprivation (OGD)-induced injury by inhibiting oxidative stress-mediated 5-lipoxygenase (5-LOX) activation in PC12 cells. METHODS: The effects of baicalin and the 5-LOX inhibitor zileuton on the changes induced by OGD/recovery or H(2)O(2) (an exogenous reactive oxygen species [ROS]) in green fluorescent protein-5-LOX-transfected PC12 cells were compared. RESULTS: Both baicalin and zileuton attenuated OGD/recovery- and H(2)O(2)-induced injury and inhibited OGD/recovery-induced production of 5-LOX metabolites (cysteinyl leukotrienes) in a concentration-dependent manner. However, baicalin did not reduce baseline cysteinyl leukotriene levels. Baicalin also reduced OGD/recovery-induced ROS production and inhibited 5-LOX translocation to the nuclear envelope and p38 phosphorylation induced by OGD/recovery and H(2)O(2). In contrast, zileuton did not show these effects. CONCLUSION: Baicalin can inhibit 5-LOX activation after ischemic injury, which may partly result from inhibition of the ROS/p38 mitogen-activated protein kinase pathway.

[Preparation and identification of polyclonal antibody against cysteinyl leukotriene receptor 2].

OBJECTIVE: To prepare and identify a polyclonal antibody against cysteinyl leukotriene receptor (CysLT(2)receptor). METHODS: Rabbits were immunized with KLH-coupled CysLT(2) receptor peptide to prepare the polyclonal antibody (pAb). The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. The tissue distribution of CysLT(2) receptor was detected by Western blot and immunohistochemistry with the prepared pAb. RESULT: The pAb showed a titer higher than 1/1047296, and was specific to CysLT(2) receptor, without cross-reaction with the antigens of CysLT(1) receptor and GPR17. A higher expression of CysLT(2) receptor in kidney, brain and lung of rats and mice was detected by Western blot analysis using the prepared pAb. The molecular weight of CysLT(2) receptor protein was about 40 kD. Immunohistochemical examination showed that CysLT(2) receptor was expressed mainly in the neuron, and partly in astrocytes in rat brain. CONCLUSION: The prepared CysLT(2) receptor pAb has high sensitivity and specificity, and can be used in Western blot and immunohistochemistry.

[Preparation and identification of a polyclonal antibody against novel cysteinyl leukotriene receptor GPR17].

OBJECTIVE: To prepare and identify a polyclonal antibody (pAb) against GPR17, a novel cysteinyl leukotriene receptor. METHODS: Rabbits were immunized with KLH-coupled GPR17 peptide to prepare the pAb. The titer of the pAb in rabbit plasma was detected by indirect ELISA, and the specificity of the pAb was tested by antigen blockade. GPR17 tissue distribution was detected by Western blot with the pAb. RESULTS: The pAb showed a titer as high as 1:16 364,and was not cross-reacted with the antigens of CysLT(1) and CysLT(2) receptors. A higher expression of GPR17 in the rat brain and heart was detected using the newly prepared pAb. The molecular weigh of GPR17 protein was about 43 kD. CONCLUSION: The prepared GPR17 pAb has high sensitivity and specificity,and can be used in Western blot for detecting GPR17.

Oxygen-glucose deprivation activates 5-lipoxygenase mediated by oxidative stress through the p38 mitogen-activated protein kinase pathway in PC12 cells.

5-Lipoxygenase (5-LOX) is a key enzyme catalyzing arachidonic acid to form leukotrienes. We have reported that ischemic-like injury activates 5-LOX in PC12 cells; however, the mechanisms are unknown. To determine whether ischemic-like injury activates 5-LOX mediated by oxidative stress through the p38 MAPK pathway, we transfected GFP-5-LOX into PC12 cells and induced ischemic-like injury by oxygen-glucose deprivation (OGD). We found that the transfected GFP-5-LOX was localized primarily in the nuclei and translocated to the nuclear envelope after OGD/recovery reaching a maximum 2 hr after a 2-hr exposure to OGD. The nonselective 5-LOX inhibitor caffeic acid, 5-LOX-activating protein inhibitor MK886, and selective 5-LOX inhibitor zileuton attenuated the cell injury and reduced the production of 5-LOX products, cysteinyl leukotrienes, after OGD/recovery. However, only caffeic acid inhibited OGD/recovery-induced 5-LOX translocation. OGD/recovery also increased reactive oxygen species (ROS), which was inhibited by caffeic acid only. Hydrogen peroxide, an exogenous ROS, evoked similar cell injury and 5-LOX translocation, and the inhibitors had effects on the changes after H(2)O(2) similar to those after OGD/recovery. Both OGD/recovery and H(2)O(2) increased the phosphorylated p38 MAPK level, which was inhibited by caffeic acid and the ROS scavenger edaravone, but not by MK886 or zileuton. Moreover, SB203580 (a p38 MAPK inhibitor) and edaravone inhibited the cell injury and 5-LOX translocation induced by OGD/recovery and H(2)O(2). Thus, we conclude that OGD/recovery-induced ischemic-like injury induces 5-LOX activation, which is mediated by oxidative stress through activating the p38 MAPK pathway.