Anesthesia triggers drug delivery to experimental glioma in mice by hijacking caveolar transport.

BACKGROUND: Pharmaceutical intervention in the CNS is hampered by the shielding function of the blood-brain barrier (BBB). To induce clinical anesthesia, general anesthetics such as isoflurane readily penetrate the BBB. Here, we investigated whether isoflurane can be utilized for therapeutic drug delivery. METHODS: Barrier function in primary endothelial cells was evaluated by transepithelial/transendothelial electrical resistance, and nanoscale STED and SRRF microscopy. In mice, BBB permeability was quantified by extravasation of several fluorescent tracers. Mouse models including the GL261 glioma model were evaluated by MRI, immunohistochemistry, electron microscopy, western blot, and expression analysis. RESULTS: Isoflurane enhances BBB permeability in a time- and concentration-dependent manner. We demonstrate that, mechanistically, isoflurane disturbs the organization of membrane lipid nanodomains and triggers caveolar transport in brain endothelial cells. BBB tightness re-establishes directly after termination of anesthesia, providing a defined window for drug delivery. In a therapeutic glioblastoma trial in mice, simultaneous exposure to isoflurane and cytotoxic agent improves efficacy of chemotherapy. CONCLUSIONS: Combination therapy, involving isoflurane-mediated BBB permeation with drug administration has far-reaching therapeutic implications for CNS malignancies.

Niosomes decorated with dual ligands targeting brain endothelial transporters increase cargo penetration across the blood-brain barrier.

Nanoparticles targeting transporters of the blood-brain barrier (BBB) are promising candidates to increase the brain penetration of biopharmacons. Solute carriers (SLC) are expressed at high levels in brain endothelial cells and show a specific pattern at the BBB. The aim of our study was to test glutathione and ligands of SLC transporters as single or dual BBB targeting molecules for nanovesicles. High mRNA expression levels for hexose and neutral amino acid transporting SLCs were found in isolated rat brain microvessels and our rat primary cell based co-culture BBB model. Niosomes were derivatized with glutathione and SLC ligands glucopyranose and alanine. Serum albumin complexed with Evans blue (67 kDa), which has a very low BBB penetration, was selected as a cargo. The presence of targeting ligands on niosomes, especially dual labeling, increased the uptake of the cargo molecule in cultured brain endothelial cells. This cellular uptake was temperature dependent and could be decreased with a metabolic inhibitor and endocytosis blockers filipin and cytochalasin D. Making the negative surface charge of brain endothelial cells more positive with a cationic lipid or digesting the glycocalyx with neuraminidase elevated the uptake of the cargo after treatment with targeted nanocarriers. Treatment with niosomes increased plasma membrane fluidity, suggesting the fusion of nanovesicles with endothelial cell membranes. Targeting ligands elevated the permeability of the cargo across the BBB in the culture model and in mice, and dual-ligand decoration of niosomes was more effective than single ligand labeling. Our data indicate that dual labeling with ligands of multiple SLC transporters can potentially be exploited for BBB targeting of nanoparticles.

Monitoring proteins using in vivo near-infrared time-domain optical imaging after 2-O-hexyldiglycerol-mediated transfer to the brain.

PURPOSE: The aim of the present study was to gain insight into the penetration, biodistribution, and fate of globulins in the brain after 2-O-hexyldiglycerol-induced blood-brain barrier opening. PROCEDURES: The spatial distribution of fluorescence probes was investigated after blood-brain barrier opening with intracarotid 2-O-hexyldiglycerol injection. Fluorescence intensity was visualized by microscopy (mice and rats) and by in vivo time-domain optical imaging. RESULTS: There was an increased 2-O-hexyldiglycerol-mediated transfer of fluorescence-labeled globulins into the ipsilateral hemisphere. Sequential in vivo measurements revealed that the increase in protein concentration lasted at least 96 h after administration. Ex vivo detection of tissue fluorescence confirmed the results obtained in vivo. CONCLUSION: Globulins enter the healthy brain in conjunction with 2-O-hexyldiglycerol. Sequential in vivo near-infrared fluorescence measurements enable the visualization of the spatial distribution of antibodies in the brain of living small animals.

Tumor localization of an anti-TGF-ß antibody and its effects on gliomas.

Even with current standard-of-care therapies, the prognosis for patients with malignant gliomas is very poor and several new treatment modalities for glioblastomas are currently under investigation. Given the role of TGF-ß in gliomas, anti-TGF-ß strategies against gliomas are currently being investigated. Biodistribution of intravenously injected AF680-labeled 1D11, a pan-neutralizing TGF-ß antibody, was monitored in mice bearing either subcutaneous or orthotopic gliomas using in vivo imaging and fluorescence microscopy. AF680-labeled 1D11 entered both the subcutaneous and intracranial tumors and the antibody was detected within the tumor tissue for several days whereas only low fluorescence was found in organs. The effects of 1D11 on subcutaneous versus orthotopic U87MG and GL261 gliomas in immunocompetent C57BL/6J versus immunodeficient CD1-Foxn1nu mice were observed by direct tumor size measurement, H&E staining and immunohistochemistry. Treatment of immunocompetent mice bearing subcutaneous GL261 tumors with 1D11 resulted in complete remission. In immune deficient mice, the growth of subcutaneous GL261 tumors was increased following treatment with 1D11. Intracranially implanted gliomas in C57Bl/6J mice showed no size reduction after 1D11 treatment but there was reduced invasion of the glioma cells into the adjacent normal brain. Together these data demonstrate that TGF-ß plays different roles in combating the tumor depending on subcutaneous versus orthotopic implantation site.

Expression of the water channel protein aquaporin-9 in malignant brain tumors.

Recently, many studies seen concerning the expression and distribution of aquaporins and K channels in the central nervous system, and their physiological and pathophysiologic roles in water and ion homeostasis. Whereas most data were collected on aquaporin-4 (AQP4) in astrocytes, only little attention was paid to AQP9 which is a water channel transporting glycerol, mannitol, and urea as well. This is the first study describing AQP9 in human brain and human brain tumors. For comparison, we also investigated the immunohistochemical distribution of AQP9 in the rat glioma RG2. Whereas in the normal rat brain AQP9 is only weakly expressed by astrocytes, the anti-AQP9 immunoreactivity was found to be increased at the tumor border, but not within the tumor. In contrast, in human glioblastoma, most glioma cells throughout the tumor revealed a strong anti-AQP9 immunoreactivity across the whole surface of the cell. In the discussion, the increase of the anti-AQP9 immunoreactivity in glioma cells is suggested to reflect an upregulation and to counteract the glioma-associated lactic acidosis by clearance of glycerol and lactate from the extracellular space. In addition, the increased level of AQP9 immunoreactivity could be involved in the energy metabolism of the glioma and/or surrounding neuronal cells.

Models of monocytic invasion into glioma cell aggregates.

BACKGROUND: In order to investigate why human gliomas are abundantly infiltrated by monocytic cells without signs of antitumor activity, experimental models were established in vitro and in vivo. MATERIALS AND METHODS: Peripheral human blood monocytes were added to A172 or U118 glioma cell spheroids and probes analyzed after 72 h by immunohistochemistry. Fluorescence-labelled peritoneal macrophages were administered to syngeneic RG2-glioma-bearing Fischer rats by intravenous or intracarotid injection. RESULTS: Spheroids of both cell lines were infiltrated by monocytes, which took on a chronic inflammatory phenotype with co-expression of MRP8 and MAC 387/MRP14 and positivity to 25F9, but not to 27E10. After both intra-arterial and intravenous injection, labelled monocytes accumulated within the tumor parenchyma of the rat gliomas, while the surrounding brain was only sparsely infiltrated. CONCLUSION: The experimental models described here allow for further investigation of the interactions between monocytes and glioma cells, both in vitro and in vivo. Moreover, monocytes that infiltrate from the peripheral blood into brain tumors may serve as carriers for targeted therapies.