Permeability of PEGylated immunoarsonoliposomes through in vitro blood brain barrier-medulloblastoma co-culture models for brain tumor therapy.

PURPOSE: Owing to restricted access of pharmacological agents into the brain due to blood brain barrier (BBB) there is a need: 1. to develop a more representative 3-D-co-culture model of tumor-BBB interaction to investigate drug and nanoparticle transport into the brain for diagnostic and therapeutic evaluation. 2. to address the lack of new alternative methods to animal testing according to replacement-reduction-refinement principles. In this work, in vitro BBB-medulloblastoma 3-D-co-culture models were established using immortalized human primary brain endothelial cells (hCMEC/D3). METHODS: hCMEC/D3 cells were cultured in presence and in absence of two human medulloblastoma cell lines on Transwell membranes. In vitro models were characterized for BBB formation, zonula occludens-1 expression and permeability to dextran. Transferrin receptors (Tfr) expressed on hCMEC/D3 were exploited to facilitate arsonoliposome (ARL) permeability through the BBB to the tumor by covalently attaching an antibody specific to human Tfr. The effect of anticancer ARLs on hCMEC/D3 was assessed. RESULTS: In vitro BBB and BBB-tumor co-culture models were established successfully. BBB permeability was affected by the presence of tumor aggregates as suggested by increased permeability of ARLs. There was a 6-fold and 8-fold increase in anti-Tfr-ARL uptake into VC312R and BBB-DAOY co-culture models, respectively, compared to plain ARLs. CONCLUSION: The three-dimensional models might be appropriate models to study the transport of various drugs and nanocarriers (liposomes and immunoarsonoliposomes) through the healthy and diseased BBB. The immunoarsonoliposomes can be potentially used as anticancer agents due to good tolerance of the in vitro BBB model to their toxic effect.

The angiogenic gene profile of circulating endothelial progenitor cells from ischemic stroke patients.

BACKGROUND: The identification of circulating endothelial progenitor cells (EPCs) has introduced new possibilities for cell-based treatments for stroke. We tested the angiogenic gene expression of outgrowth endothelial cells (OECs), an EPC subtype capable to shape vessel structures. METHODS: OECs (at colony or mature stages) from ischemic stroke patients (n=8) were characterized using the RT2 ProfilerTM human angiogenesis PCR Array, and human microvascular endothelial cells (hCMEC/D3) were used as an expression reference of endothelial cells. RESULTS: Colony-OECs showed higher expression of CCL2, ID3, IGF-1, MMP9, TGFBR1, TNFAIP2, TNF and TGFB1. However, BAI-1, NRP2, THBS1, MMP2 and VEGFC expression was increased in mature-OECs (p<0.05). ID3 (p=0.008) and TGFBR1 (p=0.03) genes remained significantly overexpressed in colony-OECs compared to mature-OECs or hCMEC/D3. MMP9 levels were significantly increased in colony-OECs (p=0.025) compared to mature-OECs. Moreover, MMP-2, VEGF-C, THBS1 and NRP-2 gene expression was also significantly increased in mature-OECs compared to hCMEC/D3 (p<0.05). Some of these genes were positively validated by RT-PCR. CONCLUSION: Our study shows that OECs from stroke patients present higher levels of pro-angiogenic factors at early stages, decreasing in mature OECs when they become more similar to mature microvascular endothelial cells.