Macrophage Delivery of Therapeutic Nanozymes in a Murine Model of Parkinson's Disease

BMM Adherence & Migratory Activities

α-4 integrin is a protein on the surface of immune cells that allows passage into the CNS.^[17] Therefore, the effect of nanozyme loading on α-4 integrin expression by BMMs was evaluated by fluorescence-activated cell sorting. BMMs were collected and incubated with catalase alone or catalase nanozyme for 2 h. After incubation, BMMs were stained with phycoerythrin-conjugated anti-CD49d antibodies (catalog number 557420); phycoerythrin-Cy5-conjugated nonspecific antibodies (catalog number 553931) were used as isotype controls to assess the level of nonspecific binding. All antibodies were purchased from BD Pharmingen (BD Biosciences, CA, USA). Following staining, BMMs were fixed in 1% BSA/4% paraformaldehyde in PBS and analyzed using LSR2 instrument by (BD Biosciences) and Diva Version 6.1.2 analysis software. Nonloaded BMMs served as a control.

To study the effect of nanozyme loading on BMM adherence and transport, confluent bovine brain microvessel endothelial cell (BMVEC) monolayers were used to reflect BBB function. The tracking of monocytes across such an artificial BBB in vitro was developed previously.^[18] Confluent primary BMVECs retain many of the morphological and biochemical characteristics of the BBB, such as formation of tight junctions and low pinocytic activity.^[19] Therefore, this system was used to closely recapitulate in vivo mechanisms of monocyte–macrophage-mediated transport of nanozyme across the BBB. For adherence studies, BMVECs were isolated from fresh cow brains by enzymatic digestion and density centrifugation, and grown on 24-well plates until confluent (typically, 12 days) as described in.^[20] BMMs, labeled with Alexa Fluor 680, were loaded with catalase nanozyme for 1 h (Z = 1), washed and then added to BMVEC monolayers (6 × 10⁵ cells/well). Nonloaded BMMs were used as a control. The monocytes were allowed to adhere for 30 min at 37°C. Afterwards, supernatants were collected, cells washed twice with PBS, solubilized with Tween X100 and the amount of Alexa Fluor-labeled BMMs was measured using a Shimadzu RF5000 fluorescent spectrophotometer as described.^[21] BMM adhesion was expressed as the number of labeled cells/cm².

For migratory activity studies, BMVECs were cultured on 24-well polycarbonate membrane inserts until confluent.^[20] Transepithelial electrical resistance values were recorded as indices of cell viability and monolayer integrity. Fluorescently labeled BMMs were loaded with catalase (1 mg/ml) nanozyme. Nonloaded BMMs were used as a control. Following preincubation with assay buffer, the solution in the upper chamber of BMVEC monolayers was replaced with the loaded BMMs at a concentration of 2 × 10⁶ cells/w; 150 ng/ml (R&D Systems, Minneapolis, MN, USA) was placed into the lower chamber and used as chemoattractant.^[18] Following 6 h incubation at 37°C in a shaker, the BMVEC inserts were removed, placed in 24-well plates and centrifuged for 10 min at 400 µg to pellet the migrating cells to the plate's bottom chamber. The pelleted cells were lysed with Triton X-100 (1%) and the amount of labeled BMMs was measured as described below. All experiments were performed in triplicate.

Nanomedicine. 2010;5(3):379-396. © 2010 Future Medicine Ltd.

Cite this: Macrophage Delivery of Therapeutic Nanozymes in a Murine Model of Parkinson's Disease - Medscape - Apr 01, 2010.