The overall goal of this procedure is to describe a validated approach to study trans-blood brain barrier delivery of nanoformulated fluorescent molecules using ferritin nano-cages loaded with FITC as proof of concept. This method can help answer key questions in the field of resistance of central level system disease to pharmacological therapies due to the low permeation of drugs across the blood brain barrier. The main advantage of this technique is the possibility to demonstrate the ability of nano formulation to improve the transmitted BBB permeation of encapsulated molecules using a approach on a single BBB model.
The implications of this technique extend toward therapy of disorders with neurological effects because it tends to overcome the high selectivity of the blood brain barrier to most drugs. This method can provided insight into the efficiency of ferritin nano cages as drug delivery system across the BBB. It has been applied with the FITC, but it can also be exploited with other carrageenan molecules or a different kind of organic or inorganic nano particles.
Begin by coating cell culture flasks with 100 micrograms per milliliter of Poly L Lysine for one hour at room temperature to promote the attachment of rat cortical astrocytes. Coat one flask with 50 micrograms per milliliter of fibronectin for one hour at 37 degrees Celsius for the culture of rat brain microvascular epithelial cells. Then thaw one times 10 to the sixth RCAs and five times 10 to the fifth RBMECs in supplemented endothelial cell medium.
Seed the astrocytes in a coated T175 flask and the endothelial cells into a coated T75 flask. Maintain the cells at 37 degrees Celsius and five percent CO2 in a humidified atmosphere for approximately six days. When the incubation time has elapsed and the desired confluence is reached, detach the cells using trypsin EDTA solution for five minutes at 37 degrees Celsius.
Stop the trypsin activity by adding supplemented endothelial cell medium. Then centrifuge the cell suspension and resuspend the pellet in the medium. Split the RBMECs into three T175 flasks.
And culture for another three days before seeding onto inserts. After detaching astrocytes from flasks as described for RBMECs, dilute a small volume of cells one-to-one with trypan blue. Load into a burker chamber and count the total number of living RCAs under an optical microscope.
To seed the cells onto an insert membrane coat the upper side with 100 micrograms per milliliter of Poly L Lysine and the lower side with 50 micrograms per milliliter of fibronectin. Place the inserts into the wells of a six well plate and add 500 microliters of fibronectin solution to the upper chamber. Incubate for one hour at 37 degrees Celsius.
Following the incubation, remove the fibronectin solution and transfer the inserts upside-down on the bottom of a 150 centimeter squared petri dish. Then gently add 800 microliters of Poly L Lysine to the bottom side of the insert and keep the solution on the inserts for one hour at room temperature. After the hour has elapsed, aspirate the solution and let the inserts dry at room temperature for 15 to 30 minutes after which time the inserts are now ready for cell seeding.
Add 800 microliters of cell suspension in drops onto the upside-down insert to seed the astrocytes at a density of 35, 000 cells per centimeter squared. Allow the cells to attach for four hours at room temperature. After attachment, aspirate the residual solution.
Place the inserts into the wells containing two milliliters of supplemented endothelial cell medium and maintain the multi-well plate in the tissue culture incubator. After three days when the astrocytes have coated the lower face of the insert, seed endothelial cells at a density of 60, 000 cells per centimeter squared in a one milliliter volume onto the upper side of the insert. Return the plate to the incubator for at least three days before measuring trans-endothelial electrical resistance.
One the third day of cold culture check the trans-endothelial electrical resistance of the developing blood brain barrier by first filling a chamber containing a pair of voltage sensing and current electrodes with four milliliters of supplemented endothelial cell medium. Then place an insert bearing the blood brain barrier system into the chamber. Fit the lid and connect the chamber to an epithelial tissue Ohm meter.
Record the resistance measurements of the insert. Then repeat for two more inserts bearing the blood brain barrier system followed by the three inserts bearing the RCAs alone. Calculate the trans-endothelial electrical resistance in Ohms per centimeter squared as shown on screen.
To measure the FD40 flux, add one milligram per milliliter FD40 diluted in media into the upper compartment of the blood brain barrier system inserts and to the three empty inserts. Then after one hour, withdraw 200 microliters of media from the lower chamber. And measure the fluorescents intensity by spectrofluorometer.
To measure the FITC ferritin flux through the blood brain barrier system add FITC loaded ferritin or free FITC to the upper portion of at least three blood brain barrier systems for each formulation. After seven hours in an incubator withdraw two milliliters of medium from the lower chambers of three sample and control inserts. Measure the fluorescents intensity of 500 microliters of the collected samples by spectrofluorometer and determine the concentration of permeated FITC or FITC ferritin according to the instructions in the written portion of the protocol.
To ascertain the location of FITC ferritin in endothelial cells, first remove the medium from the upper chamber of the blood brain barrier systems and wash with PBS. Then add 500 microliters of paraformaldehyde to the upper compartment of at least two inserts for each experimental group and allow the cells to fix for 10 minutes at room temperature. After washing the inserts three times with PBS to remove residual paraformaldehyde cut the membrane into pieces and then proceed with immunostaining as outlined in the written portion of the protocol.
After staining, mount the insert pieces onto microscope slides using an anti-fade mounting solution. Then close the sample with a cover slip for analysis by confocal microscope. The affect of ferritin encapsulation on FITC permeation across the blood brain barrier is shown here.
The FITC concentration in the lower chamber after seven and 24 hours from the addition of nanoformulated dye in the upper compartment indicates that ferritin is able to significantly increase the delivery of FITC across the blood brain barrier. The following single optical section confocal laser scanning micrographs show endothelial cells grown on the upper side of the insert at various time points after incubation with FITC loaded ferritin or free FITC. This micrograph shows the endothelial cells after seven hours of incubation with FITC loaded ferritin.
The endotheial cells are immunostained for VWF, which appears red and DAPI is shown in blue. The FITC loaded ferritin is visible as green. Here endothelial cells incubated with FITC for seven hours are shown.
The absence of green staining in the middle panel demonstrates that free FITC is not internalized by the cells. This image shows the endothelial cells after 24 hours of incubation with FITC loaded ferritin. The green FITC fluorescents is clearly visible.
Finally this image demonstrates that longer incubations with free FITC, 24 hours is shown here, does not allow the cells to take up the fluorophore. Once mastered, this technique can be done in a period of time no longer than 13 days if it is performed appropriately. Following this procedure, an indication of the status of the BBB cells upon exposure to the nanoformulation can be also obtained by recording TER variation or analyzing endothelial integrity by electromicroscopy.
After it's development this technique could pave the way for researchers in the field of nano medicine to explore the capability of nanoformulations in enhancing the permeation of fluorescent molecules into the brain as a preliminary step for furthering the investigations. After watching this video you should have a good understanding of how to validate nano systems for the delivery of drugs across the BBB. By using a good quality BBB in-vitro models and ferritin nano cages incapsulating FITC as proof of concept.
