In Vitro Permeation of FITC-loaded Ferritins Across a Rat Blood-brain Barrier: a Model to Study the Delivery of Nanoformulated Molecules

Summary

A method to establish an in vitro model of blood-brain barrier based on a co-culture of rat brain microvascular endothelial cells and astrocytes is described and validated. This system proved to be a valid tool to study the effect of nanoformulation on the trans-barrier permeation of fluorescent molecules.

Abstract

Brain microvascular endothelial cells, supported by pericytes and astrocytes endfeet, are responsible for the low permeation of large hydrosoluble drugs through the blood-brain barrier (BBB), causing difficulties for effective pharmacological therapies. In recent years, different strategies for promoting brain targeting have aimed to improve drug delivery and activity at this site, including innovative nanosystems for drug delivery across the BBB. In this context, an in vitro approach based on a simplified cellular model of the BBB provides a useful tool to investigate the effect of nanoformulations on the trans-BBB permeation of molecules. This study describes the development of a double-layer BBB, consisting of co-cultured commercially available primary rat brain microvascular endothelial cells and astrocytes. A multiparametric approach for the validation of the model, based on the measurement of the transendothelial electrical resistance and the apparent permeability of a high molecular weight dextran, is also described. As proof of concept for the employment of this BBB model to study the effect of different nanoformulations on the translocation of fluorescent molecules across the barrier, we describe the use of fluorescein isothiocyanate (FITC), loaded into ferritin nanoparticles. The ability of ferritins to improve the trans-BBB permeation of FITC was demonstrated by flux measurements and confocal microscopy analyses. The results suggest this is a useful system for validating nanosystems for delivery of drugs across the BBB.

Introduction

The resistance of central nervous system (CNS) diseases (i.e. cancer, epilepsy, depression, schizophrenia and HIV-associated neurological disorder) to pharmacological therapies is due to various different mechanisms, including arduous drug permeation across the blood-brain barrier (BBB). The BBB is the boundary that isolates brain tissues from the substances circulating in the blood. Within this barrier, a layer of brain microvascular endothelial cells (BMECs), supported by pericytes and astrocytes endfeet, is responsible for the high selectivity of the BBB to those hydrosoluble drugs with a molecular weight higher than 400 Da¹. Another drug-related resistance mechanism is linked to the presence on BMECs of drug efflux transporters (P-glycoprotein and multidrug resistance proteins), which co-operate to reduce drug penetration into the CNS and facilitate their extrusion from the brain².

In the last decade, a large number of nanotechnological approaches have been developed to meet the clinical and biological challenge of delivering drugs across the BBB^3-6. In this context, ferritin nanospheres (FnN) represent a completely innovative and promising solution. FnN are 12 nm spheres of 24 self-assembling ferritin (Fn) monomers, which are arranged in a hollow spherical structure of 8 nm inner diameter. Ferritin subunits can be disassembled at acidic pH and reassembled in a shape-memory fashion by bringing the pH to neutrality, allowing various organic molecules to be encapsulated. Therefore, FnN represent an interesting model for the development of multifunctional drug delivery systems^7,8. Moreover, FnN may interact with BMECs thanks to the specific recognition of Transferrin Receptor (TfR) 1, which is expressed on the luminal membrane of these cells⁹.

So far, different in vitro models of the BBB have been developed in order to elucidate trans-BBB permeability to various drugs, toxicity toward the BBB, or the interaction of molecules with efflux transporters. Indeed, these models are considered valid in vitro approaches for a rapid screening of active molecules before proceeding with in vivo studies. These models consist of a single endothelial layer of BMECs or co-cultured BMECs and astrocytes (more rarely pericytes), obtained from animal (rat, mouse, pig and bovine) or human cell lines^10,11,12. The TransEndothelial Electrical Resistance (TEER) and the apparent permeability (P_app) of tracers with a defined molecular weight represent two critical parameters that are used to determine the quality of the in vitro model. Here we describe the employment of a BBB in vitro model, based on a co-culture of rat BMECs (RBMECs) and rat cortical astrocytes (RCAs) to study the trans-BBB permeation of ferritin nanocages encapsulating fluorescein isothiocyanate (FITC).

Protocol

1. Establishing the BBB Model

Note: For establishing the BBB model we suggest using commercially available primary RBMECs and RCAs. All steps must be performed with sterile reagents and disposables, handled in a laminar flow hood. 

1. Cell Culture
   1. Coat cell culture flasks with poly-L-lysine 100 µg/ml (1 hr at RT) or fibronectin 50 µg/ml (1 hr at 37 °C) to promote the attachment of RCAs or RBMECs, respectively. Then, thaw 1 x 10⁶ RCAs and 5 x 10⁵ RBMECs in Endothelial Cell Medium, supplemented with 5% fetal bovine serum, 1% endothelial cell growth supplement and 1% penicillin/streptomycin (sECM). Seed RCAs in a T175 flask in 20 ml sECM and RBMECs in a T75 flask in 10 ml sECM.
      Note: The thawing and seeding passages of RCAs and RBMECs must be adjusted, in terms of cell density and time in culture, according to the number of experimental conditions to be tested with the BBB model. By starting with 1 vial of 1 x 10⁶ RCAs and 1 vial of 5 x 10⁵ RBMECs, it is possible to obtain up to 20 BBB-bearing inserts.
   2. Maintain the cells at 37 °C and 5% CO₂ in a humidified atmosphere for approximately 6 days, until about 80% confluence for RCAs and over 90% confluence for RBMECs. Detach cells using trypsin-EDTA solution (trypsin 1:250) for 5 min (1 ml trypsin for T75 flasks and 2 ml for T175 flasks). Stop trypsin activity with sECM (2:1), centrifuge cell suspensions at 750 × g for 5 min and resuspend the pellets in 60 ml sECM.
   3. Split the RBMECs into 3 T175 flasks and culture in sECM for another 3 days before seeding onto inserts. Count the total number of living RCAs, by observing a cell suspension diluted 1:1 with trypan blue under an optical microscope in a Burker chamber.
2. Cell Seeding onto Inserts
   1. Treat one side of the Polyethylene terephthalate (PET) membrane of 6 multi-well transparent inserts with poly-L-lysine 100 µg/ml and the other side with fibronectin 50 µg/ml, to allow attachment of RCAs and BMECs. Handle the inserts with tweezers in order to avoid contact with the PET membrane.
      1. Place the inserts into a 6 well plate and add fibronectin solution (minimum 500 µl) into the upper chamber. After 1 hr of incubation at 37 °C, remove the fibronectin solution, take the inserts off the multi-well plate and put them upside down on the bottom of a 150 cm² Petri dish, which is used here as a sterile support for the already coated inserts.
      2. Gently add 800 µl of poly-L-lysine onto the bottom side of the insert (as shown in Figure 1A; referred to as RCAs seeding), and keep the solution on the inserts for 1 hr at RT.
      3. Aspirate the solution and let the inserts dry at RT for 15-30 min. The inserts are now ready for cell seeding, but can also be stored in the multi-well plate for several days at 4 °C, before proceeding with the BBB construction.
         Note: Remember to keep at least 3 coated inserts free from cells, to be used as controls to validate BBB establishment by FD40 flux measurements.
   2. Seed RCAs (35,000/cm²) onto the bottom side of the poly-L-lysine-coated inserts, by dropping 800 µl of cell suspension onto the upside down insert (Figure 1A). Leave the RCA suspension on the inserts for 4 hr at RT, in order to allow efficient attachment of the cells to the membrane.
   3. Aspirate the residual solution, place the inserts into wells containing 2 ml of sECM and maintain the multi-well plate at 37 °C and 5% CO₂ in a humidified atmosphere, changing the sECM every 2 days.
   4. After 3 days, when the RCAs have coated the lower face of the insert, seed RBMECs onto the upper side of the insert, following these steps:
      1. Detach RBMECs from T175 flasks and count the total living cells, as reported in steps 1.1.2 and 1.1.3.
      2. Seed RBMECs (60,000/cm²) onto the upper surface of the insert in sECM (1,000 µl) (Figure 1B), leaving 3 inserts with the astrocytes only, to be used as TEER background. Place the multi-well plate in standard culture conditions. Maintain the system to culture for at least 3 days, changing the sECM in the inner and lower chamber every 2 days.

2. BBB Validation

1. TEER Measurement
   1. On the 3^rd day of co-culture, check the TEER by inserting the BBB-bearing inserts into an appropriate chamber(that has a cap and where both chamber and cap contain a pair of voltage-sensing and current electrodes), filled with 4 ml of sECM. Connect to an epithelial tissue volt/ohmmeter.
   2. Together with the TEER measurements of the cell BBB-systems, record the TEER values of the 3 inserts bearing the RCAs that will be subtracted from the values obtained with the BBBs. Multiply the resulting TEER values by the surface of the insert (4.2 cm²) in order to express the results as Ω x cm².
   3. From the 3^rd day of co-culture, measure the TEER every day. Note: After an initial period where the TEER increases, the recorded values should remain stable for at least two consecutive days (usually between the 5^th and the 7^th day of co-culture). At that point the BBB is ready for the second step of validation (section 2.2) and/or for the trans-BBB experiments.
      Note: The procedure described in section 2.1 can be performed on the same BBB-systems devoted to the following permeability experiments (section 3). Operate under sterile conditions.
2. Trans-BBB Flux of FITC-dextran 40 (FD40)
   1. Measure the FD40 flux from the upper to the lower chamber of the BBB models compared to that across 3 empty inserts (see the note of section 1.2.1) according to the following steps:
      1. Add 1 mg/ml FD40 (diluted in sECM) into the upper compartment of the BBBs, and after 1, 2 and 3 hr withdraw 200 µl sECM from the lower chamber and measure the fluorescence intensity by spectrofluorimeter (λ_ex 488 nm, λ_em 515 nm, slit 5).
      2. Obtain the values for sECM background fluorescenceby measuring 500 µl of virgin medium using the same analytical parameters. Subtract the sECM background fluorescence from all FD40 fluorescence values.
      3. Determine the amount of permeated FD40 by comparison of the observed fluorescence values with a calibration curve produced with known concentrations (i.e. 0.312, 0.625, 1.25, 2.5 µg/ml) of the fluorescent tracer dissolved in 500 µl of sECM.
      4. Calculate the apparent permeability coefficient (P_app) from the mean flux values according to:
         P_app = J/AC
         where J is the flux of the molecule (moles/sec), A is the permeation area (cm²) and C is the concentration of the molecule in the upper compartment (moles/cm³).
         Note: Three or more BBB-systems that have reached suitable TEER values, must be exclusively devoted to this validation procedure, and should not be employed for the following permeability experiments (section 3). Sterility is not mandatory.

3. Trans-BBB Permeation of FITC-loaded Ferritins (FnN)

Note: A recombinant variant of human ferritin (Fn), produced in Escherichia coli and assembled in nanocages (FnN) for the encapsulation of different fluorescent molecules, is available from the NanoBioLab of Prof. Prosperi (University of Milan-Bicocca, Italy). FnN are loaded with FITC, according to a previously described protocol¹³ and the concentrations of both Fn and the loaded molecules are accurately determined. 

1. Trans-BBB Flux of FITC and FITC-FnN
   1. Measure the FITC-FnN flux from the upper to the lower chamber of validated BBB models (usually at the 5^th - 7^th day of co-culture), compared to that of the free dye after 7 and 24 hr of incubation, according to the following steps:
      1. Add FITC-FnN (50 µg/ml FnN, 1.1 µM FITC) or an equal amount of free FITC into the upper chamber of at least 6 BBB systems for each formulation, in order to withdraw 2 ml of sECM solution from the lower chamber of at least 3 inserts after 7 hr, and from the lower chamber of other 3 inserts after 24 hr.
      2. Measure the fluorescence intensity of 500 µl of the collected samples by spectrofluorimeter (λ_ex 488 nm, λ_em 515 nm, slit 5).
      3. Obtain the sECM background fluorescence by measuring 500 µl of medium using the same analytical parameters. Subtract the sECM background fluorescence from all FITC or FITC-FnN fluorescence values.
      4. Determine the concentration of permeated FITC or FITC-FnN by comparing the obtained fluorescence values with two different calibration curves produced with known concentrations (i.e. 6.87, 13.75, 27.5, 55 nM) of the free or nanoformulated dye dissolved in 500 µl sECM.
2. FITC-FnN Localization in RBMECs
   1. Remove sECM from the upper chamber of the BBB systems, wash the inserts with PBS and fix the RBMECs on at least two inserts for each experimental group by adding 500 µl paraformaldehyde (4% in phosphate buffer saline-PBS) in the upper compartment for 10 min at RT.
   2. Wash the inserts three times with PBS to remove residual paraformaldehyde.
      Note: From this point on, sterility is not necessary.
   3. Cut suitable pieces of the PET membrane, and proceed with the immunodecoration of the cells according the following steps:
      1. Permeabilize RBMECs with 0.1% Triton X-100 in PBS for 10 minutes. Perform a blocking step for 1 hr at RT with a solution containing 2% bovine serum albumin (BSA), 2% goat serum in PBS. Incubate the samples with an anti-Von Willebrand Factor (VWF) at 1:20 dilution, for 2 hr at RT.
      2. After washing three times with PBS, expose the cells for 2 hr at RT to a 2% BSA, 2% goat serum solution containing a suitable secondary antibody at a 1:300 dilution in order to reveal the anti-VWR, and DAPI at a 1:1,500 dilution for nuclei detection.
   4. Mount the insert pieces onto microscope slides using an antifade mounting solution (one drop per insert fragment), then close the sample with a cover slip. Analyze by confocal microscopy using an oil immersion lens at 40X magnification, 1.5 zoom and 1,024 x 1,024 pixel resolution.

Results

During the establishment of the BBB model, cell attachment and growth on the inserts can be monitored using a light microscope thanks to the transparent nature of the PET membranes. RCAs, seeded at a density of 35,000 cells/cm², attach efficiently to the bottom side of the insert after 4 hr of incubation at RT (Figure 2A) and grow to cover the membrane surface in 3 days, taking a spindle-shaped morphology (Figure 2B). RBMECs, seeded at a densit...

Discussion

The in vitro method described here represents a useful validated approach to study the trans-BBB delivery of fluorescent molecules upon nanoformulation with nanoparticles. Here we use FnN, which represents a good candidate to study the translocation of cargo molecules across the BBB. FnN is considered the gold nanovector for trans-BBB delivery of drug/agents since it is specifically recognized by the TfR1 receptor, which is expressed on the luminal membrane of BMECs and mediates the nanoparticle uptake using a r...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Rat Brain Microvascular Endothelial Cells	Innoprot	P10308	isolated from Sprague Dawley rat brain tissue, cryopreserved at passage one and delivered frozen
Cortical Astrocytes	Innoprot	P10202	isolated from 2 days rat brain tissue, cryopreserved at passage one and delivered frozen.
Endothelial Cell Medium kit	Innoprot	P60104	ECM (500 ml) and fetal bovin serum (25 ml), endothelial cell growth supplement (5 ml) and penicillin/streptomycin (5 ml). Warm in 37 °C water bath before use and protect from light
Trypsin-EDTA without Phenol Red	EuroClone	ECM0920D	Warm in 37 °C water bath before use
Fluorescein isothiocyanate-dextran 40,000	Sigma	FD40S	protect from light
paraformaldehyde	Sigma	158127	diluition in chemical hood
Dulbecco's phosphate buffer saline w/o Ca and Mg	EuroClone	ECB4004L
Triton X-100	Sigma	T8787
bovine serum albumin	Sigma	A7906
goat serum	EuroClone	ECS0200D
mouse monoclonal anti-Von Willebrand Factor	Dako	M0616
AlexaFluor 546-conjugated antibody against mouse IgGs	ThermoFischer Scientific	A-11003	protect from light
DAPI (4’ ,6-diamidino-2-phenylindole)	ThermoFischer Scientific	D1306	protect from light
ProLong Gold Antifade Mountant	ThermoFischer Scientific	P36934
Poly-L-lysine Hydrobromide	Sigma	P1274	the same solution can be used several times
fibronectin from bovine plasma	Sigma	F1141	the same solution can be used several times
Polyethylene terephthalate (PET) inserts	Falcon	F3090	Transparent Polyethylene terephthalate (PET) membranes; surface area: 4.2 cm2; pore size 0.4 µm/surface area
T75 Primo TC flask	EuroClone	ET7076
T175 Primo TC flask	EuroClone	ET7181
EVOM2 Epithelial Tissue Volt/Ohmmeter	World Precision Instruments Germany	EVOM2
Endohm- 24SNAP cup	World Precision Instruments Germany	ENDOHM-24SNAP
Light/fluorescence microscope with camera	Leica Microsystems	DM IL LED Fluo/ ICC50 W Camera Module	inverted microscope for live cells with camera
Confocal Microscope	Leica Microsystems	TCS SPE
Spectrofluorimeter	Jasco	FP-8000