Characterizing Spions Permeability By Using An in Vitro Blood-Brain Barrier Model

Abstract: In the present study, using murine brain endothelioma cells, an in vitro blood-brain barrier model was set up and confirmed. Confirmation of the blood-brain barrier model was completed by examining the permeability of FITC-Dextran at increasing exposure times in serum-free medium and comparing such values with values in the literature. After such confirmation, the permeability of eleven different magnetic nanoparticle samples (SPIONs) was determined by this blood-brain barrier model. Through these experiments, SPIONs that would not pass through the blood brain barrier were identified for MRI usage and SPIONs which would pass through the blood brain model were identified for drug delivery usage.

Keywords:  nanoparticles, blood-brain barrier, permeability

 Introduction

The blood-brain barrier is known as a "wall" that separates somatic circulating blood from the cerebrospinal fluid in central nervous system (CNS). Consisting of tight junctions that lie between endothelial cells of capillaries in CNS, it keeps large or hydrophilic molecules and microorganisms from passing into the cerebrospinal fluid and therefore maintains the microenvironment. [1]

However, previous research shows that small lipid-soluble-molecule that has a molecular weight less than 600 Da or, 200 nm in diameter, can be transported through blood-brain barrier. Therefore it requires that nanoparticles, as effective pharmaceuticals, are able to undergo successfully transportation through the blood-brain barrier. [2]

Among several nanoparticles, superparamagnetic iron oxide nanoparticles (SPIONs) have been discovered as a contrast agent which can potentially increase image contrast and improve MRI sensitivity and specificity. Moreover, SPIONs can also be used to track brain activity and detect cellular receptors that presented in brain diseases. On the other hand, if the SPIONs inside blood-brain barrier have not been efficiently cleared, the accumulation of those nanoparticles could be toxic and harmful to normal brain function in a long term. [3]

Since there are concerns about both the application of delivering drugs across the blood-brain barrier and the accumulation of SPIONs in the body, this study set up an in vitro blood-brain barrier model to test new SPIONs' ability to pass through blood-brain barrier.

Materials And Methods [4]

<>Material Characterization

According to a patented process entitled "A biomimetic process for the synthesis of aqueous ferrofluids for biomedical applications" [5], the synthesis of ferrofluids  involved incubation of a ferrous/ferric salt solution in phosphate-buffered saline supplemented with the additives of interest using ammonium hydroxide under highly alkaline conditions. After synthesis, ferrofluids were centrifuged for a stability test. Dynamic light scattering was used to characterize hydrodynamic diameter and zeta potential was used to characterize the charge of each sample. <>Confirmation of the blood-brain barrier model

Characterization was carried out through fluorescent sugar transport to confirm the blood-brain barrier model. For the blood-brain barrier model, murine brain endothelioma cells (b.End3) were used, and cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (P/S). In the next step, 6.5 mm Transwell®-COL collagen-coated 0.4 µm pore polytetrafluoroethylene membrane inserts were combined with 24-well plates for the model. A hemocytometer and a light microscope were used to determine the concentrations of cells. After diluted down to 10⁵ cells per mL in DMEM + 10% FBS + 1% penicillin-streptomycin, cells in the previous cell culture section were pipetted onto the inserts. Then the well plates with inserts were incubated with daily changed medium in the outer well until confluency was reached. After reaching the confluency, medium should then be replaced by 1:1 DMEM/Ham's F12 with 1% penicillin-streptomycin for the next 24, 48, 72 and 96 hours respectively.

To confirm the blood-brain barrier model, the permeability of the inserts would be tested in triplicate. After each time point was reached, inserts should be transferred to wells that contain 600 µL of Hank's Balanced Salt Solution (HBSS) before adding fluorescein isothiocyanate-labeled dextran (FITC-Dextran). Immediately after the transfer, the permeability of the inserts was tested in triplicate using 10 µg/mL FITC-Dextran. Positive controls were set up by adding 100 µL of FITC-dextran solution with 600 µL of HBSS and negative controls were set up by adding 700 µL of HBSS only.

After 2 hours incubation, 100 µL were taken from each well and transferred to a black 96-well plate with a clear bottom. A fluorescent plate reader was excited at 490/20 nm and measured at 528/20 nm. Data were converted into relative fluorescence Unit (RFU) from the reader and relative permeability was then determined: <>Experimental samples in the blood-brain barrier model

The previously described in vitro blood-brain barrier model was used to test the permeability of the various nanoparticles, among which three of them were solid powder (IONP) containing bovine serum albumin (BSA), polyvinyl alcohol (PVA), collagen, and graphene and the remaining eight samples were ferrofluids (FF) containing BSA, collagen, graphene, glutamic acid, and four concentrations (0.5%, 1.5%, 2.0%, and 2.5%, w/w) of PVA.

The inserts were transferred into a 24-well plate that containing 600 µL of HBSS in each well. Nanoparticles were diluted 1:19 with HBSS to lower the concentration. Next, followed the permeability experiment talked above, the inserts were exposed to the nanoparticles for 2 hours. After 2 hours, 500 µL would be pipetted from each well into a 20 mL scintillation vial and the inserts were removed.

To determine their iron concentration, aqua regia was used to convert IONPs into ionic iron. Then inductively-coupled plasma atomic emission spectroscopy (ICP-AES) was used to determine the iron concentration.

Results and Discussion

<>        Material Characterization

The DLS results showed that hydrodynamic diameters of nanoparticles range from 20 nm to 200 nm. The results of zeta potential showed the lowest absolute zeta values for IONP with collagen is -5.36 mV, for ferrofluids with PVA and glutamic acid is -3.38 mV, and for ferrofluids with collagen is -5.39 mV. The highest absolute zeta values were from IONP with graphene and ferrofluids with graphene.

<>Confirmation of the blood-brain barrier model

The value of the experimental blood-brain barrier model was in accordance with values in the previous articles, indicating that the model was successfully established. First, the decline, which was about 30%, in permeability was similar with the decline described by Brown et al. [6] Furthermore, the decline of permeability with increasing exposure to serum-free medium also indicated the success of a tightening of the barrier model. <>Experimental samples using blood-brain barrier model

Permeability of each sample has been tested and further experiments were carried out to test whether one would be able to set up parameters to moderate permeability of nanoparticles by using zeta potential, size, or surface modification. Results showed that the highest permeability was obtained from ferrofluids (FF) with collagen (1.5% collagen-FF) and the lowest permeability was obtained from IONP with collagen (1.5% collagen-IONP). These results suggest that for nanoparticles that need to be delivered through blood-brain barrier, FF should be coated with collagen and, on the other hand, IONP should be coated with collagen to keep nanoparticles from penetrating the blood-brain barrier.

<>Conclusion

An in vitro model of blood-brain barrier was established using b.End3 cells. As the permeability decreased with increasing exposure to serum-free medium, the model was confirmed by comparing the permeability trend of FITC-dextran in serum-free medium with previous research. With the successfully established model, the permeability of eleven nanoparticle samples was then carefully examined. Results showed that in order to be delivered through blood-brain barrier, FF should be coated with collagen and IONP should be coated with collagen to avoid penetration.

Acknowledgment

The authors would like to thank the Northeastern University for funding this research.

<>References

[1]     Hamilton RD, Foss AJ, Leach L (2007). "Establishment of a human in vitro model of the outer blood–retinal barrier". Journal of Anatomy 211 (6): 707-16.

[2]     Pardridge, W. M. (1995). "Transport of small molecules through the blood-brain barrier: biology and methodology." Advanced Drug Delivery Reviews 15(1-3): 5-36.

[3]     Berry CC, Curtis ASG. Functionalisation of magnetic nanoparticles for application in biomedicine. J Phys D Appl Phys. 2003; 36:198-206.

[4]     Dan Hoff, Lubna Sheikh, Thomas J Webster. Comparison of ferrofluid and powder iron oxide nanoparticle permeability across the blood-brain barrier.International Journal of Nanomedicine. 2012; 2012:7-1.

[5]     Nayar S, Sinha A, Pramanick AK, inventors. A biomimetic process for the synthesis of aqueous ferrofluids for biomedical applications. Application number 0672DEL2010. March 22, 2010.

[6]     Brown RC, Morris AP, O'Neil RG. Tight junction protein expression and barrier properties of immortalized mouse brain microvessel endothelial cells. Brain Res. 2007; 1130:17–30.