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Kewal K. Jain (ed.), Drug Delivery System, Methods in Molecular Biology, vol. 1141, DOI 10.1007/978-1-4939-0363-4_12, © Springer Science+Business Media New York 2014
Chapter 12
A Method for Evaluating Nanoparticle Transport
CNS cause disruption in the anatomical texture of the BBB, therefore impairing its natural function [ 2 , 3 ]. On the other hand, the biomedical and pharmaceutical applications of nanotechnology have greatly facilitated the diagnosis and treatment of CNS dis-eases. A number of nanoparticulate delivery systems with promis-ing properties have been developed [ 4 ]. In particular, targeted delivery of a therapeutic cargo to the intended site of action in the brain appears to be one of the most promising noninvasive approaches to overcome the BBB, combining the advantages of brain targeting, high incorporation capacity, reduction of side effects, and circumvention of the multidrug effl ux system [ 5 – 8 ].
Several transport mechanisms across the BBB have been iden-tifi ed, including paracellular or transcellular pathways, transport proteins, receptor-mediated transcytosis, and adsorptive transcyto-sis [ 2 ]. Many studies suggest nanoparticles bind to functional molecules, like apolipoprotein [ 9 , 10 ] or transferrin [ 11 ], enabling strategies to utilize existing pathways for accessing the brain.
However, the interactions of nanoparticles at the BBB have not yet been thoroughly described, although interest in this arena is rapidly increasing [ 12 – 14 ].
Several in vitro studies have been developed in order to address some of the fundamental mechanisms involved in crossing of BBB by solutes, drugs, and nanoparticles [ 15 , 16 ]. Except their comparatively high permeability and the loss of expression of some BBB effl ux protein systems [ 17 ], in vitro models have several advantages over in vivo models, including low-cost, high- throughput screening and easiness to assess compounds and to investigate the transport mechanism at the molecular levels.
In this work, we describe in details the methods used to inves-tigate the effect of nanoparticle characteristics on the interaction with the BBB to rank them in order of effi ciency. In particular, polystyrene nanoparticles with variable size and surface charge have been tested with bEnd.3 cell monolayer, grown on a Transwell system, in order to identify the parameters regulating the nanopar-ticle/BBB interaction and, thus, the nanoparticle’s capability to pass through the endothelial barrier. This study paves the way to elucidate the mechanisms utilized by nanoparticles to cross the BBB in order to design safe and effective nanoparticles for novel diagnostic and therapeutic applications for CNS disorders.
2 Materials
1. Nanoparticles used in this work are the following:
(a) Green and red fl uorescent polystyrene nanoparticles with diameters, respectively, of 44 (NP44) and 100 (NP100) nm (Duke Scientifi c Corporation).
2.1 Polystyrene Nanoparticles
(b) Yellow–green fl uorescent carboxylate-modifi ed polystyrene nanoparticles, 100 nm (NP-COOH) (Life Technologies, USA).
(c) Orange fl uorescent amine-modifi ed polystyrene nanopar-ticles, 100 nm (NP-NH 2 ) (Sigma-Aldrich).
2. Morphology of polystyrene nanoparticles is verifi ed by trans-mission electron microscopy (TEM) (Fig. 1 ).
3. To elucidate the colloidal stability of nanoparticles, measure-ments of the zeta-potential and of the hydrodynamic diameter (D H ) are carried out with a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK) at 25 °C by using a nanopar-ticle concentration, corresponding to 0.9 × 10 10 NP/ml. At least 3–5 measurements of all samples should be performed to have a statistical signifi cance ( see Note 1 ). The size and zeta- potential of nanoparticles used in this work are reported in Table 1 . 1. Immortalized mouse cerebral endothelial cells, bEnd.3 cells
(American Type Culture Collection, Manassas, VA), are grown in Dulbecco’s Modifi ed Eagle’s Medium (DMEM) with 4.5 g/l glucose, 10 % fetal bovine serum (FBS) (Gibco), 4 mM glutamine (Gibco), 100 U/ml penicillin, and 0.1 mg/ml streptomycin (Gibco) in a 100-mm-diameter cell culture dish (Corning Incorporated, Corning, NY) in a humidifi ed atmo-sphere at 37 °C and 5 % CO 2 .
2.2 Cell Culture
Fig. 1 TEM image of 100 nm polystyrene nanoparticles. Magnifi cation bar:
100 nm
2. Cell culture medium is changed every 3–4 days, and cells are split after reaching confl uency.
3. Phosphate-buffered saline (PBS) solution and trypsin/EDTA solution are from Gibco.
4. Cells used in all experiments are at passages 28–35.
5. To evaluate nanoparticle cellular uptake, 5 × 10 4 cells are seeded in a 96-well plate.
6. For permeability experiments, cells were seeded on Transwell permeable inserts as described below.
Tight junction formation and BBB functionality are assessed by transendothelial electrical resistance (TEER) across the fi lters using an electrical resistance system (ERS) with a current-passing and voltage-measuring electrode (Millicell-ERS, Millipore Corporation, Bedford, MA). TEER (Ω⋅cm 2 ) is calculated from the displayed electrical resistance on the readout screen by subtraction of the electrical resistance of a fi lter without cells and a correction for fi lter surface area (Table 2 ).
1. Seed 3 × 10 4 cells in 150 μl of complete medium on Transwell permeable inserts (6.5 mm in diameter, 3 μm pore size) (Corning Incorporated, Corning, NY), add 400 μl of complete medium in the basal compartment of the Transwell system, and allow them to grow up to 7 days ( see Note 2 ). Change cell culture medium every 3–4 days.
2.3 TEER Measurements
2.4 Permeability Experiments
Table 1
Size and ζ-potential measurements of nanoparticles used in this work
NP44 NP100 NP-COOH NP-NH2
Size [nm] a 43.67 106.70 110.32 102.46
SD [±] 1.08 5.35 0.12 0.13
ζ-potential [mV] −25.25 −21.97 −40.42 +41.72
SD [±] 5.26 2.11 1.39 2.18
a PDI < 0.1
Table 2
Transendothelial electrical resistance of bEnd.3 cells cultured on Transwell inserts up to 12 days of culture
Days of culture 3 4 7 10 12
TEER [Ohm/cm 2 ] 122.5 338 379.33 140.5 120.33
SD [±] 3.54 17.98 19.60 6.36 8.08
2. Dilute nanoparticles in complete cell culture medium w/o phenol red at the fi nal concentration of 0.002 % solids, corre-sponding to 4.2 × 10 11 for 44 nm NPs and 3.6 × 10 10 NP/ml for NP100, NP-COOH, and NP-NH 2 , and sonicate NP suspension for 3 min prior to use.
3. A schematic representation of the system used for permeability experiments is reported in Fig. 2 .
1. Albumin from bovine serum (BSA), Alexa Fluor ® 488 conju-gate (Life Technologies, USA), is used as a standard fl uores-cent probe for permeability measurements.
2. Working solutions of fl uorescent probes are prepared by dissolving the BSA in complete medium w/o phenol red at the fi nal concentration of 0.5 mg/ml ( see Note 3 ).
3. Solution is sonicated for 2–3 min to promote BSA dissolution.
1. 4 % paraformaldehyde solution is prepared by dissolving para-formaldehyde powder in PBS at about 100 °C under stirring.
When the solution becomes transparent, make aliquots of about 4–5 ml in 15 ml conic tubes and store them at −20 °C ( see Note 4 ).
2. 0.1 % Triton X-100 solution is prepared by diluting Triton X-100 (Sigma) in PBS under stirring. Store the solution at room temperature.
3. Prepare PBS–BSA solution by dissolving 0.5 % (w/v) bovine serum albumin (BSA) (Sigma) in PBS at room temperature ( see Note 5 ).
4. Tight junctions are localized by incubating samples fi rst with mouse anti-claudin-5 primary antibodies (Invitrogen, Life Technologies) diluted 1:100 in PBS–BSA 0.5 % and then with Alexa Fluor 488 anti-mouse secondary antibodies diluted 1:500 in PBS–BSA 0.5 % (Invitrogen, Life Technologies).
2.4.1 Fluorescent Probes
2.5 Indirect Immunofl uorescence
Fig. 2 Schematic representation of the Transwell system. The apical compartment ( donor chamber ) represents the blood side where nanoparticles are dispersed.
The basolateral compartment ( acceptor chamber ) represents the brain side
5. Rabbit anti-EEA1 primary antibodies (ABR) diluted 1:200 in PBS–BSA 0.5 % are used with Alexa Fluor goat anti-rabbit sec-ondary antibodies diluted 1:500 in PBS–BSA 0.5 % (Invitrogen, Life Technologies) to label early endosomes.
6. Caveolae are localized by incubating samples fi rst with rabbit anti-caveolin 1 (Abcam) primary antibodies diluted 1:200 in PBS–BSA 0.5 % and then with Alexa Fluor goat anti-rabbit secondary antibodies diluted 1:500 in PBS–BSA 0.5 % (Invitrogen, Life Technologies).
7. The excitation wavelengths of secondary antibodies are 488 and 568 nm for samples treated with red and green fl uorescent nanoparticles, respectively.
8. Lysosomes are localized by using LysoTracker (Invitrogen, Life Technologies) diluted 1:13,000 in cell culture medium and incubating cells 30 min at 37 °C with LysoTracker solution.
9. Cell membranes are localized by using wheat germ agglutinin (WGA), Alexa Fluor 488 conjugate (Invitrogen, Life Technologies). Dilute 1 mg/ml WGA stock solution 1:200 into PBS.
1. Dilute glutaraldehyde (Sigma, Germany) in sodium cacodylate buffer 0.1 M (pH 7.2) (Electron Microscopy Sciences, USA) containing 1 % saccharose (Electron Microscopy Sciences, USA) at the fi nal concentration of 2.5 % v/v.
2. Dilute aqueous osmium tetroxide (Electron Microscopy Sciences, USA) in sodium cacodylate buffer 0.1 M (pH 7.2) at the fi nal concentration of 1 % v/v.
3. Prepare a graded series of ethanol from 30 to 100 % by diluting absolute ethanol (Sigma, Germany) in Milli-Q water.
4. EPON EMbed 812 resin is purchased by Electron Microscopy Sciences, USA.
5. Dissolve uranyl acetate (Merck, Germany) in methanol/
Milli-Q water (1:1) at the fi nal concentration of 1 % w/v.
6. Reynolds lead citrate (Merck, Germany).
3 Methods
Indirect immunofl uorescence against endocytic markers may give information about the endocytic mechanisms underlying nanopar-ticle cellular uptake (Fig. 3 ). The co-localization experiments are carried out on 70–80 % confl uent cells seeded on 12-mm- diameter glass coverslips. All the reagents and solutions are prepared as described above:
2.6 Transmission Electron
Microscopy (TEM)
3.1 Nanoparticle Uptake Mechanisms