Models and Methods to Evaluate Transport of Drug Delivery Systems Across Cellular Barriers

Summary

Many therapeutic applications require safe and efficient transport of drug carriers and their cargoes across cellular barriers in the body. This article describes an adaptation of established methods to evaluate the rate and mechanism of transport of drug nanocarriers (NCs) across cellular barriers, such as the gastrointestinal (GI) epithelium.

Abstract

Sub-micrometer carriers (nanocarriers; NCs) enhance efficacy of drugs by improving solubility, stability, circulation time, targeting, and release. Additionally, traversing cellular barriers in the body is crucial for both oral delivery of therapeutic NCs into the circulation and transport from the blood into tissues, where intervention is needed. NC transport across cellular barriers is achieved by: (i) the paracellular route, via transient disruption of the junctions that interlock adjacent cells, or (ii) the transcellular route, where materials are internalized by endocytosis, transported across the cell body, and secreted at the opposite cell surface (transyctosis). Delivery across cellular barriers can be facilitated by coupling therapeutics or their carriers with targeting agents that bind specifically to cell-surface markers involved in transport. Here, we provide methods to measure the extent and mechanism of NC transport across a model cell barrier, which consists of a monolayer of gastrointestinal (GI) epithelial cells grown on a porous membrane located in a transwell insert. Formation of a permeability barrier is confirmed by measuring transepithelial electrical resistance (TEER), transepithelial transport of a control substance, and immunostaining of tight junctions. As an example, ~200 nm polymer NCs are used, which carry a therapeutic cargo and are coated with an antibody that targets a cell-surface determinant. The antibody or therapeutic cargo is labeled with ¹²⁵I for radioisotope tracing and labeled NCs are added to the upper chamber over the cell monolayer for varying periods of time. NCs associated to the cells and/or transported to the underlying chamber can be detected. Measurement of free ¹²⁵I allows subtraction of the degraded fraction. The paracellular route is assessed by determining potential changes caused by NC transport to the barrier parameters described above. Transcellular transport is determined by addressing the effect of modulating endocytosis and transcytosis pathways.

Introduction

Cellular barriers in the body act as a gateway between the external environment and internal compartments. This is the case for the epithelial lining separating the externally-exposed surface of the gastrointestinal (GI) tract and the bloodstream^1-3. Cellular barriers also represent the interface between the bloodstream and the parenchyma and cellular components of tissues and organs. This is the case for the inner endothelial lining in blood vessels, such as the blood-lung barrier, the blood-brain barrier, etc.¹ The ability to traverse these cellular barriers in the body is crucial for efficient delivery of therapeutic and diagnostic agents into the circulation and tissues/organs where intervention is needed.

To improve delivery of therapeutic or diagnostic agents, these compounds can be loaded into sub-micrometer nanocarriers (NCs). These drug delivery vehicles can be formulated with a variety of chemistries and structures to optimize drug solubility, protection, pharmacokinetics, release, and metabolism^4,5. NCs can also be functionalized with affinity or targeting moieties (e.g. antibodies, peptides sugars, aptamers, etc.) to facilitate adhesion to areas of the body where the therapeutic action is required^2,6. Targeting of NCs to determinants expressed on the surface of cellular barriers can further facilitate transport into and/or across these linings^2,6.

The role of selectively transporting substances between two environments requires certain unique features among cell layers. One such feature is cell polarity, whereby the apical membrane facing the lumen of cavities varies from the basolateral membrane oriented towards the tissue interstitium, with respect to membrane morphology and composition of lipids, transporters, and receptors². Another feature involves intercellular junctions connecting adjacent cells. Regulation of the proteins that form tight junctions, particularly junctional adhesion molecules (JAMs), occludins, and claudins, modulate the barrier function to selectively allow or not transport of substances between cells, known as paracellular transport, allowing passage of materials from the lumen to the basolateral space³. Binding of many natural and synthetic elements (leukocytes, molecules, particles, and drug delivery systems) to cellular barriers in the body can induce cell-junction opening, which may be transient and relatively innocuous or more prolonged and, therefore, unsafe with access of undesired substances across the barrier^2,5,7-9. Consequently, this pathway can be assessed by measuring the transepithelial electrical resistance (TEER) and passive paracellular diffusion of molecules (herein called paracellular leakage), whereby decreased resistance to an electrical current or increased paracellular leakage of an inert compound into the basolateral space indicate opening of cell junctions, respectively^5,10,11. To complement these methods, any of the tight junction proteins listed above can be stained to assess their integrity, where staining should appear concentrated at the cell-cell borders all around the cell periphery^5,10,12.

Alternatively, drug delivery systems that target specific cell surface determinants, such as those associated to clathrin-coated pits or flask-shaped membrane invaginations called caveolae, may trigger vesicular uptake into cells by endocytosis, providing an avenue for drug delivery to intracellular compartments^5,13. In addition, endocytosis may lead to trafficking of vesicles across the cell body for release at the basolateral side, a phenomenon known as transyctosis, or transcellular transport¹⁴. Therefore, knowledge of the kinetics and mechanism of endocytosis may be used to exploit intracellular and transcellular drug delivery, which offers a relatively safe and controlled mode of delivery compared to the paracellular route. The mechanism of endocytosis may be evaluated with modulators of classical pathways (clathrin- and caveolin-mediated endocytosis, and macropinocytosis) or non-classical routes (such as the case of cell adhesion molecule (CAM)-mediated endocytosis)^5,13,15.

Whereas intracellular trafficking is often studied in standard wells or coverslips, the absence of a basolateral compartment precludes cell polarization and the ability to study transport across cell layers. To overcome this obstacle, transport across cell monolayers has been long studied using transwell inserts^10,11,16,17, which consist of an upper (apical) chamber, a porous permeable membrane where cells attach and form a tight monolayer, and a lower (basolateral) chamber (Figure 1). In this configuration, transport can be measured in the apical-to-basolateral direction by administering a treatment into the upper chamber, following transport through the cell monolayer and the underlying porous membrane, and finally collecting the medium in the lower chamber for quantification of transported material. Transport in the basolateral-to-apical direction can also be measured by initial administration to the lower chamber and subsequent collection from the upper chamber^5,10,12,16. Various techniques exist to verify the formation of permeability barrier on transwells, including TEER and paracellular transport assays, as described above. In addition, the permeable filter on which cells are cultured can be removed for imaging analysis (e.g. by fluorescence, confocal, electron microscopy), as further validation of the cell monolayer model as well as the mechanism of transport. Selection of the membrane type, which is available in different pore sizes, materials, and surface areas, depends on various factors such as the size of substances or objects to be transported, cell type, and imaging method^16,18-20. Transwell inserts also facilitate controlled and accurate quantification of transport compared to complex mammalian systems, as volumes of the chambers and cell surface area are known constants. While many factors involved in in vivo delivery are eliminated, including the presence of intestinal mucus, shear stress, digestive enzymes, immune cells, etc., this small scale in vitro model provides useful preliminary information regarding transport.

As an example to illustrate the adaptation of these methods to study NC transport across cellular barriers^10,11,16,17, we describe here a case where the potential for NC transport across the GI epithelium was modeled by assessing passage of a model drug delivery system through a monolayer of human epithelial colorectal adenocarcinoma (Caco-2) cells. For this purpose, cells were cultured in transwell inserts, on a 0.8 μm pore polyethylene terephthalate (PET) filter (6.4 mm diameter), which is transparent and can be used for microscopy imaging. The status of the permeability barrier is validated by measuring TEER, apical-to-basolateral transport of a control substance, albumin, and fluorescence microscopy visualization of an element of the tight junctions, occludin protein. A model of targeted polymer NC is used, consisting of 100 nm, nonbiodegradable polystyrene nanoparticles. NCs are coated by surface adsorption with a targeting antibody alone or a combination of a targeting antibody and a therapeutic cargo, where either component can be labeled with ¹²⁵I for radioisotope tracing. In the selected example, the antibody recognizes intercellular adhesion molecule-1 (ICAM-1), a protein expressed on the surface of GI epithelial (and other) cells, which has been shown to facilitate intracellular and transcellular transport of drug carriers and their cargoes²¹. The cargo is alpha-Galactosidase (α-Gal), a therapeutic enzyme used for treatment of Fabry disease, a genetic lysosomal storage disorder²².

The coated NCs, of about 200 nm in size, are added to the apical chamber over the cell monolayer and incubated at 37 °C for varying periods of time, after which ¹²⁵I on NCs can be detected associated to the cell monolayer and/or transported to the basolateral chamber below the cells. Additional determination of free ¹²⁵I allows subtraction of the degraded fraction and estimation of coated NC transport. The mechanism of transport is further assessed by examining changes in the permeability barrier pertaining to the paracellular route, through the parameters described above, while transcellular transport is determined by examining changes in transport when modulating pathways of endocytosis and transcytosis.

These methods provide valuable information regarding cellular barrier models, the extent and rate of transport of a drug delivery system, and the mechanism of such transport, altogether allowing evaluation of the potential for drug delivery across cellular barriers.

Protocol

1. Culturing a Cell Monolayer in Transwell Inserts

1. In a sterile, biosafety level 2 cell culture hood, place 0.8 mm pore PET transwell inserts into a 24-well plate (4 wells per condition, for statistical significance) with forceps. All materials entering the hood should be sterilized with ethanol.

Note: The pore size of the filter needs to be selected in accord to the mean size of the NC used, to allow transport across the membrane. Also, for statistically significant results, each experimental condition requires a minimum of four repeats (wells) within the same experiment, and a minimum of 3 independent experiments.

2. Prepare cell culture medium containing DMEM medium supplemented with 4.5 g/L glucose, 15% fetal bovine serum, and 1% Pen Strep, and heat to 37 °C in a water bath.
3. Dilute human epithelial colorectal adenocarcinoma (Caco-2) cells in cell medium and place 200-400 μl of the cell solution into the upper (apical) chamber of the transwell insert, at a density of 1.5 x 10⁵ cells/cm². Fill the lower (basolateral) chamber with 700 - 900 μl of cell medium.
4. Culture cells at 37 °C, 5% CO₂, and 95% humidity for 16-21 days, replacing the medium in the upper and lower chamber every 3-4 days, using the volumes indicated in step 1.3. Replace medium in the following order to maintain the pressure above (rather than below) the cell monolayer: aspirate the medium from the lower chamber, aspirate the medium from the upper chamber, fill the upper chamber with fresh medium, and fill the lower chamber with fresh medium.

2. Validation of the GI Epithelial Permeability Barrier Using Transepithelial Electrical Resistance (TEER) and Immunostaining of Tight Junctions

1. For short-term storage (less than 2 weeks), submerge STX100 electrodes in an electrolyte solution (0.1-0.15 M KCl or NaCl). Connect the electrode cable to the electrode port on the EVOM volt-ohm meter so that the system is internally short-circuited and electrode symmetry is maintained. For long-term storage, rinse with dH₂O and store in a dry, dark condition.
2. To sterilize the electrodes, immerse in ethanol for 15 min and allow to air dry for 15 sec. Alternatively, the electrodes can be stored in a UV hood. Rinse the electrodes in a sterile electrolyte solution (phosphate buffer saline, PBS) or 0.1-0.15 M KCl or NaCl solution, before each resistance measurement.
3. With the volt-ohm meter set to the resistance setting, vertically place electrodes in a well containing the transwell insert, with the short electrode in the upper chamber and the long electrode in the lower chamber touching the bottom of the well. Once the EVOM reading stabilizes, record the resistance value (given in ohms; Ω) for each well.
4. Calculate resistivity (resistance normalized to area; Ω×cm²) of samples by subtracting background resistance (TEER of transwell inserts without cells) and multiplying by membrane surface area. Resistivity values are most often reported in the literature. (See Discussion)
5. Repeat measurements every 1-2 days until TEER values rise to a maximum and plateau, indicating formation of a permeability barrier, which typically takes 2-3 weeks from the moment of cell platting. Plateau TEER values and time necessary to achieve barrier integrity may vary according to cell passage number.
6. To verify the presence of tight junctions in cell monolayers with high TEER (equal to or above the threshold value for barrier formation), fix cells by incubation with cold 2% paraformaldehyde for 15 min. Then, wash cells with PBS and incubate them for 30 min at room temperature with 1 μg/ml anti-occludin. Wash cells again with PBS and incubate them for 30 min at room temperature with 7.5 μg/ml fluorescently-labeled secondary antibodies. Use wells with low TEER as a negative control for barrier formation.

Note: As a substitute for anti-occludin, antibodies to alternative tight junction proteins may be used.

7. Carefully excise the filter membrane on which the fixed monolayer is attached, and mount onto slides for imaging using epifluorescence or confocal microscopy.

3. Evaluating Transepithelial Transport of Targeted Carriers

1. Label the targeting antibody (mouse monoclonal antibody against ICAM-1, anti-ICAM, in this example) with ¹²⁵I, as previously described⁷. Use Bradford assay to estimate protein concentration and a gamma counter to determine ¹²⁵I content on the antibody, then calculate the specific activity in counts per minute (CPM)/mg of labeled antibody.

Note: To control for specificity, repeat the procedure by labeling mouse IgG, which will be used to prepare non-targeted coated NCs. To study transport of a therapeutic cargo, repeat the procedure labeling the cargo, e.g, alpha-Galactosidase (α-Gal) in this example. Alternatives may be used for the targeting agent, non-specific control, or therapeutic cargo. Alternative methods may also be used to label compounds and estimate the concentration of the labeled counterparts.

2. Couple the labeled targeting antibody (anti-ICAM in our example) to the surface of NCs. In this example, incubate polystyrene nanobeads (100-nm diameter) for 1 hr at room temperature with ¹²⁵I-anti-ICAM to allow surface adsorption, as described⁷.

Note: For the non-specific control use ¹²⁵I-IgG. To trace transport of a cargo, use a combination of targeting antibody and ¹²⁵I-labeled cargo (α-Gal in our example).

3. Centrifuge at 13,000 x g for 3 min and remove the non-coated counterparts in the supernatant by aspiration. Resuspend the pellet containing coated NCs using 1% bovine serum albumin (BSA) in PBS by pipetting, and sonicate at low power to disrupt potential particle aggregates (20-30 brief pulses).
4. For NC characterization, measure size, polydispersity, and ζ-potential of coated particles using dynamic light scattering (following vendor instructions), and quantify the number of ¹²⁵I-labeled targeting antibody molecules (e.g. anti-ICAM) on the particle surface using a gamma counter.

Note: These methods can be repeated for non-specific and therapeutic counterparts. The size of coated particles ranges around 200 nm.

5. Add ¹²⁵I-antibody NCs, (e.g. ¹²⁵I-anti-ICAM NCs; 56 nCi/ml) to the upper chamber above confluent Caco-2 monolayers with TEER ≥350 Ω×cm² (see Discussion) over background (16-21 days post-seeding). Incubate at 37 °C for one or more desired time intervals. Measure TEER (Section 2.3) before and after incubation to assess the effects of NCs on the permeability barrier.

Note: This procedure can be repeated for non-specific and therapeutic counterparts.

6. Collect medium from the upper and lower chambers and wash them once with 0.5 ml DMEM at 37 °C (upper chamber) or 1 ml dH₂O (lower chamber). Collect the washes for measurement of total radioisotope content using a gamma counter.
7. For measurement of the cell fraction, excise the permeable filter, e.g. using a razor blade to cut around the edges, and incubate in a gamma counter tube with 1% Triton X-100 for 10 min (to release cell contents), before measuring cell-associated total radioactivity.
8. To measure ¹²⁵I released from NCs during transport or due to potential degradation, first mix 300 μl of sample (from the upper, lower, or cell fractions) with 700 μl of 3% BSA in PBS and 200 μl trichloroacetic acid (TCA). Incubate at room temperature for 15 min. Meanwhile this time, measure the total radioactivity of this sample in a gamma counter.
9. Centrifuge TCA samples at 3,000 x g for 5 min to separate intact protein (pellet) from the degraded protein or ¹²⁵I fraction (supernatant). Quantify the radioactivity of the free¹²⁵I fraction and subtract this value from total radioactivity measured before centrifugation. This will provide the amount of labeled-protein that is not degraded.

4. Mechanism of Transepithelial Transport of Targeted Nanocarriers

1. Label albumin with ¹²⁵I as described in Section 3.1 and previously reported⁷.

Note: Albumin is a 66.5 kDa protein and, hence, a relatively large substance. Although a valid control for passive transport of larger objects (e.g. 200 nm NCs used here), it should be substituted with smaller inert tracer molecules when studying transport of smaller drug carriers (see Discussion).

2. To assess paracellular transport using albumin paracellular leakage, culture Caco-2 monolayers on transwell inserts as described above.
3. To the upper chamber medium above the cell monolayer, add either ¹²⁵I-albumin alone (negative control showing the basal level of leakage), or ¹²⁵I-albumin and non-radiolabeled antibody-targeted NCs (as described in Section 3.5). Incubate at 37 °C for the selected time interval(s), which should match those examined when testing NC transport. Measure TEER (Section 2.3) before, during, and after incubation, then collect all fractions for total ¹²⁵I and free ¹²⁵I measurements as indicated in Section 3. Note: Concomitant addition of ¹²⁵I-albumin and non-radiolabeled control IgG NCs may be used as a control.
4. As a positive control for opening intercellular junctions, add cell media containing 5 mM H₂O₂ to the upper and lower chambers of the transwell insert, and incubate at 37 °C for 30 min. Then, measure TEER (Section 2.3) and add ¹²⁵I-albumin to the upper chamber for the selected time intervals. Measure TEER at various time points throughout the incubation to identify TEER value decay caused by H₂O₂-induced opening of the cell junctions.
5. In parallel experiments, evaluate transcellular transport, also called transcytosis, of ¹²⁵I-targeted NCs by incubating confluent Caco-2 monolayers with either 50 μM monodansylcadaverine (MDC; inhibitor of clathrin-mediated endocytosis), 1 μg/ml filipin (inhibitor of caveolar-mediated endocytosis), 0.5 μM wortmannin (inhibitor of phosphatidylinositol 3 kinase [PI3K], involved in macropinocytosis), or 20 μM [5-(N-ethyl-N-isopropyl) amiloride] (EIPA, inhibitor of macropinocytosis and CAM-mediated endocytosis¹⁵).

Note: ¹²⁵I-IgG NCs and ¹²⁵I-albumin may be used as negative controls for the effect of these inhibitors, and other methods (e.g. siRNA techniques) may provide more selective inhibition.

6. Measure TEER (Section 2.3) before, during, and after incubation of cells with inhibitors and radiolabeled materials, as an additional control for the effect of the inhibitors on the monolayer permeability.

Results

As validation of our cell model to study transepithelial transport of targeted NCs, Figure 2 shows that Caco-2 cell monolayers plated at a density of 1.5 × 10⁵ cells/cm² reached confluence ~Day 12 and maintained monolayer integrity up to Day 18, indicated by TEER (Figure 2A). This was validated by the presence of occludin-positive tight junctions (Figure 2B) in monolayers with high TEER (390 Ω×cm², Day 14), compared to poor...

Discussion

Using the methods discussed above, a cell model for studying transport of targeted NCs across cellular barriers can be established, such as the example provided for Caco-2 epithelial cells, which is relevant to evaluating transport from the GI lumen into the blood in the case of oral drug delivery systems. Culturing of GI epithelial cell monolayers in transwell inserts enabled measurement of TEER and fluorescence immunostaining of tight junctions to confirm formation of a cell permeability barrier. Subsequently, radiolab...

Materials

Name	Company	Catalog Number	Comments
Transwell inserts	BD Falcon	353095
Dulbecco's Modified Eagle's Medium (DMEM), 1x	Cellgro	10-013-CM
Fetal Bovine Serum (FBS)	Cellgro	35-015-CV
Pen Strep	Gibco	15140
Human epithelial colorectal adenocarcinoma (Caco-2) cells	ATCC	HTB-37TM
125Iodine	Perkin Elmer	NEZ033H002MC	Radioactive hazard
Phosphate Buffer Saline (PBS)	Gibco	14190-235
Bovine Serum Albumin (BSA)	Equitech Bio	BAH-66
Paraformaldehyde (16%)	Fisher Scientific	15710
Mouse Immunoglobulin G (IgG)	Jackson ImmunoResearch	015-000-003
Mouse monoclonal antibodies to human ICAM-1 (anti-ICAM)	Marlin 1987
α-Galactosidase, from green coffee beans	Sigma	G8507-25UN
FITC latex beads, 100 nm	Polysciences, Inc.	17150
Triton X-100	Sigma	234729-500ML
Trichloroacetic acid (TCA)	Fisher Scientific	SA433-500
Occludin antibody (Y-12), goat polyclonal anti-human	Santa Cruz Biotechnology	Sc-27151
Monodansylcadaverine (MDC)	Sigma	D4008
Filipin	Sigma	F9765
5-(N-ethyl-N-isopropyl) amiloride (EIPA)	Sigma	A3085
Wortmannin	Sigma	W1628
Gamma counter	Perkin Elmer	Wizard2
Volt-ohm meter	World Precision Instruments	EVOM2
TEER electrodes	World Precision Instruments	STX100	Electrodes available for different well-plates
Dynamic Light Scattering (DLS)	Malvern	Nano-ZS90