The overall goal of this procedure is to evaluate transport of a drug delivery system or the therapeutic agent. It carries across a cellular barrier such as a model of the gastrointestinal epithelium relevant to oral drug delivery. This is accomplished by first culturing caco two cells on transwell inserts to allow cell differentiation and formation of a cell monolayer.
The second step is to validate that this model represents a permeability barrier by measuring the trans epithelial electrical resistance, immuno staining cell junctions, and assessing passive permeability of a detectable tracer molecule. The next step is to prepare the drug delivery system targeted polymer nano carriers are used in this example. Carriers may contain a drug cargo or not, and controls such as non-targeted carriers or a free drug must be included.
These elements are labeled with radioisotopes to allow their detection. The carriers are then added to the apical chamber above the cell mono layer after incubation. For varying time points, carriers are collected from the apical chamber, the basolateral chamber, and the cell monolayer and tested for potential degradation of the labeled component.
Nanocar transport across the cell monolayer is then calculated finally, to investigate the mechanism of transport. Similar experiments must be conducted in the presence of pharmacological inhibitors of transcytosis across cells or with reporters of paracellular transport between cells. The technique we describe here is an adaptation of well-established methods used to evaluate the rate and mechanism of transport of drugs and drug nano carriers across cell monolayers in culture, which represents cell barriers in the body.
Although the example that we have focused on here validates transport of i a targeted polymer nanoparticles across a model of gastrointestinal epithelium. Similar methods can be also applied to examine the passage of different drug delivery systems across these or different cell monolayers, such as endothelial cells that typically form a barrier between the bloodstream and tissues and organs. For example, in the case of the blood brainin barrier, Generally individuals new to this method will struggle because of the difficulty selecting labels to properly trace drug delivery systems through these studies, particularly if they're transported across cells and small quantities.
For this aspect, we'll use radioisotopes to label drug delivery systems in our studies, another major concern is validating the integrity of the model cell barrier and using the proper controls to validate this through the experiments. This procedure is performed in a sterile biosafety level two cell culture hood, and all materials entering the hood must be sterilized with ethanol using forceps place 0.8 micrometer. Poor polyethylene tariff, phthalate transwell inserts into a 24 well plate for statistically significant results.
Each experimental condition requires a minimum of four repeats. Dilute human epithelial colorectal adenocarcinoma or caco two cells in previously prepared cell medium. Place 200 to 400 microliters of the cell solution into the upper or apical chamber of the transwell.
Insert at a density of 1.5 times 10 to the five cells per centimeter squared. Fill the lower or basolateral chamber with 700 to 900 microliters of cell medium culture cells at 37 degrees Celsius, 5%carbon dioxide and 95%humidity for 16 to 21 days every three to four days. Replace the medium in the upper and lower chambers.
Replace medium in the following order to maintain the pressure above the cell mono layer, aspirate the medium from the lower chamber, aspirate the medium from the upper chamber. Fill the upper chamber with 200 to 400 microliters of fresh medium and fill the lower chamber with 700 to 900 microliters of fresh medium to validate the epithelial permeability barrier by tier. Set the volt oh meter to the resistance setting and vertically place electrodes in a well containing the transwell insert.
The short electrode should be in the upper chamber and the long electrode in the lower chamber touching the bottom of the well. Once the volt O meter reading stabilizes record the resistance value in ohms for each. Well repeat measurements every one to two days until tear values rise to a maximum and plateau indicating formation of a permeability barrier, which typically takes two to three weeks from the moment of cell plating.
To verify the presence of tight junctions in cell monolayers with high tier first, fix the cells, aspirate the media from both chambers and wash the cells three times with warm DMEM incubate with cold 2%paraform aldehyde for 15 minutes. After 15 minutes, remove the paraform aldehyde and wash the cells three times with PBS. Next, add one microgram per milliliter anti occludin to each.
Well use wells with low tear as a negative control for barrier formation. Incubate for 30 minutes at room temperature After 30 minutes, wash the cells again with PBS and then at 7.5 micrograms per milliliter labeled secondary antibodies to each well incubate for 30 minutes at room temperature in the dark. Finally, wash the cells with PBS and carefully excise the filter membrane on which the fixed monolayer is attached mount onto slides for imaging using epi, fluorescence or confocal microscopy.
This procedure begins with labeling the targeting antibody with iodine 1 25 and calculating the specific activity of the labeled antibody in counts per minute per microgram using previously published protocols. In this example, the targeting antibody is a mouse monoclonal antibody against human intercellular adhesion molecule, one or anti ICAM one to control for specificity. Mouse IgG labeled with iodine 1 25 will be used to prepare non-targeted coated nano carriers to study transport of a therapeutic cargo.
The cargo is also labeled with iodine 1 25. In this example, the cargo is alpha galacto SASE or Alpha G, a therapeutic enzyme used for treatment of Fabry disease, a genetic lysosomal storage disorder. One of the most difficult aspects of this procedure is ensuring adequate detection of transported materials.
To ensure success, nanocar components must be labeled with a sufficient amount of isotopes at high specific activity. The next step is to couple the labeled anti ICAM to the surface of nano carriers. In this case, we simply mix the iodine 1 25 labeled anti ICAM with 100 nanometer diameter polystyrene nano beads as a model for the non-specific control.
Use iodine 1 25 labeled IgG and to trace transport of a cargo, use a combination of anti ICAM and iodine 1 25 labeled Alpha G.Incubate the mixtures for one hour at room temperature to allow surface absorption after one hour centrifuge at 13, 000 GS for three minutes, and then remove the non coated counterparts in the supernatant by aspiration pipette to suspend the pellet containing coated nano carriers in 1%Bovine serum albumin in PBS subsequently sonicate at low power to disrupt potential particle aggregates. Add iodine 1 25 anti ICAM nano carriers to the upper chamber above confluent caco two monolayers with high tier. Repeat the procedure for the non-specific and therapeutic counterparts after measuring tear.
Incubate at 37 degrees Celsius for one or more desired time intervals. Measure tear again after incubation. The tear measurements are for assessing the effects of nano carriers on the permeability barrier.
Collect medium from the upper and lower chambers. Wash the upper chamber with 0.5 milliliters of DMEM at 37 degrees Celsius and the lower chamber with one milliliter of deionized water. Collect the washes for measurement of total radioisotope content using a gamma counter for measurement of the cell fraction.
Excise the permeable filter by using a razor blade to cut around the edges. Incubate it in a gamma counter tube with 1%Triton X 100 for 10 minutes to release the cell contents before measuring cell associated total radioactivity to measure iodine 1 25 released from nano carriers during transport or due to potential degradation. First mix 300 microliters of each sample with 700 microliters of 3%BSA in PBS and 200 microliters of trichloroacetic acid or TCA incubate at room temperature for 15 minutes.
During this 15 minute incubation, measure the total radioactivity of the samples in a gamma counter centrifuge TCA samples at 3000 GS for five minutes to separate the intact protein from the degraded protein or iodine 1 25 fraction. Quantify the radioactivity of the free iodine 1 25 fraction and subtract this value from the total radioactivity measured before centrifugation. This will provide the amount of labeled protein versus the fraction of radiotracer that detached from it to assess paracellular transport using albumin paracellular leakage culture KCO two monolayers on transwell inserts as demonstrated earlier to the upper chamber medium above the cell monolayer at either iodine 1 25 labeled albumin alone as a negative control showing the basal level of leakage or iodine.
1 25 labeled albumin and non radio labeled antibody. Targeted nano carriers incubate at 37 degrees Celsius for the selected time intervals which should match those examined when testing nz.Transport. Measure tear as demonstrated earlier before, during and after incubation.
Collect all fractions after incubation for measurements of total iodine 1 25 and free iodine 1 25 as a positive control for opening intercellular junctions. Add cell media containing five millimolar hydrogen peroxide to the upper and lower chambers of the transwell. Insert and incubate at 37 degrees Celsius for 30 minutes.
Measure tear and add iodine 1 25 albumin to the upper chamber for the selected time intervals. Measure tear at various time points throughout the incubation to identify tear value decay caused by hydrogen peroxide induced opening of the cell junctions. In parallel experiments, evaluate transcellular transport or transcytosis of iodine 1 25 targeted nano carriers by incubating confluent Caco two Monolayers with each of these inhibitors.
50 Micromolar MDC an inhibitor of clathrin mediated endocytosis. One microgram per milliliter filipin an inhibitor of clar mediated endocytosis. 0.5 Micromolar WT manin an inhibitor of Phosphatidylinositol three kinase, which is involved in macropinocytosis and 20 micromolar, EIPA.
An inhibitor of macropinocytosis and chem mediated endocytosis measure tear before, during and after incubation of cells with inhibitors and radio labeled materials as an additional control for the effect of the inhibitors on the monolayer permeability to validate CACO two monolayers As a model of trans epithelial transport of targeted nano carriers, caco two cells were grown on transwell inserts at 1.5 times 10 of the five cells per centimeter squared and trans epithelial electrical resistance or tear was measured to assess monolayer integrity as shown in this graph, the monolayer reached confluence at day 12 and maintained monolayer integrity up to day 18. This result was further validated by the presence of occludin positive tight junctions in monolayers with a high tier of 380 ohms per centimeter squared on day 14 compared to port tight junction labeling at a low tier of 17 ohms per centimeter squared. On day five to assess transport of anti ICAM nano carriers across caco two cell monolayers confluent Caco two Monolayers grown on transwell inserts were incubated with iodine 1 25 anti ICAM nano carriers added to the apical chamber.
Iodine 1 25 content in the basal lateral chamber was measured at the indicated time points to calculate the amount of nano carriers transported per cell as shown in the upper panel in the lower panel, the percent of transported nano carriers was calculated as the ratio of carriers found in the basolateral fraction to that in the combined basolateral and cell fractions. These parameters estimated in this example for iodine 1 25 anti ICAM nano carriers were also compared to those of control. Iodine 1 25 IgG nano carriers to demonstrate the transport efficiency relative to a non-targeted counterpart and the results are shown in the upper panel.
Apparent permeability coefficients were also calculated reflecting rates of transport of iodine 1 25 anti ICAM nano carriers or iodine 1 25 IgG nano carriers as shown in the lower panel when the radioactive label is incorporated on the nanocar cargo. The described parameters reflect transport of the cargo, which in this example is alpha GA enzyme used for treatment of a genetic lysosomal storage disorder known as Fabry Disease. Radioisotope content of apical basolateral and cell fractions were measured at three hours and 24 hours and converted into various values relevant to transport as shown in this table.
In addition to calculating nanocar transport by tracing the cargo, trans epithelial delivery of this therapeutic enzyme can be estimated by expressing it as molecules or picograms of Alpha-gal transported per cell. The mechanism of transport of anti ICAM nano carriers across Caco Monolayers was also investigated Transcellular Transport of iodine 1 25 anti ICAM nano carriers across confluent Caco two Monolayers was assessed at 24 hours in the absence or presence of the following inhibitors, EIPA, Philippine MDC or Wirt manin, EIPA is an inhibitor of macropinocytosis and can mediated endocytosis. Philippine, MDC and WT menin are inhibitors of clar mediated endocytosis, clathrin mediated endocytosis and macropinocytosis respectively.
As indicated in this graph, EIPA, reduced transport of iodine 1 25 anti IAM nano carriers across CACO two cell monolayers. Whereas Philippine MDC and Wirt Menin did not reduce transport levels with respect to the control condition. This suggests that anti ICAM nano carriers utilize chem mediated endocytosis for transcellular transport, but not clar, clathrin or macropinocytosis related transcytosis.
To assess Paracellular transport tear was measured during transport of iodine 1 25 anti ICAM nano carriers across caco. Two monolayers tear values in the absence of nano carriers are shown as controls and the dashed interval marks SEM of the mean incubation with five millimolar. Hydrogen peroxide was a positive control for opening of intercellular junctions as reflected in the decrease of tear to basal levels.
In addition, paracellular protein leakage measured as the apparent permeability coefficient of iodine 1 25, albumin crossing the cell monolayer in the absence or presence of five millimolar hydrogen peroxide, IgG nano carriers or anti ICAM nano carriers was measured and calculated hydrogen peroxide markedly enhanced iodine 1 25 albumin leakage to the basal lateral chamber and served as a positive control as shown in these graphs. Incubation with anti ICAM nano carriers did not alter tear or iodine. 1 25 albumin paracellular leakage over a period of 48 hours similar to control IgG nano carriers that are not transported.
These results rule out paracellular transport and are consistent with the previous results suggesting that anti ICAM nano carriers utilize cam mediated endocytosis for transcellular transport. While performing this procedure, it's important to select one or more suitable cell types and a permeable filter with appropriate pore size, material diameter, et cetera for the Transwell model, and labeling techniques for effective quantification of your drug delivery system following this procedure. Other methods like fluorescence or transmission electron microscopy can be used to determine the location of transported materials and in addition to check the status of transported materials.
Western blot can be used to measure protein degradation. EISA can be used to determine the targeting ability of transported carriers and the activity of the drug cargo can be measured using spectro photometric assays or other suitable techniques.
