Improving Caco-2 cell permeability assay using phospholipid covered silica beads

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Improving Caco-2 cell permeability assay using phospholipid covered silica beads

Author: Lana Faradj

Degree Project in Drug Delivery, 30hp. Fall semester 2020

Supervisor: Patrik Lundquist Examiner: Per Artursson

Drug Delivery Group Department of Pharmacy Uppsala University

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Summary

The Caco-2 cell assay is widely used for in vitro permeability measurements. However, a draw back with the assay that this study will focus on improving, is compound adsorption to the plastic material. Lipophilic compounds such as Cyclosporin A and Peptide J, that will be used in this study, tend to bind to the plastic material in the assay. This can result in poor recovery and misleading permeability predictions. Bovine serum albumin (BSA) is an alternative used today to prevent this but is not always successful.

The aim of this study is therefore to improve the Caco-2 permeability assay by adding

phospholipid covered silica beads (PLB) to the basolateral chamber. The role of the PLB is to bind the compound of interest and decrease the amount of compound bound to the plastic material and thus better predict the permeability of the compound of interest.

The PLB was produced using phosphatidylcholine and silica beads. Caco-2 cells were seeded and maintained for 21-29 days ahead of the experiment. PLB concentration of 20, 60 and 100 mg/ml were prepared. Samples were analyzed with HPLC-MSMS. The results showed that with increasing PLB concentration we had a significantly decrease in non-specific plastic binding resulting in reliable permeability predictions, concluding that the hypothesis was correct.

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1. Introduction

1.1 Background

The Caco-2 cell assay is a widely used model for in vitro permeability studies. Caco-2 cells originates from human colon carcinoma cells and their most favorable property is their ability to differentiate and form polarized epithelial cell monolayers that can mimic the human intestinal epithelial barrier (Sambuy et al., 2005; Ölander et al., 2016). The Caco-2

permeability assay is made up of two chambers separated by a cell monolayer that rests on a permeable filter (Fig. 1). The upper chamber is referred to as the apical side and the lower chamber is called the basolateral side. Compound solution is added to the apical chamber, from where the compound will then permeate to the basolateral chamber (Hubatsch et al., 2007).

Fig. 1: Schematic diagram of Caco-2 monolayer cultivation on permeable filter.

1.2 Plastic adsorption and recovery

A draw back with the assay that this study will focus on improving is compound adsorption to the plastic material used in the experiment (Awortwe et al., 2014). A compounds permeability is determined from the concentration of the compound on the basolateral side. If the

compound binds the plastic material, then the free unbound compound concentration will be lower than the concentration that would be seen if all of the compound was free in solution.

This non-specific binding to the material will thus lead to misleading results and an

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underestimated permeability (Awortwe et al., 2014). This is particularly true for lipophilic compounds that tends to adsorb to most types of surfaces (Chemburu et al., 2010; Goebel- Stengel et al., 2011). Previous studies have reported that the recovery for many lipophilic compounds is low, resulting in unreliable permeability measurements. For the permeability results to be reliable a recovery >75% is needed (Krishna et al., 2001; Neuhoff et al., 2006).

Today one of the solutions to plastic adsorption is to rinse the wells with buffer and do an extraction with organic solvent e.g. methanol or acetonitrile (ACN) after the experiment (Krishna et al., 2001). Another solution that has been used in previous studies is adding bovine serum albumin (BSA) to the basolateral chamber. BSA binds many drug compounds, so with BSA in the chamber compound can bind to the protein instead of the plastic (Cai et al., 2019; Neuhoff et al., 2007). In a study they looked at the recovery of the lipophilic

compound Chlorpromazine with and without BSA. This study was able to show that including BSA could increase the compound recovery, but not enough to exceed the 75% recovery limit. They found that the majority of the compound had been adsorbed to the plastic material (Broeders et al., 2012). For other compounds, such as lipophilic peptides with high BSA binding, previous experiments have shown that they can sometimes precipitate with BSA upon denaturation with organic solvents leading to low recovery (Lundquist, unpublished observation).

For this study the permeability and recovery of two lipophilic compounds, Cyclosporine A (CsA) and Peptide J, will be studied. CsA is a cyclic peptide used in the medical field to prevent allograft rejection in patients that has undergone organ transplantation. Peptide J is a stapled α-helical peptide with promising anti-cancer effect. Peptide J has not been published yet, and therefore its structure cannot be shown in this paper. However, it is closely related to

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another peptide called Peptide ATSP-7041 which structure can be seen in figure 2 (Chang et al., 2013). An issue with Peptide J is that this compound binds strongly to BSA resulting in the peptide precipitating with BSA during sample preparation when organic solvent is added, this has been seen previously in experiments in our research group.

a b

Fig. 2: (a) The molecular structure of Cyclosporine A. (b) The molecular structure of Peptide ATSP-7041.

1.3 Phospholipid covered beads

In this study, phospholipid covered silica beads (PLB) will be added to the basolateral chamber (Fig. 3). The role of PLB is to bind the peptide in the basolateral chamber and prevent the peptides from binding the plastic material. When peptide solution is added to the apical chamber, there is a higher unbound peptide concentration on the apical side than the basolateral. The peptide will gradually permeate to the basolateral side to even out the concentration difference. However, if there are PLB in the basolateral chamber the peptide will be able to bind to them. Unbound peptide in the solution and peptide bound to PLB will quickly reach equilibrium. This results in a lower free unbound peptide concentration on the basolateral chamber which will improve sink conditions. Sink conditions in when the concentration on the basolateral side builds up so much that is significantly affect the

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permeability of the compound. This concentration limit is drawn when the concentration on the basolateral side exceeds 10% of the concentration on the apical side.

If PLB binds the peptide, the amount of non-specific peptide binding to the plastic material could potentially decrease resulting in a higher compound recovery and better predict the permeability. The additional step where the wells are washed and extracted would therefore not be necessary, simplifying the experiment. The aim of the study is thus improving the Caco-2 cell permeability assay by adding PLB to the basolateral chamber. The PLB will potentially bind our lipophilic peptides and decrease the amount of peptide bound to the plastic and thus lead to better prediction of permeability.

Fig. 3: A schematic illustration of a PLB.

3. Materials and method

3.1 Materials

Cell culture medium Dulbecco's modified Eagle's medium (DMEM) and additives,

Phosphate-buffered saline (PBS) and Hanks' Balanced Salt Solution (HBSS) were all bought from Gibco/Invitrogen. All other chemicals were purchased from Sigma-Aldrich. The

compound Cyclosporin A (CsA) was bought from Sigma-Aldrich and Peptide J was obtained from Merck Sharpe & Dohme (MSD).

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3.2 Cell culture

The Caco-2 cells were obtained from American Type Culture Collection (ATCC), all cells were from a high passage (96-103). The cells were maintained using culture medium DMEM that consisted of 500 ml DMEM high glucose (4500 mg/L glucose) with L-glutamine

(without pyruvate), 50 ml fetal calf serum (FCS), 5 ml (100x) nonessential amino acids, and 10,000 U m^-1 penicillin and 10,000 μg/mL^-1 streptomycin (PEST).

Caco-2 cells were sub-cultured every seventh day in 75 cm²cell flasks with DMEM. When the cells reached a confluency of 90% they were seeded on permeable supporters (Transwell polycarbonate filters) of a 12-well culture plate (diameter = 1.131 cm², pore size = 0.4 µm) as per following. Culture medium in the 75 cm²cell flask was removed and the cells were then rinsed with 15 ml PBS. To detach the Caco-2 cells from the flask, the cells were trypsinized with 1 ml trypsin/EDTA composed of 40 ml PBS (without Ca/Mg), 5 ml trypsin (10x stock solution 2.5%, wt/vol) and 5 ml of EDTA (2% EDTA-sodium salt). The cells were incubated for 5-10 min at 37 ^oC and 5% CO2. When taken out of the incubator the side of the flask is knocked with the palm of the hand to ensure that the cells are detached from the flask surface.

When the cells were detached 10 ml of DMEM was added to stop the trypsinization and resuspend the Caco-2 cells. An aliquot of cell solution was taken to count the cells using nucleocounter (ChemoMetec A/S). The cell solution was centrifuged for 5 min at 260 relative centrifugal force (rcf) and the supernatant was removed. The pellet was resuspended with DMEM to a final concentration of 1.0 million cells ml^-1. The cells were then seeded on pre- wetted filters by dispending 0.5 ml of the cell solution to each filter (seeding density of 0.5 x10⁶ cells per ml). To the basolateral side 1.5 ml DMEM was added. The culture medium was changed three times a week until the cell monolayers were formed (21-29 days).

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3.3 Productions of PLB

The silica beads were covered with the phospholipid (PL) phosphatidylcholine (PC). First the PL membrane vesicles were made. The PL forms a double membrane with the lipophilic tails facing inwards and the hydrophilic heads facing outwards, resembling a biological cell membrane. The membrane vesicles were then added to a silica bead solution. In the solution the vesicles folded over the particles producing the PLB.

3.4 Caco-2 permeability assay

Basolateral and apical solutions were prepared and pre-warmed to 37 ^oC. Basolateral

solutions consisted of HBSS with 20, 60 or 100 mg/ml PLB concentration and HBSS with 4%

BSA. HBSS was used as a control in all experiments. Apical solution consisted of HBSS, 10 μM Lucifer Yellow (LY) and 10 μM of the compound of interest.

The cell monolayers on filters were washed three times with PBS and transferred to a new 12- well cluster plate. To the basolateral side 1.5 ml basolateral solution was added and to the apical side 0.5 ml HBSS. The plate was then incubated (37 ^oC) under shaking (calibrated orbital shaker) at 270 r.p.m for 10 min. After incubation the HBSS from the apical side was removed and 0.5 ml compound solution was added. Samples were taken between 0-2h (0, 5, 30, 60, 90, 120 min), 10 μl from the apical side and 100 μl from the basolateral side that was also replaced with same amount. The apical samples were diluted 10x with HBSS.

At the end of the experiment the wells and filters were rinsed two times with HBSS to get rid of any excess medium. Then 250 μl 50% ACN/HBSS including 100 nM warfarin as internal standard (IS) was added to each well and filter to release any bound compound. The well and filters were incubated for 30 min before 200 μl samples were taken. For quantification a

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standard curve was prepared with the compound of interest and HBSS at 5000, 1000, 200, 40, 8 and 1.6 μM.

3.5 Sample preparation and analysis

All apical, basolateral and standard curve samples were prepared by diluting them with 100%

ACN (including 100 nM IS) and centrifuged for 20 min at 3500 rpm and 4 ^oC. The organic solvent denatured the BSA and dissolved PLB freeing the peptides that was bound. Denatured proteins and other solid materials pellet to the bottom of the wells leaving free peptides in the solution.

Prior to the HPLC-MSMS analysis the fluorescence of LY in the samples was measured with a plate reader (Saffire², Tecan). The samples were then analyzed using UPLC-MSMS with a Sciex 6500 Qtrap coupled to a Waters Acquity UPLC-system. Mobile phase consisting of A (0.1% formic acid in water)/B (0.1% formic acid in ACN) was used for the elution gradient for each compound.

3.6 Data analysis

The apparent permeability coefficients (Papp) were determined by following equation (eq. 1):

𝑃_𝑎𝑝𝑝 = (

𝑑𝑄⁄𝑑𝑡

𝐶₀×𝐴) (1)

where A is the area of the filter (1.131 cm²), C0 is the initial concentration (nM) and the dQ/dt is the steady state flux (nmol s^-1). The recovery (%M) of the compounds was calculated at the end of the experiment with eq. 2 where the amount of compound after 2h on the basolateral side (nb,2h), apical side (na,2h), well (nw,2h) and filter (nl,2h) were summarized and divided by the

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initial amount of compound after 5 min (nb,5min). The calculated value show how much of the compound was lost during the experiment.

𝑀% = ^𝑛^𝑏,2ℎ^+𝑛^𝑎,2ℎ^+𝑛^𝑤,2ℎ^+𝑛^𝑙,2ℎ

𝑛_{𝑏,5𝑚𝑖𝑛} × 100 (2)

3.7 Statistics

The values are presented as the mean ±the standard deviation (SD) of triplicates. The statistical difference of the permeability, recovery and plastic binding between the different basolateral solutions was assessed using one-way ANOVA, with Dunnet’s multiple

comparisons test. P-values less then 0.05 was considered as statistically significant, (p <0.05).

4. Results

The permeability assay was performed in two experimental setups under same conditions, only the basolateral solutions differed. In a 12-well plate, four different basolateral solutions were added to 3 wells each. In the first experimental setup control, 20 and 100 mg/ml PLB concentration and 4% BSA was used (Fig. 4a), and in the second experimental setup control, 20, 60 and 100 mg/ml PLB concentration was used (Fig. 4b). All data for the experimental setups can be found in appendix A and B for CsA and appendix C and D for Peptide J.

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a b

Fig. 4: (a) Schematic illustration of experimental setup 1 in a 12-well plate. (b) Schematic illustration of experimental setup 2 in a 12-well plate.

4.1 Integrity of the cell monolayers

The integrity of the Caco-2 cell monolayers was evaluated using the fluorescent dye LY.

Figure 5a and 5b presents the results from experiment setup 1 and experimental setup 2, respectively for CsA. Same results are presented for Peptide J in figure 5c for experimental setup 1 and in figure 5d for experimental setup 2.

Based on the results the integrity of the cell monolayers is disturbed with increasing PLB concentration compared to the control. The results suggest that BSA disturb the monolayer integrity the most out of all solutions, but BSA has an autofluorescence in the same

wavelength as LY. The disturbance of the cell monolayers integrity with BSA presence can therefore not be evaluated.

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a b

c d

Fig. 5: (a) and (b) illustrates the fluorescence (AU) of LY for CsA over time. (c) and (d) illustrates the fluorescence (AU) of LY for Peptide J over time. Data is presented as mean and SD from triplicates.

The permeability of LY for CsA is presented in figure 6a for experimental setup 1 and in figure 6b for experimental setup 2. No significant difference in LY permeability could be observed in the experiments with CsA. Figure 6c and 6d presents the LY permeability with Peptide J for experiment setup 1 and experimental setup 2, respectively. The only significant difference in LY permeability observed is between 100 mg/ml PLB concentration and the control with peptide J.

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Fluorescence (AU) Control

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a b

c d

Fig. 6: (a) and (b) presents the LY permeability for the two experimental setups with CsA. (c) and (d) presents the LY permeability for the two experimental setups with Peptide J. Data is presented as mean and SD from triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test:

ns = not significance, *P < 0.05 versus control.

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4.2 CsA concentration in the chambers (experimental setup 1)

The concentration of the CsA is the highest on the apical chamber where the compounds solution was added. Over time the CsA concentration decreases on the apical side at the same time as the concentration increases on the basolateral side (Fig. 7a and 7b). The results show that the peptide permeates the cell monolayer from a higher concentration on the apical side to a lower concentration on the basolateral side. Figure 7c presents the concentrations changes in both chambers from start to end of the experiment.

a b

c

Fig. 7: (a) Concentration change of CsA on the apical side over time. (b) Concentration change of CsA on the basolateral side over time. (c) Concentration change of CsA in the basolateral and apical chamber over time.

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Time (s)

Concentration (nM)

Apical chamber

Control 20 mg/ml 100 mg/ml 4% BSA

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Basolateral chamber

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Time (s)

Concentration (nM)

Basolateral and Apical chamber

Control-B 20 mg/ml-B 100 mg/ml-B 4% BSA-B Control-A 20 mg/ml-A 100 mg/ml-A 4% BSA-A

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4.3 Effects of PLB on Papp and recovery of CsA 4.3.1 Permeability: experimental setup 1

In figure 8 the permeability measurements of CsA transport across the Caco-2 cell monolayer from the apical to the basolateral chamber is presented. The first experimental setup includes control, 20 and 100 mg/ml PLB concentration and 4% BSA. Figure 8a shows the permeability results for samples taken from the basolateral chamber, while figure 8b displays the

permeability measurements based on the same samples but including the compound bound to the wells. The later permeability value represents the true permeability more accurately as it takes into account all the amount of CsA that have permeated the cell monolayer.

When adding the CsA extracted from the wells the permeability for the control and PLB concentration reach about the same permeability. There is however no significant different in permeability between the PLB solutions and control. In both permeability measurements the lowest permeability for CsA was measured in the samples with BSA, it is also the solution which show a significant difference in permeability.

a b

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Fig. 8: (a) Permeability of CsA in Caco-2 cells. Transport of CsA from apical to basolateral chamber. (b) Permeability of CsA in Caco-2 cells with included data from the amount of CsA bound to the wells. Transport of CsA from apical to basolateral chamber. Data is presented as mean and SD from triplicates. Statistically

significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance,

**P < 0.01, ***P < 0.001 versus control.

4.3.2 Recovery: experimental setup 1

In figure 9a the amount of CsA extracted from the wells after the experiment (2h) is

presented. There is a significant reduction of CsA bound to the plastic material of the wells in all basolateral solutions in comparison with the control. The highest amount of compound was found in the wells with the control and BSA and the lowest amount of compound was found in the wells with the PLB solutions. The amount of CsA bound to the Caco-2 cell monolayer is presented in figure 9b. There are no significant differences in the amount of CsA extracted from the monolayers.

a b

Fig. 9: (a) Amount of CsA (nmol) bound to the wells in the basolateral chamber after 2h. (b) Amount of CsA (nmol) bound in the cell monolayer after 2h. Data is presented as the mean and SD of triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance,

****P < 0.0001 versus control.

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The amount of CsA recovered from the wells, cell monolayers, and basolateral and apical chambers after 2h is shown in figure 10. A recovery >75% is necessary for the permeability measurements to be reliable and as seen in the figure, all but BSA solution exceed 75%. There is otherwise no significant difference in compound recovery for the different experimental groups.

Fig. 10: Amount (M%) of CsA that was recovered after 2h from the basolateral chamber, apical chamber, wells and cell monolayers. The data is presented as the mean and SD of triplicate. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance versus control.

4.3.3 Permeability: experimental setup 2

The permeability of CsA was also assessed in a second experimental setup with control and PLB concentrations of 20, 60 and 100 mg/ml. Figure 11a represents the permeability based on the basolateral samples and figure 11b present the same permeability but including the

amount of CsA extracted from the wells in the calculations. As seen in the figure the control has the highest permeability but has also a very wide SD range. A high LY signal was detected for one of the control replicates. This indicates that there was a leakage in the cell

Control 20 mg/ml

100 mg/ml 4% BSA 0

50 100 150

80 78 78 68

Compound recovery (M%)

ns ns

ns

Basolateral Apical Wash Lysate

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monolayer for that sample, which was therefore excluded from the calculations. No significant differences in permeability measurements could be observed.

a b

Fig. 11: (a) Permeability of CsA in Caco-2 cells. Transport of CsA from apical to basolateral chamber. (b) Permeability of CsA in Caco-2 cells with included data from the amount of CsA bound to the wells. Transport of CsA from apical to basolateral chamber. Data is presented as mean and SD from triplicates. Statistically

significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance versus control.

4.3.4 Recovery: experiment setup 2

The highest amount of CsA was extracted from the wells with the control as seen in figure 12a. The smallest amount of compound bound to the wells was found in the PLB

concentrations 60 and 100 mg/ml, which was about the same amount. There is a significant reduction of CsA bound to the plastic material in all basolateral solutions compared to the control. The amount of CsA bound into the cell monolayer is shown in figure 12b. The only significant difference was observed between the control and 100 mg/ml PLB concentration.

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a b

Fig. 12: (a) Amount of CsA (nmol) bound to the wells in the basolateral chamber after 2h. (b) Amount of CsA

(nmol) bound in the cell monolayer after 2h. Data is presented as the mean and SD of triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance,

*P < 0.05, ****P < 0.0001 versus control.

The total amount of CsA recovered after 2h in the wells, lysate, basolateral and apical chamber is presented in figure 13. A significant difference between the control and the three PLB solutiona can be seen. The PLB concentrations of 20 and 60 mg/ml both exceed 75% but the 100 mg/ml PLB concentration is under the acceptable recovery limit.

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Fig. 13: Amount (M%) of CsA after 2h in the basolateral chamber, apical chamber, wells and cell monolayers.

The data is presented as the mean and SD of triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: *P < 0.05 versus control.

4.4 Peptide J concentration in the chambers (experimental setup 1)

The concentration of the Peptide J is the highest on the apical chamber where the compounds solution was added to as seen in figure 14a. Though there is not a big change in the

concentration on the apical side, Peptide J do permeate the cell monolayer from a high concentration (apical side) to a low concentration (basolateral side) as can be observed in figure 14b. In fgure 14c the concentrations changes in both chambers from start to end of the experiment is presented.

a b

c

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Concentration (nM)

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Basolateral and Apical chamber

Control-B 20 mg/ml-B 100 mg/ml-B 4% BSA-B Control-A 20 mg/ml-A 100 mg/ml-A 4% BSA-A

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Fig. 14: (a) Concentration change of Peptide J on the apical side over time. (b) Concentration change of Peptide J on the basolateral side over time. (c) Concentration change of Peptide J in the basolateral and apical chamber over time. Transport of Peptide J from apical to basolateral chamber. Data is presented as mean and SD from triplicates.

4.5 Effects of PLB on Papp and recovery Peptide J 4.5.1 Permeability: experimental setup 1

In this experiment setup with Peptide J control, 20 and 100 mg/ml PLB concentration and 4%

BSA was used. As seen in the figure 15a the permeability measurements for only the PLB solutions could be calculated. Figure 15b shows the permeability including the compound that was bound to the wells in the calculations. There is a significant difference in permeability between the control and the PLB solutions. The permeability for the 100 mg/ml PLB concentration was the highest.

a b

Fig. 15: (a) Permeability of Peptide J in Caco-2 cells. Transport of Peptide J from apical to basolateral chamber.

(b) Permeability of Peptide J in Caco-2 cells with included data from the amount of Peptide J bound to the wells.

Transport of Peptide J from apical to basolateral chamber. Data is presented as mean and SD from triplicates.

Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance, ****P < 0.0001 versus control.

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4.5.2 Recovery: experimental setup 1

No Peptide J could be detected from the control or BSA well samples (Fig. 16a). There was about the same amount of peptide bound to the wells with 20 and 100 mg/ml PLB

concentrations. A significant difference between the PLB solutions and the control can be seen. Figure 16b shows the amount of Peptide J found in the Caco-2 cell monolayer which was about the same for all basolateral solutions.

a b

Fig. 16: (a) Amount of Peptide J (nmol) bound to the wells in the basolateral chamber after 2h. (b) Amount of Peptide J (nmol) bound in the cell monolayer after 2h. Data is presented as the mean and SD of triplicates.

Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance, *P < 0.05, ****P < 0.0001 versus control.

The recovery for Peptide J slightly exceeds 100% for all but BSA, but all solutions are above the recovery limit of 75% (Fig 17). As seen in the figure most of the compound was found on the apical side. As seen earlier only a relatively small amount of Peptide J permeated the cell monolayer from the apical to the basolateral chamber after 2h. No significant difference between the experimental groups could be observed.

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Fig. 17: Amount (M%) of Peptide J that was recovered after 2h from the basolateral chamber, apical chamber, wells and cell monolayers. The data is presented as the mean and SD of triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance versus control.

4.5.3 Permeability: experimental setup 2

In the second experiment setup with Peptide J, 20, 60 and 100 mg/ml PLB concentration was included. Permeability for only the PLB solutions could be measured and there is a significant difference compared to the control (fig 18). Because no peptide J could be detected in the basolateral samples for control the permeability could not be calculated.

Control

20 mg/ml 100 mg/ml

4% BSA 0

50 100 150 200

128 103 109 96

Compound recovery (M%)

ns ns

ns

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Fig. 18: Permeability of Peptide J in Caco-2 cells. Transport of Peptide J from apical to basolateral chamber.

Data is presented as mean and SD from triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: **P < 0.01, ***P < 0.001 versus control.

4.5.4 Recovery: experiment setup 2

The result over the wash extraction in this experimental setup could not be obtained as no compound could be detected in the wash samples for any of the basolateral solutions. Figure 16 presents the amount of Peptide J that was excreted from the cell monolayers and there is a significant difference between the experimental groups.

Fig. 16: Amount of Peptide J (nmol) bound in the cell monolayer after 2h. Data is presented as the mean and SD of triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: *P < 0.05, **P < 0.01, ***P < 0.001 versus control.

Figure 17 present the recovery from the second experiment setup. Only the PLB solutions exceeded a recovery of 75% indicating that the measured permeability rates for these solutions are reliable. No peptide could be detected from the well samples which could explain the low recovery in the control solution. No significant difference of the total recovery could be observed between the experimental groups.

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Fig. 17: Amount (nmol) of Peptide J after 2h in the basolateral chamber, apical chamber, wells and cell monolayers. The data is presented as the mean and SD of triplicates. Statistically significant was assessed by one-way ANOVA with Dunnet’s multiple comparisons test: ns = not significance versus control.

5. Discussion

The aim of this study was to investigate if adding PLB to the basolateral chamber of the Caco-2 assay could decrease the amount of compound binding the plastic material and so better predict the permeability of lipophilic compounds.

The inclusion of PLB in the basolateral chamber resulted in a decrease of CsA bound to the plastic which is in agreement with what was expected. What is significant here is that all PLB concentrations were able to reduce the non-specific binding compare to the control as well as BSA. This could be observed from the wash results. The higher the PLB concentration was the less peptide was bound to the wells. Which shows that the more of the peptide that bound the PLB the less amount of free unbound peptide was in the solution to be able to bind the plastic. This resulted in a higher PLB-bound peptide concentration in the basolateral chamber which allowed for better permeability prediction. The lower peptide concentration we had in the basolateral chamber due to non-specific binding the lower permeability prediction we

Control 20 mg/ml

60 mg/ml 100 mg/ml 0

50 100 150

68 78 74 80

Compound recovery (M%)

ns ns

ns

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measured. We are able to see the effect of this in the results where the permeability is compared with the same permeability measurements but have included the compound extracted from the wells into the calculations. This goes to show the effect on non-specific binding to the plastic material in presence of PLB.

In contrast to the CsA wash results, the wash results for Peptide J could not be acquired for most wash samples. This was most likely because the Peptide concentration was too low to be detected due to the low permeability of the compound after 2h. Even in the wash samples where Peptide J was detected the amount of the peptide extracted was very low compare to the CsA. This suggest that the concentration on the basolateral chamber was either slightly under or over the lower detection limit. We still had an overall compound recovery that exceeded 75% for Peptide J as well as CsA in all PLB concentrations, suggesting that the predicted permeability is reliable. Poor recovery is otherwise an issue for lipophilic compounds which has been reported in several studies.

The fact that we were not able to predict the permeability with BSA for Peptide J was expected, as the peptide binds very well to and precipitate with BSA when ACN is added in during sample preparation for the LC-MSMS analysis. What we did not expect was for CsA to also bind BSA in some extent resulting in a poor recovery. This goes to show what inclusion of PLB could accomplish. PLB would be a good alternative for permeability prediction of lipophilic compounds such as Peptide J and CsA which would otherwise not be possible due to the strong interaction with BSA. BSA has been used in previous studies with the intention of minimize the adsorption of lipophilic compounds to the plastic material and so better predict the permeability. Though these studies were able to improve the recovery for lipophilic compounds, the results obtain from this study indicates that BSA is not an option

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for all lipophilic compounds. Furthermore, the additional step where the wells are washed and extracted would not be necessary which simplifies the method.

When analyzing the integrity of the Caco-2 cell monolayers no significant difference could be observed in terms of what PLB concentration would be the better option. What was noticeable though was that the highest PLB concentration (100 mg/ml) significantly affect the epithelial integrity. Though 100 mg/ml reduced the plastic binding the most out of all three PLB solution, it is not a suitable choice if the cell monolayer integrity is disturbed. The

recommendation would therefore be to use the second highest PLB concentration (60 mg/ml), to ensure the integrity of the cell monolayer as wells as minimizing the compound adsorption to the material.

An issue with using these PLB was that they were too big to remain suspended in solution.

The PLB kept sedimenting in between sampling creating a pellet in the bottom and leaving the solution clear. Because of this problem the 12-well plate had to be taken from the orbital shaker and shaken by hand 10-15s before taking the samples in order to resuspend the PLB.

This created an extra step in the overall method. For future experiments, using smaller PLB could be a solution to keep the PLB suspended in the solution at all time overcoming this issue.

6. Conclusion

In conclusion this study showed that the presence of PLB is able to reduces the amount non- specific binding of lipophilic compound to the plastic material of the method. Which resulted in better permeability prediction for both CsA and Peptide J. This would otherwise not be possible, even by using BSA as in previous studies, because both peptides precipitated with

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BSA resulting in a poor recovery. This shows that the PLB is a better alternative for lipophilic compounds and proves that the hypothesis was correct. The recommended PLB concentration is 60 mg/ml with regard to reduction of plastic binding and the integrity of cell monolayer.

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Supplementary material:

Appendix A – Permeability and recovery CsA

Table 1A: Papp and recovery measures. Data is presented as mean and SD of triplicates

Experiment Compound

Cyclosporine A Nr. Basolateral

solution

Papp

(x10^-6 cm/s)

SD Papp+wash

(x10^-6 cm/s)

SD Recovery

(M%)

SD

1

Ctrl 2.3 ±2.92 x10^-7 2.9 ±2.92 x10^-6 80% ±2.23x 10^-1

20 mg/L PLB 2.6 ±2.81 x10^-7 2.8 ±2.81 x10^-6 78% ±5.63 x10^-2

100 mg/ml PLB 2.7 ±7.79 x10^-7 2.8 ±7.79 x10^-6 78% ±6.23 x10^-2

4% BSA 1.3 ±1.56 x10^-7 1.6 ±1.56 x10^-6 68% ±2.52 x10^-2

2

Ctrl 16.4 ±1.72 x10^-5 18.6 ±1.95 x10^-5 472% ±2.04 x 10^-0

20 mg/L PLB 4.4 ±8.90 x10^-7 4.61 ±9.20 x10^-7 126% ±2.12 x10^-1

100 mg/ml PLB 33.2 ±2.17 x10^-6 25.1 ±2.11 x10^-6 991% ±8.51 x10^-1

4% BSA 3.0 ±9.84 x10^-8 3.2 ±1.02 x10^-7 91% ±1.25 x10^-2

3

Ctrl 3.75 ±1.13 x10^-6 5.5 ±1.57 x10^-6 117% ±3.04 x 10^-1

20 mg/ml PLB 2.67 ±4.55x10^-6 2.9 ±4.55x10^-7 85% ±4.83 x10^-2

60 mg/ml PLB 2.13 ±4.04 x10^-7 2.2 ±4.20 x10^-7 77% ±1.23 x10^-2

100 mg/ml PLB 1.75 ±1.29 x10^-6 1.8 ±1.34 x10^-6 66% ±1.20 x10^-1

4

Ctrl 4.5 ±2.75 x10^-7 6.7 ±4.08 x10^-7 239% ±1.37x 10^-0

20 mg/ml PLB 3.9 ±2.94x10^-8 4.1 ±2.88x10^-8 196% ±2.13 x10^-1

60 mg/ml PLB 4.0 ±9.12 x10-⁸ 4.1 ±9.12 x10-⁸ 228% ±2.53 x10^-1

100 mg/ml PLB 3.3 ±2.24 x10^-8 3.4 ±2.63 x10^-7 143% ±1.25 x10^-1

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Appendix B – CsA extracted from wells and cell monolayers

Table 2B: Wash and lysate results presented as mean and SD of triplicates.

Experiment Compound

Cyclosporine A Nr. Basolateral

solution ---

Well bound (nmol)

SD Lysate bound

(nmol)

SD

1

Ctrl 0.14 ±1.03 x10^-2 0.86 ±3.77 x10^-2

20 mg/L PLB 0.05 ±8.70 x10^-3 0.87 ±3.75 x10^-2

100 mg/ml PLB 0.032 ±4.25 x10^-3 0.88 ±6.19 x10^-2

4% BSA 0.082 ±6.19 x10^-3 0.76 ±5.08 x10^-2

2

Ctrl 0.097 ±3.21 x10^-2 0.87 ±7.90 x10^-2

20 mg/L PLB 0.04 ±4.0 x10^-3 0.73 ±2.7 x10^-1

100 mg/ml PLB 0.03 ±1.13 x10^-3 0.73 ±7.10 x10^-2

4% BSA 0.044 ±3.27 x10^-3 0.67 ±1.45 x10^-2

3

Ctrl 0.31 ±4.04 x10^-2 0.21 ±1.24 x10^-1

20 mg/ml PLB 0.075 ±7.05 x10^-3 1.4 ±7.72 x10^-2

60 mg/ml PLB 0.033 ±2.39 x10^-3 1.32 ±6.60 x10^-2

100 mg/ml PLB 0.034 ±1.16 x10^-1 0.79 ±5.59 x10^-1

4

Ctrl 0.13 ±2.96 x10^-2 1.16 ±1.44 x10^-1

20 mg/ml PLB 0.044 ±5.16 x10^-3 1.2 ±7.33 x10^-2

60 mg/ml PLB 0.021 ±1.35 x10^-3 1.15 ±2.39 x10^-2

100 mg/ml PLB 0.018 ±4.64 x10^-3 1.14 ±1.22 x10^-1

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Appendix C – Permeability and recovery Peptide J

Table 3C: Papp and recovery measures. Data is presented as mean and SD of triplicates

Experiment Compound

Peptide J Nr. Basolateral

solution

Papp

(x10^-6 cm/s)

SD Papp+bound

(x10^-6 cm/s)

SD Recovery

(M%)

SD

1

Ctrl 0 ±0 0 ±0 128% ±1.25 x10^-1

20 mg/L PLB 1.1 ±5.09 x10^-8 1.2 ±1.60 x10^-7 103% ±1.05 x10^-1

100 mg/ml PLB 1.4 ±1.70 x10^-7 1.5 ±1.70 x10^-7 109% ±1.93 x10-¹

4% BSA 0 ±0 0 ±0 96% ±1.20 x10^-1

2

Ctrl 0 ±0 0 ±0 68% ±4.47 x10^-2

20 mg/ml PLB 16.7 ±4.75 x10^-8 16.7 ±4.75 x10^-8 78% ±1.30 x10^-1

60 mg/ml PLB 12.5 ±1.45 x10^-8 12.5 ±1.45 x10^-8 74% ±4.18 x10^-2

100 mg/ml PLB 16.9 ±4.24 x10^-4 16.9 ±4.24 x10^-4 80% ±1.17 x10^-4

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Appendix D – Peptide J extracted from wells and cell monolayer

Table 4D: Wash and lysate results presented as mean and SD of triplicates

Experiment Compound

Peptide J Nr. Basolateral

solution

Well bound (nmol)

SD Lysate bound

(nmol)

SD

1

Ctrl 0 ±0 0.025 ±3.9 x10^-4

20 mg/L PLB 0.017 ±7.03 x10^-5 0.024 ±1.02 x10^-3

100 mg/ml PLB 0.017 ±3.84 x10^-5 0.024 ±1.09 x10^-4

4% BSA 0 ±0 0.025 ±2.30 x10^-4

2

Ctrl 0 ±0 0.025 ±7.91 x10^-4

20 mg/ml PLB 0 ±0 0.024 ±1.78 x10^-4

60 mg/ml PLB 0 ±0 0.024 ±2.56 x10^-4

100 mg/ml PLB 0 ±0 0.023 ±1.85 x10^-4

Improving Caco-2 cell permeability assay using phospholipid covered silica beads