Application Note 20

Diffusion rates for small drug molecules
studied in GrowDex® hydrogel
Lauri Paasonen1, Anne-Mari Moilanen2
1
UPM, Finland2
Admescope, Finland

INTRODUCTION

Diffusion of various molecules, such as nutrients and oxygen, in a 3D cell culture matrix
is crucial for sustained viability and functionality of the cells. In cell-based drug efficacy
screening and toxicity evaluation assays, it is essential that the drug/compound under test
is able to reach the cells in the hydrogel layer easily. Typical set up for a 3D cell-based
assay involves the drug/compound to be administered either on top of or below the cellcontaining hydrogel layer. The compound will then be left to diffuse through the culture
matrix. Physicochemical properties such as molecular weight, charge and lipophilicity of thedrug molecules may affect the rate of diffusion. Earlier it has been reported that GrowDex®
supports 3D growth of various cell types, indicating that adequate diffusion of nutrients andgases within the hydrogel has occured. It has also been shown that the rate at which large
molecules diffuse through GrowDex is dependent on the molecular weight of the molecule
under test (Bhattacharya et al. 2012, Laurén et al. 2014).
In this study diffusion rates of small drug molecules, namely nadolol, ketoprofen andmetronidazole were evaluated. Test drugs were administered on top of a 1% GrowDex layerthat had been dispensed into a Transwell insert. Samples from bottom well were collected
and analysed using UPLC-MS to determine permeability of GrowDex.

MATERIALS• GrowDex 1.5% (Cat No. 100 100 005, UPM)
• PBS (Amresco, VWR)
• 24-well Transwell® plate (Cat No. 3398, Corning)• Nadolol (N1892, Sigma-Aldrich), metronidazole (M3761, Sigma Aldrich), ketoprofen(K1751 Sigma-Aldrich)• DMSO (Cat No. 1029311000, Merck)• 96-deepwell plates (Cat No. 186002481, Waters)• Acetonitrile (Cat No. 1000291000, Merck)

METHODS

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The cumulative permeability of nadolol, ketoprofen and metronidazole during 48 hours ispresented in Fig 1. Diffusion was slightly slower with GrowDex during the first 8 h whencompared to control without GrowDex. The cumulative permeability of the drugs throughthe GrowDex layer reached the control levels by the 24 h time point. The actual inputs
were 1904 pmol/well for nadolol, 1614 pmol/well for ketoprofen and 1366 pmol/wellfor metronidazole. The recovery-% of all compounds was high, 90 % for nadolol, 95 % forketorofen and 125 % for metronidazole, the values corresponded well with those recorded
for the control samples minus the GrowDex layer.

Compound recovery (pmol)

RESULTS

GrowDex stock hydrogel (1.5% w/v) was diluted to 1% w/v with PBS.
100 μl 1% GrowDex was plated to the apical chamber of a 24-well Transwell plate,centrifuged for 3 min at 700 rpm, and the hydrogel layer structural uniformity waschecked by visual inspection.
Nadolol, ketoprofen and metronidazole were dissolved in DMSO and dilutions
were prepared in PBS buffer to final concentration of 10 μM. Diluted drug solutions
contained 1% DMSO.
150 μl of drug solution was pipetted on top of the GrowDex layer, and 750 μl PBS
was placed in the basolateral compartment of the Transwell plate. Permeation of drug
molecules through the Transwell insert without GrowDex was used as a control.
The plates were placed at 37°C with 5 % CO2. 100 μl samples were collected frombasolateral compartment and the PBS solution replaced at 7 time points during the 48 hincubation: 1, 2, 4, 6, 8, 24, and 48 h.
Samples were transferred to a 96-well UPLC plate (Waters) and stored at -80°C until
the day of analysis.
The samples were thawed at room temperature, mixed well then centrifuged at
13,000g for 10 min. An equal volume of ice cold acetonitrile was added to eachsample, which were subsequently analysed using a Waters Acquity UPLC + Thermo
TSQ Endura triple quadrupole mass spectrometer.

TimeFigure 1. Cumulative diffusion rates of test drugs (pmol) with and without GrowDex
hydrogel layer studied over 48 h incubation period at 37°C.

CONCLUSIONSREFERENCES

In this study we show that GrowDex readily allows the permeation of small drug molecules.
The high recovery-% in both set-ups indicated that the drugs were not degraded during theprocess and indeed, obtaining similar values for samples with or without GrowDex layer
after 48 h, indicated that the drugs were not bound to the hydrogel. The charge on eachdrug molecule (nadolol=cationic, metronidazole=neutral, ketoprofen=anionic) did notappear to affect the diffusion rate significantly. In conclusion 3D cell models using GrowDex
provide a suitable platform for e.g. cell-based drug efficacy and toxicity assays, where drug
diffusion in the culture matrix is specially relevant.

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Bhattacharya M, Malinen M et al. 2012. Nanofibrillar cellulose hydrogel promotes
three-dimensional liver cell culture, Journal of Controlled Release, 164:3, pp. 291–298.
Laurén P. et al. 2014. Technetium-99m-labeled nanofibrillar cellulose hydrogel for invivo drug release. Eur J Pharm Sci. Dec 18;65:79-88.

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