'Stirred not Shaken!' Comparing Agitation Methods for Permeability Studies Using a Novel Type of 96-Well Sandwich-Plates

In order to achieve a high sample throughput, permeation experiments are often carried out using 96-well sandwich plates. Even though agitation is regarded as important, permeation studies in 96-well format are often carried out without agitation since orbital shaking, the most common agitation method for 96-well plates, has been reported to create difficulties (e.g., well-to-well cross-talk), and high cost and low availability limits the use of other agitation techniques (e.g., magnetic stirring). This study investigates how orbital shaking and magnetic stirring affect the apparent permeability of model compounds with different water-solubilities (methylene blue, carbamazepine, and albendazole) using a novel 96-well sandwich plate comprising a cellulose-hydrate membrane (PermeaPlain (R) plate). Orbital shaking was found less efficient than magnetic stirring in terms of homogeneously distributing a small volume of dye within the donor compartment. Furthermore, in terms of achieving maximum trans-barrier flux, magnetic stirring was found a more effective agitation method than orbital shaking. Obviously, with orbital shaking the medium in the bottom compartment of the sandwich plates never was mixed in-phase. The impact of insufficient mixing on permeation was found strongest with the most lipophilic compound, which correlates with literature reports that the contribution of the unstirred water layer towards the overall resistance of the barrier is most expressed in case of lipophilic drugs. Finally, it was tested how different liquid volumes in the bottom compartment of the plates affect the well-to-well cross-talk during permeation experiments under orbital shaking. This study revealed that 250-300 mu L should be used in the bottom compartment of the sandwich plates to reduce well-to-well cross-talk when using orbital shaking for agitation. (C) 2021 Published by Elsevier Inc. on behalf of American Pharmacists Association.

Automated summary

To achieve high sample throughput, permeation experiments are often conducted using 96-well sandwich plates. However, agitation is often omitted in these experiments due to difficulties associated with orbital shaking and limited availability of other agitation techniques. This study compared the effects of orbital shaking and magnetic stirring on the permeability of model compounds with different water-solubilities. The results showed that magnetic stirring was more efficient in distributing dye within the donor compartment and achieving maximum trans-barrier flux compared to orbital shaking. Additionally, insufficient mixing was found to have the greatest impact on permeation, especially for lipophilic compounds. The study also investigated the well-to-well cross-talk during permeation experiments with orbital shaking and found that using a liquid volume of 250-300 μL in the bottom compartment of the plates reduced cross-talk.