Membrane arrays were fabricated by capturing biMEM at concentrations in the range of 0.02–0.2 mg/mL on SpotReady™ chips. The arrays were then exposed to six drugs (Figure 2). The drugs were chosen because they are commercially available; they are highly soluble in aqueous solvent; their absorption to liposomes had previously been evaluated by SPR; and they represent different categories of drugs with respect to charge and lipophilicity.
The chip was assembled into the SPRimager II and an image of the array was obtained—both to evaluate probe density on each spot, and to serve as a reference image for calculating reflectivity changes as binding occurs.
The array was then exposed sequentially to the six different drug solutions (1 mM in HBS). Drug absorption was considered complete when a plateau in SPR signal was reached. The drug was then washed from the membrane with HBS, and the next drug was introduced. As drug absorption to the membranes was reversible, several drugs could be tested in series on the same array.
A typical drug absorption profile plotted as absolute change in reflectivity (D%R) with time is presented in Figure 2. For five of the six drugs, the binding plateaus were rapidly achieved in the presence of drug followed by rapid release when the array was washed with buffer. The sixth drug, verapamil, did not reach equilibrium during the five minute exposure used.Verapamil also did not completely dissociate after buffer wash, a behavior consistent with previous reports.
As expected, the amount of drug absorbed was higher where the amount of immobilized biMEM was greater. The absorption profiles (Figure 2) were used to calculate D%RDRUG, the normalized signal due to drug binding on the biMEM.
D%RDRUG =D%RbiMEM - D%RSA
D%RSA is the reflectivity change for the streptavidin control (i.e., no membrane) spots.
When specific absorption, D%RDRUG, is plotted as a function of the spotted biMEM concentrations, the resulting absorption response is linear with a distinct slope for each drug (Figure 3).
Slopes increase as drug absorption increases. The slope values are reproducible indicating that this slope value can be used as a specific measure of a drug’s membrane absorption characteristics. The rank order measured for drug absorption in this pilot study correlates well with conventional SPR drug absorption to liposomes (Table).
In summary, we have developed a protocol for fabricating natural membrane arrays and demonstrated that these arrays are suitable for measuring drug absorption using SPRi. The two main advantages of this method are: drug binding to natural membranes and not synthetic lipids is measured and the array format of the sensor surface allows interrogation of multiple membrane samples simultaneously on the same chip.
Although the pilot study presented here interrogated few samples simultaneously, higher density arrays fabricated by robotic spotting can be analyzed with the same instrument, providing a platform for a highly efficient screen for drug absorption characteristics.