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Assay Tutorials : Mar 1, 2009 ( )
Drug Absorption on Membrane Arrays
A Novel Method Using SPR Imaging!--h2>
Surface plasmon resonance (SPR) detectors monitor molecular interactions without labels and in real time. SPR imaging (SPRi) has the additional advantage of increased throughput since it monitors binding to arrays of molecules. Indeed, the coupling of quantitative, label-free, mass-sensitive SPR technology with array capabilities has been used successfully in many diverse applications such as characterizing antibody–antigen interactions, proteins binding to aptamers, transcription factors binding to DNAs, and profiling cell surface receptors on ligand arrays.
This tutorial describes a novel SPRi-based assay for measuring drug absorption to natural membrane arrays. This general protocol can be used in drug evaluation since it can investigate absorption of a drug to cell membranes from multiple tissue sources arrayed on a single chip.
The bioavailability of a drug is influenced by its ability to cross cell membranes, so a drug’s absorption profile is a crucial factor affecting drug efficacy. A number of approaches to evaluate drug absorption and lipid permeability are in use including tissue diffusion assays, cell-based permeability assays, noncell-based assays such as liposome chromatography and parallel artificial membrane permeability assays. These applications, however, are not readily amenable to multiplexing; rely on downstream analytical procedures such as UV spectroscopy or mass spectrometry; and often require synthesis of radio- or fluorescently labeled marker compounds.
SPR Imaging Technology
The SPRimager®II from GWC Technologies measures binding events that occur on the surface of a gold-coated chip. Binding of an analyte to probe molecules immobilized on a chip results in an increase in reflectivity (D%R). A CCD camera captures this change in reflectivity by imaging the whole array in real time.
In a typical experiment, a reference image of the array is collected prior to exposure to analyte. Then, images are collected as the array is exposed to a flow of analyte solution. For each time point, the reference image is subtracted and D%R for each element on the array is plotted as a function of time. An increase in reflectivity indicates binding has occurred. A decrease in reflectivity indicates dissociation.
Fabrication of Arrays
Drug-lipid interactions have been measured in a label-free manner using synthetic lipids in the form of liposomes. In this assay, natural cell membranes from Spodoptera frigiperda Sf9 insect cells (Invitrogen) were immobilized on gold-coated SPRi chips. The core of this approach is the incorporation of biotinylated PEG-phospholipids into the membrane (using double sonication) to generate biotin-tagged, small membrane vesicles.
Membrane fractions of Sf9 insect cells were sonicated with polymeric micelles composed of biotin-PEG-DSPE (Avanti Polar Lipids). After washing and centrifugation, the supernatant containing the biotinylated cell membranes (biMEM) was diluted and spotted on streptavidin-coated SPRi sensors where they were immobilized via the biotin-containing anchors.
An image of a typical biMEM array on a SpotReady16™ chip before exposure to analyte is shown in Figure 1A. biMEM capture on the chip surface is specific, and probe density of tethered membranes increases as the concentration of spotted biMEM increases (Figure 1B).
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.
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