In Vitro Validation of Targeted Ultrasound Contrast Agent Binding Specificity Using Alpha Technology

Introduction:: Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease and end-stage kidney disease [1,2]. As such, there is a need for early and reliable testing methods able to detect DKD. The current testing methods for detecting DKD have limitations that oftentimes make it difficult to clinically detect DKD before tissue damage and functional changes have begun [3]. A reliable predictive tool would allow for patients and physicians to implement early intervention strategies to mitigate disease progression. Molecular imaging using ultrasound can serve as an early indicator of DKD, however developing and characterizing molecularly targeted contrast agents can be time consuming. Amplified luminescent proximity homogeneous assay (Alpha) technology can help shorten this process by offering an efficient method for validating potential molecular probes. For example, early stages of DKD are characterized by increased VEGFR2 expression [4]. We demonstrate how Alpha technology can be optimized to validate targeted nanodroplet (ND) interaction with disease biomarkers such as VEGFR2.

Materials and Methods:: Biotin and cMyc-A7R were incorporated into the lipid shell of the ultrasound contrast agent at an equal molar percentage (0.2 mol %) to make targeted NDs (A7R-ND). Next, the lyophilized His-tagged VEGFR2 protein was reconstituted in 100 µL of 1x PBS 0.1% BSA (w/v). A quarter-log dilution series was performed to generate A7R-ND concentrations from 38.7 pM to 688 pM and VEGFR2 concentrations spanning 68.6 pM to 1.22 nM for use in a cross-titration assay. The A7R-NDs were pipetted onto a 384-well microplate, followed by His-tagged VEGFR2, nickel chelate acceptor beads, and streptavidin donor beads. Bead concentrations of 10 µg/mL and 20 µg/mL were tested to optimize signal generation. Alpha fluorescence from the A7R-ND and VEGFR2 interaction at different molar concentrations was recorded over 20 hours. From the Alpha signal, optimal concentrations for each component were determined according to the expected K_d (~9 nM) [5]. The selected concentrations should meet the requirements to use the Cheng-Prusoff equation to calculate K_d, such that the IC50 value can be equated with the K_d value in the competitive inhibition assay [6]. For the competitive inhibition assays, set concentrations of A7R-NDs (38.7 pM) and VEGFR2 (0.688 nM) determined from the cross titration were pipetted onto a 384-well microplate. This was followed by untagged, free A7R with concentrations ranging from 0.09 nM to 900 nM, nickel chelate acceptor beads, and streptavidin donor beads. The decline in Alpha fluorescence for increasing concentrations of untagged A7R was recorded over 24 hours.

Results, Conclusions, and Discussions:: First, the effect of bead concentration was observed: as the bead concentration increased from 10 µg/mL to 20 µg/mL, the fluorescence also increased from 915 cps to 3010 cps for 3.87 nM of VEGFR2 and 38.7 pM of A7R-NDs (Figure 1A). In the cross-titration assay, the fluorescence increased until optimal A7R-ND and VEGFR2 concentrations were achieved. Once an optimal concentration was reached, the fluorescence dropped dramatically (Figure 1B). VEGRF2 had two concentrations that performed the best - the first was at 1.22 nM, which exceeds the K_d requirements for the receptor (Figure 1B) and the second best was at 0.688 nM, which falls within the K_d requirements for the receptor (Figure 1B). For the A7R-NDs, the optimal concentration in the cross titration was 38.7 pM, which suits the ligand concentration range based on the K_d. The optimal concentrations found from the cross titrations assays were used to complete the competitive inhibition assays. At 0.688 nM of VEGFR2 and 38.7 pM of A7R-NDs, the maximum signal achieved was 2101 cps. At 1.22 nM of VEGFR2 and 38.7 pM of A7R-NDs, the maximum signal achieved was 2416 cps. By selecting concentrations that suit the K_d requirements, we can validate the dissociation constant found in previous studies and demonstrate the binding strength of A7R with VEGFR2. Preliminary results for the competitive inhibition assay are based on a single experiment. Initially, at 24 hours the measured fluorescence remained steady around approximately 1000 cps, and then steadily declined over time as the concentrations of untagged A7R increased (Figure 1C). Currently, the competitive inhibition experiments are being further optimized to increase Alpha fluorescence and reliably use the calculated IC50 value as a representation of the dissociation between A7R and VEGFR2.

As we move forward with the competitive inhibition assays, these steps can be repeated to characterize and validate targeted ND interaction with other important kidney receptors such as RAGE, TNFR1, and TNFR2. This data currently supports the development of potential biomarkers used for early DKD detection.


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References (Optional): : [1] Benjamin O, et al. StatPearls Publishing, 2021. [2] Koye DN, et al. Adv. Chronic Kidney Dis., 2018. [3] Lin CH, et al. World J. Diabetes, 2016 [4] Deng B, et al. Int J Mol Sci, 2023 [5] Ying M, Shen Q, Liu Y, et al.. ACS Appl Mater Interfaces, 2016[6] Cheng Y, Prusoff WH. Biochemical Pharmacology, 1973