Background and PurposeA monoclonal antibody (PF-00547659) against mucosal addressin cell adhesion molecule (MAdCAM), expressed as both soluble (sMAdCAM) and trans-membrane (mMAdCAM) target forms, showed over 30-fold difference in antibody-target KD between in vitro (Biacore) and clinically derived (KD,in-vivo) values. Back-scattering interferometry (BSI) was applied to acquire physiologically relevant KD values which were used to establish in vitro and in vivo correlation (IVIVC).Experimental ApproachBSI was applied to obtain KD values between PF-00547659 and recombinant human MAdCAM in buffer or CHO cells and endogenous MAdCAM in human serum or colon tissue. CHO cells and tissue were minimally processed to yield homogenate containing membrane vesicles and soluble proteins. A series of binding affinities in serum with various dilution factors was used to estimate both KD,in-vivo and target concentrations; MAdCAM concentrations were also measured using LC–MS/MS.Key ResultsBSI measurements revealed low KD values (higher affinity) for sMAdCAM in buffer and serum, yet a 20-fold higher KD value (lower affinity) for mMAdCAM in CHO, mMAdCAM and sMAdCAM in tissue. BSI predicted KD,in-vivo in serum was similar to clinically derived KD,in-vivo, and the BSI-estimated serum sMAdCAM concentration also matched the measured concentration by LC–MS/MS.Conclusions and ImplicationsOur results successfully demonstrated that BSI measurements of physiologically relevant KD values can be used to establish IVIVC, for PF-00547659 to MAdCAM despite the lack of correlation when using Biacore measured KD and accurately estimates endogenous target concentrations. The application of BSI would greatly enhance successful basic pharmacological research and drug development.

Interaction/reaction assays have led to significant scientific discoveries in the biochemical, medical, and chemical disciplines. Several fundamental driving forces form the basis of intermolecular and intramolecular interactions in chemical and biochemical systems (London dispersion, hydrogen bonding, hydrophobic, and electrostatic), and in the past three decades the sophistication and power of techniques to interrogate these processes has developed at an unprecedented rate. In particular, label-free methods have flourished, such as NMR, mass spectrometry (MS), surface plasmon resonance (SPR), biolayer interferometry (BLI), and backscattering interferometry (BSI), which can facilitate assays without altering the participating components. The shortcoming of most refractive index (RI)-based label-free methods such as BLI and SPR is the requirement to tether one of the interaction entities to a sensor surface. This is not the case for BSI. Here, our hypothesis is that the signal origin for free-solution, label-free determinations can be attributed to conformation and hydration-induced changes in the solution RI. We propose a model for the free-solution response function (FreeSRF) and show that, when quality bound and unbound structural data are available, FreeSRF correlates well with the experiment (R2 > 0.99, Spearman rank correlation coefficients >0.9) and the model is predictive within ∼15% of the experimental binding signal. It is also demonstrated that a simple mass-weighted dη/dC response function is the incorrect equation to determine that the change in RI is produced by binding or folding event in free solution.

Realizing personalized medicine, which promises to enable early disease detection, efficient diagnostic staging, and therapeutic efficacy monitoring, hinges on biomarker quantification in patient samples. Yet, the lack of a sensitive technology and assay methodology to rapidly validate biomarker candidates continues to be a bottleneck for clinical translation. In our first direct and quantitative comparison of backscattering interferometry (BSI) to fluorescence sensing by ELISA, we show that BSI could aid in overcoming this limitation. The analytical validation study was performed against ELISA for two biomarkers for lung cancer detection: Cyfra 21-1 and Galectin-7. Spiked serum was used for calibration and comparison of analytical figures of merit, followed by analysis of blinded patient samples. Using the ELISA antibody as the probe chemistry in a mix-and-read assay, BSI provided significantly lower detection limits for spiked serum samples with each of the biomarkers. The limit of quantification (LOQ) for Cyrfa-21-1 was measured to be 230 pg/mL for BSI versus 4000 pg/mL for ELISA, and for Galectin-7, it was 13 pg/mL versus 500 pg/mL. The coefficient of variation for 5 day, triplicate determinations was <15% for BSI and <10% for ELISA. The two techniques correlated well, ranging from 3–29% difference for Cyfra 21-1 in a blinded patient sample analysis. The label-free and free-solution operation of BSI allowed for a significant improvement in analysis speed, with greater ease, improved LOQ values, and excellent day-to-day reproducibility. In this unoptimized format, BSI required 5.5-fold less sample quantity needed for ELISA (a 10 point calibration curve measured in triplicate required 36 μL of serum for BSI vs 200 μL for ELISA). The results indicate that the BSI platform can enable rapid, sensitive analytical validation of serum biomarkers and should significantly impact the validation bottleneck of biomarkers.

Aptamers are segments of single-strand DNA or RNA used in a wide array of applications, including sensors, therapeutics, and cellular process regulators. Aptamers can bind many target species, including proteins, peptides, and small molecules (SM) with high affinity and specificity. They are advantageous because they can be identified in vitro by SELEX, produced rapidly and relatively economically using oligonucleotide synthesis. The use of aptamers as SM probes has experienced a recent rebirth, and because of their unique properties they represent an attractive alternative to antibodies. Current assay methodology for characterizing small molecule–aptamer binding is limited by either mass sensitivity, as in biolayer interferometry (BLI) and surface plasmon resonance (SPR), or the need for using a fluorophore, as in thermophoresis. Here we report that backscattering interferometry (BSI), a label-free and free-solution sensing technique, can be used to effectively characterize SM–aptamer interactions, providing Kd values on microliter sample quantities and at low nanomolar sensitivity. To demonstrate this capability we measured the aptamer affinity for three previously reported small molecules; bisphenol A, tenofovir, and epirubicin showing BSI provided values consistent with those published previously. We then quantified the Kd values for aptamers to ampicillin, tetracycline and norepinephrine. All measurements produced R2 values >0.95 and an excellent signal to noise ratio at target concentrations that enable true Kd values to be obtained. No immobilization or labeling chemistry was needed, expediting the assay which is also insensitive to the large relative mass difference between the interacting molecules.

Advances in probes for cellular imaging have driven discoveries in biology and medicine. Primarily, antibodies and small molecules have been made for contrast enhancement of specific proteins. The development of new dendrimer-based tools offers opportunities to tune cellular internalization and targeting, image multiple modalities in the same molecule and explore therapeutics. The translocator protein (TSPO) offers an ideal target to develop dendrimer tools because it is well characterized and implicated in a number of disease states. The TSPO-targeted dendrimers reported here, primarily ClPhIQ-PAMAM-Gd-Liss, are cell membrane permeable nanoparticles that enable labeling of TSPO and provide contrast in fluorescence, electron microscopy and magnetic resonance imaging. The molecular binding affinity for TSPO was found to be 0.51 μM, 3 times greater than the monomeric agents previously demonstrated in our laboratory. The relaxivity per Gd3+ of the ClPhIQ23-PAMAM-Gd18 dendrimer was 7.7 and 8.0 mM−1 s−1 for r1 and r2 respectively, approximately double that of the clinically used monomeric Gd3+ chelates. In vitro studies confirmed molecular selectively for labeling TSPO in the mitochondria of C6 rat glioma and MDA-MB-231 cell lines. Fluorescence co-registration with Mitotracker Green® and increased contrast of osmium-staining in electron microscopy confirmed mitochondrial labeling of these TSPO-targeted agents. Taken collectively these experiments demonstrate the versatility of conjugation of our PAMAM dendrimeric chemistry to allow multi-modality agents to be prepared. These agents target organelles and use complementary imaging modalities in vitro, potentially allowing disease mechanism studies with high sensitivity and high resolution techniques.

Chemotherapeutics such as doxorubicin (DOX) and paclitaxel (PXL) have dose-limiting systemic toxicities, including cardiotoxicity and peripheral neuropathy. Delivery strategies to minimize these undesirable effects are needed and could improve efficacy, while reducing patient morbidity. Here, DOX and PXL were conjugated to a nanodendron (ND) through an MMP9-cleavable peptide linker, producing two new therapies, ND2DOX and ND2PXL, designed to improve delivery specificity to the tumor microenvironment and reduce systemic toxicity. Comparative cytotoxicity assays were performed between intact ND-drug conjugates and the MMP9 released drug in cell lines with and without MMP9 expression. While ND2DOX was found to lose cytotoxicity due to the modification of DOX for conjugation to the ND; ND2PXL was determined to have the desired properties for a prodrug delivery system. ND2PXL was found to be cytotoxic in MMP9-expressing mouse mammary carcinoma (R221A-luc) (53%) and human breast carcinoma (MDA-MB-231) (66%) at a concentration of 50 nM (in PXL) after 48 h. Treating ND2PXL with MMP9 prior to the cytotoxicity assay resulted in a faster response; however, both cleaved and intact versions of the drug reached the same efficacy as the unmodified drug by 96 h in the R221A-luc and MDA-MB-231 cell lines. Further studies in modified Lewis lung carcinoma cells that either do (LLCMMP9) or do not (LLCRSV) express MMP9 demonstrate the selectivity of ND2PXL for MMP9. LLCMMP9 cells were only 20% viable after 48 h of treatment, while LLCRSV were not affected. Inclusion of an MMP inhibitor, GM6001, when treating the LLCMMP9 cells with ND2PXL eliminated the response of the MMP9 expressing cells (LLCMMP9). The data presented here suggests that these NDs, specifically ND2PXL, are nontoxic until activated by MMP9, a protease common in the microenvironment of tumors, indicating that incorporation of chemotherapeutic or cytostatic agents onto the ND platform have potential for tumor-targeted efficacy with reduced in vivo systemic toxicities.Keywords: dendrons; drug delivery; MMP9; nanomaterials; paclitaxel; prodrug;

Intermolecular interactions involving hydrogen bonds are responsible for catalysis and recognition. Traditional methods used to study hydrogen-bonding interactions are generally limited to relatively large volumes and high substrate concentrations. Backscattering Interferometry (BSI) provides a microfluidic platform to study these interactions in nonaqueous media at micromolar to nanomolar concentrations in picoliter volumes by monitoring changes in the refractive index.

We report the quantitative measurement of aptamer–protein interactions using backscattering interferometry (BSI) and show that BSI can determine when distinct binding regions are accessed. As a model system, we utilized two DNA aptamers (Tasset and Bock) that bind to distinct sites of a target protein (human α-thrombin). This is the first time BSI has been used to study a multivalent system in free solution wherein more than one ligand binds to a single target. We measured aptamer equilibrum dissociation constants (Kd) of 3.84 nM (Tasset–thrombin) and 5.96 nM (Bock–thrombin), in close agreement with the literature. Unexpectedly, we observed allosteric effects such that the binding of the first aptamer resulted in a significant change in the binding affinity of the second aptamer. For example, the Kd of Bock aptamer binding to preformed Tasset–thrombin complexes was 7-fold lower (indicating higher affinity) compared to binding to thrombin alone. Preliminary modeling efforts suggest evidence for allosteric linkage between the two exosites.

Methylation of lysine residues in histones has been known to serve a regulatory role in gene expression. Although enzymatic removal of the methyl groups was discovered as early as 1973, the enzymes responsible for their removal were isolated and their mechanism of action was described only recently. The first enzyme to show such activity was LSD1, a flavin-containing enzyme that removes the methyl groups from lysines 4 and 9 of histone 3 with the generation of formaldehyde from the methyl group. This reaction is similar to the previously described demethylation reactions conducted by the enzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase, in which protein-bound tetrahydrofolate serves as an accepter of the formaldehyde that is generated. We now show that nuclear extracts of HeLa cells contain LSD1 that is associated with folate. Using the method of back-scattering interferometry, we have measured the binding of various forms of folate to both full-length LSD1 and a truncated form of LSD1 in free solution. The 6R,S form of the natural pentaglutamate form of tetrahydrofolate bound with the highest affinity (Kd = 2.8 μM) to full-length LSD1. The fact that folate participates in the enzymatic demethylation of histones provides an opportunity for this micronutrient to play a role in the epigenetic control of gene expression.

Backscattering interferometry enables the detection of syphilis antibody–antigen interactions in the presence of human serum, showing promise as a diagnostic tool for the serological diagnosis of infectious disease with potentially quantitative capabilities.

To quantitatively evaluate the utility of a translocator protein (TSPO)-targeted near-infrared (NIR) probe (NIR-conPK11195) for in vivo molecular imaging of TSPO in breast cancer.NIR-conPK11195 uptake and TSPO-specificity were validated in TSPO-expressing human breast adenocarcinoma cells (MDA-MB-231). In vivo NIR-conPK11195 biodistribution and accumulation were quantitatively evaluated in athymic nude mice bearing MDA-MB-231 xenografts.Fluorescence micrographs illustrated intracellular labeling of MDA-MB-231 cells by NIR-conPK11195. Quantitative uptake and competition assays demonstrated dose-dependent (p < 0.001) and TSPO-specific (p < 0.001) NIR-conPK11195 uptake. In vivo, NIR-conPK11195 preferentially labeled MDA-MB-231 tumors with an 11-fold (p < 0.001) and 7-fold (p < 0.001) contrast enhancement over normal tissue and unconjugated NIR dye, respectively.NIR-conPK11195 appears to be a promising TSPO-targeted molecular imaging agent for visualization and quantification of breast cancer cells in vivo. This research represents the first study to demonstrate the feasibility of TSPO imaging as an alternative breast cancer imaging approach.

We report the quantitative, label-free analysis of protein−protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BSI traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein α-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target βB1-Crystallin. The results recapitulate the selectivity of αB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an αA-Crystallin mutant linked to hereditary cataract has activated binding to βB1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.

Carbohydrate−protein binding is important to many areas of biochemistry. Here, backscattering interferometry (BSI) has been shown to be a convenient and sensitive method for obtaining quantitative information about the strengths and selectivities of such interactions. The surfaces of glass microfluidic channels were covalently modified with extravidin, to which biotinylated lectins were subsequently attached by incubation and washing. The binding of unmodified carbohydrates to the resulting avidin-immobilized lectins was monitored by BSI. Dose−response curves that were generated within several minutes and were highly reproducible in multiple wash/measure cycles provided adsorption coefficients that showed mannose to bind to concanavalin A (conA) with 3.7 times greater affinity than glucose consistent with literature values. Galactose was observed to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were much higher than monovalent glycans, with increases of 60−200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage Qβ. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption, which was consistent with the expected density of lectins on the surface. The sensitivity of BSI matches or exceeds that of surface plasmon resonance and quartz crystal microbalance techniques, and is sensitive to the number of binding events, rather than changes in mass. The operational simplicity and generality of BSI, along with the near-native conditions under which the target binding proteins are immobilized, make BSI an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.

Cannabinoid CB2 receptor is a particularly attractive target for noninvasive imaging of neuroinflammation and monitoring of therapeutic efficacy. Its expression is low to undetectable in healthy brain and induced in resident microglial cells (the macrophage of the brain) after cerebral ischemia, injury, and in neuroinflammatory disease. Additionally, immune cells migrating across the blood−brain barrier typically express CB2 receptors, which adds to the expression pool of this target and provides a reliable indicator of inflammation in the brain. Here, we synthesized a novel conjugable CB2 receptor ligand, mbc94, which has a terminal amino group that allows for facile conjugation to imaging moieties. A near-infrared (NIR) dye labeled mbc94, NIRmbc94, was developed for CB2 targeted imaging. Preliminary evidence, including in vitro fluorescence imaging and a competition study, showed that NIRmbc94 specifically labeled CB2-expressing cells.

The translocator protein (TSPO) is an attractive target for tumor imaging due to its up-regulation in numerous cancer cell types. Here, we report a series of functional TSPO ligands, n-TSPOmbb732, which can be conjugated to a variety of signaling moieties and are widely applicable in TSPO-targeted molecular imaging. Two fluorescent dye-labeled 6-TSPOmbb732 displayed nanomolar binding affinities to TSPO and were successfully imaged in vitro.