Membrane curvature based lipid sorting using a nanoparticle patterned substrate.

Cellular membranes contain a variety of shapes that likely act as motifs for sorting lipids and proteins. To understand the sorting that takes place within cells, a continuous, fluid bilayer with regions of membrane curvature was designed and characterized using confocal fluorescence and total internal reflection fluorescence microscopy techniques. A supported lipid bilayer was formed over fluorescently labelled nanoparticles deposited on a glass surface. The lipid composition and membrane shape are separately controlled and the nanoparticle dimensions (d = 40-200 nm) determine the extent of curvature. The bulk membrane is fluid as demonstrated by fluorescence recovery after photobleaching (FRAP) using dye labelled lipids. In bilayers that contain fluorescently labelled, single-tailed lipids, accumulation is observed at regions of curvature, yet the molecules retain fluidity. Using single particle imaging methods, lipids are observed to visit regions of curvature and exchange with the surrounding flat membrane. The nanoparticle patterned substrate described here allows for quantitative measurement of the transient interactions between fluorescently labelled biomolecules and regions of membrane curvature.

Evaluation of a triple-helical peptide with quenched FluorSophores for optical imaging of MMP-2 and MMP-9 proteolytic activity.

Matrix metalloproteinases (MMP) 2 and 9, the gelatinases, have consistently been associated with tumor progression. The development of gelatinase-specific probes will be critical for identifying in vivo gelatinoic activity to understand the molecular role of the gelatinases in tumor development. Recently, a self-assembling homotrimeric triple-helical peptide (THP), incorporating a sequence from type V collagen, with high substrate specificity to the gelatinases has been developed. To determine whether this THP would be suitable for imaging protease activity, 5-carboxyfluorescein (5FAM) was conjugated, resulting in 5FAM3-THP and 5FAM6-THP, which were quenched up to 50%. 5FAM6-THP hydrolysis by MMP-2 and MMP-9 displayed kcat/KM values of 1.5 × 104 and 5.4 × 103 M-1 s-1, respectively. Additionally 5FAM6-THP visualized gelatinase activity in gelatinase positive HT-1080 cells, but not in gelatinase negative MCF-7 cells. Furthermore, the fluorescence in the HT-1080 cells was greatly attenuated by the addition of a MMP-2 and MMP-9 inhibitor, SB-3CT, indicating that the observed fluorescence release was mediated by gelatinase proteolysis and not non-specific proteolysis of the THPs. These results demonstrate that THPs fully substituted with fluorophores maintain their substrate specificity to the gelatinases in human cancer cells and may be useful in in vivo molecular imaging of gelatinase activity.

Application of a Best Practice Approach Using Resonant Mass Measurement for Biotherapeutic Product Characterization.

Characterizing and quantifying subvisible particles in protein drug products is critical to ensuring product quality. A variety of analytical methods are used to detect and make meaningful measurements of subvisible particles. Resonant mass measurement (RMM) is a novel technology that characterizes the subvisible particle content of samples on a particle-by-particle basis. The technology presents great promise in the study of therapeutic protein products. As an emerging tool in the biopharmaceutical field, the best practices and limitations of RMM for protein products have not been well established. One key challenge of particle analysis is producing robust and reliable data, with high precision and accuracy, for particle characterization. In this study, we develop a set of possible best practices for RMM using a model protein system. We test the effects of these practices on the repeatability and reproducibility of particle measurements. Additionally, we present the data collected under a rigorously controlled set of operating conditions at 3 collaborating sites as well as a summary of the resulting optimal practices. In employing these practices, we successfully obtained improved relative standard deviation values and achieved high reproducibility and repeatability in both sizing and concentration measurement results over a broad range of sample volumes.

Single Lipid Molecule Dynamics on Supported Lipid Bilayers with Membrane Curvature.

The plasma membrane is a highly compartmentalized, dynamic material and this organization is essential for a wide variety of cellular processes. Nanoscale domains allow proteins to organize for cell signaling, endo- and exocytosis, and other essential processes. Even in the absence of proteins, lipids have the ability to organize into domains as a result of a variety of chemical and physical interactions. One feature of membranes that affects lipid domain formation is membrane curvature. To directly test the role of curvature in lipid sorting, we measured the accumulation of two similar lipids, 1,2-Dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and hexadecanoic acid (HDA), using a supported lipid bilayer that was assembled over a nanopatterned surface to obtain regions of membrane curvature. Both lipids studied contain 16 carbon, saturated tails and a head group tag for fluorescence microscopy measurements. The accumulation of lipids at curvatures ranging from 28 nm to 55 nm radii was measured and fluorescein labeled DHPE accumulated more than fluorescein labeled HDA at regions of membrane curvature. We then tested whether single biotinylated DHPE molecules sense curvature using single particle tracking methods. Similar to groups of fluorescein labeled DHPE accumulating at curvature, the dynamics of single molecules of biotinylated DHPE was also affected by membrane curvature and highly confined motion was observed.

Wide-field quantitative imaging of tissue microstructure using sub-diffuse spatial frequency domain imaging.

Localized measurements of scattering in biological tissue provide sensitivity to microstructural morphology but have limited utility to wide-field applications, such as surgical guidance. This study introduces sub-diffusive spatial frequency domain imaging (sd-SFDI), which uses high spatial frequency illumination to achieve wide-field sampling of localized reflectances. Model-based inversion recovers macroscopic variations in the reduced scattering coefficient [Formula: see text] and the phase function backscatter parameter (γ). Measurements in optical phantoms show quantitative imaging of user-tuned phase-function-based contrast with accurate decoupling of parameters that define both the density and the size-scale distribution of scatterers. Measurements of fresh ex vivo breast tissue samples revealed, for the first time, unique clustering of sub-diffusive scattering properties for different tissue types. The results support that sd-SFDI provides maps of microscopic structural biomarkers that cannot be obtained with diffuse wide-field imaging and characterizes spatial variations not resolved by point-based optical sampling.

Utilizing the Multiradionuclide Resolving Power of SPECT and Dual Radiolabeled Single Molecules to Assess Treatment Response of Tumors.

PURPOSE: Single photon emission computed tomography (SPECT) radionuclide pairs having distinct decay rates and different energy maxima enable simultaneous detection of dual gamma signals and real-time assessment of dynamic functional and molecular processes in vivo. Here, we report image acquisition and quantification protocols for a single molecule labeled with two different radionuclides for functional SPECT imaging. PROCEDURES: LS370 and LS734 were prepared using modular solid phase peptide synthesis. Each agent has a caspase-3 cleavable reporting motif, flanked by a tyrosine residue and a chelator at the opposite end of molecule. Cell uptake and efflux were assessed in human MDA-MB-231 breast cancer cells. Biodistribution studies were conducted in tumor naive and orthotopic 4T1 metastatic breast cancer tumor mice. NanoSPECT dual-imaging validation and attenuation correction parameters were developed using phantom vials containing varying radionuclide concentrations. Proof-of-principle SPECT imaging was performed in MMTV-PyMT transgenic mice. RESULTS: LS370 and LS734 were singly or dually radiolabeled with (125)I and (111)In or (99m)Tc. Cell assays demonstrated 11-fold higher percent uptake (P < 0.001) of [(125)I]LS734 (3.6 ± 0.5) compared to [(125)I]LS370 (0.3 ± 0.3) at 2 h. Following chemotherapy, cellular retention of [(125)I]LS734 was 3-fold higher (P < 0.05) than untreated cells. Pharmacokinetics at 1 h postinjection demonstrated longer blood retention (%ID/g) for [(125)I]LS734 (3.2 ± 0.9) compared to [(125)I]LS370 (1.6 ± 0.1). In mice bearing bilateral orthotopic 4T1 tumors, the uptake (%ID/g) was 2.4 ± 0.3 for [(125)I]LS734 and 1.2 ± 0.03 for [(125)I]LS370. The iodinated tyrosine peptide residue label was stable under in vitro conditions for up to 24 h; rapid systemic deiodination (high thyroid uptake) was observed in vivo. Phantom studies using standards demonstrated deconvolution of radionuclide signals based on different gamma ray energies. In MMTV-PyMT mice imaged with dual-labeled [(111)In]-[(125)I]LS734, the gamma signals were separable and quantifiable. CONCLUSIONS: Image processing protocols were developed for quantitative signal separation resulting from a caspase-3 responsive dual-radiolabeled SPECT probe. Crosstalk unmixing was obtained for multiradionuclide NanoSPECT imaging. In vitro and in vivo data demonstrated structure-activity relationships for developing functional agents for ratiometric SPECT imaging.

Single molecule tracking of P-glycoprotein in live cells reveals dynamic heterogeneity.

P-glycoprotein transports chemotherapy drugs from the plasma membrane and allows cancer cells to survive treatment. We transiently transfected PGP labeled with enhanced green fluorescent protein (PGP-EGFP) into MES-SA cells and used single molecule tracking techniques to characterize the dynamics on the surface of live cells. PGP exhibits freely diffusive behavior at short times and is confined at long times with a transition to anomalous diffusion at 0.7 s.

Multimodal imaging of integrin receptor-positive tumors by bioluminescence, fluorescence, gamma scintigraphy, and single-photon emission computed tomography using a cyclic RGD peptide labeled with a near-infrared fluorescent dye and a radionuclide.

Integrins, particularly the alpha(v)beta(3) heterodimers, play important roles in tumor-induced angiogenesis and invasiveness. To image the expression pattern of the alpha(v)beta(3) integrin in tumors through a multimodality imaging paradigm, we prepared a cyclic RGDyK peptide analogue (LS308) bearing a tetraazamacrocycle 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA) and a lipophilic near-infrared (NIR) fluorescent dye cypate. The alpha(v)beta(3) integrin binding affinity and the internalization properties of LS308 mediated by the alpha(v)beta(3) integrin in 4t1luc cells were investigated by receptor binding assay and fluorescence microscopy, respectively. The in vivo distribution of (111)In-labeled LS308 in a 4t1luc tumor-bearing mouse model was studied by fluorescence, bioluminescence, planar gamma, and single-photon emission computed tomography (SPECT). The results show that LS308 has high affinity for alpha(v)beta(3) integrin and internalized preferentially via the alpha(v)beta(3) integrin-mediated endocytosis in 4t1luc cells. We also found that LS308 selectively accumulated in alpha(v)beta(3)-positve tumors in a receptor-specific manner and was visualized by the four imaging methods. Whereas the endogenous bioluminescence imaging identified the ensemble of the tumor tissue, the fluorescence and SPECT methods with the exogenous contrast agent LS308 reported the local expression of alpha(v)beta(3) integrin. Thus, the multimodal imaging approach could provide important complementary diagnostic information for monitoring the efficacy of new antiangiogenic drugs.

Complementary optical and nuclear imaging of caspase-3 activity using combined activatable and radio-labeled multimodality molecular probe.

Based on the capability of modulating fluorescence intensity by specific molecular events, we report a new multimodal optical-nuclear molecular probe with complementary reporting strategies. The molecular probe (LS498) consists of tetraazacyclododecanetetraacetic acid (DOTA) for chelating a radionuclide, a near-infrared fluorescent dye, and an efficient quencher dye. The two dyes are separated by a cleavable peptide substrate for caspase-3, a diagnostic enzyme that is upregulated in dying cells. LS498 is radiolabeled with (64)Cu, a radionuclide used in positron emission tomography. In the native form, LS498 fluorescence is quenched until caspase-3 cleavage of the peptide substrate. Enzyme kinetics assay shows that LS498 is readily cleaved by caspase-3, with excellent enzyme kinetic parameters k(cat) and K(M) of 0.55+/-0.01 s(-1) and 1.12+/-0.06 microM, respectively. In mice, the initial fluorescence of LS498 is ten-fold less than control. Using radiolabeled (64)Cu-LS498 in a controlled and localized in-vivo model of caspase-3 activation, a time-dependent five-fold NIR fluorescence enhancement is observed, but radioactivity remains identical in caspase-3 positive and negative controls. These results demonstrate the feasibility of using radionuclide imaging for localizing and quantifying the distribution of molecular probes and optical imaging for reporting the functional status of diagnostic enzymes.

Activatable molecular systems using homologous near-infrared fluorescent probes for monitoring enzyme activities in vitro, in cellulo, and in vivo.

We have developed a generic approach to determine enzyme activities in vitro and monitor their functional status in vivo. Specifically, a method to generate donor (CbOH)-acceptor (Me2NCp) near-infrared (NIR) fluorescent dye pairs for preparing enzyme activatable molecular systems were developed based on the structural template of heptamethine cyanine dyes. Using caspase-3 as a model enzyme, we prepared two new caspase-3 sensitive compounds with high fluorescence quenching efficiency: Me2NCp-DEVD-K(CbOH)-OH (4) and AcGK(Me2NCp)-DEVD-APK(CbOH)-NH2 (5). The mechanism of quenching was based on combined effects of direct (classical) and reverse fluorescence resonance energy transfer (FRET). Caspase-3 cleavage of the scissile DEVD amide bond regenerated the NIR fluorescence of both donor and acceptor dyes. While both compounds were cleaved by caspase-3, substrate 5 was cleaved more readily than 4, yielding k(cat) and K(M), values of 1.02 +/- 0.06 s(-1) and 15 +/- 3 microM, respectively. Treatment of A549 tumor cells with paclitaxel resulted in > 2-fold increase in the fluorescence intensity by NIR confocal microscopy, suggesting the activation of pro-caspase-3 to caspase-3. A similar trend was observed in a mouse model, where the fluorescence intensity was nearly twice the value in caspase-3-rich tissue relative to the control. These results demonstrate the use of the same NIR activatable molecular systems for monitoring the activities of enzymes across a wide spatial scale ranging from in vitro kinetics measurements to in cellulo and in vivo localization of caspase-3 activation. The NIR activatable molecular probes provide an effective strategy to screen new drugs in vitro and monitor treatment response in living organisms.

Agonist-antagonist dilemma in molecular imaging: evaluation of a monomolecular multimodal imaging agent for the somatostatin receptor.

The combination of different imaging modalities, each providing information according to its strengths, can be a powerful method for diagnosing diseases. We have synthesized a monomolecular multimodal imaging agent (MOMIA), LS172, containing a subtype-2 somatostatin receptor (SSTr2)-avid peptide (Y3-octreotate or Y3-TATE), a radiometal chelating group (DOTA) and a near-infrared (NIR) fluorescent dye (cypate). In addition to optical methods, radiolabeling LS172 with 64Cu and 177Lu provides a strategy for in vitro evaluation or in vivo multimodal imaging by positron emission tomography (PET) and single photon emission computed tomography (SPECT), respectively. Determination of the binding affinity of LS172, nat Cu- and nat Lu-LS172 in SSTr2-transfected A427 cells (A427-7) showed that they all displayed high binding affinity toward SSTr2 with K i values of 0.234 nM, 11.5 nM, and 2.15 nM respectively. In contrast to cypate-labeled Y3-TATE (cytate), fluorescence microscopy showed that LS172 and nat Cu-LS172 accumulate modestly in A427-7 cells by SSTr2-mediated endocytosis, in spite of their relatively high binding affinity. In vivo, the biodistribution of the SSTr2 receptor specific 64Cu- and 177Lu-LS172 in AR42J tumor-bearing rats exhibited low (90% ID/liver). Both optical and radionuclear biodistribution studies showed a similar in vivo distribution profile. Surprisingly, the strong binding of LS172 to SSTr2 did not translate into high SSTr2-mediated endocytosis in cells or uptake in tumor in vivo. Considering that LS172 is a putative antagonist, the poor accumulation of the labeled MOMIAs in SSTr2 positive tumor tissue supports the paradigm that agonists with their concomitant internalization favors appreciable target tissue accumulation of receptor-specific ligands.