Preparation and Characterization of Targeted Microbubbles.

Targeting of microbubbles (ultrasound contrast agents for molecular imaging) has been researched for more than two decades. However, methods of microbubble preparation and targeting ligand attachment are cumbersome, complicated, and lengthy. Therefore, there is a need to simplify the targeted microbubble preparation procedure to bring it closer to clinical translation. The purpose of this publication is to provide a detailed description and explanation of the steps necessary for targeted microbubble preparation, functional characterization and testing. A sequence of the optimized and simplified procedures is presented for two systems: a biotin-streptavidin targeting pair model, and a cyclic RGD peptide targeting the recombinant αvß3 protein, which is overexpressed on the endothelial lining of the tumor neovasculature. Here, we show the following: covalent coupling of the targeting ligand to a lipid anchor, assessment of the reagent quality, and tests that confirm the successful completion of the reaction; preparation of the aqueous precursor medium containing microbubble shell components, followed by microbubble preparation via amalgamation; assessment of the efficacy of lipid transfer onto the microbubble stabilizer shell; adjustment of microbubble size distribution by flotation at normal gravity to remove larger microbubbles that might be detrimental for in vivo use; assessment of microbubble size distribution by electrozone sensing; evaluation of targeted binding of the microbubbles to receptor-coated surface in a static binding assay test (in an inverted dish); and evaluation of targeted binding of the microbubbles to receptor-coated surface in a parallel plate flow chamber test.

Targeted Ultrasound Contrast Imaging of Tumor Vasculature With Positively Charged Microbubbles.

PURPOSE: Molecular ultrasound imaging of tumor vasculature is being actively investigated with microbubble contrast agents targeted to neovasculature biomarkers. Yet, a universal method of targeting tumor vasculature independent of specific biomarkers, or in their absence, would be desirable. We report the use of electrostatic interaction to achieve adherence of microbubbles to tumor vasculature and resulting tumor delineation by ultrasound imaging. METHODS AND MATERIALS: Microbubbles were prepared from decafluorobutane gas by amalgamation of aqueous micellar medium. Distearoyl phosphatidylcholine (DSPC) and polyethylene glycol (PEG)-stearate were used as microbubble shell-forming lipids; cationic lipid distearoyl trimethylammoniumpropane (DSTAP) was included to introduce positive electrostatic charge. Microbubbles were subjected to flotation in normal gravity, to remove larger particles. Murine colon adenocarcinoma tumor (MC38, J. Schlom, National Institutes of Health) was inoculated in the hind leg of C57BL/6 mice. Contrast ultrasound imaging was performed under isoflurane anesthesia, using a clinical imaging system in low power mode, with tissue signal suppression (contrast pulse sequencing, 7 MHz, 1 Hz; Mechanical Index, 0.2). The ultrasound probe was positioned to monitor the tumor and contralateral leg muscle; microbubble contrast signal was monitored for 30 minutes or more, after intravenous bolus administration of 2.10 microbubbles. Individual time point frames were extracted from ultrasound video recording and analyzed with ImageJ. RESULTS: Mean bubble diameter was ~1.6 to 2 µm; 99.9% were less than 5 µm, to prevent blocking blood flow in capillaries. For cationic DSTAP-carrying microbubbles, contrast signal was observed in the tumor beyond 30 minutes after injection. As the fraction of positively charged lipid in the bubble shell was increased, adherent contrast signal in the tumor also increased, but accumulation of DSTAP-microbubbles in the normal muscle increased as well. For bubbles with the highest positive charge tested, DSTAP-DSPC molar ratio 1:4, at 10 minutes after intravenous administration of microbubbles, the contrast signal difference between the tumor and normal muscle was 1.5 (P < 0.005). At 30 minutes, tumor/muscle contrast signal ratio improved and reached 2.1. For the DSTAP-DSPC 1:13 preparation, tumor/muscle signal ratio exceeded 3.6 at 10 minutes and reached 5.4 at 30 minutes. Microbubbles with DSTAP-DSPC ratio 1:22 were optimal for tumor targeting: at 10 minutes, tumor/muscle signal ratio was greater than 7 (P < 0.005); at 30 minutes, greater than 16 (P < 0.01), sufficient for tumor delineation. CONCLUSIONS: Cationic microbubbles are easy to prepare. They selectively accumulate in the tumor vasculature after intravenous administration. These microbubbles provide target-to-control contrast ratio that can exceed an order of magnitude. Adherent microbubbles delineate the tumor mass at extended time points, at 30 minutes and beyond. This may allow for an extension of the contrast ultrasound examination time. Overall, positively charged microbubbles could become a universal ultrasound contrast agent for cancer imaging.

Formation of Microbubbles for Targeted Ultrasound Contrast Imaging: Practical Translation Considerations.

For preparation of ligand-decorated microbubbles for targeted ultrasound contrast imaging, it is important to maximize the amount of ligand associated with the bubble shell. We describe optimization of the use of a biocompatible cosurfactant in the microbubble formulation media to maximize the incorporation of targeting ligand-lipid conjugate into the microbubble shell, and thus reduce the fraction of ligand not associated with microbubbles, following amalgamation preparation. The influence of the concentration of a helper cosurfactant propylene glycol (PG) on the efficacy of microbubble preparation by amalgamation and on the degree of association of fluorescent PEG-lipid with the microbubble shell was tested. Three sets of targeted bubbles were then prepared: with VCAM-1-targeting peptide VHPKQHRGGSK(FITC)GC-PEG-DSPE, cyclic RGDfK-PEG-DSPE, selective for αVß3, and control cRADfK-PEG-DSPE, without such affinity. Microbubbles were prepared by 45 s amalgamation, with DSPC and PEG stearate as the main components of the shell, with 15% PG in aqueous saline. In vitro microbubble targeting was assessed with a parallel plate flow chamber with a recombinant receptor coated surface. In vivo targeting was assessed in MC-38 tumor-bearing mice (subcutaneous tumor in hind leg), 10 min after intravenous bolus of microbubble contrast agent (20 million particles per injection). Ultrasound imaging of the tumor and control nontarget muscle tissue in a contralateral leg was performed with a clinical scanner. Amalgamation technique with PG cosurfactant produced microbubbles at concentrations exceeding 2 × 109 particles/mL, and ∼50-60% or more of the added fluorescein-PEG-DSPE or VCAM-1-targeted fluorescent peptide was associated with microbubbles, about 2 times higher than that in the absence of PG. After intravenous injection, peptide-targeted bubbles selectively accumulated in the tumor vasculature, with negligible accumulation in nontumor contralateral leg muscle, or with control nontargeted microbubbles (assessed by contrast ultrasound imaging). For comparison, administration of RGD-decorated microbubbles prepared by traditional sonication, and purified from free peptide-PEG-lipid by repeated centrifugation, resulted in the same accumulation pattern as for translatable amalgamated microbubbles. Following amalgamation in the presence of PG, efficient transfer of ligand-PEG-lipid to microbubble shell was achieved and quantified. Purification of microbubbles from free peptide-PEG-lipid was not necessary, as proven by in vitro and in vivo targeting studies, so PG cosurfactant amalgamation technique generated peptide-targeted microbubbles are amenable for bedside preparation and clinical translation. The pathway to clinical translation is simplified by the fact that most of the materials used in this study either are on the United States Food and Drug Administration GRAS list or can be procured as pharmaceutical grade substances.

Plectin-targeted liposomes enhance the therapeutic efficacy of a PARP inhibitor in the treatment of ovarian cancer.

Advances in genomics and proteomics drive precision medicine by providing actionable genetic alterations and molecularly targeted therapies, respectively. While genomic analysis and medicinal chemistry have advanced patient stratification with treatments tailored to the genetic profile of a patient's tumor, proteomic targeting has the potential to enhance the therapeutic index of drugs like poly(ADP-ribose) polymerase (PARP) inhibitors. PARP inhibitors in breast and ovarian cancer patients with BRCA1/2 mutations have shown promise. About 10% of the patients who received Olaparib (PARP inhibitor) showed adverse side effects including neutropenia, thrombocytopenia and in some cases resulted in myelodysplastic syndrome, indicating that off-target effects were substantial in these patients. Through proteomic analysis, our lab previously identified plectin, a cytolinker protein that mislocalized onto the cell surface during malignant transformation of healthy ovarian tissue. This cancer specific phenotype allowed us to image pancreatic cancer successfully using plectin targeted peptide (PTP) conjugated to nanoparticles or displayed on capsid protein of adeno-associated virus (AAV) particles. Objective: The goal of this study was to integrate the available pharmacogenomics and proteomic data to develop effective anti-tumor therapies using a targeted drug delivery approach. Methods: Plectin expression and localization in human ovarian tumor specimens were analyzed followed by in vitro confirmation of cell surface plectin localization in healthy and ovarian cancer cell lines. PTP-conjugated liposomes were prepared and their specificity for plectin+ cells was determined in vitro and in vivo. A remote loading method was employed to encapsulate a PARP inhibitor (AZ7379) into liposomes. An ideal buffer exchange method and remote loading conditions were determined based on the amount of lipid and drug recovered at the end of a remote loading process. Finally, in vivo tumor growth studies were performed to determine the efficacy of PTP liposomes in preventing PARP activity in mice bearing OVCAR8 (high grade epithelial ovarian cancer (EOC)) tumors. Results: PTP liposomal AZ7379 delivery not only enhanced PARP inhibition but also resulted in decelerated tumor growth in mice bearing subcutaneous and intraperitoneal OVCAR8 tumors. In mice bearing subcutaneous or intraperitoneal tumors, treatment with PTP liposomes resulted in a 3- and 1.7-fold decrease in tumor volume, respectively, compared to systemic drug treatment. Conclusion: Targeted drug delivery assisted by genomic and proteomic data provides an adaptable model system that can be extended to effectively treat other cancers and diseases.

Ultra-Low-Dose Ultrasound Molecular Imaging for the Detection of Angiogenesis in a Mouse Murine Tumor Model: How Little Can We See?

OBJECTIVES: The objective of this study was to evaluate the minimum microbubble dose for ultrasound molecular imaging to achieve statistically significant detection of angiogenesis in a mouse model. MATERIALS AND METHODS: The preburst minus postburst method was implemented on a Verasonics ultrasound research scanner using a multiframe compounding pulse inversion imaging sequence. Biotinylated lipid (distearoyl phosphatidylcholine-based) microbubbles that were conjugated with antivascular endothelial growth factor 2 (VEGFR2) antibody (MBVEGFR2) or isotype control antibody (MBControl) were injected into mice carrying adenocarcinoma xenografts. Different injection doses ranging from 5 × 10 to 1 × 10 microbubbles per mouse were evaluated to determine the minimum diagnostically effective dose. RESULTS: The proposed imaging sequence was able to achieve statistically significant detection (P < 0.05, n = 5) of VEGFR2 in tumors with a minimum MBVEGFR2 injection dose of only 5 × 10 microbubbles per mouse (distearoyl phosphatidylcholine at 0.053 ng/g mouse body mass). Nonspecific adhesion of MBControl at the same injection dose was negligible. In addition, the targeted contrast ultrasound signal of MBVEGFR2 decreased with lower microbubble doses, whereas nonspecific adhesion of MBControl increased with higher microbubble doses. CONCLUSIONS: The dose of 5 × 10 microbubbles per animal is now the lowest injection dose on record for ultrasound molecular imaging to achieve statistically significant detection of molecular targets in vivo. Findings in this study provide us with further guidance for future developments of clinically translatable ultrasound molecular imaging applications using a lower dose of microbubbles.

The magnetic field dependence of water T1 in tissues.

The magnetic field dependence of the composite (1)H(2)O nuclear magnetic resonance signal T(1) was measured for excised samples of rat liver, muscle, and kidney over the field range from 0.7 to 7 T (35-300 MHz) with a nuclear magnetic resonance spectrometer using sample-shuttle methods. Based on extensive measurements on simpler component systems, the magnetic field dependence of T(1) of all tissues studied are readily fitted at Larmor frequencies above 1 MHz with a simple relaxation equation consisting of three contributions: a power law, A*ω(-0.60) related to the interaction of water with long-lived-protein binding sites, a logarithmic term B*τ(d) *log(1+1/(ωτ(d))(2)) related to water diffusion at macromolecular interfacial regions, and a constant term associated with the high frequency limit of water-spin-lattice relaxation. The parameters A and B include the concentration and surface area dependences respectively. The logarithmic diffusion term becomes significant at high magnetic fields and is consistent with rapid translational dynamics at macromolecular surfaces. The data are fitted well with translational correlation times of approximately 15 ps for human brain white matter, but with a B value three times larger than gray matter tissues. This analysis suggests that the water-surface translational correlation time is approximately three times longer than in gray matter.

Extreme-values statistics and dynamics of water at protein interfaces.

Immobilized proteins present a unique interface with water. The water translational diffusive motions affect the high-frequency dynamics and the nuclear spin-lattice relaxation as with all surfaces; however, rare binding sites for water in protein systems add very low-frequency components to the dynamics spectrum. Water binding sites in protein systems are not identical, thus distributions of free energies and consequent dynamics are expected. (2)H(2)O spin-lattice relaxation rate measurements as a function of magnetic field strength characterize the local rotational fluctuations for protein-bound water molecules. The measurements are sensitive to dynamics down to the kilohertz range. To account for the data, we show that the extreme-values statistics of rare events, i.e., water dynamics in rare binding sites, implies an exponential distribution of activation energies for the strongest binding events. In turn, for an activated dynamical process, the exponential energy distribution leads to a Pareto distribution for the reorientational correlation times and a power law in the Larmor frequency for the (2)H(2)O spin-lattice relaxation rate constants at low field strengths. The most strongly held water molecules escape from rare binding sites in times on the order of microseconds, which interrupts the intramolecular correlations and causes a plateau in the spin-lattice relaxation rate at very low magnetic field strengths. We examine the magnetic relaxation dispersion (MRD) data using two simple but related models: a protein-bound environment for water characterized by a single potential well and a protein-bound environment characterized by a double potential well where the potential functions for the local motions of the bound-state water are of different depth. This analysis is applied to D(2)O deuterium spin-lattice relaxation on cross-linked albumin and lysozyme, which is dominated by the intramolecular relaxation driven by the dynamical modulation of the nuclear electric quadrupole coupling. We also separate the intramolecular from the intermolecular contribution to water proton spin-lattice relaxation by isotope dilution and show that the intramolecular proton data map onto the deuterium relaxation by a scale factor implied by the relative strength of the quadrupole and dipolar couplings. The temperature and pH dependence of the magnetic relaxation dispersion are complex and accounted for by changing only the weighting factors in a superposition of contributions from single-well and double-well contributions. These experiments show that the reorientational dynamics spectrum for water, in and on a protein, is characterized by a strongly asymmetric distribution with a long-time tail that extends at least to microseconds.

Water-proton-spin-lattice-relaxation dispersion of paramagnetic protein solutions.

The paramagnetic contributions to water-proton-spin-lattice relaxation rate constants in protein systems spin-labeled with nitroxide radicals were re-examined. As noted by others, the strength of the dipolar coupling between water protons and the protein-bound nitroxide radical often appears to be larger than physically reasonable when the relaxation is assumed to be controlled by 3-dimensional diffusive processes in the vicinity of the spin label. We examine the effects of the surface in biasing the diffusive exploration of the radical region and derive a relaxation model that incorporates 2-dimensional dynamics at the interfacial layer. However, we find that the local 2-dimensional dynamics changes the shape of the relaxation dispersion profile but does not necessarily reproduce the low-field relaxation efficiency found by experiment. We examine the contributions of long-range dipolar couplings between the paramagnetic center and protein-bound-water molecules and find that the contributions from these several long range couplings may be competitive with translational contributions because the correlation time for global rotation of the protein is approximately 1000 times longer than that for the diffusive motion of water at the interfacial region. As a result the electron-proton dipolar coupling to rare protein-bound-water-molecule protons may be significant for protein systems that accommodate long-lived-water molecules. Although the estimate of local diffusion coefficients is not seriously compromised because it derives from the Larmor frequency dependence, these several contributions confound efforts to fit relaxation data quantitatively with unique models.

Water and backbone dynamics in a hydrated protein.

Rotational immobilization of proteins permits characterization of the internal peptide and water molecule dynamics by magnetic relaxation dispersion spectroscopy. Using different experimental approaches, we have extended measurements of the magnetic field dependence of the proton-spin-lattice-relaxation rate by one decade from 0.01 to 300 MHz for (1)H and showed that the underlying dynamics driving the protein (1)H spin-lattice relaxation is preserved over 4.5 decades in frequency. This extension is critical to understanding the role of (1)H(2)O in the total proton-spin-relaxation process. The fact that the protein-proton-relaxation-dispersion profile is a power law in frequency with constant coefficient and exponent over nearly 5 decades indicates that the characteristics of the native protein structural fluctuations that cause proton nuclear spin-lattice relaxation are remarkably constant over this wide frequency and length-scale interval. Comparison of protein-proton-spin-lattice-relaxation rate constants in protein gels equilibrated with (2)H(2)O rather than (1)H(2)O shows that water protons make an important contribution to the total spin-lattice relaxation in the middle of this frequency range for hydrated proteins because of water molecule dynamics in the time range of tens of ns. This water contribution is with the motion of relatively rare, long-lived, and perhaps buried water molecules constrained by the confinement. The presence of water molecule reorientational dynamics in the tens of ns range that are sufficient to affect the spin-lattice relaxation driven by (1)H dipole-dipole fluctuations should make the local dielectric properties in the protein frequency dependent in a regime relevant to catalytically important kinetic barriers to conformational rearrangements.

Dimensionality of diffusive exploration at the protein interface in solution.

The dynamics of water are critically important to the energies of interaction between proteins and substrates and determine the efficiency of transport at the interface. The magnetic field dependence of the nuclear spin-lattice relaxation rate constant 1/T(1) of water protons provides a direct characterization of water diffusional dynamics at the protein interface. We find that the surface-average translational correlation time is 30-40 ps and the magnetic field dependence of the water proton 1/T(1) is characteristic of two-dimensional diffusion of water in the protein interfacial region. The reduced dimensionality substantially increases the intermolecular re-encounter probability and the efficiency of the surface exploration by the small molecule, water in this case. We propose a comprehensive theory of the translational effects of a small diffusing particle confined in the vicinity of a spherical macromolecule as a function of the relative size of the two particles. We show that the change in the apparent dimensionality of the diffusive exploration is a general result of the small diffusing particle encountering a much larger particle that presents a diffusion barrier. Examination of the effects of the size of the confinement relative to the macromolecule size reveals that the reduced dimensionality characterizing the small-molecule diffusion persists to remarkably small radius ratios. The experimental results on several different proteins in solution support the proposed theoretical model, which may be generalized to other small-particle-large-body systems like vesicles and micelles.

Changes in protein structure and dynamics as a function of hydration from (1)H second moments.

We report the proton second moment obtained directly from the Free Induction Decay (FID) of the NMR signal of variously hydrated bovine serum albumin (BSA) and hen egg white lysozyme (HEWL) and from the width of the NMR Z-spectrum of the cross-linked protein gels of different concentrations. The second moment of the proteins decreases in a continuous stepwise way as a function of increasing water content, which suggests that the structural and dynamical changes occur in small incremental steps. Although the second moment is dominated by the short range distances of nearest neighbors, the changes in the second moment show that the protein structure becomes more open with increasing hydration level. A difference between the apparent liquid content of the sample as found from decomposition of the FID and the analytically determined water content demonstrates that water absorbed in the early stages of hydration is motionally immobilized and magnetically indistinguishable from rigid protein protons while at high hydration levels some protein side-chain protons move rapidly contributing to liquid-like component of the NMR signal.

Relaxation of protons by radicals in rotationally immobilized proteins.

Proton spin-lattice relaxation by paramagnetic centers may be dramatically enhanced if the paramagnetic center is rotationally immobilized in the magnetic field. The details of the relaxation mechanism are different from those appropriate to solutions of paramagnetic relaxation agents. We report here large enhancements in the proton spin-lattice relaxation rate constants associated with organic radicals when the radical system is rigidly connected with a rotationally immobilized macromolecular matrix such as a dry protein or a cross-linked protein gel. The paramagnetic contribution to the protein-proton population is direct and distributed internally among the protein protons by efficient spin diffusion. In the case of a cross-linked-protein gel, the paramagnetic effects are carried to the water spins indirectly by chemical exchange mechanisms involving water molecule exchange with rare long-lived water molecule binding sites on the immobilized protein and proton exchange. The dramatic increase in the efficiency of spin relaxation by organic radicals compared with metal systems at low magnetic field strengths results because the electron relaxation time of the radical is orders of magnitude larger than that for metal systems. This gain in relaxation efficiency provides completely new opportunities for the design of spin-lattice relaxation based contrast agents in magnetic imaging and also provides new ways to examine intramolecular protein dynamics.

Paramagnetic relaxation of protons in rotationally immobilized proteins.

The proton magnetic relaxation dispersion profiles are reported over the proton Larmor frequency range from 0.01 to 30 MHz for cross-linked gels and for the dry lyophilized bovine serum albumin covalently labeled at lysine with diethylenetriaminepentaacetic acid chelates of either Gd(III) or Mn(II) ions. The proton spin-lattice relaxation dispersion for the cross-linked paramagnetic protein gel is accurately represented as a sum of two major relaxation contributions. The diamagnetic term is a power law from the magnetic field dependence of the protein protons. The paramagnetic term is approximately described by the Solomon-Bloembergen-Morgan class of models. However, the paramagnetic relaxation mechanism in the dry lyophilized protein is fundamentally different and we develop a new quantitative description of the dispersion profile. In the dry case, no peak in the proton relaxation dispersion profile is detected from the field dependence of the electron spin relaxation times. The high-field paramagnetic relaxation dispersion is a power law in the Larmor frequency with an exponent of -0.8, which results from modulation of the electron-nuclear coupling by the intramolecular dynamics of the protein which primarily propagates along the primary structure of the protein. The low-field plateau is caused by the interruption of the electron-nuclear spin correlation by electron spin relaxation. This new quantitative description provides a simple approach to the measurement of electron spin-lattice relaxation times in paramagnetic protein systems at room temperature based on the magnetic field dependence of the proton spin-lattice relaxation rate constant.

The aqueous reference for ESR oximetry.

The interaction of molecular oxygen with derivatives of nitroxide EPR spin labels has been investigated using nuclear spin-relaxation spectroscopy in aqueous and nonaqueous solvents. The proton spin-lattice relaxation rate induced by oxygen provides a measure of the local concentration of oxygen, which we find is dependent on solvent. In water, the hydrophobic effect increases the local concentration of oxygen in the nonpolar portions of solute molecules. For nitroxides reduced to the hydroxylamine in aqueous solutions, we find that the local concentration of oxygen is approximately twice that associated with a free diffusion hard sphere limit, while in octane, this effect is absent. These results show that nitroxide based ESR oximetry may suffer a reference concentration shift of order a factor of two if the aqueous nitroxide spectrum or relaxation is used as the reference.

Chromium(III) complexes as intermolecular probes.

Metal ion complexes provide flexible paramagnetic centers that may be used to define intermolecular contacts in a variety of solution phase environments because both the charge and electronic relaxation properties of the complex may be varied. For most complex ions, there are several proton equilibria that may change the effective charge on the complex as a function of pH which in turn affects the efficacy of application for defining the electrostatic surfaces of co-solute molecules. We report here spectrophotometric and nuclear spin relaxation studies on aqueous solutions of chromium(III) complexes of EDTA, DTPA, and bis-amides of both. The effective charges available from these paramagnetic centers range from -3 to +1 and we report the pH ranges over which the effective charge is defined with confidence for application in magnetic relaxation experiments.