NON-INVASIVE METHOD FOR DETECTION OF ENZYME ACTIVITY IN VIVO, SUBSTRATES AND A DEVICE THEREFORE

Abstract

Methods for the detection of enzymatic activity, in particular, to in vivo methods. A non-invasive method for in vivo enzyme activity detection, such as activity of proteinases, to substrates specifically developed for these methods and to a device detecting product formation of the enzyme to be tested based on determination of signals produced by the substrates and/or its products.

Claims

1-15. (canceled)

16. A responsive microbubble comprising a substrate for an enzyme of interest and wherein the microbubble alters its interaction with ultrasound waves in response to substrate conversion by the enzyme of interest.

17. The responsive microbubble according to claim 16 made of lipids, phospholipids, surfactants, proteins, biocompatible synthetic polymers, or combinations thereof and being coupled to substrates for an enzyme of interest.

18. The responsive microbubble according to claim 16 for use in a method of diagnosis in vivo of a disease associated with or caused by an altered activity of the enzyme of interest.

19. The responsive microbubble according to claim 16, wherein the microbubbles may be suitable as an ultrasound contrast agent showing enzyme activity.

20. The responsive microbubble according to claim 16, wherein the microbubbles have a size have a size of 0.5-4 micrometers in diameter.

21. The responsive microbubble according to claim 16 being an aggregate of molecules comprising a shell and a gas core and wherein the substrate for an enzyme of interest is part of the shell or the shell is covered by the substrate for an enzyme of interest.

22. The responsive microbubble according to claim 21, wherein cleavage of the substrate results in an observable change in ultrasonic acoustic properties due to a change of the mechanical properties of the microbubble shell.

23. A method for detection of enzymatic activity comprising the following steps: a) providing a sample containing an enzyme of interest b) adding microbubbles as defined in claim 16 to the sample, and c) determining enzyme activity by measurement of altered ultrasound signal of the microbubbles

Description

BRIEF DESCRIPTION OF THE INVENTION

[0089] The subject matter of the invention will be explained in more detail in the following text with reference to exemplary embodiments which are illustrated in the attached drawings, in which:

[0090] FIG. 1 illustrates the principle of the inventive method.

[0091] FIG. 2 further illustrates the principle of the inventive method, including determination of inductive detection using a pulsed field magnetometer.

[0092] FIG. 3 illustrates a magnetometer according to the invention, wherein the pulse coil, sense coil and compensation coil consist of electrically conductive spirals patterned in the layers of a printed circuit board or other planar geometry to which conductive features can be added with high resolution and geometric symmetry. The simplified schematic of the detection circuit shows that the sense and compensation coil are inductively coupled to the applied field with opposite polarity. A variable resistor is used as indicated to finely adjust the cancellation of the sense and compensation coils or aid in background subtraction and the signal is amplified for detection. Schematics of possible pulse circuits are also shown, with the one on the left based on a silicon controlled rectifier and the one on the right based on a gas discharge tube. Depending on the particular components and values used, different types of pulse forms are possible, several of which have been simulated and are shown at the bottom of the figure.

[0093] FIG. 4 shows an embodiment of the invention wherein a magnetostatic gating field is imposed by permanent magnets or electromagnets to produce spatially selective measurement of magnetic material. As an illustrative example, field lines are shown for a finite element simulation of two oppositely aligned bar magnets, and the expected field magnitude is plotted at positions along a dashed line. Two positions are considered, represented by two small vials at different positions on this line, however this strategy could equivalently be employed to isolate signal from a subvolume of a larger shape, such as a target of interest in a patient. Below, the plot on the left shows a simulated pulsed field versus time and a representative magnetization versus field curve for chains RIMAs produced from a 2D Monte Carlo simulation. At bottom center, the expected magnetization versus time is shown for the same RIMAs at these two positions. To the right, the resulting induced voltage signals show that a signal is only expected to arise from the RIMAs located at the point of vanishing magnitude of the field.

[0094] FIG. 5 illustrates variations on the arrangement of pulse, sense and compensation coils, some of which may be used to achieve increased penetration depth. In one variation, the sense and compensation coils are geometric mirror images and the pulsed field is symmetric, whereas in another the pulsed field is antisymmetric and the sense and compensation coils are identical. An antisymmetric pulse coil geometry may offer the advantage of reducing the inductance of the pulse coil, allowing for higher dH/dt values, and thus greater detection signal. Preferred pulse coil designs have few turns (as little as a single turn) to limit inductance and maximize dH/dt. Sense and compensation coils could consist of tens to hundreds of spiral turns embedded in a multilayer circuit board. While it is advantageous to have as many turns as possible in the sense coil to increase signal magnitude, the influence of parasitic capacitance limits the total number of allowable turns. Thus, the exact number of turns in the sense and compensation coils would be adapted for each setup. A concept for coupling to remote targets by moving the sense and compensation coils out of the plane of the pulse coil is shown at middle left. At middle right, a more general example is shown in which the compensation coil is reduced in diameter, but contains a different number of windings (40 times more than the sense coil in the example shown) and finely adjustable distance ensures cancellation of the voltage from the pulsed field. Such an arrangement could make remote measurement more feasible by permitting a large measurement coil that is as close to the target as the pulse coil, while reducing coupling of the compensation coil with the sample due to its smaller diameter.

[0095] FIG. 6 illustrates examples of voltage signals and their dependence of the conformation of assemblies from magnetic nanoparticles and pulse type. In 6A, at the top, two simulated pulsed fields are shown, an asymmetrically damped pulse and a monophasic pulse. At left, 2D Monte Carlo simulations of magnetization versus field curves for two different types of assemblies (chain and ring) are shown, along with the expected curves for their fully disassembled state. In the center of the figure, expected voltage signal versus time plots are shown for the different assemblies, paired with the two pulse types shown at the top. In 6B, alternative methods for detecting responses are sketched, including the observation of magnetic remanence or a measurable shift in complex susceptibility measured from a decaying oscillatory pulse.

[0096] FIG. 7 illustrates the basic concept of physical background subtraction possible within the present invention, respectively using the magnetometer of the invention. At top, the magnetometer is shown with no sample. Consistent with the concept of cancellation using the compensation coil and fine adjustment with the amplification circuit, no signal arises in this state. When a sample is introduced, its signal is formed from the superposition of two main components: diamagnetic contributions from the vial and solution, and the superparamagnetic signal of the magnetic assemblies. In the limit of small quantities of magnetic assemblies, the diamagnetic signal is expected to be considerably larger. By introducing a blank sample into the compensation coil, which does not contain magnetic assemblies, this comparatively large diamagnetic signal is physically subtracted, isolating the signal from the magnetic assemblies.

[0097] FIG. 8 shows the basic principle of responsive aggregates in the results of 2D Monte Carlo models of equilibrium magnetization versus field curves for chains and rings.

[0098] FIG. 9 shows a variety of possible structures for responsive magnetic nanoassemblies.

[0099] FIG. 10 A illustrates an example of chemical functionalization resulting in crosslinking of magnetic nanoparticles including a cleavable substrate suitable for the method of the invention. [0100] B shows examples of functional groups suitable for bioconjugation of cleavable substrates to magnetic nanoparticles suitable for the method of the invention.

[0101] FIG. 11 illustrates a shift of resonance frequency after cleavage of a substrate from a microbubble.

[0102] FIG. 12 illustrates exemplarily how cleavage of a substrate can result in changed ultrasound signals. Shown is, that the cleavage causes release of an auxiliary chain.

[0103] FIG. 13 illustrates exemplarily how cleavage of a substrate can result in changed ultrasound signals. Shown is that the cleavage causes an altered crosslinking, where auxiliary chains are de-crosslinked upon cleavage of the linker.

[0104] FIG. 14 shows possible arrangements of a cleavable linker and auxiliary components as parts of a microbubble shell.

[0105] FIG. 15 shows possible arrangements of a cleavable linker and auxiliary components as parts of a microbubble shell.

[0106] FIG. 16 illustrates determination of microbubble resonance frequency using a custom-made attenuation measurement system.

[0107] FIG. 17 illustrates inductive detection of magnetic nanoparticles using a ferrite electromagnet.

[0108] FIG. 18 illustrates inductive detection of magnetic nanoparticles that have been crosslinked using an LCR meter.

[0109] FIG. 19 depicts a possible layout of a pulsed field magnetometer, accompanied by an analysis of the order of magnitude of the induced voltage signals as a function of particle concentration.

[0110] FIG. 20 shows acoustically measured stiffening effects of the microbubble shell as a function of crosslinker concentration.

[0111] FIG. 21 depicts an ultrasound phantom used to collect ultrasound images of crosslinked and non-crosslinked microbubbles showing a stiffness-dependent change in brightness.

DETAILED DESCRIPTION OF THE INVENTION

[0112] The reference symbols used in the drawings, and their meanings, are listed in summary form in the list of reference symbols. In principle, identical parts are provided with the same reference symbols in the figures.

EXAMPLES

[0113] All explanations of specific embodiments are included for illustrative purposes and not intended to be limiting. Reference is made to the accompanying figures in the application.

Responsive Magnetic Nanoassemblies and an Accompanying Device that Detects them Via Magnetic Induction

Example of Synthesis and Application:

Synthesis of Iron and Cobalt Oleate

[0114] The synthesis of magnetic nanoparticles is well known to the art and many practical methods exist. The inventors used a previously described method based on metal-oleate decomposition, with slight modifications (Chen et al., DOI: 10.1021/acs.nanolett.5b04761, Nano Lett. 2016, 16, 1345-1351). 30 mmol of iron chloride hexahydrate are dissolved in 50 mL ultrapure (milliQ®) water and thoroughly degassed under vacuum. Atop this solution, the following are added sequentially: sodium oleate (90 mmol), hexane (100 mL), and finally ethanol (50 mL). The reaction is brought to reflux for 30 minutes under nitrogen and gentle stirring. Upon cooling, the hexane rich phase is isolated and washed 3 times with ultrapure (milliQ®) water (100 mL per wash) and finally kept at 135° C. under atmosphere while stirring for 12 to 16 hours to remove volatiles. The final product is a dark brown, extremely viscous liquid. For cobalt doped magnetic nanoparticles, the only departure from the described procedure is a modification of the starting components. For a Co:Fe ratio of 1:11 (i.e. for Co.sub.xFe.sub.3-xO.sub.4, x=0.25), 27.5 mmol of iron chloride hexahydrate, 2.5 mmol anhydrous cobalt chloride, and 87.5 mmol sodium oleate are combined.

Synthesis of Magnetic Nanoparticles

[0115] In brief, 4.51 g of the iron oleate or cobalt/iron oleate product described above is combined with 90% oleic acid and 15 mL of a co-solvent mixture of 1-octadecene and dibenzyl ether. The size of the particles is controlled by varying the amount of oleic acid, ratio of the co-solvents, and temperature ramp rate. Typically, a 1 to 2 dibenzyl ether to 1-octadecene ratio is used. The quantity of oleic acid added ranges from 1 mmol to 15 mmol. In each reaction, this mixture is degassed under stirring at 90° C. for 30 minutes and an ultimate pressure of 0.1 mbar or less. The mixture is then heated rapidly to 200° C. under nitrogen, typically ramped at a typical rate of 3.33° C./min to its reflux temperature, and maintained there for 30 minutes. Reflux temperature varies between approximately 290° C. and 330° C. depending on the composition of the mixture and on nitrogen pressure.

[0116] After synthesis, the particle-containing mixture is collected and washed from the flask using approximately 5 mL hexane. The total solution is brought to 45 mL with ethanol to help precipitate the particles and centrifuged at an RCF of 9000 g for 10 minutes. After decanting, the pellet is redispersed under vortexing and sonication in 20 mL hexane with 1% oleic acid by volume, followed by the addition of 25 mL of ethanol, further vortexing, and centrifugation. This process is repeated 4 to 5 times. Chloroform is used for the final resuspension of the pellet and this nanoparticle stock solution is stored at 4° C.

Synthesis of PEG-PMAO and Phase Transfer

[0117] Synthesis of the amphiphilic polymer PEG-PMAO has been reported in many sources as an effective and biocompatible means to phase transfer nanoparticles (e.g., Yu et al., J. Am. Chem. Soc. 2007129102871-2879). In a preferred method, 0.50 g of PMAO (1.4 mmol anhydride groups), 27.10 g of Jeffamine PEG diamine (14.3 mmol), and 0.87 g triethylamine (8.4 mmol) were combined in 175 mL dichloromethane under rapid stirring and allow the solution to react for 1 hour. The solution is then heated under nitrogen to its boiling point with the dichloromethane collected for disposal. Finally, the polymer rich residual solution is dried in vacuum overnight and the resulting powder is collected and stored under nitrogen.

[0118] For phase transfer, the typical method proceeds as follows: 1 mL of chloroform solution containing MNPs at a concentration of 1 mg of metal ion content per mL is prepared by diluting the stock solution with chloroform. 1.0 g of the solid product described above, containing both PEG-PMAO and excess unbound PEG is dissolved in 3 mL of chloroform. These solutions are bath sonicated separately for 15 minutes before mixing, and the combined mixture is sonicated for 30 minutes. The solution is then transferred to a large glass petri dish and dried overnight under vacuum. The polymer film containing evenly distributed nanoparticles is then dissolved in TRIS-acetate-EDTA buffer, sonicated for 15 minutes, and eluted through a 200 nm filter. Excess PEG and PEG-PMAO is washed away and the particles are transferred to their desired reaction buffers using MACS columns or other magnetic separation techniques and afterward sonicated for 15 minutes to ensure proper redispersion.

Synthesis of Flux Closure IMAs and RIMAs

[0119] Upon phase transfer, the polymer surfaces of the particles contain free amine groups. To functionalize them with alkyne and azide moieties, the particles are reacted with propargyl-N-hydroxysuccinimidyl ester and azidoacetic acid NHS ester, respectively. For these reactions, the particles are transferred into 0.1 M sodium phosphate buffer with a pH value of 7.9. Approximately 1 mg of the desired NHS ester, enough to provide a substantial molar excess to the estimated available amine groups, is dissolved in a volume of DMSO equal to 10% of the total volume of the anticipated reaction mixture, which ranged from 200 μL to 1000 μL. Immediately after dissolution, the buffer containing the magnetic nanoparticles is combined and mixed thoroughly. After 4 hours at room temperature, dialysis or magnetic separation is used to remove excess click reagents and transfer the particles to 0.2M triethylammonium acetate buffer adjusted to a pH value of 7.0, and the particles are sonicated for 15 minutes to ensure proper redispersion.

[0120] To assemble the core shell structures, alkyne functionalized particles (e.g., 20 nm iron oxide) are combined with a 10-fold or greater excess of azide functionalized particles (e.g., 7.5 nm cobalt doped iron oxide). The following components are added to these solutions to initiate the click reaction: DMSO, a stock solution with 10 mM TBTA and copper (II) sulfate in 55% DMSO, and L-ascorbic acid freshly dissolved at 5 mM in water. The final reaction mixture contained 50% DMSO and overall concentrations of 0.1 M triethylammonium acetate buffer, 0.5 mM TBTA-Cu complex, and 0.5 mM ascorbic acid. Nitrogen is bubbled through the mixture and the vial containing the reaction mixture is flushed with nitrogen, sealed, and kept under agitation overnight.

[0121] To separate the assemblies from excess unbound azide-functionalized nanoparticles, magnetic separation is employed, for instance using MACS columns with a water rinse step, a method that retains primarily the assemblies due to their larger magnetic moments, and elutes the smaller particles. After magnetic separation, the particles are sonicated for 15 minutes to ensure proper dispersion. To produce photo-responsive magnetic aggregates of this type, the NHS esters indicated above are substituted with photocleavable analogues containing the same end groups.

Synthesis of Flux Reinforcement Particles

[0122] In one method, particles phase transferred as described above are similarly coated in azide or alkyne groups and placed in click chemistry buffers. This time, they are combined with a crosslinker containing endgroups complimentary to their surface coatings such as PEG bis(azide), PEG bis(alkyne) or multi-arm variants of these. In a volume of 200 μL, and a concentration on the order of 100 μg to 1 mg per mL, the particles are placed in a uniform 0.8 T field. The concentration and time necessary for the formation of well controlled chains has to be determined on a case by case basis, but upon final combination of the TBTA-Cu catalyst, crosslinking is initiated and the structures are held in the assemblies they formed under the influence of the field.

[0123] Alternatively, flux reinforcement structures can be formed through the use of microfluidic device with permanent magnets incorporated to generate strong fields and gradients tangential to a 100 m diameter PTFE tube. The crosslinking solution and particles are combined just before they enter the magnet, fed by syringe pumps at the rates ranging from 0.1 μL to 10s of L per minute.

Incorporation of Peptides into Flux Closure and Flux Reinforcement Particles

[0124] In order to incorporate peptides, the above methodology requires modification. Rather than functionalizing the surface of the particles with click reactive moieties, the same procedure is adapted to 3-Mercaptopropanyl-N-hydroxysuccinimide ester by using a 0.1 M sodium phosphate buffer of the same pH, but also incorporating 10 mM EDTA and bubbling nitrogen through the solution beforehand. The reaction of this NHS ester provides thiol groups on the polymer surface of the nanoparticles. After magnetic separation, transfer to a 0.1 M sodium phosphate buffer of neutral pH with 10 mM EDTA, and sonication, 1 mg of a customized peptide is dissolved in a volume of DMSO sufficient for 10% of the total reaction volume and combined with the particles for 4 hours. The customized peptide includes a maleimide linker on one end that binds it selectively to the surface of the particles, as well as an alkyne containing unnatural amino acid at the opposite end. Synthesis of flux closure particles can then proceed as described above using click chemistry to affix particles directly functionalized with azide groups. Synthesis of the flux reinforcement particles proceeds as described above with the application of a uniform field and the use of a bis-azide, bis-alkyne, or multi arm crosslinker terminated with click groups.

Use of Magnetic Particles to Detect Proteolytic Activity in Ex Vivo Samples

[0125] A trained medical professional is able to use established methods to collect a small liquid sample (less than 100 μL), e.g., of synovial fluid, which may or may not contain proteases of interest in their active form as a biomarker relevant to some disease or risk of disease. The ex vivo detection setup would be miniaturized so that the minimal dilution of the ex vivo sample would be required. An identical sample container, containing a physiological buffer, would be placed in the compensation coil. A general measuring procedure would begin by recording pulsed measurements using the sample without magnetic particles in the measurement coil and a blank in the compensation coil and either adjusting values of components in the amplification circuit or determining relevant values for digital background subtraction. The goal of this initial background calibration would be to eliminate background signal as fully as possible. A small quantity of particles would then be added to the physiological solution, in a concentration depending on the predetermined detection threshold of the setup, but likely ranging from 1 to 100 μg of material. The M vs H curve, or relevant property thereof, would then be inductively detected repeatedly for pulses spaced over minutes or hours, in effect measuring the time dependence of the change in the aggregation state of the particles. In each case, these curves could be compared against a control sample of particles (containing only physiological buffer) and a standard curve for known concentrations of the protease of interest to estimate the concentration of active protease. It would be possible to design a device in which the circuitry for pulse generation was shared such that these calibration curves could be run alongside measurement of the sample.

Use of the Magnetic Particles In Vivo

[0126] The magnetic particles with predetermined characteristics can be introduced into the body, either systemically in cases where accumulation in the site of interest is expected, or locally as in cases such as implant coatings or in situ analysis of synovial fluid. The necessary concentrations vary widely depending on the geometry of the detection apparatus, but are restricted to a total dose of mg scale or lower. For measurement, the site of interest should be placed in or near the pulse and detection coils. If necessary, background subtraction calibration could be performed on an analogous body part not expected to exhibit a signal from the used particles, such as a second hand or location of the body remote from the site of injection. With the pulse and detection coils in position over the site of interest, a magnetostatic selection field could additionally be applied to restrict inductively detectable signal to a point of vanishing magnitude. Repeated measurements over time indicate the aggregation state of the particles, and their rate of disassembly can be used to infer the activity of the protease of interest. It is worth noting that if properties of the M vs H curve such as saturation-normalized low field susceptibility are used, the relevant features are concentration independent. I.e., provided that a sufficient quantity of particles were being detected to provide a measurable signal, changes in their concentration through slow diffusion or other processes do not necessarily impact the ability of the device to measure a signal indicating their state of assembly.

Responsive Microbubbles with Acoustic Readout

Example of Synthesis and Application:

Synthesis of Microbubbles

[0127] The synthesis of lipid-based microbubbles is well known to the art and many practical methods exist. The inventors used a previously described method were primary and secondary lipids are added to a 25 mL borosilicate glass vial according to the ratios mentioned in the “Lipid Molar Ratios used for Microbubble Synthesis”-section and dissolved in chloroform. Chloroform is evaporated via a continuous stream of nitrogen to form a lipid thin film at the vial bottom. Remaining chloroform is then removed via desiccation overnight under house vacuum. The resulting lipid film is rehydrated using phosphate buffered saline (pH 7.4) to yield a final total lipid concentration of either 2 mg/mL (in case of sonication or shaking based synthesis) or 10 mg/mL (in case of flow focusing synthesis). The solution is stirred at 75° C. for at least 1 h and then bath sonicated for 20 min at RT. This process should yield a liposome solution with a mean liposome size of approximately 100 nm (measured by DLS). The formation of microbubbles is subsequently carried out with one of the following approaches:

[0128] 1) Synthesis of Microbubbles by Sonication [0129] The liposome solution can be heated above the phase transition temperature (approximately 65° C.) and then probe sonicated (3 mm microtip, Branson Sonifier) for 10 s at 70% amplitude under a continuous flow of perfluorobutane gas (PFB). After sonication, the white microbubble solution needs to be rapidly cooled below the phase transition temperature by placing the vial in an ice bath.

[0130] 2) Synthesis of Microbubbles by Shaking [0131] In this methods, a 2 mL aliquot of the liposome solution is transferred to a borosilicate vial (3.75 mL, 17 mm, septum lid) and heated above phase transition temperature (approximately 65° C.). A 27-gauge needle, attached to a T-valve (connected to house vacuum and perfluorocarbon gas) needs to be inserted through the septum and the vial head space is exchanged five times with PFB. The vial is fixed in an amalgamator device (CapMix ESPE, 3M) and vigorously shaken for 30 s. After shaking, the white microbubble solution is rapidly cooled below the phase transition temperature by placing the vial in an ice bath.

[0132] 3) Synthesis of Microbubbles by Flow Focusing [0133] As reported previously by Segers et al. (DOI: 10.1021/acs.langmuir.6b00616 Langmuir 2016, 32, 3937-3944), a flow focusing microfluidic device can be utilized to synthesize monodisperse solutions of microbubbles. Here, the liposome solution is transferred to a 1 mL Hamilton syringe and connected to the liquid inlet of the mentioned microfluidic device using PTFE tubing. A syringe pump is used to control the liquid flow rate. The PFB gas pressure also needs to be controlled and connected to the gas inlet of the microfluidic chip by PTFE tubing. Depending on the desired size of microbubbles, liquid flow rate and gas pressure are adjusted based on empirical testing and verification for the given device.
Lipid Molar Ratios used for Microbubble Synthesis

[0134] a) Synthesis of Poly(Acrylic Acid) Microbubbles [0135] Lipid A: DSPC [0136] Lipid B: DSPE-PAA (synthesis see below) [0137] Ratio of Lipid A:Lipid B=170:1

[0138] b) Synthesis of DSPE-mPEG2k and DSPE-mPEG5k Microbubbles [0139] Lipid A: DSPC [0140] Lipid B: DSPE-mPEG2k or DSPE-mPEG5k [0141] Ratio of Lipid A:Lipid B=99:1 for mushroom regime, 9:1 for brush regime, 3:1 for dense brush regime

[0142] c) Synthesis of Microbubbles with Streptavidin Coating [0143] Lipid A: DSPC [0144] Lipid B: DSPE-PEG2k-biotin [0145] Ratio of Lipid A:Lipid B=9:1 [0146] After microbubble synthesis, streptavidin (excess to cover the microbubble surface, A.sub.streptavidin=19.63 nm.sup.2) is added to the microbubble solution and incubated for 1 h at RT to form a crystalline streptavidin protein shell.

[0147] d) Synthesis of Microbubbles with Interconnected 4-Arm PEG DSPE [0148] Lipid A: DSPC [0149] Lipid B: 4-arm-PEG-DSPE 20 kDa (4-Arm PEG-DSPE is a 4-Arm PEG with each of the four PEG arms terminated with a DSPE). It was purchased from Creative PEG Works. [0150] Ratio of Lipid A:Lipid B=36:1

Synthesis of DSPE-PAA

[0151] Following earlier protocols by Nakatsuka et al. (DOI: 10.1002/adma.201102677, Adv. Mater. 2011, 23, 4908-4912), a 50 wt % aqueous solution of poly(acrylic acid) (PAA, 200 mg, MW 5000 g/mol) is mixed with dimethylsulfoxide (DMSO, 4 mL) in a 25 mL borosilicate glass vial with a PTFE lid equipped with a magnetic stirbar. If necessary, 1 M aqueous hydrochloric acid solution is titrated dropwise to enhance solubility of the polymer. N-hydroxysuccinimide (NHS, 46 mg, 0.40 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 38 mg, 0.20 mmol) are added and dissolved by stirring the solution for 30 min at room temperature. In a separate vial, a 5 mM solution of 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (DSPE, 15 mg, 0.02 mmol) in chloroform (4 mL) is prepared and mixed with trimethylamine (TEA, 1 mL). The mixture needs to be incubated at 60° C. until it appears as a clear solution. The two solutions are then combined under stirring, heated to 60° C. and finally allowed to react for 1 h. After cooling to room temperature, the chloroform and the TEA are removed via a continuous stream of nitrogen and transferred to a dialysis tube (MWCO 1 kDa). Dialysis was preferably performed during 2 days against distilled water with 8 changes of water. The dialyte is then lyophilized to obtain a white powder.

Crosslinking of Poly(Acrylic Acid) Emulsifier Microbubbles

[0152] In order to create a crosslinked network on the surface of the microbubble, and therefore turn of the microbubble resonance (“OFF” mode), post processing of the microbubble in the “ON” mode needs to be carried out. After poly(acrylic acid) microbubble synthesis, excess of 4-arm PEG-amine (2 kDa, Creative PEG Works) and EDC are added to the microbubble solution. The solution is then transferred via a syringe to a septum lid vial filled with PFB gas and equipped with a venting needle. The solution is then set aside to react at RT overnight. To separate the microbubbles from unreacted material, the solution is transferred to a syringe and the microbubble solution is washed five times with PBS by centrifugation at 300 g for 10 min.

Incorporation of Peptides into Microbubble Shells to Produce Responsive Microbubbles

[0153] In order to achieve microbubbles that are functionally responsive to proteolytic activity, selectively cleavable peptides need to be incorporated into the shell of the microbubble. Therefore, the above described methodologies require modifications.

a) Synthesis of a Responsive 4-armPEG-Peptide Crosslinker

[0154] The selectively cleavable peptide needed for this synthesis has an unnatural amino acid with an alkyne group (at the C terminus). The responsive crosslinker can be synthesized for example by combining a 4-arm PEG-azide linker with the peptide via click chemistry. For this, the 4-arm PEG-azide is combined with an eight-fold molar excess of the peptide. The following components are added to initiate the click reaction: DMSO, a stock solution with 10 mM TBTA and copper (II) sulfate in 55% DMSO, and L-ascorbic acid freshly dissolved at 5 mM in water. The final reaction mixture contained 50% DMSO and overall concentrations of 0.1 M triethylammonium acetate buffer, 0.5 mM TBTA-Cu complex, and 0.5 mM ascorbic acid. Nitrogen is bubbled through the mixture and the vial containing the reaction mixture is flushed with nitrogen, sealed, and kept under agitation overnight.

[0155] To separate unreacted material from the responsive crosslinker, the solution is transferred to a dialysis tube (MWCO 3.5 kDa). Dialysis is performed during 2 days against distilled water with 4 changes of water. The dialyte is lyophilized to obtain a white powder. MALDI-TOF can be used to verify if the functionalization of all 4 arms was successful.

[0156] Finally, the formation of responsive, crosslinked microbubbles can be performed in the same way as described above for the crosslinking of non-responsive microbubbles.

Synthesis of a Responsive DSPE-PEG2k-Peptide-PEG3k Emulsifier Component

[0157] The selectively cleavable peptide needed for this synthesis has an additional cysteine at the N-terminus and an unnatural amino acid with an alkyne group at the C-terminus.

[0158] In the first step of this two-step functionalization reaction the peptide is attached to a DSPE-PEG2k via sulfhydryl-reactive crosslinker chemistry. For example, DSPE-PEG2k-MAL is reacted with the cysteine of the peptide at the N-terminus with a 3:2 molar ratio in DMSO. The reaction is let run under nitrogen at room temperature overnight. To separate unreacted components from the product, the reaction solution is transferred into a dialysis tube (MWCO 3.5 kDa). Dialysis is performed during two days with four changes of water. The dialyte is lyophilized to obtain a white powder.

[0159] In the second step of this synthesis, DSPE-PEG2k-peptide is attached to a PEG3k-alkyne via click chemistry. For this, DSPE-PEG2k-peptide is combined with a two-fold molar excess of PEG3k-azide. Click chemistry is then performed as described above in section a). Unreacted materials are separated using a dialysis tube (MWCO 6-8 kDa).

[0160] Finally, responsive microbubbles can be synthesized as described above, but containing the responsive emulsifier component instead of the non-responsive DSPE-PEG2k or DPES-PEG5k.

Synthesis of Responsive DSPE-PEG2k-Peptide-Biotin

[0161] The selectively cleavable peptide needed for this synthesis has an additional cysteine at the N-terminus and a biotin functionalization at the C-terminus. Similarly, as described in section b), DSPE-PEG2k-MAL is reacted with the cysteine of the peptide via sulfhydryl-reactive crosslinker chemistry with a 3:2 molar ratio in DMSO. The reaction is let run under nitrogen at room temperature overnight. To separate unreacted components from the product, the reaction solution is transferred into a dialysis tube (MWCO 3.5 kDa). Dialysis is performed during two days with four changes of water. The dialyte is lyophilized to obtain a white powder.

[0162] Finally, responsive microbubbles with a streptavidin coating could be synthesized as described above, but containing the responsive DSPE-PEG2k-peptide-biotin instead of the non-responsive DSPE-PEG2k-biotin.

Synthesis of Responsive Interconnected 4-Arm PEG-Peptide-PEG-DSPE

[0163] The selectively cleavable peptide needed for this synthesis has an additional cysteine at the N-terminus and an unnatural amino acid with an alkyne group at the C-terminus

[0164] The first step of this two-step synthesis is similar to the one described in section a). For example, a 4-arm PEG-azide crosslinker can be combined with the peptide via click chemistry as described above. To separate unreacted material from the product, the solution is transferred to a dialysis tube (MWCO 3.5 kDa). Dialysis is performed during 2 days against distilled water with four changes of water. The dialyte is lyophilized to obtain a white powder. It is recommended to use MALDI-TOF to verify whether the functionalization of all 4 arms was successful.

[0165] For the second step of the synthesis 4-armPEG-peptide is combined with DSPE-PEG2k-MAL via sulfhydryl-reactive crosslinker chemistry with a 1:8 molar ratio in DMSO. The reaction is let run under nitrogen at room temperature overnight. To separate unreacted components from the product, the reaction solution is transferred into a dialysis tube (MWCO 6-8 kDa). Dialysis is performed during two days with four changes of water. The dialyte is lyophilized to obtain a white powder.

[0166] Finally, responsive microbubbles with a responsive interconnected shell can be synthesized as described above, but containing the responsive 4-arm PEG-peptide-PEG-DSPE instead of the non-responsive 4-arm PEG-DSPE.

Measurements and Analysis of Microbubble Resonance Spectra

[0167] After synthesis, microbubble resonance frequency can be determined by any method known in the art. For our invention, a custom-made attenuation measurement system is used. The set up consists of 250 mL glass chamber to which a transmitting and a receiving transducer are attached coaxially (FIG. 16). The intensity of the received signal is collected between 0.1 and 15 MHz. Intensities recorded from a suspension of microbubbles are compared to reference measurements and according to the following equation it is possible to calculate the attenuation coefficient. The resonance frequency of the microbubble suspension is then equal to the driving frequency at which the attenuation coefficient is the highest.

Use of Responsive Microbubbles to Detect Proteolytic Activity in Ex Vivo Samples

[0168] A general measuring procedure can begin by diluting a small sample of synovial fluid (less than 100 μL) in PBS and transferring the solution to the ultrasound analysis chamber. First the acoustic background response of the solution needs to be measured by sending half-gaussian tapered sinusoidal waves at frequencies ranging from 0.1 to 15 MHz and with a total length of 12 μs through the measuring chamber in the absence of the responsive microbubbles. Following, an appropriate amount of microbubbles is added to the chamber. Appropriate concentrations of microbubbles need to be balanced between sufficient sensitivity for a given sample and attenuation of the ultrasound signal with increasing microbubble concentration. The measurement is repeated with the same signal as used for the background measurement. By subtracting the received signal intensities of the solution in presence of the microbubbles from the background, the resonance frequency of the microbubbles is determined. The sample with the responsive microbubbles should be incubated for several minutes and while repeated measurements are taken. In case of proteolytic activity, a shift in resonance frequency of the microbubbles due to changes in mechanical properties of the microbubble shell will be observed. In principle, it is also possible to determine the rate or reaction and therefore the concentration of the protease present in the chamber.

Use of Responsive Microbubbles In Vivo

[0169] For example, for diagnostics of inflammatory diseases of the joints, the microbubbles can be introduced locally into the synovial capsule of the affected joint via an intraarticular injection. The necessary concentration of microbubbles depends on the joint under examination and the distance of the ultrasound probe to the joint. After injection, the microbubbles can be detected using a clinical ultrasound imaging device. Using signal post-processing the frequency response of the microbubbles can be analyzed and visually encoded for image construction. A change in resonance frequency is visually detectable via a shift in contrast or color in respective areas of the ultrasound image. In some cases, the changes in mechanical shell properties should be so drastic that, proteolytic activity could also be visualized with harmonic imaging. For example, microbubbles with a responsive streptavidin coating are expected to be stiff enough to completely turn off harmonic response. After cleavage of the streptavidin, the shell softens and therefore an onset of harmonic responses should be observable. Similar effects are expected from the PAA-emulsifier microbubbles.

Further Examples and Embodiments

[0170] An example of an embodiment of inductive detection of the aggregation state of aqueously dispersed nanoparticles is shown in FIG. 17. The gapped ferrite electromagnet can be used to generate a pulsed magnetic field of variable duration and amplitude using the circuit depicted. Plots are shown of field versus time, measured by integrating inductively generated voltages. The pulse duration and the maximum field amplitude can be adjusted by varying the value of the pulse capacitor and the charge voltage, respectively. A continuous alternating field is another driving condition suitable for inductive detection, and representative voltage signals are shown for an uncompensated field pickup and nanoparticle sample driven continuously at 9 Hz and 5 mT. The nanoparticle sample consists of cobalt doped iron oxide nanoparticles and has a concentration of less than 1 mgFe/mL. Its resultant voltage signal is amplified in two stages and averaged 16 times. The frequency dependence of the nonlinear contribution to the sample signal (extracted from the third harmonic of representative traces) is shown for three samples of magnetic nanoparticles identical except for exposure to different crosslinking conditions. “Ctrl” denotes the control sample that was exposed to a constant field overnight without chemical crosslinking. “SCM” and “SG” refer to 8 arm polyethylene glycol succinimidyl carboxyl methyl ester and 8 arm polyethylene glycol succinimidyl glutarate ester, respectively. Both types of crosslinker have been reacted to propargylamine and used to crosslink azide-functionalized particles with a Cu-catalyzed click reaction under continuous exposure to a 15 mT uniform magnetic field.

[0171] Another suitable scheme for inductive detection of nanoparticle assemblies is shown in FIG. 18 using the same assemblies as those represented in FIG. 17. In brief, this approach consists of using an LCR meter or similar variable frequency 4-point probe system to supply a minute driving current to an inductor and measure impedance. The inductor is suitably designed so that the susceptibility of the sample detectably modifies its inductance. The “coupling factor” shown in FIG. 18 is defined as the fractional change in inductance (or magnetostatic field energy) divided by the fractional increase in the magnetic permeability of the sample. This quantity is shown for a solenoid surrounding a 5 mm sample tube, as calculated from finite element simulations, both for the overall coil and for each additional layer of wire. Dynamic light scattering data demonstrate the altered aggregation state of the previously mentioned crosslinked particle samples, and this difference is shown to be detectable via fractional changes in inductance.

[0172] The capacitive discharge circuit shown in FIG. 19 is a simplified representation of a capacitive discharge circuit for a rapidly pulsed field embodiment that includes a circuit layout. The pulse coil has a small number of turns to limit the inductance of the coil, and ensure a rapid rise rate of the pulsed field, dH/dt. To dampen the oscillation of the field without reducing peak amplitude, power film resistors in series with silicon carbide Schottky diodes are envisioned to draw current only when the current in the pulse coil is dropping. The result is an oscillating pulse with high dH/dt that dissipates nearly all the energy released from the capacitor in the damping resistors.

[0173] Sense and compensation coils will be formed by looped traces in multilayer circuit boards that inductively couple the PRIMAs to a detection circuit (Figure. 19). In such a design, the cancellation arising from geometric symmetry can be supplemented with additional fine tuning by adjusting a potentiometer as shown in the schematic. One or more amplification stages are possible, and measurements will likely consist of a sequence of pulses to allow for signal averaging that improve the signal to noise ratio. Estimates of the magnitude of the induced voltage magnitude are shown as a function of particle concentration and dH/dt, assuming 100 turn pickup coils and approximately spherical samples.

[0174] An example of an application of a suitable embodiment of the invention is shown in FIG. 20. The data shows ultrasound signal attenuation as a function of transmitted frequency at constant microbubble concentration. The “control” shows the attenuation profile for freely oscillating microbubbles, which compared to all other samples had the highest signal attenuation indicating a soft shell without constraining crosslinkers. “3 links”, “6 links” and “12 links” donate different degrees of stiffening crosslinking on the microbubble shell. All of the samples show lower attenuation compared to the control suggesting that those samples consist of microbubbles with a crosslinked shell with higher stiffness. The data suggest that full stiffening was reached only if the number of interconnections between elements on the microbubble were 6 or higher. Stiffness values can be estimated using mathematical models for microbubble oscillations known in the art and can be compared to theoretical simulations of interconnected networks. The simulations show that a fully interconnected network (more than 99% of connecting elements are part of the network) is only achieved after 6 links per connecting element. This is a logical explanation for the experimental observation also shown in this figure.

[0175] Another example of an application of a suitable embodiment of the invention is shown in FIG. 21. It shows an example of an ultrasound phantom that was used to generate the B-mode ultrasound images below. The ultrasound images show that differentiation between crosslinked and freely oscillating microbubbles at the same concentration can occur for example by analyzing the pixel brightness of the ultrasound image within a uniform region of interest.