Method for magnetic resonance molecular imaging of nanoconstructs and uses thereof

Abstract

Described herein are methods for imaging magnetic resonance imaging (MRI) agents containing nanoconstructs therein using diffusion weighted fast spin echo (FSE) or gradient and spin echo (GRASE) methods.

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit and priority of U.S. Provisional Application No. 63 / 522,007, filed June 20, 2023, the entire contents of which are incorporated herein by reference for all purposes. Technical Field

[0003] This invention generally belongs to the field of imaging magnetic resonance imaging (MRI) agents using diffusion-weighted fast spin echo (FSE) or gradient and spin echo (GRASE) methods. Background Technology

[0004] When generating images using NMR, a technique is employed to obtain NMR signals from specific locations on the subject. Typically, the region to be imaged (the region of interest) is scanned through a series of NMR measurement cycles, which vary depending on the specific localization method used. The resulting set of received NMR signals is digitized and processed to reconstruct an image using one of many well-known reconstruction techniques. For such scanning to be performed, it is necessary to induce NMR signals from specific locations on the subject. This is achieved by employing a magnetic field having the same orientation as the polarization field B0 but with gradients along the corresponding x, y, and z axes. By controlling the intensity of these gradients during each NMR cycle, the spatial distribution of the spin frequency can be controlled, and the location of the resulting NMR signal can be identified. However, scan time is a crucial consideration in imaging, as reducing scan time increases patient throughput, improves patient comfort, and enhances image quality by reducing motion artifacts. Therefore, various techniques have been developed to acquire NMR image data within short time intervals.

[0005] One such technique is called "fast spin echo" (FSE), which uses a pulse sequence to acquire multiple k-space lines under a single excitation. Compared to, for example, echo-plane imaging, FSE is much less sensitive to field inhomogeneities and gradient timing errors. Furthermore, unlike echo-plane imaging, gradient fidelity is not an issue because the readout gradient is always positive. Diffusion-weighted FSE imaging employs a gradient pulse at the beginning of the pulse sequence to make the acquired NMR signal sensitive to spin motion. Conventional and diffusion-weighted spin echo imaging techniques are known in the art. Clinical diffusion-weighted imaging relies heavily on the commercialization of single-shot echo-plane imaging (EPI), which in turn requires high-performance gradient coils to reduce non-resonance-related artifacts to diagnostically acceptable levels. See Le Bihan D. Molecular diffusion, tissue microdynamics and microstructure. NMR Biomed. 1995; 8(7-8):375-86.

[0006] Another such technique is Gradient and Spin Echo (GRASE), which employs and integrates various aspects of EPI within FSE. GRASE can collect multiple gradient echoes within a Fast Spin Echo (FSE) echo train. GRASE employs multiple refocusing RF pulses after an excitation RF pulse, similar to Fast Spin Echo (FSE), but with a series of gradient echo readings, analogous to EPI inserted in the echo intervals between successive refocusing pulses. Each gradient echo is preceded by an appropriate phase-coded gradient, which fills the k-space at the corresponding location to form an image. See Chu et al., “Gradient- and spin-echo (GRASE) MR imaging: a long-existing technology that may find wide applications in modern era,” Quant Imaging Med Surg. 9(9): 1477-1484 (September 2019); Bihan D. Molecular diffusion, tissue microdynamics and microstructure. Magnetic Resonance in Biomedicine 1995; 8(7-8):375-86; Park S et al., Highly accelerated submillimeter resolution 3D GRASE with controlled T2 blurring in T2-weighted functional MRI at 7 Tesla: A feasibility study. Magnetic Resonance Imaging. May 2021; 85(5):2490-2506; and Prasloski T et al., Rapid whole cerebrum myelin water imaging using a 3D GRASE sequence. Neuroimage. October 15, 2012; 63(1):533-9.

[0007] There is a need to develop improved methods for using imaging agents that employ this type of technology. Summary of the Invention

[0008] This article describes methods for imaging magnetic resonance imaging (MRI) agents using diffusion-weighted fast spin echo (FSE) or gradient and spin echo (GRASE) methods.

[0009] In various examples, the present invention provides improved methods for imaging different agents of interest using techniques such as FSE and GRASE.

[0010] In another instance, the present invention provides a method for determining the concentration of different drugs in vivo.

[0011] In yet another instance, the present invention provides an improved method for visualizing various aspects of a tumor in a subject.

[0012] In a non-limiting embodiment, a method for imaging a magnetic resonance imaging (MRI) agent is provided, wherein the method includes the following steps:

[0013] (a) Administering the MRI agent to the subject,

[0014] The MRI agent comprises nanostructures (NCs), each having multiple equivalent protons and an average molecular weight in the range of about 300 Da to about 300 kDa, and each nanostructure having a T2 relaxation time greater than at least about 250 milliseconds.

[0015] (b) Using an MRI apparatus to obtain diffusion-weighted fast spin echo (FSE) nuclear magnetic resonance scans of the nanostructure, wherein step (b) comprises:

[0016] (i) Application at approximately 500 s / mm 2 From approximately 10,000 s / mm 2 Diffusion filtering within a certain range is used to suppress water signals from the scan and to selectively image the nanostructure;

[0017] (ii) A signal acquisition bandwidth applicable in the range of at least about 2 kHz to about 50 kHz; and

[0018] (iii) Obtaining more than 8 spin echoes, optionally 9 to 256 or 9 to 128 spin echoes; optionally, step (b) further comprises modifying the gain of the receiver-amplifier of the MRI device.

[0019] In another non-limiting embodiment, a method for imaging a magnetic resonance imaging (MRI) agent is provided, wherein the method relates to the GRASE method and includes the following steps:

[0020] (a') Administer the MRI agent to the subject.

[0021] The MRI agent comprises nanostructures, each having multiple equivalent protons and an average molecular weight in the range of about 300 Da to about 300 kDa, and each nanostructure having a T2 relaxation time greater than at least about 250 milliseconds.

[0022] (b') Using an MRI apparatus to obtain gradient and spin echo (GRASE) nuclear magnetic resonance scans of the nanostructure, wherein step (b') comprises:

[0023] (i') Applied at approximately 500 s / mm 2 From approximately 10,000 s / mm 2 Diffusion filtering within a certain range is used to suppress water signals from the scan and to selectively image the nanostructure;

[0024] (ii') The signal acquisition bandwidth is applied in the range of at least about 2 kHz to about 200 kHz; and

[0025] (iii') Obtain more than 8 spin echoes, optionally 9 to 1024 spin echoes; and obtain at least 3 gradient echoes, optionally 3 to 7 gradient echoes;

[0026] Optionally, step (b') further comprises modifying the gain of the receiver-amplifier of the MRI device.

[0027] In some cases of the methods described herein, the NMR apparatus is operated and settings and / or parameters are selected to provide sufficient water and lipid suppression, thereby enabling selective imaging of the MRI agent and nanostructure while suppressing all or substantially all water and / or lipid signals, where “substantially all” means at least about 95%, 96%, 97%, 98%, or 99% or more of the suppressed water and lipid signals from the scan. For example, diffusion-based MRI imaging methods can be employed, in which at least about 95% or more of the tissue and water-related signals are suppressed or eliminated (i.e., 100% absent or otherwise undetectable). Residual water can be removed by other water suppression methods known to those skilled in the art in imaging techniques. For example, for additional water suppression, spectral imaging can be used, where each voxel consists of an NMR spectrum containing water, PEG, and lipids.

[0028] In the case of the methods described above, the selective detection of MRI agents and nanostructures can be achieved by using specific imaging and / or diffusion parameters such as diffusion-weighted FSE and GRASE. The signal strength of MRI agents and nanostructures is proportional to their concentration.

[0029] The nanostructure comprises a backbone or platform, which may have one or more additives associated with it, and the NC acts as an imaging agent. Such additives, which may be present on the nanostructure, may provide other functions, such as targeted selection of specific cell types, specific biological tissues or structures, or specific membrane-related properties, such as cell membrane permeability.

[0030] Each nanostructure comprises a plurality of equivalent or substantially equivalent protons. In some cases, the plurality of equivalent or substantially equivalent protons ranges from about 30 to 27,000 protons, from 200 to about 15,000 protons, or from about 200 to about 10,000 protons, as well as any subranges or individual values ​​disclosed therein. In some cases, the plurality of equivalent or substantially equivalent protons are selected from methylene protons, methyl protons, and / or methoxy protons.

[0031] In some cases, the T2 relaxation time of each nanostructure is greater than at least about 250 ms, 300 ms, 350 ms, 400 ms, 450 ms, or 500 ms. In some cases, the T2 relaxation time is at least about 250 ms to about 900 ms or about 500 ms to about 900 ms, and any subranges or individual values ​​disclosed therein.

[0032] In some cases, when measured at 25°C or 35°C, the translational diffusion coefficients of the individual nanostructures were less than approximately 2 × 10⁻⁶. -10 m 2 s -1 Less than approximately 3 × 10 -11 m 2 s -1 Less than approximately 3.5 × 10 -11 m 2 s -1 Less than approximately 4 × 10 -11 m 2 s -1 Less than approximately 4.5 × 10 -11 m 2 s -1 or less than about 5 × 10 -11 m 2 s -1 In some other cases, when measured at 25°C or 35°C, the translational diffusion coefficients of the individual nanostructures are approximately 2 × 10⁻⁶. -10 m 2 s -1 To approximately 5 × 10 -11 m 2 s -1 Any subrange or individual value disclosed within or within the scope of.

[0033] In the methods described herein, the nanostructure may have one or more additives associated with it. These additives include activators, which may be selected from the group consisting of: targeting agents, therapeutic agents, chemotherapeutic agents, photosensitizers, sonosensitizers, imaging agents, diagnostic agents, photoacoustic agents, therapeutic diagnostic agents, and combinations thereof. Attached Figure Description

[0034] The accompanying drawings described below depict various aspects of the systems and methods disclosed herein. It should be understood that each drawing depicts an embodiment of a specific aspect of the disclosed systems and methods, and each drawing is intended to conform to one or more possible embodiments thereof. Furthermore, wherever possible, the following description refers to the reference numerals included in the following drawings, wherein features depicted in the plurality of drawings are indicated by consistent reference numerals.

[0035] Figure 1A This is a non-limiting formula representation of a nanostructure (NC) having multiple arms (i.e., 8-arm PEG) associated with one or more additives thereon. Form (1) depicts a target agent (TA) conjugated or associated with the multi-arm PEG. Form (2) depicts an active agent, such as a therapeutic molecule, conjugated or associated with the multi-arm PEG. Forms (3) and (4a, b) depict alternative structures of the NC, wherein both the TA and the active agent are conjugated or associated with the multi-arm PEG. These representations are not intended to limit the number or ratio of agents thereon.

[0036] Figure 1B These are non-limiting formula representations of nanostructures (NCs) having linear PEG conjugated or associated with it. Form (1) depicts a target agent (TA) conjugated or associated with PEG. Form (2) depicts an active agent, such as a therapeutic molecule, conjugated or associated with PEG. Forms (3) and (4a, b) depict alternative structures of NCs in which both the TA and the active agent are conjugated or associated with PEG. These representations are not intended to limit the number or ratio of agents on them.

[0037] Figure 1C An exemplary flowchart is shown for the synthesis process of preparing nanostructures having one or more additives associated therewith.

[0038] Figure 2 A graph showing the 1H NMR signal versus PEG concentration for fast spin echo and conventional spin echo images is presented. Figure 2 The signal depicted in the fast spin echo image is 30 times that detected using conventional spin echo techniques.

[0039] Figure 3A schematic diagram of an exemplary MRI system 2501 is shown. The exemplary system 2501 includes a magnet 2502, a gradient coil 2503, a radio frequency coil 2504, an aperture 2505, and a stage 2506.

[0040] Figure 4A The image shows NMR imaging of water with a high signal intensity in the central vial due to manganese (Mn) doping, with manganese providing the tissue T2 value.

[0041] Figure 4B This study demonstrates MRI of rapid spin-echo selective imaging using PEG without the application of fat suppression, wherein the central vial consists of water and contains fat at 1:00, 5:00, and 9:00, while the vials contain PEG dissolved in water at 3:00, 7:00, and 11:00 (10 mg / mL, 4 mg / mL, and 2 mg / mL, respectively).

[0042] Figure 4C The MRI of fast spin-echo selective imaging using PEG with fat suppression is shown, wherein the central vial is composed of water and contains fat at 1:00, 5:00 and 9:00, while the vial contains PEG dissolved in water at 3:00, 7:00 and 11:00 (10 mg / mL, 4 mg / mL and 2 mg / mL, respectively). Detailed Implementation

[0043] The following describes methods for imaging magnetic resonance imaging (MRI) agents using diffusion-weighted fast spin echo (FSE) or gradient and spin echo (GRASE) methods.

[0044] I. Definition

[0045] The terms "nanoparticle" and "nanostructure" are used interchangeably to refer to particles having a volumetric shape having at least one dimension ranging in size from about 1 nm to about 100 nm. In some cases, these volumetric shapes may have a maximum cross-section of about 1 nm to about 100 nm. Such particles have a main chain material (e.g., a cage, carrier, matrix, or polymer material), and one or more additives, such as pharmaceuticals, portions, compositions, biological agents, and molecules, optionally associated with the main chain. The main chain material itself may be a nanoparticle.

[0046] The term "additive" refers to an active material having targeting, therapeutic, imaging, diagnostic, therapeutic, or other capabilities, as well as combinations and variations of these capabilities.

[0047] The term “therapeutic diagnostic” refers to any suitable agent or material with multiple capabilities and functions, including both imaging and therapeutic capabilities, both diagnostic and therapeutic capabilities, and combinations and variations of these and other features, such as targeting.

[0048] The terms “imaging,” “imaging agent,” “imaging device or apparatus,” and similar terms generally encompass the ability to enhance, provide, or enable the detection, analysis, and visualization of structures, and specifically the size, shape, location, composition, and combinations and variations thereof, as well as other characteristics of structures in animals, mammals, and humans. Imaging agents include contrast agents, dyes, and similar materials known in the field of imaging. Examples of imaging devices or apparatuses include those used in a variety of imaging methods, including: X-ray imaging; magnetic resonance imaging; computed axial computed tomography (CAT); positron emission tomography (PET); ultrasound imaging; fluorescence imaging; and photoacoustic imaging.

[0049] The term “diagnosis” generally refers to the identification, determination, definition, combination, and variation of these symptoms, diseases (including those in animals, mammals, and humans) or both.

[0050] The terms “therapeutic” and “treatment” refer to the resolution, treatment, management, relief, cure, prevention, combination, and variation of these conditions and diseases (including those in animals, mammals, and humans).

[0051] As used herein, unless otherwise specified, room temperature is 25°C or approximately 25°C. Standard ambient temperature and pressure are 25°C and 1 atmosphere. Unless otherwise explicitly stated, all tests, test results, physical properties, and temperature-dependent, pressure-dependent, or both-dependent values ​​are provided at standard ambient temperature and pressure, including viscosity.

[0052] The term "subject" can refer to any animal. Animals can be mammals, such as humans.

[0053] As used herein, the term "spin echo" refers to the application of 180° radio frequency (RF) pulses to refocus transverse magnetization that has dephased over time due to magnetic field inhomogeneities. In standard spin echo imaging, a single 180° RF pulse is used for each k-space line. For fast spin echo imaging (FSE), a sequence of multiple 180° RF pulses is used to acquire the desired number of k-space lines.

[0054] As used herein, the term "gradient echo" refers to the application of a magnetic field gradient to fully dephase and rephase the transverse magnetization. Gradient echoes are created by applying a pre-phase gradient to position the transverse magnetization at the edge of k-space, followed by a rephase gradient. A gradient echo is generated when the transverse magnetization crosses k = 0; the point where all spatial components of the transverse magnetization are in phase and the signal is strongest. In conventional gradient echo imaging, a single gradient echo is acquired for each k-space line. In other cases, such as gradient spin echo (GRASE), multiple gradient echoes can be acquired within a given spin echo. Typically, each spin echo corresponds to at least three gradient echoes, but up to seven.

[0055] The numerical ranges disclosed in this application include, but are not limited to, relaxation time ranges, bandwidth ranges, integer ranges, time ranges, and echo ranges. Each disclosed range of any type individually discloses every possible number that such a range can reasonably cover, as well as any sub-ranges and combinations thereof. For example, consistent with the disclosure herein, the disclosure of integer ranges is intended to individually disclose every possible integer value that such a range can cover.

[0056] The term "about" is used to describe values ​​that are higher or lower than the value modified by the term "about" within a range of approximately + / - 10%; in other cases, the range of these values ​​may be higher or lower than the value within a range of approximately + / - 5%. When the term "about" precedes a range of numbers (i.e., about 1-5) or a series of numbers (i.e., about 1, 2, 3, 4, etc.), it is intended to modify both ends of the range of numbers or each number in the series, unless otherwise specified.

[0057] II. Methods for molecular imaging of magnetic resonance imaging (MRI) agents

[0058] This article describes methods for imaging magnetic resonance imaging (MRI) agents using diffusion-weighted fast spin echo (FSE) or gradient and spin echo (GRASE) methods.

[0059] In a non-limiting case, a method for imaging a magnetic resonance imaging (MRI) agent includes the following steps:

[0060] (a) Administering the MRI agent to the subject,

[0061] The MRI agent comprises nanostructures, each having multiple equivalent protons and an average molecular weight in the range of about 300 Da to about 300 kDa, and each nanostructure having a T2 relaxation time greater than at least about 250 milliseconds.

[0062] (b) Using an MRI apparatus to obtain diffusion-weighted fast spin echo (FSE) nuclear magnetic resonance scans of the nanostructure, wherein step (b) comprises:

[0063] (i) Application at approximately 500 s / mm 2 From approximately 10,000 s / mm 2 Diffusion filtering within a certain range is used to suppress water signals from the scan and to selectively image the nanostructure;

[0064] (ii) A signal acquisition bandwidth applicable in the range of at least about 2 kHz to about 50 kHz; and

[0065] (iii) Obtaining more than 8 spin echoes, optionally 9 to 256 or 9 to 128 spin echoes; optionally, step (b) further comprises modifying the gain of the receiver-amplifier of the MRI device.

[0066] In the methods described above, the translational diffusion coefficient of each nanostructure is typically less than approximately 2 × 10⁻⁶. -10 m 2 s -1 .

[0067] In some cases of the diffusion-weighted fast spin echo (FSE) method described above, one or more additives are associated with the nanostructure, said one or more additives including targeting agents, such as tumor targeting agents; and the method further includes: after step (b),

[0068] (c) Provide data to identify the shape, structure and / or location of tumors in the subject.

[0069] In some cases, the data in step (c) further include data on the relative abundance of a specific receptor or other genotypic information related to the tumor in the subject. For example, the intensity of the MRI scan and the proton signal generated by imaging with the nanoconstruct can be used to target and infer the relative abundance of the receptor, thereby determining its genotype. Exemplary receptor targets that can be used to target receptors may include, but are not limited to: folic acid, iRGD, iNGR, PSMA, cRGD, gastrin-releasing peptide receptor (GRPR), human epidermal growth factor receptor 2 (HER2), insulin-like growth factor 1 receptor (IGF-1R), fibroblast growth factor receptor 3 (FGFR3), EGFR antibody, p53, fucoidan, and combinations thereof. Other receptor targets may include integrin-binding targets, which represent a family of targets that have attracted attention due to their reliable prevalence in tumors and their association with progression / prognosis.

[0070] In some cases of the aforementioned diffusion-weighted fast spin echo (FSE) method, the method further includes:

[0071] Following step (b), (d) provides data on the concentration of the nanostructure in the subject.

[0072] In another non-limiting case, a method for imaging a magnetic resonance imaging (MRI) agent, the method relating to the GRASE method, the method comprising the following steps:

[0073] (a') Administer the MRI agent to the subject.

[0074] The MRI agent comprises nanostructures, each having multiple equivalent protons and an average molecular weight in the range of about 300 Da to about 300 kDa, and each nanostructure having a T2 relaxation time greater than at least about 250 milliseconds.

[0075] (b') Using an MRI apparatus to obtain gradient and spin echo (GRASE) nuclear magnetic resonance scans of the nanostructure, wherein step (b') comprises:

[0076] (i') A diffusion filter is applied in the range of about 500 s / mm2 to about 10,000 s / mm2 to suppress water signals from the scan and to selectively image the nanostructure;

[0077] (ii') The signal acquisition bandwidth is applied in the range of at least about 2 kHz to about 200 kHz; and

[0078] (iii') Obtain more than 8 spin echoes, optionally 9 to 1024 spin echoes; and obtain at least 3 gradient echoes, optionally 3 to 7 gradient echoes;

[0079] Optionally, step (b') further comprises modifying the gain of the receiver-amplifier of the MRI device.

[0080] In the method described above, the translational diffusion coefficient of each nanostructure is typically less than about 2 × 10⁻⁶. -10 m 2 s -1 .

[0081] In some cases of the GRASE method described above, one or more additives are associated with the nanostructure, said one or more additives including targeting agents, such as tumor targeting agents; and the method further includes: after step (b'),

[0082] (c') Provide data to identify the shape, structure and / or location of tumors in the subject.

[0083] In some cases, the data in step (c') further include data on the relative abundance or other genotypic information of a specific receptor associated with the tumor in the subject. For example, the intensity of the MRI scan and the proton signal generated by imaging with the nanoconstruct can be used to target and infer the relative abundance of the receptor, thereby determining its genotype. Exemplary receptor targets that can be used to target receptors may include, but are not limited to: folic acid, iRGD, iNGR, PSMA, cRGD, gastrin-releasing peptide receptor (GRPR), human epidermal growth factor receptor 2 (HER2), insulin-like growth factor 1 receptor (IGF-1R), fibroblast growth factor receptor 3 (FGFR3), EGFR antibody, p53, fucoidan, and combinations thereof. Other receptor targets may include integrin-binding targets, which represent a family of targets that have attracted attention due to their reliable prevalence in tumors and their association with progression / prognosis.

[0084] In some cases of the GRASE method described above, the method may further include:

[0085] Following step (b'), (d') provides data on the concentration of the nanostructure in the subject.

[0086] In the case of the first method described above, diffusion-weighted FSE depends on the diffusion constant and transverse spin relaxation rate of the nanostructure of the MRI agent, wherein the slow diffusion constant and transverse spin relaxation rate are combined to allow the diffusion-weighted MRI sequence to suppress the surrounding water signal, thereby providing a clear image of the MRI agent and the nanostructure.

[0087] In some cases of the methods described above, the NMR apparatus is operated and settings and / or parameters are selected to provide sufficient water and fat suppression, thereby enabling selective imaging of the MRI agent and nanostructures while suppressing all or substantially all water and / or fat signals, where substantially all means at least about 95%, 96%, 97%, 98%, or 99% or more of the suppressed water and lipid signals from the scan. For example, diffusion-based MRI imaging methods can be employed, in which at least about 95% or more of the tissue and water-related signals are suppressed or eliminated (i.e., 100% absent or otherwise undetectable). Residual water can be removed by other water suppression methods known to those skilled in the art in imaging techniques. For example, to provide additional water suppression, spectral imaging can be used, where each voxel consists of an NMR spectrum containing water, PEG, and fat. In some cases, fat suppression or fat saturation techniques can be applied to remove any imaging residues due to fat, and the NMR apparatus used for the methods described above can incorporate fat suppression techniques known to those skilled in the art. For example, fat suppression techniques in magnetic resonance imaging (MRI) are used to selectively suppress signals from adipose tissue to improve visualization of structures or pathologies that may be obscured by the presence of fat. Fat suppression techniques used in MRI can include, but are not limited to: chemical fat saturation; inversion recovery (IR) with fat suppression; spectral fat saturation; saturation bands; and / or oil-water separation techniques. Advantageously, fat protons resonate with chemical shifts different from those of water and PEG, and can be effectively suppressed by a variety of methods and techniques, as described elsewhere above. Fat suppression is commonly performed in MRI, particularly in body MRI, to provide clearer diagnostic images of water protons. One method of fat suppression involves applying RF pulses at fat frequencies to suppress signals generated by fat protons. These methods can be combined in vivo to create PEG-selective images with fat suppression.

[0088] In the aforementioned methods, the selection of specific imaging and / or diffusion parameters, such as diffusion-weighted FSE and GRASE, can be used to selectively detect MRI agents and nanostructures. The signal strength of MRI agents and nanostructures is proportional to their concentration. In some cases, the combination of a high b-value (e.g., at least about 5000 s / mm²) and a long TE time (e.g., at least about 245 ms) effectively suppresses water signals but not sufficiently suppresses long T2 fat signals, thus creating an image of superimposed PEG and fat signals. However, MRI images taken without such high b-values ​​and long TE times will display a conventional water proton image. In some cases, by adding such high b-values ​​and long TE times and combining them with fat suppression, both water and fat signals can be sufficiently reduced, thus creating a PEG-selective image.

[0089] In some cases, the method can be used as a therapeutic diagnostic method. For example, when the above method is used as a therapeutic diagnostic method, a targeted therapeutic diagnostic nanoconstruct is delivered to the subject and carried by the bloodstream, associating with a target structure (e.g., a tumor). In step (b) or (b'), the tumor is scanned with MRI, and the resulting image is enhanced by the presence of the nanoconstruct in the MRI agent. The location and shape of the target structure within the subject's body are obtained and stored. Very precise images, along with location data and information, of the target structure within the subject's body can be provided using subsequent imaging techniques (e.g., photoacoustic imaging), modeling techniques (e.g., computer enhancement and rendering of the initial MRI images), and combinations thereof. An illumination pattern can then be developed based on this image and location data. This illumination pattern can be predetermined, customized, and specific to the target structure.

[0090] The described MRI agents include multiple nanostructures. MRI agents can be provided as pharmaceutical compositions, in kits, and in systems. Compositions containing MRI agents can be in the form of pharmaceutical-grade compositions or solutions. Compositions or solutions can be prepared in suitable pharmaceutical-grade solvents and may include one or more known pharmaceutical excipients, as appropriate. They may also be in the form of pellets or tablets rather than liquid compositions. Such liquid or solid compositions include MRI agents comprising multiple nanostructures at concentrations of about 1 mg / kg to 200 mg / kg body weight, 10-100 mg / kg, or 20-50 mg / kg, and any subranges or individual concentrations within the foregoing ranges. Such MRI agents can be administered to subjects by any suitable manner, including orally, percutaneously, or intravenously (using injection, catheter, or other intravenous or intra-arterial delivery methods).

[0091] When provided as a pharmaceutical composition for administration to a subject, there are no particular limitations on the concentration of the nanostructures in the MRI agent. In some cases, the concentration of the nanostructures in the pharmaceutical composition may be, for example, about 10 wt% in saline, which, when administered at 25 mg / kg, corresponds to 1750 mg for a 70 kg individual. The composition may include additional inactive ingredients, including but not limited to: buffers (such as citrate, phosphate, and carbonate buffers), preservatives, vitamins, surfactants, liposome materials (e.g., DSPC), proteins (e.g., albumin, lactose), alcohols, sugars, antioxidants (e.g., butylated hydroxyanisole), anti-inflammatory drugs, salts, cholesterol, or amino acids. Other excipients may also be present, such as inactive or active additives known for pharmaceutical use.

[0092] A. Diffusion-weighted FSE and GRASE imaging parameters

[0093] In the cases of the first and second methods, the diffusion-weighted FSE and GRASE methods involve the selection of several parameters, as discussed below.

[0094] For example, diffusion-weighted FSE methods include those applied at approximately 500 s / mm. 2 From approximately 10,000 s / mm 2 The FSE method further includes applying a diffusion filter within or a sub-range disclosed therein to suppress water signals from the scan and selectively image the nanostructures of the MRI agent. The FSE method further includes applying a signal acquisition bandwidth within a range of at least about 2 kHz to about 50 kHz or about 5 kHz to about 50 kHz, and sub-ranges and individual bandwidth values ​​contained therein. In some cases, the signal acquisition bandwidth is about 10 kHz. The FSE method further includes obtaining more than 8 spin echoes, such as 9 to 256 or 9 to 128 spin echoes, and sub-ranges and individual echo values ​​contained therein. In some cases, at least 16, 32, 64, or 128 spin echoes are obtained during the method. In some cases, the FSE method only obtains spin echoes and does not include obtaining any gradient echoes. In some cases, the FSE method optionally includes modifying the gain of the receiver-amplifier of the MRI apparatus. For example, the gain can be modified by increasing the receiver gain of the MRI system by 6 dB to 50 dB to deliver an analog characteristic greater than 0.1 V and less than 1.0 V to the digitizer. In the case of selective PEG imaging, the water signal is sufficiently attenuated to allow the detection of the PEG signal, thus allowing the receiver gain to be increased to digitize the remaining PEG signal. Increasing the receiver gain improves the sensitivity of selective PEG imaging.

[0095] In some cases, one or more imaging parameters for an FSE-based magnetic resonance imaging (MRI) scan can be selected from the following:

[0096] (i) Repeat time (TR), which can be about 2 seconds to about 10 seconds, about 2 seconds to about 4 seconds, or about 3 seconds to about 5 seconds, and the subranges and individual values ​​disclosed therein;

[0097] (ii) Spin echo time (TE), which can be from about 50 milliseconds to about 100 milliseconds, and subranges and individual values ​​disclosed within the specified range;

[0098] (iii) Gain, which can be from about 6 dB to 50 dB, and the sub-ranges and individual values ​​disclosed therein;

[0099] (iv) Spectral width (sw), which may be from about 2,000 Hz to about 50,000 Hz, from about 4,000 Hz to about 10,000 Hz, and the subranges and individual values ​​disclosed therein; or, in some cases, the spectral width may be about 5,000 Hz;

[0100] (v)b value, which can be approximately 500 s / mm 2 Up to 10,000 s / mm 2 , and the subranges and individual values ​​disclosed therein; or where the value of b can be approximately 4,000 s / mm 2 Or 5,000 s / mm 2 ;

[0101] (vi) Voxel size, which can be from about 1.5 μL to about 15 μL;

[0102] (vii) Spin echo train length, which can be in the range of 9 to 256 or 9 to 128 spin echoes;

[0103] (viii) Echo train segments, which can be 1 to 8 segments, and disclosed subranges and individual values ​​therein; or, where the echo train segment is 1; and

[0104] The combination of the above imaging parameters.

[0105] In some cases, one or more diffusion parameters for nuclear magnetic resonance scanning using the FSE method can be selected from the following:

[0106] (ix) Incremental values ​​in the range of approximately 6 milliseconds to approximately 40 milliseconds, or subranges or individual values ​​contained therein;

[0107] (x) A diffusion gradient, which can be from about 3 Gauss / cm to about 25 Gauss / cm, and the disclosed subranges and individual values ​​thereof; or, where the diffusion gradient is about 12 Gauss / cm; and combinations thereof.

[0108] Alternatively, a combination of two or more of the imaging and diffusion parameters described above can be selected to perform a magnetic resonance scan during the described FSE method.

[0109] In diffusion-weighted fractional electron scanning (FSE), the nanostructure of the MRI agent comprises multiple equivalent or substantially equivalent protons that are directly detected and measured. This differs from MRI using metals such as gadolinium, which alter the relaxation time of water protons interacting with them, thereby producing visible contrast during scanning. Therefore, the FSE method provides images based on direct measurements of protons within the nanostructure of the MRI agent and does not depend on or utilize variations in the relaxation time of water protons.

[0110] The diffusion-weighted FSE method described above can generate and detect signals that are at least approximately 5, 10, 15, 20, 25, or 30 times greater than those detected by conventional spin-echo techniques using the same MRI agent. Furthermore, the same method allows for MRI scanning of the MRI agent even at concentrations as low as 0.05 mg / mL. The ability to suppress water signals and selectively image low-concentration nanostructures using the claimed method is not feasible with other known MRI techniques.

[0111] For the GRASE method, the selection of the following parameters is related to the method described. For example, GRASE acquires several gradient echoes in each of many spin echoes. This contrasts with FSE methods that do not include gradient echoes (as described above). The GRASE parameters will be adjusted to include the number of gradient echoes, the number of data points per echo, and the digitization rate for acquiring each echo. In some cases, the GRASE method may include applications at approximately 500 s / mm. 2 Approximately 10,000 s / mm 2 The GRASE method further includes applying diffusion filtering within or a sub-range disclosed therein to suppress water signals from the scan and selectively image the nanostructures of the MRI agent. The GRASE method further includes applying a signal acquisition bandwidth within a range of at least about 2 kHz to about 200 kHz or about 5 kHz to about 100 kHz, and sub-ranges and individual bandwidth values ​​contained therein. The GRASE method further includes obtaining at least three gradient echoes, such as three to seven gradient echoes, and sub-ranges and individual echo values ​​contained therein. In some cases, at least eight and possibly up to 1024 spin echoes are obtained during the GRASE method. The GRASE method further includes obtaining at least three gradient echoes, such as three to seven gradient echoes, and sub-ranges and individual echo values ​​contained therein. In some cases, at least three to seven gradient echoes are obtained during the GRASE method. In some cases, the GRASE method optionally includes modifying the gain of the receiver-amplifier of the MRI apparatus. For example, the gain can be modified by increasing the receiver gain of the MRI system by 6 dB to 50 dB to deliver an analog characteristic greater than 0.1 V and less than 1.0 V to the digitizer. In the case of selective PEG imaging, the water signal is sufficiently attenuated to allow the detection of the PEG signal, thus allowing the receiver gain to be increased to digitize the remaining PEG signal. Increasing the receiver gain improves the sensitivity of selective PEG imaging.

[0112] In some cases, one or more imaging parameters for GRASE method magnetic resonance imaging can be selected from the following:

[0113] (i) Repeat time (TR), which can be about 2 seconds to about 10 seconds, about 2 seconds to about 4 seconds, or about 3 seconds to about 5 seconds, and the subranges and individual values ​​disclosed therein;

[0114] (ii) Spin echo time (TE), which can be from about 50 milliseconds to about 100 milliseconds, and the subranges and individual values ​​disclosed therein;

[0115] (iii) Gain, which can be from about 6 dB to 50 dB, as well as sub-ranges and individual values ​​disclosed within the specified range;

[0116] (iv) Spectral width (sw), which may be from about 2,000 Hz to about 50,000 Hz, from about 4,000 Hz to about 10,000 Hz, and the subranges and individual values ​​disclosed therein; or, in some cases, the spectral width may be about 5,000 Hz;

[0117] (v)b value, which can be approximately 500 s / mm 2 Up to 10,000 s / mm 2 , and the subranges and individual values ​​disclosed therein; or where the value of b can be approximately 4,000 s / mm 2 Or 5,000 s / mm 2 ;

[0118] (vi) Voxel size, which can be from about 1.5 μL to about 15 μL;

[0119] (vii) Spin echo train length, which can range from 9 to 1024 spin echoes;

[0120] (viii) Gradient echo train length, which can be in the range of 9 to 1024 gradient echoes;

[0121] (ix) an echo string segment, which can be 1 to 8 segments, and disclosed subranges and individual values ​​therein; or, where the echo string segment is 1; and

[0122] The combination of the above imaging parameters.

[0123] In some cases, one or more diffusion parameters for GRASE method nuclear magnetic resonance scanning can be selected from the following:

[0124] (x) Incremental values ​​in the range of approximately 6 milliseconds to approximately 40 milliseconds, or subranges or individual values ​​contained therein;

[0125] (xi) A diffusion gradient, which can be from about 3 Gauss / cm to about 25 Gauss / cm, and the disclosed subranges and individual values ​​therein; or, where the diffusion gradient is about 12 Gauss / cm; and

[0126] The combination of the above diffusion parameters.

[0127] Alternatively, a combination of two or more of the imaging and diffusion parameters described above can be selected to perform a magnetic resonance scan during the described GRASE method.

[0128] Use an MRI device to apply diffusion-weighted fast spin echo (FSE) or GRASE MRI scans. Figure 3 A non-limiting schematic diagram of a non-limiting example of an MRI system 2501 is shown. System 2501 includes a magnet 2502, a gradient coil 2503, a radio frequency coil 2504, an aperture 2505, and a stage 2506. It should be understood that... Figure 3 This is a schematic representation of an MRI system that can employ other components, configurations, and types. In some cases, system 2501 is configured to generate three magnetic fields. The first field is a strong static magnetic field that creates energy level differences in cell nuclei with spin angular momentum and induces global nuclear magnetization. The second field is a radio frequency field and is used to tilt the resulting nuclear magnetization so that it can be detected by the RF coil 2504. The third field is a set of magnetic field gradients used to spatially encode the signal to create a nuclear magnetization map. Thus, in some cases, the magnetic fields are configured to generate images of non-aqueous protons present in an additive placed in the subject to be imaged; wherein the magnetic field gradients can be pulsed in a specific manner to make the cell nuclei sensitive to motion due to flow or diffusion.

[0129] In some cases of the described method, an MRI agent consisting of nanoconstructs is administered to the subject (i.e., the patient). The patient with the administered imaging agent is then placed on stage 2506, the stage is moved into aperture 2505, and system 2501 performs a scan to obtain MRI images of, for example, the type disclosed herein. The nanoconstructs are directly imaged to provide detailed images and data regarding their localization within the patient.

[0130] In some cases, the MRI system 2501 has a control system 2507, which includes operator inputs and other control features, as well as operating instructions, such as computer code. The control system 2507 is in control communication with the device 2510, as shown by dashed line 2508. Control communication refers to the transmission of data, information, control commands, and other instructions between the control system 2507 and the device 2510. The control system 2507 may be separate from the device 2510, or it may be part of the device 2510, for example, within the structure of the device 2510. In one embodiment, instructions for operating the MRI to provide operating parameters for imaging PEG-based nanoparticles are provided to the system 2501. This can be, for example, a software upgrade. In this way, existing MRI systems can be easily upgraded to take advantage of the benefits of imaging MRI agents using the method of the present invention, as described.

[0131] B. Nanostructures

[0132] Nanostructures (NCs) comprise a backbone or platform, which may have one or more additives associated with it, and the NCs act as imaging agents. The additives, when present on the nanostructure, can provide other functions, such as targeted selection of specific cell types, specific biological tissues or structures, or possessing specific membrane-related properties, such as cell membrane permeability. NCs associated with the backbone or platform can be covalently or non-covalently linked to the backbone or platform.

[0133] In some cases, nanostructures comprise a backbone or platform and contain no additives associated with it. In such cases, NCs themselves are capable of bioaccumulating in tissues and acting as imaging agents. It is believed that the ability to bioaccumulate in tissues is related to the molecular weight of the NCs, with increased molecular weight leading to increased bioaccumulation in tissues.

[0134] The NC described in this article is non-toxic and biocompatible, and NC can be used as a molecular imaging agent in various magnetic resonance imaging (MRI) techniques.

[0135] For the described methods, the average molecular weight of each of the nanostructures may be in the range of about 300 Da to about 300 kDa, about 3 kDa to about 300 kDa, about 10 kDa to about 300 kDa, or about 3 kDa to about 200 kDa, and any subranges or individual values ​​disclosed therein. In some cases, the average molecular weight of each of the nanostructures is in the range of about 10 kDa to about 250 kDa, and any subranges or individual values ​​disclosed therein. In some cases, the nanostructure (NC) does not contain any additives associated therewith and does not bioaccumulate in tissues. Optionally, the NC contains a polymer without additives associated therewith, and the molecular weight of the NC is about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, or about 100 kDa to about 200 kDa, and any subranges or individual values ​​disclosed therein. Optionally, the NC contains a multi-armed PEG, and each arm of the PEG is free of additives associated therewith, and the NC has a molecular weight of about 50 kDa to about 300 kDa, about 50 kDa to about 250 kDa, about 50 kDa to about 200 kDa, or about 100 kDa to about 200 kDa, and any subranges or individual values ​​disclosed therein.

[0136] In some cases, the average hydrodynamic diameter of the nanostructures of the MRI agent is less than about 50 nm. In other cases, the nanostructures of the MRI agent may be selected to have an average hydrodynamic diameter of less than about 25 nm, less than about 15 nm, or less than about 7 nm or smaller. Subranges or individual values ​​disclosed above are possible. In some cases, the size or dimension of the nanostructure is defined as a cross-section of about 5 nm to about 20 nm, about 5 nm to about 15 nm, about 10 nm to about 15 nm, and about 9 nm to about 12 nm. Relatively small nanostructures (i.e., < 20 nm) can allow for easy penetration of target tissues or structures, including, for example, very dense tissues such as muscle.

[0137] Each nanostructure comprises a plurality of equivalent or substantially equivalent protons. As used herein, “substantially equivalent protons” means protons that undergo the same chemical shift due to molecular symmetry. In some cases, the plurality of equivalent or substantially equivalent protons ranges from about 30 to 27,000 protons, 30 to 25,000 protons, 30 to 20,000 protons, 30 to 15,000 protons, 30 to 10,000 protons, 200 to about 15,000 protons, or about 200 to about 10,000 protons, and any subranges or individual values ​​disclosed therein. In some cases, the plurality of equivalent or substantially equivalent protons is selected from methylene protons, methyl protons, and / or methoxy protons.

[0138] In some cases, the T2 relaxation time of each nanostructure is greater than at least about 250 ms, 300 ms, 350 ms, 400 ms, 450 ms, or 500 ms. In some cases, the T2 relaxation time is at least about 250 ms to about 900 ms or about 500 ms to about 900 ms, and any subranges or individual values ​​disclosed therein.

[0139] In some cases, when measured at 25°C or 35°C, the translational diffusion coefficients of the individual nanostructures were less than approximately 2 × 10⁻⁶. -10 m 2 s -1 Less than approximately 3 × 10 -11 m 2 s -1 Less than approximately 3.5 × 10 -11 m 2 s -1 Less than approximately 4 × 10 -11 m 2 s -1 Less than approximately 4.5 × 10 -11 m 2 s -1 or less than about 5 × 10 -11 m 2 s -1 In some other cases, when measured at 25°C or 35°C, the translational diffusion coefficients of the individual nanostructures are approximately 2 × 10⁻⁶. -10 m 2 s -1 To approximately 5 × 10 -11 m 2 s -1 Any subrange or individual value disclosed within or within the scope of.

[0140] The following describes various other aspects of nanostructures.

[0141] 1. Main chain or platform

[0142] As described above, a nanostructure (NC) comprises a main chain or platform that may have one or more additives associated with it. In some cases, the NC has multiple ends associated with the additives. The additives present on, in, or covalently linked to the NC can be all the same or can be a combination of different additives. Figure 1A Various forms of multi-arm NCs with one or more additives are shown. Figure 1B Various forms of linear NCs with one or more additives are shown.

[0143] In some cases, the main chain or platform is selected from polyethylene glycol (PEG), polyethyleneimine (PEI), polypropylene glycol, polyvinyl alcohol, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polymethacrylate, polyacrylamide, polymethacrylamide, polylactic acid, polyglycolic acid, polyamide amine, and copolymers thereof. Depending on its structure, the main chain or platform can be considered symmetrical or asymmetrical. These main chains or platforms can be modified as needed to include reactive functional groups, such as, but not limited to, amine groups, maleimide groups, N-hydroxysuccinimide, sulfonic acids, carboxylic acid esters, thiols, alcohols, ketones, esters, alkenes, alkynes, DBCO, isothiocyanates, acyl halides, alkyl halides, aldehydes, and aromatic compounds. Such reactive functional groups can be used to associate one or more additives with the main chain or platform, such as through covalent association.

[0144] In some cases, the nanostructure includes a main chain or platform that is multi-armed and optionally has one or more additives associated with it. In some cases, the multi-armed NC has one or more additives associated with it, and at least one arm of the multi-armed NC does not contain said one or more additives, meaning that at least one arm does not contain additives associated with the other arms of the multi-armed NC. In some cases, the multi-armed NC has at least one free arm; at least two free arms; at least three free arms; at least four free arms; at least five free arms; at least six free arms; at least seven free arms; at least eight free arms; or at least nine free arms; it should be understood that at least one arm has an additive associated with it.

[0145] In some cases, the one or more additives associated with the multi-arm NC comprise at least two additives, such as a targeting agent (TA) and a therapeutic agent (i.e., a photosensitizer, a chemotherapeutic agent, a radiotherapy agent, etc.). In some such cases, the ratio of the at least two additives may be selected as 2:1, 3:1, 4:1, and 5:1, depending on the number of arms present on the multi-arm NC.

[0146] Those skilled in the art are familiar with methods for synthesizing nanostructures having such a backbone or platform, as well as methods for chemically modifying the backbone or platform to include selected reactive functional groups thereon. In some cases, such nanostructures are available directly from commercial sources.

[0147] a. Polyethylene glycol (PEG) backbone or platform

[0148] In some cases, the backbone or platform of each nanostructure is or comprises linear or multi-arm polyethylene glycol (PEG). In some cases, the nanostructure comprises only multi-arm PEG. Such multi-arm PEGs can have 2, 3, 4, 5, 6, 7, 8, 9, 10 or more arms. In some cases, bi-arm PEGs include a connector or linking group between the two arms. In some cases, the nanostructure comprises only multi-arm PEGs having the same number of arms, such as 8 arms (i.e., 8PEG). 8PEG may have additional functions thereon, such as at the ends of the arms, wherein 8PEG can be 8PEGamine (8PEGA) or 8PEG maleimide (8PEGMAL).

[0149] In some embodiments, the multi-arm PEG used for imaging can be defined as a nanocomposition in which individual PEG chains are covalently linked or associated via an intermediate molecular anchor group in the form of PEG1-X-PEG2, where X represents the molecular anchor group. PEG1 and PEG2 can be the same or different PEGs of any suitable length. These PEGs described can be combined in this form and can be individually branched or linear. In this way, two or more branched or linear PEGs can be combined or associated together. The molecular anchor group can be, for example, but not limited to, block copolymers, antibodies, small molecules, heterofunctional or bifunctional linkers, or peptides.

[0150] In some cases based on the PEG main chain or platform, depending on the nanostructure, the number of protons can be greater than or less than 3,600, and can be about 30 to about 30,000, 200 to about 30,000, greater than about 500, greater than about 1,500, greater than about 4,000, greater than about 5,000, about 200 to about 10,000, about 3,000 to about 7,000, and all values ​​and subranges contained in these ranges.

[0151] In some cases, a multi-arm PEG has one or more additives associated with it. In some cases, a multi-arm PEG has one or more additives associated with it, and at least one arm of the multi-arm PEG does not contain said one or more additives, meaning that at least one arm does not contain additives associated with the other arms of the multi-arm PEG. In some cases, a multi-arm PEG has at least one free arm; at least two free arms; at least three free arms; at least four free arms; at least five free arms; at least six free arms; at least seven free arms; at least eight free arms; or at least nine free arms; it should be understood that at least one arm has an additive associated with it.

[0152] In some cases, the one or more additives present on a multi-arm NC include at least two additives, such as a targeting agent (TA) and a therapeutic agent (i.e., a photosensitizer, a chemotherapeutic agent, a radiotherapy agent, etc.). In some such cases, the ratio of the at least two additives can be selected as 2:1, 3:1, 4:1, and 5:1, depending on the number of arms present on the multi-arm NC.

[0153] In some cases, the main chain of the nanostructure is itself made of or primarily of polyethylene glycol (PEG), for example, at least about 85% PEG, at least 90% PEG, at least 95% PEG, at least 99% PEG, at least 99.9% PEG, and up to 100% PEG.

[0154] In one example, the multi-arm PEG is a polyethylene glycol amine, such as an 8-arm polyethylene glycol amine (8PEGA), which is a non-limiting example allowing for a range of modified biocompatible polymers. Typically, the amine group can be used to covalently anchor a range of additives. In some other cases, the other arms of the polymer can be converted to maleimide groups to provide, for example, multi-arm polyethylene glycol maleimides, such as 8-arm polyethylene glycol maleamide (8PEGMAL), allowing for the linkage of various peptides, such as cysteine-terminated peptides. Exemplary processes are shown in... Figure 1C middle.

[0155] In some non-limiting cases, for PEG-based NC, the inherent flexibility of the polyethylene oxide chain and the slow translational diffusion create a set of available physical and dynamic conditions for selective MR imaging of such PEG protons using 1H NMR. Specifically, the rapid chain motion of PEG with a correlation time of approximately 0.1 nanoseconds provides sufficient averaging of proton dipole-dipole interactions to produce a long nuclear spin transverse relaxation time T2. In contrast to the rapid internal chain dynamics, the high molecular weight of PEG molecules produces a translational diffusion constant that is two orders of magnitude slower than that of water molecules. Therefore, the water signal can be effectively suppressed by a large diffusion gradient, allowing only the ethylene oxide signal to be preserved due to its long T2 time and slow diffusion. For example, the MR signal intensity attenuation is:

[0156] M xy (b, TE) = M xy (0)e^(-bD e -TE / T2)

[0157] Here, the b-value is determined by the magnitude and duration of the magnetic field gradient, D is the translational diffusion constant of water or PEG, such as 8PEG, TE is the echo time, and T2 is the transverse spin relaxation time. Furthermore, the symmetry of the ethylene oxide monomer causes a single chemical shift of all four protons, and when evaluating 8PEG, each 40 kDa polymer molecule carries approximately 3,600 protons, resulting in a large molar amplification of the NMR or MRI signal. By performing diffusion-weighted fast spin echo (FSE) with a high b-value and a long TE time, the water signal due to rapid diffusion and the lipid signal due to the short T2 time can be effectively suppressed, and selective imaging of PEG-based NC signals is possible. In vivo, due to the limited diffusion of water molecules within cells, a small fraction of the water signal intensity at high b-values ​​may remain unchanged, but due to conventional... 1 There is a -1 ppm difference between water and ethylene glycol protons in ¹H NMR, and these signals can be removed or distinguished from PEG-based NC signals.

[0158] 2. Additives

[0159] In the described method, the nanostructure may have one or more additives associated with it. These additives include activators, which may be selected from the group consisting of: targeting agents, therapeutic agents, chemotherapeutic agents, photosensitizers, sonosensitizers, imaging agents, diagnostic agents, photoacoustic agents, therapeutic diagnostic agents, and combinations thereof.

[0160] For one or more additives associated with the nanostructure of the MRI agent, this association with the NC backbone or platform allows for easy quantification of the additive via imaging of the MRI agent. In other words, the known amount of additive present on the NC of the MRI agent and the concentration of NC in the agent allow for the measurement of how much NC and how much additive are present in the image of the MRI agent. In some cases, it also allows for real-time tracking of treatment, thereby enabling imaging of the applied MRI agent at different times.

[0161] In some cases, the one or more additives can associate with the main chain or platform of the nanostructure through, but not limited to, chemical bonds (e.g., covalent, ionic, van der Waals bonds); spatially or mechanically, such as by spatial hindrance or physical capture within the main chain or platform; or through the main chain or platform. The one or more additives can be part of the molecular structure constituting the main chain or platform of the NC; and combinations and variations of the above are also possible. The one or more additives can be added before, during, or after the formation of the nanostructure, as well as combinations and variations thereof.

[0162] Methods for covalently binding one or more additives to the main chain are known to those skilled in the art. These methods may include, but are not limited to, click chemistry, and reactions between functional groups on the main chain and functional groups on the additive, wherein these functional groups may include, but are not limited to, alkyl halides, acyl halides, aromatic phenyl groups, aromatic halides (such as iodine), carboxylic acids, sulfonic acids, phosphoric acids, alcohols (such as primary alcohols), maleimides, esters, thiols, azides, aldehydes, alkenes (mono- or dienes), isocyanates, isothiocyanates, amines, acid anhydrides, or thiols. The methods and conditions required for selecting suitable reactive functional groups to achieve covalent binding, and for forming a covalent link between the main chain or platform and the additive, are well known to those skilled in the art.

[0163] The one or more additives may be known surfactants, which may include or be modified to include reactive functional groups capable of coupling with reactive functional groups present on the nanostructure, as described above. Those skilled in the art are familiar with the selection of reactive functional groups that allow such covalent coupling and synthetic methods for covalently linking surfactants to nanostructures.

[0164] In some cases, the one or more additives are active agents selected from the following: methylene blue, dihydroporphyrin e6 (Ce6), Coomassie blue, gold, tetrapyrrole compounds, cyanine dyes, porphyrin, dihydroporphyrin, phthalocyanine (such as IR700 and its derivatives), bacterial dihydroporphyrin, HPPH, TOOKAD, LUZ 11, BC19 porphyrin, phenothiazine onion salts, benzophenothiazine onion salts, oxanthracene halides, squaric acid, toluidine blue O, PP9004, EtNBS, rose bengal, ASQI, BODIPY (such as zinc(II) dimethylpyridinium diiodophosphate BODIPY or BIMPy-BODIPY), transition metal coordination compounds, and combinations thereof.

[0165] In some cases, the active agent is a photosensitizer, such as a phthalocyanine. Exemplary phthalocyanines include IR700 and its derivatives. Phthalocyanine dyes, such as IR700 and its derivatives, are described in paragraphs

[0074] -

[0080] and

[0093] -

[0113] of U.S. Publication No. 2021 / 0085790 A1, which is incorporated herein by reference. As described in U.S. Publication No. 2021 / 0085790 A1, such photosensitizers can be used as one or more additives in the nanostructures described herein.

[0166] In some cases, the one or more additives are activators of transition metal coordination compounds, and the transition metal coordination compounds include metals selected from the group consisting of: ruthenium, rhodium, platinum, gold and iridium, zinc, copper and palladium.

[0167] Other exemplary additives that act as activators may form part of nanostructures and formulations including nanostructures, including but not limited to agents that induce apoptosis; polynucleotides (e.g., antisense, ribozymes, siRNA); peptides (e.g., enzymes and antibodies (and fragments thereof)); agents that bind to (e.g., oligomerize or complex) Bcl-2 family proteins such as Bax; alkaloids; alkylating agents; antibiotics; antimetabolites; hormones; monoclonal or polyclonal antibodies (e.g., antibodies conjugated to anticancer drugs, toxins, defensins), toxins; and radioactive substances. Nucleotides; biological response modulators (e.g., interferons (e.g., IFN-α) and interleukins (e.g., IL-2); adoptive immunotherapy agents; hematopoietic growth factors; agents that induce cell differentiation (e.g., all-trans retinoic acid); gene therapy agents (e.g., antisense therapy agents and nucleotides); angiogenesis inhibitors; proteasome inhibitors; NF-κB modulators; anti-CDK compounds; HDAC inhibitors; chemotherapeutic agents (e.g., doxorubicin or cisplatin), etc. Many other examples are known to those skilled in the art.

[0168] Examples of sound-sensitizing agents include, but are not limited to: porphyrins (e.g., hematoporphyrin, diacetylhematoporphyrin-mitomycin-C conjugate, photosensitizing protein II, mesoporphyrin, protoporphyrin IX, copper protoporphyrin, tetraphenylporphyrin tetrasulfonate, ATX-70, ATX-S10, pheophytic acid-a, CIA1-phthalocyanine tetrasulfonate, and chloroPAD-S31), tenoxicam, piroxicam, Bengal rose red, erythrosine B, anthocyanin 540, dimethylformamide, Cytarabine, pyridocarbazole, 2,2'-azobis(2-aminopropane), 5,5'-dimethyl-1-pyrrolidone X-oxide, e-pyridyl-1-oxide-N-tert-butylnitrone, and anticancer agents (e.g., nitrogen mustard, cyclophosphamide, bleomycin, doxorubicin, FAD104, amphotericin B, mitomycin C, daunorubicin, cisplatin, etoposide, diaziquone, dihydroxy(oxobiguanidinyl)boron, and 5-fluorouracil) (see also, for example, Rosenthal et al., Ultrasonics Sonochemistry 11(2004) 349; incorporated herein by reference in its entirety).

[0169] Exemplary active agents that can induce or stimulate apoptosis include, but are not limited to, tumor-derived growth factor ligands, receptors, and analogues; kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitors, angiogenic growth factor receptor (VGFR) kinase inhibitors, fibroblast growth factor receptor (FGFR) kinase inhibitors; platelet-derived growth factor receptor (PDGFR) kinase inhibitors and Ber-Ab! kinase inhibitors (such as GLEEVEC)); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, BEXXAR, and AVASTIN); and antiestrogens (e.g., raloxifene and tamoxifen). Tamoxifen); anti-androgens (e.g., flutamide, bicalutamide, finasteride, aminoglutethamide, ketoconazole, and corticosteroids); cyclooxygenase-2 (COX-2) inhibitors (e.g., celecoxib, meloxicam, NS-398, and nonsteroidal anti-inflammatory drugs); anti-inflammatory drugs (e.g., phenbuprofen, DECADRON, DELTASONE, dexamethasone, and dexamethasone oral concentrate). Intensol), DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, oxyphenbutazone, PEDIAPRED, phenylbutazone, PLAQUENIL, prednisolone, prednisone, PRELONE, and TANDEARIL; and cancer chemotherapy drugs (e.g., irinotecan (CAMPTOSAR), CPT-11, fludarabine) FLUDARA, dacarbazine, dexamethasone, mitoxantrone, MYLOTARG, VP-16, cisplatin, carboplatin, oxaliplatin, 5-FU, doxorubicin, gemcitabine, bortezomib, gefitinib, bevacizumab, TAXOTERE or TAXOL; cell signaling molecules; ceramides and cytokines; astrocytocin, etc.

[0170] Exemplary alkylating agents include, but are not limited to: 1) nitrogen mustard (e.g., dichloromethyldiethylamine, cyclophosphamide, ifosfamide, melphalan (L-saccharin); and chlorambucil); 2) ethyleneimine and methylmelamine (e.g., hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine (methyl-CCNU); and streptozotocin (streptozomycin)); and 5) triazine (e.g., dacarbazine (dimethyltriazolamide)).

[0171] Exemplary antimetabolites include, but are not limited to: 1) folic acid analogs (e.g., methotrexate); 2) pyrimidine analogs (e.g., fluorouracil (5-fluorouracil), fluorouridine (fluorodeoxyuridine), and cytarabine (cytarabine); and 3) purine analogs (e.g., mercaptopurine (6-mercaptopurine), thioguanine (6-thioguanine), and pentostatin (2'-deoxycofomycin)).

[0172] Exemplary chemotherapeutic agents include, but are not limited to: 1) vinca alkaloids (e.g., vincristine, vinblastine); 2) epipodophyllotoxin (e.g., etoposide and teniposide); 3) antibiotics (e.g., daunorubicin (actinomycin D), doxorubicin (daunorubicin; erythromycin), doxorubicin, bleomycin, procainoxam (photomycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modulators (e.g., interferon-α); 6) platinum coordination complexes (e.g., cisplatin and carboplatin); 7) anthraquinones (e.g., mitoxantrone); 8) substituted ureas (e.g., hydroxyurea). ;9) Methylhydrazine derivatives (e.g., methylbenzylhydrazine (N-methylhydrazine)); 10) Adrenocortical inhibitors (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11) Adrenocortical steroids (e.g., prednisone); 12) Progesterone (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) Estrogens (e.g., diethylstilbestrol and ethinylestradiol); 14) Antiestrogens (e.g., tamoxifen); 15) Androgens (e.g., testosterone propionate and flumethasone); 16) Antiandrogens (e.g., flutamide); and 17) Gonadotropin-releasing hormone analogs (e.g., leuprorelin).

[0173] In some cases, imaging agents may be selected from magnetic materials (e.g., iron for MRI); proteins that catalyze luminescent reactions (e.g., luciferin, such as luciferase for bioluminescent imaging); fluorescent dyes (e.g., rhodamine or luciferin isothiocyanate for fluorescence imaging); fluorescent proteins (e.g., green fluorescent protein); and radioactive elements (e.g., for autoradiography).

[0174] In some cases, the one or more additives are activators, which are targeting agents that, when administered to a subject, allow the nanostructure to target specific tissues or lumens. In some cases, the targeting agents target cancer or solid tumors.

[0175] In some cases, the one or more additives are activators, i.e., peptides. In some cases, the peptides are cancer-targeting peptides, without particular limitation, such as those selected from the group consisting of RGD, NGR peptides, and iNGR; the RGD includes cRGD, iRGD, and F3. In some other cases, the peptides are cardiac-targeting peptides, without particular limitation, such as those selected from SEQ ID NO: 1 to SEQ ID NO: 48. The CTPs in Table 1 are shown below and further defined as follows. In one embodiment, there is a twelve-amino acid CTP (CTP12aa) having the sequence Ala-Pro-Trp-His-Leu-SerSer-Gln-Tyr-Ser-Arg-Thr (SEQ ID NO: 1). In one embodiment, there is a six-amino acid CTP (CTP6aa) having the sequence SQYSRT (SEQ ID NO: 5), or a twelve-amino acid CTP having the sequence AA WHLSSQYSRT (SEQ ID NO: 2 (CTP-P2A)). In some embodiments, the sequence Xaa1 Xaa2 Y Xaa3 Xaa4 T (SEQ ID NO: 4) is used, wherein Xaa1, Xaa2, Xaa3, and Xaa4 are any naturally occurring amino acids. In some embodiments, Xaa1 in CTP6aa of SEQ ID NO: 4 is serine (S). In some embodiments, Xaa2 in CTP6aa of SEQ ID NO: 4 is glutamine (Q). In some embodiments, Xaa3 in CTP6aa of SEQ ID NO: 4 is serine (S). In some embodiments, Xaa4 in CTP6aa of SEQ ID NO: 1 is arginine (R). In some embodiments, Xaa1 and Xaa2 in CTP6aa of SEQ ID NO: 4 are serine (S) and glutamine (Q), respectively. In some embodiments, both Xaa1 and Xaa3 in CTP6aa of SEQ ID NO: 4 are serine (S). In some embodiments, Xaa1 and Xaa4 in CTP6aa of SEQ ID NO: 4 are serine (S) and arginine (R), respectively. In some embodiments, Xaa2 and Xaa3 in CTP6aa of SEQ ID NO: 4 are glutamine (Q) and serine (S), respectively. In some embodiments, CTP6aa contains the sequence SQYSRT (SEQ ID NO: 5). The CTPs in Table 1 are further defined as follows. In one aspect, CTP6aa contains the sequence SQ Xaa1 SR Xaa2 (SEQ ID NO: 6).In some embodiments, Xaa1 in CTP6aa of SEQ ID NO: 6 is alanine (A), and CTP6aa contains the sequence of SQASRXaa2 (SEQ ID NO: 7), or optionally, Xaa1 in CTP6aa of SEQ ID NO: 6 is tryptophan (W), and CTP6aa contains the sequence of SQWSRXaa2 (SEQ ID NO: 8), or Xaa1 in CTP6aa of SEQ ID NO: 6 is tyrosine (Y), and CTP6aa contains the sequence of SQYSRXaa2 (SEQ ID NO: 8). In some embodiments, Xaa2 in CTP6aa of SEQ ID NO: 6 is threonine (T), and Xaa1 in CTP6aa of SEQ ID NO: 6 is alanine (A), tryptophan (W), or tyrosine (Y), comprising the sequence SQASRT (SEQ ID NO: 10), SQWSRT (SEQ ID NO: 11), or SQYSRT (SEQ ID NO: 5), respectively. In some embodiments, Xaa2 in CTP6aa of SEQ ID NO: 6 is alanine (A). In some embodiments, Xaa1 in CTP6aa of SEQ ID NO: 6 is tyrosine (Y), and Xaa2 is alanine (A). In some embodiments, CTP6aa comprises the sequence SQYSRT (SEQ ID NO: 5). The CTPs in Table 1 are further defined as follows. In some embodiments, Xaal in CTP6aa of SEQ ID NO: 4 is serine (S). In some embodiments, Xaa2 in CTP6aa of SEQ ID NO:4 is glutamine (Q). In some embodiments, Xaa3 in CTP6aa of SEQ ID NO:4 is serine (S). In some embodiments, Xaa4 in CTP6aa of SEQ ID NO:4 is arginine (R). In some embodiments, Xaal and Xaa2 in CTP6aa of SEQ ID NO:4 are serine (S) and glutamine (Q), respectively. In some embodiments, Xaa1 and Xaa3 in CTP6aa of SEQ ID NO:4 are both serine (S). In some embodiments, Xaal and Xaa4 in CTP6aa of SEQ ID NO:4 are serine (S) and arginine (Q), respectively. In some embodiments, Xaa2 and Xaa3 in CTP6aa of SEQ ID NO:4 are glutamine (Q) and serine (S), respectively. In some embodiments, CTP6aa contains the sequence SQYSRT (SEQ ID NO: 5).In some embodiments, the peptide comprises CTP6aa, wherein the CTP6aa contains the sequence Xaa1Xaa2WXaa3Xaa4T (SEQ ID NO: 23), wherein Xaa1, Xaa2, Xaa3, and Xaa4 are any naturally occurring amino acids. In some embodiments, Xaa1 in the CTP6aa of SEQ ID NO: 6 is alanine (A), and the CTP6aa contains the sequence SQASRXaa2 (SEQ ID NO: 7), or optionally, Xaa1 in the CTP6aa of SEQ ID NO: 6 is tryptophan (W), and the CTP6aa contains the sequence SQWSRXaa2 (SEQ ID NO: 8), or Xaa1 in the CTP6aa of SEQ ID NO: 6 is tyrosine (Y), and the CTP6aa contains the sequence SQYSRXaa2 (SEQ ID NO: 9). In some embodiments, Xaa2 in CTP6aa of SEQ ID NO: 6 is threonine (T), and Xaai in CTP6aa of SEQ ID NO: 6 is alanine (A), tryptophan (W), or tyrosine (Y), comprising the sequence SQASRT (SEQ ID NO: 10), SQWSRT (SEQ ID NO: 11), or SQYSRT (SEQ ID NO: 5), respectively. In some embodiments, Xaa2 in CTP6aa of SEQ ID NO: 6 is alanine (A). In some embodiments, Xaa1 in CTP6aa of SEQ ID NO: 6 is tyrosine (Y), and Xaa2 is alanine (A). In some embodiments, CTP6aa comprises the sequence SQYSRT (SEQ ID NO: 5). In some embodiments, the peptide comprising CTP6aa of SEQ ID NO: 4 and SEQ ID NO: 6, such as SEQ ID NO: 5, is a recombinant or synthetically prepared peptide.

[0176] Table 1. Cardiac-targeting peptides used as targeting agents

[0177]

[0178] SEQ ID NO: 1-35 is disclosed and taught in WO 2019 / 226785.

[0179] SEQ ID NO: 36-48 (and corresponding to sequence numbers 1-13 of U.S. Patent No. 9,249,184) are disclosed and taught.

[0180] In some cases, the described methods and the one or more additives do not include imaging dyes or reagents, or other such materials or compounds that may be considered undesirable (especially for human use). Such undesirable dyes and reagents may be heavy metal-based and use or contain metals, metal oxides, or metal compounds or complexes, such as iron, iron-platinum, magnesium, and manganese. Such less desirable materials or compounds may include gadolinium-based materials. Therefore, nanostructures may be free of or substantially free of heavy metals (such as gadolinium), for example, nanostructures containing less than about 10 ppm of heavy metals, less than about 1 ppm of heavy metals, less than about 0.1 ppm of heavy metals, and zero heavy metals. Heavy metals will include titanium and all heavier metals.

[0181] For some of the additives described, such as photosensitizers and sound sensitizers, it is understood that these active agents may require the use of an activator to activate such reagents. Any activator that activates an active agent can be used. Typically, the activator provides an energy source that causes, for example, the active agent to release energy (e.g., in the form of free radicals), thereby causing cell death or destruction. Exemplary activators include...

[0182] However, it is not limited to light, heat, radiation, sound, etc. In some cases, activators can be pharmaceutical agents that activate therapeutic agents (such as chemotherapy agents). For example, verapamil can be used to activate or improve the efficacy of chemotherapy agents (such as doxorubicin).

[0183] Various activators, activation systems, and methods and conditions for applying them to additives to activate them are known in the art. For example, an activator can be sound (e.g., sonodynamic therapy). Sonodynamic therapy is based on the synergistic effect of ultrasound with a sonosensitive agent. The effect can be localized by focusing ultrasound onto a defined area (e.g., a target tissue region). Ultrasound can be delivered percutaneously to a specific area of ​​the target tissue. In sonodynamic therapy, the ultrasound-dependent enhancement of the cytotoxic activity of certain compounds (sonic sensitizers) can be based on the application of ultrasound, a mechanical wave in which particles periodically vibrate in a continuous elastic medium at a frequency equal to or greater than 20 kHz. In liquids, its velocity of approximately 1000–1600 m / s translates to a wavelength range of micrometers to centimeters. In some cases, sound is generated in vitro and targeted to a desired treatment area via tissue, where a nanostructure containing a sonosensitive agent is present.

[0184] In some cases of the described methods, the aforementioned additives may be administered concurrently during the method without associating with the nanostructure. Methods for administering such additives to subjects are known to those skilled in the art.

[0185] Example

[0186] Example 1: Fast spin echo (FSE) imaging of PEG nanostructures

[0187] Materials and methods:

[0188] 7.5 mm NMR tubes were prepared using 40 kDa 8PEG (JenKem, Inc.) surrounded by agarose gel (Sigma) at different concentrations (0, 0.05, 0.1, 0.5, 1.0, 3.0 and 5.0 mg / mL).

[0189] Selective spin echo and selective fast spin echo (not shown) images of different concentrations of PEG were obtained using a 7T animal imaging system manufactured by Varian. These images show that the noise in the spin echo images is much higher than that in the fast spin echo images. Both conventional diffusion-weighted spin echo and diffusion-weighted fast spin echo NMR images of PEG protons were acquired within a total imaging time of 6 minutes.

[0190] Results and discussion:

[0191] 8PEG diffusion filtering and T2 filtering fast spin echo imaging allow for single k-space sampling of the PEG proton signal. Six minutes of signal averaging produces a PEG proton image with a signal 30 times stronger than that of conventional spin echo imaging (see [link to image]). Figure 2 Imaging of PEG concentrations using fast spin echo is possible at concentrations as low as 0.05 mg / ml.

[0192] Example 2: Measurement of T2 and translational diffusion coefficient (TDC) of PEG

[0193] Materials and methods:

[0194] Samples were prepared by adding the described PEG (see Table 2 below) to separate tubes and dissolving it in 99.9% D2O. More specifically, samples were prepared by dissolving deuterium oxide (D2O) at a concentration of 10 mg / mL and scanned on a 700 MHz Varian / Agilent high-resolution NMR spectrometer at 25 °C.

[0195] As part of the Varian DOSY toolbox, diffusion was estimated using a convection-compensated stimulated echo NMR pulse sequence. Diffusion delay times of D = 100 ms and d = 2 ms were used at magnetic field gradients sufficient to produce b = 59,320 s / mm². Specifically, twenty-one b values ​​were used: 8.98, 2972, 5935, 8902, 11865, 14830, 17794, 20757, 23728, 26690, 29657, 32619, 35594, 38551, 41519, 44487, 47443, 50410, 53380, 56347, and 59320 s / mm². The diffusion decay data were fitted to an exponential function in Matlab R200b using the curve fitting toolbox, and the translational diffusion coefficient (TDC) was estimated.

[0196] The T2 value was estimated using a CPMGT2 pulse sequence provided by Varian, with data acquired as a function of TE time. The experiment was repeated for 10 seconds, and the TE time varied between 37.5 ms and 9.6 ms. Four transients were acquired and averaged. The data were analyzed on a Varian scanner using an exponential decay model and the Levenberg-Marquardt nonlinear least squares algorithm to provide an estimate of the T2 value.

[0197] The T1 value was estimated using an INVREC (Inversion Recovery) pulse sequence provided by Varian, to acquire data as a function of the inversion recovery time. The repetition times of the acquisition experiments ranged from 10 seconds to 62 milliseconds, with inversion recovery times ranging from 8 seconds to 10 seconds. The data were fitted to the inversion recovery model using the Levenberg-Marquardt nonlinear least squares algorithm on a Varian scanner, and the T1 time was estimated.

[0198] result:

[0199] Table 2 below shows the T2 and TDC values ​​for the various PEGs evaluated:

[0200] Table 2. Results of analysis of T1, T2 and TDC values ​​for various PEGs

[0201]

[0202] Example 3: Inhibition of fat signaling in DWFSE of PEG in a phantom

[0203] Materials and methods:

[0204] A series of materials (i.e., imaging phantoms) with MRI properties mimicking those found in vivo were prepared. These materials consist of water in a central and surrounding vial, fat in the vials at 1:00, 5:00, and 9:00, and PEG dissolved in water at 3:00, 7:00, and 11:00, as shown in the figure. Figures 4A-4C In the middle. A conventional water image is shown in Figure 4C This shows the high signal intensity in the central vial because it is doped with Mn to provide the tissue T2 value.

[0205] In a mouse study, Matrigel implants containing 100 μL of Matrigel were placed in two flank locations on a single mouse weighing 25.4 g. Each implant contained 10 mg / ml PEG (in one implant) and 2 mg / ml PEG (in the other). Standard water proton fast spin-echo MRI scans were performed at TR = 2000 ms and TE = 10 ms to visualize the Matrigel implants.

[0206] Results and discussion:

[0207] Figure 4B The images in the figure illustrate the selective PEG imaging method without fat suppression. The fat signal in this phantom was derived from peanut oil, which has been shown to have properties similar to in vivo adipose tissue. This indicates that the fat signal is dominant in the PEG images.

[0208] Figure 4C The images in the figure show selective PEG imaging with the application of fine-tuned fat suppression signals. The intensity and frequency of the RF pulses were fine-tuned to provide fat saturation, thereby revealing clear PEG signals at concentrations of 2, 4, and 10 mg / ml.

[0209] In vivo, signals from adipose tissue, namely fat signals, are not adequately suppressed by current PEG imaging protocols and are clearly visible by creating images of PEG and fat proton superposition.

[0210] In mouse studies, PEG images of water and fat suppression were acquired (images not shown). The combination of a b-value of 5000 s / mm² and fat suppression also allowed for selective PEG imaging. MRI images taken without such a b-value and TE time would show a conventional water proton image. Adding a b-value of 5000 s / mm² and TE = 245 ms effectively suppressed the water signal but not sufficiently suppressed the long T2 fat signal, thus creating an image with superimposed PEG and fat signals. Adding fat suppression in combination with a high b-value and long TE time sufficiently reduced both water and fat signals, thus creating a PEG-selective image.

[0211] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. All publications cited herein and the materials referenced therein are specifically incorporated herein by reference.

[0212] Those skilled in the art will recognize, or be able to determine, the specific features of the invention described herein using only conventional experiments. Such equivalents are intended to be covered by the appended claims.

Claims

1. A method of imaging a magnetic resonance imaging (MRI) agent, the method comprising the steps of: (a) administering the MRI agent to a subject, wherein the MRI agent comprises nanoconstructs each having a plurality of equivalent protons and an average molecular weight in a range of about 300 Da to about 300 kDa, and a T2 relaxation time of at least about 250 milliseconds for each of the nanoconstructs; (b) using an MRI device to obtain a nuclear magnetic resonance scan of the nanoconstructs, wherein step (b) comprises: (i) applying diffusion filtering to suppress water signals from the scan and selectively image the nanoconstructs; (ii) applying a signal acquisition bandwidth in a range of at least about 2 kHz to about 200 KHz; and (iii) obtaining greater than 8 spin echoes, optionally 9 to 64 or 9 to 128 spin echoes; optionally wherein step (b) further comprises modifying a gain of a receiver-amplifier of the MRI device.

2. The method of claim 1, wherein step (b) further comprises modifying a gain of a receiver-amplifier of the MRI device.

3. The method of claim 1, wherein step (b) does not further comprise modifying a gain of a receiver-amplifier of the MRI device.

4. The method of any one of claims 1 to 3, wherein the translational diffusion coefficient of each of the nanoconstructs is less than about 2 x 10 -10 m 2 s -1 .

5. The method of claim 1, wherein step (iii) further comprises obtaining at least 3 gradient echoes, optionally 3 to 7 gradient echoes.

6. The method of claim 5, wherein step (iii) further comprises obtaining 3 to 7 gradient echoes.

7. The method of claim 5, wherein step (iii) further comprises obtaining more than 7 gradient echoes.

8. The method of any one of claims 1 to 7, wherein each of the nanoconstructs has an average molecular weight in a range of about 3 kDa to about 300 kDa, about 10 kDa to about 300 kDa, or about 3 kDa to about 250 kDa.

9. The method of any one of claims 1 to 8, wherein each of the nanoconstructs has an average molecular weight in a range of about 10 kDa to about 50 kDa.

10. The method of any one of claims 1 to 5, wherein the T2 relaxation time is at least about 550 milliseconds to about 900 milliseconds or about 600 milliseconds to about 900 milliseconds.

11. The method of claim 1, wherein the signal acquisition bandwidth is about 2 kHz to about 50 kHz.

12. The method of claim 5, wherein the signal acquisition bandwidth is about 5 kHz to about 100 kHz.

13. The method of any one of claims 1 to 12, wherein at least 16, 32, 64, or 128 spin echoes are obtained during step (b).

14. The method of any one of claims 1 to 13, wherein the plurality of equivalent protons comprises about 30 to 27,000 protons, 200 to about 15,000 protons, or about 200 to about 10,000 protons.

15. The method of any one of claims 1 to 14, wherein each of the nanoconstructs comprises a linear or multi-armed polyethylene glycol (PEG); optionally wherein the multi-armed PEG has 2, 3, 4, 5, 6, 7, 8, 9, 10, or more arms.

16. The method of claim 15, wherein the multi-armed PEG has 2, 3, 4, 5, 6, 7, 8, 9, 10, or more arms.

17. The method of claim 15 or 16, wherein the multi-armed PEG is an 8PEG, and the 8PEG is optionally selected from the group consisting of 8PEG A and 8PEG MAL.

18. The method of any one of claims 15 to 17, wherein the 8PEG is selected from the group consisting of 8PEG A and 8PEG MAL.

19. The method of any one of claims 15 to 18, wherein each of the nanoconstructs further comprises one or more additives associated with the multi-armed PEG.

20. The method of claim 19, wherein the one or more additives comprise an active agent selected from the group consisting of a targeting agent, a therapeutic agent, a chemotherapeutic agent, a photosensitizing agent, a sonosensitizing agent, an imaging agent, a diagnostic agent, a photoacoustic agent, a theranostic agent, and combinations thereof; optionally wherein the active agent is covalently associated with the multi-armed PEG.

21. The method of claim 20, wherein the active agent is covalently associated with the multi-armed PEG.

22. The method of claim 19, wherein the one or more additives comprise an active agent selected from the group consisting of methylene blue, chlorin e6 (Ce6), coomassie blue, gold, a tetrapyrrole compound, a cyanine dye, a porphyrin, a chlorin, a phthalocyanine, IR700 and derivatives thereof, a bacteriochlorin, HPPH, TOOKAD, LUZ 11, BC19 porphyrin, a phenothiazinium salt, a benzophenothiazinium salt, a halogenated xanthene, squaraine, toluidine blue O, PP9004, EtNBS, rose bengal, ASQI, a BODIPY (such as zinc(II) dimethylpyridinum diiodide BODIPY or BIMPy-BODIPY), a transition metal coordination compound, and combinations thereof.

23. The method of claim 22, wherein the BODIPY is zinc(II) dimethylpyridinum diiodide BODIPY or BIMPy-BODIPY.

24. The method of claim 19, wherein the one or more additives comprise an active agent that is a transition metal coordination compound, and wherein the transition metal coordination compound comprises a metal selected from the group consisting of ruthenium, rhodium, platinum, gold, and iridium, zinc, copper, and palladium.

25. The method of claim 19, wherein the one or more additives comprise a targeting agent, optionally wherein the targeting agent is a cancer targeting agent; optionally wherein the cancer targeting agent is selected from the group consisting of RGDs, including cRGD, iRGD, and F3, and NGR peptides and iNGRs.

26. The method of claim 25, wherein the targeting agent is a cancer targeting agent.

27. The method of claim 26, wherein the cancer targeting agent is selected from the group consisting of RGDs, NGR peptides, and iNGRs.

28. The method of claim 27, wherein the RGD is cRGD, iRGD, or F3.

29. The method of claim 19, wherein the one or more additives comprise a targeting agent, optionally wherein the targeting agent is a heart targeting agent; optionally wherein the heart targeting agent is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

48.

30. The method of claim 29, wherein the targeting agent is a heart targeting agent.

31. The method of claim 30, wherein the heart targeting agent is selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO:

48.

32. The method of claim 19, wherein the one or more additives comprise a photosensitizer that is a phthalocyanine dye, optionally wherein the phthalocyanine dye is IR700.

33. The method of claim 32, wherein the phthalocyanine dye is IR700.

34. The method of any one of claims 15 to 33, wherein at least one arm of the multi-armed PEG does not contain the one or more additives.

35. The method of any one of claims 1 to 34, wherein the average diameter of each of the nanoconstructs is less than about 50 nm.

36. The method of any one of claims 19 to 35, wherein the one or more additives comprise a targeting agent that is a tumor targeting agent; and wherein the method further comprises, after step (b), step (c) providing data identifying the shape, structure, and / or location of a tumor in the subject.

37. The method of any one of claims 1 to 36, further comprising: after step (b), step (d) providing data regarding the concentration of the nanoconstructs in the subject.

38. The method of any one of claims 19 to 35, further comprising: after step (b), step (e) providing data regarding the concentration of the nanoconstructs in the subject and the one or more additives associated with the multi-armed PEG in the subject.

39. The method of any one of claims 1 to 38, wherein step (b) further comprises applying one or more fat saturation techniques.

Images