Small, highly uniform nanoparticle drug compositions for therapeutic, imaging, and theranostic applications

Abstract

A targetable nanoconstruct capable of serving as both a therapeutic platform for photodynamic therapy and an MR molecular imaging agent without heavy metal atoms. A F3‑cys targeting agent nanoconstruct containing 8PEGA‑Ce6NC. A label-free 8PEGA nanoconstruct that can be directly and selectively imaged by MRI using a standard spin echo imaging sequence with a large diffusion magnetic field gradient to suppress water signals.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of the filing date of U.S. Provisional Application Serial No. 62 / 730,882, filed on September 13, 2018, under 35 U.S.C. §119(E)(1), the entire disclosure of which is incorporated herein by reference.

[0003] Government Interest Statement

[0004] This invention was made with government support under Grant CA 186769 from the National Institutes of Health. The government has certain rights in this invention. Technical Field

[0005] The present invention generally relates to nanoconstructs and the use of these constructs in dynamic therapy, imaging, diagnosis, theranostic and other applications.

[0006] Unless otherwise indicated, the terms "nanoparticle", "nanomaterial", "nanoparticles", "nanoproduct", "nanoplatform", "nanoconstruct", "nanocomposite", "nano" and similar such terms will be given their broadest possible meaning and include particles, materials and compositions having a certain volume shape, at least one dimension of which is about 1 nanometer (nm) to about 100 nm. Preferably, in embodiments, the maximum cross-section of these volume shapes is about 1 nm to about 100 nm.

[0007] Unless otherwise indicated, the terms "nanoconstruct", "nanoplatform", "nanocomposite" and "nanoconstruct" and similar such terms will be given their broadest possible meaning and include particles having a backbone material (e.g., a scaffold, support or matrix material) and one or more additives (e.g., agents, parts, compositions, biological agents and molecules) associated with the backbone. Typically, the backbone material can be a nanoparticle. Typically, the additive is an active material with targeting, treatment, imaging, diagnosis, theranostic or other capabilities and combinations and variations of these capabilities. In embodiments, the backbone material can be an active material with targeting, treatment, imaging, diagnosis, theranostic or other capabilities and combinations and variations of these capabilities. In embodiments, both the additive and the backbone material are active materials. One, two, three or more different types of backbone materials, additives and combinations and variations of these are contemplated.

[0008] Unless otherwise indicated, the term "theranostic" is to be given its broadest possible meaning and encompasses particles, agents, constructs or materials with a variety of capabilities and functions, including imaging and therapeutic capabilities, diagnostic and therapeutic capabilities, and combinations and variations of these capabilities and functions and other features (such as targeting).

[0009] Unless otherwise indicated, the terms "imaging," "imaging agents," "imaging devices," and similar such terms should be given their broadest possible meaning and will include devices, agents, and materials that enhance, provide, or enable the ability to detect, analyze, and visualize structures, and specifically the size, shape, location, composition, and combinations and variations of these, and other characteristics of structures in animals, mammals, and humans. Imaging agents will include contrast agents, die, and similar types of materials. Examples of imaging devices and methods include: X-ray; magnetic resonance; computer axial tomography (CAT scan); proton emission tomography (PET scan); ultrasound; fluorescence; and photoacoustic.

[0010] Unless otherwise indicated, the term "diagnosis" shall be given its broadest possible meaning and shall encompass identification, determination, definition, and combinations and variations of conditions, diseases, and both, including conditions and diseases in animals, mammals, and humans.

[0011] Unless otherwise indicated, the terms "therapeutic" and "therapy" and similar such terms are to be given the broadest possible meaning and are to encompass addressing, treating, managing, alleviating, curing, preventing, and combinations and variations of these of conditions and diseases, including those of animals, mammals, and humans.

[0012] Unless otherwise expressly stated, the terms "photodynamic therapy", "PDT", "photosensitizer", "PS" and similar such terms will be given their broadest possible meaning and will include methods for ablating (e.g., killing) biological tissue by photooxidation using photosensitizer (PS) molecules. When the photosensitizer is exposed to light of a specific wavelength, it produces a form of oxygen that kills nearby cells, such as reactive oxygen species ("ROS"), including any form of oxygen that is cytotoxic to cells. It is understood that although light across all wavelengths (e.g., UV to visible light to IR) is generally used as an activator for PS.

[0013] The terms "activated dynamic therapy," "dynamic therapy," "dynamic therapeutic agent," and similar such terms should be given their broadest possible meaning and will include PDT and PS as well as agents that trigger the production of reactive oxygen species, such as reactive oxygen species (ROS) or other active therapeutic materials that can act as activators when exposed to energy other than light. These will include materials or substances that are activated by energy such as radio waves, other electromagnetic radiation, magnetism, and sound waves (e.g., sonodynamic therapy or SDT).

[0014] As used herein, room temperature is 25° C. unless otherwise specified. Also, standard ambient temperature and pressure are 25° C. and 1 atmosphere. Unless otherwise expressly stated, all tests, test results, physical properties, and temperature-related, pressure-related, or both-related values ​​are provided at standard ambient temperature and pressure, which will include viscosity.

[0015] Generally, unless otherwise indicated, the term "about" and the symbol It is meant to encompass a variance or range of ±10%, the experimental or instrument error associated with obtaining the stated value, and preferably the greater of these.

[0016] As used herein, unless otherwise indicated, recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each separate value within the range is incorporated into the specification as if it were individually recited herein.

[0017] This background section is intended to introduce various aspects of the art that may be associated with embodiments of the present invention. Therefore, the foregoing discussion in this section provides a framework for a better understanding of the present invention and should not be regarded as an admission of prior art. Summary of the invention

[0018] There is a long-standing and unmet need for new and innovative drugs, medical products and imaging agents that address pathological conditions in animals, mammals and humans. Specifically, this long-standing and unmet need exists in the use of heavy metals in cancer diagnosis and treatment, other pathological conditions in mammals and humans, and MRI imaging agents.

[0019] The present invention addresses these needs, among other things, by providing the compositions, materials, articles, apparatus, methods and processes taught, disclosed and claimed herein.

[0020] A composition having therapeutic, imaging, diagnostic or therapeutic diagnostic applications is provided, the composition comprising: a plurality of nanoparticles, wherein the nanoparticles include a backbone material; an active agent, the active agent being attached to the backbone; and wherein the plurality of nanoparticles have a predetermined particle size distribution defined by D10=n-5, D50=n, D90=x+5.

[0021] In addition, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided, which have one or more of the following features: wherein n is a number in the range of about 5 nm to about 25 nm; wherein n is a number in the range of 7 nm to 22 nm; wherein n is a number in the range of about 10 nm to about 20 nm; wherein n is a number in the range of about 11 nm to about 15 nm; wherein the active agent is a photosensitizer; wherein the active agent is a photoacoustic agent; wherein the active agent is a sonosensitizer; having a second active agent; having a second active agent, wherein the second active agent is different from the active agent; wherein the active agent is selected from the group consisting of methylene blue, dihydrochlorin e6 (Ce6), Coomassie blue and gold; wherein the active agent is a tetrapyrrole; wherein the active agent is selected from the group consisting of porphyrin, dihydrochlorin, phthalocyanine and bacteriochlorin; wherein the active agent is selected from the group consisting of HPPH, TOOKAD, LUZ 11 and BC19 porphyrins; wherein the active agent is selected from the group consisting of: phenothiazine salts, benzophenothiazine salts, halogenated xanthenes, and squaric acids; wherein the active agent is selected from the group consisting of dyes: methylene blue, toluidine blue O, PP9004, EtNBS, Bengal rose red, ASQI, zinc (II) dimethylpyridinium amine diiodo-BODIPY and BIMPy-BODIPY; wherein the active agent is a transition metal coordination compound; wherein the active agent is a transition metal coordination compound having a metal selected from the group consisting of ruthenium, rhodium, platinum, gold and iridium, zinc, copper and palladium; wherein the nanoparticle is 8PEGA; wherein the nanoparticle is BiPEG; wherein the nanoparticle includes a targeting agent; wherein the nanoparticle includes a targeting agent, wherein the targeting agent is F3-cys; and, wherein the nanoparticle is 8PEGA, wherein the active agent is Ce6, and wherein the nanoparticle includes a targeting agent, wherein the targeting agent is F3-cys.

[0022] Further, a composition having therapeutic, imaging, diagnostic and therapeutic diagnostic applications is provided, the composition having: a plurality of nanoparticles, wherein the nanoparticles include a backbone material composed of PEG; an active agent, wherein the active agent is attached to the backbone, thereby defining a plurality of nanoconstructs; wherein the plurality of nanoconstructs have a narrower particle size distribution defined by D10=n-5, D50=n, D90=x+5; and wherein the plurality of nanoconstructs are capable of performing therapeutic, imaging, diagnostic and therapeutic diagnostic applications.

[0023] Still further, a composition for destroying tumor cells is provided, the composition comprising: an excipient having a plurality of nanoparticles; a photosensitizer associated with the excipient; wherein the excipient has a backbone substantially consisting of PEG; and wherein the excipient has a particle size distribution defined by D10=n-5, D50=n, D90=x+5.

[0024] In addition, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided, which have one or more of the following features: wherein n is a number in the range of about 5 nm to about 25 nm, wherein the nanoparticle is 8PEGA, and wherein the active agent is Ce6; having a targeting agent; and wherein the targeting agent is F3-cys.

[0025] Further, a method for obtaining data for guiding therapeutic applications is provided, the method having the following steps: administering an imaging agent having a plurality of nanoparticles to a subject; the nanoparticles do not contain heavy metals; and, performing a magnetic resonance imaging scan on the subject after administering the imaging agent; wherein the nanoparticles are directly imaged; thereby providing MRI of the nanoparticles and data related to the nanoparticles and the subject.

[0026] Further, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided, having one or more of the following features: wherein the nanoparticles include PEG; wherein the nanoparticles include 8PEGA; wherein the nanoparticles define a therapeutic nanoconstruct; wherein the nanoparticles define a PDT nanoconstruct; wherein the data identifies the shape and location of the tumor; the data is further used, at least in part, to provide PDT; the data is further used, at least in part, to provide PDT; and an MRI of the nanoparticles is obtained after providing the PDT; the data is further provided to a PDT system; and, the data is further provided to a medical record.

[0027] Still further, a method of providing PDT is provided, the method having: obtaining data from an MRI of nanoparticles in a subject; and, using, at least in part, the data to provide PDT; wherein the nanoparticles are substantially free of heavy metals.

[0028] In addition, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided, which have one or more of the following characteristics: wherein the nanoparticles have less than 1 ppm of heavy metals; wherein the nanoparticles have less than 0.1 ppm of heavy metals; wherein the nanoparticles have less than 0.01 ppm of heavy metals; wherein the nanoparticles have less than 0.001 ppm of heavy metals.

[0029] Additionally, a method of providing PDT is provided, the method having: obtaining data from an MRI of nanoparticles in a subject; and, using the data, at least in part, to provide PDT; wherein the nanoparticles are substantially free of gadolinium.

[0030] Still further, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided, having one or more of the following features: wherein the nanoparticles have less than 1 ppm heavy metals; wherein the nanoparticles have less than 1 ppm gadolinium; wherein the nanoparticles have less than 0.1 ppm gadolinium; wherein the nanoparticles have less than 0.01 ppm gadolinium; wherein the nanoparticles have less than 0.001 ppm gadolinium.

[0031] In addition, a method for obtaining data for guiding therapeutic applications is provided, the method comprising: administering an imaging agent having a plurality of nanoparticles to a subject; the nanoparticles being substantially free of gadolinium; and performing a magnetic resonance imaging scan on the subject after administration of the imaging agent; wherein the nanoparticles are directly imaged; thereby providing MRI of the nanoparticles and data related to the nanoparticles and the subject.

[0032] Additionally, a method of developing PDT is provided, the method having: obtaining data from MRI of nanoparticles in a subject; and, using the data, at least in part, to develop PDT; wherein the nanoparticles are substantially free of heavy metals.

[0033] Still further, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided, having one or more of the following features: wherein the development of the PDT has an evaluation of photosensitizers; wherein the development of the PDT has an evaluation of targeting agents; wherein the development of the PDT has an evaluation of nanoconstructs; wherein the data has direct NMR images of nanoparticles; and wherein the subject is selected from the group consisting of animals, mammals and humans.

[0034] Additionally, a method of developing a therapy is provided, the method having: obtaining data from an MRI of a nanoparticle; and, using the data, at least in part, to develop a therapy; wherein the nanoparticle has less than 1 ppm of gadolinium.

[0035] Still further, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided having one or more of the following features: wherein the development of the therapy has an assessment selected from the group consisting of: drug development, cancer treatment progress, cardiac condition development, genetic material analysis, reaction pathway analysis and pharmacology; wherein the nanoparticles are imaged in vivo; and, wherein the nanoparticles are imaged in vitro.

[0036] Additionally, a method of developing a material is provided, the method comprising: obtaining data from MRI of nanoparticles; and, using the data, at least in part, to develop a material; wherein the nanoparticles have less than 1 ppm of gadolinium.

[0037] Further, a method of evaluating a subject is provided, the method comprising: obtaining data from an MRI of nanoparticles; and, using the data, at least in part, to evaluate the subject; wherein the nanoparticles have less than 1 ppm of gadolinium.

[0038] Still further, provided are these nanoconstructs, nanoparticles, agents, compositions, methods and devices having one or more of the following features: wherein the subject is selected from the group consisting of a material, a drug, a process, a reaction pathway and a manufacturing method.

[0039] Furthermore, a nuclear magnetic resonance imaging agent is provided, which has: a plurality of nanoparticles, wherein the nanoparticles are substantially free of heavy metals; and nanoparticles having PEG; wherein the nanoparticles can be directly imaged by a magnetic field generated by a magnetic resonance imaging system.

[0040] In addition, a magnetic resonance imaging agent is provided, which has: a plurality of nanoparticles, wherein the nanoparticles have PEG; wherein the nanoparticles are capable of being directly imaged by static, gradient, and radio frequency (RF) magnetic fields generated by a magnetic resonance imaging system, and thereby generating images of the nanoparticles; and, wherein the imaging agent is substantially free of heavy metals.

[0041] Still further, a nuclear magnetic resonance imaging agent is provided, the imaging agent comprising: a plurality of nanoparticles, the nanoparticles having less than 1 ppm of gadolinium; nanoparticles having PEG; wherein the nanoparticles can be directly imaged by a magnetic field generated by a magnetic resonance imaging system.

[0042] Furthermore, a nuclear magnetic resonance imaging agent is provided, which has: a plurality of nanoparticles, wherein the nanoparticles have PEG; wherein the nanoparticles can be directly imaged by a magnetic field generated by a magnetic resonance imaging system, thereby generating an image of the nanoparticles; and wherein the imaging agent has less than 1 ppm of gadolinium.

[0043] Still further, an imaging agent having nanoparticles is provided, wherein the nanoparticles can be directly imaged by a magnetic field in a magnetic resonance imaging device, wherein the nanoparticles have: a nanoconstruct having a skeleton material, wherein the skeleton material is non-paramagnetic; and the nanoconstruct can be directly imaged by a magnetic field.

[0044] Still further, these nanoconstructs, nanoparticles, agents, compositions, methods and devices are provided, having one or more of the following features: wherein the nanoconstruct has about 3,600 protons; and, wherein the nanoconstruct is less than 25 nm; wherein the nanoconstruct has a photosensitizer; wherein the nanoconstruct has a targeting agent; wherein the nanoconstruct has a targeting agent and an imaging agent; and wherein the nanoconstruct is pro-tumorigenic.

[0045] Still further, a method of performing therapy in an MRI while directly obtaining images of at least one of the imaging agents or the nanoparticles is provided.

[0046] Still further, provided are these nanoconstructs, nanoparticles, agents, compositions, methods, and devices having one or more of the following features: wherein the therapy comprises surgery; and wherein the therapy comprises PDT.

[0047] In an embodiment, MRI imaging of nanoparticles with PEG is accomplished by using conventional MR pulse sequences, wherein components are added to selectively visualize the nanoparticle MRI signals and suppress other proton signals caused by water or fat. These sequences include, but are not limited to, spin echo imaging methods, gradient echo imaging methods, stimulated echo imaging methods, echo planar imaging (EPI) methods, spiral imaging methods, back-projection imaging methods, and chemical shift imaging (CSI) or voxel-based spectroscopy methods.

[0048] In an embodiment, other single terms are filtered out. In order to isolate the PEG nanoparticle signal from other proton signals, certain filtering components will be added to the selected pulse sequence. These MRI signal filtering methods include but are not limited to: magnetic field gradient b value greater than 1,000 s / mm 2Pulse sequences that preferentially reduce tissue water MRI signal, conventional fat suppression schemes using radiofrequency (RF) fat suppression or T1 fat suppression, pulse sequences with sufficiently long TE times to take advantage of the long T2 times of PEG protons and reduce tissue water and tissue fat signals, pulse sequences based on or using specific chemical shifts of PEG protons, pulse sequences that utilize fat suppression and water suppression as previously described and then use a Dixon-type imaging sequence to produce separate water and PEG proton images, pulse sequences that use magnetization transfer (MT) to further suppress water in tissue without suppressing PEG protons, CSI pulse sequences that produce low resolution 1D, 2D, or 3D proton images at specific chemical shifts of water, fat, and PEG protons, single-voxel or multi-voxel localized spectroscopy pulse sequences that use water and fat suppression methods to produce voxel nuclear NMR spectra. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 1 is a diagram (not drawn to scale) showing the difference in structural representation of embodiments of Ce6 delivery and ROS generation efficacy, wherein the left diagram shows how Ce6 generates ROS alone, while the right diagram shows an embodiment of encapsulated Ce6 according to the present invention and an embodiment anchored to 8PEGA. This difference in the way ROS are mobilized shows a clear increase in efficacy.

[0050] Figure 2 is a graphical representation of an example of a time-varying k-value curve for Ce6 encapsulated in PAAm NPs tracked by ADPA fluorescence quenching. In PBS, 660 nm OD = 0.12; the slope of the curve is the k-value.

[0051] Figure 3A is a schematic representation of an example of modification of 8PEGA-Ce6 with F3-cys peptide according to the present invention.

[0052] Figure 3B is a graphical representation of an example of UV / VIS spectra of 8PEGA-Ce6 and F3-8PEGA-Ce6 in PBS at 0.1 mg / ml according to the present invention.

[0053] Figure 4 1 is a graph of an example of the results of blood cell counting cell populations. Incubation conditions: 200ug / mL F3-8PEGA-Ce6 is placed in a well inoculated with 200,000 cells for 24 hours; N=3x3 for the control group and the test group (3 plates per group, 3 tests per group). Under the same conditions, the cell control group does not contain the F3-8PEGA-Ce6 nanostructure. The results show almost identical cell populations according to the present invention.

[0054] Figures 5A to 5E This is a photograph of a PDT test image of HeLa 229 cells. Figure 5A ) Calcein AM fluorescence of PDT control cells (without F3-8PEGA-Ce6). Figure 5B ) Calcein AM fluorescence of PDT control cells 2 h after irradiation. Figure 5C ) Calcein AM fluorescence of cells (with F3-8PEGA-Ce6) was tested before PDT. Figure 5D ) Calcein AM fluorescence of cells was tested 2 hours after PDT. Figure 5E ) Fluorescence 2 hours after PDT. Before PDT, the PDT test plate was incubated with 200ug / mL F3-8PEGA-Ce6 for 2 hours and illuminated with a 692+ / -20nm filter and an arc lamp at 50mW / cm 2 All cells were irradiated for 10 minutes with a total flux of .

[0055] Figure 6 So b=10 8 s / m 2 (A) and b = 10 10 s / m 2 (B) Image of a diffusion-weighted spin echo MR image of an embodiment of 8PEGA obtained. As shown in (A), the 110M water proton signal dominates in conventional MR images, but at b=10 10 The imaging was suppressed by 10 s / m2 -10 The diffusion constant of PEGA was measured by stimulated echo pulsed field gradient NMR at 25°C to be 3.572 10 -11 m 2 / s, thus allowing 70% of the initial magnetization to occur at b = 10 10 s / m 2 The color bar in (B) shows the concentration of 8PEGA detected according to the present invention.

[0056] Figure 7 8PEGA is a graph of concentration-dependent MRI signals for an example. A region of interest (ROI) was selected for each of the 5 vials, and the mean (circle) and standard deviation (error bars) of the signal were calculated. According to the present invention, a linear equation was fitted to the 5 measured vials, and the results are shown in dashed lines.

[0057] Figure 8 is a graph showing the particle size distribution of an example of a nanocomposite material according to the present invention.

[0058] Fig. 9 is a schematic diagram showing an example of PDT according to the present invention.

[0059] Fig.10is a diagram showing an exemplary embodiment of 8-arm-PEG-amine (8PEGA) according to the present invention.

[0060] Figure 11-18 is a graph illustrating the performance and properties of an embodiment according to the present invention.

[0061] Fig.19 is a graph showing an example of a K value curve for PAAm-Ce6 NPs at an OD equal to that used in 8 PEGA-Ce6 experiments according to the present invention.

[0062] Fig. 20 is a spectrum of an example of Ce6 on 8PEGA according to the present invention, showing that adding Ce6 to 8PEGA with or without targeting does not cause aggregation, fluorescence is maintained, and it still generates ROS.

[0063] Fig.21 is an example of a graph showing the signal from 8PEGA as a function of the applied gradient (b-value); the slope gives the diffusion constant D according to the invention.

[0064] Fig. 22 is an image showing the selective uptake of CTP-targeted 8PEGA-Ce6 in myocytes. Ce6 fluorescence is only present in myocytes.

[0065] Fig.23 is a TEM of an example of 8PEGA according to the present invention. 8PEGA is a hydrogel and TEM uses vacuum for measurements, therefore the grid is inserted while still wet, with some 8PEGA likely remaining in the hydration confirmation due to surface tension on the plate. The average range is about 10-12 nm.

[0066] Fig.24 PI fluorescence is from myocytes only.

[0067] Fig.25 is an image showing an example of non-targeted PDT with Ce 6 according to the present invention. PI fluorescence is no longer confined to myocytes and appears in coronary vascular cells, indicating damage to the vasculature.

[0068] Fig.26 is a perspective schematic diagram of an embodiment of an MRI and a method of using an MRI according to the present invention. DETAILED DESCRIPTION

[0069] In general, the present invention relates to nanoconstructs, methods of making these nanoconstructs, and therapeutic, imaging, diagnostic, theranostic, and other applications of these nanoconstructs.

[0070] Typically, in embodiments, nanoconstructs (NCs) can be used as both therapeutic platforms and imaging agents. These nanoconstructs can also have other active ingredients that provide other capabilities, such as targeting agents for selecting specific cell types, specific structures, or have specific membrane-related properties, such as cell membrane permeability.

[0071] Typically, the preferred embodiment is a multifunctional, ultra-small nano-platform with many desired therapeutic, imaging, diagnostic, therapeutic diagnostic properties and combinations and variations of these properties. Compared with individual photosensitizers and other PDT nano-constructs, this preferred embodiment has excellent photodynamic efficacy. In use, this preferred nano-construct has excellent photodynamic efficacy when applied to cancer cells. In addition to its excellent efficacy, this NC is non-toxic and is a molecular imaging agent for MRI.

[0072] 8-arm polyethylene glycol amine (8PEGA) is a biocompatible polymer that can be modified in a variety of ways. Typically, amine groups can be used to covalently anchor a range of photosensitizers (PS) in photodynamic therapy (PDT). In addition, in embodiments, the other arms of the polymer can then be converted to maleimide groups, allowing the attachment of various cysteine-terminated peptides. For example, a peptide can be attached with an additional amino acid (cysteine) to obtain a free thiol, which is then utilized. In such embodiments, there is a peptide + 1 amino acid, where the functionality of the peptide is retained. 8PEGA as an embodiment of NC can be flexibly customized to target and apply PDT in many biological environments, such as cancer, arrhythmias, and choroidal neovascularization, to name a few. 8PEGA also has a long T2 lifetime for MRI, and is used as an imaging agent in vivo, for example, by conventional diffusion-weighted imaging (DWI) at high b values, where the water signal can be fully suppressed by a combination of methods to produce clean, direct images.

[0073] The NCs, systems and methods of the present invention provide many improvements over previous treatments, imaging systems, previous nanoparticles (NPs) and treatments. These improvements include, for example: optimized reactive oxygen species (ROS) generation; small size to allow penetration into any desired biological region; easy modifiability to achieve targeting and NC accumulation in various biological systems; increased bioelimination rate due to the small size and biodegradability of 8PEGA; and improved DWI compared to traditional methods.

[0074] In embodiments used as NMR imaging or contrast agents, in embodiments of NC, 10 8 with 10 9 s / m 2However, the longer T2 lifetime and higher MW (40 kDa) of 8PEGA allows the use of b values ​​up to b = 10 10 s / m 2 , thereby fully suppressing the water / fat signal, leaving only 8PEGA. In addition, for MRI, it will allow the use of 8PEGA in DWI without the heavy metal atoms that have always caused safety concerns.

[0075] These nano constructs can be formulated into drug delivery systems, such as for: direct delivery into the bloodstream, e.g., prepackaged IV formulations or disposable prepackaged syringes; ingestion, such as pills, tablets or liquids; suction, such as by a metered dose inhaler or nebulizer; topical use, such as an ointment or liquid for transdermal delivery or in vivo administration, as part of an insufflation gas. These drug delivery systems can have one, two, three or more different nano constructs, each of which is specially designed or dedicated. These drug delivery systems can also contain other additives and activating agents that are not nano constructs and serve as, for example, other imaging agents and therapeutic agents.

[0076] Typically, additives can be associated with the nanostructure framework (e.g., nanoparticles) by chemical bonds (e.g., covalent bonds, ionic bonds, Van der Waals bonds); spatial or mechanical means, such as by spatial hindrance or physical capture within or within the framework; it can be a part of the molecular structure of the framework material; and combinations and variations of these. Additives can be added before, during, after, and combinations and variations of nanoparticle formation.

[0077] Many imaging dyes or agents and other diagnostic tools have used materials that may be considered undesirable, especially for use in humans. These dyes and agents may be metal-based and use or contain metals, metal oxides, or metal compounds or complexes, such as iron, iron-platinum, magnesium, and manganese. MRI imaging agents use gadolinium-based materials.

[0078] The embodiment of the nanoconstruct of the present invention that does not contain heavy metals (for example, nanoconstruct, drug delivery system for nanoconstruct or both) contains less than about 10ppm heavy metals, less than about 1ppm heavy metals and less than about 0.1ppm heavy metals and zero heavy metals.Heavy metals will include titanium and all heavier metals.These nanoconstructs that do not contain heavy metals can serve as imaging agents and diagnostic agents.The embodiment of these imaging nanoconstructs provides a kind of MRI imaging agent, and the imaging agent does not contain gadolinium, for example, has less than 0.1ppm and preferably has zero gadolinium.NPs, NCs, drug delivery systems that do not contain heavy metals and these combinations and variations can be imaged directly by MRI, and therefore serve as direct MRI imaging agents, diagnostic agents and both.

[0079] Embodiments of targetable NCs can be used as both a therapeutic platform for photodynamic therapy (PDT) and an MR molecular imaging agent. In embodiments, the nanoconstructs are free of heavy metal atoms, particularly gadolinium.

[0080] In the embodiments, ultra-small 8-arm polyethylene glycol amine (8PEGA) NCs with attached chlorin e6 (Ce6) PS and CTP-cys (cardiac targeting peptide) targeting moieties produced therapeutic results for PDT of cardiac arrhythmias in vivo and ex vivo on living rat and sheep hearts, respectively, when targeting peptides were used for cell-specific destruction of cardiomyocytes. This NC is an octopus-like, ultra-compact and highly biocompatible polymer 8-arm polyethylene glycol amine (8PEGA). The amine-terminated arms anchor the algae-derived PS, chlorin e6 (Ce6) and cardiomyocyte targeting moieties. This nanoconstruct can be used as an MRI imaging agent and can also be used as an MRI diagnostic therapeutic agent for cardiac conditions and other pathologies.

[0081] In an embodiment, this nanostructure can be configured so that it provides PDT for cancer. For this purpose, the targeting peptide can be F3-cys. Compared with traditional Ce6 encapsulated polyacrylamide (PAAm) NC, 8PEGA-Ce6 NC has excellent reactive oxygen species (ROS) production. When compared with PAAm NC, in addition to other aspects and in some applications, this provides the advantage of PDT for 8PEGANC.

[0082] The superior reactive oxygen species demonstrated by 8PEGA-Ce6 NCs is singlet oxygen, as Ce6 produces singlet oxygen. The production is defined as superior compared to NP encapsulation where ROS may not always escape the NC matrix, so that more of the produced product can be effectively used for oxidative stress through wider use in cells. In addition, NCs like 8PEGA or PAA act as an effective tool to prevent PS aggregation, which free PS may produce and thus reduce the yield due to quenching of excited states. When making this comparison, it is preferred to compare the k values ​​of Ce6-8PEGA and Ce6-PAA at the same optical density.

[0083] 8PEGA-Ce6 NCs are also cytocompatible and provide chemical flexibility for attaching a variety of targeting peptides. Finally, this label-free 8PEGA NC can be directly and selectively imaged by MRI using a standard spin-echo imaging sequence with a large diffusion magnetic field gradient to suppress the water signal. Notably, this NC has improved in vivo penetration and bioclearance due to its ultrasmall size.

[0084] Generally, in theory, any peptide can be attached to PEG nanoparticles, and it is believed that all peptides with cysteine ​​as the end can be attached to 8PEGA. F3-cys peptide is a specific cancer targeting peptide. In an embodiment, it is used to apply 8PEGA-Ce6 to cancer. Due to its robust characteristics and known nucleolar protein (specific target of F3-cys peptide) overexpression, HeLa cells are selected as the model system of concern. In addition to the advantages of small size, uniformity and biocompatibility of 8PEGA, the present invention has also developed other advantages, which include: using a given PS to optimize ROS generation and use it as a molecular imaging agent for MRI. In theory, compared with being encapsulated in a standard model matrix (such as polyacrylamide hydrogel nanoparticles (PAAmNP)), since, for example, PS Ce6 is in direct contact with an oxygen-containing environment, it is expected that the ROS generation of this NC reaches the best. In this way, in an embodiment, a nanostructure with increased ROS generation efficacy, biocompatibility and targeting flexibility than existing NC is provided.

[0085] In embodiments of the present invention, nanoparticles and nanoconstructs based on 8PEGA are used as MRI imaging agents for imaging applications, diagnostic applications, therapeutic diagnostic applications, and combinations and variations of these. These 8PEGA MRI imaging agents can be used in combination with other therapeutic agents, imaging agents, or contrast agents and applications. Theoretically, the high molecular weight (e.g., 40 kDa) of the embodiments of the 8PEGA group, the flexible chain dynamics, and its specific structure partially create favorable conditions for the highly selective molecular imaging MRI agent of the present invention. Further, the slow diffusion constant and transverse spin relaxation rate combination of 8PEGA allow diffusion-weighted MRI sequences that suppress surrounding water signals, thereby providing clear images of 8PEGA. In embodiments of the 8PEGA MRI imaging agent and 8PEGA MRI imaging application of the present invention, the 8PEGA MR signal is selectively detected and proportional to its concentration. 8PEGA MRI imaging agents and applications are superior to current MRI imaging agents and applications because, among other aspects, compared with current heavy metal atom MRI imaging agents, they have excellent biocompatibility. Furthermore, 8PEGA MRI imaging agents are also therapeutic diagnostic agents or have therapeutic diagnostic capabilities, which have additional functions and advantages compared to current MRI imaging agents and applications.

[0086] Figure 1 Two competing nanoconstructs are presented, comparing ROS production when Ce6 is attached to 8PEGA versus encapsulated in polyacrylamide (PAAm). Figure 1 shows that when Ce6 102 is exposed to light with a wavelength of 660nm, it is converted from molecular oxygen ( 3 O2) triplet ground state 101 produces ROS 100 excited singlet state ( 1 The generation of ROS 100 when Ce6 102 is encapsulated within PAAm NCs (Ce6 / PAAm NPs) 105 and the pathway of ROS are compared to the generation of ROS 100 when Ce6 102 is part of 8PEGA NCs (8PEGA-Ce6) 106.

[0087] Figure 2 is a graph of an example of a "k value" curve for relative ROS generation of PAAm encapsulated Ce6. The k value is a measure of the kinetic rate of ROS generation by Ce6, as measured by the first-order decay of ADPA fluorescence. To generate comparable k values, the optical density (OD) of PAAm-Ce6 NPs was adjusted to the OD of 8PEGA-Ce6 at 660nm (0.12) by UV / VIS. Table 1 shows the relative results of the two NCs when normalized to their literature OD.

[0088] Table 1

[0089]

[0090] Table 1: k values ​​at 660 nm for the two discussed NCs at OD = 0.12.

[0091] The diameter of NC 8PEGA was also calculated using the Stokes-Einstein approximation and TEM, as Fig.23 shown.

[0092] Figure 3A The absorption spectra of F3-8PEGA-Ce6 conjugate 301 as targeted NC and 8PEGA-Ce6 conjugate 302 as non-targeted NC are shown. Both NCs retain the characteristic peak at 660 nm.

[0093] Figure 3B A diagram showing chemical modification of 8PEGA-Ce6 with F3-cys is provided. Thus, NC 310 having 8PEGA 311 and Ce6 312 is modified with BiPEG (eg, 313) to provide NC 310a, which is then modified with F3-cys (eg, 314) to provide targeted NC 310b.

[0094] Flow cytometry was used as a method to test the biocompatibility of this F3-cys-8PEGA-Ce6 NC to test dark toxicity. Cells were tested at a concentration of 200ug / mL F3-8PEGA-Ce6; control cells represent the data set without the addition of F3-8PEGA-Ce6 NC. Figure 4 As can be seen, no significant toxicity was observed.

[0095] As a control test to eliminate the possibility of simple cell stress caused by the excitation light, HeLa cells without F3-8PEGA-Ce6 were plated and PDT was performed at the same length of time and power (50 mW / cm 2 , 10 minutes) for irradiation. Figure 5A , B), there was no significant change in cell morphology, no change in cytoplasmic staining calcein AM fluorescence counts, and no signs of plasma membrane blebbing (a sign of apoptosis).

[0096] After light irradiation of cells with F3-8PEGA-Ce6, significant differences were observed in Calcein AM fluorescence (Figure 2A). Figure 5C , D). Although no propidium iodide (PI) fluorescence was observable before irradiation (data not shown), after irradiation, cell nuclei were stained by PI, which is impermeable to the cell membrane (as shown in FIG. Figure 5E shown).

[0097] The translational diffusion constant D and relaxation times T1 and T2 of 8PEGA were measured at 25°C and 35°C (Table 2, Fig.21 Images were collected in 8PEGA (unmodified) solution to demonstrate that when water / fat is suppressed (as shown in Figure 6 The generation of clear images and their concentration-dependent responses (as shown) Figure 7 Therefore, Figure 6 The image 601 shows the 8 s / m 2 8PEGA obtained, and image 602 shows the 10 s / m 2 As shown in image 601, the 110M water proton signal dominates in conventional MR images; however, by 10 s / m 2 The image is suppressed by 10 -10 Bar 603 is associated with the concentration of 8PEGA detected in image 602. It can be seen that this response is linearly related to the concentration (e.g. Figure 7 ), which is consistent with images collected using water suppression techniques (e.g. Figure 6 B). In H2O, the measured concentrations were 0, 2.38, 4.77, 9.54 and 19.08 mg / mL 8PEGA. In addition, the Stokes-Einstein equation was used:

[0098] r=kT / 6πηD trans

[0099] The size of 8PEGA was calculated to confirm the TEM results; the diameter was calculated to be 10.96 nm at 35 °C, consistent with the range of about 10-12 nm found in 13 measured NCs (e.g. Fig.23 shown).

[0100] Table 2

[0101]

[0102] Table 2. Relaxation times and translational diffusion constants of 8PEGA at 25°C and 35°C measured at 16.4 T. Sample was 5 mg / ml L8PEGA in 25 / 75 (V / V) H2O / D2O.

[0103] Compared to hydrogel NPs, Ce6-8PEGA is a more efficient ROS generation platform based on, among other aspects, the direct contact of the Ce6 group in 8PEGA with the oxidative environment of the cell. Figure 1This configuration is shown in Figure 1. Therefore, oxygen does not need to diffuse into the PAAm NP matrix 105 encapsulating PS 102, and ROS 101 does not need to diffuse out and will not suffer losses due to reactions with the matrix. The k value test confirmed this feature by showing that the k value of Ce6-8PEGA is about 50% higher than that of Ce6 encapsulated PAAmNP when adjusted to the same OD (Table 1).

[0104] Since 8PEGA is a star polymer, it is expected to measure its size by the Stokes-Einstein equation assuming a spherical material shape. After measuring the translational diffusion coefficient D (Table 21, Table 2), the size of the polymer was found to be about 11 nm at 35°C. As a secondary analytical method, 8PEGA was stained with uranyl acetate and visualized by TEM (see Figure 2). Fig.23 The sizes of 13 selected points within circles 2301 (13.1 nm), 2302 (11.3 nm), 2303 (10.4 nm), 2304 (12.5 nm), 2305 (10.8 nm), 2306 (12.2 nm), 2307 (10.1 nm), 2308 (11.5 nm), 2309 (11.9 nm), 2310 (10.8 nm), 2311 (10.9 nm), 2312 (11.6 nm), 2313 (12.5 nm) were found to be approximately 10-12 nm, which is in good agreement with the Stokes-Einstein equation measurement results.

[0105] Unlike the surface of PEGylated nanoparticles, in embodiments of the present invention, the nanoparticle skeleton itself is mainly made of PEG, such as at least about 85% PEG, at least 90% PEG, at least 95% PEG, at least 99% PEG, at least 99.9% PEG and preferably 100% PEG. Therefore, the targeting carrier is not only helpful for in vivo applications, but also helps to accelerate in vitro cellular uptake. For example, for cancer targeting, the nucleolar protein targeting peptide F3-cys was chosen to be transplanted onto 8PEGA-Ce6. It is worth noting that after F3-cys was attached, the spectral characteristics of Ce6 were largely unaffected, as shown in FIG. Figure 3B As shown, this indicates that the photophysical properties are retained when switching peptides (from CTP for targeting cardiomyocytes to F3 for targeting cancer cells). It was noted that Ce6 absorption was slightly reduced by 0.1 mg / mL compared to before and after modification, but this is expected because BiPEG and F3-cys will increase the MW of NC. BiPEG is a 2-arm bifunctional PEG (e.g., 2kDa). An embodiment of BiPEG can have an NHS ester at one end and a maleimide at the other end. In an embodiment, this serves to convert the amine into the maleimide of the peptide to be attached.

[0106] An important aspect of embodiments of the NC of the present invention is their biocompatibility. In embodiments, the overall construction is composed of PEG, Ce6 and the homing peptide F3-cys. PEG is a highly biocompatible substance, and F3-cys is a good targeting agent that has no toxic effects in vitro or in vivo. Algae-derived Ce6 is an example of a PDT agent. Embodiments of the NC having these three parts do not have significant biocompatibility issues, for example, the NC is biocompatible. This is achieved by, for example, Figure 4 The blood cell count results shown were confirmed in vitro.

[0107] Before starting the PDT test with F3-8PEGA-Ce6, the selected laser conditions (50 mW / cm 2 for 10 minutes) to ensure that irradiation is not a source of significant cellular stress. Figure 5A as shown) and afterwards (as Figure 5B The calcein AM images of the cells (shown in Figure 2) showed little morphological change and no signs of apoptosis. Therefore, the light irradiation source did not cause much stress to the cells. PDT was then initiated in the presence of 200 ug / mL F3-8PEGA-Ce6 (shown in Figure 2). Figure 5C / D / E). Calcein AM fluorescence was significantly reduced after PDT, indicating a loss of cytoplasmic content, which only occurs when the cell membrane is ruptured. Membrane rupture was shown by staining the nucleus with the cell-impermeable dye PI (Figure 2A). Figure 5E PDT test results and Figure 4 Combined with the cytocompatibility in the mitochondria, it is clear that cell death is mediated by PDT.

[0108] In addition to more efficient PDT (50% greater k-value), the use of 8PEGA NCs, therapy, and theranostics is believed to have many additional advantages, including, for example: (1) the smaller size of the NCs provides the potential for rapid renal clearance from the body, a feature that larger NPs cannot offer; and (2) the ability to penetrate tumor areas where angiogenesis has not occurred, in contrast to traditional larger NPs that require a porous / leaky vasculature to penetrate the tumor.

[0109] Prior to the present invention, it was believed that PEG needed to be 13 C labeling can then be used for selective imaging in vivo using heteronuclear MR methods. Embodiments of the PEG NPs and NCs of the present invention demonstrate surprising capabilities as NMR, MRI, imaging agents, contrast agents, and combinations and variations of these. In the PEG embodiment, among other things, the inherent flexibility of the polyethylene oxide chain and the slow translational diffusion of 8PEGA provide a means for using 1Selective MR imaging of 8PEGA protons by HNMR creates a set of exploitable physical and dynamic conditions. Specifically, the fast chain motion of 8PEGA with a correlation time of approximately 0.1 nanoseconds provides sufficient averaging of proton dipole-dipole interactions to produce long nuclear spin transverse relaxation times, T2, measured here as 586 and 769 milliseconds at 25°C and 35°C, respectively. In contrast to the fast internal chain dynamics, the high molecular weight of the molecule 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, so that only the ethylene oxide signal will remain due to its long T2 time and the slow diffusion of 8PEGA. Notably, the MR signal intensity decays as:

[0110]

[0111] where the b value is determined by the magnetic field gradient amplitude and duration, D is the translational diffusion constant of water or 8PEGA, TE is the echo time, and T2 is the transverse spin relaxation time. In addition, the symmetry of the ethylene oxide monomer causes a single chemical shift for all four protons, and 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 spin echo MR imaging experiments with high b values ​​and long TE times, the water signal due to rapid diffusion and the fat signal due to the short T2 time are effectively suppressed, and the 8PEGA signal is selectively imaged. In vivo, due to the limited diffusion of water molecules in cells, a small portion of the water signal intensity at high b values ​​will remain unchanged, but due to the conventional 1 There is a difference of about 1 ppm between water and ethylene glycol protons in HNMR, and these signals can be removed or distinguished from the 8PEGA signal. It should be noted that the number of protons can be greater or less than 3,600, can be from about 2,000 to about 30,000, greater than about 4,000, greater than about 5,000, about 2,000 to about 10,000, about 3,000 to about 7,000, and all values ​​within these ranges, as well as greater or lesser amounts, depending on the nanoparticle.

[0112] So, for example, in Figure 6 As can be seen in the 8PEGA image 601, when combined with the above-described suppression techniques, 8PEGA functions very well as an MR imaging agent. This provides a significant advancement and the ability to replace or eliminate other MRI contrast agents that have less favorable, problematic, or dangerous materials like gadolinium salts or chelates that are the subject of health concerns, safety issues, and present health risks for certain patient populations. The image 601 without the suppression technique applied is significantly different from the image 602 with the suppression technique applied. The 8PEGA imaging signal also has a linear relationship with its concentration (e.g., Figure 7 When the above-described embodiments of imaging agents and imaging techniques are applied, 8PEGA restores clear and well-defined images, clearly demonstrating its feasibility as an in vivo imaging agent. Under proper calibration, this provides accurate imaging and diagnosis, including allowing quantification of 8PEGA in biological tissues (e.g., tumor regions and filter organs).

[0113] Thus, 8PEGA-Ce6 NCs provide NCs with one or more, preferably all, of the following features: excellent reactive oxygen species, MRI imaging capability, free of heavy metals, and capable of having cancer targeting agents.

[0114] The ability of the targeted nano construct that is both an imaging agent and a PDT agent has produced efficacy in treating conditions that have been unheard of so far. This targeted therapeutic nano construct uses a targeting agent to target a specific structure, for example, cell type, tumor, etc. In this way, the targeted therapeutic nano construct will selectively associate with the targeting structure by the effect of the targeting agent. Independent targeting agent can provide good specificity, wherein about 80%, about 90% and about 95% of the nano construct associates with the targeting structure. However, targeting agent can not provide the absolute specificity to the targeting structure. Therefore, when delivering activation energy, it is desirable to image the target structure and preferably determine the precise pattern for delivering activation energy (for example, light) thus.

[0115] In this way, in an embodiment of the therapeutic diagnosis method, the targeted therapeutic diagnosis nano construct is delivered to the body and is carried by the blood and associated with the target structure (e.g., a tumor). An MRI image is taken of the tumor, and this image is enhanced due to the presence of the nano construct. The position and shape of the target structure in the body are obtained and stored. Subsequent image techniques (e.g., photoacoustic imaging), modeling techniques (e.g., computer enhancement and rendering of initial MRI images) and both can be used to provide very accurate images and position data and information for the target structure in the body. An irradiation pattern can then be developed based on this image and position data. This irradiation pattern can be predetermined, customized and specific to the target structure.

[0116] In an embodiment, this predetermined irradiation pattern can be a small diameter laser spot. The energy of the laser beam delivered in this pattern is sufficient to activate the PS, thereby causing the generation of ROS. However, the energy delivered by the laser beam is below the threshold for laser-induced optical breakdown of the tissue (LIOB), and preferably below the threshold for heating the tissue. In an embodiment, the properties of the laser beam, such as wavelength, focal length, scanning time or duration, power, pulse, pulse length or continuous, and spot size, can be determined so that the PS is activated at a very precise location (z, x and y coordinates) down to the cellular and subcellular levels; and direct interaction with the laser causes little damage to the tissue of the target structure. In this way, the PS can be activated with cellular precision and ROS can be provided to the target structure without damaging the adjacent cells of the structure. (It will be appreciated that the duration of ROS after generation is very limited, and if generated within or near a cell, it may not migrate to or affect non-adjacent cells.)

[0117] In the nanoconstruct embodiment, spatial (laser focusing) and biological (cell selectivity) selectivity are achieved by employing nanoconstructs (NCs) with targeting antibodies or peptides, which also extends PDT treatment to subsurface tumors. In general, the use of NCs can protect PS from the biological environment and vice versa, thereby bypassing the immune system.

[0118] The relatively small size (<20 nm) of 8PEGA-derived NCs penetrated target tissues, including very dense tissues (such as muscle), selectively accumulated in specific cell types (e.g., myocytes), and thus allowed their photodynamic destruction under mild near-infrared irradiation. In cancer therapy, the small size of these NCs will provide tumor-promoting NCs, which alone or in combination with targeting moieties will be cell-selective for tumors.

[0119] Any type of active dynamic therapy part can be used with nanoparticles, and preferably used with targeting agents to form nanoconstructs; and for example, therapeutic nanoconstructs. In an embodiment, any currently known or later developed dynamic therapeutic agent is combined with a nanoparticle formed at least in part by PEG, PEG-based materials, and combinations and variations of these. In an embodiment, any currently known or later developed dynamic therapeutic agent is combined with a nanoparticle having a cross section less than 50nm and an embodiment less than 40nm. In an embodiment, any currently known or later developed dynamic therapeutic agent is combined with a nanoparticle having a cross section of about 5nm to about 20nm, about 5nm to about 15nm, about 10nm to about 15nm, and about 9nm to about 12nm. In an embodiment, any currently known or later developed dynamic therapeutic agent is combined with a nanoparticle having a cross section less than 50nm, less than 40nm, less than 30nm, less than 20nm, less than 15nm, and less than 10nm. In an embodiment, any currently known or later developed dynamic therapeutic agent is combined with a nanoparticle 8PEGA. All of these embodiments of nanoconstructs may also have targeting agents that have targeting capabilities or characteristics (eg, tumor affinity) as well as combinations and variations of these.

[0120] Combinations and variations of the above embodiments on the nanoconstructs and other embodiments taught in this specification are used as drug products or as part of drug products. In embodiments of these drug products, the nanoconstructs are uniform in size and highly uniform in size. Therefore, in embodiments, the nanoconstructs in the drug product, particularly the particles of the drug product dose for a subject or patient (animal, mammal or human) have a particle size difference of no more than about 1%, no more than about 5% and no more than about 10%. In embodiments, the particle size distribution of the nanoconstructs in the drug product can be: D10=n-5, D50=n, D90=x+5 (where n=5 to 25nm); D10=n-10, D50=n, D90=x+10 (where n=5 to 25nm); D50 is about 10, D50 is about 15nm, D50 is about 20nm, D50 is about 50nm, D50 is about 8nm to about 15nm, and larger and smaller values. ( Figure 8 Calculation and distribution of D10, D50, and D90 values ​​are shown. D50 is the value representing the size of nanoconstructs that account for 50% of the cumulative amount in the drug product. D-90 is the value representing the size of nanoconstructs that account for 90% of the cumulative amount in the drug product. D-10 is the value representing the size of nanoconstructs that account for 10% of the cumulative amount in the drug product.) In an embodiment, the drug product has a particle size distribution of nanoconstructs that are no greater than about 10 nm, no greater than about 5 nm, and no greater than about 1 nm.

[0121] Typically, for PDT, the wavelength of the light source needs to be suitable for exciting the photosensitizer to generate reactive oxygen species. These reactive oxygen species generated by PDT are free radicals or highly reactive oxygen species known as singlet oxygen. Typically, in embodiments, the photosensitizer can generate a triplet state of appropriate energy (approximately 0.95 eV), which is a suitable energy for converting molecular oxygen ( 3 O2) is excited from its triplet ground state to its excited singlet state ( 1 O 2) required. Other cytotoxic species that may be produced include, for example, other ROS, type 1 ROS, hydroxyl radicals, peroxides, and superoxide anions.

[0122] In general, there are three mechanisms, which in embodiments may be interrelated, by which PDT mediates destruction of targeted tissues (e.g., tumor destruction): direct cytotoxic effects on tumor cells, destruction of tumor vasculature, and induction of an inflammatory immune response leading to the development of systemic immunity.

[0123] Incorporating photosensitizers into smaller nanoconstructs (e.g., 8PEGA) provides the ability to have photosensitizers in lower energy states. In theory, the smaller size of the NCs provides the NCs, and therefore the photosensitizers, with the ability to generate RO species closer to tissues or structures affected by ROS, among other reasons, because the photosensitizers are at or near the surface of the NCs and therefore have more tissue oxygen available to form ROS.

[0124] In an embodiment, if the quantum yield of singlet oxygen or other reactive oxygen species is high enough (> 0.4), a typical photosensitizer can be, for example, an efficient PDT agent. That is, at least 40% of the excited photosensitizer molecules will produce singlet oxygen or reactive oxygen species, rather than distributing energy by fluorescence, phosphorescence or other means. In addition, the longer the lifetime of the triplet state (> 1 millisecond) of the excited photosensitizer, the better the interaction with the surrounding molecules, resulting in the production of more cytotoxic substances. Embodiments of the present invention utilizing nanostructures of smaller sizes and pharmaceutical products having a highly uniform nanostructure particle size distribution provide the ability to make photosensitizers with lower potency as effective PDT, and greatly improve the therapeutic efficacy of existing photosensitizers used in PDT. Incorporating photosensitizers into smaller nanostructures (e.g., 8PEGA) provides the ability to have photosensitizers with lower energy states. Theoretically, among other reasons, since the photosensitizer is on or near the surface of the NC, it has more tissue oxygen available to form ROS, and the smaller size of the NC provides the photosensitizer with the ability to generate ROS species closer to the tissue or structure affected by the ROS.

[0125] Notably, the mechanism of PDT differs from other light-based therapies and laser therapies, such as laser wound healing and regeneration or intense pulsed light hair removal, in that it does not require photosensitizers, fluorescence, phosphorescence, or other means of generating, for example, active moieties that are generated or required to be generated in vivo.

[0126] In an embodiment, usually, the example of the structure targeted by PDT can be mitochondria, lysosomes or endoplasmic reticulum. The effect on cells (such as cyttid effect) is theoretically through such as apoptotic cell death mechanism, necrosis pathway and both and occurs. In theory, the enzyme required for apoptosis is destroyed, and there will be enough cell damage to cause necrosis results (plasma membrane is damaged). Another major cause of tumor destruction is theoretically to limit the supply of oxygen and nutrients to the tumor through vascular closure, thereby causing tissue hypoxia and cell death. Other theoretical mechanisms are that PDT activates immune response, thereby immune cells (such as lymphocytes, leukocytes and macrophages) infiltrate into the target tissue. Another reason for tumor destruction is to limit the supply of oxygen and nutrients to the tumor through vascular closure, thereby causing tissue hypoxia and tumor cell death. In the embodiment of 8PEGA NC in tumor destruction, this is the main way of tumor cell death.

[0127] Embodiments of the invention utilizing smaller sized nanoconstructs, drug products with highly uniform nanoconstruct size distributions, and combinations of these provide the ability to use new photosensitizers, older less popular photosensitizers, and photosensitizers that have been previously overlooked in PDT. These embodiments can use these currently theoretical mechanisms of tumor cell death, as well as other methods or approaches that will occur as a result of the invention or that may be discovered later, including the imaging capabilities and theranostics of the invention.

[0128] The present invention is not limited to a specific photosensitizer. In some embodiments, the agent is methylene blue (MB), chlorin e6 (Ce6), Coomassie blue (acting as a PTT (photothermal therapy) agent in embodiments), gold or other suitable photosensitizer. In some embodiments, the photosensitizer is also suitable for imaging (e.g., MB).

[0129] Embodiments of the present invention are not limited to photodynamic therapy. Additional therapeutic agents that can form part of the nanoconstructs and nanoconstruct drug products of the present invention can be utilized in embodiments of the present invention. Examples include, but are not limited to: agents that induce apoptosis; sonosensitizers; polynucleotides (e.g., antisense, ribozymes, siRNA); polypeptides (e.g., enzymes and antibodies); agents that bind (e.g., oligomerize or complex) to Bcl-2 family proteins such as Bax; alkaloids; alkylating agents; antibiotics; antimetabolites; hormones; platinum compounds; monoclonal or polyclonal antibodies (e.g., antibodies conjugated to anticancer drugs, toxins, defensins), toxins; radionuclides; biological response modifiers (e.g., interferons (e.g., IFN-α) and interleukins (e.g., IL-2); adoptive Immunotherapeutic agents; hematopoietic growth factors; agents that induce cell differentiation (e.g., all-trans retinoic acid); gene therapy agents (e.g., antisense therapeutic agents and nucleotides); angiogenesis inhibitors; proteomic inhibitors: NF-KB regulators; anti-CDK compounds; HDAC inhibitors; heavy metals (e.g., barium, gold, or platinum); chemotherapeutic agents (e.g., doxorubicin or cisplatin), etc. Many other examples of toxic compounds are known to those skilled in the art, and these compounds can be used through smaller sized NCs, highly uniform NC particle size distribution in the drug product, and combinations of these.

[0130] In some embodiments, the toxic agent is a sonosensitizer. Examples of sonosensitizers include, but are not limited to, porphyrins (e.g., hematoporphyrin, diacetyl hematoporphyrin-mitomycin-C conjugate, photosensitive protein II, mesoporphyrin, protoporphyrin IX, copper protoporphyrin, tetraphenylporphyrin tetrasulfonate, ATX-70, ATX-S10, pheophorbide-a, CIA1-phthalocyanine tetrasulfonate, and chloroPAD-S31), tenoxicam, piroxicam, rose bengal, erythrosine B, merocyanidin 540, dimethylformamide, cytarabine, pyridocarbazole, 2,2'-azobis(2-amino)-1,4-dihydropyridine, 1,2 ... , 5,5'-dimethyl-1-pyrroline 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, diazaquinone, dihydroxy(oxo-guanidinoamino)borane, and 5-fluorouracil) (see, e.g., Rosenthal et al., Ultrasonics Sonochemistry 11 (2004) 349; incorporated herein by reference in its entirety).

[0131] In some embodiments, toxic agents include agents that induce or stimulate apoptosis. Factors that induce apoptosis include, but are not limited to, radiation (e.g., x-rays, gamma rays, UV); tumor-derived growth factor ligands, receptors, and analogs; kinase inhibitors (e.g., epidermal growth factor receptor (EGFR) kinase inhibitors, vascular growth factor receptor (VGFR) kinase inhibitors, fibroblast growth factor receptor (FGFR) kinase inhibitors, platelet-derived growth factor receptor (PDGFR) kinase inhibitors, and Bcr-Abl kinase inhibitors (such as GLEEVEC); antisense molecules; antibodies (e.g., HERCEPTIN, RITUXAN, ZEVALIN, BEXXAR, and AVASTIN); anti-estrogen Hormones (e.g., raloxifene and tamoxifen); antiandrogens (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., phenbutazone butazolidin, DECADRON, DELTASONE, dexamethasone, dexamethasone intensol, DEXONE, HEXADROL, hydroxychloroquine, METICORTEN, ORADEXON, ORASONE, hydroxyphenylbutazone, 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; staurosporine, etc.

[0132] Alkylating agents suitable for use in the compositions and methods of the invention include, but are not limited to: 1) nitrogen mustards (e.g., dichloromethane, cyclophosphamide, ifosfamide, melphalan (L-sarcolysin); and chlorambucil); 2) ethyleneimines and methylmelamines (e.g., hexamethylmelamine and thiotepa); 3) alkyl sulfonates (e.g., busulfan); 4) nitrosoureas (e.g., carmustine (BCNU); lomustine (CCNU); semustine (methyl-CCNU); and streptozotocin (streptozotocin)); 5) triazenes (e.g., dacarbazine (dimethyltriazene imide-azolecarboxamide).

[0133] In some embodiments, antimetabolites suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) folic acid analogs (e.g., methotrexate (methotrexate)); 2) pyrimidine analogs (e.g., fluorouracil (5-fluorouracil), floxuridine (fluorodeoxyuridine) and cytarabine (cytosine arabinoside)); 3) purine analogs (e.g., mercaptopurine (6-mercaptopurine), thioguanine (6-thioguanine) and pentostatin (2'-deoxycoformycin).

[0134] In still further embodiments, chemotherapeutic agents suitable for use in the compositions and methods of the present invention include, but are not limited to: 1) vinca alkaloids (e.g., vinblastine, vincristine); 2) epipodophyllotoxins (e.g., etoposide and teniposide); 3) antibiotics (e.g., dactinomycin (actinomycin D), daunomycin (daunorubicin; erythromycin), doxorubicin, bleomycin, plicamycin (mithramycin), and mitomycin (mitomycin C)); 4) enzymes (e.g., L-asparaginase); 5) biological response modifiers (e.g., interferon-α); 6) platinum coordination complexes (e.g., cisplatin and carboplatin); 7) anthracenediones (e.g., mitoxantrone); 8) substituted urea (e.g., hydroxyurea); 9) methylhydrazine derivatives (e.g., methylbenzalazine (N-methylhydrazine)); 10) adrenocortical suppressants (e.g., mitotane (o,p'-DDD) and aminoglutethimide); 11) adrenocortical steroids (e.g., prednisone); 12) progestins (e.g., hydroxyprogesterone caproate, medroxyprogesterone acetate, and megestrol acetate); 13) estrogens (e.g., diethylstilbestrol and ethinyl estradiol); 14) antiestrogens (e.g., tamoxifen); 15) androgens (e.g., testosterone propionate and fluoxymesterone); 16) antiandrogens (e.g., flutamide); and 17) gonadotropin-releasing hormone analogs (e.g., leuprolide).

[0135] In some alternative embodiments, nanoparticles, nanoconstructs and both include other agents for imaging purposes. In some embodiments, imaging agents are, for example, 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 fluorescein isothiocyanate for fluorescent imaging); fluorescent proteins (e.g., green fluorescent protein); and radioactive elements (e.g., for autoradiography).

[0136] In some embodiments, nanoparticles, nanoconstructs, and both include nanomaterials used as X-ray / CT contrast agents, or MRI utilizing photosensitive properties, absorbance of X-rays, or paramagnetic properties of T1 magnetic resonance imaging. Exemplary contrast agents include, but are not limited to, gadolinium contrast agents, fluorescent agents (e.g., Alizarin Red S), and contrast agents described in U.S. Pat. Nos. 7,412,279 or 6,540,981, each of which is incorporated herein by reference in its entirety.

[0137] Embodiments of the present invention provide an activator that activates a toxic agent, thereby causing local cell and tissue damage in target cells in a cell-specific manner. The present invention is not limited to specific activators. Any activator that activates a toxic agent can be used in embodiments of the present invention. Typically, the activator provides an energy source that causes the toxic agent to release energy (e.g., in the form of free radicals), thereby causing cell death or destruction. Exemplary activators include, but are not limited to, light, heat, radiation, sound, etc.

[0138] In some embodiments, photodynamic therapy is used to illustrate the present invention. However, the present invention is not limited to the use of photodynamic therapy. A variety of toxic agents and activation systems can be used in embodiments of the present invention.

[0139] Go to Fig. 9 , embodiments of photodynamic therapy (PDT) generally include the use of a chemical reaction whereby a photosensitizer is activated by light energy and releases reactive oxygen species. PDT comprises two stages. First, a photosensitizer is applied by passive or active targeting and is accumulated on or in a tissue. Then, the photosensitized tissue is exposed to light of a wavelength consistent with the absorption spectrum of the photosensitizer, which is activated after irradiation. In the case of a photodynamically efficient photosensitizer, energy is transferred to molecular oxygen (available in cells) and reactive oxygen species (ROS) are produced, mainly singlet oxygen (O2). Subsequent oxidation of lipids, amino acids, and proteins of cells induces cell damage, such as necrosis and / or apoptosis of tissues. Since ROS have extremely limited lifespan and diffusion length and have strong local toxicity, their release can lead to irreversible but strictly limited cell damage and tissue necrosis. Therefore, the damage induced by PDT is limited to cells that have been photosensitized, while adjacent cells that have not been photosensitized are not affected.

[0140] Embodiments of the nanoplatforms of the present invention are conjugated photosensitizers and targeting moieties with hydrogels, in this way, targeted cell-specific PDT can be used for a variety of applications with greatly improved efficacy and controllability. For example, cell-specific and spatially specific cell death approaches encompass the synergistic implementation of two agents, both conjugated to biodegradable nanoparticles: a peptide targeting myocytes (e.g., CTP) and a photodynamic therapy thereby enabling the photosensitizer (e.g., chlorin e6).

[0141] In some embodiments, the activator is sound (e.g., sonodynamic therapy). Sonodynamic therapy is an ultrasound-dependent enhancement of the cytotoxic activity of certain compounds (sonosensitizers). Ultrasound is a mechanical wave whose particles vibrate periodically in a continuous elastic medium at a frequency equal to or greater than 20kHz. In a liquid, its speed of about 1000-1600m / s is converted to a wavelength range of microns to centimeters. Therefore, the sound field cannot be directly coupled to the energy level of the molecule, including the biological environment at the molecular level. Therefore, this radiation is not only considered safe, but also has very good tissue penetration ability without significantly reducing its energy. In some embodiments, sound is generated in vitro and targeted to the desired treatment area through tissue.

[0142] Sonodynamic therapy is based on the synergistic effect of ultrasound with compounds known as "sonosensitizers." The effect can be localized by focusing the ultrasound on a defined area (e.g., a target tissue area). In some embodiments, ultrasound is delivered transdermally to a specific area of ​​the target tissue.

[0143] In some embodiments, the activating agent is an agent that activates a therapeutic agent (e.g., a chemotherapeutic agent). For example, in some embodiments, verapamil is used to activate or improve the efficacy of a chemotherapeutic agent (e.g., doxorubicin).

[0144] Embodiments of the present invention provide compositions, kits and systems including nanoconstructs and nanoconstruct drug products as described herein. In certain embodiments, the system includes nanoparticles, nanoconstructs and both, and instruments or devices (e.g., lasers, ultrasonic devices, radiation delivery devices, etc.) for delivering activators. In certain embodiments, the system further includes instruments for imaging nanoparticles, nanoconstructs and both in targeted tissues and computer systems to control activator delivery, imaging, data analysis and data display.

[0145] Then, after local delivery of an activator (e.g., laser or sound), cell-specific death is induced (e.g., by a toxic agent embedded or on the surface of the nanoparticle), followed by local release of ROS. In an embodiment, specifically targeted cell types are killed only in the area where the activator is delivered, while the number of untargeted cells remains constant after the activator is delivered.

[0146] The present invention is not limited to a specific method of delivering the activator. In some embodiments, the activator is delivered directly to the target tissue area in need of treatment by surgery (e.g., endoscopic or open surgery). In some embodiments, an automated system (e.g., computer control) is used to target and control the activator.

[0147] In some embodiments, the active agent is delivered locally to a targeted area in need of treatment using a catheter or other intravenous or intraarterial delivery or percutaneously (eg, by ultrasound).Such methods avoid the need for open surgery.

[0148] In some embodiments, the therapy is sonodynamic therapy. Sonodynamic therapy has the advantage of percutaneous delivery, thus allowing the entire procedure to be performed without invasive means. A toxic agent (e.g., a sonosensitizer) is delivered (e.g., intravenously), and then a targeted area of ​​tissue is treated with ultrasound.

[0149] In some embodiments, the therapy is photodynamic therapy. Photodynamic therapy has the ability to bring spatial specificity, because only the irradiated area is treated, while other areas are not treated.

[0150] In some embodiments, the treatment targets and ablates or kills myocytes. However, the present invention is not limited to targeted myocytes. The advantage of implementing therapeutic nano platforms is the versatility of these carriers to be conjugated with a variety of optional targeting agents for different target tissues. In fact, any other targeting moiety (e.g., antibodies, peptides, etc.), functional dyes or bioactive agents can be easily implemented by these nano platforms.

[0151] In some embodiments, the therapeutic uses described herein are used in conjunction with or in place of existing therapies. In some embodiments, nanoparticle-based therapies are used as follow-up therapies to failed or incomplete therapies (e.g., non-nanoparticle therapies).

[0152] In some embodiments, nanoparticles, nanoconstructs, and both are used for imaging (e.g., in vivo imaging) applications. In some embodiments, photosensitizers (e.g., chlorin e6) or particles that are also fluorescent or imageable are utilized. As described herein, important embodiments of the present invention are nanoparticles, nanoconstructs, and both, which themselves add other agents or materials to act as MRI imaging agents. In other embodiments, nanoparticles, nanoconstructs, and both further include separate imaging agents. For example, as described herein, in some embodiments, nanoparticles, nanoconstructs, and both include contrast agents for imaging (e.g., X-rays, computed tomography (CT) imaging, PET imaging, ultrasound, photoacoustic imaging, or MRI imaging). For example, in some embodiments, 157 Gd, gold, iodine, iron oxide or other suitable agents for imaging coated nanoparticles, nanoconstructs and both.

[0153] In some embodiments, nanoparticles, nanoconstructs, and both are used to detect biological targets in vivo or in vitro by bioluminescent imaging. In some embodiments, nanoparticles, nanoconstructs, and both include enzymes that catalyze bioluminescent reactions. Enzymes that catalyze bioluminescent reactions include, but are not limited to, the following luciferases: bacterial luciferase (U.S. Pat. No. 4,548,994), firefly luciferase (U.S. Pat. Nos. 5,670,356 and 5,674,713), sea renilla luciferase, click beetle luciferase, Luciola cruciata luciferase (Masuda et al., Gene 77: 265-70

[1989] ), Luciola lateralis luciferase (Tatsumi et al., Biochim. Biophys Acta 1131: 161-65

[1992] ), and Latia neritoides luciferase. The foregoing publications and patents are expressly incorporated herein by reference.

[0154] In some embodiments, imaging is performed in situ. Nanoparticles, nanoconstructs, and both containing bioluminescent enzymes are provided to animals intravenously, and time is allowed for the molecular recognition element to bind to its biological target. In some embodiments, the substrate of the bioluminescent enzyme (e.g., bacteria or insect luciferin) is then provided to the animal (e.g., delivered intravenously, intraperitoneally, intravesically, or intracerebrally). In some embodiments, the bioluminescence produced by the action of the enzyme on the substrate is then detected by a bioluminescence detection system. In some embodiments, the bioluminescence detection system includes a Hamamatsu enhanced CCD (ICCD, model C2400-32). In other embodiments, the bioluminescence detection system further includes other devices for enhancing weak signals (e.g., a microchannel plate enhancer and a Peltier or liquid nitrogen cooling device for a detector and / or enhancer). In some embodiments, a grayscale image of the animal is obtained by opening the darkroom door where the animal is placed. The door is then closed, and the gain on the enhancer is adjusted to the maximum to detect the bioluminescent signal. Then, the signal is covered by a pseudo-colored grayscale image.

[0155] In other embodiments, nanoparticles, nanoconstructs and both are used to detect biological targets by magnetic resonance imaging (MRI). In certain embodiments, the biological target of imaging is in situ. Nanoparticles, nanoconstructs and both (e.g., intravenously) comprising magnetic materials are provided to animals, and time is allowed for the molecular recognition element to bind to its biological target. In certain embodiments, the biological target is then imaged with a magnetic resonance system (e.g., a 7-Tesla magnetic resonance system). In certain embodiments, T1-weighted or T2-weighted images are obtained.

[0156] In some embodiments, diagnostic and imaging applications are performed in conjunction with therapeutic applications. For example, in some embodiments, imaging agents are used to visualize target tissues to monitor cell death before and after photodynamic therapy. For example, in some embodiments, imaging enables clinicians to see the location of nanoparticles, nanoconstructs, and the combination of the two (e.g., before, during, or after activation). In some embodiments, imaging is used to visualize target tissues after treatment to determine the extent or location of cell killing. Therefore, imaging allows real-time monitoring of the progress of dynamic therapy.

[0157] In some embodiments, imaging methods are utilized to determine the course of active treatment. For example, in some embodiments, imaging is used after treatment to determine whether additional treatment is needed, for example in the form of additional active agents in the same or different areas or in the form of delivery of additional nanoparticles, nanoconstructs, and both.

[0158] In some embodiments, nanoparticles, nanoconstructs, and both are used in research (eg, imaging in animal models, structural studies, DNA-protein binding interactions, protein capture, etc.), during surgery or drug screening applications.

[0159] Various recording and monitoring techniques and equipment can be used to evaluate the current NCs, methods of making these NCs, and methods of using these NCs. For example, a Shimadzu UV-1601 UV / visible spectrophotometer can be used to record and adjust the optical density (OD) of the NPs. Fluoromax-3 can be used to obtain fluorescence spectra.

[0160] The starting materials, reagents and components used to make the NC of the present invention can be from available commercial sources, and preferably FDA-approved materials for medical products. For example, the following are the sources of materials that can be used in the embodiments in the examples. Chlorin e6 (Ce6) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) are derived from Frontier Technology. 8PEGA (40kDa) and Bi-PEG (maleimide-PEG-succinimide ester, 2kDA) are derived from Creative PEG Works. F3-Cys peptide (KDEPQRRSARLSAKPAPPKPEPKPKKAPAKKC) is derived from SynBioSci. Natural ethanol with 190 proof alcohol is from Decon Labs. 10kDa and 300kDa filters and 10kDa centrifugal filters for Amicon cells are derived from Amicon. DMEM [(+) glutamine, sugar, sodium pyruvate, penicillin-streptomycin and fetal bovine serum are derived from Life Technologies. All other chemicals were from Sigma Aldrich. Acrylamide, 3-(acryloyloxy)-2-hydroxypropyl methacrylate (AHM), aminopropyl methacrylamide hydrochloride (APMA), N-hydroxysuccinimide (NHS), N,N'-dicyclohexylcarbodiimide (DCC), Brij L4, dioctyl sodium sulfosuccinate (AOT), dimethyl sulfoxide (DMSO), dimethylformamide (DMF), ammonium persulfate (APS), tetramethylethylenediamine (TEMED), phosphate buffered saline (0.01 M, PBS), hexane, cysteine, anthracene dipropionic acid (ADPA), calcein AM, propidium iodide (PI).

[0161] In an embodiment, the NMR is operated and analyzed to provide sufficient water and fat suppression to provide clear PEG images. For example, a diffusion-based method that eliminates 95% of tissue water can be used. The residual water can be removed by other water suppression methods known to those skilled in the art. For example, in order to have additional water suppression, spectral imaging can be used, where each voxel consists of an NMR spectrum with water, PEG, and fat. This suppression method is also suitable for tissue distribution studies.

[0162] In embodiments, MRI of 8PEGA directly picks up the signal of ethoxy protons and measures them. This is different from metals such as gadolinium, which change the relaxation time of water protons that interact with them, thereby creating visible contrast during scanning. Therefore, embodiments of the present invention provide images based on direct measurement of protons in NPs, and do not rely on or use changes in the relaxation time of water protons.

[0163] Embodiments of NCs, particularly PEG-based NCs, can have a variety of different sizes and weights, and can include, for example, monomers (e.g., a single macromolecule that is not composed of multiple PEGs), multi-arm PEGs (e.g., 2, 4, 6, 8, 10 or more arms), and combinations and variations of these. The total MW of the NCs can be <1 MDa, about 10 kDA to about 1,000 kDa, about 20 kDa to about 500 kDa, about 30 kDa to about 200 kDa, about 40 kDa to about 750 kDa, about 35 kDa to about 75 kDa, about 50 kDa to about 800 kDa, and all values ​​within these ranges, as well as greater or lesser amounts.

[0164] Go to Fig.10 , provides a diagram showing an embodiment of 8-arm-PEG-amine (8PEGA) 1010, which is a generally applicable ultra-small nanoplatform for therapeutic diagnosis: for example, photodynamic therapy (PDT, "thera") and as a molecular MR agent ("nostic"). The left side of image 1011 shows the flexibility of attaching a photosensitizer (PS) through different functional groups, followed by a targeting agent (e.g., a peptide) attached through a simple "click" chemistry reaction with a bifunctional PEG (such as a maleimide-thiol reaction for cell-specific targeting approaches). The right side shows how 8PEGA 1010 contains various physical properties ideal for MRI imaging. These properties include a longer T2 lifetime and a slower diffusion constant due to its higher MW of 40kDa, which are ideal properties for diffusion-weighted MRI.

[0165] Go to Fig.11, shows a general flow chart for assembling 8PEGA with Ce6 (PS) and CTP (TA). This can generally be applied using any suitable TA and PS. The top diagram 1101 is a general route description of how PDT works: the nanoplatform receives the light it absorbs and transfers it to local oxygen to produce ROS. The ROS then damage the cells. The bottom diagram 1102 is a schematic reaction path for making targeted NCs.

[0166] Go to Fig.12 , a slide showing the in vitro performance of CTP-8PEGA-Ce6 NPs on rat and human cell cultures; the cultures contained fibroblasts and cardiomyocytes. It can be seen that in the case of rats, only myocytes were killed even in direct contact with fibroblasts. The calcein AM fluorescence of dead cells decreased, and propidium iodide increased. Human cell testing showed that the same selectivity could be achieved in humans without changing the platform (targeted and non-targeted testing was performed, showing that targeting was both preferred and effective).

[0167] Go to Fig.13 , shows tissue isolated from a rat heart after NP injection (1 hour after injection). DAPI stains the nuclei of all cells. It can be seen that Ce6 fluorescence localizes to myocytes with extreme selectivity, indicating that the in vitro selectivity results directly translate to in vivo selectivity results.

[0168] Go to Fig.14 , shows a graphic representation of a surgical setting subject 1401, heart 1402, and the resulting electrograms from PDT (targeted, non-targeted, or sham). The reduction in electrogram amplitude indicates that the signal is blocked; the remaining signal is due to far-field activity. Both targeting and non-targeting have an effect, while laser alone has no effect. This indicates that PDT can generally induce changes.

[0169] Go to Fig.15 , showing that PI (nuclear stain for dead cells) is present only in myocytes (3 different magnified areas). D and E show that PDT occurs only in the irradiated area by showing that PI fluorescence decreases as the examined area moves away from the treated area.

[0170] Go to Fig.16 , showing the Fig.15 Similar tissue sampling was tested, but non-targeted PDT was used. As can be seen in the bar graph (and quantified in the figure), the untargeted Ce6 PDT killed everything, compared to the target which only killed the myocytes.

[0171] Go to Fig.17 , shows the electrocardiograms of mice that received either targeted PDT or laser alone. Only targeted PDT could normalize the differences in heartbeat.

[0172] Go to Fig.18 , shows the depth of PDT penetration into tissue (tracked by PI fluorescence through the tissue). Also shows that laser alone has no effect.

[0173] In an embodiment, a new targetable nanoconstruct (NC) that can be used as a therapeutic platform for photodynamic therapy (PDT) and a MR molecular imaging agent that does not contain heavy metal atoms is provided. In an embodiment, a NC-based PDT for cancer is provided. In an embodiment, a F3-cys targeting agent NC is provided. In an embodiment, 8PEGA-Ce6 NC has excellent reactive oxygen species (ROS) production compared to traditional Ce6 encapsulated polyacrylamide (PAAm) NC. This embodiment of NC is also cell-compatible and provides chemical flexibility for the attachment of selected targeting peptides. A label-free 8PEGA NC is also provided, which can be directly and selectively imaged by MRI using a standard spin echo imaging sequence with a large diffusion magnetic field gradient to suppress water signals. It is worth noting that due to its ultra-small size, this NC may have improved in vivo penetration and biological clearance.

[0174] Go to Fig.26 , a schematic diagram of an embodiment of an MRI system 2501 is shown. The system 2501 has a magnet 2502, a gradient coil 2503, a radio frequency coil 2504, a bore 2505, and a table 2506. It should be understood that this figure is a schematic representation of an MRI system that can employ other components and other configurations and types. In an embodiment, the system 2501 is configured to generate three magnetic fields. The first field is a strong static magnetic field that produces energy level differences in cell nuclei having spin angular momentum and causes overall nuclear magnetization. The second field is a radio frequency field and is used to tilt the generated nuclear magnetization so that it can be detected by the RF coil 2504. The third field is a set of magnetic field gradients for spatially encoding signals to create a nuclear magnetization map. Therefore, in an embodiment, the magnetic field is configured to generate an image of non-water protons that are present in an additive placed in a subject to be imaged; wherein the magnetic field gradients can be pulsed in a specific manner to make the cell nuclei sensitive to movement due to flow or diffusion.

[0175] In an embodiment, an imaging agent consisting of nano constructs of the present invention (e.g., nanoparticles based on PEG) is administered to a patient. The patient with the administered imaging agent is then placed on a workbench 2506, the workbench is moved to hole 2505, and system 2501 performs a scan to obtain an MR image of a type such as disclosed in this specification. Nanoparticles are imaged directly to provide detailed images and data about their position in the patient's body. A therapy can be designed, improved, and implemented based on this information. For example, if the nano construct is a targeted PDT nano construct, surgery, surgical PDT, or a separate PDT therapy can be developed and implemented to remove targeted tissue.

[0176] MRI system 2501 has control system 2507, and described control system comprises operator input and other control features, and operation instruction, such as computer code.Control system 2597 is in control communication with device 2510, as shown in dotted line 2508.Control communication refers to the transmission of data, information and control command and other instructions between control system 2507 and device 2510.Control system 2507 can be separated from device 2510, or can be a part of device 2510, such as within the structure of device 2510.In an embodiment, the instruction for operating MRI to provide the operation parameter for imaging of nanoparticles based on PEG is provided to system 2501.For example, this can be the mode of software upgrade.In this way, existing MRI system can be easily upgraded to take full advantage of the benefit of nanoparticle imaging agent based on PEG of the present invention.

[0177] Examples

[0178] The following examples are provided to illustrate various embodiments of the systems, therapies, methods, compositions, applications and materials of the present invention. These examples are for illustrative purposes, which may be prophetic, and should not be considered or would not otherwise limit the scope of the present invention. Unless otherwise expressly specified, the percentages used in the examples are weight percentages of the total amount of, for example, formulations, compositions, mixtures, products or structures, unless otherwise expressly specified.

[0179] Example 1

[0180] Compared with encapsulated PAAm-Ce6 NPs, the examples of Ce6-8PEGA NCs are ultra-small and have excellent ROS production. The successful exchange of the targeting peptide from CTP to F3-cys demonstrates the chemical flexibility of this NC in changing the target. F3-8PEGA-Ce6 NCs have good in vitro and in vivo biocompatibility.

[0181] Example 2

[0182] 8PEGANP and NC embodiments are molecular imaging agents in MRI. These imaging agents can be further combined with techniques to suppress water and fat signals to provide even more enhanced imaging capabilities.

[0183] Example 3

[0184] F3-8PEGA-Ce6 provides an attractive universal NC for theranostics (imaging and PDT) ranging from arrhythmias to cancer and potentially other pathologies. Benefits include among others rapid renal clearance of early tumor accumulation (even before angiogenesis) and MRI results.

[0185] Example 4

[0186] 8PEGA-Ce6 conjugate: Ce6 was conjugated to 8PEGA in DMF via DCC / NHS coupling. Briefly, 448uL of Ce6 solution (20mg / mL, DMF) was stirred (20mg / mL, DMF) for activation for 30 minutes with 154.8uL DCC and 172.8uL NHS. 500mg 8PEGA was dissolved in DMF at a concentration of 50mg / mL using ultrasonic treatment. After dissolution, the Ce6 solution was added to the 8PEGA solution and stirred overnight. The next day, unconjugated Ce6 was removed using a 50% ethanol / PBS mixture in an Amicon cell filtration system using a 10kDa membrane. After purification, the solvent was exchanged with Millipore ultrapure water, the material was filtered using a 0.45um syringe filter, and freeze-dried for storage.

[0187] Example 5

[0188] F3-8PEGA-Ce6 conjugate: 8PEGA-Ce6 was modified with F3 by the same method reported by our laboratory for many years. After modification with F3-cys, UV / VIS was then used to ensure that Ce6 did not aggregate during the process. Briefly, 20 mg of Bi-PEG was added to 1 mL of 8PEGA-Ce6 (20 mg / mL, PBS) and stirred for 30 minutes. The solution was then washed 4x15 minutes in PBS using a 10 kDa centrifugal filter. The resulting solution was concentrated to 20 mg / mL (based on original mass), 22 mg of F3-cys (220 uL, 11 mg / 110 uL DMSO) was added, and stirred overnight. The next day, excess cysteine ​​was added and stirred for 2 hours to cap any unreacted maleimide groups. The solution was then filtered again using a 10 kDa centrifugal filter and Millipore ultrapure water and freeze-dried for storage.

[0189] Example 6

[0190] Ce6 encapsulated polyacrylamide nanoparticles (PAAm NPs): Ce6 was encapsulated in PAA NPs by a previously reported method with slight modifications. Briefly, 5 mg of Ce6 was added to 930 uL of PBS and 100 uL of DMSO with 28 mg APMA, 19 mg NHS and 16 mg EDC. The solution was stirred at 37 ° C for 2 hours. Acrylamide and AHM were then added to the solution (368 mg and 52.6 uL, respectively) and ultrasonicated to produce a uniform solution. This solution was added to a 100 mL round-bottom flask containing 31 mL of hexane, 2.2 mL of Brij L4 and 1.07 g of AOT under stirring. The stirring was adjusted to the extent that the vortex generated just touched the stirring bar (about 500 RPM). The contents of the flask were then purged with nitrogen for 15 minutes. The nitrogen stream was removed from contact with the contents of the flask and kept in the flask. 15 mg of APS was added dropwise in 100 uL of water to initiate polymerization, and 100 uL of TEMED was added dropwise to catalyze the process. The polymerization was allowed to proceed for 2 hours. The hexane was then removed by rotary evaporation. The resulting contents were redispersed in ethanol and washed using 10x150 mL of ethanol and 5x150 mL of Millipore ultrapure water using a 300 kDa filter in an Amicon cell. The purified material was syringe filtered using a 0.45 um filter and freeze dried for storage.

[0191] Example 7

[0192] The drug product contains F3-8PEGA-Ce6 NCs. The NCs have an average particle size of about 10 nm to 15 nm and are uniform with a very narrow particle size distribution of less than about 5% of the mean distance.

[0193] Example 8

[0194] The drug product contains F3-8PEGA-Ce6 NCs. The particle size distribution of NCs is D10=10 nm, D50=12 nm, D90=15 nm.

[0195] Example 9

[0196] The drug product contained F3-8PEGA-Ce6 NCs. The average particle size of the NCs ranged from about 10 nm to 15 nm and was uniform with a very narrow particle size distribution of less than about 10% of the mean distance.

[0197] Example 9A

[0198] The drug product contained F3-8PEGA-Ce6 NCs. The average particle size of the NCs ranged from about 8 nm to 18 nm and was uniform with a very narrow particle size distribution of less than about 10% of the mean distance.

[0199] Example 9B

[0200] The drug product contained F3-8PEGA-Ce6 NCs. The average particle size of the NCs ranged from about 8 nm to 10 nm and was uniform with a very narrow particle size distribution of less than about 10% of the mean distance.

[0201] Example 9C

[0202] The drug product contained F3-8PEGA-Ce6 NCs. The average particle size of the NCs ranged from about 10 nm to 12 nm and was uniform with a very narrow particle size distribution of less than about 10% of the mean distance.

[0203] Example 9D

[0204] The drug product contained F3-8PEGA-Ce6 NCs. The average particle size of the NCs ranged from about 11 nm to 14 nm and was uniform with a very narrow particle size distribution of less than about 10% of the mean distance.

[0205] Example 9E

[0206] The drug product contained F3-8PEGA-Ce6 NCs. The average particle size of the NCs ranged from about 15 nm to 18 nm and was uniform with a very narrow particle size distribution of less than about 10% of the mean distance.

[0207] Example 10

[0208] The drug product contains F3-8PEGA-Ce6 NCs. The particle size distribution of NCs is D10 = about 8 nm, D50 = about 10 nm, D90 = about 12 nm.

[0209] Example 11

[0210] The drug product contains F3-8PEGA-Ce6 NCs. The particle size distribution of NCs is D10 = about 5 to 12 nm, D50 = about 8 to 15 nm, D90 = about 12 to 22 nm.

[0211] Example 11A

[0212] The drug product contains F3-8PEGA-Ce6 NCs. The particle size distribution of NCs is D10 = about 6 to 10 nm, D50 = about 8 to 15 nm, D90 = about 12 to 20 nm.

[0213] Example 11B

[0214] The drug product contains F3-8PEGA-Ce6 NCs. The particle size distribution of NCs is D10 = about 10 to 12 nm, D50 = about 11 to 13 nm, D90 = about 10 to 15 nm.

[0215] Example 12

[0216] The drug products of Examples 7 to 12 are useful as imaging agents, contrast agents, or both for NMR or MRI.

[0217] Example 13

[0218] The drug products of Examples 7 to 12 include other NCs, imaging agents or therapeutic agents.

[0219] Example 14

[0220] The drug products of Examples 7 to 12 may be used together with other drug products, such as other NCs, imaging agents or therapeutic agents.

[0221] Example 15

[0222] The drug product contained TA (targeting agent)-8PEGA-AA (active agent) NCs. The average particle size of the NCs was about 10 nm and was uniform with a very narrow particle size distribution of less than about 5% of the mean distance.

[0223] Example 16

[0224] The drug product contains TA-8PEGA-AA NCs. The particle size distribution of NCs is D10=10 nm, D50=12 nm, D90=15 nm.

[0225] Example 17

[0226] The drug product contained TA-8PEGA-AA NCs. The average particle size of the NCs ranged from about 10 nm to 15 nm and was uniform with a very narrow particle size distribution of less than about 10% of the mean distance.

[0227] Example 18

[0228] The drug product contains TA-8PEGA-AA NCs. The particle size distribution of NCs is D10 = about 8 nm, D50 = about 10 nm, D90 = about 12 nm.

[0229] Example 19

[0230] The drug product contains TA-8PEGA-AA NCs. The particle size distribution of NCs is D10 = about 6 to 10 nm, D50 = about 8 to 15 nm, D90 = about 12 to 20 nm.

[0231] Example 20

[0232] The drug products of Examples 15 to 19 are useful as imaging agents, contrast agents, or both for NMR or MRI.

[0233] Example 21

[0234] The drug products of Examples 15 to 19 include other NCs, imaging agents or therapeutic agents.

[0235] Example 22

[0236] The drug products of Examples 15 to 19 may be used together with other drug products, such as other NCs, imaging agents or therapeutic agents.

[0237] Example 23

[0238] The drug products of Examples 15 to 22, wherein the TA is one or more of the following, for example: Any peptide ending in cysteine ​​and not containing any additional free thiol groups can be attached to 8PEG and used, for example, RGD, cRGD, iRGD, F3, and CTP. Additional examples are contained in publicly available phage libraries. In general, any cell in which a small molecule or peptide selectively accumulates can be attached to or is part of the NC and thus act as a TA, including a TA that is 8PEGA.

[0239] Example 24

[0240] The pharmaceutical products of Examples 15 to 22, wherein AA is one or more of the following: photosensitizers, sonosensitizers, photoacoustic agents and other products disclosed in this specification, known to those skilled in the art or later developed.

[0241] Example 25

[0242] A NC with a particle size of about 8-15 nm and having AA from the group of Example 24.

[0243] Example 26

[0244] A NC with a particle size of about 8-15 nm and having a TA from the group of Example 23.

[0245] Example 27

[0246] A NC with a particle size of about 10-20 nm and having AA from the group of Example 24.

[0247] Example 28

[0248] A NC with a particle size of about 10-20 nm and a TA from the group of Example 23.

[0249] Example 29

[0250] A drug product having NCs from Examples 25, 26, 27 and 28 has a particle size distribution with D10 and D90 values ​​within 5 nm of the D50 value.

[0251] Example 30

[0252] A drug product having NCs from Examples 25, 26, 27 and 28 has a particle size distribution with D10 and D90 values ​​within 10 nm of the D50 value.

[0253] Example 31

[0254] A drug product having NCs from Examples 25, 26, 27 and 28 has a particle size distribution with D10 and D90 values ​​within 2 nm of the D50 value.

[0255] Example 32

[0256] MRI calibration for quantitative imaging of embodiments of the imaging agents of the present invention. Images and spectra produced by selective 8PEGA NMR imaging and spectral sequences and protocols will have signal intensities proportional to the local concentration of the PEG nanostructure. If it is desired to quantitatively measure the absolute concentration of the PEG nanostructure in mg / ml or molar concentration, the MRI system can be calibrated by performing a selective PEG pulse sequence in a specific MRI scanner with a model of known PEG concentration or by simultaneously imaging a patient and a model of known PEG concentration. In addition, concentration calibration curves can be as shown in FIG. Figure 6 and 7 Build as shown, Figure 6 and 7 It is shown that the signal intensity generated by PEG-selective MRI is proportional to the concentration of 8PEGA.

[0257] Example 32A

[0258] MRI is configured to image protons in PEG-based imaging agents. MRI is an imaging tool that is often used to provide physicians with diagnostic medical information to aid in patient care management. MRI uses three magnetic fields. First, a strong static magnetic field creates energy level differences in cell nuclei that have spin angular momentum and causes overall nuclear magnetization. Second, a radio frequency (RF) field is used to tilt the resulting nuclear magnetization so that it can be detected by the RF coil. Finally, a set of magnetic field gradients is used to spatially encode the signal to create a nuclear magnetization map. In addition, the magnetic field gradients can be pulsed in a specific manner to make the cell nuclei sensitive to motion due to flow or diffusion. MRI pulse sequences consist of a series of RF and gradient pulses to produce MR images that are sensitive to T1, T2, diffusion, and other parameters.

[0259] Example 32B

[0260] exist Figure 6 and 7An example of an MRI pulse sequence for producing 8PEGA-specific images can be seen in FIG. 1 . In this particular case, a diffusion-weighted spin echo imaging sequence with a repetition time TR = 500 milliseconds, an echo time TE = 200 milliseconds, an amplitude G = 100, and a pulse width of 100 milliseconds is used. diff = 126 mT / m, with a duration of δ = 7.1 ms and an interval of Δ = 180 ms, to produce 10 10 s / m 2 The diffusion b value. The magnetic field gradient reduces the MR signal intensity by S(b) = exp(-bD), where

[0261] The diffusion constant D of water at 25°C is 2.2 10 -9 m 2 s -1 , and 8PEGA is 3.5 10 -11 m 2 s -1 , two orders of magnitude slower than water. Under these specific parameters, the pure water signal will be reduced by exp(-bD) = 2.7 10 -10 , effectively placing the water signal at the noise floor. In contrast, 60% of the PEG8A signal remains at b = 10 10 s / m 2 Background images of both PEG8A and water were obtained using the same parameters, except that the gradient intensity was reduced to 12.5 mT / m, yielding a 10 8 s / m 2 With these parameters, water decays to 80% of its initial value and PEG8A decays to 99%, but the water proton concentration is significantly higher and dominates the MRI signal.

[0262] In vivo, water diffusion is restricted by cell walls, and it is estimated that approximately 5% of the water signal will remain at b = 10 10 s / m 2 The water proton T2 time is typically <80 milliseconds, while the 8PEGA T2 time is >500 milliseconds. Therefore, additional water suppression can be achieved by imaging at long TE times. The signal decay will be as S(b,TE)=exp(-TE / T2)exp(-bD), so additional water attenuation can be achieved by imaging at long TE times. In addition, conventional water suppression methods based on chemical shift or water proton relaxation time can be used simultaneously with the gradient suppression method and provide even more water signal attenuation.

[0263] In clinical MRI systems, the magnitude of the magnetic field gradient is limited to a value of approximately 40 mT / m. 10 s / m 2For example, with a gradient amplitude of 40 mT / m, δ = 60 milliseconds and Δ = 200 milliseconds resulting in a b value of 7 × 10 10 s / m 2 , which is sufficient for selective imaging of 8PEGA.

[0264] In general, any MRI pulse sequence that provides a high b-value and a TE time >100 msec will provide adequate attenuation of the water and fat signals.

[0265] Example 33

[0266] Photoacoustic imaging - Photoacoustic imaging (PAI) provides greater depth limits than other optical imaging systems while also improving resolution. PAI uses acoustic waves generated in response to the absorption of pulsed laser light and provides non-invasive images of the absorbed light energy density at depths of several centimeters with a resolution of, for example, about 100 μm and potentially greater.

[0267] 8PEGA photoacoustic imaging NC (PAI-NC) has an active agent. The particle size of PAI-NC is about 10nm to 20nm. PAI-NC can also have a targeting agent, including one of the targeting agents from Example 23.

[0268] Active agents (eg, imaging agents, contrast agents) for PAI-NC can be, for example, small molecule dyes, gold, carbon, liposome encapsulation, heptamethine cyanine dyes (eg, indocyanine green), azo dyes (eg, methylene blue), and naphthalocyanine dyes.

[0269] Typically, when selecting an active agent, if there is a hydrophilic R group in the compound structure, it should be possible to label PEG (the most common groups are sulfonic acid and carboxylate). Cyanine dyes typically contain a sulfonic acid group (SO3-), which can be derived into a chlorinated intermediate that will be highly reactive to the amines on the PEG. ICG (indigocyanine green, obtained under the trade name CARDIOGREEN) is an example of a spectral absorption (780nm) agent for 8PEGA PAI-NCs approved by the FDA. Other substances that can be used in PAI-NCs include, for example, Coomassie Brilliant Blue (absolute maximum = 595-610nm), alexa fluor 750, IR780 and IRDye 800, Ce6, methylene blue and SiNc.

[0270] Example 34

[0271] A system and method for high resolution and accurate diagnosis. The system uses a drug product with targeted 8PEGA Nc to obtain MRI of NC located in a target tissue (e.g., a tumor), and in this way obtains images of the targeted tissue and the location, concentration, content, and combinations and changes of NC in the targeted tissue. The image provides data and information about the shape, location, and position of the target tissue and NC, as well as combinations and changes of these, and other information. The image is then stored and transferred to a resolution-enhanced photoacoustic imaging device for MRI. Based at least in part on or using enhanced PAI images of NC and targeted tissue, a customized laser delivery pattern for delivering energy to activate an active agent on NC is developed. The customized laser delivery pattern is then delivered to the targeted tissue. In this way, very precise treatment of the condition can be performed. The combination of enhanced imaging, targeted NC, and a predetermined laser delivery pattern provides the ability to remove tissue (including cellular levels) very precisely. This system and method essentially provides a cell scalpel.

[0272] This system can be in an integrated unit. It can be in several different units, where the data in each unit is passed to the other units. In this way, the units can be configured in a network.

[0273] Additionally, the system can monitor the effectiveness of laser delivery patterns and, based on historical data, refine and enhance predetermined laser delivery patterns for specific situations, conditions, or tissue types.

[0274] Additionally, the system provides the ability to perform various operations at different times. 8PEG NCs can remain in the targeted tissue and remain active or viable as a theranostic material for about 12 hours to about 1 week, about 1 day to about 4 days, about 12 hours to about 3 days, and all values ​​within these ranges, as well as longer or shorter times.

[0275] Example 35

[0276] An 8PEGA therapeutic NC having a dynamic therapeutic agent (eg, an active agent) that is activated upon exposure to acoustic energy.

[0277] Example 35A

[0278] An 8PEGA therapeutic NC having a sonodynamic therapeutic agent (eg, an active agent) that is activated upon exposure to sonic energy.

[0279] Example 36

[0280] An 8PEGA theranostic NC with a dynamic therapeutic agent that is activated upon exposure to an energy source. The agent may be selected to be activated by acoustic energy, light energy, or any electromagnetic energy source.

[0281] Example 37

[0282] The systems and methods of Examples 32, 32A, 32B, 33, and 34 may further use models and algorithms to further enhance the resolution of images, location, shape, and position of targeted tissue and NCs, and laser delivery patterns.

[0283] Example 38

[0284] The systems of Examples 32, 32A, 32B, 33 and 34 are used to provide increased layering and data modeling of images. These layered and modeled images are valuable for diagnostic, therapeutic and theranostic purposes. This data, layered images and both can be further used with machine learning to provide enhanced systems of these examples.

[0285] Example 39

[0286] The systems of Examples 32, 32A, 32B, 33, and 34 provide for quantification of 8PEGA NCs in biological tissues (eg, tumor regions and filter organs).

[0287] Example 40

[0288] An example of applied 8PEGA would require the following: addition of PS via a DCC / NHS coupling reaction in DMF to yield approximately 1.5 PS per 8PEGA; conversion of the amine arms to maleimide using NHS-PEG-MAL (2 kDa); addition of a cysteine-terminated peptide anchored to 8PEGA via the known thiol-maleimide reaction; and no further modification of DWI using 8PEGA.

[0289] Title and Examples

[0290] It should be understood that the use of headings in this specification is for the purpose of clarity and is not limiting in any way. Therefore, the processes and disclosures described under the headings should be read in the context of the entire specification (including various examples). The use of headings in this specification should not limit the scope of protection provided by the present invention.

[0291] The various embodiments of the systems, therapies, processes, compositions, applications and materials described in this specification can be used in various other fields and various other activities, uses and embodiments. In addition, for example, these embodiments can be used with the following: existing systems, therapies, processes, compositions, applications and materials; can be used with systems, therapies, processes, compositions, applications and materials that may be developed in the future; and systems, therapies, processes, compositions, applications and materials that can be modified in part based on the teachings of this specification. Further, the various embodiments and examples described in this specification can be used in whole or in part and in different and various combinations. Therefore, for example, the configurations provided in the various embodiments and examples of this specification can be used with each other. For example, according to the teachings of this specification, components of embodiments with A, A' and B and components of embodiments with A ", C and D can be used with each other in various combinations (for example, A, C, D and A, A ", C and D, etc.). Therefore, the scope of protection provided by the present invention should not be limited to the specific embodiments, examples, configurations or arrangements described in the embodiments in specific embodiments, examples or specific figures.

[0292] The present invention may be embodied in other forms than those specifically disclosed herein without departing from the spirit or essential characteristics of the invention.The described embodiments are to be considered in all respects only as illustrative and not restrictive.

Claims

1. A method of imaging in a subject, the method comprising the steps of: (i) administering to a subject an imaging agent comprising a plurality of nanoconstructs; wherein each of the nanostructures comprises a multi-arm PEG and one or more additives associated with the multi-arm PEG, wherein the one or more additives comprise a photosensitizer PS; and wherein when scanned in step (ii), the multi-arm PEG in the plurality of nanostructures alone is sufficient to provide a nuclear magnetic resonance image; (ii) after step (i), performing an imaging scan of the subject, wherein the imaging scan is a magnetic resonance imaging scan; (iii) providing a nuclear magnetic resonance image of the multi-armed PEG in the plurality of nanostructures; (iv) applying light energy to the nanoconstruct to activate the PS; and (v) after step (iv), obtaining a second imaging scan of the subject, wherein the second imaging scan is selected from the group consisting of: a nuclear magnetic resonance scan, a computerized axial tomography scan, and a proton emission tomography (PET) scan.

2. The method according to claim 1, wherein the method further comprises the steps of performing a computerized axial tomography scan; a proton emission tomography (PET) scan; and / or a fluorescence imaging scan.

3. The method of claim 1, wherein the NMR scan comprises spin echo imaging or diffusion weighted spin echo imaging, and wherein the NMR scan suppresses proton signals from water in tissue and / or fat present during the imaging scan.

4. method according to claim 1, wherein in step (iii), described nuclear magnetic resonance image does not include water matrix molecules in described experimenter.

5. The method of claim 1, wherein the multi-arm PEG has 2, 4, 6, 8, 10 or more arms.

6. The method of claim 1, wherein the one or more additives further comprises an active agent selected from the group consisting of a targeting agent, a therapeutic agent, a photosensitizer, an imaging agent, a diagnostic agent, a theranostic agent, and combinations thereof, and wherein the active agent is covalently associated with the backbone material.

7. The method of claim 1, wherein step (iii) further comprises providing data identifying the shape, structure and / or location of a tumor in the subject.

8. The method of claim 1, wherein step (iii) further comprises providing data identifying the concentration of the imaging agent in a tumor in the subject.

9. The method of claim 1, wherein the nanoconstruct is a photodynamic therapy (PDT) nanoconstruct and wherein the one or more additives comprise a PDT agent.

10. The method of claim 6, wherein the framework material is non-paramagnetic.

11. The method of claim 1, wherein the nanoconstruct comprises 2,000 to 5,000 protons, and wherein the nanoconstruct has a size of less than 25 nm.

12. The method of claim 1, wherein the nanoconstruct comprises 5,000 to 15,000 protons, and wherein the nanoconstruct has a size of less than 50 nm.

13. The method of claim 1, wherein the one or more additives comprise a tetrapyrrole compound or a phthalocyanine dye, or a combination thereof.

14. The method of claim 1, wherein the one or more additives further comprises a porphyrin, a chlorin, a phthalocyanine, or a bacteriochlorin, or a combination thereof.

15. The method of claim 1, wherein the nanoconstruct has a molecular weight in the range of 35 kDa to 75 kDa.

16. The method of claim 1, wherein the one or more additives further comprises a targeting agent.

17. The method of claim 16, wherein the targeting agent is a cardiac targeting peptide.

18. The method of claim 16, wherein the targeting agent is a cancer targeting agent.

19. The method of claim 18, wherein the cancer targeting agent is selected from the group consisting of RGD, cRGD, iRGD and F3.

20. The method of claim 6, wherein the imaging agent is a contrast agent.

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