CN114057773A - Near-infrared two-region aggregation-induced emission molecule and application thereof - Google Patents
Near-infrared two-region aggregation-induced emission molecule and application thereof Download PDFInfo
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Abstract
The invention relates to the design of organic fluorescent materials, in particular to a small-molecule fluorescent compound with polymerization-induced light-emitting characteristics, which can display emitted light in a second near-infrared window (1000nm-1700nm) and can provide imaging for deep tissues with ultrahigh signal-to-noise ratio. For example, neutrophils carrying the compounds of the invention can penetrate the brain and observe inflammation deep in the brain tissue through the intact scalp and skull.
Description
Technical Field
The invention relates to the development of organic fluorescent materials, in particular to a molecule with polymerization-induced light-emitting characteristics and application of the molecule in near-infrared two-region (1000nm-1700nm) fluorescence imaging.
Background
Fluorescence imaging with high temporal and spatial resolution and sensitivity provides a powerful tool for the direct visualization of dynamic biological processes. Fluorescent molecules capable of emitting light in the near infrared two region (NIR-II, 1000nm-1700nm) show significant advantages of deeper tissue penetration, higher spatial resolution and better signal-to-noise ratio due to the reduced light scattering and autofluorescence in tissue at longer wavelengths. Therefore, near-infrared fluorescence imaging offers great promise for accurate diagnosis of deep diseases.
Although deep diseases such as brain tumors can be observed using fluorescent molecules emitting in the near infrared II region, achieving excellent imaging quality with high signal-to-noise ratio can be challenging. This requires the development of near infrared fluorescent molecules with high Quantum Yields (QY). To date, most near infrared fluorescent molecules from organic molecules have shown low quantum yields (about 2%) in aqueous dispersions or solutions due to the predominance of non-radiative decay pathways, which reduces contrast and increases the sensitivity of optical detectors and the imaging requirements during in vivo imaging.
At present, there are two main methods for designing near-infrared fluorescent molecules: enhanced conjugation length and donor-acceptor engineering. In the former case, increasing the conjugation length shifts the absorption and emission red of polymethine cyanine dyes (e.g., IR-26) into the near infrared region, while fluorescence is effectively attenuated by quenching due to solvatochromism. Polymerization of small molecules into the corresponding conjugated polymers provides another strategy; however, strong intermolecular interactions and entanglement can cause damage to fluorescence. Alternatively, digital to analog engineering provides an efficient way to reduce the energy band gap and red-shift the emission. In order to emit fluorescence in the near infrared window, deoxyribonucleic acid based fluorescent molecules typically employ a widely conjugated backbone. The strong intermolecular interactions resulting from the formation of excimers often cause aggregation-induced quenching (ACQ) problems. In addition, non-radiative decay in the dark-Twisted Intramolecular Charge Transfer (TICT) state, typically observed in polar environments such as water, also destroys fluorescence quantum yield.
To address these problems, electron donating groups with steric hindrance are typically grafted onto a strong acceptor, such as benzobisthiadiazole (BBTD), to distort the conjugated backbone, which can reduce intermolecular interactions and thus the formation of molecular excitons. In addition, dialkyl substituted fluorenes are incorporated into the central DAD core to act as shielding units to prevent interaction with water. These strategies open up new avenues for developing a series of high brightness near infrared fluorescent molecules. Although long side chains can reduce intermolecular interactions, they can also initiate molecular motion in the aggregates, and nonradiative decay of the aggregates can disrupt fluorescence quantum yield. Therefore, effectively increasing the radiation attenuation is an important bottleneck for high-brightness near-infrared fluorescent molecules.
Molecules with aggregation-induced emission (AIE) properties have great potential to solve this problem. Aggregation-induced emission molecules (AIEgen) generally fill the molecular rotor like a propeller, and thus this deeply twisted structure can effectively reduce the intermolecular interactions. It is for this reason that AIEgen is more emissive as a nanoparticle than other molecules. According to the AIEgen molecular design philosophy, many near-infrared secondary emission type light emitting diodes have been developed. The optimized sample had a slightly enhanced quantum yield of 6.2% with reduced fluorescence emission (975nm <1000 nm). To date, achieving high quantum yields of near-infrared fluorescent molecules using AIE molecular design principles remains a challenge.
Disclosure of Invention
As described above, it is desired in the art to develop an aggregation-inducing luminescent molecule having a higher emission intensity in the near-infrared region, which is capable of imaging deep tissues with an ultra-high signal background ratio.
Based on this, the inventors of the present invention synthesized a series of compounds having aggregation-induced emission properties through rational molecular design, and found that: these compounds may exhibit emission in the near infrared region II (1000nm-1700nm), providing imaging of deep tissues with ultra-high signal-to-background ratios. For example, neutrophils carrying the compounds of the invention can penetrate the brain and allow observation of inflammation deep in brain tissue through the intact scalp and skull.
Accordingly, in a first aspect of the present invention, there is provided a compound represented by the following formula (I) or (II):
wherein
R1Is a molecular rotor, each R1Each independently selected from:
n is an integer ranging from 1 to 20,
each R2Each independently selected from:
wherein X is an integer ranging from 0 to 20, m is an integer ranging from 6 to 22, and Y is an integer ranging from 10 to 22; and the number of the first and second electrodes,
two R2Each independently located at 3 or 4 bits of the label, but not at 3 bits at the same time.
The compounds of the present invention have Aggregation Induced Emission (AIE) properties, can exhibit emission in the near infrared region II (1000nm-1700nm), and can provide imaging of deep tissues with ultra-high signal-to-background ratios.
In a second aspect of the invention, there is provided a conjugate comprising a compound of the first aspect of the invention conjugated to a protein.
In a third aspect of the invention, there is provided a composition comprising a compound of the first aspect of the invention or a conjugate of the second aspect of the invention, and a matrix for encapsulating the compound or the conjugate.
In a fourth aspect of the invention there is provided a labelled immune cell labelled with a compound of the first aspect of the invention, a conjugate of the second aspect of the invention or a composition of the third aspect of the invention.
In a fifth aspect of the invention, there is provided a probe for in vivo or in vitro fluorescence imaging comprising: a compound of the first aspect of the invention, a conjugate of the second aspect of the invention, or a composition of the third aspect of the invention.
In a sixth aspect of the invention, there is provided a method of performing biological imaging, the method comprising:
administering a probe of the fifth aspect of the invention to a subject in need thereof.
In a seventh aspect of the invention, there is provided a kit for in vivo or in vitro imaging, the kit comprising:
a probe according to the fifth aspect of the invention; and
instructions for directing imaging.
In an eighth aspect of the invention there is provided use of a probe of the fifth aspect of the invention in the manufacture of a detection agent for detecting tissue inflammation in a subject.
In a ninth aspect of the invention, there is provided a method of detecting tissue inflammation in a subject, the method comprising: administering a probe of the fifth aspect of the invention to the subject.
In a tenth aspect of the invention there is provided a probe of the fifth aspect of the invention for use in detecting tissue inflammation in a subject.
The invention has the advantages that:
the invention provides a series of compounds with aggregation-induced emission properties, which can display emission in a near-infrared II region (1000nm-1700nm) and can provide imaging for deep tissues with ultrahigh signal-to-background ratio. For example, neutrophils carrying the compounds of the invention can penetrate the brain and allow observation of inflammation deep in brain tissue through the intact scalp and skull.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, provide a further explanation of the invention. It is to be understood that the drawings in the following description are directed to only some embodiments of the invention and are not to be construed as limiting the invention. Other embodiments can be derived from these figures without inventive effort for a person skilled in the art.
FIG. 1 shows the chemical structure (a), optimized ground state (S) of 2TT-oC6B, 2TT-m, oC6B and 2TT-mC6B0) Geometry (b), calculated HOMO and LUMO (c), absorption and emission spectra (d), alpha AIE curves (e), calculated reorganization energy (reorganisation energy) versus normal mode wave number (f), bond length (bond length), bond angle (bond angle) and dihedral angle (dihedral angle) contributions to the total recombination energy (g), S0(Black) and S1(red) electron state computed Density Functional Theory (DFT) minimum energy geometry (h).
Figure 2 shows a schematic representation of AIE nanoparticles for Neutrophil (NE) -mediated brain inflammation IR-II imaging (a), Dynamic Light Scattering (DLS) spectra of AIE nanoparticles (2TT-oC6B) (with inset representing transmission electron microscopy images at 100nm scale) (b), and normalized absorption and emission spectra of AIE nanoparticles (2TT-oC6B) (c).
FIG. 3 shows the integrated intensity of the fluorescence spectra of five different concentrations of 2TT-oC6B, 2TT-m, oC6B and 2TT-mC6B nanoparticles and IR-26 in the 900-1500nm interval.
Fig. 4 shows photographs of NIR-II fluorescence imaging of blood vessels in hind limbs (a) and scalp (c) using 2TT-m, oC6B nanoparticles and ICG and fluorescence signals (b and d) at sections along the red dashed lines in hind limbs and scalp.
FIG. 5 shows a photograph of NIR-II fluorescence imaging of scalp vessels using 2TT-oC6B nanoparticles and ICG.
FIG. 6 in vivo NIR-II fluorescence imaging of lymphatic system using 2TT-m, oC6B nanoparticles, where a is bright field photograph, b-e is fluorescence photograph captured at 10min, 2h, 4h, 24h, f is bright field (left panel) and fluorescence photograph (right panel) of sentinel lymph node removed from mouse under NIR-II fluorescence guidance.
Figure 7 shows tumor resection with 2TT-m, oC6B nanoparticle imaging guidance and without 2TT-m, oC6B nanoparticle imaging guidance, where a is bioluminescence and NIR-II imaging of the abdominal cavity before and after tumor resection, b is bioluminescence and NIR-II imaging of tumor nodules resected from the unguided and 2TT-m, oC 6B-guided groups, and c is a histogram of tumor nodule diameters resected from the unguided and 2TT-m, oC 6B-guided groups.
FIG. 8 shows the optical density at 808nm (20.6 mW/cm)2) NIR-II fluorescence images (1000nm LP, 50ms) (a), 5X 10 of different numbers of AIE @ NE and ICG @ NE cells under laser irradiation5Mean fluorescence signals of individual AIE @ NE and ICG @ NE cells (b), fluorescence images of different numbers of AIE @ NE and ICG @ NE cells following subcutaneous injection (c), mean fluorescence signals of AIE @ NE and ICG @ NE cells of the data provided in c (d), non-invasive time-dependent in vivo near-infrared fluorescence images of brain inflammation through the intact scalp and skull (1000nm LP, 100ms) (e), mean fluorescence signals of AIE @ NE and ICG @ NE cells as a function of time in the affected area (f), and mean signal-to-background ratio in inflamed brain at 12 hours (SBR) (5 mm scale) (g).
FIG. 9 shows the tissue distribution and fluorescence intensity of AIE @ NE and ICG @ NE in a mouse model of brain inflammation.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings. It is to be understood that the following description is intended to illustrate the present invention by way of example only and is not intended to limit the scope of the invention, which is defined by the appended claims. Also, it is understood by those skilled in the art that modifications may be made to the technical aspects of the present invention without departing from the spirit and gist of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.
As used herein, the term "λ ex" refers to the excitation wavelength.
As used herein, the phrase "aggregation-induced quenching" or "ACQ" refers to the phenomenon in which aggregation of a pi-conjugated fluorescent molecule significantly reduces the fluorescence intensity of the fluorescent molecule. Aggregate formation can result in "quenching" of the light emission of the fluorescent molecule.
As used herein, the phrase "aggregation-induced emission" or "AIE" refers to the phenomenon of a compound exhibiting significantly enhanced light emission when aggregated in an amorphous or crystalline (solid) state, while exhibiting weak or little emission in dilute solutions.
As used herein, the term "emission intensity" refers to the magnitude of fluorescence/phosphorescence, typically obtained by fluorescence spectroscopy or fluorescence microscopy measurements; the term "fluorescent molecule" as used herein refers to a molecule that exhibits fluorescence; the term "luminescent molecule" as used herein refers to a molecule that exhibits luminescence; the term "AIEgen" or "aggregation-induced emission molecule" as used herein refers to a molecule that exhibits aggregation-induced emission (AIE) characteristics.
As used herein, a "donor" material refers to an organic material, such as an organic nanoparticle material, having holes as the predominant current or charge carrier.
As used herein, an "acceptor" material refers to an organic material, such as an organic nanoparticle material, having electrons as the predominant current or charge carrier.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this invention belongs.
Where a range of values is provided, such as a concentration range, a percentage range, or a ratio range, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the subject matter described. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also encompassed within the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.
Throughout this application, many embodiments use the expressions "comprising", "including" or "consisting essentially of … …". The terms "comprises," "comprising," or "consisting essentially of … …," are generally to be construed as open-ended terms that include not only the elements, components, assemblies, method steps, etc., specifically recited below in the term, but also other elements, components, assemblies, method steps. In addition, the expressions "comprising", "including" or "consisting essentially of … …" may in some cases also be understood as a closed expression in the present context, meaning that only the elements, components, assemblies, method steps specifically listed after the expression are included, but not any other elements, components, assemblies, method steps. At this time, the expression is equivalent to the expression "consisting of … …".
For a better understanding of the present teachings and not to limit the scope of the present teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions used in the specification and claims, as well as other numerical values, are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
As described above, there is a need in the art for a small molecule fluorescent compound having polymerization-induced emission characteristics, capable of exhibiting emission in the near-infrared two-region (1000nm to 1700nm), and capable of providing imaging for deep tissues with an ultra-high signal-to-background ratio (SBR).
Accordingly, in a first aspect of the present invention, there is provided a compound represented by the following formula (I) or (II):
wherein
R1Is a molecular rotor, each R1Each independently selected from:
n is an integer ranging from 1 to 20,
each R2Each independently selected from:
wherein X is an integer ranging from 0 to 20, m is an integer ranging from 6 to 22, and Y is an integer ranging from 10 to 22; and the number of the first and second electrodes,
two R2Each independently located at 3 or 4 bits of the label, but not at 3 bits at the same time.
Herein, n may be specifically an integer of 0, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
Herein, X may be specifically an integer of 0, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. In a preferred embodiment, X is 5, i.e. R2In the case of hexyl.
Herein, m may be specifically an integer of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, and Y may be specifically an integer of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22.
In a specific embodimentIn the compound, each R2Each independently selected from:
wherein X is an integer ranging from 0 to 20.
As defined above, X may specifically be an integer 0, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
In one embodiment, the compound is represented by the following formula (I):
wherein
R1Is a molecular rotor, each R1Each independently selected from:
each R2Each independently selected from:
wherein X is an integer ranging from 0 to 20.
Herein, for example, X may be specifically an integer of 0, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.
In yet another specific embodiment, the compound is represented by any one of the following formulas (III) - (VI):
wherein n is an integer ranging from 1 to 20, such as the integer 0, 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
In a second aspect of the invention, there is provided a conjugate comprising a compound of the first aspect of the invention conjugated to a protein.
First, it will be appreciated that the description hereinbefore with respect to the compounds of the first aspect of the invention also applies to the compounds of this aspect of the invention. Therefore, for the sake of brevity, no further description of the compounds will be provided herein.
Further, in the present invention, the conjugate specifically means a compound in which a compound of the present invention and a protein are covalently bonded to each other. The protein may have a function such as a targeting function (e.g., targeting peptide) or a cell penetrating function (e.g., cell penetrating peptide), whereby the compound is imparted with such a function such as targeting or cell penetrating by conjugation to the compound.
In a specific embodiment, the protein is a cell penetrating peptide, such as a transcribed transactivator protein. In this way, the compounds of the invention can be more easily and conveniently introduced into cells, such as immune cells, when the cells are labeled with the conjugates.
In a third aspect of the invention, there is provided a composition comprising a compound of the first aspect of the invention or a conjugate of the second aspect of the invention, and a matrix for encapsulating the compound or the conjugate.
Herein, the term "matrix" refers to a polymer having amphiphilicity (amphophathy). By "amphiphilic" is meant that the molecule has both hydrophilicity and lipophilicity, where hydrophilicity and lipophilicity are properties of a particular group. The amphiphilic polymer is a macromolecular compound containing a hydrophilic segment and an oleophilic segment in the same molecular chain, and can be also called a macromolecular surfactant because it can reduce the surface tension of water. The incompatibility of the hydrophilic chain segment and the lipophilic chain segment can cause the micro-phase separation, so that the amphiphilic polymer shows the self-assembly characteristic in selective solvents, bodies, surface and interface structures.
Thus, in one embodiment, the matrix is an amphiphilic polymer. By way of example, the amphiphilic polymer may include 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000] and Pluronic F127, but is not limited thereto. Any other useful amphiphilic polymer is also within the scope of the present invention.
In a preferred embodiment, the compound or conjugate of the invention is encapsulated within the matrix. By encapsulating the compounds or conjugates of the invention in a matrix, fluorescence imaging can be better performed using the aggregation-induced emission properties of the compounds of the invention.
In one embodiment, the compound is in the form of nanoparticles. It is noted that, as used herein, the term "AIE nanoparticle" is used interchangeably with the expression "AIE dot" or "AIE-dot(s)", and refers to AIE particles having a size within 1000nm (≦ 1000 nm). In a preferred embodiment, the nanoparticles are 20-500nm, more preferably 20-200nm, most preferably 50-120nm nanoparticles.
In a fourth aspect of the invention there is provided a labelled cell labelled with a compound of the first aspect of the invention, a conjugate of the second aspect of the invention or a composition of the third aspect of the invention.
In this context, by "labeling" is meant the process of allowing a compound, conjugate or composition of the invention to enter the interior of a cell by incubating the cell with the compound, conjugate or composition of the invention for a period of time. It will be appreciated that in order to allow better labelling of the cells by the compounds of the invention, in other words better entry into the cells, it is preferred to use conjugates conjugated with cell-penetrating peptides or compositions comprising said conjugates. Thus, the compound of the present invention can be more easily taken into cells by the action of a cell-penetrating peptide, thereby exerting its effect such as fluorescence imaging.
In a preferred embodiment, the cell is an immune cell, such as a neutrophil. As can be appreciated by those skilled in the art, immune cells such as neutrophils have a tendency to migrate to sites of inflammation. Thus, after immune cells, such as neutrophils, labeled with a compound, conjugate or composition of the invention are administered into a subject by injection (e.g., intravenous injection), they migrate to the site of inflammation. When a subject is subjected to biological imaging, such as near infrared II-zone fluorescence imaging, the luminescent site indicates the presence of inflammation at the site.
In a fifth aspect of the invention, there is provided a probe for in vivo or in vitro fluorescence imaging comprising: it includes: a compound of the first aspect of the invention, a conjugate of the second aspect of the invention, or a composition of the third aspect of the invention.
As described above, the compound of the present invention is an aggregation-inducing luminescent molecule and is capable of fluorescence imaging in the near-infrared region. Thus, in one embodiment, the probe may be used for near infrared two-zone fluorescence imaging.
In yet another embodiment, the probe may be an injectable formulation, such as an intravenous or subcutaneous injectable formulation. Of course, it is to be understood that any other suitable type of formulation may be within the scope of the present invention.
In a sixth aspect of the invention, there is provided a method of performing biological imaging, the method comprising:
administering a probe of the fifth aspect of the invention to a subject in need thereof.
In one embodiment, the administration is by injection, e.g., intravenous or subcutaneous injection. Of course, other suitable modes of administration are also possible.
In one embodiment, the subject is bioimaged during a period of 10 hours to 24 hours after administration of the probe. Preferably, the subject is bioimaged 12 hours after administration of the probe.
In a preferred embodiment, the biological imaging is fluorescence imaging; more preferably, the biological imaging is near infrared two-zone fluorescence imaging.
In one embodiment, bioimaging is performed by irradiating the subject with laser light. In a preferred embodiment, the laser wavelength used for said laser irradiation is 700-2Preferably 20.6mW/cm2Each irradiation is for 50 to 100ms, preferably 50 ms.
In one embodiment, the subject is a mammal. For example, the mammal is a human or non-human animal such as a mouse, rat, rabbit, primate such as monkey, chimpanzee, and the like.
In yet another embodiment, the bioimaging is bioimaging of tissue within the subject at least 3 mm from its surface. In a preferred embodiment, the tissue is brain tissue within the brain of the subject at least 3 mm from the scalp.
In one embodiment, the tissue is a blood vessel in the body, such as a blood vessel in the limbs, scalp and brain.
In a seventh aspect of the invention, there is provided a kit for in vivo or in vitro imaging, the kit comprising:
a probe according to the fifth aspect of the invention; and
instructions for directing imaging.
In a preferred embodiment, the imaging is near infrared two-zone fluorescence imaging.
In an eighth aspect of the invention there is provided use of a probe of the fifth aspect of the invention in the manufacture of a detection agent for detecting tissue inflammation in a subject.
In one embodiment, the detection agent is in the form of an injectable formulation, such as an intravenous or subcutaneous formulation. Of course, other forms of formulation are possible.
In one embodiment, the subject is a mammal. For example, the mammal may be a human and non-human animal such as a mouse, rat, rabbit, primate such as monkey, chimpanzee.
In yet another embodiment, the detection agent is used to detect inflammation in brain tissue of the subject, for example, at least 3 mm from the scalp in the brain of the subject.
In a ninth aspect of the invention, there is provided a method of detecting tissue inflammation in a subject, the method comprising the steps of:
(a) administering the labeled cell of the fourth aspect of the invention to a subject;
(b) inflammation in living tissue in a subject is detected by fluorescence imaging of the labeled cells.
In one embodiment, the administration may be by injection, more specifically, intravenous injection.
In one embodiment, the labeled immune cells are neutrophils. Neutrophils have a tendency to migrate to inflammatory tissues, and thus by fluorescence imaging of the labeled immune cells, the presence or absence of inflammation of tissues in a subject can be detected.
In one embodiment, bioimaging is performed by irradiating the subject with laser light. In a preferred embodiment, the laser wavelength used for said laser irradiation is 700-2Preferably 20.6mW/cm2Each irradiation is for 50 to 100ms, preferably 50 ms.
In yet another embodiment, the bioimaging is bioimaging of tissue within the subject at least 3 mm from its surface.
In a preferred embodiment, the tissue is brain tissue within the brain of the subject at least 3 mm from the scalp.
In a tenth aspect of the invention there is provided a probe of the fifth aspect of the invention for use in detecting tissue inflammation in a subject.
In one embodiment, the probe may be administered into the subject by injection, e.g., intravenous injection.
In one embodiment, the probe is a compound of the first aspect, a conjugate of the second aspect or a composition of the third aspect of the invention. In this embodiment, it is desirable to first load the compound, conjugate or composition on immune cells, such as neutrophils, and then to detect tissue inflammation in the subject.
In one embodiment, bioimaging is performed by irradiating the subject with laser light. In a preferred embodiment, the laser wavelength used for said laser irradiation is 700-2Preferably 20.6mW/cm2Each irradiation is for 50 to 100ms, preferably 50 ms.
In yet another embodiment, the bioimaging is bioimaging of tissue within the subject at least 3 mm from its surface.
In a preferred embodiment, the tissue is brain tissue within the brain of the subject at least 3 mm from the scalp.
The invention has the advantages that:
the invention provides a series of compounds with aggregation-induced emission properties, which can display emission in a near-infrared II region (1000nm-1700nm) and can provide imaging for deep tissues with ultrahigh signal-to-background ratio. For example, neutrophils carrying the compounds of the invention can penetrate the brain and allow observation of inflammation deep in brain tissue through the intact scalp and skull.
Examples
The following examples will demonstrate the high-brightness emission of aggregation-induced emission molecules designed according to the present invention in the near infrared two region, with aggregation-induced emission molecules 2TT-oC6B, 2TT-m, oC6B, and 2TT-mC6B as subjects. Unless otherwise specified, the test methods employed therein were all conventional methods, and, unless otherwise specified, the test materials used in the following examples were all purchased from a conventional reagent store. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It should be noted that the terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The foregoing summary, as well as the following detailed description, is intended merely to be illustrative of the invention and is not intended to be in any way limiting. The scope of the invention is to be determined by the appended claims without departing from the spirit and scope of the invention.
Material
All chemicals and reagents were purchased from chemical sources and the solvents used for the chemical reactions were distilled prior to use.
Measuring method
Ultraviolet-visible-near infrared absorption spectrum:measurements were made using a PerkinElmer Lambda 365 spectrophotometer.
Nuclear Magnetic Resonance (NMR) spectrum:recording was performed using a Unity-400 nuclear magnetic resonance spectrometer with cadmium chloride as the solvent and Tetramethylsilane (TMS) as the reference at room temperature1H and13and (4) C spectrum.
Mass Spectrum (MS):measurements were performed using a GCT premier CAB048 mass spectrometer in MALDI-TOF mode.
Photoluminescence (PL) spectrum:the measurement was performed using a Horiba Fluorolog-3 fluorescence spectrophotometer.
Dynamic Light Scattering (DLS):the measurement was performed using a 90+ particle size analyzer.
Transmission Electron Microscope (TEM)Image: obtained using a JEM-2010F transmission electron microscope at an acceleration voltage of 200 kV.
The Density Functional Theory (DFT) calculation was performed using the Gaussian 09 software package, using the B3LYP functional in combination with the 6G (d) basis set.
Example 1: synthesis of compounds
Synthetic scheme for 2TT-oC 6B:
synthesis of Compound 2TT-oC6B
To synthesize the compound 2TT-oC6B, organotin (1, 0.7g, 1mmol), dibromo-BBT (2, 87mg, 0.25mmol), Pd were added to a 10mL tube2(dba)3(22mg，0.025mmol)、P(o-tol)3(66mg, 0.21mmol) and degassed dry toluene (1.5mL) and sealed with a Teflon cap. In N2The reaction mixture was heated to 130 ℃ under stirring and held for 48 hours. After cooling, the crude product was quenched with potassium fluoride (KF) solution, extracted with Dichloromethane (DCM), and taken up with Na2SO4The combined organic phases were dried. After removal of the solvent, the product was purified on a silica gel column to give a dark green solid (35% yield).1H NMR(400MHz,CDCl3),δ(ppm)＝7.59-7.56(4H,m),7.37(2H,s),7.31-7.26(8H,m),7.16-7.12(8H,m),7.11-7.03(8H,m),2.61-2.57(4H,t,J＝8Hz),1.63,(4H,m),1.15-1.10(12H,m),0.73(6H,m).13C NMR(100MHz,CDCl3),δ(ppm):152.6,146.9,146.8,146.3,145.0,128.7,127.5,126.1,124.1,124.0,122.7,122.5,115.4,99.3,30.8,29.8,29.6,28.4,21.8,13.3.MS:m/z:[M]+C62H56N6S4Calculated value of 1012.3, found 1012.3.
Compound 2TT-mC6B was synthesized in a similar manner.
Synthetic scheme for 2TT-m, oC 6B:
synthesis of Compound 2TT-m, oC6B
In N2Under the atmosphere, BBT-Br2(1,170mg,0.25mmol), organotin (2 and 3,400mg,0.75mmol), Pd (PPh)3)4(22mg,0.025mmol) and 20mL of toluene were charged to a 100mL pre-dried two-necked flask. The mixture was refluxed for 24 hours. After cooling to room temperature, the solvent was removed by rotary evaporation. The crude product was purified by silica gel column to obtain the target molecule in 29% yield.1H NMR(400MHz,CDCl3),δ(ppm)＝8.95(1H,s),7.61(2H,d,J＝8Hz),7.54(2H,d,J＝8Hz),7.37-7.26(9H,m),7.23-7.06(16H,m),2.88(2H,d,J＝8Hz),2.57(2H,d,J＝8Hz),1.75(2H,m),1.4-1.1(14H,m),0.91(3H,m),0.73(3H,m).13C NMR(100MHz,CDCl3),δ(ppm):153.9,150.4,147.6,147.4,146.3,145.2,145.1,139.7,136.5,135.1,129.8,129.4,129.3,128.6,128.4,128.0,126.7,124.8,124.6,123.4,123.3,123.1,122.9,115.4,113.0,31.7,31.6,31.5,31.0,30.5,29.3,29.1,29.0,22.7,22.5,14.1,14.0.MS:m/z:[M]+C62H56N6S4Calculated value of 1012.3449, found 1012.3325.
Synthesis of Polymer (V) of Compound 2TT-oC6B and Polymer (VI) of 2TT-m, oC 6B:
in N2Under the atmosphere, BBT-Br2(0.25mmol), organotin (0.25mmol), Pd (PPh)3)4(22mg,0.025mmol) and 20mL of toluene were charged to a 100mL pre-dried two-necked flask. The mixture was refluxed for 24 hours. Cooling to room temperature, adding into methanol dropwise for precipitation, and drying for later use.
Example 2: production of AIE nanoparticles (AIE-dot)
A mixture of compounds 2TT-oC6B, 2TT-mC6B or 2TT-m, oC6B (1mg), DSPE-polyethylene glycol 2000-maleimide (1.5mg) and Tetrahydrofuran (THF) (1mL) was sonicated (12W output, XL2000, Misonix Incorporated, NY) to obtain a clear solution. The solution was quickly poured into 9mL of water and vigorously sonicated in water for 2 minutes. The mixture was then stirred in a fume hood for 12 hours to remove THF. AIE nanoparticle suspensions were subjected to ultrafiltration (molecular weight cut-off of 100kDa) at 3000 × g for 30 min. As a result, it was found that the compounds 2TT-oC6B, 2TT-mC6B or 2TT-m, oC6B were successfully encapsulated in DSPE-polyethylene glycol 2000-maleimide matrix in the form of AIE nanoparticles.
Example 3: determination of fluorescence Quantum Yield (QY) of dye
The QY of the dye was measured using NIR-II fluorescent IR-26 dye as reference (QY ═ 0.5%). For reference calibration, IR-26 in 1, 2-Dichloroethane (DCE) was diluted to DCE solution to prepare five samples each having an absorbance value at 808nm of &0.1, -0.08, -0.06, -0.04, and-0.02, since these highly diluted samples can minimize secondary optical processes such as re-absorption and re-emission effects. Then, a total of five concentrations of linearly spaced IR-26 solutions in DCE were transferred once to 10mm path fluorescent tubes. The excitation source is a diode laser at 808 nm. The emitted light was filtered with a 900nm long pass filter to exclude other light interference and an emission spectrum in the region of 900nm to 1500nm was acquired. For DCE and H2The same procedure was performed with the AIE dye in O. All emission spectra of the reference and sample were then integrated in the NIR-II region from 900nm to 1500 nm. The integrated NIR-II fluorescence intensity is plotted against absorbance at the excitation wavelength of 808nm and fitted to a linear function. The quantum yield of the sample was calculated using two slopes, one obtained from IR-26 in the reference DCE and the other from the sample (AIE dye) based on the following formula:
wherein, QYsampleAnd QYrefQuantum yields, Slope, of the samples and of the reference, respectivelysampleAnd SloperefSlope, n, of sample and reference, respectivelysampleAnd nrefAre each H2Refractive indices of O and DCE.
Example 4: preparation of AIE nanoparticle-TAT (AIE-dot-TAT)
A mixture of compound 2TT-oC6B or 2TT-m, oC6B (1mg), DSPE-polyethylene glycol 2000-maleimide (1.5mg) and THF (1mL) was sonicated (12W output, XL2000, Misonix Incorporated, NY) to obtain a clear solution. The solution was quickly poured into 9mL of water and vigorously sonicated in water for 2 minutes. To couple cell penetrating peptides (transcribed HIV-1 transactivator (Tat) proteins) to AIE nanoparticles, 1. mu. mol of TAT peptide was added to the AIE nanoparticle suspension described above and allowed to react for 12 hours. Free TAT peptide was subsequently removed by ultrafiltration.
Example 5: extraction and purification of Neutrophils (NE) and uptake of AIE-dot-TAT/ICG
Mature Neutrophils (NE) were isolated from mouse bone marrow using a modified procedure. Briefly, after muscle and tendon removal, bone was immersed in RPMI 1640 medium. Bone marrow was washed with phosphate buffered saline, centrifuged at 200 Xg for 3 minutes, and resuspended in phosphate buffered saline. The single cell suspension was added to a Percol mixed solution prepared in 55%, 65% and 78% (by volume) of Percol in PBS, and then centrifuged at 500 Xg for 30 minutes. Mature neutrophils were recovered at the interface of the 65% and 78% fractions and washed three times with ice-cold PBS. The yield was quantified with a hemocytometer (Bright-Line, Sigma-Aldrich). The viability of the obtained mature neutrophils was calculated by trypan blue exclusion and purified with Fluorescein Isothiocyanate (FITC) -conjugated Ly-6G/Ly-6C (Gr-1) antibody (250ng mL)-1) (BioLegend) and Phycoerythrin (PE) conjugated MAIR-IV (CLM-5) antibody (1. mu.g.mL)-1) (BioLegend) double immunofluorescence staining was performed to determine purity. The morphology of Wright-Giemsa (established organism) stained neutrophils was observed with an optical microscope (Ts2R, Nikon).
Then, 1 × 106The neutrophils were treated with medium and AIE-dot-TAT (containing 2TT-oC6B, 1mg/mL) or ICG (3mg/mL) prepared in example 4 at 37 ℃ for 1 hour. After incubation, neutrophils were washed three times with ice-cold PBS, trypsinized and resuspended in culture medium. The content of AIE spots and ICG spots in the cells can be determined by measuring the absorbance at an excitation wavelength of 745nm and 780nm, respectively.
Example 6: in vitro and in vivo NIR-II imaging
Imaging was performed on a homemade imaging device consisting of a 2D InGaAs camera (Princeton Instruments, 2D OMA-V). The excitation source was a 808nm laser. The power density of the excitation laser on an imaging plane is 20.6mW/cm2Obviously lower than 329mW cm at 808nm-2Safe exposure limits of. The emitted fluorescence is allowed to pass through 810nm, 880nm, 1000nm, 1250nm long pass filters to take advantage of near NIR-II fluorescence imaging. For in-vitro and in-vivo near infrared fluorescence imaging, the long-pass filter is fixed at 1000nm。
For the study of in vitro near-infrared fluorescence imaging, excitation at 808nm (20.6 mW/cm)2) And collecting near-infrared fluorescence signals by using a near-infrared fluorescence imaging system under the exposure time of 50 ms.
For the in vivo penetration depth of near infrared fluorescence imaging of AIE points, the following experimental procedures are carried out: first, adult female mice (6-8 weeks old) were anesthetized with abamectin (2,2, 2-tribromoethanol, 250mg/kg, intraperitoneal Injection (IP)) and placed in a stereotaxic apparatus (Stoelting co.). Then, AIE dots and indocyanine green (ICG) were injected directly subcutaneously in mouse hind limbs (500 μ M), respectively. Immediately after AIE-site and ICG injection, mice were imaged from prone (thickness 8mm) and supine (thickness 0.5mm) positions.
In vivo NIR II fluorescence imaging of hindlimb blood vessels, scalp blood vessels, and cerebral blood vessels of mice was performed according to the following experimental procedure:
after intravenous injection of AIE spots and ICG, respectively, in mice, images were collected at different time points using near infrared fluorescence imaging techniques at these sites. Continuous 808nm irradiation (20.6 mW/cm)2) Used as a light source in an NIR II fluorescence imaging system equipped with a 1000nm long pass filter (1000LP) with an exposure time of 50 ms. The fluorescence intensity is the average fluorescence intensity in the blood vessels in these sites. When cerebral vascular imaging was performed, the skull of the mouse was opened for better visualization.
Example 7: AIE-dot-TAT (comprising 2TT-m, oC6B) NIR-II fluorescence imaging for guiding surgical operations
First, 2TT-M, oC6B nanoparticles (1mM,200 μ L) were injected directly subcutaneously in the hind limb (500 μ M) of mice, followed by observation at different time points (10min, 2h, 4h and 24h) and the sentinel lymph nodes of mice were removed under NIR-II fluorescence guidance.
Secondly, 2TT-m, oC6B nanoparticles (1mM,200 μ L) were injected into the peritoneal tumor mice by tail vein injection, NIR-II fluorescence imaging was performed on the peritoneal cavity, and tumor resection surgery was performed according to the guidance of NIR-II fluorescence signals.
Example 8: in vivo NIR II fluorescence imaging of brain inflammation
First, a mouse model of brain inflammation was prepared according to the following experimental procedure: female mice (6-8 weeks old) were first anesthetized with abamectin (2,2, 2-tribromoethanol, 250mg/kg, intraperitoneal Injection (IP)) and placed in a stereotaxic apparatus (Stoelting co.). Coli (E.coli) Lipopolysaccharide (LPS) (serotype O111: B4, type S. Enzo Life Sciences, ALX581-M005) was then administered to the right hemisphere (AP 0.0mm, ML +2.5mm, DV-4.0mm from bregma) to induce acute neuroinflammation. Each animal received 3. mu.g of E.coli lipopolysaccharide (in 2. mu.LPBS) over 5 minutes. The non-injected contralateral hemisphere served as a control.
Then, NIR II fluorescence imaging was performed on the brain inflammation of the mice, and the specific experimental procedure was as follows: first, mice were anesthetized in the prone position using avermectin (2,2, 2-tribromoethanol, 250mg/kg, intraperitoneal Injection (IP)). All in vivo data was collected in parallel experiments using three mice. According to animal ethics approved by Shenzhen high-grade technical research institute, the anesthesia time of mice used for experiments should not exceed 24 hours. The study therefore monitored in vivo imaging from pre-nanoparticle injection to 24 hours post-injection. Mice were injected intravenously with AIE @ NE and ICG @ NE (2X 10)6Individual neutrophils), images of different time points were collected using near infrared fluorescence imaging techniques. Continuous 808nm irradiation (20.6 mW/cm)2) Used as a light source in a near-infrared two-zone fluorescence imaging system equipped with a 1000nm long-pass filter (1000LP) with an exposure time of 100 ms. The signal/background ratio was processed using Image J software by counting six points and obtaining an average.
Example 9: in vivo distribution study
Tissue distribution of AIE @ NE and ICG @ NE in a mouse model of brain inflammation was monitored separately. Mice injected with samples were sacrificed at designated time intervals and various tissues including brain, heart, liver, spleen, lung, kidney, stomach, intestine, skin, muscle, bone were isolated and subjected to NIR-II fluorescence imaging.
The experimental results are as follows:
the structure of 2TT-mC6B is shown in fig. 1a, with near NIR-I absorption (about 808nm) and NIR-II emission (1063nm) (fig. 1d), highly fluorescent in solution (when the poor solvent, e.g. water, is less than 50%), but hardly (predominantly as non-radiative decay) in the aggregated state (when the poor solvent, e.g. water, is greater than or equal to 50%), showing a typical aggregation induced quenching (ACQ) effect (fig. 1 e). It is speculated that the ACQ effect in 2TT-mC6B comes from its coplanar benzodithiadiazole (TBT) core (fig. 1b), which hardly limits strong intermolecular interactions even in the presence of the molecular rotor Triphenylamine (TPA) (fig. 1 c).
However, the resulting 2TT-m, oC6B and 2TT-oC6B (structures are also shown in fig. 1a) molecules show significantly enhanced TBT (48 °) dihedral angles (fig. 1b) and more distorted structures, simply by shifting one or both hexyl units of 2TT-mC6B from meta to ortho position. As expected, 2TT-m, oC6B and 2TT-oC6B were weakly emissive in solution, but were highly fluorescent in the aggregated state, showing typical AIE properties (FIG. 1 e). The low frequency vibrational modes predominate in the AIE active 2TT-m, oC6B and 2TT-oC6B molecules, manifested as dynamic twisting motions of the twisted TBT backbone and twisted TPA rotors (FIG. 1 f). Whereas in ACQ activity 2TT-m, oC6B and 2TT-mC6B, despite the presence of inherently distorted TPA, the high frequency mode dominates the total combined energy due to the stretching and bending motion of the bond. Most importantly, 2TT-m, oC6B and 2TT-oC6B showed maximum fluorescence emission at 1059nm and 1014nm, respectively (FIG. 1d), with quantum yields of 3.7% and 8.4%, respectively (FIG. 3), of which 2TT-oC6B is one of the highest aggregated luminescent molecules to date. Furthermore, S in 2TT-m, oC6B and 2TT-oC6B0And S1The conformational overlap between was lower than 2TT-mC6B (FIG. 1 h). These results indicate that the low frequency torsional motion in 2TT-m, oC6B and 2TT-oC6B contributes significantly to the nonradiative decay channel, thus leading to low fluorescence quantum yields of 2TT-m, oC6B and 2TT-oC6B in solution (1.5% for 2TT-m, oC6B and 1.1% for 2TT-oC6B) (FIG. 1 g). In the aggregate state, the presence of a twisted backbone and twisted TPA rotors in 2TT-m, oC6B and 2TT-oC6B reduced the intermolecular interactions and gave high fluorescence quantum yields.
The size distribution of 2TT-m, oC6B and 2TT-oC6B nanoparticles was measured by Dynamic Light Scattering (DLS) method using a 90Plus particle size analyzer. The dynamic light scattering results show that 2TT-m in nanoparticle form, oC6B, had an average diameter of 80 nanometers (not shown), while 2TT-oC6B in nanoparticle form, had an average diameter of 70 nanometers (FIG. 2 b).
FIG. 3 shows the integrated intensity of the fluorescence spectra of 2TT-oC6B, 2TT-m, oC6B and 2TT-mC6B nanoparticles at different concentrations in the 900nm-1500nm region. As shown in the figure, the integrated intensity of the fluorescence spectrum of 2TT-m, oC6B and 2TT-oC6B in the form of nanoparticles in the region of 900nm-1500nm is in linear relation with the absorbance at the excitation wavelength of 808nm under different concentrations, and the slope of the linear relation is 2TT-m, and the fluorescence quantum yield of oC6B and 2TT-oC6B in the region of 900nm-1500nm is 3.7 percent and 8.4 percent respectively. In contrast, the fluorescence quantum yields of 2TT-mC6B and IR26 were very low, with the former being only 0.75% and the latter being only 0.5%. Therefore, the compounds 2TT-m, oC6B and 2TT-oC6B, particularly 2TT-oC6B have excellent fluorescence quantum yield and can be effectively used in near-infrared II-region biological imaging.
Fig. 4 shows a photograph of NIR-II fluorescence imaging of blood vessels in hind limbs (a) and scalp (b) using 2TT-m, oC6B nanoparticles and ICG and fluorescence signals (b and d) at sections along the red dashed line in hind limbs and scalp. It can be seen from this figure that the oC6B nanoparticles successfully visualized blood vessels in hind limbs as well as in the scalp relative to the control ICG, 2TT-m, the resulting fluorescence intensity was significantly stronger compared to the ICG, 2TT-m, oC6B nanoparticles.
FIG. 5 shows photographs of NIR-II fluorescence imaging of scalp vessels using 2TT-oC6B nanoparticles (AIE dots) and ICG. As can be seen from this figure, AIE dots more significantly visualized the scalp vessels relative to IGC throughout the experiment. Also, scalp blood vessels can be clearly shown from 5 minutes after AIE dots injection, with very high fluorescence intensity up to 5 hours; from the 8 th hour onward to the 24 th hour, the fluorescence intensity gradually decreased.
In addition, the inventors also verified the imaging of the lymphatic system in vivo by the AIE nanoparticles of the present invention and tumor resection under image guidance, and the results are shown in fig. 6-7. FIG. 6 shows in vivo NIR-II fluorescence imaging of the lymphatic system using 2TT-m, oC6B nanoparticles, where a is a bright field photograph, b-e are fluorescence photographs captured at 10min, 2h, 4h, 24h, and f is a bright field (left panel) and fluorescence photograph (right panel) of sentinel lymph nodes removed from mice under NIR-II fluorescence guidance. Figure 7 shows tumor resection with 2TT-m, oC6B nanoparticle imaging guidance and without 2TT-m, oC6B nanoparticle imaging guidance, where a is bioluminescence and NIR-II imaging of the abdominal cavity before and after tumor resection, b is bioluminescence and NIR-II imaging of tumor nodules resected from the unguided and 2TT-m, oC 6B-guided groups, and c is a histogram of tumor nodule diameters resected from the unguided and 2TT-m, oC 6B-guided groups. From these two figures, it can be seen that 2TT-m, oC6B can image the lymphatic system and by means of 2TT-m, oC6B imaging, the target tissue in vivo can be successfully and completely removed, providing a new method for the precise excision of the target tissue by surgery, such as an in vivo tumor.
In addition, Neutrophils (NE) were used in the present invention to penetrate brain tissue since they are the most abundant immune cell type and have a tendency to migrate to areas of inflammation (fig. 2 a).
To assess the quality of NIR-II fluorescence imaging of AIE @ NE, cell imaging was first performed in vitro (1000nm LP, 50 ms). As shown in FIG. 8a, NIR-II fluorescence intensity increases with increasing number of AIE @ NE cells, demonstrating that NE successfully internalizes AIE sites. The NIR-II fluorescence intensity was even stronger in 500 AIE @ NE cells than in 5000 ICG @ NE cells, indicating that the AIE spot has excellent sensitivity and brightness.
To more intuitively compare the fluorescence emission of AIE-bearing Neutrophils (NE), we measured 5X 105Photoluminescence intensity of individual NE cells. As shown in FIG. 8b, AIE @ NE showed an emission intensity of about 4.8 times ICG @ NE.
To evaluate the imaging performance of AIE @ NE in vivo, subcutaneous imaging was performed. As shown in fig. 8c, 1000 AIE @ NE cells showed strong fluorescence signals at a depth of about 1mm, which is particularly valuable for bioimaging. In addition, 500 AIE @ NE cells showed higher brightness than 2000 ICG @ NE cells. Quantitative data of Photoluminescence (PL) support these results (fig. 8 d).
Then, AIE @ NE is used to pass integrityThe skull and scalp of the mice to determine brain inflammation. Injection of AIE @ NE (2X 10)6Individual cells) no fluorescence signal was detected 1 hour after (1000nm LP, 100ms), whereas at 4 hours a weak fluorescence delineation of the inflammatory area was achieved (fig. 8 e). The fluorescence signal at the site of inflammation increases over time and becomes strongest and clearest 12 hours after injection. However, in mice treated with ICG @ NE, the site of inflammation is difficult to distinguish from healthy tissue due to its weak fluorescence. These results indicate that the AIE dots are deeper penetrating and have higher brightness than the ICG dots. Quantitative studies of sites of brain inflammation showed that mice treated with AIE @ NE had 6.5 times higher fluorescence intensity than ICG @ NE (FIG. 8 f). Furthermore, during the 24 hour study period, the near infrared II-zone fluorescence intensity at the site of inflammation reached a maximum at 12 hours post-injection, with signal to background ratios (SBR) as high as 30.6 for the AIE @ NE treated group, while SBR values for the ICG @ NE treated group were only 5.6 (fig. 8g), indicating that AIE @ NE can be used for accurate diagnosis of brain inflammation (fig. 8 e).
The results show that the near infrared-II imaging quality of the 2TT-oC6B nano particle is far better than indocyanine green (ICG) in the aspect of imaging the hindlimb vascular system and the scalp vascular system of the mouse; moreover, neutrophils (AIE @ NE) carrying 2TT-oC6B nanoparticles can easily migrate into inflamed brains; for example, AIE @ NE can non-invasively identify sites of inflammation within the brain of mice through the intact scalp and skull, which are about 3 mm in depth.
FIG. 9 shows the tissue distribution and fluorescence intensity of AIE @ NE and ICG @ NE in a mouse model of brain inflammation. It can be seen from the figure that AIE @ NE and ICG @ NE have similar distribution in vivo, both being mainly distributed in the liver, spleen and lung, but, relatively, AIE @ NE may additionally be distributed in other tissues, such as brain, heart, kidney, skin and bone marrow, etc., with some difference relative to ICG @ NE.
The inventors also verified the effects of the polymer (V) of the compound 2TT-mC6B and the polymer (VI) of the compound 2TT-m, oC6B, and found that they had the same or similar effects as the compound monomers 2TT-mC6B and 2TT-m, oC 6B.
The subject matter thus described is susceptible of modification or variation in many ways. Such modifications and variations are not to be regarded as a departure from the spirit and scope of the present subject matter, and all such modifications and variations are intended to be included within the scope of the following claims.
Claims (20)
1. A compound represented by the following formula (I) or (II):
wherein
R1Is a molecular rotor, each R1Each independently selected from:
n is an integer ranging from 1 to 20,
each R2Each independently selected from:
wherein X is an integer ranging from 0 to 20, m is an integer ranging from 6 to 22, and Y is an integer ranging from 10 to 22; and is
Two R2Each independently located at 3 or 4 bits of the label, but not at 3 bits at the same time.
2. The compound of claim 1, wherein each R2Each independently selected from:
wherein X is an integer ranging from 0 to 20.
3. The compound of claim 1 or 2, wherein the compound is represented by the following formula (I):
wherein
R1Is a molecular rotor, each R1Each independently selected from:
each R2Each independently selected from:
wherein X is an integer ranging from 0 to 20.
4. The compound of claim 3, wherein the compound is represented by formula (III) or formula (IV):
5. a conjugate comprising a compound according to any one of claims 1-4 and a protein conjugated thereto, e.g. a cell penetrating peptide such as a transcribed transactivator protein.
6. A composition comprising a compound according to any one of claims 1 to 4 or a conjugate according to claim 5, and a matrix for encapsulating the compound or the conjugate, for example an amphiphilic polymer such as 1, 2-distearoyl-sn-glycerol-3-phosphoethanolamine-N- [ maleimide (polyethylene glycol) -2000] or Pluronic F127; preferably, the compound or the conjugate is encapsulated in the matrix.
7. The composition of claim 6, wherein the composition is in the form of nanoparticles; preferably, the nanoparticles are 20-500nm, more preferably 20-200nm, most preferably 50-120nm nanoparticles.
8. A labeled cell labeled with the compound of any one of claims 1-4, the conjugate of claim 5, or the composition of claim 6; preferably, the cell is an immune cell, such as a neutrophil.
9. A probe for in vivo or in vitro fluorescence imaging, comprising: the compound of any one of claims 1-4, the conjugate of claim 5, or the composition of claim 6; preferably, the fluorescence imaging is near-infrared two-zone fluorescence imaging.
10. The probe according to claim 9, wherein the probe is an injectable formulation, such as an intravenous or subcutaneous injectable formulation.
11. A method of performing biological imaging, the method comprising:
administering the probe of claim 9 or 10 to a subject in need thereof, e.g., by injection, e.g., intravenous or subcutaneous injection.
12. The method of claim 11, wherein the method further comprises: bioimaging the subject during a period of 10 to 24 hours (preferably at 12 hours) after administration of the probe; preferably, the biological imaging is fluorescence imaging such as near infrared two-zone fluorescence imaging.
13. The method of claim 11 or 12, wherein bioimaging is performed by irradiating the subject with laser light; for example, the laser wavelength used for the laser irradiation is 700-850nm, preferably 808nm, and the laser intensity is 10-30mW/cm2Preferably 20.6mW/cm2Each irradiation is for 50 to 100ms, preferably 50 ms.
14. The method according to any one of claims 11-13, wherein the subject is a mammal, e.g. human and non-human animals such as mice, rats, rabbits, primates such as monkeys, chimpanzees.
15. The method of any of claims 11-14, wherein the bioimaging is bioimaging of tissue within the subject at least 3 mm from its surface; preferably, the tissue is brain tissue within the subject's brain at least 3 mm from the scalp; preferably, the tissue is a blood vessel in the body, such as a blood vessel in the limbs, scalp and brain.
16. A kit for in vivo or in vitro imaging, the kit comprising:
the probe of claim 9 or 10; and
instructions for directing imaging;
wherein the imaging is fluorescence imaging such as near-infrared two-zone fluorescence imaging.
17. Use of the probe of claim 9 or 10 in the preparation of a detection agent for detecting tissue inflammation in a subject.
18. Use according to claim 17, wherein the detection agent is an injectable formulation, such as an intravenous or subcutaneous formulation.
19. Use according to claim 17 or 18, wherein the subject is a mammal, e.g. a human and a non-human animal such as a mouse, rat, rabbit, primate such as monkey, chimpanzee.
20. Use according to any one of claims 17 to 19, wherein the detection agent is for detecting inflammation in brain tissue of a subject, for example at least 3 mm from the scalp in the brain of a subject.
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