TREM-1 targeted fluorescent molecular probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to a TREM-1 targeted fluorescent molecular probe as well as a preparation method and application thereof, and discloses a fluorescent molecular probe named as' TREM-1 targeted fluorescent molecular probe as well as a preparation method and application thereof '. The invention aims to solve the problems of large molecular weight, poor pharmacokinetic performance, limited tissue penetration, high imaging background and poor imaging timeliness of the existing antibody fluorescent probe. The probe comprises a TREM-1 targeting module (TDM), a near-infrared fluorescence reporter group, a dark quenching group and a conformational sensitive connecting arm (CSL). The fluorescence of the probe is quenched before the probe is combined with TREM-1, and after the probe is combined with a target spot, TDM specific binding induces CSL conformation rearrangement, so that a reporter group and a quenching group are spatially separated, fluorescence resonance energy transfer (FRET) is relieved, and fluorescence activation is realized. According to the invention, rapid, high-contrast and low-background fluorescence imaging of atherosclerotic plaques is realized, and the probe is small in molecular size, strong in tissue penetrability and rapid in-vivo clearing.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a TREM-1-targeted fluorescent molecular probe, its preparation method, and its application. Background Technology

[0002] This invention relates to the field of biomedical imaging technology, specifically to a fluorescent molecular probe for specifically recognizing and labeling myeloid cell triggering receptor-1 (TREM-1), its preparation method, and its applications. In modern medical diagnostics and life science research, molecular imaging technology, especially fluorescence imaging technology, is playing an increasingly crucial role due to its unique advantages of high sensitivity, high spatiotemporal resolution, and the ability to achieve real-time, non-invasive visualization of vital activities. By designing fluorescent probes that can specifically target specific biomarkers, researchers and clinicians can gain insights into the occurrence and development of diseases at the molecular level, providing strong technical support for early disease diagnosis, disease monitoring, and treatment efficacy evaluation.

[0003] In numerous complex physiological and pathological processes, the inflammatory response, as a fundamental defense mechanism of the body against injury or infection, is closely related to the development and progression of many major diseases such as atherosclerosis, sepsis, rheumatoid arthritis, inflammatory bowel disease, and even tumors. Therefore, precise and dynamic monitoring of the inflammatory response has significant clinical implications. Myeloid trigger receptor-1 (TREM-1), a member of the immunoglobulin superfamily, is mainly expressed on the surface of myeloid cells such as neutrophils, monocytes / macrophages, and is a key upstream amplifier in the inflammatory signaling pathway. Under the stimulation of infection or injury, TREM-1 expression is significantly upregulated and can synergistically work with pattern recognition receptors such as Toll-like receptors to dramatically amplify the release of downstream pro-inflammatory cytokines and chemokines, thereby driving a strong inflammatory cascade effect. Given its central role in the inflammatory cascade and its low expression at rest, TREM-1 has been recognized as a highly promising inflammation-specific diagnostic biomarker and drug intervention target. Against this backdrop, the development of specific imaging probes targeting TREM-1 shows broad application prospects for achieving precise localization and activity assessment of inflammatory lesions.

[0004] Currently, fluorescent probes used for in vivo targeted imaging in this field largely rely on antibodies or their functional fragments (such as Fab and scFv) as target recognition modules. Specifically, these probes typically covalently couple high-affinity and high-specificity anti-TREM-1 monoclonal antibodies with suitable fluorescent groups (such as near-infrared fluorescent dyes) using chemical methods. The core advantage of this whole-antibody-based design lies in its ability to fully utilize the naturally occurring, precise antigen-binding sites of antibody molecules, achieving sub-nanomolar affinity for the TREM-1 target and unparalleled recognition specificity, thus ensuring the probe's targeting accuracy in complex biological environments. In past studies, these probes have indeed successfully achieved preliminary imaging of inflammatory sites in animal disease models, validating the feasibility of using TREM-1 as a target for inflammatory imaging.

[0005] However, with a deeper understanding of the complexity of the inflammatory microenvironment and increasingly stringent requirements for imaging timeliness and signal-to-noise ratio in clinical applications, the inherent characteristics of traditional fluorescent probes based on intact antibodies are gradually revealing significant limitations in addressing new challenges. The root cause of this deep-seated contradiction lies in the physicochemical properties of antibody molecules themselves. As a large protein molecule, antibodies typically have a molecular weight of around 150 kDa. This enormous molecular size directly leads to a series of unfavorable characteristics in pharmacokinetics and biodistribution. First, the large molecular size severely hinders the probe molecules' transendothelial penetration and effective diffusion in the interstitial tissue, making it difficult for the probe to efficiently reach and penetrate the core of deep inflammatory lesions with dense structures or poor blood supply, potentially leading to an underestimation of the extent and intensity of inflammation. Second, large molecular probes are cleared from the body extremely slowly, with plasma half-lives lasting several days. While this slow clearance rate is beneficial for the sustained action of therapeutic antibodies, it is a significant drawback for imaging applications. It results in prolonged, high-intensity background fluorescence signals in the bloodstream, severely reducing the signal-to-noise ratio (SNR) between the target and non-target areas. To obtain a clear, diagnostically valuable image, researchers often need to wait hours or even one to two days for most unbound probes to be cleared from the blood before imaging can begin. This greatly limits its application in diagnosing acute inflammation (such as septic shock) requiring rapid decision-making. Furthermore, the protein properties of antibody molecules pose a potential risk of immunogenicity; repeated use may trigger an immune rejection response.

[0006] Therefore, existing technologies face an inherent technical contradiction: the complete antibody structure adopted in pursuit of ultimate targeting specificity and affinity has become the fundamental bottleneck limiting probe pharmacokinetic performance, tissue penetration, and imaging immediacy. How to overcome the inherent limitations of traditional antibody probes in terms of molecular size, biodistribution, and clearance rate while ensuring high specificity recognition of the TREM-1 target, and develop a novel probe with significantly reduced molecular weight, stronger tissue penetration, faster in vivo clearance, and lower background signal to achieve rapid, high-contrast fluorescence imaging of inflammatory lesions, has become a key challenge and an urgent technical problem for those skilled in the art. Summary of the Invention

[0007] The purpose of this invention is to overcome the technical defects of existing fluorescent probes based on intact antibodies, such as poor pharmacokinetic performance, limited tissue penetration, high imaging background signal, and poor imaging timeliness due to their large molecular weight. To address the inherent contradiction in the prior art—the use of large molecular recognition elements in pursuit of target specificity at the expense of imaging kinetic performance—this invention aims to provide a novel molecular probe that, while ensuring high specificity and high affinity recognition of the myeloid cell trigger receptor-1 (TREM-1) target, significantly reduces molecular size, enhances tissue penetration, accelerates in vivo clearance, and possesses target-binding-dependent fluorescence activation characteristics, thereby achieving rapid, high-contrast fluorescence imaging of inflammatory lesions.

[0008] To achieve the above-mentioned objective, this invention provides a TREM-1-targeted fluorescent molecular probe, characterized in that the probe has a molecular switch conformation that is in a fluorescence quenched state before binding to the TREM-1 target and transforms into a fluorescence-activated state after binding to the TREM-1 target. The chemical structure of the molecular probe consists of a TREM-1 targeting module (TDM), a near-infrared fluorescent reporter group, a dark quenching group, and a conformation-sensitive linker (CSL) connecting the targeting module, the reporter group, and the quenching group. The specific binding event of the targeting module to the TREM-1 target induces a conformational rearrangement of the probe molecule through the conformation-sensitive linker, thereby causing spatial separation of the reporter group and the quenching group, which are pre-configured to be closely adjacent in space, relieving the fluorescence resonance energy transfer (FRET) effect between them and achieving an activation response of fluorescence signal from absence to presence.

[0009] Specifically, the TREM-1 targeting module (TDM) is a highly specific non-antibody recognition scaffold with a predetermined three-dimensional structure. In one embodiment of the invention, the TDM is a synthetic bicyclic peptide. The molecular weight of the bicyclic peptide is precisely controlled within the range of 2.0 kDaltons to 3.5 kDaltons, and it is prepared via solid-phase peptide synthesis. The bicyclic peptide comprises two peptide rings composed of amino acid residues, which are covalently linked by a non-peptide chemical crosslinking core molecule to form a stable, rigid saddle-shaped structure. The chemical crosslinking core molecule is 1,3,5-tris(bromomethyl)benzene. The amino acid sequence of the bicyclic peptide is a specific sequence obtained through multiple rounds of affinity screening of a phage display library targeting the extracellular domain of recombinant human TREM-1 protein. This sequence contains three cysteine ​​residues for nucleophilic substitution reactions with the chemical crosslinking core molecule. The dissociation constant (Kd) of the bicyclic peptide with human TREM-1 protein is less than 5 nanomoles / L, ensuring high affinity binding to the target in complex biological environments.

[0010] Furthermore, the fluorescence signal system of the probe, namely the near-infrared fluorescent reporter group and the dark quencher group, is a pair of energy donor-acceptor pairs precisely selected and matched based on their spectral characteristics. The near-infrared fluorescent reporter group is a heptamethrin dye derivative with high quantum yield and excellent photostability. Its maximum excitation wavelength is located in the range of 740 nm to 760 nm, and its maximum emission wavelength is located in the range of 770 nm to 800 nm. This spectral characteristic makes it suitable for deep tissue penetration imaging and can effectively avoid the interference of autofluorescence in biological tissues. The dark quencher group is a non-fluorescent black hole quencher (BHQ) derivative with a broad and efficient absorption spectrum. The wavelength range of its maximum absorption peak completely covers the emission spectrum of the near-infrared fluorescent reporter group, thereby ensuring efficient quenching of the reporter group fluorescence at sufficiently close spatial distances. The Foster radius (R0) between the dark quencher group and the reporter group was calculated and determined to be 6.0 nm.

[0011] The conformation-sensitive linker (CSL), one of the core technical features of this invention, is a key actuator for switching the probe from an "off" to an "on" state. The CSL is not a simple flexible polymer chain, but a short peptide with a specific amino acid sequence and a predetermined secondary structure. This short peptide sequence consists of a core flexible region composed of glycine-serine repeating units, with proline residues strategically introduced at both ends to induce and stabilize a specific bending conformation. In free aqueous solution, due to internal hydrogen bonding and hydrophobic interactions, the CSL preferentially forms a U-shaped or hairpin-shaped fold conformation with the lowest energy. This predetermined fold conformation forces the near-infrared fluorescent reporter group and the dark quencher group, covalently linked at both ends of the CSL, to be spatially closer, with their average distance precisely controlled below 4.0 nanometers. This distance is much smaller than the Foster radius between them, resulting in over 98% of the fluorescence energy being transferred to the quencher group via the FRET mechanism and dissipated as heat, causing the entire probe molecule to exhibit a highly quenched "dark" state when not bound to the target.

[0012] The molecular probe of this invention exhibits high site specificity and spatial geometric precision in its overall structural assembly. The near-infrared fluorescent reporter group is site-coupled via a stable amide bond to the ε-amino group of a specific lysine residue on the first peptide ring of the bicyclic peptide TDM. One end (e.g., the C-terminus) of the conformation-sensitive linker (CSL) is covalently linked to the dark quencher group, while its other end (e.g., the N-terminus) is site-coupled via another amide bond to the ε-amino group of another specific lysine residue on the second peptide ring of the bicyclic peptide TDM. This "bridging" connection strategy across the two peptide rings mechanically locks the binding behavior of the TDM to the conformational state of the CSL. When the saddle-shaped domain of the bicyclic peptide TDM binds with high affinity to a specific antigenic epitope on the surface of the TREM-1 protein, the two peptide rings of the TDM are forced into a fixed spatial arrangement determined by the target protein surface topology. This binding-induced conformational locking imposes an unavoidable tension on the "bridging" CSL, sufficient to overcome the intramolecular forces maintaining its folded conformation within the CSL. This forces it to unfold from its original U-shaped hairpin conformation and extend into a linear, stretched conformation. In this stretched conformation, the spatial distance between the near-infrared fluorescent reporter group and the dark quencher group is significantly increased to over 10 nanometers, a distance much larger than the Foster radius between them. This completely blocks the FRET effect, allowing the previously quenched fluorescence to recover. This process results in a more than 20-fold increase in the probe's fluorescence quantum yield, achieving highly specific, signal-activated detection of the TREM-1 target, thus fundamentally solving the problem of high background signal.

[0013] This invention also provides a method for preparing the above-mentioned TREM-1-targeted fluorescent molecular probe, which ensures the precise synthesis of each functional module and site-specific assembly, and includes the following steps:

[0014] The first step involved the synthesis and cyclization of the TREM-1 targeting module (TDM). Using the standard Fmoc solid-phase peptide synthesis strategy, amino acids were sequentially coupled onto Rink Amide AM resin according to a pre-defined amino acid sequence (containing three spaced cysteine ​​residues and two orthogonally protecting lysine residues for subsequent coupling) to synthesize a linear precursor peptide. After the complete sequence synthesis, the linear peptide was cleaved from the resin using a cleavage reagent containing trifluoroacetic acid, triisopropylsilane, and water, and the side-chain protecting groups were removed. The crude product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) to obtain a high-purity linear precursor peptide. Subsequently, the purified linear peptide was dissolved in ammonium bicarbonate buffer at pH 8.0, and under highly diluted conditions (concentration less than 0.1 mg / mL), a solution of 1,3,5-tris(bromomethyl)benzene dissolved in acetonitrile was added dropwise. The reaction was carried out at room temperature for 16 hours to promote intramolecular cyclization. After the reaction was completed, the reaction mixture was separated and purified by RP-HPLC, the components of the target bicyclic peptide TDM were collected, and its molecular weight was accurately identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), confirming the successful cyclization.

[0015] The second step involves the preparation of the conformation-sensitive linker-quencher conjugate (CSL-Quencher). Using the Fmoc solid-phase peptide synthesis method, the specific amino acid sequence of the CSL was synthesized on a 2-chlorotriphenylmethylchloro resin pre-loaded with Fmoc-protected lysine. After sequence synthesis, while the peptide chain remained on the resin, the N-terminal Fmoc protecting group was removed, and then a carboxyl-activated dark quencher group (e.g., BHQ-3-COOH activated by HBTU / DIPEA) was coupled to the N-terminus of the CSL peptide chain. After coupling, the CSL-Quencher conjugate was cleaved from the resin using a weakly acidic cleavage reagent (e.g., a 1% (v / v) solution of trifluoroacetic acid in dichloromethane), while preserving the protecting groups on the amino acid side chains. The crude product was purified by RP-HPLC to obtain the high-purity CSL-Quencher conjugate for use.

[0016] The third step involves a two-step site-specific coupling method. First, the first coupling step, namely the labeling of the fluorescent reporter group, is performed. The purified bicyclic peptide TDM obtained in step one is dissolved in borate buffer at pH 8.5, and a near-infrared fluorescent dye (NIR-NHS ester) activated with N-hydroxysuccinimide ester at a stoichiometric ratio of 1:1.2 is added. The TDM molecule has only one lysine residue with its ε-amino group in a deprotected state and exhibits high reactivity, thus ensuring site-specific labeling of the fluorescent group. The reaction is carried out at room temperature in the dark for 2 hours. The reaction product is purified by RP-HPLC to separate the single-labeled TDM-NIR intermediate, and its molecular weight is confirmed by mass spectrometry.

[0017] Next, a second coupling step, namely bridging of the CSL-Quencher conjugate, is performed. The purified TDM-NIR intermediate is dissolved in dimethylformamide (DMF), and an excess (e.g., 5 equivalents) of the CSL-Quencher conjugate prepared in step two, along with a coupling agent (e.g., HATU) and an organic base (e.g., diisopropylethylamine), are added. The goal of this reaction is to form an amide bond between the free carboxyl group of the CSL-Quencher conjugate and the ε-amino group of the second, currently deprotected lysine residue on the TDM-NIR intermediate. The reaction is carried out at room temperature for 4 hours.

[0018] The fourth step is the purification and characterization of the final product. After the reaction is complete, the reaction mixture is directly loaded onto a size exclusion column for preliminary purification to remove unreacted small molecule reagents. The collected high molecular weight fraction is then finely purified by preparative RP-HPLC to obtain the final, high-purity TREM-1-targeted fluorescent molecular probe. Analytical HPLC confirmed its purity to be higher than 95%, and high-resolution mass spectrometry (such as ESI-Q-TOF MS) accurately determined its molecular weight to verify the chemical structure of the final product. Finally, its function was verified by fluorescence spectroscopy: the baseline fluorescence intensity in the buffer solution and the change in fluorescence intensity after the addition of excess recombinant TREM-1 protein were measured, and the activation factor of the fluorescence signal was calculated and confirmed to be no less than 20-fold.

[0019] This invention also provides the application of the aforementioned TREM-1-targeted fluorescent molecular probe in the preparation of imaging agents for the diagnosis or monitoring of inflammation-related diseases. Specifically, the application involves mixing the fluorescent molecular probe with a pharmaceutically acceptable carrier (such as physiological saline or phosphate-buffered saline) to prepare an injectable imaging agent.

[0020] This invention further provides a method for applying the probe in biomedical imaging, comprising the following steps: administering an effective dose of a pharmaceutical composition containing the fluorescent molecular probe of this invention to a target organism via an enteric route, particularly by intravenous injection. The effective dose is calculated based on the organism's body weight and ranges from 0.5 nanomoles to 15 nanomoles of probe molecules per kilogram of body weight. Within a predetermined time window following probe administration, non-invasive fluorescence imaging of the target region of the organism is performed using an in vivo fluorescence imaging system with an appropriate excitation source and emission filters. Due to the small molecule characteristics, rapid pharmacokinetic clearance, and signal activation mechanism of the probe of this invention, the predetermined time window is significantly shortened, and imaging can be performed between 30 minutes and 4 hours after probe administration. Precise localization and visualization of TREM-1-expressing inflammatory cell aggregation regions are achieved by detecting the intensity and spatial distribution of the emitted fluorescence signal in the wavelength range of 770 nm to 800 nm. High fluorescence signal regions presented in the image indicate the presence, extent, and activity intensity of inflammatory lesions. This method can be applied to the preclinical or clinical diagnosis and disease monitoring of a variety of inflammation-related diseases, including but not limited to sepsis, assessment of atherosclerotic plaque instability, assessment of the degree of synovitis in rheumatoid arthritis, delineation of the lesion extent in inflammatory bowel disease, and assessment of pro-inflammatory cell infiltration in the tumor microenvironment. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of the TREM-1-targeted fluorescent molecular probe described in this invention.

[0022] Figure 2 This is a schematic diagram of the conformation of the probe described in this invention in a fluorescence quenching state before binding to the target.

[0023] Figure 3 This is a schematic diagram of the conformation of the probe described in this invention after binding to the TREM-1 target and changing to a fluorescently activated state.

[0024] Figure 4 This is a schematic flowchart of the probe preparation method described in this invention.

[0025] Figure 5 This is a schematic flowchart of the method for applying the probe described in this invention in biomedical imaging.

[0027] The attached figures are labeled as follows: 1. TREM-1 targeting module; 2. Near-infrared fluorescent reporter group; 3. Dark quenching group; 4. Conformation-sensitive linker arm; 5. TREM-1 target site; Detailed Implementation

[0028] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are only for explaining this invention and not for limiting it. Unless otherwise specified, the conditions in the embodiments are conventional experimental conditions.

[0029] This specification aims to provide a detailed implementation path for a TREM-1-targeted fluorescent molecular probe, its preparation method, and its applications, ensuring that those skilled in the art can unambiguously reproduce the technical content of this invention and verify its beneficial effects. (Refer to...) Figures 1 to 3 The fluorescent molecular probe disclosed in this invention is a precisely designed chemical entity with target-binding-dependent signal activation function. Its overall molecular structure, as shown... Figure 1 As shown, it is organically integrated from four core functional modules: a myeloid cell trigger receptor-1 (TREM-1) targeting module 1, a near-infrared fluorescent reporter group 2, a non-fluorescent dark quencher group 3, and a conformation-sensitive linker arm 4. These four modules, through precise chemical bonding, together constitute a reversible "on-off" system operating at the molecular level.

[0030] Specifically, the initial state of the probe, i.e., when it is free in biological fluids and has not encountered the TREM-1 target site 5, has the following conformation: Figure 2 As shown. In this state, the conformation-sensitive linker 4 exhibits a pre-defined, energy-optimal folded conformation, such as a U-shape or hairpin structure. This folded conformation forces the near-infrared fluorescent reporter group 2 and the dark quencher group 3, covalently linked within its domain, to a very small distance in three-dimensional space. This distance is significantly smaller than the effective radius of the Foster resonance energy transfer (FRET) effect between them, resulting in the energy generated by the excitation of the reporter group 2 being transferred almost entirely non-radiatively to the quencher group 3 and dissipated as thermal energy. Macroscopically, this manifests as the entire probe molecule being non-fluorescent or exhibiting extremely low fluorescence intensity, remaining in a "off" or "dark" state. This design fundamentally eliminates the background signal caused by the diffuse distribution of unbound probes in the circulatory system and tissues.

[0031] When the probe molecule reaches the inflammatory lesion area through blood circulation and encounters TREM-1 target 5 expressed on the surface of specific immune cells, a crucial conformational change occurs in the probe, the process of which is as follows: Figure 3As shown, TREM-1 targeting module 1 binds to the extracellular domain of TREM-1 target 5 with high affinity and specificity. This binding event is not a simple attachment, but is accompanied by a fine fit and locking of the target module 1's own conformation. This conformational locking, induced by the target protein's surface topology, transfers a mechanical tension to the conformation-sensitive linker arm 4 connected to it via chemical bonds. This tension is sufficient to overcome the weak intramolecular interactions that maintain the folded state of linker arm 4, forcing it to unfold from its original U-shaped conformation and stretch into a linear, stretched conformation. In this stretched state, the spatial distance between the previously closely spaced near-infrared fluorescent reporter group 2 and the dark quencher group 3 is drastically increased, far exceeding the effective range of the FRET effect. Therefore, the energy transfer process is effectively blocked, and reporter group 2 can normally emit fluorescent photons after being excited by an external light source. Macroscopically, this manifests as the probe's fluorescence signal being "lit up" or "activated," with its fluorescence quantum yield increasing by tens of times. This signal activation, which occurs only at the target site, ensures a high degree of specificity in the imaging signal source, thereby enabling rapid, high-contrast, and high-signal-to-noise-ratio non-invasive visualization of inflammatory lesions.

[0032] As a specific embodiment of the present invention, the chemical nature, preparation parameters and assembly process of each module constituting the fluorescent molecular probe will be described in detail below.

[0033] The core of the TREM-1 targeting module 1 is a synthetic bicyclic peptide. To maximize pharmacokinetic performance while ensuring high targeting affinity, the molecular weight of this bicyclic peptide is strictly controlled within the range of 2.0 kDaltons to 3.5 kDaltons. In this embodiment, the molecular weight of the bicyclic peptide used is 2685.2 Daltons. Its amino acid sequence was identified through multiple rounds of biopanning targeting the extracellular domain of recombinant human TREM-1 protein in a large and diverse phage display library. The specific sequence is:

[0034] H-Lys(Mtt)-Gly-Trp-Cys-His-Glu-Asn-Cys-Val-Pro-Leu-Lys(Boc)-Tyr-Arg-Cys-CONH2. This sequence is strategically designed: it contains three cysteine ​​(Cys) residues, whose side chain thiol groups react with the chemically crosslinked core molecule to form two stable peptide rings; it also contains a lysine (Lys) residue with a 4-methyltriphenylmethyl (Mtt) protecting group and a lysine residue with a tert-butyloxycarbonyl (Boc) protecting group. These two protecting groups have orthogonal deprotection conditions, thus providing a chemical basis for the site-specific, stepwise coupling of subsequent functional groups. The chemically crosslinked core molecule is 1,3,5-tris(bromomethyl)benzene, which undergoes a nucleophilic substitution reaction with the thiol groups of the three cysteine ​​residues to form three stable thioether bonds, thereby locking the linear peptide chain into a saddle-shaped structure with a predetermined three-dimensional rigid conformation. The structure was determined by surface plasmon resonance (SPR) technology, and its dissociation constant (Kd) with recombinant human TREM-1 protein was 3.8 nanomoles / liter, showing extremely high binding affinity.

[0035] The near-infrared fluorescent reporter group 2 and the dark quencher group 3 are a pair of FRET energy donor-acceptor pairs with precisely matched spectral parameters. In this embodiment, the selected near-infrared fluorescent reporter group 2 is a water-soluble sulfonated heptamethrin dye derivative, specifically the N-hydroxysuccinimide ester (NHS ester) activated form of Sulfo-Cyanine 7.5. This dye has a maximum excitation wavelength of 755 nm and a maximum emission wavelength of 778 nm in phosphate-buffered saline (PBS, pH 7.4). This wavelength range falls within the longer band of the "near-infrared I region window" of biological tissues, effectively reducing tissue absorption and scattering, and avoiding the absorption peaks of hemoglobin and water, achieving deeper tissue penetration and lower autofluorescence background. The selected dark quencher group 3 is the black hole quencher BHQ-3. The quencher itself does not emit fluorescence, but it possesses a broad and intense absorption spectrum in the 500 nm to 800 nm range. Its absorption peaks perfectly cover the emission spectrum of Sulfo-Cyanine 7.5, ensuring efficient energy reception. Calculations show that the Foster radius (R0, the distance at which 50% energy transfer efficiency) of the FRET combination of Sulfo-Cyanine 7.5 and BHQ-3 is 6.0 nm.

[0036] The conformation-sensitive linker arm 4 functions as an intramolecular mechanosensor and signal transducer. It is not a randomly coiled polymer chain, but rather a short peptide with a specific amino acid sequence. In this embodiment, the sequence of this short peptide is: H-Pro-Gly-Ser-Gly-Gly-Ser-Gly-Gly-Ser-Pro-OH. The core of this sequence is a flexible region composed of alternating or repeating glycine (Gly) and serine (Ser) residues. Glycine provides the greatest rotational freedom of the main chain, while the hydroxyl side chains of serine can participate in stabilizing the specific conformation by forming intramolecular hydrogen bonds. A proline (Pro) residue is introduced at each end of the sequence. The unique cyclic side chain structure of proline can induce a fixed twist angle (phi angle approximately -60°) in the peptide main chain, thereby applying a structural constraint at both ends, causing the entire linker arm to preferentially fold into a hairpin conformation with the lowest energy under no external force. Molecular dynamics simulations and circular dichroism spectroscopy experiments confirmed that, in an aqueous environment, the average spatial distance between the fluorophore and the quencher group attached to the linker arm was precisely maintained at 3.5 ± 0.8 nm. This distance is much smaller than the Foster radius of 6.0 nm, and the theoretically calculated FRET efficiency is as high as 98.7%, thus achieving deep fluorescence quenching of the probe in the unbound state.

[0037] This invention also provides a preparation method capable of achieving precise assembly of the above-mentioned fluorescent molecular probes, the process of which is as follows: Figure 4 As shown, this ensures the chemical structural integrity of each functional module and the site specificity of the connections.

[0038] The first step involved the synthesis and purification of the bicyclic peptide TREM-1 targeting module (TDM). A standard Fmoc solid-phase peptide synthesis (SPPS) protocol was employed, using Rink Amide AM resin (0.5 mmol / g) as the solid support. Following the sequence H-Lys(Mtt)-Gly-Trp(Boc)-Cys(Trt)-His(Trt)-Glu(OtBu)-Asn(Trt)-Cys(Trt)-Val-Pro-Leu-Lys(Boc)-Tyr(tBu)-Arg(Pbf)-Cys(Trt)-CONH2, amino acids were sequentially coupled using an automated peptide synthesizer. The coupling reagents were a combination of benzotriazol-1-yl-oxytripyrrolidinephosphine hexafluorophosphate (PyBOP) and N,N-diisopropylethylamine (DIPEA), with N,N-dimethylformamide (DMF) as the solvent. Each coupling cycle consisted of two steps: Fmoc deprotection (using 20% ​​v / v piperidine / DMF solution) and amino acid coupling. After synthesis, the resin carrying the intact linear peptide chain was washed alternately with dichloromethane (DCM) and methanol and dried under vacuum. Subsequently, the resin was treated with a cleavage reagent mixture of trifluoroacetic acid (TFA) / water / triisopropylsilane (TIS) / dithiothreitol (DTT) in a ratio of 94:2.5:2.5:1.0 (v / v / v / v) to cleave the peptide chain from the resin and remove all side chain protecting groups except Mtt. The reaction solution was dried under nitrogen and concentrated, precipitated with ice-cold anhydrous diethyl ether, and the crude peptide was collected by centrifugation and washed three times with diethyl ether. The obtained crude linear peptide was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The purification conditions were as follows: an Agilent Zorbax SB-C18 preparative column (21.2 x 250 mm, 5 μm), mobile phase A was an aqueous solution containing 0.1% TFA, and mobile phase B was an acetonitrile solution containing 0.1% TFA. Elution was performed using a linear gradient of 10% to 50% of phase B over 30 minutes at a flow rate of 15 mL / min. The detection wavelength was 220 nm. The target peak was collected, and after rotary evaporation to remove most of the acetonitrile, the sample was freeze-dried to obtain a linear precursor peptide with a purity greater than 98%. The molecular weight was identified by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS), and the measured value was consistent with the theoretical calculation (2066.4 Da).

[0039] The second step involved the cyclization of the bicyclic peptide TDM. The purified linear precursor peptide was dissolved in an aqueous solution of ammonium bicarbonate (50 mmol / L, pH 8.2) containing 50% acetonitrile, adjusting the peptide concentration to an extremely dilute 0.1 mg / mL to maximize intramolecular cyclization and inhibit intermolecular polymerization. Under vigorous stirring, a solution of 1,3,5-tris(bromomethyl)benzene (1.05 equivalents) dissolved in acetonitrile was slowly added dropwise to the peptide solution over 4 hours using a syringe pump. After the addition was complete, the reaction was continued for 12 hours at room temperature in the dark. The reaction progress was monitored by analytical HPLC. After the reaction was complete, a small amount of glacial acetic acid was added to terminate the reaction, and the sample was then directly loaded onto a preparative RP-HPLC for purification. The separation conditions were similar to the previous step, but with a gentler gradient (e.g., 20% to 45% B phase over 60 minutes). The chromatographic peaks corresponding to the bicyclic peptide product were collected, and after freeze-drying, the purified bicyclic peptide TDM was obtained, with a purity confirmed by analytical HPLC to be greater than 98%. The molecular weight was determined by MALDI-TOF-MS, and the measured value was 2198.6 Da, which is highly consistent with the theoretical value (2198.5 Da) after the reaction of the linear peptide and the cross-linking agent, confirming that the cyclization was successful.

[0040] The third step involved the preparation of the conformation-sensitive linker-quencher conjugate (CSL-Quencher). Using the Fmoc-SPPS method, the conjugate was synthesized on a 2-chlorotriphenylmethylchloro resin pre-loaded with Fmoc-Pro-OH, following the sequence H-Pro-Gly-Ser(tBu)-Gly-Gly-Ser(tBu)-Gly-Gly-Ser(tBu)-Pro-OH. After synthesis, while the peptide chain remained on the resin, the N-terminal Fmoc protecting group was removed using 20% ​​piperidine / DMF. Then, BHQ-3-COOH (1.5 equivalents) was pre-activated in DMF for 5 minutes with O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 1.5 equivalents) and DIPEA (3.0 equivalents) before being added to the resin. The reaction was carried out at room temperature for 4 hours to covalently couple BHQ-3 to the N-terminus of the peptide chain. After coupling, the resin was treated with very weak acidic conditions, i.e., a 1% TFA solution in DCM, for 30 minutes to cleave the CSL-Quencher conjugate from the ultrasensitive 2-CTC resin. This mild condition ensured that the carboxyl group at the C-terminus of the peptide chain was retained, while the protecting groups (tBu) of all amino acid side chains remained intact. The crude product was purified by flash chromatography to obtain the high-purity CSL(tBu)-BHQ3 conjugate for use.

[0041] Step 4: Two-step site-specific conjugation assembly. First, fluorescent reporter labeling was performed. The bicyclic peptide TDM obtained in step 2 was dissolved in bicarbonate buffer at pH 8.8. Since the Boc protecting group had been removed during the TFA cleavage step, only the ε-amino group of the Lys12 residue was free and highly reactive. 1.1 equivalents of Sulfo-Cyanine 7.5 NHS ester dye (dissolved in anhydrous DMSO) were added to the solution, and the reaction was stirred for 2 hours at room temperature in the dark. The reaction product was purified by RP-HPLC to obtain a single-labeled TDM-Cy7.5 intermediate. Next, bridging of the CSL-Quencher conjugate was performed. The purified TDM-Cy7.5 intermediate was treated with a small amount of 2% TFA in DCM solution for 10 minutes to selectively remove the Mtt protecting group of Lys1, exposing its ε-amino group. It was then rapidly purified by RP-HPLC and lyophilized. The deprotected TDM-Cy7.5 was dissolved in anhydrous DMF, and 5 equivalents of the CSL(tBu)-BHQ3 conjugate prepared in step three, along with HATU (4.5 equivalents) and DIPEA (10 equivalents), were added. The reaction aimed to form a stable amide bond between the free carboxyl group of CSL(tBu)-BHQ3 and the newly exposed ε-amino group of Lys1 on TDM-Cy7.5. The reaction was carried out at room temperature for 6 hours.

[0042] Step 5: Deprotection and purification of the final product. The reaction mixture from the previous step was directly treated with the aforementioned TFA / water / TIS / DTT cleavage reagent for 2 hours to remove all tBu side-chain protecting groups on the CSL. After precipitation with diethyl ether, the crude final probe was purified using a two-step method. First, size exclusion chromatography (SEC, using a Sephadex G-25 column with PBS as the eluent) was used to remove unreacted small molecule reagents and salts. The collected high molecular weight fraction was then subjected to preparative RP-HPLC for final fine purification, with optimized elution conditions to achieve the best separation. The purified target product peak was collected, freeze-dried, and the final TREM-1 targeted fluorescent molecular probe was obtained as a loose blue powder. Analytical HPLC confirmed its purity to be greater than 99%. High-resolution mass spectrometry (ESI-Q-TOF MS) accurately determined its molecular weight, and the measured value (m / z) perfectly matched the theoretical calculation, verifying the chemical structure of the final product. Finally, its function was characterized by fluorescence spectroscopy: its basic fluorescence was found to be extremely low in PBS buffer, but after adding an excess (10-fold molar ratio) of recombinant human TREM-1 protein and incubating for 15 minutes, its fluorescence intensity at 778 nm increased by 28-fold, confirming its excellent target binding-dependent fluorescence activation performance.

[0043] This invention also provides a method for applying the probe in biomedical imaging, such as... Figure 5 As shown, it aims to achieve dynamic and accurate localization and assessment of atherosclerotic plaques in the body through non-invasive means.

[0044] Example 1: In vivo fluorescence imaging in an ApoE gene knockout mouse model of atherosclerosis

[0045] To verify the in vivo imaging capability and diagnostic potential of the probe of this invention, a classic high-fat diet ApoE gene knockout mouse model of atherosclerosis (AS) was constructed. Eight-week-old male ApoE gene knockout mice, weighing approximately 20-22 grams, were fed a high-fat diet for 16 weeks. The control group (Ctrl) consisted of ApoE gene non-knockout mice fed a normal diet for 16 weeks. Gross Oil Red staining and HE staining of aortic sections revealed significant lipid deposition and plaque formation in the vessel wall, indicating that the atherosclerosis model had been successfully established.

[0046] The TREM-1 targeted fluorescent molecular probe prepared in the examples was diluted with sterile saline to prepare an injection solution with a concentration of 100 μmol / L. The probe was administered via tail vein injection to mice in the AS model group and the Ctrl group (n=5 in each group) at a dose of 5 nanomoles per kilogram of body weight. Whole-body in vivo fluorescence imaging of the mice was performed using the IVISSpectrum in vivo imaging system at 30 minutes, 1 hour, 2 hours, 4 hours, and 24 hours post-injection. The imaging parameters were set as follows: excitation filter 745 nm, emission filter 800 nm, exposure time 1 second, and F / stop = 2.

[0047] Imaging results showed that in AS model mice, significant fluorescence signal enrichment was observed in the carotid artery region 1 hour after probe injection following exposure of the carotid artery. This signal peaked at 2 hours and then gradually weakened. In contrast, no significant fluorescence signal accumulation was observed in the carotid artery region of the Ctrl group mice at any time point. Quantitative analysis indicated that at the 2-hour imaging time point, the mean fluorescence radiation efficiency of the carotid artery in the AS group mice was significantly higher than that in the Ctrl group (p<0.001). More importantly, the fluorescence signal intensity ratio (target / background ratio, TBR) between the thoracic and abdominal cavities and adjacent non-inflammatory muscle regions in the AS group was as high as 5.6±0.7, exhibiting extremely high imaging contrast. The rapid clearance characteristics of the probe resulted in a significant reduction in background signal after 4 hours, further improving the signal-to-noise ratio.

[0048] After imaging, the mice were euthanized, and major organs such as the aorta, heart, liver, spleen, lungs, and kidneys were rapidly harvested for in vitro fluorescence imaging. The imaging results of the ex vivo organs were highly consistent with the in vivo imaging, confirming that the fluorescence signal indeed originated primarily from the carotid artery and aorta. Subsequent histopathological analysis, including hematoxylin and eosin (H&E) staining, Oil Red O staining, and immunohistochemical (IHC) staining, further validated the imaging results. H&E and Oil Red O staining showed extensive lipid plaque infiltration in the carotid artery and aorta of AS group mice, while IHC staining confirmed that macrophages expressed high levels of TREM-1 protein, the probe target, in these lipid plaque regions. This pathological evidence highly correlated with the spatial distribution of the fluorescence signal, strongly demonstrating that the probe of this invention can specifically and sensitively target and visualize atherosclerotic plaques constructed from ApoE gene knockout mice.

[0049] Comparative Example 1: Imaging effect compared with the "always-on" non-activating control probe

[0050] To highlight the superiority of the probe signal activation mechanism of this invention, a control probe was designed and synthesized. This control probe possesses the same bicyclic peptide TREM-1 targeting module 1 and near-infrared fluorescent reporter group 2 (i.e., the TDM-Cy7.5 intermediate) as the probe of this invention, but its molecular structure does not contain the conformation-sensitive linker arm 4 and the dark quencher group 3. Therefore, this control probe is an "always-on" fluorescent probe, and its fluorescence signal is independent of target binding.

[0051] Using the same AS mouse model and experimental procedure as in Example 1, another group of AS model mice (n=5) were injected with an equimolar dose of the "always-on" control probe. In vivo fluorescence imaging results showed that immediately after injection of the control probe, the mice exhibited extremely strong diffuse fluorescence signals throughout their bodies, due to the probe being in a fluorescently activated state in the bloodstream. Although some signal enrichment was also present in the inflamed areas of the lungs, the imaging contrast was extremely poor due to the very high background signal from blood and tissues. At the 2-hour imaging time point, the TBR was only 1.8±0.4, far lower than the 5.6 of the probe of this invention. The high background signal severely interfered with the accurate identification and quantitative analysis of the target organs.

[0052] Performance Comparison Summary

[0053] To more intuitively demonstrate the advancement of the technical solution of the present invention, the key performance parameters of the probe of the present invention in Example 1 and the control probe in Comparative Example 1 are quantitatively compared, as shown in the table below.

[0054]

[0055] In summary, this invention provides a TREM-1-targeted fluorescent molecular probe that, through its ingenious molecular design, successfully combines the high tissue penetration and excellent pharmacokinetic properties of a small molecule targeting module with a target-binding-dependent signal activation mechanism. Experimental data fully demonstrate that, compared with traditional non-activating probes, the probe of this invention can achieve rapid, high-contrast, and high signal-to-noise ratio imaging of atherosclerotic plaques in an extremely short time, greatly enhancing the practical value and clinical translational potential of fluorescent molecular imaging technology in the diagnosis and monitoring of atherosclerosis-related diseases. Its preparation method is highly controllable and reproducible, laying the foundation for the large-scale production of this type of advanced molecular probe.

Claims

1. A TREM-1-targeted fluorescent molecular probe, characterized in that, The probe includes: A TREM-1 targeting module (1); A near-infrared fluorescent reporter group (2); A dark quenching group (3); and a conformation-sensitive linker (4) for connecting the TREM-1 targeting module (1), the near-infrared fluorescent reporter group (2) and the dark quenching group (3); When the probe does not bind to the target site (5) of TREM-1, the trigger receptor-1 of myeloid cells, the conformation-sensitive linker (4) presents a preset folded conformation, which makes the near-infrared fluorescent reporter group (2) and the dark quencher group (3) spatially close to each other, and the distance between them is less than the Foster radius of their Foster resonance energy transfer, thereby achieving fluorescence quenching of the near-infrared fluorescent reporter group (2) through the fluorescence resonance energy transfer effect; Furthermore, when the TREM-1 targeting module (1) specifically binds to the TREM-1 target (5), the binding event induces a conformational change in the TREM-1 targeting module (1). This conformational change applies a mechanical tension to the conformation-sensitive connecting arm (4), forcing the conformation-sensitive connecting arm (4) to unfold from the preset folded conformation and stretch into a linear stretched conformation. This causes the near-infrared fluorescent reporter group (2) and the dark quencher group (3) to be spatially separated, with the distance between them being greater than their Foster radius. This relieves the fluorescence resonance energy transfer effect and achieves fluorescence activation of the probe.

2. The fluorescent molecular probe according to claim 1, characterized in that, The TREM-1 targeting module (1) is a synthetic bicyclic peptide; the molecular weight of the bicyclic peptide ranges from 2.0 kDalton to 3.5 kDalton, and the bicyclic peptide contains a first peptide ring and a second peptide ring composed of amino acid residues, the first peptide ring and the second peptide ring being covalently linked by a non-peptide chemical cross-linking core molecule to form a saddle-shaped structure with a predetermined three-dimensional rigid conformation.

3. The fluorescent molecular probe according to claim 2, characterized in that, The core molecule of the chemical crosslinking is 1,3,5-tris(bromomethyl)benzene; the amino acid sequence of the bicyclic peptide contains three cysteine ​​residues, and the side chain thiol groups of the three cysteine ​​residues undergo a nucleophilic substitution reaction with the 1,3,5-tris(bromomethyl)benzene to form a thioether bond that cyclizes the peptide chain into the bicyclic peptide.

4. The fluorescent molecular probe according to claim 1, characterized in that, The near-infrared fluorescent reporter group (2) is a heptamethrin dye derivative, with its maximum excitation wavelength in the range of 740 nm to 760 nm and its maximum emission wavelength in the range of 770 nm to 800 nm; and the dark quenching group (3) is a black hole quencher derivative, the wavelength range of which covers the emission spectrum of the near-infrared fluorescent reporter group (2).

5. The fluorescent molecular probe according to claim 1, characterized in that, The conformation-sensitive linker (4) is a short peptide with a specific amino acid sequence; the sequence of the short peptide contains a core flexible region composed of glycine-serine repeating units, and a proline residue is introduced at each end of its sequence to induce and stabilize the preset folding conformation, which is a U-shaped or hairpin conformation.

6. The fluorescent molecular probe according to claim 2, characterized in that, The molecular assembly structure of the probe is as follows: The near-infrared fluorescent reporter group (2) is covalently linked to the ε-amino group of a lysine residue on the first peptide ring of the bicyclic peptide; The dark quenching group (3) is covalently attached to one end of the conformation-sensitive linker arm (4); The other end of the conformation-sensitive linker (4) is covalently linked to the ε-amino group of another lysine residue on the second peptide ring of the bicyclic peptide, thereby forming a bridging link structure that spans the first peptide ring and the second peptide ring.

7. A method for preparing a TREM-1-targeted fluorescent molecular probe as described in any one of claims 1 to 6, characterized in that, The method includes the following steps: Step 1, Synthesis and Cycling of TREM-1 Targeting Module: A linear precursor peptide containing three cysteine ​​residues and two lysine residues with orthogonal protecting groups was synthesized by solid-phase peptide synthesis. After purification, the linear precursor peptide was subjected to intramolecular cyclization reaction by chemical cross-linking core molecules under dilution conditions. After purification, the TREM-1 targeting module with the bicyclic peptide structure (1) was obtained. Step 2, Preparation of conformation-sensitive linker-quencher conjugate: The peptide sequence of the conformation-sensitive linker (4) is synthesized by solid-phase peptide synthesis and the dark quencher group (3) is covalently coupled to one end of it to obtain the conformation-sensitive linker-quencher conjugate. Step 3, two-step site-specific coupling: First, selectively remove the protecting group of the first lysine residue on the TREM-1 targeting module (1) and react it with the activated near-infrared fluorescent reporter group (2) to purify and obtain a single-labeled intermediate; then, selectively remove the protecting group of the second lysine residue on the single-labeled intermediate and react it with the free end of the conformation-sensitive linker-quencher conjugate to form a bridging link; Step 4, Purification and Characterization of the Final Product: The reaction product from Step 3 is purified to obtain the TREM-1-targeted fluorescent molecular probe.

8. The method according to claim 7, characterized in that, In step one, the two lysine residues of the linear precursor peptide are orthogonally protected by a 4-methyltriphenylmethyl (Mtt) protecting group and a tert-butyloxycarbonyl (Boc) protecting group, respectively. In step three, the Boc protecting group is first removed under acidic conditions to couple the near-infrared fluorescent reporter group (2), and then the Mtt protecting group is removed under weakly acidic conditions to couple the conformation-sensitive linker-quencher conjugate.

9. The use of the TREM-1-targeted fluorescent molecular probe as described in any one of claims 1 to 6 in the preparation of imaging agents for the diagnosis or monitoring of inflammation-related diseases.

10. The application according to claim 9, characterized in that, The inflammation-related diseases are selected from at least one of sepsis, atherosclerosis, rheumatoid arthritis, inflammatory bowel disease, and pro-inflammatory cell infiltration in the tumor microenvironment.

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