Dyeing-free AIE nano probe for tumor hypoxia instant detection as well as preparation and application of staining-free AIE nano probe

Abstract

The invention discloses a staining-free AIE nano probe for tumor hypoxia instant detection and preparation and application thereof, and belongs to the technical field of new auxiliary materials and new dosage forms for pharmaceutical preparation combined treatment. The dye-free AIE nanoprobe is formed by self-assembly of dimer AIE fluorophores bridged by hypoxia-related enzyme sensitive chemical bonds through intermolecular force, the surface of the dye-free AIE nanoprobe is modified by a polyethylene glycol modifier, and the mass ratio of the drug to the PEG modifier is 10: 90-90: 10. The intermolecular acting force comprises electrostatic interaction and hydrophobic acting force. The preparation method of the dye-free AIE nanoprobe is simple and reliable, specific imaging can be realized at a tumor hypoxia part, and meanwhile, the dye-free AIE nanoprobe has very high safety. The staining-free AIE nanoprobe provides a new strategy and more choices for developing delivery of the probe, and meets the urgent requirements of imaging and detection strategies in a tumor hypoxia region in the clinical field on preparations.

Description

Technical Field

[0001] The present invention belongs to the technical field of new excipients and new dosage forms for combined drug therapy, and particularly relates to a non-staining AIE nanoprobe for instant detection of tumor hypoxia, and its preparation and application. Background Art

[0002] In recent years, tumor microenvironment-responsive probes have become a research hotspot in the field of biomedicine because they can provide real-time and accurate feedback of pathological information. The rapid proliferation of solid tumors and abnormal vascular networks together lead to the formation of a heterogeneous hypoxic microenvironment within the tumor, and the degree of hypoxia is closely related to tumor progression and treatment resistance. Therefore, it has become an urgent task to develop highly sensitive fluorescent probes for accurate detection of the tumor hypoxic microenvironment. Based on the tumor hypoxia region-specific highly expressed bioreductases (such as azoreductase, nitroreductase), researchers have developed a variety of hypoxia-responsive probes. However, existing probes face significant bottlenecks in clinical translation: small molecule probes have a low tumor accumulation rate due to rapid systemic clearance after intravenous administration, making it difficult to effectively target the hypoxic regions inside solid tumors.

[0003] With the progress of nanotechnology, functionalized nanocarriers have significantly prolonged the blood circulation time of probes and improved tumor targeting efficiency through the enhanced permeability and retention (EPR) effect. However, traditional fluorescent probes are prone to fluorescence quenching due to aggregation (Aggregation-caused quenching, ACQ) after being encapsulated into nanocarriers, severely restricting imaging performance. In 2001, the aggregation-induced emission (Aggregation-induced emission, AIE) pioneered by the Tang team provided a breakthrough direction for this problem. Aggregation-induced emission fluorogens (AIEgens) show significantly enhanced fluorescence in the aggregated state, which highly coincides with the self-assembly characteristics of nanocarriers. Although AIE-based nanoprobes have shown potential in hypoxia detection, their clinical applications still face two major challenges: First, the fluorescence signal of AIEgens is continuously activated in the aggregated state, resulting in high background noise and lack of lesion specificity; Second, the biocompatibility of nanocarrier materials is different from the clinical pharmaceutical excipient standards, restricting the transformation process.

[0004] Currently, instant detection of tumor hypoxia is urgently needed for clinical pathological analysis. However, although in vivo imaging technology can trace tumors as a whole, it cannot accurately locate the hypoxic microregions; while traditional tissue section staining relies on antibody labeling and complex incubation processes, and the time-consuming operation is likely to expose the samples to an oxygen-rich environment, resulting in distortion of in situ hypoxia information. Summary of the Invention

[0005] In response to the above problems, the present invention innovatively proposes to combine the advantages of both in vivo detection and tissue sectioning strategies, designing a hypoxia-responsive AIE nanoprobe that efficiently accumulates in the tumor site through the EPR effect after intravenous injection; fresh tumor sections are then obtained and directly fluorescently imaged without staining or incubation. This model combines the targeting of in vivo detection with the spatiotemporal resolution of in vitro detection, and is expected to achieve instant, label-free analysis of the tumor hypoxic microenvironment.

[0006] The present invention designs a method based on AIE, A real-time hypoxia detection nanoprobe (p-TNNT NAs) based on the resonance energy transfer (FRET) effect and hypoxia-sensitive azobenzene bond (Azo). The core design strategy of the probe is as follows: First, an azobenzene bridged dimer (TNNT) is constructed by covalently linking two tetraphenylethylene (TPE) units. The aromatic groups in its molecular structure give it excellent self-assembly ability to form stable nanoassemblies (NAs). The selection of TPE as a fluorescent group has two considerations: first, an efficient FRET energy transfer pair can be formed between TPE and Azo, providing a molecular basis for the construction of a fluorescent switch; second, although the short-wavelength emission characteristics of TPE limit its in vivo imaging application, its strong AIE effect is compatible with conventional fluorescence microscopy, making it particularly suitable for rapid imaging of ex vivo tissue sections. The working mechanism of the probe exhibits a unique "hypoxia activation" mode: under normoxic conditions, the FRET effect between TPE and Azo puts the AIE fluorescence in the "off" state; while in the tumor hypoxic microenvironment, the Azo bond is specifically cleaved, the FRET effect is released, and the AIE fluorescence of TPE is triggered to "turn on". Based on this mechanism, p-TNNT NAs are efficiently enriched in the tumor site through the EPR effect after intravenous injection, and then fresh tumor slices are imaged without staining, successfully achieving precise localization of hypoxic micro-regions in the tumor of 4T1 breast cancer model mice. Compared with the commercially available hypoxia probe pimonidazole hydrochloride (HP3), p-TNNT NAs show stronger fluorescence signals in tumor slices. In addition, this nanoprobe can accurately detect the hypoxia micro-regions in tumors ranging from 40 to 200 mm 3 CD31 staining further confirmed that fluorescence illumination mainly occurred in areas away from blood vessels. Notably, p-TNNT NAs allow stain-free hypoxia detection immediately after slice acquisition, eliminating the time-consuming staining procedure. This will undoubtedly improve the accuracy of hypoxia detection.

[0007] The present invention achieves the above object through the following technical solutions:

[0008] In the first aspect of the present invention, a non-staining AIE nanoprobe for instant detection of tumor hypoxia is provided. The nanoprobe is self-assembled by intermolecular forces from AIE dimeric molecules of hypoxia-responsive FRET, and its surface is modified with a polyethylene glycol modifier. The mass ratio of the AIE dimeric molecule to the PEG modifier is 10:90 to 90:10.

[0009] In the above technical solution, further, the intermolecular forces include π-π stacking force, hydrophobic force, hydrogen bond, and electrostatic force.

[0010] In the above technical solution, further, the AIE dimeric molecule of hypoxia-responsive FRET is formed by connecting two AIE molecules through a hypoxia-sensitive chemical bond. Among them, the AIE molecule is an AIE fluorophore containing active hydroxyl, amino, or carboxyl groups, selected from tetraphenylethylene-based and triphenylamine-based AIE fluorophores; the hypoxia-sensitive chemical bond is selected from azoreductase-sensitive bonds such as azobenzene, nitroreductase-sensitive bonds, and quinone reductase-sensitive bonds.

[0011] In the above technical solution, further, the AIE molecule is preferably 4-(1,2,2-triphenylvinyl)phenyl methanol ((4-(1,2,2-triphenylvinyl)phenyl)Methanol, TPE); the tumor site-specific response bond is preferably a hypoxia-responsive bond, that is, an azo bond. Specifically, two AIE molecules (TPE) are coupled together through an azo bond to obtain a TPE homodimer prodrug. Its structural formula is:

[0012]

[0013] In the above technical solution, further, the preparation method of the AIE dimeric molecule (AIE dimeric prodrug TNNT) with FRET effect includes: dissolving carboxyazobenzene in an organic solvent, adding TPE and a catalyst to react, and finally purifying and separating; the organic solvent is one or any combination of dichloromethane, chloroform, dimethyl sulfoxide, and N,N-dimethylformamide, and the catalyst is one or any combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDCI), 1-hydroxybenzotriazole (HOBT), N,N-dicyclohexylcarbodiimide (DCC), N,N-carbonyldiimidazole (CDI), and 4-dimethylaminopyridine (DMAP).

[0014] In the above technical solution, further, the carboxyazobenzene is one or any combination of compounds with CAS numbers 101351-18-2, 586-91-4, 71987-42-3, 1562-93-2, 6925-48-0, and 37790-20-8.

[0015] In the above technical solution, further, the preparation method of the tumor site hypoxia-responsive AIE dimer prodrug TNNT includes the following steps:

[0016] Dissolve carboxyazobenzene in a mixed solvent of dichloromethane and N,N-dimethylformamide and stir evenly. Dissolve 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDCI) and 1-hydroxybenzotriazole (HOBT) in an appropriate amount of dichloromethane and stir evenly. Slowly add the solution dropwise to the above system. Under nitrogen protection, stir at 0 °C for 2 h. Separately, dissolve TPE and 4-dimethylaminopyridine (DMAP) in an appropriate amount of dichloromethane, and slowly add it to the reaction system. Under nitrogen protection, continue to stir at room temperature for 24 h. The obtained product is separated and purified by preparative liquid chromatography.

[0017] In the above technical solution, further, the polyethylene glycol modifier includes one or more of DSPE-PEG, PLGA-PEG, PCL-PEG, and PE-PEG, and the molecular weight of PEG is 200-20000.

[0018] In the above technical solution, further, the polyethylene glycol modifier is preferably DSPE-PEG 2K .

[0019] In the above technical solution, further, the present invention also provides a preparation method of a stain-free AIE nanoprobe for instant detection of tumor hypoxia, including the following steps:

[0020] Dissolve the hypoxia-responsive FRET AIE dimer molecule in an organic solvent, mix well under stirring, and slowly add the mixed solution dropwise to deionized water. Under the action of magnetic stirring, uniform self-assembled nanoparticles are spontaneously formed. Remove the organic solvent to obtain a non-polyethylene glycol-modified AIE nanoprobe; slowly add a mixed organic solvent of the hypoxia-responsive FRET AIE dimer molecule and a polyethylene glycol modifier dropwise to deionized water under magnetic stirring, and spontaneously form uniform self-assembled nanoparticles. Remove the organic solvent to obtain a polyethylene glycol-modified AIE nanoprobe.

[0021] In the above technical solution, further, the organic solvent is one or any combination of two of anhydrous ethanol, tetrahydrofuran, methanol, acetone, acetonitrile, dioxane, dimethyl sulfoxide, and N,N-dimethylformamide.

[0022] In the above technical solution, further, the organic solvent is preferably a mixed solvent of tetrahydrofuran and anhydrous ethanol.

[0023] In the above technical solution, further, the method for removing the organic solvent includes solvent evaporation, ultrafiltration, dialysis, or membrane permeation.

[0024] In the second aspect of the present invention, there is provided a stain-free AIE nanoprobe for instant detection of tumor hypoxia prepared by the above method.

[0025] In the third aspect of the present invention, there is provided the application of the aforementioned stain-free AIE nanoprobe for instant detection of tumor hypoxia in the preparation of a drug delivery system.

[0026] In the fourth aspect of the present invention, there is provided the application of the aforementioned stain-free AIE nanoprobe for instant detection of tumor hypoxia in the preparation of a tumor imaging probe.

[0027] In the fifth aspect of the present invention, there is provided the application of the aforementioned stain-free AIE nanoprobe for instant detection of tumor hypoxia in the preparation of an injection administration, oral administration or topical administration system.

[0028] The beneficial effects of the present invention compared with the prior art are as follows:

[0029] 1. The present invention prepares self-assembled nanoparticles of a hypoxia-responsive AIE small molecule dimer prodrug (preferably the TPE dimer prodrug TNNT), which are modified with a PEG modifier to obtain a stain-free AIE nanoprobe for instant detection of tumor hypoxia, specifically imaging at the tumor hypoxia site, and can achieve precise detection of the tumor hypoxia region.

[0030] 2. The carrier-free nano-assembly formed by self-assembly of TNNT of the present invention achieves technical effects such as high drug loading, good stability, and low toxicity and side effects, meets the urgent needs of high-efficiency and low-toxic tumor probe preparations in clinical practice, and provides an innovative idea for the precise detection of tumor hypoxia regions. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly introduced below.

[0032] Figure 1 HNMR spectrum of the hypoxia-responsive AIE dimer fluorescence probe (TNNT) of Example 1 of the present invention 1 HNMR spectrum.

[0033] Figure 2 Malvern particle size distribution diagrams of the non-PEGylated (TNNT) nanoparticles and PEGylated (p-TNNT) nanoparticles of Example 2 of the present invention.

[0034] Figure 3 Transmission electron microscope images of the TNNT nanoparticles and p-TNNT nanoparticles of Example 2 of the present invention.

[0035] Figure 4Stability diagram of TNNT nanoparticles and p-TNNT nanoparticles of Example 3 of the present invention under PBS (pH 7.4) conditions.

[0036] Figure 5 Stability diagram of TNNT nanoparticles and p-TNNT nanoparticles of Example 3 of the present invention under PBS (pH 7.4) containing 10% FBS conditions.

[0037] Figure 6 Molecular docking diagram of p-TNNT of Example 4 of the present invention.

[0038] Figure 7 Diagram of molecular force disruption of TNNT nanoparticles of Example 4 of the present invention under sodium chloride (10 mM), sodium dodecyl sulfate (10 mM), and urea (10 mM) conditions.

[0039] Figure 8 Diagram of the overlap of the absorption spectrum of azobenzene and the emission spectrum of TPE-OH of Example 5 of the present invention.

[0040] Figure 9 Ultraviolet spectrum diagram of p-TNNT nanoparticles of Example 5 of the present invention before and after the reduction reaction.

[0041] Figure 10 Fluorescence spectrum diagram of p-TNNT nanoparticles of Example 5 of the present invention before and after the reduction reaction.

[0042] Figure 11 Diagram of fluorescence changes of p-TNNT nanoparticles of Example 5 of the present invention at different times under sodium dithionite (20 mM) conditions.

[0043] Figure 12 Diagram of particle size changes of p-TNNT nanoparticles of Example 5 of the present invention at different times under sodium dithionite (20 mM) conditions.

[0044] Figure 13 Mass spectrometry diagram of the cleavage of TPE-OH from p-TNNT nanoparticles of Example 5 of the present invention under sodium dithionite (20 mM) conditions.

[0045] Figure 14 Cytotoxicity diagram of p-TNNT nanoparticles of Example 6 of the present invention against 4T1 under normoxia and hypoxia conditions.

[0046] Figure 15 Cytotoxicity diagram of p-TNNT nanoparticles of Example 6 of the present invention against MCF-7 under normoxia and hypoxia conditions.

[0047] Figure 16Cytotoxicity graphs of p-TNNT nanoparticles of Example 6 of the present invention against 3T3 under normoxia and hypoxia conditions.

[0048] Figure 17 Cytotoxicity graphs of p-TNNT nanoparticles of Example 6 of the present invention against L02 under normoxia and hypoxia conditions.

[0049] Figure 18 Hypoxia lighting-up graphs of p-TNNT nanoparticles of Example 7 of the present invention against 4T1 cells under normoxia and hypoxia conditions.

[0050] Figure 19 Semi-quantitative graphs of hypoxia lighting-up of p-TNNT nanoparticles of Example 7 of the present invention against 4T1 cells under hypoxia conditions.

[0051] Figure 20 Semi-quantitative graphs of hypoxia lighting-up of p-TNNT nanoparticles of Example 7 of the present invention against 4T1 cells under normoxia conditions.

[0052] Figure 21 Hypoxia lighting-up graphs of p-TNNT nanoparticles of Example 7 of the present invention against MCF-7 cells under normoxia and hypoxia conditions.

[0053] Figure 22 Semi-quantitative graphs of hypoxia lighting-up of p-TNNT nanoparticles of Example 7 of the present invention against 4T1 cells under hypoxia conditions.

[0054] Figure 23 Semi-quantitative graphs of hypoxia lighting-up of p-TNNT nanoparticles of Example 7 of the present invention against 4T1 cells under normoxia conditions.

[0055] Figure 24 Hypoxia lighting-up graphs of p-TNNT nanoparticles and PBS of Example 8 of the present invention against 4T1 tumor spheres.

[0056] Figure 25 Quantitative graphs of hypoxia lighting-up of p-TNNT nanoparticles and PBS of Example 8 of the present invention against 4T1 tumor spheres.

[0057] Figure 26 Pharmacokinetic curves of administering DiR solution and DiR-labeled p-TNNT nanoparticles in Example 9 of the present invention.

[0058] Figure 27 Photos taken by an in vivo imager of pharmacokinetic blood samples of administering DiR solution and DiR-labeled p-TNNT nanoparticles in Example 9 of the present invention.

[0059] Figure 28 In vivo imaging graphs of mice administered with DiR solution and DiR-labeled p-TNNT nanoparticles in Example 10 of the present invention.

[0060] Figure 29 This is the quantitative in vivo imaging graph of mice given DiR solution and DiR-labeled p-TNNT nanoparticles in Example 10 of the present invention.

[0061] Figure 30 This is the fluorescence imaging graph of excised organs of mice given DiR solution and DiR-labeled p-TNNT nanoparticles in Example 10 of the present invention.

[0062] Figure 31 This is the fluorescence quantitative graph of excised organs of mice given DiR solution and DiR-labeled p-TNNT nanoparticles in Example 10 of the present invention.

[0063] Figure 32 This is the fluorescence graph of p-TNNT nanoparticles in Example 11 of the present invention and commercially available hypoxia probe HP3 in tumor sections of 4T1 tumor-bearing mice.

[0064] Figure 33 This is the fluorescence graph of p-TNNT nanoparticles in Example 11 of the present invention in tumor sections of 4T1 tumor-bearing mice with different tumor volumes.

[0065] Figure 34 This is the fluorescence graph of p-TNNT nanoparticles in Example 11 of the present invention in tumor sections of 4T1 tumor-bearing mice. Detailed implementation manners

[0066] The present invention will be described in detail below in conjunction with the embodiments. However, the implementation manners of the present invention are not limited thereto. Obviously, the embodiments described below are only partial embodiments of the present invention. For those skilled in the art, without creative efforts, obtaining other similar embodiments will fall within the protection scope of the present invention.

[0067] The carboxyazobenzene used in the examples, with the CAS number 101351-18-2, was purchased from Zhengzhou Aike Chemical Co., Ltd.

[0068] Example 1: Synthesis of chemically bonded TPE dimer prodrug

[0069] Dissolve carboxyazobenzene (1 mmol) in dichloromethane, and then successively add EDCI (3 mmol) and HOBT (2 mmol). After stirring for 2 h under nitrogen protection at 0 °C, add TPE (3 mmol) and DMAP (0.5 mmol) to the above system, and continue the reaction for 24 h under nitrogen protection at room temperature. The reaction product was purified by preparative liquid chromatography to obtain the azo-bonded TPE dimer prodrug.

[0070] The prodrug structure was determined by nuclear magnetic resonance hydrogen spectrum (1 confirmed by \(^1\)H NMR or mass spectrometry. The results are as Figure 1 shown. For the \(^1\)H NMR of the azo-bridged TPE dimer (TNNT), deuterated chloroform was used as the solvent. The results of the NMR spectrum analysis are as follows: 1 \(^1\)H NMR (600 MHz, CDCl\(_3\)) δ 8.22 (dd, \(J\) = 8.4, 1.3 Hz, 4H), 8.00–7.96 (m, 4H), 7.21–7.02 (m, 38H), 5.31 (s, 4H), 3.35 (d, \(J\) = 5.9 Hz, 4H).

[0071] Example 2: Preparation of TNNT self-assembled nanoparticles

[0072] In this example, a carrier-free nano-assembly self-assembled from TNNT was prepared by a one-step nanoprecipitation method. Briefly, TNNT was dissolved in a mixed solution of tetrahydrofuran and absolute ethanol (1:1, v / v) to obtain a drug solution with a concentration of 5 mg / mL. Under magnetic stirring (1200 rpm), 200 μL of the drug solution was slowly added dropwise to 2 mL of deionized water, and stirring was continued evenly for 3 min. TNNT could spontaneously form uniform nanoparticles. Then, the organic solvent in the nano-preparation was removed by rotary evaporation at 30 °C for 3 min, and the nano-preparation was made up to 2 mL with deionized water to obtain a nano-colloidal solution without any organic solvent.

[0073] DSPE-PEG 2K Preparation method of PEGylated nanoparticles modified with DSPE-PEG (p-TNNT nanoparticles): Weigh accurately 1.24 mg of TNNT and dissolve it with 248 μL of a mixed solution of tetrahydrofuran and absolute ethanol; weigh accurately 0.25 mg of DSPE-PEG 2K , and dissolve it with 25 μL of a mixed solution of tetrahydrofuran and absolute ethanol to prepare a stock solution with a concentration of 10 mg / mL. Mix 200 μL of the drug mixed solution with 25 μL of the DSPE-PEG 2K solution evenly. Under magnetic stirring (1200 rpm), slowly add 225 μL of the mixed solution dropwise to 2 mL of deionized water, and continue to stir evenly for 3 min. Then, remove the organic solvent in the nano-preparation by rotary evaporation at 30 °C for 3 min, and make up the nano-preparation to 2 mL with deionized water to obtain a nano-colloidal solution without any organic solvent. The particle size, particle size distribution, Zeta potential, drug loading and morphology of the prepared TNNT nanoparticles and p-TNNT nanoparticles were investigated by dynamic light scattering and transmission electron microscopy (Table 1, Figures 2 - 3 ).

[0074] Table 1. Particle size, particle size distribution and Zeta potential of TNNT nanoparticles and p-TNNT nanoparticles

[0075] Nanoparticle Particle size (nm) Particle size distribution (PDI) Zeta potential (mV) Drug loading TNNT Nanoparticle 118.40±1.30 0.15±0.02 -17.37±5.25 100％ p - TNNT Nanoparticle 63.50±1.36 0.14±0.04 -22.83±2.04 75％

[0076] As shown in Table 1 and Figure 2 as shown, the particle size of TNNT nanoparticles is about 118 nm, and the Zeta potential is about -17 mV; the particle size of p-TNNT nanoparticles is about 64 nm, and the Zeta potential is about -23 mV.

[0077] The morphologies of TNNT nanoparticles and p-TNNT nanoparticles prepared in Example 2 were determined by transmission electron microscopy, and the results are as Figure 3 shown. The transmission electron micrograph shows that the shape of the nanoparticles is a uniform sphere.

[0078] Example 3: Colloidal stability experiment of nanoparticles

[0079] The TNNT nanoparticles and p-TNNT nanoparticles (0.5 mg / mL) prepared in Example 2 were incubated in phosphate buffer (PBS, pH 7.4), and their particle size changes were measured at predetermined time points (0, 0.5, 1, 2, 4, 8, and 12 h). The results are as Figure 4 shown. The non-PEG-modified TNNT nanoparticles have poor colloidal stability, and the particle size increases significantly during incubation. Under the same conditions, the particle size of p-TNNT nanoparticles does not change significantly within 12 h. In addition, p-TNNT nanoparticles (0.5 mg / mL) were placed in PBS (pH 7.4) containing 10% FBS, incubated in a shaker at 37 °C for 12 h, and their particle size changes were measured by dynamic light scattering at predetermined time points (0, 0.5, 1, 2, 4, 8, and 12 h). The results are as Figure 5 shown. p-TNNT nanoparticles have good colloidal stability in PBS (pH 7.4) containing 10% FBS.

[0080] Example 4: Analysis of TNNT self-assembly mechanism

[0081] Using computer simulation technology, the self-assembly mechanism of TNNT was explored, and the molecular docking calculation was completed using the AutodockVina program of the YinFu cloud computing platform. The results are as Figure 6 shown. The π-π interaction and hydrophobic force jointly drive the TNNT nano-assembly process. In addition, sodium chloride (10 mM), sodium dodecyl sulfate (10 mM), and urea (10 mM) were used to disrupt the interactions respectively to further verify the intermolecular forces of TNNT. The results are as Figure 7As shown, the particle size of the nanoparticles incubated with sodium chloride (10 mM) and urea (10 mM) remained stable, indicating that the effects of electrostatic interaction and hydrogen bond interaction on nanoparticle formation were negligible. Notably, in the presence of sodium dodecyl sulfate (10 mM), the particle size of the nanoparticles increased sharply within a short time, further confirming that hydrophobic interaction played a dominant role in nanoassembly.

[0082] Example 5: Optical Properties of Nanoparticles

[0083] First, the fluorescence emission spectrum of TPE-OH and the absorption spectrum of azobenzene were measured. As Figure 8 shown, the spectra of the two overlapped, confirming that the two substances met one of the conditions for generating FRET. After being covalently linked into one molecule, the distance was shortened, further meeting the conditions for generating FRET. This proved the generation of the FRET effect. Next, Na2S2O4 (20 mM) was used to simulate the in vitro hypoxic environment, and the absorption spectrum and fluorescence emission spectrum of p-TNNT nanoparticles before and after incubation with Na2S2O4 (20 mM) were measured respectively. The results are as Figure 9 and Figure 10 shown. After the reduction reaction, the absorption peak of azobenzene disappeared and fluorescence appeared; the p-TNNT nanoparticles of the present invention could specifically respond in a hypoxic environment and had hypoxia-activated fluorescence characteristics, which were suitable for detecting hypoxic microenvironments.

[0084] 100 μg of p-TNNT nanoparticles were dispersed in an aqueous solution of Na2S2O4 (20 mM) and incubated at 37 °C for 0, 5, 30, 60, and 90 min. The overall fluorescence change was monitored using a fluorescence spectrometer (Ex = 320 nm, Em = 400 - 600 nm). The particle size change was measured at predetermined time points by dynamic light scattering.

[0085] The results are as Figure 11 and Figure 12 shown. The nanoparticles exhibited a time-dependent fluorescence recovery behavior, and within 90 minutes of fluorescence recovery, the particle size of the nanoparticles did not change significantly and the morphology of the nanoparticles remained intact. The p-TNNT nanoparticles of the present invention had structural stability in a hypoxic environment and did not show obvious aggregation or disassembly during the fluorescence recovery process.

[0086] Figure 13 The mass spectrometry results confirmed that after incubation with Na2S2O4 (20 mM), TNNT was successfully cleaved into TPE-OH. These results indicated that the p-TNNT nanoparticles had excellent hypoxia-responsive fluorescence recovery behavior.

[0087] Example 6: Cytotoxicity of Nanoparticles

[0088] The cytotoxicity of p-TNNT nanoparticles against mouse breast cancer (4T1) cells, human breast cancer (MCF-7) cells, mouse embryonic fibroblasts (3T3) cells and human normal liver (L02) cells was investigated by the MTT method. Briefly, 4T1 cells, MCF-7 cells, 3T3 cells and L02 cells were seeded into 96-well plates at a density of 2×10 3 cells / well and incubated for 12 h at 37 °C and 5% CO2 to allow cell attachment. Then, the original medium was replaced with fresh medium containing a series of concentrations of p-TNNT nanoparticles. Cells cultured in fresh blank medium served as negative controls. After incubation for 48 h under normoxic or hypoxic conditions, 20 μL of MTT solution (5 mg / mL) was added to each well and incubated in a 37 °C incubator for 4 h. The liquid in the 96-well plates was discarded, and 200 μL of DMSO was added to each well and shaken on an oscillator for 10 min to dissolve the formed formazan crystals. The ultraviolet absorbance value at 490 nm was measured using a microplate reader.

[0089] The cytotoxicity results are as Figures 14 - 17 shown. Among the four types of cells, p-TNNT nanoparticles showed high safety. These results well confirmed the superiority of the dimer prodrug design in enhancing tumor-specific antitumor effects and reducing off-target toxicity.

[0090] Example 7: Hypoxic imaging of nanoparticles in cells

[0091] The fluorescence hypoxic lighting of the p-TNNT nanoparticles prepared in Example 2 in mouse breast cancer cells (4T1) and human breast cancer (MCF-7) cells was evaluated using a confocal laser scanning microscope (CLSM). For the qualitative analysis by CLSM, 4T1 and MCF-7 cells were seeded in 35-mm culture dishes at a density of 2×10 5 cells / dish and cultured for 12 h to allow cell attachment. Then, the old medium was discarded and replaced with fresh medium containing p-TNNT nanoparticles. After incubation for 4 h under hypoxic and normoxic conditions respectively, the medium was discarded, and the cells were washed 3 times with cold PBS. Subsequently, the intracellular fluorescence signals were observed by CLSM. The experimental results are as Figures 18 - 23 shown. The above experimental results all showed that the cells treated with nanoparticles under hypoxic conditions had higher intracellular fluorescence signals, and the cells treated under normoxic conditions had almost no fluorescence signals. The p-TNNT nanoparticles prepared in Example 2 could specifically respond to the intracellular hypoxic environment.

[0092] Example 8: Formation and hypoxic imaging of 3D tumor spheroids

[0093] 4T1 cells (1×10 5The cells were placed in a 96-well agarose-coated culture plate and cultured at 37 °C for 7 days to construct three-dimensional tumor spheroids. Then, PBS and the p-TNNT nanoparticles prepared in Example 2 were added to the culture wells respectively and incubated for 12 h. Fluorescence images were taken using a confocal laser scanning microscope (CLSM, C2, Nikon, Japan). The experimental results are as Figure 20 shown. Compared with the negative control group, obvious fluorescence signals were shown in the sections at different depths of the tumor spheroids in the nanoparticle group, indicating that the nanoparticles could specifically label the hypoxic regions within the tumor spheroids, and the nanoparticles could effectively penetrate into the tumor interior to achieve visualization of the hypoxic regions. Figure 25 is Figure 24 a quantitative presentation.

[0094] Example 9: Pharmacokinetic study of nanoparticles

[0095] The near-infrared fluorescent dye DiR was used as a fluorescent probe to prepare DiR-labeled p-TNNT nanoparticles (DiR / p-TNNT nanoparticles). Male Sprague-Dawley (SD) rats weighing between 180 - 220 g were randomly grouped and fasted for 12 h before drug administration and allowed free access to water. DiR solution and DiR / p-TNNT nanoparticles (DiR equivalent dose was 1 mg / kg) were injected intravenously respectively. Blood was collected from the orbital cavity at predetermined time points (0.033, 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h), and plasma was separated. Then, DiR was extracted by the protein precipitation method, and finally, the concentration of DiR in the plasma was measured using a multifunctional microplate reader (excitation 748 nm, emission 780 nm).

[0096] The experimental results are as Figures 26 - 27 and Table 2 shown. Due to the short half-life, the DiR solution was cleared from the blood relatively quickly. In contrast, DiR / p-TNNT nanoparticles showed obvious advantages over the DiR solution in terms of prolonging the blood circulation time, and the area under the plasma concentration-time curve (AUC 0-24h ) was significantly increased, which laid a good foundation for the specific accumulation of the drug at the tumor site in vivo.

[0097] Table 2. Pharmacokinetic parameters of DiR solution and DiR / p-TNNT nanoparticles (n = 3)

[0098] Group <![CDATA a) AUC 0-24h > <![CDATA b) C 0.5 > DiR Solution 0.96±0.19 0.04±0.03 DiR / p - TNNT Nanoparticle 99.98±6.07 10.97±0.67

[0099] a) Area under the plasma concentration-time curve (nmol / mL*h).

[0100] b) Plasma drug concentration at the 0.5 h time point (nmol mL-1).

[0101] Example 10: Tissue Distribution Experiment of Nanoparticles

[0102] A 4T1 subcutaneous tumor-bearing mouse model was constructed to study the in vivo biodistribution of p-TNNT nanoparticles. DiR was also used as a fluorescent probe to prepare DiR / p-TNNT nanoparticles. Briefly, a 4T1 cell suspension was inoculated into BALB / c mice. When the tumor volume reached approximately 300 mm 3 , the mice were randomly divided into two groups. DiR solution and DiR / p-TNNT nanoparticles (DiR equivalent dose of 1 mg / kg) were injected via the tail vein, respectively. The mice were anesthetized at predetermined time points (1, 2, 4, 8, 12, and 24 h), and in vivo imaging analysis was performed using an IVIS small animal imaging system. The ROI tool was used to quantify the fluorescence signal. The results are as Figures 28 - 29 shown. At 12 h after administration, the mice were sacrificed and the major organs (heart He, liver Li, spleen Sp, lung Lu, kidney Ki) and tumor tissues Tu were collected for ex vivo fluorescence imaging. The ROI tool was used to quantify the fluorescence signal. The results are as Figures 30 - 31 shown.

[0103] The above results showed that compared with the DiR solution, the fluorescence intensity of DiR / p-TNNT nanoparticles at the tumor site was significantly increased and reached the maximum accumulation at 12 h. In addition, the ex vivo imaging results were consistent with the in vivo imaging results. DiR / p-TNNT nanoparticles could effectively accumulate at the tumor site, which could be attributed to the significant advantages of DiR / p-TNNT nanoparticles in terms of colloidal stability and pharmacokinetic behavior. In summary, the PEG-modified carrier-free nano-assembly (p-TNNT nanoparticles) could not only significantly prolong the circulation time of drugs in the blood, but also showed tumor-specific drug accumulation.

[0104] Example 11: In Vivo Fluorescence Sections of Nanoparticles

[0105] A 4T1 tumor-bearing BALB / c mouse model was used to further evaluate the hypoxia imaging effect of the fluorescent probe in vivo. The tumor volume calculation formula was length × width × width / 2. When the tumor volume reached 200 mm 3 , HP3 and p-TNNTNAs were injected systemically via the tail vein. After 12 h, the tumors were resected for sectioning and staining ( Figure 32 ). In addition, tumors of different sizes were selected and divided into groups 1, 2, and 3 as shown in Table 3. p-TNNT NAs were injected via the tail vein. After 12 h of injection, the tumors were resected for sectioning and staining ( Figure 33 ). When the tumor volume of the mice reached approximately 200 mm 3At that time, p-TNNT nanoparticles were administered by tail vein injection. After 12 hours, the mice were sacrificed, and tumor tissues were collected for fixation and embedding. CD31 was used to stain blood vessels and PI was used to stain cell nuclei, and the hypoxia in tumor tissues was evaluated using the blue fluorescence inherent in TNNT ( Figure 34 ). Figure 32 It can be seen that p-TNNT nanoparticles showed stronger fluorescence signals in tumor sections, had higher sensitivity and specificity, and could more accurately reflect the hypoxia state in tumors. Its response sensitivity to the hypoxic environment was higher than that of HP3. Figure 33 It can be seen that p-TNNT nanoparticles can detect hypoxic regions in tumors of different volumes, all showing good hypoxic response characteristics, and can be applied to tumor detection at different stages. Figure 34 It can be seen that combining CD31 staining and PI staining can more intuitively show the relative positions of hypoxic regions, blood vessels and cells, demonstrating the hypoxic response characteristics of the nanoparticles of the present invention. The AIE nanoprobe is superior to HP3 in in vitro hypoxia detection. In addition, it can also report different degrees of hypoxia in tumors of different sizes.

[0106] Table 3 Length, width and volume of tumors

[0107] Group Length (mm) Width (mm) <![CDATA[Volume (mm 3 )]]> 1 7.40 3.34 41.28 2 5.85 5.77 97.38 3 7.66 7.24 200.76

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An undyed AIE nanoprobe for instant detection of tumor hypoxia, characterized in that, The nano-probe is self-assembled by AIE dimeric molecules with FRET effect through intermolecular forces, and polyethylene glycol is used as a modifier; the mass ratio of the AIE dimeric molecules to the PEG modifier is 10:90 to 90:

10.

2. The non-staining AIE nanoprobe for tumor hypoxia instant detection according to claim 1, wherein The intermolecular forces include π-π stacking force, hydrophobic force, hydrogen bond, and electrostatic force.

3. The stain-free AIE nanoprobe for on-site detection of tumor hypoxia according to claim 1, wherein The AIE dimeric molecule with FRET effect is a dimeric AIE fluorophore bridged by a hypoxia-related enzyme-sensitive chemical bond. The AIE fluorophore is selected from tetraphenylethylene-based and triphenylamine-based AIE fluorophores containing active hydroxyl, amino, or carboxyl groups; the hypoxia-related enzyme-sensitive chemical bond is an azoreductase-sensitive bond, including azobenzene, nitroreductase-sensitive bond, or quinone reductase-sensitive bond.

4. The non-staining AIE nanoprobe for tumor hypoxia instant detection according to claim 3, wherein The AIE fluorophore is 4-(1,2,2-triphenylethynyl)benzyl alcohol TPE; the hypoxia-related enzyme-sensitive chemical bond is an azo bond; the AIE dimeric molecule with FRET effect is obtained by coupling two TPE molecules through an azo bond.

5. The non-staining AIE nanoprobe for on-site detection of tumor hypoxia according to claim 4, wherein The preparation method of the AIE dimeric molecule with FRET effect includes: dissolving carboxyazobenzene in an organic solvent, adding TPE and a catalyst for reaction, and finally purifying and separating; preferably, the organic solvent is one or any combination of two of dichloromethane, chloroform, dimethyl sulfoxide, N,N-dimethylformamide, and the catalyst is one or any combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide, 1-hydroxybenzotriazole, N,N-dicyclohexylcarbodiimide, N,N-carbonyldiimidazole, 4-dimethylaminopyridine; the carboxyazobenzene is one or any combination of those with CAS numbers 101351-18-2, 586-91-4, 71987-42-3, 1562-93-2, 6925-48-0, 37790-20-8.

6. The stain-free AIE nanoprobe for on-site detection of tumor hypoxia according to any one of claims 1-5, characterized in that, The preparation method includes the following steps: Slowly drop the organic solvents of the AIE dimeric molecule with FRET effect and the PEG modifier into deionized water under stirring to spontaneously form uniform self-assembled nanoparticles, and remove the organic solvents to obtain the PEG-modified AIE nano-assembly.

7. The non-staining AIE nanoprobe for tumor hypoxia instant detection according to claim 6, wherein The organic solvent is one or any combination of two of absolute ethanol, tetrahydrofuran, methanol, acetone, acetonitrile, dioxane, dimethyl sulfoxide, N,N-dimethylformamide.

8. The non-staining AIE nanosensor for tumor hypoxia instant detection according to claim 1, characterized in that, The PEG modifiers described above include one or more of PCL-PEG, DSPE-PEG, DSPE-SS-PEG, PLGA-PEG, and PE-PEG, and the molecular weight of PEG is 200-20000; preferably, the PEG modifier is DSPE-PEG 2K .

9. Use of the stain-free AIE nano-probe for tumor hypoxia instant detection according to any one of claims 1-8 in the preparation of a drug delivery system; use in an injection administration, oral administration, or local administration system.

10. Use of the stain-free AIE nano-probe for tumor hypoxia instant detection according to any one of claims 1-8 in the preparation of a tumor imaging probe.

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