Fluorescent probe for detecting brain glioma

Abstract

The invention relates to the field of brain glioma detection, and particularly discloses a fluorescent probe for brain glioma detection, which takes a carbon quantum dot as a fluorescent matrix, co-modifies an antibody targeting a brain glioma stem cell marker CD133 and RGD peptide targeting alpha v beta 3 integrin on the surface, and coats a pH response type polymer shell layer; the brain glioma stem cell marker and the tumor neovascularization integrin are synchronously recognized through the double-targeting synergistic effect of the CD133 antibody and the RGD peptide, the single-target leak detection limitation caused by tumor heterogeneity is overcome, and the capacity of capturing malignant cells is remarkably improved; the pH response type polymer shell layer keeps fluorescence silence in a normal physiological environment, dissociates and releases strong near-infrared signals only in a tumor acidic microenvironment, background noise interference is eliminated from the source, and double-lock activated high-signal-to-noise-ratio imaging is achieved. The probe efficiently penetrates through the blood brain barrier by means of the RGD peptide, and double receptor anchoring and microenvironment response activation are completed at the tumor site.

Description

Technical Field

[0001] This invention belongs to the field of glioma detection, specifically a fluorescent probe for glioma detection. Background Technology

[0002] The malignant progression of gliomas is highly dependent on the driving role of tumor stem cells (GSCs). CD133, a surface marker of GSCs, is a key receptor for maintaining stem cell characteristics and is directly related to tumor recurrence and drug resistance. Simultaneously, integrin αvβ3, specifically highly expressed by tumor neovascular endothelial cells, promotes glioma invasion and metastasis by mediating cell adhesion and signal transduction. These two types of targets together constitute the biological basis for molecular diagnostics of gliomas.

[0003] Current fluorescent probes for gliomas mostly rely on single-targeting strategies (such as targeting only CD133 or αvβ3), failing to simultaneously identify the dual targets of tumor stem cells and new blood vessels, resulting in a significantly increased false negative rate for highly heterogeneous tumor cells. At the same time, due to the lack of microenvironment response mechanisms, probes are prone to non-specific activation under normal physiological conditions, generating background fluorescence interference. Furthermore, traditional linear peptide-modified probes have poor serum stability, making it difficult to efficiently cross the blood-brain barrier, which severely restricts the signal-to-noise ratio and localization accuracy of in vivo imaging. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a fluorescent probe for glioma detection, thereby resolving the issues of high single-target false negative rate, severe background noise interference, and low blood-brain barrier penetration efficiency in the prior art.

[0005] A fluorescent probe for glioma detection uses carbon quantum dots as a fluorescent matrix, with its surface co-modified with an antibody targeting the glioma stem cell marker CD133 and an RGD peptide targeting αvβ3 integrin, and coated with a pH-responsive polymer shell.

[0006] Preferably, the probe must simultaneously meet the following conditions to activate near-infrared fluorescence:

[0007] Dual receptor binding: CD133 antibody and RGD peptide bind to tumor cell surface receptors respectively;

[0008] Acidic microenvironment trigger: The pH of the environment is ≤6.5.

[0009] The preparation method of the fluorescent probe includes the following steps:

[0010] S1: Dissolve citric acid, CD133 antibody, and RGD peptide in dimethyl sulfoxide at a molar ratio of (10:1:1)-(10:1:3) and mix by sonication;

[0011] S2: Add dimethylaminoethyl methacrylate monomer and polymerize under nitrogen to form a pH-responsive shell;

[0012] S3: Transfer the mixture to a high-pressure reactor and react at 160-220℃ for 6-24 hours. Cool to obtain the crude product.

[0013] Preferably, the reaction temperature in S3 is 180°C and the reaction time is 12 hours.

[0014] Preferably, the following steps are also included:

[0015] S4: The crude product was separated by silica gel column chromatography, with the eluent being a mixed solution of methanol and ethyl acetate in a volume ratio of (1:3) to (1:5), and the near-infrared fluorescent component was collected.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] By leveraging the dual-target synergistic effect of CD133 antibody and RGD peptide, the simultaneous identification of glioma stem cell markers and tumor angiogenesis integrins overcomes the limitation of single-target missed detection caused by tumor heterogeneity and significantly enhances the ability to capture malignant cells.

[0018] The pH-responsive polymer shell remains fluorescence-silent in a normal physiological environment, and only dissociates and releases a strong near-infrared signal in the acidic microenvironment of the tumor, thus eliminating background noise interference from the source and achieving high signal-to-noise ratio imaging with "dual-lock activation".

[0019] The probe efficiently penetrates the blood-brain barrier via RGD peptides, completing dual receptor anchoring and microenvironment response activation at the tumor site. Its near-infrared fluorescence signal has deep tissue penetration and can be seamlessly compatible with clinical imaging equipment, providing a new solution for intraoperative real-time navigation and efficacy assessment. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the preparation method of the present invention. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] like Figure 1 As shown:

[0023] Example 1: A method for preparing a fluorescent probe for glioma detection, comprising the following steps:

[0024] S1: Raw material mixing:

[0025] Take 10.0 mmol of citric acid, 1.0 mmol of CD133 antibody, and 1.0 mmol of RGD peptide and place them in a 50 mL pressure-resistant glass bottle;

[0026] Add 20 mL of dimethyl sulfoxide (DMSO), seal and place in an ultrasonic bath (40 kHz, 300 W) for 45 minutes to mix.

[0027] S2: Polymerizes to form a pH-responsive shell:

[0028] Add 5.0 mmol of dimethylaminoethyl methacrylate monomer to the mixture;

[0029] High-purity nitrogen (99.99%) was introduced for 15 minutes to remove oxygen, and the reaction was carried out at a constant temperature of 60°C with stirring (500 rpm) for 2 hours under a nitrogen atmosphere.

[0030] S3: Hydrothermal reaction synthesis probe:

[0031] Transfer the reaction solution to a 100 mL polytetrafluoroethylene-lined autoclave, with a filling degree of 70%.

[0032] React at 160°C for 6 hours in a forced-air drying oven, then allow to cool naturally to 25°C.

[0033] The precipitate was removed by filtration, yielding a brownish-red crude product solution.

[0034] S4: Purification:

[0035] Silicone columns (200-300 mesh, column size 2.5×40cm) are used;

[0036] Eluent: Methanol / ethyl acetate = 1:5 (v / v);

[0037] The flow rate was 1.0 mL / min, and the near-infrared fluorescent components were collected using an ultraviolet detector (λ = 780 nm).

[0038] The solid probe was obtained by rotary evaporation concentration and freeze-drying.

[0039] Example 2: A method for preparing a fluorescent probe for glioma detection, comprising the following steps:

[0040] S1: Raw material mixing:

[0041] Take 10.0 mmol of citric acid, 1.0 mmol of CD133 antibody, and 2.0 mmol of RGD peptide and place them in a 50 mL pressure-resistant glass bottle;

[0042] Add 20 mL of dimethyl sulfoxide (DMSO), seal and place in an ultrasonic bath (40 kHz, 300 W) for 45 minutes to mix.

[0043] S2: Polymerizes to form a pH-responsive shell:

[0044] Add 5.0 mmol of dimethylaminoethyl methacrylate monomer to the mixture;

[0045] High-purity nitrogen (99.99%) was introduced for 15 minutes to remove oxygen, and the reaction was carried out at a constant temperature of 60°C with stirring (500 rpm) for 2 hours under a nitrogen atmosphere.

[0046] S3: Hydrothermal reaction synthesis probe:

[0047] Transfer the reaction solution to a 100 mL polytetrafluoroethylene-lined autoclave, with a filling degree of 70%.

[0048] React at 180°C for 12 hours in a forced-air drying oven, then allow to cool naturally to 25°C.

[0049] The precipitate was removed by filtration, yielding a brownish-red crude product solution.

[0050] S4: Purification:

[0051] Silicone columns (200-300 mesh, column size 2.5×40cm) are used;

[0052] Eluent: Methanol / ethyl acetate = 1:4 (v / v);

[0053] The flow rate was 1.0 mL / min, and the near-infrared fluorescent components were collected using an ultraviolet detector (λ = 780 nm).

[0054] The solid probe was obtained by rotary evaporation concentration and freeze-drying.

[0055] Example 3: A method for preparing a fluorescent probe for glioma detection, comprising the following steps:

[0056] S1: Raw material mixing:

[0057] Take 10.0 mmol of citric acid, 1.0 mmol of CD133 antibody, and 3.0 mmol of RGD peptide and place them in a 50 mL pressure-resistant glass bottle;

[0058] Add 20 mL of dimethyl sulfoxide (DMSO), seal and place in an ultrasonic bath (40 kHz, 300 W) for 45 minutes to mix.

[0059] S2: Polymerizes to form a pH-responsive shell:

[0060] Add 5.0 mmol of dimethylaminoethyl methacrylate monomer to the mixture;

[0061] High-purity nitrogen (99.99%) was introduced for 15 minutes to remove oxygen, and the reaction was carried out at a constant temperature of 60°C with stirring (500 rpm) for 2 hours under a nitrogen atmosphere.

[0062] S3: Hydrothermal reaction synthesis probe:

[0063] Transfer the reaction solution to a 100 mL polytetrafluoroethylene-lined autoclave, with a filling degree of 70%.

[0064] React at 220°C for 24 hours in a forced-air drying oven, then allow to cool naturally to 25°C.

[0065] The precipitate was removed by filtration, yielding a brownish-red crude product solution.

[0066] S4: Purification:

[0067] Silicone columns (200-300 mesh, column size 2.5×40cm) are used;

[0068] Eluent: Methanol / ethyl acetate = 1:3 (v / v);

[0069] The flow rate was 1.0 mL / min, and the near-infrared fluorescent components were collected using an ultraviolet detector (λ = 780 nm).

[0070] The solid probe was obtained by rotary evaporation concentration and freeze-drying.

[0071] Application Example 1

[0072] Experimental subjects:

[0073] Positive group: CD133+ / αvβ3+ human glioma stem cells (U87MG cell line)

[0074] Negative group 1: CD133- / αvβ3- normal human astrocytes (HA cell line)

[0075] Negative group 2: CD133+ / αvβ3- hepatocellular carcinoma cells (HepG2 cell line)

[0076] Experimental steps:

[0077] Probe processing:

[0078] The probe prepared in Example 2 was diluted to 5 μM with PBS.

[0079] Co-incubate with three groups of cells (37°C, 2 hours).

[0080] Set the pH gradient: pH 6.0 (simulating lysosomes), pH 7.4 (normal tissue).

[0081] Fluorescence detection:

[0082] Group Fluorescence intensity at pH 6.0 (820nm) pH 7.4 fluorescence intensity U87MG (double positive) 2850±120a.u. 85±10a.u. HA (double negative) 110±15a.u. 70±8a.u. HepG2 (single positive) 420±30a.u. 95±12a.u.

[0083] in conclusion:

[0084] The probe fluorescence intensity is significantly activated (>2000 a.u.) only when both dual receptor binding (CD133+ & αvβ3+) and an acidic environment (pH≤6.5) are simultaneously met, which meets the specificity requirements of in vitro diagnostic reagents (sensitivity 92.3%, specificity 98.1%).

[0085] Application Example 2

[0086] Animal models:

[0087] Experimental group: nude mice with orthotopic glioma (U87MG transplantation, n=6)

[0088] Control group: Healthy nude mice (n=3)

[0089] Imaging process:

[0090] Probe injection:

[0091] Example 2: Probe (2 mg / kg) administered via tail vein injection

[0092] Control group: Injection of unmodified RGD probe (all other parameters the same)

[0093] Live animal imaging (near-infrared imaging system for small animals):

[0094]

[0095] Tissue section verification:

[0096] Brain tissue was collected from euthanized animals and frozen into sections (10 μm thick).

[0097] Tumor area: The probe showed strong red fluorescence in a pH 6.2 microenvironment (excitation / emission: 785 / 820nm).

[0098] Normal brain tissue: No fluorescent signal (pH 7.4)

[0099] in conclusion:

[0100] The probe penetrates the blood-brain barrier through RGD-mediated targeting and dual receptors, specifically enriching at the tumor site (24hT / NT ratio 18.5:1), and activates near-infrared fluorescence in an acidic microenvironment (pH 6.2-6.8), meeting the requirements of in vivo imaging reagents.

[0101] Experimental example:

[0102] Reference group: Probe prepared in Example 2 (citric acid:CD133 antibody:RGD peptide = 10:1:2, synthesized at 180℃ / 12h)

[0103] Comparative design:

[0104] Group Modify parameters Purpose Comparative Example 1 CD133-only modified antibody (without RGD) Verify the necessity of dual targeting Comparative Example 2 pH-insensitive polymer shell Verify the specificity of acidic microenvironment triggering Comparative Example 3 RGD is replaced with a linear peptide (acyclic). Verify blood-brain barrier penetration efficiency

[0105] Experimental protocol

[0106] I. In vitro diagnostic performance testing

[0107] Cell model:

[0108] Positive cells: CD133+ / αvβ3+ U87MG glioma stem cells

[0109] Negative cells: CD133- / αvβ3-HA astrocytes

[0110] Processing conditions:

[0111] Probe concentration: 5 μM

[0112] Environmental pH: 6.5 (simulating tumor lysosomes) vs 7.4 (normal tissue)

[0113] Incubation time: 2 hours

[0114] Testing indicators:

[0115] Fluorescence intensity (820nm excitation / 850nm emission)

[0116] Cell binding rate (flow cytometry)

[0117] II. Live Imaging Performance Test

[0118] Animal models:

[0119] Experimental group: nude mice with U87MG orthotopic glioma (n=5 / group)

[0120] Control group: Healthy nude mice (n=3)

[0121] Dosage regimen:

[0122] Tail vein injection probe (2 mg / kg)

[0123] Imaging time points: 0h, 2h, 12h, 24h

[0124] Testing indicators:

[0125] Tumor / normal tissue fluorescence intensity ratio (T / NT)

[0126] Fluorescence localization of brain tissue sections

[0127] Experimental results

[0128] Table 1: Comparison of in vitro diagnostic performance (fluorescence intensity, au)

[0129] probe group U87MG (pH 6.5) HA (pH 6.5) U87MG (pH 7.4) Example 2 2850±120 110±15 85±10 Comparative Example 1 1050±90 100±12 80±8 Comparative Example 2 2900±130 980±85 900±75 Comparative Example 3 2700±110 120±20 95±12

[0130] Key data:

[0131] Dual-target loss (Comparative Example 1): Tumor fluorescence intensity decreased by 62% (due to loss of αvβ3 binding).

[0132] No pH-responsive shell (Comparative Example 2): Normal cells showed severe false positives (intensity > 900 a.u. at pH 7.4).

[0133] Table 2: Comparison of in vivo imaging performance (24hT / NT ratio)

[0134]

[0135] Key mechanism verification:

[0136] The necessity of dual targeting (Comparative Example 1):

[0137] Tumor enrichment efficiency decreased (T / NT from 18.5 to 5.3) due to the lack of RGD-mediated αvβ3 binding sites.

[0138] Flow cytometry data: Double-positive cell binding rate decreased from 98% to 45%.

[0139] pH-responsive shell value (Comparative Example 2):

[0140] Increased background fluorescence throughout the body (signal intensity in normal tissue reached 47.6% of that in tumor tissue).

[0141] The probe was pre-activated in the blood (fluorescence leakage >800 a.u. at pH 7.4).

[0142] The role of RGD ring structure (Comparative Example 3):

[0143] The blood-brain barrier penetration efficiency decreased by 54% (because linear peptides are easily degraded by proteases).

[0144] Tumor fluorescence peak delay (12h vs. 6h in Example 2)

[0145] Analysis of Experimental Conclusions

[0146]

[0147] Overall benefits:

[0148] The probe of this invention (Example 2) utilizes a dual-targeting-acidic microenvironment dual-locking mechanism:

[0149] The in vitro diagnostic sensitivity reached 92.3% (compared to only 68.7% in Comparative Example 1).

[0150] The signal-to-noise ratio of live imaging is improved by 8.8 times (vs. Comparative Example 2).

[0151] Its blood-brain barrier penetration efficiency is 3.2 times that of clinically commonly used probes (Gd-DTPA).

[0152] The embodiments of the present invention are given for the purposes of illustration and description. Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Any changes, modifications, substitutions and variations made by those skilled in the art to the above embodiments within the scope of the present invention should be included within the protection scope of the present invention.

Claims

1. A fluorescent probe for detecting gliomas, characterized in that, Using carbon quantum dots as the fluorescent matrix, the surface is co-modified with an antibody targeting the glioma stem cell marker CD133 and an RGD peptide targeting αvβ3 integrin, and coated with a pH-responsive polymer shell.

2. The fluorescent probe for glioma detection as described in claim 1, characterized in that, The probe must simultaneously meet the following conditions to activate near-infrared fluorescence: Dual receptor binding: CD133 antibody and RGD peptide bind to tumor cell surface receptors respectively; Acidic microenvironment trigger: The pH of the environment is ≤6.

5.

3. The method for preparing the fluorescent probe according to any one of claims 1-2, characterized in that, Includes the following steps: S1: Dissolve citric acid, CD133 antibody, and RGD peptide in dimethyl sulfoxide at a molar ratio of (10:1:1)-(10:1:3) and mix by sonication; S2: Add dimethylaminoethyl methacrylate monomer and polymerize under nitrogen to form a pH-responsive shell; S3: Transfer the mixture to a high-pressure reactor and react at 160-220℃ for 6-24 hours. Cool to obtain the crude product.

4. The method for preparing the fluorescent probe as described in claim 3, characterized in that, The reaction temperature in S3 is 180℃, and the reaction time is 12 hours.

5. The method for preparing the fluorescent probe as described in claim 3, characterized in that, It also includes the following steps: S4: The crude product was separated by silica gel column chromatography, with the eluent being a mixed solution of methanol and ethyl acetate in a volume ratio of (1:3) to (1:5), and the near-infrared fluorescent component was collected.

6. The application of the fluorescent probe according to any one of claims 1-2, characterized in that, This probe is used to prepare in vitro diagnostic reagents for gliomas. It specifically recognizes tumor cells by binding to two receptors and activates fluorescence in the acidic lysosomal microenvironment.

7. The application of the fluorescent probe according to any one of claims 1-2, characterized in that, This reagent is used to prepare in vivo imaging reagents for gliomas. The probe crosses the blood-brain barrier via RGD peptide, targets and accumulates at the tumor site through dual receptors, and generates near-infrared fluorescence signals in the acidic tumor microenvironment.

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