Fluorescent probe and preparation method thereof
阅读说明：本技术 一种荧光探针及其制备方法 (Fluorescent probe and preparation method thereof ) 是由 卞旺青 卢宇靖 龙威 何燕 张焜 张智 陈泽丰 王亚坤 陈霓平 黄艺斌 于 2020-07-03 设计创作，主要内容包括：本发明涉及生物医药技术领域,尤其涉及一种荧光探针及其制备方法。本发明公开了一种荧光探针,包括：式(I)所示的化合物和转铁蛋白；式(I)所示的化合物与转铁蛋白通过氨基和羧基的缩合反应进行结合。本发明提供的荧光探针优选以转铁蛋白作为转运配体,式(I)所示的化合物作为成像剂,将转铁蛋白与式(I)所示的化合物进行自组装,得到以全铁的转运蛋白作为载体偶联小分子的荧光探针,该荧光探针可以通过配体-受体的介导与血脑屏障和脑胶质瘤上过度表达的受体TFR结合,实现成像剂有效地靶向输送,从而到达脑胶质瘤组织进行荧光成像,可以对脑胶质瘤患者进行早期的精确诊断以及术后的跟踪。(The invention relates to the technical field of biological medicines, in particular to a fluorescent probe and a preparation method thereof. The invention discloses a fluorescent probe, which comprises: a compound of formula (I) and transferrin; the compound shown in the formula (I) is combined with transferrin through condensation reaction of amino and carboxyl. The fluorescent probe provided by the invention preferably takes transferrin as a transport ligand, a compound shown in a formula (I) as an imaging agent, and the transferrin and the compound shown in the formula (I) are self-assembled to obtain the fluorescent probe which takes the transport protein of total iron as a carrier and couples small molecules.)
1. A fluorescent probe, comprising: a compound of formula (I) and transferrin;
the compound shown in the formula (I) is connected with the transferrin through a peptide bond;
wherein R is2Selected from aliphatic or aromatic carboxylic acids, R3Is halogenated C2-C10 straight-chain alkyl.
2. The fluorescent probe of claim 1, wherein the transferrin is HOLO-iron transferrin, HOLO-TF.
3. The fluorescent probe according to claim 1, wherein the compound of formula (I) is prepared by a method comprising the steps of:
reacting a compound shown in a formula (II) with a compound shown in a formula (III) to obtain a compound shown in a formula (I);
wherein R is1Is methyl, R2Selected from aliphatic or aromatic carboxylic acids, R3The halogen-containing linear alkyl is C2-10 halogen-containing linear alkyl.
4. The fluorescent probe of claim 3, wherein R is2Selected from C3-C8 straight chain carboxylic acid or aromatic carboxylic acid with 1-3 benzene rings, R3Is halogenated C4-C8 straight-chain alkyl.
5. The fluorescent probe according to claim 3, wherein the molar ratio of the compound represented by the formula (II) to the compound represented by the formula (III) is (1: 1-1.2).
6. The fluorescent probe according to claim 3, wherein the reaction temperature is room temperature and the reaction time is 18-26 h.
7. A preparation method of a fluorescent probe is characterized by comprising the following steps:
step 1: carrying out coupling reaction on a compound shown as a formula (I) and transferrin to obtain a fluorescent probe;
wherein R is2Selected from aliphatic or aromatic carboxylic acids, R3The halogen-containing linear alkyl is C2-10 halogen-containing linear alkyl.
8. The preparation method according to claim 7, wherein the temperature of the coupling reaction is 4 ℃ and the time is 36-54 h.
9. The production method according to claim 7, wherein the mass ratio of the compound represented by the formula (I) to the transferrin is (1: 10) to (6: 10).
10. The method according to claim 7, further comprising, after the coupling reaction and before obtaining the fluorescent probe:
and dialyzing the product obtained by the coupling reaction to remove free small molecules.
Technical Field
The invention relates to the technical field of biological medicines, in particular to a fluorescent probe and a preparation method thereof.
Background
Glioma is a tumor derived from neuroepithelium, accounts for 40% -50% of craniocerebral tumors, is the most common intracranial malignant tumor, has an annual incidence rate of 3-8 persons/10 ten thousand of people, and has 14000 new increasing cases. The age groups of 10-20 years and 40-50 years are at the peak stage of onset of disease, the treatment means is mainly surgical treatment and assisted by radiotherapy and chemotherapy, and the treatment effect is limited. Therefore, in order to accurately and timely treat patients with glioma, a better means for advanced diagnosis and a better method for tracking after operation are needed. However, glioma mainly occurs in the neuroectoderm of the brain, and is relatively special in position, and most of the existing fluorescent probes cannot directly penetrate through the Blood Brain Barrier (BBB) to enter the brain, so that the diagnosis accuracy is affected.
Disclosure of Invention
The invention provides a fluorescent probe and a preparation method thereof, and solves the problem that the accuracy of diagnosis is affected because the existing fluorescent probe cannot directly penetrate through a blood brain barrier to enter the brain.
The specific technical scheme is as follows:
the present invention provides a fluorescent probe comprising: a compound of formula (I) and transferrin;
the compound shown in the formula (I) is connected with the transferrin through a peptide bond;
wherein R is2Selected from aliphatic carboxylic acid or aromatic carboxylic acid, preferably C3-C8 straight chain carboxylic acid or aromatic carboxylic acid with 1-3 benzene rings, R3The linear alkyl is halogenated C2-10, preferably halogenated C4-8.
It should be noted that human Transferrin (TF) is a single-chain glycoprotein consisting of 679 amino acid residues, contains 19 disulfide bonds, and has a molecular weight of 79 KD. In addition, transferrin receptor is expressed in various cells, but is expressed more abundantly in BBB under tumor cells and pathological conditions, and TFR can mediate TF to realize unidirectional transport of transferrin from peripheral blood to brain tissue through endocytosis, thereby crossing BBB. HOLO-iron transferrin (HOLO-TF), one of the most important members of the Tf family, contains two iron atoms and has a higher affinity for transferrin receptor TFR, which is overexpressed in glioma tissues, than iron-free TF (APO-TF) and monoiron TF, and thus HOLO-TF can be used as a specific ligand for tumor-targeted imaging and therapy. The fluorescent probe provided by the invention preferably takes transferrin as a transport ligand, a compound (organic micromolecule PROP) shown in a formula (I) as an imaging agent, TF and PROP are subjected to self-assembly to obtain the fluorescent probe (TPP) which takes the transport protein of total iron as a carrier and couples with micromolecules, the fluorescent probe can penetrate through a blood brain barrier through mediation of a ligand-receptor, can be actively targeted and positioned to a brain glioma region, is specifically combined with the transferrin receptor on the glioma region, and can emit red fluorescence in the glioma region under irradiation of near infrared light, so that accurate positioning of brain glioma is realized. In the present invention, the wavelength of the near infrared light is preferably 650-750 nm.
HOLO-iron transferrin (HOLO-TF) is one of the most important members of the Tf family, contains two iron atoms, has higher affinity with transferrin receptor TFR overexpressed in glioma tissues than iron-free TF (APO-TF) and monoiron TF, and thus, HOLO-TF can be preferably used as a specific ligand for tumor-targeted imaging and therapy.
In the invention, the preparation method of the compound shown in the formula (I) comprises the following steps:
reacting a compound shown in a formula (II) with a compound shown in a formula (III) to obtain a compound shown in a formula (I);
wherein R is1Is methyl, R2Selected from aliphatic carboxylic acid or aromatic carboxylic acid, preferably C3-C6 straight chain carboxylic acid or aromatic carboxylic acid with 1-3 benzene rings, R3The linear alkyl is halogenated C2-10, preferably halogenated C4-8.
In the present invention, the molar ratio of the compound represented by the formula (II) to the compound represented by the formula (III) is (1: 1 to 1.2), and preferably 1: 1; the reaction temperature is room temperature, the reaction time is 18-26 h, and preferably 24 h.
The process for producing the compound represented by the formula (II) in
the preparation method of the compound represented by the formula (III) is preferably: reacting 2-methylthiobenzothiazole with 1, 4-dibromobutane under a catalyst to obtain a compound shown in a formula (III); the catalyst is preferably triethylamine, the solvent for the reaction is preferably DMF, and the reaction is preferably carried out at room temperature for 12 h.
In the invention, the room temperature is 25 +/-5 ℃.
The invention also provides a preparation method of the fluorescent probe, which comprises the following steps:
step 1: carrying out coupling reaction on a compound shown as a formula (I) and transferrin to obtain a fluorescent probe;
wherein R is2Selected from aliphatic carboxylic acid or aromatic carboxylic acid, preferably C3-C6 straight chain carboxylic acid or aromatic carboxylic acid with 1-3 benzene rings, R3The linear alkyl is halogenated C2-10, preferably halogenated C4-8.
In the present invention, the mass ratio of the compound represented by the formula (I) to the transferrin is (1: 10) to (6: 10), and is preferably 3: 10.
in the invention, the temperature of the coupling reaction is 4 ℃, the time is 36-54 h, and the reaction is preferably carried out for 48h at 4 ℃;
after the coupling reaction, the method further comprises the following steps: dialyzing a product obtained by the coupling reaction to remove free small molecules to obtain a fluorescent probe;
the dialysis time is 24-48 h, preferably 36 h.
According to the technical scheme, the invention has the following advantages:
the present invention provides a fluorescent probe comprising: a compound of formula (I) and transferrin; the compound shown in the formula (I) is combined with transferrin through intermolecular hydrogen and hydrophobic interaction.
The fluorescent probe provided by the invention preferably takes transferrin as a transport ligand, a compound shown in a formula (I) as an imaging agent, and the transferrin and the compound shown in the formula (I) are self-assembled to obtain the fluorescent probe which takes the transport protein of total iron as a carrier and couples with micromolecules. Due to the endogenesis of transferrin, the biocompatibility and biodegradability of the fluorescent probe are good; because the PROP is absorbed in a near infrared region, compared with X rays, ultraviolet light and the like, the near infrared has better tissue penetration capacity, so that the depth of the fluorescent probe penetrating through the tissue is considerable; the low toxicity of the fluorescent probe minimizes the risk to the body, thereby ensuring that the detection is performed without any concern for any side effects on the body.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to these drawings without inventive exercise.
FIG. 1 is a nuclear magnetic resonance hydrogen spectrum of Compound 3 in example 1 of the present invention;
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum of Compound 5 in example 1 of the present invention;
FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of Compound 6 in example 1 of the present invention
FIG. 4 is a NMR spectrum of Compound 6 in example 1 of the present invention;
FIG. 5 is a graph showing the results of the cell viability of U87 cells incubated with PROP and TPP measured by the MTT method in example 3 of the present invention;
FIG. 6 is an image of confocal laser scanning cells of U87 cells in example 4 of the present invention, wherein (a) is an image of fluorescent probes of TPP, (b) is a cell stain of DAPI, (c) Merge, and (d) is a bright field image of TPP.
Detailed Description
The embodiment of the invention provides a fluorescent probe and a preparation method thereof, which are used for solving the problem that the accuracy of diagnosis is influenced because the conventional fluorescent probe cannot directly penetrate through a blood brain barrier to enter a brain.
In order to make the objects, features and advantages of the present invention more obvious and understandable, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it should be apparent that the embodiments described below are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Glioma cell U87 cells in the examples of the present invention were provided by the institute of biomedicine and medicine, Guangdong university of Industrial science.
Other raw materials and reagents used in the examples of the present invention were all commercially available.