A subcellular organelle active targeting imaging probe and a preparation method thereof

Abstract

The application belongs to the technical field of biological diagnosis, and relates to a subcellular organelle active targeting imaging probe and a preparation method thereof. The subcellular organelle active targeting imaging probe of the application is formed into nanoparticles (NPs) by coupling a fluorescent imaging probe with a special sequence of bombesin peptide and a derivative thereof; on one hand, the NPs can be surface-functionalized with lipophilic cations related to the NPs; on the other hand, the NPs can be passively targeted to tumor sites, and have high permeability and retention effect for solid tumors.

Description

Technical Field

[0001] This invention belongs to the field of biodiagnostic technology and relates to a subcellular organelle active targeting imaging probe and its preparation method. Background Technology

[0002] Studies have shown that gastrin-releasing peptide (GRPR), a growth factor, is abnormally expressed in various tumor tissues and acts on its specific receptor (GRPR) through autonomous secretion, abnormal secretion, and neuroendocrine mechanisms, thereby affecting multiple signal transduction pathways and directly stimulating tumor growth. GRPR expression is closely related to the nature, type, differentiation degree, grade, and invasiveness of the tumor. GRPR activation in both normal and tumor tissues has significant growth effects, and its expression is more prevalent in malignant tumors than in normal human tissues. It is abnormally or excessively expressed in various human cancer cells, including small cell lung cancer, non-small cell lung cancer, prostate cancer, breast cancer, glioblastoma, and lung cancer. Therefore, GRPR has become a popular tumor marker for the early diagnosis and treatment of cancer.

[0003] Currently, most imaging probes used in clinical practice are small molecule probes. These fluorescent probes are designed to be highly sensitive and specific to biomarkers. The main problem they face is that they are not very efficient at reaching the disease site and can cause unavoidable side effects to other organs. They are also difficult to locate subcellular organelles. It is evident that the complexity of tumors makes the development of highly sensitive tumor imaging probes a challenging task. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned problems in the existing technology by proposing a subcellular organelle active targeting imaging probe. This probe is directed to the target cell by utilizing molecular affinity such as receptor ligands, and accumulates in the subcellular organelle site by utilizing the charge properties and lipophilicity of bufotin and its derivatives, thereby achieving subcellular organelle targeting.

[0005] The objective of this invention can be achieved through the following technical solution: a subcellular organelle active targeting imaging probe, the imaging probe comprising an active targeting peptide and an imaging probe group connected in sequence, wherein the active targeting peptide is bufotin and its derivatives.

[0006] In the above-mentioned subcellular organelle active targeting imaging probe, bufotoxin and its derivatives are at least one of SEQ ID No. 1-17.

[0007] Preferably, bufotoxin and its derivatives are at least one of SEQ ID No. 1-7.

[0008] In the above-mentioned subcellular organelle active targeting imaging probe, the imaging probe group is selected from at least one of fluorescent probes, CT probes, radionuclide probes, and magnetic resonance imaging probes.

[0009] Preferably, the fluorescent probe is selected from at least one of FITC, Cy3, Cy5, Cy3.5, Cy5.5, Cy7, Cy7.5, ICG, IR-780, IR-783, IR775, IR-792, IR-808, IR-820, IR-825, IR-1048, IR-1064 and their derivatives.

[0010] Preferably, the CT probe is selected from at least one of iopamidol, iohexol, iopentol, iopromide, iodoxane, iodoxane, iodixanol, and iodomeprazole.

[0011] Preferably, the nuclide probe is selected from at least one of 18F, 99mTc, 131I, 133Xe, 32P, 3H, 109Cd, 111Zn, 141Ce, 153Sm, and 198Au.

[0012] Preferably, the magnetic resonance imaging probe is selected from at least one of gadodiamine, disodium gadoxetate, gadopentetate dimeglumine, gadodiamine and sodium carbides, gadotate dimeglumine, gadotetrol, and iron tetroxide.

[0013] In the aforementioned subcellular organelle active targeting imaging probe, a linker is included between the active targeting peptide and the imaging probe group. The subcellular organelle active targeting imaging probe of the present invention may include any linker between the targeting peptide and the fluorescent probe, or may not include a linker.

[0014] In the above-mentioned active targeting imaging probe for subcellular organelles, the subcellular organelles include at least one of mitochondria, lysosomes, and endoplasmic reticulum.

[0015] Mitochondria are double-membrane subcellular organelles located in the cytoplasm of eukaryotic cells. They consist of four parts: the outer mitochondrial membrane, the inner mitochondrial membrane, the intermembrane space, and the mitochondrial matrix. Proton pumps located within the inner mitochondrial membrane pump protons from the mitochondrial matrix into the intermembrane space, resulting in a large positive charge in the intermembrane space and a large negative charge in the mitochondrial matrix. This creates the mitochondrial transmembrane potential (MTP), which spans the inner mitochondrial membrane. The MTP is negative inside and positive outside. In normal cells, the MTP value is 130–150 millivolts, while it is increased in tumor cells. Therefore, when a positively charged agent enters a cell, it is targeted to the mitochondria under the drive of high MTP in tumor cells. The short peptide portion of the subcellular organelle active targeting probe of the present invention is derived from bufotoxin and its derivatives. The present invention makes a series of substitutions, additions and subtractions to its amino acid sequence, retaining the positive charge and lipophilicity of the short peptide sequence, so that after it enters tumor cells by targeting GRPR, it can be targeted to the mitochondria under the drive of high MTP in tumor cells.

[0016] Lysosomes are dynamic systems composed of acidic vesicles (pH ≈ 4.8) that receive substances from the plasma membrane or cytoplasm through endocytosis and autophagy, and then degrade and recycle them. Lysosomes can also digest localized cytoplasm or organelles within the cell itself. When cells age, their lysosomes rupture, releasing hydrolytic enzymes that digest the entire cell, leading to cell death. In tumor cells, these degradation pathways of lysosomes are unrestricted, making them more susceptible to exogenous or endogenous stimuli that trigger lysosomal membrane permeation (LMP). The positively charged bufotin and its derivatives coupled with a fluorescent imaging probe of this invention interact with the cell membrane through strong electrostatic attraction, causing cell membrane depolarization or the creation of hydrophilic membrane pores, ultimately altering plasma membrane permeability and leading to lysosomal accumulation.

[0017] The endoplasmic reticulum (ER), a crucial membranous organelle in eukaryotic cells, is the primary site for protein folding and transport, lipid synthesis, vesicle transport, and calcium ion storage, and participates in the regulation of various signal transduction pathways. Furthermore, through its well-developed extended structures, the ER establishes connection sites with other membranous structures such as the plasma membrane, mitochondria, endosomes, and lysosomes, playing a vital role in the exchange of information, matter, and energy between intracellular organelles and the plasma membrane. This invention utilizes positively charged bufotin and its derivatives coupled with fluorescent imaging probes that enter the cell via selective endocytosis (such as foveolar protein-mediated endocytosis) and directly and passively target the ER through intracellular transport.

[0018] The present invention also provides a method for preparing the above-mentioned subcellular organelle active targeting imaging probe, the method comprising the following steps:

[0019] S1. Resin 1 is obtained by coupling an active targeting peptide onto resin 0 using an automated peptide synthesizer.

[0020] S2. Add imaging groups to resin 1 and react to obtain resin 2;

[0021] S3. Resin 2 is cut to remove the amino acid side chain protection, and the purified product is a subcellular organelle active targeting multifunctional imaging probe.

[0022] The present invention also provides an application of the above-mentioned subcellular organelle active targeting imaging probe in the preparation of antitumor drugs.

[0023] As a preferred application, it is used in the preparation of drugs for breast cancer, prostate cancer, colorectal cancer, kidney cancer, and ovarian cancer.

[0024] The present invention also provides an imaging agent comprising the above-described subcellular organelle active targeting imaging probe.

[0025] The present invention also provides an application of the above-mentioned imaging agent in the preparation of drugs for diagnosing tumor diseases.

[0026] As a preferred application, it is used in the preparation of drugs for diagnosing breast cancer, prostate cancer, colorectal cancer, kidney cancer, and ovarian cancer.

[0027] Compared with the prior art, the present invention has the following beneficial effects: The subcellular organelle active targeting imaging probe of the present invention uses a special sequence of bufotoxin and its derivatives coupled with a fluorescent imaging probe to form nanoparticles (NPs). On the one hand, it can actively target the surface functionalization of lipophilic cations involved in NPs. On the other hand, it can passively target tumor sites and has a high permeability and retention effect for solid tumors.

[0028] Among them, the amino acid sequence fragment of SEQ ID No. 1 is MLHGVAWQ;

[0029] The amino acid sequence fragment of SEQ ID No. 2 is MHQLGVAW;

[0030] The amino acid sequence fragment of SEQ ID No. 3 is HQMGVALW;

[0031] The amino acid sequence fragment of SEQ ID No. 4 is HHHGVAWQ;

[0032] The amino acid sequence fragment of SEQ ID No. 5 is RRRGVAWQ;

[0033] The amino acid sequence fragment of SEQ ID No. 6 is MLHGVARWQR;

[0034] The amino acid sequence fragment of SEQ ID No. 7 is MLRGVARWQR;

[0035] The amino acid sequence fragment of SEQ ID No. 8 is MLHGVAWQN;

[0036] The amino acid sequence fragment of SEQ ID No. 9 is WLVMAHQ;

[0037] The amino acid sequence fragment of SEQ ID No. 10 is MLHGVAWQNGLRQE;

[0038] The amino acid sequence fragment of SEQ ID No. 11 is XXMLHGVAWQ, where X stands for Cha, Nle, and so on.

[0039] The amino acid sequence fragment of SEQ ID No. 12 is MLHGVAWQNGLKQE;

[0040] The amino acid sequence fragment of SEQ ID No. 13 is XFHAVAWQY, where X stands for Nle;

[0041] The amino acid sequence fragment of SEQ ID No. 14 is MLHGVAWQN;

[0042] The amino acid sequence fragment of SEQ ID No. 15 is MLHGVAWHNGRPYMKTLVTGGGAPLPV;

[0043] The amino acid sequence fragment of SEQ ID No. 16 is MFHGTAWLNGRSHVRIKSARSRPEPLDWSLPA;

[0044] The amino acid sequence fragment of SEQ ID No. 17 is MLHGVAWHNG. Attached Figure Description

[0045] Figure 1 The mass spectrum of the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1.

[0046] Figure 2 The UV-Vis absorption spectrum of the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1.

[0047] Figure 3 The particle size distribution of the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1.

[0048] Figure 4 Laser confocal imaging results of human breast cancer cells using the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1.

[0049] Figure 5 The results show the average fluorescence intensity of 10,000 individual breast cancer cells examined using the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1. Detailed Implementation

[0050] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0051] Example 1:

[0052] S1. Configure active targeting peptides and imaging groups according to Table 1;

[0053] S2. Based on the first amino acid at the C-terminus of the polypeptide sequence, select 0.5 mmol of the corresponding Fmoc protected amino acid - WangResin, add it to the solid-phase reactor, add DCM swelling resin for 30 min, dry it, wash it three times with DMF, add a 20% hexahydropyridine DMF solution, react for 5 min, add another 20% hexahydropyridine DMF solution and react for 10 min, wash with DMF once in between, dry it after the reaction is finished, and wash it with DMF three times.

[0054] S3. Following the sequence of the peptide from C-terminus to N-terminus, condensation and Fmoc removal reactions were carried out alternately in a reaction amount of 1.5 mmol each time, until all the remaining amino acids of the peptide were condensed onto the resin. After the last amino acid was condensed, a 20% (v / v) solution of hexahydropyridine (DMF) was added, and the reaction was carried out for 5 min. Then, another 20% (v / v) solution of hexahydropyridine (DMF) was added, and the reaction was carried out for 10 min. The DMF was washed once in between. After the reaction was completed, the solution was dried and washed with DMF three times.

[0055] S4. Add 1 mmol of FITC and N-methylmorpholine to the reactor and react for 5-10 min. Check whether the reaction is complete with ninhydrin. After the reaction is complete, wash three times alternately with DMF and DMC, and then wash with methanol to shrink and obtain dried polypeptide-resin.

[0056] S5. Place the peptide-resin in a round-bottom flask and slowly add the prepared lysis buffer (the reagent formula for the lysis buffer has a volume ratio of TFA: benzyl sulfide: phenol: triisopropylsilane: water = 82.5: 7.5: 5: 3: 2) at 0°C. Stir slowly and react at low temperature for 0.5 h, then at room temperature for 2 h. Filter to obtain the lysis buffer. Slowly add the lysis buffer to anhydrous ice-cold ether and stir. Filter to separate the crude peptide. Wash the crude peptide three times with ice-cold ether to obtain the crude peptide.

[0057] S6. The molecular weight of the crude product was determined by mass spectrometry, and finally purified and separated by high performance liquid chromatography and lyophilized to obtain pure peptides.

[0058] Table 1: Results of active-targeting peptide and imaging group configuration in Example 1

[0059]

[0060] The particle size of the prepared probe compound sample was measured, and the specific steps are as follows:

[0061] The prepared probe compound sample was dissolved in PBS at a concentration of 10 μmol and tested using a nanoparticle size analyzer.

[0062] Table 2: Particle size test results of the probe compound samples prepared in Example 1

[0063]

[0064] Figure 1 The mass spectrum of the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1 is shown. The structure of FITC-Acp-MLRGVARWQR was determined.

[0065] Figure 2 The image shows the UV-Vis absorption spectrum of the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1. The image indicates that the short peptide MLRGVARWQR was successfully linked to FITC.

[0066] Figure 3 The particle size distribution of the FITC-Acp-MLRGVARWQR imaging probe prepared in Example 1 is shown in the figure. As can be seen from the figure, the compound has a uniform particle size of 146 nanometers.

[0067] The prepared probe compound sample FITC-Acp-MLRGVARWQR was used for subcellular organelle imaging experiments on human breast cancer cells. The specific steps are as follows:

[0068] Fifty thousand MCF-7 cells were cultured in glass-bottomed dishes and incubated overnight at 37°C with 5% CO2. After washing with PBS, the cells were incubated with 20 μmol of the probe compound sample at 37°C with 5% CO2 for 1 h. After rinsing three times with PBS, the cells were fixed with 4% paraformaldehyde for 30 min. Following this, the cells were rinsed three times with PBS and stained with 200 nmol Mito-Tracker Red CMXRos for 30 min, then rinsed three times with PBS and stained with 10 μg / ml hoechst 33342 for 30 min. Finally, after rinsing three times with PBS, the cells were imaged using a confocal microscope. Figure 4It can be seen that the fluorescence distribution of the probe compound at the same time showed different degrees of colocalization with the fluorescence of mitochondria, lysosomes, and endoplasmic reticulum, indicating that the probe compound has a targeting effect with mitochondria, lysosomes, and endoplasmic reticulum, respectively.

[0069] The prepared probe compound sample FITC-Acp-MLRGVARWQR was subjected to an uptake experiment in human breast cancer cells. The specific steps are as follows:

[0070] 400,000 MCF-7 human breast cancer cells were seeded in 6-well plates and incubated overnight at 37°C with 5% CO2. Cells were then incubated with a probe sample prepared at a concentration of 20 μmol for 1 hour, followed by cell collection. The cells were washed three times with PBS and resuspended. The mean fluorescence intensity of 10,000 human breast cancer cells was examined using a flow cytometer (BD FACSCalibur, USA), with excitation at 488 nm and signal collection at 520 nm. Results are as follows: Figure 5 As shown, the fluorescence intensity in the cells reached about 5000 after 1 hour, compared with the fluorescence intensity of about 2000 for conventional probes, which fully demonstrates that the probe of the present invention was effectively taken up by the cells.

[0071] In summary, the subcellular organelle active targeting imaging probe of the present invention uses a special sequence of bufotin and its derivatives coupled with a fluorescent imaging probe to form nanoparticles (NPs). On the one hand, it can actively target the surface functionalization of lipophilic cations involved in NPs. On the other hand, it can passively target tumor sites and has a high permeability and retention effect on solid tumors.

[0072] The embodiments herein cover any points not exhaustively within the scope of the technical claims of this invention, as well as new technical solutions formed by equivalent substitutions of one or more technical features in the embodiments. These are all within the scope of the claims of this invention. Furthermore, in all listed or unlisted embodiments of this invention, each parameter in the same embodiment merely represents an instance (i.e., a feasible solution) of its technical solution, and there is no strict coordination or limitation relationship between the parameters. The parameters can be substituted for each other without violating axioms and the claims of this invention, unless otherwise stated.

[0073] The technical means disclosed in this invention are not limited to those described above, but also include technical solutions composed of any combination of the above technical features. The above descriptions are specific embodiments of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

[0074] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A subcellular organelle active targeting imaging probe, characterized in that, The imaging probe comprises an active targeting peptide and an imaging probe group connected in sequence, wherein the active targeting peptide is bufotin and its derivatives; Bufotenoids and their derivatives are MLRGVARWQR, as shown in SEQ ID No. 7; The imaging probe group is FITC; A linker is present between the active targeting peptide and the imaging probe group; Subcellular organelles include at least one of mitochondria, lysosomes, and endoplasmic reticulum; The imaging probe has a diameter of 146 nm.

2. The method for preparing a subcellular organelle active targeting imaging probe according to claim 1, characterized in that, The method includes the following steps: S1. Resin 1 is obtained by coupling an active targeting peptide onto resin 0 using an automated peptide synthesizer. S2. Add imaging groups to resin 1 and react to obtain resin 2; S3. Resin 2 is cut to remove the amino acid side chain protection, and the purified product is a subcellular organelle active targeting multifunctional imaging probe.

3. The application of a subcellular organelle active targeting imaging probe as described in claim 1 in the preparation of antitumor drugs.

4. An imaging agent, characterized in that, The imaging agent includes the subcellular organelle active targeting imaging probe of claim 1.

5. The application of the imaging agent according to claim 4 in the preparation of drugs for diagnosing tumor diseases.

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