Preparation method and application of bifunctional fluorescent probe for marking drug conjugate

Abstract

The invention belongs to the field of biological medicine, and provides a preparation method and application of a fluorescent probe molecule. The synthesized fluorescent probe molecule can be combined with an intramolecular fluorescence quenching principle to realize real-time monitoring of drug molecule release of the polypeptide drug conjugate in serum, and can also realize tracing of polypeptide in the polypeptide drug conjugate at a cellular level and monitoring of drug molecule release and dynamic distribution. The comprehensive visual research on the hybrid peptide is realized. The preparation method of the fluorescent probe is simple and easy to implement. The method provides an effective visual tool molecule for research and development of the polypeptide drug conjugate. The monitoring method disclosed by the invention is relatively simple and convenient to operate, good in monitoring result stability, strong in monitoring capability and mild in condition, provides an efficient tool molecule for development of the polypeptide drug conjugate, and has relatively high practical application value.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the preparation of a fluorescent probe based on intramolecular quenching and visualization and its application in peptide conjugates. Background Technology

[0002] Camptothecin (CPT) is a small-molecule natural alkaloid that exerts its effective antitumor effect by inhibiting DNA topoisomerase I (TOP I). CPT has a rigid backbone structure composed of five rings, which leads to problems such as low water solubility and poor stability of the lactone ring. Furthermore, severe toxic side effects and significant individual variability limit the clinical application of CPT. Peptide drug conjugates based on the tumor-targeting peptide camptothecin are an effective way to improve the pharmacokinetic properties of camptothecin-based chemotherapy drugs.

[0003] Peptides are protein fragments composed of fewer than 50 amino acids, characterized by high selectivity, strong penetration, and low immunogenicity. Preparing peptide-drug conjugates (PDCs) through covalent conjugation of peptides with small-molecule antitumor drugs is an effective method to improve the antitumor activity of small-molecule drugs. Tracing and detecting PDC-type hybrid peptides released from drugs can effectively promote the development and research of this class of drugs. Intrinsically quenched fluorescent (IQF) is a fluorescent probe technique developed based on the principle of fluorescence resonance energy transfer. When the emission spectrum of a donor fluorescent molecule overlaps with the absorption spectrum of a acceptor molecule, and the distance between the two molecules is appropriate, the fluorescence energy of the donor molecule is absorbed by the acceptor molecule. Specifically, when the acceptor molecule is a non-fluorescent molecule, fluorescence will be absorbed, resulting in the IQF phenomenon.

[0004] Based on this, this invention, for the first time, combines the IQF principle to design and synthesize a hybrid peptide based on stapled peptide-CPT coupled fluorescent molecules, which is used for real-time detection of CPT-based PDC drug release while realizing the visual monitoring of the hybrid peptide. Summary of the Invention

[0005] The purpose of this invention is to provide a fluorescent hybrid peptide that enables real-time monitoring of CPT release in PDC while simultaneously achieving visual monitoring of the hybrid peptide using fluorescence detection.

[0006] To achieve the objective of this invention, the following technical solution is adopted:

[0007] In a first aspect, the present invention provides a fluorescent tool molecule for visualizing peptide-conjugated drugs. The tool molecule includes dual tracer functions of IQF and covalently coupled fluorescent molecules on the peptide.

[0008] In the fluorescent hybrid peptide provided by this invention, the small molecule drug is preferably CPT and its derivatives. The polypeptide is preferably a membrane-cleaving stapler peptide A4K14-Citropin 1.1, which exhibits high protease stability and antitumor activity. The small molecule drug is coupled to the polypeptide via a linker, preferably 2-(2-(2-aminoethoxy)ethoxy)acetic acid (AEEA), and an IQF combination is formed by introducing a quenching group, preferably 2,4-dinitrophenol (Dnp). Simultaneously, a fluorescent group, preferably Rhodamine B (RhB), is introduced onto the polypeptide to achieve fluorescence monitoring of the hybrid peptide. This hybrid peptide is named 855 in this invention, and its structural formula is as follows:

[0009]

[0010] R1: A luminescent drug molecule, preferably CPT and its derivatives; R2: A fluorescence quencher group, preferably Dnp; R3: A fluorescent molecule; R4: A linker, preferably AEEA; R5: A polypeptide, specifically a stapling peptide with membrane-breaking function provided by this invention, the abbreviated amino acid sequence of which is... K is the abbreviation for the amino acid form of lysine, including L or D-type lysine.

[0011] Secondly, the method for preparing the polypeptide-conjugated drug of the present invention includes synthesizing the polypeptide by a polypeptide solid-phase synthesis method, and then conjugating a small molecule drug and a fluorescent group to the polypeptide, preferably through the following steps:

[0012] (1) RinkAmide AM resin is preferred for solid-phase synthesis.

[0013] (2) The preferred catalysts are the HCTU / DIEA condensation system and the HATU / HOAt / DIEA condensation system.

[0014] (3) The preferred peptide cleavage reagent is a mixed solution of trifluoroacetic acid / phenol / deionized water / triisopropylsilane with a volume ratio of 88:5:5:2.

[0015] (4) CPT modified with succinic acid, a small molecule drug, is preferred, and the coupling method is preferably the HATU / HOAt / DIEA condensation reaction system.

[0016] (5) Dnp is the preferred quenching agent, and the HATU / HOAt / DIEA condensation system is the preferred coupling method.

[0017] (6) RhB is the preferred fluorescent molecule, and the HCTU / DIEA condensation system is the preferred coupling method.

[0018] Thirdly, the present invention provides molecular tracing and application of the above-mentioned hybrid peptide molecules in anti-tumor activity.

[0019] (1) Monitoring the release of small molecule drugs at serum and cellular levels. The hybrid peptide was incubated with serum or tumor cells at 37°C, and samples were collected at different time points. The fluorescence signal of CPT was monitored by a fluorescence spectrophotometer or ELISA reader. Due to the release of the drug, the covalent coupling of IQF was destroyed, and the quenching group Dnp could not absorb the fluorescence of CPT. Therefore, the release of the small molecule antitumor drug CPT in the hybrid peptide can be evaluated based on the fluorescence intensity of the released CPT.

[0020] (2) Achieve visualized monitoring of hybrid peptides at the cellular level. HepG2 tumor adherent cells were selected, and the hybrid peptides were co-incubated with the cells. At different time points, the fluorescence of the fluorescent molecule in the hybrid peptides, preferably rhodamine, was traced using a fluorescence microscope to achieve tracking of the peptides within the hybrid peptides. Simultaneously, fluorescence monitoring was performed using free CPT to achieve real-time monitoring of drug release and distribution.

[0021] The beneficial effects of this invention are as follows: the strategy provides comprehensive monitoring capabilities for PDC drug molecules, and the synthesis method is relatively mature. It possesses the ability to monitor the release of small molecule drugs in real time while also monitoring peptides, providing an effective visualization tool molecule for the development of PDC drugs. The monitoring method of this invention is simple to operate, and the monitoring results are stable and highly reproducible, providing an efficient tool molecule for PDC drug development. Attached Figure Description

[0022] Figure 1 The structure, primary amino acid sequence, reversed-phase high-performance liquid chromatography (RP-HPLC) chromatogram, and ESI-MS mass spectrometry of the fluorescent hybrid peptide 855 of this invention are shown.

[0023] Figure 2 The fluorescence intensity curves of the fluorescent hybrid peptide 855 of the present invention at the levels of A) serum and B) tumor cells, λex = 366 nm, λem = 440 nm, and C) the corresponding CPT release.

[0024] Figure 3This is a dynamic distribution tracer of the fluorescent hybrid peptide 855 at the cellular level. A) Fluorescence images (10×) of hybrid peptide 855, a non-covalent mixture (Dnp / CPT / RhB / peptide), and a blank control group in HepG2 cells; B) and C) Bar charts showing the quantitative analysis of RhB and CPT fluorescence signals in the cell nucleus, respectively. ***P<0.001, **P<0.01; D) Pearson correlation coefficients between RhB and CPT signals in the hybrid peptide and the nuclear dye SYBR Green I, respectively.

[0025] Specific real-time methods

[0026] To better illustrate the present invention, the following embodiments are provided:

[0027] Example 1: The preparation method of a real-time monitoring multifunctional fluorescent molecular probe is achieved through the following steps:

[0028] RinkAmide AM resin was preferred as the carrier for solid-phase peptide synthesis, and the target peptide was synthesized using a solid-phase peptide synthesis method based on the 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group.

[0029] Resin pretreatment: The resin was washed alternately with N,N-dimethylformamide (DMF) and dichloromethane (DCM). DMF was added to allow swelling at room temperature for 1-2 hours. Then, a DMF / DCM mixed solvent (v:v = 4:1) was added to the peptide synthesis tube, and the tube was placed in a constant-temperature shaker with the shaking parameters set to 28℃ and 120 rpm for 30-60 minutes to activate the resin. The Fmoc protecting group was removed using a 20% piperidine DMF solution. Finally, a Kaiser colorimetric reaction was performed to monitor whether the Fmoc groups were completely removed.

[0030] Condensation reactions of amino acids in solid-phase peptide synthesis: The reactant ratio for ordinary amino acid condensation was Fmoc-amino acid:HCTU:DIEA = 3 eq: 2.8 eq: 6 eq, with two condensation reaction times of 40 min and 60 min, respectively. For Fmoc-(S)-2-(4-pentenyl)alanine (Fomc-(S)-2-(4-pentenyl)alanine, S5) and AEEA, a HATU / HOAt / DIEA condensation reaction system was used (Fmoc-amino acid:HATU:HOAt:DIEA = 1.5 eq: 1.3 eq: 1.5 eq: 3 eq, Fmoc-AEEA:HATU:HOAt:DIEA = 2 eq: 1.8 eq: 2 eq: 4 eq), with two condensation reaction times of 60 / 80 min and 60 / 120 min, respectively. The synthesized peptide chains were labeled as 1 eq and 2.5 eq. The first-generation Grubb catalyst was dissolved in anhydrous dichloroethane, added to the peptide synthesis tube, and shaken at 28°C for 4 h to achieve site-directed cyclization of the intramolecular side chains of the peptides on the resin.

[0031] Introduction of fluorescent group (RhB), quencher group (Dnp), and small molecule drug (CPT): Lysine containing an Alloc special protecting group and lysine containing a Dnp side chain protecting group were sequentially coupled to the cyclized polypeptide chain at a condensation ratio of Fmoc-amino acid:HCTU:DIEA = 3 eq:2.8 eq:6 eq, with two condensation reactions taking 50 min and 60 min respectively. Then, a condensation reaction was carried out with RhB to remove the Alloc special protecting group from the lysine side chain. The lysine was then dissolved in DMF solvent at a ratio of RhB:HCTU:DIEA = 4 eq:3.7 eq:8 eq, and a two-step reaction was performed (70 min, 90 min), maintaining a constant temperature of 28 °C throughout. Finally, the coupling of CPT with the main chain was completed using the HATU / HOAt / DIEA condensation reaction system. The reactant ratio was CPT-COOH:HATU:HOAt:DIEA = 2eq:1.8eq:2:eq:4eq, and the reaction times were 70 min and 90 min, respectively.

[0032] Crude peptide shearing and precipitation: First, prepare 5 mL of peptide cleavage reagent at a volume ratio of 88:5:5:2 for trifluoroacetic acid (TFA):phenol:deionized water:triisopropylsilane. Place the reagent in an insulated box with crushed ice for at least 20 minutes to lower the temperature. Then, wash the target peptide with a standard solution followed by three DCM washes. Filter the resin using a water pump and an oil pump for approximately 5 minutes each to ensure thorough drying. Add the chilled peptide cleavage reagent to the peptide synthesis tube and immediately place it in a constant-temperature shaker for 2.5-3 hours. After the peptide cleavage reaction is complete, transfer the reaction solution to a clean three-necked round-bottom flask. Connect the flask necks with three sealed rubber tubes, introducing high-purity nitrogen gas through the middle neck to expel volatile components such as TFA from both ends. Finally, add pre-cooled anhydrous ether to the concentrate and sonicate to dissolve any residues on the flask walls. The mixture was transferred to a pre-weighed 50 mL centrifuge tube and centrifuged at 3000 rpm for 3 min at room temperature, with the centrifugation repeated three times. The product obtained after centrifugation was dried under ventilation to remove residual ether solvent.

[0033] Lyophilization and purification of crude peptides: The crude peptide solution was reconstituted by sonication using an acetonitrile / water mixture containing 0.08-0.1% TFA. 1 mL of the crude peptide solution was retained for analysis by reversed-phase high-performance liquid chromatography (RP-HPLC) and positive ion electrospray ionization mass spectrometry (ESI-MS). The remaining solution was lyophilized using a vacuum freeze dryer to obtain the crude peptide product. The obtained product was dissolved in an acetonitrile solution containing 0.1% TFA, followed by the addition of an aqueous solution containing 0.1% TFA. The solution was sonicated until a particle-free and transparent crude peptide solution was obtained. This solution was then filtered through a 0.22 μm microporous membrane before injection. The target peptide purified by RP-HPLC was lyophilized, and the final sample was stored at -20°C.

[0034] Figure 1 The images show the structural formula, amino acid sequence, and RP-HPLC chromatogram of the synthesized 855 molecules. The ESI-MS mass spectrometry results indicate that the molecular weight of the synthesized peptide is consistent with that of the target peptide, further verifying the accuracy of the synthesized peptide.

[0035] Example 2: Detection of the ability of hybrid peptides to inhibit tumor cell growth

[0036] HepG2 and HeLa tumor cells in logarithmic growth phase were seeded into 96-well plates at a density of 5000 cells per well and incubated overnight in a cell culture incubator. 50 μL of basal medium with peptide concentrations of 0.3, 1, 3, 10, 30, and 100 μM were added to each well, and incubation was continued for 72 h. After incubation, 15 μL of MTT (5 mg / mL) was added to each well, and incubation was continued for 4 h. The supernatant was removed, and 150 μL of dimethyl sulfoxide (DMSO) was added to each well. After incubation for 1 h, absorbance was measured at 492 nm using a microplate reader.

[0037] The IC50 of AEEA covalently coupled binder peptide in HepG2 and Hela tumor cells 50 The values ​​were 4.1±0.1 and 3.3±0.4 μM, respectively, for the 855 IC. 50 The values ​​were 3.1 ± 0.2 and 0.9 ± 0.2 μM, respectively. After 72 h of IC50... 50 The results showed that the inhibitory effect of 855 on HepG2 and Hela cancer cells was comparable to that of the original peptide, indicating that the modification based on the bifunctional fluorescent molecule did not reduce the inhibitory effect of the original peptide on tumor cell proliferation.

[0038] Example 3: CPT release assay of hybrid peptides at cellular and serum levels

[0039] To monitor CPT release in real time, this invention constructs a "turm on" type fluorescent probe hybrid peptide 855 based on the IQF principle. 855 suppresses the intrinsic fluorescence of CPT by covalently linking the fluorescence quencher group Dnp to CPT; when esterase hydrolysis triggers ester bond cleavage, free CPT is released from 855 accompanied by fluorescence recovery, thereby achieving real-time monitoring of the released CPT.

[0040] Specifically, the release of CPT from peptide 855 in tumor cells was studied as follows: Tumor cells in the logarithmic growth phase, preferably HeLa cells, were used at a density of 5 × 10⁶ cells per well. 3 Cells were seeded at a density of [number] cells per well in a 96-well plate. 100 μL of DMEM medium containing 10% fetal bovine serum was added, and the plate was incubated overnight at 37°C with 5% CO2 to ensure proper adhesion. Subsequently, 5 μM of sample (855, non-covalent mixture of CPT / Dnp / RhB, DMSO) was added to each well in the experimental groups. At nine pre-defined time points (0, 2, 4, 8, 12, 24, 36, 48, 60, and 72 h), the intensity of intracellular CPT characteristic fluorescence signals was measured using a multi-mode microplate reader under specific spectral parameters (excitation wavelength 366 nm, emission wavelength 440 nm). To ensure data reliability, three replicates were performed for each experiment, and all procedures were completed under light-protected conditions to eliminate photodegradation interference.

[0041] In addition, the release of CPT from peptide 855 at human serum levels was investigated: First, 5% human serum solution was uniformly mixed with peptide 855 and CPT+Dnp+RhB mixed solution to prepare reference systems with a molar concentration of 35 μM for each component. Subsequently, dynamic monitoring was performed at 14 time points (0, 2, 4, 8, 12, 24, 36, 48, 60, 72, 96, 120, 132 and 144 h) within the range of 0-144 h: 50 μL of reaction solution was aspirated into a pre-cooled centrifuge tube for each sampling. 20 μL of acetonitrile (containing 0.1% TFA) was added, the tube was shaken three times, and the mixture was allowed to stand on ice for 2 min. Then, 30 μL of acetonitrile (containing 0.1% TFA) and 250 μL of water (containing 0.1% TFA) were added, the tube was shaken three times, and the mixture was immediately transferred to -20℃ for temporary storage. After sampling at all time points, the samples were thawed on ice and centrifuged at 12,000 rpm for 5 min at 4°C. The supernatant was collected and transferred to pre-cooled sample tubes. The characteristic fluorescence intensity of CPT was detected using an ELISA reader at an excitation wavelength of 366 nm and an emission wavelength of 440 nm. All residual samples after detection were sealed and stored at -80°C for subsequent validation experiments.

[0042] The results are as follows Figure 2 As shown, in human serum ( Figure 2 A) and tumor cells ( Figure 2 In (B), the fluorescence intensity of 855 increased with prolonged incubation time over 144 hours, eventually reaching a stable plateau. Quantitative analysis showed that after 144 hours of incubation, the cumulative release rate of CPT in tumor cells was 40.6%, while the serum CPT release rate reached as high as 63.8%. Figure 2 C).

[0043] Example 4: Tracing and monitoring of PDCs drug release at the cellular level

[0044] HepG2 adherent cells were selected. Logarithmic growth phase HepG2 cell suspension was evenly seeded into 24-well plates, with 1 mL of cell suspension added to each well (approximately 2.5 × 10⁶ cells per well). 4(Number of cells). The well plates were incubated overnight at 37°C in a 5% CO2 incubator. Experiments began after cell attachment. Serum-free medium containing 5 μM peptide 855, 5 μM CPT / Dnp / RhB / peptide non-covalent mixture, and DMSO (solvent control) was added to the well plates, with three replicates for each group. Incubation was performed at 0 h, 2 h, 24 h, 48 h, and 72 h. Before detection, 1×SYBR Green I working solution was added to each well, and incubation was performed for approximately 15 min in the dark, followed by washing the cells twice. After incubation at each time point, intracellular fluorescence distribution was observed using a confocal microscope. SYBR Green I: excitation wavelength 497 nm, emission wavelength 520 nm, showing green fluorescence, used for labeling cell nuclei. CPT: excitation wavelength 366 nm, emission wavelength 440 nm (CPT release monitoring, showing blue fluorescence). RhB: Excitation wavelength 560nm, emission wavelength 580nm (peptide localization signal, displayed as red fluorescence).

[0045] Figure 3 A showed that the characteristic red fluorescence signal of RhB in group 855 was significantly higher than that in the non-covalently mixed group, and both groups showed a time-dependent increase. Quantitative analysis of red-green fluorescence intensity (…) Figure 3 B) showed that the intracellular accumulation rate of 855 reached 34.3% at 4 h, while only 4.0% of free RhB entered the cells in the non-covalent mixture group; within 72 h, up to 94.1% of the hybrid peptide 855 accumulated intracellularly and emitted strong red fluorescence, while only 31.6% of non-covalent free RhB entered the cells. The red-green fluorescence ratio in the covalent group was significantly higher than that in the non-covalent mixture group. Simultaneously, analysis of the colocalization coefficient with the cell nucleus (…) Figure 3 D) It was found that 855 showed a co-localization level of 0.4 with the cell nucleus after 4 hours of culture, and this coefficient increased to over 0.9 by 72 hours. These data indicate that the hybrid peptide can be effectively taken up by cells and accumulate in the cell nucleus in a short period of time.

[0046] Regarding CPT release and dynamic real-time monitoring, 855 showed characteristic blue fluorescence of CPT (blue-green fluorescence ratio of 3.4%) after 4 hours of incubation in HeLa cells. With prolonged incubation, the blue fluorescence significantly and continuously increased, reaching a prominent blue fluorescence level of 72.3% at 72 hours, indicating that 855 can rapidly penetrate the cell membrane and effectively release CPT. Figure 3 C). Colocalization coefficients with nuclear dyes indicate that released CPT primarily accumulates around the cell nucleus. Figure 3 D). In the non-covalently mixed group, almost no blue fluorescence was observed throughout the process, and the blue-green fluorescence ratio tracer was less than 1%, indicating that when monomeric CPT and peptide are non-covalently mixed, the ability of CPT to enter cells cannot be promoted, further proving the necessity of covalent coupling.

[0047] In summary, this probe reveals that when peptides, RhB, and CPT form a covalent complex, it significantly accelerates the entry of CPT and RhB into cells, leading to the release of CPT within the cell, its aggregation in the cell nucleus, and the full exertion of CPT's antitumor activity. This invention is the first to successfully design a dual monitoring system for simultaneous hybrid peptide imaging and real-time monitoring of the release and dynamic distribution of covalently coupled small molecule drugs, providing an effective tool molecule for the development of PDC (proton pump inhibitor) drugs.

Claims

1. The preparation and application of a fluorescent molecular probe based on intramolecular quenching and visualization of peptide drug conjugates, characterized in that, The combination of intramolecular quenching probes enables real-time monitoring of the release of small molecule drugs in peptide drug conjugates at the serum level. In addition, it enables tracing of peptides in peptide drug conjugates at the cellular level, as well as monitoring of the release and dynamic distribution of drug molecules, thus achieving comprehensive monitoring of peptide drug conjugates.

2. The fluorescent molecule based on intramolecular quenching and visualization of peptide drug conjugates according to claim 1 comprises the following components: R1-K(R2)-K(R3)-R4-R5 R1 is a luminescent drug molecule, preferably CPT and its derivatives; R2 is a fluorescence quencher group, preferably Dnp; R3 is a fluorescent molecule; R4 is a linker, preferably AEEA; R5 is a polypeptide, preferably a membrane-permeable stapled peptide, with the abbreviated amino acid sequence as follows: K is the abbreviation for lysine.

3. The method for preparing the fluorescent molecular probe as described in claim 1, characterized in that... The preparation method includes: synthesizing a polypeptide using a solid-phase polypeptide synthesis method, and condensing and linking the polypeptide with a linking group, a fluorescent group, a fluorescence quenching group, and a drug molecule.

4. The application of the fluorescent molecular probe according to claim 1, preferably, is for the release of drug molecules in peptide drug conjugates at cellular and serum levels; for in vivo and in vitro fluorescence imaging of peptide drug conjugate molecules; and for the tracing of covalently conjugated small molecule drugs.

5. A pharmaceutical composition, characterized in that, It includes the use of the fluorescent probe molecule of claim 1 in a medicament for the prevention and / or treatment of tumor-related diseases.

6. A pharmaceutical preparation, characterized in that, It comprises the fluorescent probe molecule of claim 1 and pharmaceutically acceptable excipients; Preferably, the excipients include diluents, fillers, disintegrants, surfactants, suspending agents, binders, lubricants, colorants, and flavorings.

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