A mitochondria-targeting polypeptide-drug conjugate, and a preparation method and application thereof

Abstract

The application discloses a kind of mitochondrial targeting polypeptide-drug conjugate and its preparation method and application, belong to the field of biological medicine technology.The antitumor drug lonidamide is covalently connected with mitochondrial penetrating peptide by solid phase synthesis method to obtain drug-polypeptide conjugate, the drug-polypeptide conjugate can target tumor cell mitochondria, and the tumor cell killing activity and solubility are significantly higher than lonidamide.The drug-polypeptide conjugate can self-assemble to form hydrogel, fill in the slow release of tumor resection cavity, efficiently kill residual tumor cells, while reducing the systemic toxic side effects of drug.The polypeptide hydrogel can also be used as a local drug depot, co-loaded with antitumor drugs, synergistic effect, and wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a mitochondrial-targeting polypeptide-drug conjugate, its preparation method, and its application. Background Technology

[0002] Malignant tumors pose a serious threat to human health, with rapid disease progression, high mortality, and low five-year survival rates. Currently, surgical resection is the preferred treatment for malignant solid tumors such as lung cancer, glioma, and breast cancer. However, residual tumor cells after surgery increase the risk of tumor recurrence and metastasis.

[0003] Lonidamine (LND) is a broad-spectrum antitumor drug that selectively acts on the mitochondria of tumor cells, inhibiting glycolysis and interfering with their energy metabolism. It also inhibits mitochondrial complex II, which promotes the generation of reactive oxygen species (ROS), and induces tumor cell apoptosis. LND exhibits inhibitory activity against lung cancer, brain cancer, breast cancer, and prostate cancer, without the adverse reactions of traditional chemotherapy drugs such as bone marrow suppression and hair loss. However, its extremely poor water solubility and low bioavailability limit its clinical use. Therefore, improving the water solubility of lonidamine, increasing its effective concentration at the tumor site, and enhancing its antitumor efficacy are urgent problems to be solved.

[0004] Mitochondrial-penetrating peptides (MPPs) are a class of highly permeable mitochondrial-targeting peptides that target mitochondria via a membrane potential-driven mechanism. Mitochondria play a crucial role in the regulation of tumor growth and metastasis, and using MPPs to specifically deliver mitochondrial-targeting drugs to tumor cell mitochondria is a highly promising tumor suppression strategy.

[0005] Peptide-drug conjugates (PDCs) are conjugates formed by covalently linking peptides to drug molecules. Different functionalizations can be achieved by designing different amino acid sequences. Conjugating mitochondrial-active drugs to MPPs can achieve high cell permeability and mitochondrial localization, thereby increasing the drug concentration within the mitochondria and enhancing its efficacy.

[0006] Amphiphilic drug-derived polymeric substances (PDCs) formed by the coupling of low molecular weight hydrophobic drugs and hydrophilic peptides can self-assemble into supramolecular hydrogels through intermolecular non-covalent interactions. PDC hydrogels exhibit strong shape adaptability and good biocompatibility. A local drug delivery system for PDC hydrogels, injectable into the tumor resection cavity, was constructed to achieve long-term sustained release and accumulation of drugs at the target site, effectively inhibiting and killing residual tumor cells, preventing tumor recurrence, enhancing the therapeutic effect while reducing systemic toxicity.

[0007] However, currently reported PDC hydrogel carriers suffer from drawbacks such as long hydrogel formation time and large dosage requirements. Heating or the addition of strong alkalis like NaOH solution are commonly used to trigger gelation, which affects drug efficacy. Furthermore, traditional PDC hydrogels, as local sustained-release carriers, require additional drug delivery systems, increasing metabolic burden. Self-delivering PDC hydrogels, on the other hand, involve directly modifying drugs with peptides to form amphiphilic PDC molecules, improving drug physicochemical properties and enhancing drug activity. Simultaneously, they can rapidly self-assemble into hydrogels under physiological conditions, achieving local self-delivery and further enhancing efficacy while reducing toxicity, making them of significant research value. Summary of the Invention

[0008] The purpose of this invention is to provide an injectable mitochondrial-targeting peptide-drug conjugate, its preparation method, and its application in the preparation of tumor therapeutic drugs. This peptide-drug conjugate is a chlordamine-mitochondrial-penetrating peptide conjugate, which can self-assemble into a supramolecular hydrogel and can also co-load other antitumor drugs for synergistic effect and toxicity reduction. This peptide-drug conjugate improves the solubility and bioavailability of chlordamine, reduces systemic toxicity, and exhibits short gelation time, small drug dosage, good biocompatibility, injectability, tumor-killing activity, and sustained drug release. The peptide hydrogel formed by the self-assembly of this peptide-drug conjugate can be used in tumor treatment directions such as in situ drug delivery after tumor resection, showing broad application prospects.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] This invention provides a mitochondrial-targeting polypeptide-drug conjugate, the structural formula of which is shown in Formula I:

[0011]

[0012] Ⅰ,

[0013] n = 5, 6, 7, 8 or 9.

[0014] The present invention also provides a polypeptide hydrogel, which is formed by the self-assembly of the above-mentioned polypeptide-drug conjugate.

[0015] Furthermore, the polypeptide hydrogel is composed of the above-mentioned polypeptide-drug conjugate and buffer solution; the preparation method of the polypeptide hydrogel is as follows: dissolve the above-mentioned polypeptide-drug conjugate in buffer solution, mix well, and let stand at room temperature to obtain polypeptide hydrogel.

[0016] The present invention also provides a tumor treatment drug, comprising the above-mentioned polypeptide-drug conjugate or polypeptide hydrogel.

[0017] Furthermore, the tumor therapeutic drug also includes other co-loaded drugs. The preparation method of the tumor therapeutic drug is as follows: the above-mentioned polypeptide-drug conjugate and other co-loaded drugs are dissolved together in a buffer solution, mixed well, and allowed to stand at room temperature to obtain a drug-loaded hydrogel.

[0018] Furthermore, the buffer solution is PBS or other alternative buffer solutions whose components are safe for human use.

[0019] Furthermore, the standing time at room temperature is 10-30 minutes, preferably 15-30 minutes at 37°C.

[0020] The present invention also provides a method for preparing a polypeptide-drug conjugate, wherein the polypeptide-drug conjugate is synthesized by the Fmoc solid-phase method.

[0021] This invention also provides the role of the above-mentioned polypeptide-drug conjugate in the preparation of antitumor drugs.

[0022] Beneficial effects of this invention:

[0023] 1. This invention synthesizes a chlordamine-mitochondrial penetrating peptide conjugate. The conjugate has high solubility, targets tumor cell mitochondria, and exhibits significantly higher penetration and killing activity against tumor cells than chlordamine.

[0024] 2. The chlordamine-mitochondrial penetrating peptide conjugate of the present invention can self-assemble into a hydrogel with a nanofiber structure to achieve localized long-term sustained release;

[0025] 3. The chlordamine-mitochondrial penetrating peptide conjugate of the present invention can co-load other co-loaded drugs to form a drug-loaded hydrogel, achieving local co-release and exerting a synergistic effect;

[0026] 4. The hydrogel of the present invention has a short gelation time, small drug dosage, good biocompatibility, injectability and shape adaptability. It can be used for local precise drug delivery in the tumor resection cavity, increase the drug concentration at the tumor site, significantly reduce systemic toxicity, improve the short-term tumor killing effect, and prolong the long-term tumor recurrence effect. It has great clinical application prospects.

[0027] 5. The hydrogel drug synthesis and preparation process of the present invention is simple and has great potential for industrial-scale production. Attached Figure Description

[0028] Figure 1 This is the mass spectrum of LND-mito;

[0029] Figure 2 This is the high-performance liquid chromatogram of LND-mito;

[0030] Figure 3 This is a TEM image of the LND-mito hydrogel;

[0031] Figure 4 It is the result of the critical micelle concentration (CMC) measurement;

[0032] Figure 5 This is the in vitro release curve of LND-mito hydrogel;

[0033] Figure 6 LND and LND-mito are effective against human alveolar basal epithelial cells A549 in lung cancer. Figure 6 A) Rat glioma cells C6 ( Figure 6 B) Mouse glioma cells GL261 ( Figure 6 C) and human astrocytoma U87 ( Figure 6 D) Graph showing the results of in vitro toxicity tests;

[0034] Figure 7 This is a schematic diagram of mitochondrial targeting verification using a mixture of LND-mito (n=5) and pyrene-mito under a laser scanning confocal microscope. Detailed Implementation

[0035] A peptide-drug conjugate, with the structural formula shown in Formula I, was obtained by covalently linking the antitumor drug chlordamine to a mitochondrial-penetrating peptide using a solid-phase synthesis method. This peptide-drug conjugate exhibits high solubility, targets tumor cell mitochondria, and demonstrates significantly superior killing effects against tumor cells (such as rat glioma cells C6, mouse glioma cells GL261, lung cancer cells A549, and human astrocytoma U87) compared to chlordamine. When prepared at room temperature, the peptide hydrogel exhibits suitable gelation time and a flowing liquid state, thus possessing good injectability and shape adaptability, making it suitable for precise local drug delivery within tumor resection cavities. The peptide-drug conjugate self-assembles into a hydrogel upon standing at room temperature, allowing for slow release by injection into the tumor resection cavity, providing long-term killing of residual tumor cells and reducing systemic drug toxicity. Furthermore, the peptide hydrogel can serve as a local drug reservoir, co-loading antitumor drugs for synergistic local co-release.

[0036] In this invention, LND-mito represents structural formula I.

[0037] In this invention, unless otherwise specified, polypeptide hydrogel refers to LND-mito hydrogel. The invention is further illustrated below with reference to the accompanying drawings and specific embodiments. These embodiments should be understood as illustrative only and not as limiting the scope of protection of the invention. After reading the contents of this invention, those skilled in the art can make various modifications or alterations to the invention, and these equivalent changes and modifications also fall within the scope defined by the claims of this invention. Experimental methods and reagents not specified in the embodiments are performed according to conventional conditions in the art.

[0038] Example 1: Fmoc-solid-phase synthesis of LND-mito peptide-drug conjugate

[0039] Using a solid-phase synthesis tube as the reaction vessel, the specific operating steps are as follows:

[0040] 1. Resin Treatment: Rink Amide-AM Resin was swollen with dichloromethane (DCM) and dried after swelling. 20% piperidine was added, and the mixture was placed in a constant-temperature shaking incubator at 35 °C for 5 min to remove the Fmoc protecting groups from the resin. The resin was then washed three times each with N,N-dimethylformamide (DMF), DCM, and DMF sequentially. The ninhydrin test was positive.

[0041] 2. Amino Acid Coupling: Fmoc-protected lysine Fmoc-Lys-OH and 1-hydroxybenzotriazole (HOBt) were dissolved in DMF, and N,N-diisopropylcarbodiimide (DIC) was added. The mixture was then placed in a gas bath constant-temperature shaking incubator and activated at 35 °C for 5-10 min. The mixture was added to a solid-phase synthesis tube and reacted with the resin in a gas bath constant-temperature shaking incubator at 60 °C for 20 min. After the reaction, the resin was washed with DMF, DCM, and DMF again. A negative ninhydrin test indicated that all sites on the resin had reacted. 20% piperidine was added, and the mixture was reacted in a gas bath constant-temperature shaking incubator at 35 °C for 5 min. This process was repeated once to remove the Fmoc protecting group from the amino acid. The resin was washed three times each with DMF, DCM, and DMF. Following the reaction method for Fmoc-Lys-OH, the amino acids were sequentially coupled according to the Fmoc-Lys-OH reaction. D RFKF D RFK sequence joining.

[0042] 3. Alkyl chain coupling: Connect 6-aminohexanoic acid, 7-aminoheptanoic acid, 8-aminononanoic acid, 9-aminooctanoic acid or 10-aminononanoic acid according to the above amino acid coupling steps, that is, connect an alkyl chain with n=5-9.

[0043] 4. LND Coupling: After the alkyl chain reaction is complete, the Fmoc group is removed using 20% ​​piperidine. LND and HOBt are dissolved in DMF, DIC is added, and the mixture is placed in a gas bath constant temperature shaking incubator and activated at 35 °C for 5-10 min. The mixture is then added to a solid-phase synthesis tube and reacted overnight. The resin is washed with DMF, DCM, and DMF again; a negative result for ninhydrin testing indicates that all sites on the resin have reacted.

[0044] 5. Resin removal: Remove the Fmoc protecting group on the last amino acid of the polypeptide sequence using 20% ​​piperidine. Transfer the resin from the solid-phase synthesis tube to a sealing tube, add 20 mL of trifluoroacetic acid / phenol / water / triisopropylsilane = 88 / 5 / 5 / 2 (volume ratio), and place in a gas bath constant temperature shaking incubator at 35 °C for 3 h before filtering.

[0045] 6. Post-processing: Trifluoroacetic acid was purged with argon gas, cold diethyl ether was added and shaken well, centrifuged, and the supernatant was discarded. This process was repeated 3 times. The diethyl ether was evaporated, and the mixture was freeze-dried under vacuum for 24 h to obtain the LND-mito peptide-drug conjugate, which was stored at -20°C.

[0046] Results Analysis: The theoretical exact molecular weights of LND-mito (n=5-9) were 1737.94, 1751.98, 1766.00, 1780.03, and 1794.05, respectively. The actual ESI-MS values ​​were 1738.22, 1752.40, 1767.35, 1780.84, and 1794.13, respectively, which are consistent with the target molecular weights, proving that the synthesis of LND-mito compounds was correct. Figure 1 HPLC results showed that the retention times of the LND-mito (n=5-9) compounds were 13.655 min, 16.621 min, 16.910 min, 17.294 min, and 17.683 min, respectively. The purity of all compounds was above 98%. Figure 2 ).

[0047] Example 2: Preparation of polypeptide hydrogels

[0048] Peptide hydrogel (using 145 mM LND-mito (n=5) as an example): Weigh 2 mg of LND-mito peptide powder and dissolve it in 8 µL of pH 7.4 PBS. Mix well and let stand at room temperature for 10-30 minutes to form a gel, thus obtaining the peptide hydrogel. Specifically, let it stand at 37°C for 15 minutes to form a gel.

[0049] Example 3: Preparation of polypeptide hydrogels

[0050] Peptide hydrogel (using 145 mM LND-mito (n=5) as an example): Weigh 2 mg of LND-mito peptide powder and dissolve it in 8 µL of physiological saline at pH 6.8. Mix well and let stand at room temperature for 10-30 minutes to form a gel, thus obtaining the peptide hydrogel. Specifically, let it stand at 37°C for 15 minutes to form a gel.

[0051] In addition to PBS and physiological saline, other alternative buffer solutions with safe components for human use can also be used to prepare the polypeptide hydrogels of this invention.

[0052] Example 4: Microstructure characterization of peptide hydrogels

[0053] 4 mg of LND-mito peptide powder was dissolved in 16 µL of pH 7.4 PBS and mixed thoroughly to prepare a 145 mL LND-mito solution. 10 µL of this LND-mito solution was added dropwise onto a carbon-coated copper grid. Excess liquid was absorbed with filter paper, and the solution was allowed to stand for 15 min to solidify. Subsequently, 10 µL of uranium acetate was added for staining, and the solution was allowed to stand for 30 s. Microscopic morphology was observed using a transmission electron microscope (TEM). Figure 3 As shown, LND-mito (n=5-9) self-assembles to form a hydrogel, and a dense fibrous network structure can be observed.

[0054] Example 5: Determination of Critical Micelle Concentration (CMC)

[0055] Following Example 2, LND-mito peptide hydrogels with gradient concentrations were prepared, with a maximum concentration of 6 mM. Hydrophobic pyrene was used as a fluorescent probe to determine the critical micelle concentration of the self-assembled LND-mito. Using a fluorescence spectrophotometer, the ratio of the peak intensities of the first vibrational band (370-373 nm) and the third vibrational band (381-384 nm) of pyrene (I1 / I3) was plotted against the logarithm of the LND-mito solution concentration (LogC). The intersection point represents the critical micelle concentration. Experimental results are as follows: Figure 4 As shown in Table 1, the CMC is between 460 and 630 µM when using LND-mito (n=5-9).

[0056] Table 1. CMC values ​​of LND-mito (n=5-9)

[0057]

[0058] Example 6: In vitro release experiment

[0059] 15 µL of a 145 mM LND-mito peptide hydrogel was prepared according to Example 2. Then, 400 µL of release medium (PBS, pH 7.4) was added to the surface of the peptide hydrogel, and the gel was placed in a constant temperature shaker at 37 ℃ and 100 rpm. The upper layer of release medium was removed on days 1, 3, 4, 5, 6, 8, 9, 10, 11, 13, 15, 16, 17, 19, 21, 23, 25, 26, 28, 31, 34, 39, and 41, and 400 µL of PBS was added to the surface of the hydrogel. The LND-mito content in each sample was determined using high-performance liquid chromatography (HPLC), the cumulative release rate was calculated, and release curves were plotted. The cumulative release curves are shown below. Figure 5 As shown. By Figure 5 It can be seen that the cumulative release rate of LND-mito (n=5-9) within 40 days is between 35% and 65%, and it has good stability and sustained-release ability under physiological conditions.

[0060] Example 7: In vitro antitumor experiment

[0061] The specific experimental steps are as follows:

[0062] (1) Human alveolar basal epithelial cells A549 (lung cancer), human astrocytoma cells U87 (brain astrocytoma), rat glioma cells C6 (rat glioma), and mouse glioma cells GL261 (mouse glioma) were seeded into 96-well plates, with approximately 4 × 10⁶ cells per well. 3 Each well contains 100 μL and is incubated overnight at 37°C in a 5% CO2 incubator.

[0063] (2) After the cells were cultured overnight, 100 μL of LND solution and LND-mito solution of different concentrations were added to each well. After culturing for 24 h at 37 °C in a 5% carbon dioxide incubator, 10 μL of CCK8 reagent was added to each well. The absorbance value was measured at 490 nm using an ELISA reader, and the measurement was repeated three times in parallel.

[0064] Different culture media are used for different cell types. Specifically, in step (1), the culture medium for A549 cells is F12K medium containing 10% FBS and 1% penicillin-dextrose antibody; the culture medium for U87 cells and GL261 cells is DMEM medium containing 10% FBS and 1% penicillin-dextrose antibody; and the culture medium for C6 cells is medium containing 15% horse serum, 2.5% FBS, and 82.5% F12K. Of course, this invention does not exclude other culture media, as long as they can meet the purpose of cell culture.

[0065] Specifically, in step (2), the concentration gradient of the LND solution is 15-1920 µM. The preparation method is as follows: weigh a certain amount of LND powder and dissolve it in an appropriate amount of PBS with pH 7.4, and mix them evenly.

[0066] Specifically, in step (2), the concentration gradient of the LND-mito solution is 7.5-240 µM, and the preparation method is as follows: weigh a certain amount of LND-mito peptide powder and dissolve it in an appropriate amount of PBS with pH 7.4, and mix it evenly.

[0067] Experimental results are as follows Figure 6 As shown, within the concentration range, all experimental groups exhibited concentration-dependent cytotoxic effects. LND-mito showed significantly higher cytotoxicity against tumor cells than LND.

[0068] Calculations showed that the IC50 values ​​of LND-mito and LND on different tumor cells were [values ​​to be filled in]. 50 The values ​​are shown in Table 2. When the treatment time is 24 hours, the IC50 values ​​of LND-mito on different tumor cells are... 50 The values ​​were all significantly lower than those of LND, indicating that LND-mito, which is linked to the mitochondrial penetrating peptide, significantly enhanced the killing effect on tumor cells.

[0069] Table 2. Effects of LND and LND-mito (n=5-9) on the IC50 of different tumor cells 50 value

[0070]

[0071] Example 7: In vitro mitochondrial targeting verification experiment

[0072] To verify mitochondrial targeting using laser confocal microscopy, the LND in the LND-mito structure was replaced with the fluorescent group pyrene to synthesize pyrene-mito. Pyrene-mito was added to the LND-mito solution at a ratio of 50:1. GL261 cells in logarithmic growth phase were used at a concentration of 2 × 10⁻⁶ cells / year. 5 Cells / well were seeded at a density equal to that of laser confocal microscopy and incubated at 37°C for 24 h. A high-glucose DMEM medium solution with 30 µM LND-mito (n=5) and 0.6 μM pyrene-mito was prepared in the dark. The cells were incubated in this medium for 24 h, then the medium was discarded. The cells were washed with PBS, and then stained with the MitoTracker Red CMXRos (100 nM) probe at 37°C in the dark for 15 min. After rinsing twice with PBS, the cells were kept moist with PBS and imaged using a laser scanning confocal microscope. Results are shown below. Figure 7 As shown, Mitotracker Red is localized to mitochondria. The blue fluorescence localization of pyrene-mito indicates that pyrene-mito can be well taken up by cells and co-localizes with mitochondria. This demonstrates that the polypeptide sequence has good mitochondrial targeting, and the drug-peptide conjugate exhibits good mitochondrial targeting.

Claims

1. A mitochondrial-targeting polypeptide-drug conjugate, the structural formula of which is shown in Formula I: Ⅰ, n = 5, 6, 7, 8 or 9.

2. A polypeptide hydrogel, formed by self-assembly of the polypeptide-drug conjugate of claim 1.

3. The polypeptide hydrogel according to claim 2, characterized in that, The polypeptide hydrogel is composed of a polypeptide-drug conjugate and a buffer solution.

4. A tumor treatment drug, characterized in that, The tumor treatment drug includes the polypeptide-drug conjugate as described in claim 1 or the polypeptide hydrogel as described in any one of claims 2-3.

5. The tumor treatment drug according to claim 4, characterized in that, It also includes other co-loaded drugs.

6. A method for preparing a polypeptide hydrogel, characterized in that, The preparation method is as follows: dissolve the polypeptide-drug conjugate of claim 1 in a buffer solution, mix well, and let stand at room temperature to obtain the polypeptide hydrogel.

7. A method for preparing a tumor therapeutic drug, characterized in that, The preparation method is as follows: the polypeptide-drug conjugate of claim 1 and other co-loaded drugs are dissolved in a buffer solution, mixed well, and allowed to stand at room temperature to obtain the drug-loaded hydrogel.

8. The method according to claim 6, characterized in that, The buffer solution is PBS or physiological saline.

9. The method according to claim 7, characterized in that, The buffer solution is PBS.

10. The method according to claim 6 or 7, characterized in that, The settling time at room temperature is 10-30 minutes.

11. The method for preparing the polypeptide-drug conjugate according to claim 1, characterized in that, The peptide-drug conjugate was synthesized using the Fmoc solid-phase method.

12. The role of the polypeptide-drug conjugate of claim 1 in the preparation of an antitumor drug for treating lung cancer, brain cancer, breast cancer, or prostate cancer.

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