CN114533894A - Self-assembly polypeptide medicine with mitochondrion targeting function and preparation method and application thereof - Google Patents

Self-assembly polypeptide medicine with mitochondrion targeting function and preparation method and application thereof Download PDF

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CN114533894A
CN114533894A CN202210190346.7A CN202210190346A CN114533894A CN 114533894 A CN114533894 A CN 114533894A CN 202210190346 A CN202210190346 A CN 202210190346A CN 114533894 A CN114533894 A CN 114533894A
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polypeptide
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王浩
宋本利
乔增莹
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Zhengzhou University
National Center for Nanosccience and Technology China
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Zhengzhou University
National Center for Nanosccience and Technology China
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Abstract

The invention relates to a self-assembly polypeptide drug with a mitochondrial targeting function, a preparation method and an application thereof. The self-assembly polypeptide drug can form nanoparticles through self-assembly under the hydrophobic effect in the body fluid circulation process to be passively targeted to tumor cells and target mitochondria, as the mitochondria in the tumor cells can generate a large amount of Reactive Oxygen Species (ROS), the nanoparticles can further form fibers by virtue of the environment of endogenous ROS, the synergistic effect of the fibers and multivalent bonds of the mitochondria is enhanced, so that the interaction sites of the nanoparticles and the mitochondria are more, the binding force with the mitochondria is firmer, and the nanoparticles are subsequently damaged by activating a sonosensitizer through exogenous Ultrasound (US) to generate singlet oxygen, thereby inducing the apoptosis of the tumor cells.

Description

Self-assembly polypeptide medicine with mitochondrion targeting function and preparation method and application thereof
Technical Field
The invention belongs to the technical field of biomedical medicines, relates to a self-assembled polypeptide medicine with a mitochondrion targeting function, and a preparation method and application thereof, and particularly relates to a self-assembled polypeptide medicine with a mitochondrion targeting function for precise treatment of acoustodynamic mediated tumor, and a preparation method and application thereof.
Background
Cancer is one of three major diseases threatening human health, and has been receiving more and more attention in recent years. How to rapidly and accurately kill cancer cells and completely cure cancers becomes important content of research in the medical field. Research shows that tumor cells have vigorous metabolism and strong reproductive capacity, and various physicochemical properties of the tumor microenvironment are different from those of the normal in vivo environment, such as hypoxia, low pH, abnormal blood vessels and the like. The characteristics of the tumor microenvironment are beneficial to the proliferation, invasion, adhesion and angiogenesis of the tumor on one hand, and promote the formation of malignant tumor; meanwhile, the method also becomes an important basis for distinguishing tumor tissues from healthy tissues, and a new idea is provided for specific diagnosis and treatment of tumors.
The current clinical main means for treating cancer include surgery, chemotherapy, radiotherapy, immunotherapy, etc. However, since the microscopic lesions are invisible to the naked eye in the surgical treatment, it is difficult to distinguish between normal cells and tumor cells, and it is difficult to achieve complete tumor resection with the risk of recurrence and metastasis; chemotherapeutic drugs, such as cisplatin, adriamycin and paclitaxel, are common star drugs and have good therapeutic effect on middle and late stage cancers. However, chemotherapy drugs cannot effectively distinguish between normal cells and cancer cells, and their treatment is accompanied by damage to normal cells and susceptibility to drug resistance, and mucositis, alopecia, bone marrow suppression and severe gastrointestinal adverse reactions occurring during chemotherapy limit their current applications. Therefore, the key to treat cancer is to improve the targeting of the medicament and reduce the toxic and side effects of the medicament.
The nano material has the advantages of small size, low immunotoxicity, easy synthesis and the like, and through different polypeptide material designs, the nano material with a certain particle size is gathered in a tumor area through passive targeting in the in vivo circulation process, so that the tumor can be accurately treated.
The acoustic dynamic therapy has the advantages of small damage, high selectivity, strong penetrating power (more than 10cm) and the like, and is widely concerned in the field of tumor therapy. At present, the sonodynamic therapy of tumors has a plurality of mechanisms, wherein the theory of free radicals is widely accepted and applied to practical therapy, and is an important research hotspot in various medical fields in recent years. For example, sonodynamic therapy utilizes a porphyrin sensitizer to generate active compounds such as superoxide radical and the like under ultrasonic stimulation, can realize tumor tissue killing, and has the advantages of local precision, no wound, low side effect and the like. The development of nanotechnology brings new ideas and changes to tumor treatment. At present, the ROS response type nano delivery system mainly loads a drug in a carrier, and realizes controlled release of the drug after entering a tumor tissue.
However, some toxicity is often generated during drug delivery due to stability and other factors. Thus, there is still a need for improvement in drug delivery system design.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a self-assembled polypeptide medicament with a mitochondrion targeting function and a preparation method and application thereof, and particularly provides a self-assembled polypeptide medicament with the mitochondrion targeting function for precise sonodynamic mediated tumor treatment and a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a self-assembled polypeptide drug with a mitochondrial targeting function, wherein the self-assembled polypeptide drug with a mitochondrial targeting function comprises a mitochondrial targeting unit, a self-assembled unit, and an acoustodynamic response unit, which are sequentially connected.
The self-assembled polypeptide drug can form nanoparticles through self-assembly under the hydrophobic effect in the circulation process of body fluid to passively target tumor cells and target mitochondria, because the nanoparticles are self-assembledMitochondria in tumor cells can generate a large amount of Reactive Oxygen Species (ROS), the nano-particles can further form fibers by virtue of the environment of endogenous ROS, the synergistic effect of the fibers and multivalent bonds of the fibers and the mitochondria is enhanced, so that the sites of action of the fibers and the mitochondria are more, the bonding force with the mitochondria is firmer, and subsequently, exogenous Ultrasound (US) is used for activating a sound-sensitive agent to generate singlet oxygen to damage the mitochondria, thereby inducing the apoptosis of the tumor cells. The medicine can accurately position focus position, realize long-acting retention effect on mitochondrial surface, enhance stability of combination with mitochondria, selectively kill tumor cells, has strong tumor inhibiting effect in vivo, provides new non-invasive ultrasonic treatment strategy for diagnosis and treatment of tumor diseases, and can be used for treating tumor diseases with ultrasonic wave (1.0MHz, 1.25 Wcm)-250% duty cycle), can induce 90% of tumor cell apoptosis. And the medicine has good biological safety.
Preferably, the self-assembly unit is a polypeptide having a self-assembly function.
Preferably, the sequence of the polypeptide comprises any one of YFF, KLVFF, DEVD, GFLG or RVRR, or a combination of at least two thereof.
Preferably, the mitochondrial targeting unit is a mitochondrial targeting polypeptide.
Preferably, the sequence of the mitochondrial targeting polypeptide comprises CGKRK, [ KLAKLAK]2Any one or a combination of at least two of TPP, Szeto-Schiller, or DLP.
Preferably, the mitochondrial targeting unit is linked to the self-assembling unit by an amide bond.
Preferably, the sonodynamic response unit is a sonosensitizer.
Preferably, the sonosensitizer comprises meso-tetra (4-carboxyphenyl) porphin, purpurin, hematoporphyrin monomethyl ether, IR-780Iodide, DVDMS-Mn-LPs or MnTTP-HSA.
The acoustic dynamic response unit and the self-assembly unit are directly connected through an amido bond or connected through a polypeptide sequence.
Preferably, the polypeptide sequence is a sequence containing acidic amino acids, and more preferably a sequence containing acidic amino acids with the number of amino acids of 1-3, such as GG and GGK, wherein GG is a common connecting bond for connecting polypeptide amino acids or small molecules and has the function of regulating and controlling spatial conformation, and amino groups of a K amino acid main chain and a side chain have the function of simultaneously connecting the small molecules and functional molecules.
In a second aspect, the present invention provides a method for preparing the self-assembled polypeptide drug with a mitochondrial targeting function according to the first aspect, wherein the method for preparing the self-assembled polypeptide drug with a mitochondrial targeting function comprises:
and obtaining a mitochondrion targeting unit through a polypeptide solid phase synthesis method, and connecting the self-assembly unit with the electrokinetic response unit to obtain the self-assembly polypeptide medicament with the mitochondrion targeting function.
Preferably, the preparation method comprises:
(1) fixing the C end of the first amino acid in the mitochondrion targeting unit on resin, performing Fmoc protection on the N end, performing Dde protection on a side chain, and then performing swelling treatment;
(2) removing Fmoc protection at the N end of the first amino acid by using a deprotection agent, washing, adding the next amino acid for reaction, and sequentially connecting the amino acids to obtain a polypeptide sequence fixed on the resin;
(3) and mixing the obtained polypeptide sequence fixed on the resin with a sound sensitive agent for reaction, then shrinking the resin, splitting, and drying by nitrogen to obtain the self-assembled polypeptide medicament with the mitochondrion targeting function.
The preparation process of the self-assembled polypeptide medicament with the mitochondrion targeting function is simple and easy to operate, and is suitable for industrial production.
Preferably, the swelling treatment is carried out using activated DMF for 1-5h, e.g. 1h, 1.5h, 2h, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, etc.
Preferably, the reaction of step (3) is carried out under protection from light.
Preferably, the lysis is performed in an ice-water bath for 2.5-3h, such as 2.5h, 2.6h, 2.7h, 2.8h, 2.9h, 3h, etc.
Other specific point values within the above numerical range can be selected, and are not described in detail herein.
Preferably, the nitrogen is blown dry and washed with diethyl ether.
The polypeptide solid phase synthesis method related by the invention is to synthesize from the C end of the polypeptide chain to the N end, adopts Fmoc to protect the amino acid of the N end, detects whether the existence of primary amino group by Kaiser test method, and under the condition that the primary amino group is naked, the color of the resin containing the detection reagent in a 1.5mL centrifuge tube is dark purple; in the presence of Fmoc protected amino groups, the resin was colorless. Wherein, the Kaiser text detection method comprises three reagents which are respectively named as A, B, C, and the formulas of the three reagents are as follows:
a: 0.5g of ninhydrin in 10mL of absolute ethanol;
b: 20g of phenol was dissolved in 5mL of absolute ethanol;
c: 0.1g ascorbic acid was dissolved in 5mL absolute ethanol.
Illustratively, the synthesis method of tcpp-KGGFFYCGKRK is specifically as follows:
(1) swelling resin: weighing 300mg of Fmoc-D-Lys (BOC) -Wang Resin, putting the Fmoc-D-Lys (BOC) -Wang Resin into a polypeptide synthesis tube, adding activated N, N-Dimethylformamide (DMF), and putting the mixture into a shaking table to shake and swell for 1-5 h;
(2) deprotection: washing with DCM and DMF repeatedly (3 times), adding deprotection agent (piperidine: DMF 1:4) into the polypeptide synthesis tube, shaking on a shaker for 10min, and removing Fmoc group on amino acid;
(3) and (3) detection: after repeated washing with DCM and DMF (3 times) alternately, A, B, C and a small amount of resin were added to a 1.5ml centrifuge tube and the tube was heated in boiling water for 1 min. If the color of the resin is changed into purple, the Fmoc protecting group is removed;
(4) coupling: weighing 10 times equivalent of amino acid and HBTU relative to resin loading capacity in a 15mL centrifuge tube, adding 10mL of coupling agent (DMF: N-methylmorpholine (NMM) ═ 19:1) for pre-reaction for 10 minutes, pouring into a polypeptide synthesis tube, and placing on a shaking table for reaction for 2 hours;
(5) and (3) detection: washing with DCM and DMF repeatedly, each for 3 times, adding Kaiser text detection reagent, if the color of the resin has not changed, proving that the amino acid is coupled;
(6) cyclic coupling: repeating the steps (2), (3), (4) and (5) to completely couple all amino acids, and removing the Fmoc protecting group by using a deprotection agent after the last amino acid is completely coupled;
(7) and (3) detection: repeating the second step;
(8) coupled sonodynamic response molecule of-tetrakis (4-carboxyphenyl): weighing 4 times of equivalent TCPP and 10 times of equivalent benzotriazole-N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HBTU) in a 15mL centrifuge tube, adding 10mL of coupling agent for reaction for 10 minutes, pouring into a polypeptide synthesis tube, and placing on a shaking table for overnight reaction;
(9) shrinking: shrinking the resin by using methanol for 15min, transferring the shrunk resin to a 10mL serum bottle, and adding magnetons into the bottle;
(10) cracking: adding 3mL of lysate (2.5% ultrapure water, 2.5% triisopropylsilane, 92.5% trifluoroacetic acid and 2.5% ethanedithiol), magnetically stirring in ice bath for 2.5-3h, suction-filtering, rinsing the serum bottle with TFA, drying TFA with nitrogen, adding glacial ethyl ether to precipitate polypeptide in ethyl ether, transferring the polypeptide to a centrifuge tube at 4 deg.C and 8000r min-1Centrifuging under the condition, repeating for three times, transferring the polypeptide to a 1.5ml centrifuge tube, sealing with a sealing film, and standing overnight to completely volatilize diethyl ether to finally obtain the mitochondrion-targeted assembled polypeptide medicament.
In a third aspect, the invention provides an application of the self-assembled polypeptide drug with a mitochondrion targeting function in preparing an anti-tumor drug mediated by sonodynamic therapy.
Compared with the prior art, the invention has the following beneficial effects:
the self-assembly polypeptide drug can form nanoparticles through self-assembly under the hydrophobic effect in the body fluid circulation process to passively target tumor cells and target mitochondria, as mitochondria in the tumor cells can generate a large amount of Reactive Oxygen Species (ROS), the nanoparticles can further form fibers under the environment of endogenous ROS, thereby enhancing the synergistic effect of the fibers and multivalent bonds of the mitochondria, leading the interaction sites of the fibers and the mitochondria to be more, and leading the fibers and the mitochondria to have more interaction sitesThe binding force is firmer, and the exogenous Ultrasound (US) is used for activating the sonosensitizer to generate singlet oxygen to destroy mitochondria so as to induce the apoptosis of tumor cells. The medicine can accurately position focus position, realize long-acting retention effect on mitochondrial surface, enhance stability of combination with mitochondria, selectively kill tumor cells, has strong tumor inhibiting effect in vivo, provides new non-invasive ultrasonic treatment strategy for diagnosis and treatment of tumor diseases, and can be used for treating tumor diseases with ultrasonic wave (1.0MHz, 1.25 Wcm)-250% duty cycle), can induce 90% of tumor cell apoptosis. And the medicine has good biological safety.
Drawings
FIG. 1 is a MALDI-TOF-MS validation of self-assembly products of self-assembly polypeptide drugs in PBS solution;
FIG. 2 shows self-assembly of a polypeptide drug at H2O2MALDI-TOF-MS verification of self-assembly products in solution;
FIG. 3 shows the self-assembly of polypeptide drugs in PBS solution and H, respectively2O2TEM images of the self-assembled product in solution;
FIG. 4 is a graph showing the results of the verification of co-localization of self-assembled polypeptide drug and mitochondria (20 μ M on the scale of the graph);
FIG. 5 is an SEM image of self-assembled polypeptide drug after assembly on mitochondria (scale 200nm in the figure);
FIG. 6 is pre-and post-sonication production of self-assembled polypeptide drugs1O2A signal diagram;
FIG. 7 is a graph of the cytotoxicity statistics of self-assembled polypeptide drugs before and after sonication;
FIG. 8 is a graph of in vivo imaging of the distribution of self-assembling polypeptide drugs in mice;
FIG. 9 is a statistical chart of the results of in vivo anti-tumor experiments with self-assembled polypeptide drugs.
Detailed Description
The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.
The animal mice referred to in the following test examples were female BALB/c nude mice (6-8 weeks, 17-19g) purchased from Vantonlifys, Beijing.
Examples
This example provides a self-assembling polypeptide drug with mitochondrion targeting (TCPP-KGGFFYCGKRK, TY for short) having a structural formula represented by formula I:
Figure BDA0003524972020000081
the preparation method comprises the following steps:
(1) weighing 300mg of Fmoc-D-Lys (BOC) -Wang Resin, adding the Fmoc-D-Lys (BOC) -Wang Resin into a polypeptide solid phase synthesis tube, adding DMF, swelling for 3h, filtering off the DMF by a suction pump, adding a deprotection agent, and placing on a shaking table for 10 min; pumping out the deprotection solution, washing with DMF and DCM for 3 times, taking Wang resin from a polypeptide solid phase synthesis tube, detecting the deep blue color by an indetrione method to obtain a positive result, preparing to insert a second amino acid (R), and performing amino acid condensation reaction;
(2) respectively taking 10 times of equivalent of amino acid and HBTU according to TY amino acid sequence order, dissolving with 10mL of reaction solution, putting into a polypeptide solid phase synthesis tube, stirring for reaction, after 1h, taking a proper amount of Wang resin from the polypeptide solid phase synthesis tube into a test tube, and after detecting that the color is not changed by an indetrione method, the condensation reaction is successful; extracting liquid in the polypeptide solid phase synthesis tube, washing with DMF and DCM for 3 times to obtain a second amino acid condensed peptide resin;
(3) repeating the Fmoc deprotection-amino acid condensation reaction steps on the obtained peptide resin until the last K amino acid reaction is finished, washing the obtained peptide resin with DMF and DCM for 3 times respectively, adding TCPP coupling solution, placing the obtained peptide resin on a shaking table in a dark place for reacting for 12 hours, washing the obtained peptide resin with DMF and DCM for 3 times respectively after the reaction is finished, washing the obtained peptide resin with methanol for 3 times, and drying the obtained peptide resin by pumping;
(4) taking out the synthesized peptide resin from the polypeptide solid phase synthesis tube, and cracking for 3h in ice water bath; filtering the resin, blowing dry with nitrogen, washing with anhydrous ether for 3 times under ice bath condition, and purifying with preparative reverse phase HPLC to obtain the polypeptide drug, which is stored at-20 deg.C for use.
Test example 1
MALDI-TOF-MS verification:
the self-assembled polypeptide drug TY prepared in the example was dissolved in PBS solution or 100. mu. M H, respectively2O2In aqueous solution (500. mu.g/mL), reacted at 25 ℃ for 8H to allow for sufficient assembly, and a small amount of the solution was identified by Mass Spectrometry (MS), as shown in FIG. 1(PBS solution) and FIG. 2 (H)2O2Aqueous solution). As can be seen from fig. 1 and 2: TY self-assembly polypeptide nano-drug is assembled into nano-particles in PBS solution, the molecular weight of the nano-particles is 2063.687, and the nano-particles are H2O2After the reaction in aqueous solution, crosslinking occurs, the molecular weight is 4125.732, and the coupling is further proved to be diploid by mass spectrum.
Test example 2
TEM verification:
the self-assembled polypeptide drug TY prepared in the example was dissolved in PBS solution or 100. mu. M H2O2In an aqueous solution (500. mu.g/mL), the mixture was reacted at 25 ℃ for 8 hours to sufficiently assemble the mixture, 10. mu.L of the two solutions were dropped on a 230-mesh carbon film copper net, the mixture was allowed to stand for 10 minutes to allow the sample to settle on the copper net, and then the excess sample was blotted dry with filter paper. Dyeing with uranyl acetate solution for 5min, washing with deionized water 2 times, and blotting with filter paper to clean the copper mesh, standing, and drying. Real-time CCD imaging by Transmission Electron Microscopy (TEM) as shown in FIG. 3 (a is sample dissolved in PBS solution; b is dissolved in H2O2Samples in aqueous solution), as can be seen from fig. 3: the invention discloses a self-assembly polypeptide drug with a mitochondrion targeting function, which is 100 mu M H2O2In (b) is a nanofiber other than a nanoparticle.
Test example 3
Verification of co-localization of drug and mitochondria:
will be 1 × 104HeLa cells were placed in confocal microscope culture dishes for 24h, incubated with TY (10 μm) for 8h and then washed 3 times with PBS. Cells were then incubated with a Mitotracker Red fluorescent probe (abbreviated MT-Red) for 30 minutes. Finally, the cells were washed 3 times with PBS and with Zeiss LSM710 laser confocal microscope (Zeiss LSM710) and oily 63 x objective imaging. As shown in fig. 4, it can be seen that: when the time is 8 hours, the self-assembly polypeptide drug of the targeted mitochondria and the mitochondria have good co-localization phenomenon, and the Pearson's coefficient is 0.71.
Test example 4
Verification of the assembled form of the drug on mitochondria:
the drug TY prepared in the example is incubated with mitochondria on a silicon wafer for 8h, fixed by 4% paraformaldehyde for 2h, dehydrated by 50%, 70%, 80%, 90% and 100% ethanol for 15min in sequence, fully dried and sprayed with gold for 20s to be shot by a scanning electron microscope. As shown in fig. 5, it can be seen that: the self-assembled polypeptide drug with the mitochondrion targeting function can form nano-fibers on the mitochondrion.
Test example 5
Functional verification of the drug ultrasound response unit:
the examples provide a medicament wherein TCPP is produced by ultrasound1O2The production of (d) was determined by Bruker EMX ESR spectroscopy. 50. mu.L of 2,2,6, 6-tetramethylpiperidine (TEMP, 97. mu.M) and then 1mL of TY (10. mu.M) were added to a 4mL centrifuge tube and irradiated (1.0MHz, 1.25W cm) onto the ultrasonic treatment probe-250% duty cycle) 3 minutes later, it was immediately detected by an electron spin resonance spectrometer1O2A signal. Detection of pre-and post-sonication under the same ultrasonic irradiation (1.0M Hz, 1.5W/cm-2, 50% duty cycle) after 3 minutes1O2A signal. As shown in fig. 6, it can be seen that: a triplet characteristic peak signal of 1:1:1 was observed, from which it could be demonstrated1O2Without ultrasound, TY nano-drugs1O2The signal is low.
Test example 6
And (3) cytotoxicity verification:
the CCK-8 colorimetric method is utilized to determine the cytotoxic effect of the mitochondrion-targeted assembled polypeptide drug on HeLa cells. HeLa cells were seeded in 96-well culture plates (6000/well) containing 100. mu.L of DMEM and cultured for 24 hours, and then different concentrations (20, 10, 5, 2.5, 1.2)0.6, 0. mu.M) were incubated with the cells for 8h, and ultrasound (1.0MHz, 1.25W. cm) was given-250% duty cycle), adding 100 μ L of CCK-8 solution to each well, incubating for 2h, and determining the concentration of proliferating cells in each well at a test wavelength of 450nm and a reference wavelength of 690nm using a microplate reader, respectively. As shown in fig. 7, it can be seen that: the self-assembled polypeptide medicament with the mitochondrion targeting function has no toxicity to cells when ultrasonic is not added, and the cell activity is about 100 percent; IC of TY-US group under ultrasound conditions50The value is 9.8 mu M, and the anti-tumor effect is obvious. The self-assembly polypeptide drug can be covalently bound with mitochondria and can better induce apoptosis under the action of ultrasound.
Test example 7
In vivo targeting verification of the medicament:
(1) weighing an appropriate amount of TY, preparing into 200 mu M PBS solution, injecting the sample into mice by tail vein administration, wherein the administration volume of each mouse is 200 mu L;
(2) the biodistribution of TY in mice was observed by anesthetizing the mice with isoflurane at different time points designed by a small animal imaging instrument (model BioSpec70/20USR, manufactured under the name Bruker, inc.) and imaging experiments were performed on the mice.
The results are shown in FIG. 8, where we chose to fluorescently image mice at different time points, with TY having a high enrichment at the tumor site at 8h until slowly beginning to diminish after 24h, with the ROS-responsive deformed TY polypeptide nanopharmaceuticals having a long enrichment in the tumor region.
Test example 8
In vivo antitumor effect verification:
the nude mice were divided into 6 groups (n ═ 10), PBS, Only-US, TF-US, TY, TY-US groups. When the subcutaneous tumor volume of the nude mice reaches 100mm3About the same time, tail vein injection TY (200 μ M) or PBS (PBS) is adopted, each mouse is injected with 200 μ L, and ultrasonic treatment is given or not given for 8h after tail vein injection;
the specific ultrasonic experimental scheme is as follows: firstly, turning on the ultrasonic therapeutic apparatus, adjusting the power of the ultrasonic therapeutic apparatus to 1.25w/cm2, uniformly coating an ultrasonic coupling agent on a probe of the ultrasonic therapeutic apparatus, and starting ultrasonic therapy. After the treatment period, the tumor part of the nude mice was dissected and weighed.
The results are shown in FIG. 9: compared with a control group, the self-assembly polypeptide drug has a remarkable anti-tumor effect.
The applicant states that the present invention is illustrated by the above examples to show a self-assembled polypeptide drug with a mitochondrion targeting function, and a preparation method and application thereof, but the present invention is not limited by the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

Claims (10)

1. The self-assembled polypeptide drug with the mitochondrial targeting function is characterized by comprising a mitochondrial targeting unit, a self-assembled unit and an acousto-dynamic response unit which are sequentially connected.
2. The self-assembled polypeptide drug with a mitochondrion targeting function according to claim 1, wherein the self-assembly unit is a polypeptide with a self-assembly function;
preferably, the sequence of the polypeptide comprises any one of YFF, KLVFF, DEVD, GFLG or RVRR, or a combination of at least two thereof.
3. The self-assembled polypeptide drug with a mitochondrial targeting function of claim 1 or 2, wherein the mitochondrial targeting unit is a mitochondrial targeting polypeptide;
preferably, the sequence of the mitochondrial targeting polypeptide comprises CGKRK, [ KLAKLAK]2Any one or a combination of at least two of TPP, Szeto-Schiller or DLP;
preferably, the mitochondrial targeting unit is linked to the self-assembling unit by an amide bond.
4. The self-assembling polypeptide agent with mitochondrial targeting function of any of claims 1-3, wherein the sonodynamic response unit is a sonosensitizer;
preferably, the sonosensitizer comprises meso-tetra (4-carboxyphenyl) porphin, purpurin, hematoporphyrin monomethyl ether, IR-780Iodide, DVDMS-Mn-LPs or MnTTP-HSA.
5. The self-assembled polypeptide drug with mitochondrial targeting function of claim 4, wherein the sonodynamic response unit is directly linked to the self-assembled unit through an amide bond or through a polypeptide sequence.
6. The method for preparing the self-assembled polypeptide drug with the function of mitochondrial targeting according to any one of claims 1 to 5, wherein the method for preparing the self-assembled polypeptide drug with the function of mitochondrial targeting comprises the following steps:
and obtaining a mitochondrion targeting unit through a polypeptide solid phase synthesis method, and connecting the self-assembly unit with the electrokinetic response unit to obtain the self-assembly polypeptide medicament with the mitochondrion targeting function.
7. The method for preparing the self-assembled polypeptide drug with the mitochondrion targeting function as claimed in claim 6, wherein the preparation method comprises:
(1) fixing the C end of the first amino acid in the mitochondrion targeting unit on resin, performing Fmoc protection on the N end, performing Dde protection on a side chain, and then performing swelling treatment;
(2) removing Fmoc protection at the N end of the first amino acid by using a deprotection agent, washing, adding the next amino acid for reaction, and sequentially connecting the amino acids to obtain a polypeptide sequence fixed on the resin;
(3) and mixing the obtained polypeptide sequence fixed on the resin with a sound sensitive agent for reaction, then shrinking the resin, splitting, and drying by nitrogen to obtain the self-assembled polypeptide medicament with the mitochondrion targeting function.
8. The method for preparing the self-assembled polypeptide drug with the mitochondrial targeting function of claim 7, wherein the swelling treatment is performed for 1-5h by using activated DMF;
preferably, the reaction of step (3) is carried out under protection from light.
9. The method for preparing a self-assembled polypeptide drug with a mitochondrial targeting function according to claim 7 or 8, wherein the cleavage is performed in an ice-water bath for 2.5-3 h;
preferably, the nitrogen is blown dry and washed with diethyl ether.
10. Use of the self-assembled polypeptide drug with mitochondrion targeting function as claimed in any one of claims 1-5 in the preparation of sonodynamic therapy-mediated antitumor drugs.
CN202210190346.7A 2022-02-28 2022-02-28 Self-assembly polypeptide medicine with mitochondrion targeting function and preparation method and application thereof Pending CN114533894A (en)

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