An enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function encapsulating siRNA and preparation method thereof

Abstract

The present invention discloses an enzyme-responsive polypeptide nano-drug with mitochondrial targeting function encapsulating siRNA and a preparation method thereof, and relates to the technical field of nanomaterials. The preparation method comprises the following steps: dissolving a carrier and an enzyme-responsive polypeptide in an organic solvent, adding siRNA and mixing evenly to obtain a mixed solution, adding the mixed solution into water for self-assembly, and obtaining the enzyme-responsive polypeptide nano-drug with mitochondrial targeting function encapsulating siRNA. The polypeptide nano-drug designed by the present invention uses a novel and effective combination mechanism to biphasically intervene in the energy metabolism of tumor cells and induce apoptosis of tumor cells. It is expected to become a new type of treatment method targeting organelles in the future, and can be used for anticancer, antibacterial, anti-inflammatory and other drug treatments and tracing.

Description

Technical Field

[0001] The invention relates to the technical field of nanomaterials, and in particular to an enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function and encapsulating siRNA, and a preparation method thereof. Background Art

[0002] At present, the main treatments for cancer in clinical practice include surgery, chemotherapy, radiotherapy, immunotherapy and targeted therapy. Due to the differences in tumor specificity and the easy generation of acquired drug resistance, the first four therapies have large toxic side effects and poor long-term treatment effects on cancer. Targeted therapy kills cancer cells by targeting specific cancer cell targets, which improves the targeting of drugs, reduces damage to normal cells, and reduces the toxic side effects of drugs. Among them, subcellular organelle-targeted cancer treatment strategies have the advantages of precise active targeting, which have attracted extensive research attention in the fields of cancer and precision medicine and have shown broad application prospects. Mitochondria play a key role in cells and participate in regulating multiple basic physiological processes such as energy metabolism and cell apoptosis. The growth and metastasis of cancer cells depend on efficient energy production, and targeting mitochondria can interfere with the energy metabolism pathways of cancer cells, directly cut off their energy supply, and induce apoptosis. This process does not rely on the cell cycle regulation of traditional chemotherapy drugs, can effectively overcome the problem of drug resistance, and thus improve the effect of cancer treatment. Therefore, mitochondria have become one of the important subcellular targets of nanomedicine targeted delivery strategies. However, how to improve the delivery efficiency, stability and targeting accuracy of mitochondrial targeted drugs in vivo remains challenging and needs to be solved urgently.

[0003] Nanotechnology can achieve efficient drug delivery and controlled release by adjusting the size and structure of nanomaterials, and has played a huge role in cancer treatment. At present, a variety of nano drug delivery systems have been successfully developed and achieved good cancer treatment effects. Among them, the performance of peptide assemblies is also particularly impressive. Peptides are highly specific and can be designed to bind to specific receptors or target molecules on cancer cells, thereby achieving efficient targeted delivery of drugs. At the same time, compared with traditional chemotherapy drugs, peptides generally have lower toxic side effects and can reduce the impact on normal cells. In addition, peptides can be used as multifunctional carriers to carry a variety of therapeutic molecules (such as platinum metal drugs, nucleic acid drugs, and responsive substances such as sonosensitizers and photosensitizers) to achieve combined therapy and enhance the therapeutic effect. Despite its high-efficiency targeting potential, mitochondrial targeting peptides may still face problems such as clearance by the immune system and poor stability during in vivo delivery, and their therapeutic effect on cancer is not satisfactory. Therefore, the design of drug delivery systems still needs to be further improved. By rationally designing a peptide assembly system, a peptide nano-delivery system with good biocompatibility, strong mitochondrial targeting and rich biological functions is developed. Summary of the invention

[0004] The purpose of the present invention is to provide an enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function encapsulating siRNA and a preparation method thereof, so as to solve the problems existing in the above-mentioned prior art. The polypeptide nanomedicine has a novel and effective combination mechanism, biphasically intervenes in the energy metabolism of tumor cells, induces apoptosis of tumor cells, and is expected to become a new type of treatment method targeting organelles in the future, and can be used for anticancer, antibacterial, anti-inflammatory and other drug treatments and tracing.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an enzyme-responsive polypeptide with mitochondrial targeting function, wherein the amino acid sequence of the enzyme-responsive polypeptide is RGD-RRRRRRRRR-DEVD-Kd(KLAKLAK)2-K.

[0007] Furthermore, the 17th amino acid K at the N-terminus of the enzyme-responsive polypeptide is connected to the photosensitizer through an amide bond; and the 32nd amino acid K at the N-terminus of the enzyme-responsive polypeptide is connected to stearic acid through an amide bond.

[0008] Furthermore, the photosensitizer is dihydrochlorin e6.

[0009] The present invention also provides the use of the enzyme-responsive polypeptide in preparing an enzyme-responsive polypeptide nanomedicine encapsulating siRNA and having a mitochondrial targeting function.

[0010] The present invention also provides a method for preparing an enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function encapsulating siRNA, comprising the following steps:

[0011] The carrier and the enzyme-responsive polypeptide are dissolved in an organic solvent, and then siRNA is added and mixed evenly to obtain a mixed solution, and the mixed solution is added into water for self-assembly to obtain the enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function encapsulating siRNA.

[0012] Furthermore, the carrier is phospholipid.

[0013] Furthermore, the volume ratio of the mixed solution to the water is 1:20.

[0014] Furthermore, the molar ratio of the siRNA, the enzyme-responsive polypeptide and the phospholipid is 1:87.8:175.6.

[0015] Furthermore, the siRNA is an siRNA that silences the Glut1 gene.

[0016] The present invention also provides an enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function and encapsulating siRNA, which is prepared according to the above preparation method.

[0017] The present invention discloses the following technical effects:

[0018] (1) The polypeptide sequence with mitochondrial targeting function designed by the present invention not only plays a role in mitochondrial targeting and tumor killing, but also has the advantages of good biodegradability, biocompatibility, low immunogenicity and low toxicity, thereby reducing the toxic side effects on normal tissues.

[0019] (2) The polypeptide delivery system provided by the present invention can efficiently deliver siRNA into cells, and has a significant gene silencing effect.

[0020] (3) The present invention obtains a polypeptide nanomedicine by connecting a mitochondrial targeting peptide and a photosensitizer to a polypeptide sequence with enzyme-responsive cleavage, and then co-assembling with siRNA. The nanomedicine can damage the mitochondrial membrane structure of tumor cells with the help of mitochondrial targeting peptides, interfere with energy metabolism, and promote tumor cell apoptosis; at the same time, Caspase 3 enzyme in apoptotic cells is activated, cleaves polypeptides, releases siRNA, silences the expression of proteins related to energy metabolism, reduces the intake of energy-generating raw material glucose, thereby interfering with energy metabolism and further promoting cell apoptosis. In addition, the photosensitizer connected to the polypeptide is transported to the vicinity of the mitochondria through the functional polypeptide, and can enhance the mitochondrial destruction effect after illumination. The synergistic effect of multiple parties achieves a self-amplified tumor killing effect.

[0021] (4) The preparation method of the enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function encapsulating siRNA provided by the present invention is simple to operate, has mild reaction conditions, and is suitable for industrial production.

[0022] The polypeptide nanomedicine designed in the present invention uses a novel and effective combination mechanism to biphasically intervene in the energy metabolism of tumor cells and induce tumor cell apoptosis. It is expected to become a new therapeutic method targeting organelles in the future and can be used for anti-cancer, antibacterial, anti-inflammatory and other drug treatments and tracing. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 The mass spectrum of the enzyme-responsive peptide with mitochondrial targeting function;

[0025] Figure 2This is a transmission electron microscopy image of an enzyme-responsive peptide nanomedicine with mitochondrial targeting function that encapsulates siRNA; the scale bar is 200 nm;

[0026] Figure 3 This is the result of gel retardation experiment;

[0027] Figure 4 Figure 1 is a flow cytometric analysis of the cell uptake experiment;

[0028] Figure 5 This is the result of in vitro transfection gene silencing experiment;

[0029] Figure 6 The figure is the result of in vitro protein immunoblotting experiment;

[0030] Figure 7 The results of in vitro mitochondrial co-localization experiments are shown in Figure 1. The scale bar is 50 μm.

[0031] Figure 8 This is the result of the in vitro mitochondrial membrane potential detection experiment; the scale bars are all 50μm;

[0032] Fig. 9 This is the result of in vitro Caspase 3 enzyme activity detection experiment;

[0033] Fig.10 This is a statistical graph of cell survival rate in cytotoxicity experiments;

[0034] Fig.11 This is the flow cytometry analysis of the cytotoxicity experiment;

[0035] Fig.12 This is the in vivo drug enrichment result diagram of the in vivo biodistribution imaging experiment;

[0036] Fig.13 This is a diagram showing the enrichment results of drugs in isolated organs and tumors from in vivo biodistribution imaging experiments;

[0037] Fig.14 This is a statistical chart of mouse body weight in the in vivo anti-tumor experiment;

[0038] Fig.15 It is a statistical diagram of tumor volume in the in vivo anti-tumor experiment. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1

[0045] An enzyme-responsive polypeptide with mitochondrial targeting function comprises a tumor targeting unit, an enzyme-responsive cleavage unit and a mitochondrial targeting unit connected in sequence, and the polypeptide sequence is as follows:

[0046] RGD-RRRRRRRRR-DEVD-K(Ce6)-d(KLAKLAK)2-K(stearic acid) (SEQ ID NO.1);

[0047] Among them, Ce6 and stearic acid are branched chains;

[0048] Tumor targeting unit (peptide with tumor targeting and cell penetrating abilities): RGD-RRRRRRRRRR;

[0049] Enzyme-responsive cleavage unit (peptide with Caspase 3 enzyme-responsive properties): DEVD;

[0050] Mitochondrial targeting unit (polypeptide with mitochondrial targeting function): d(KLAKLAK)2;

[0051] The carboxyl terminus of the tumor targeting unit is connected to the amino terminus of the enzyme-responsive cleavage unit via an amide bond;

[0052] The enzyme-responsive cleavage unit is connected to the mitochondrial targeting unit via amino acid K, and the amino acid K is connected to the photosensitizer chlorin e6 (Ce6) via an amide bond;

[0053] The first amino acid K at the C-terminus is linked to stearic acid via an amide bond.

[0054] According to the peptide sequence, coupling is performed sequentially from the C-terminus to the N-terminus until the first amino acid Boc-Arg(Pbf)-OH at the N-terminus, and then the Dde protecting group of the first amino acid Lys side chain at the C-terminus is removed by hydrazine hydrate to prepare an enzyme responsive peptide with mitochondrial targeting function. The specific method is as follows:

[0055] (1) Weigh Ramage Amide MBHAResin resin and add it to a peptide solid phase synthesis tube. Add DCM to swell for 0.5 h. Filter out DCM with a suction pump. Add a deprotecting agent and react with nitrogen for 20 min. Remove the deprotecting agent and wash repeatedly with DMF and DCM three times. Take an appropriate amount of resin from the peptide solid phase synthesis tube and put it in a test tube. Use the ninhydrin detection method to detect. The resin turns dark blue, which is a positive result, indicating the removal of the Fmoc protecting group.

[0056] The side chain protecting groups of Arg, Asp, Glu, Lys and D-Lys are Pbf, OtBu, OtBu, Alloc and Boc respectively; except for the α-amino group of the first amino acid Arg at the N-terminus which is protected by Boc, the α-amino groups of all amino acids are protected by Fmoc.

[0057] (2) Prepare to access the first amino acid (Fmoc-Lys(Dde)-OH) and carry out amino acid condensation reaction. According to the amino acid sequence of the peptide, take 3 times the equivalent of amino acid and HBTU relative to the resin load, dissolve them in the reaction solution, put them into the peptide solid phase synthesis tube, fill with nitrogen to react for 1 hour, then add the DMF solution containing stearic acid to the reaction column and continue to react for 1 hour at room temperature. Take an appropriate amount of resin from the peptide solid phase synthesis tube into a test tube, and verify that the resin does not change color by the ninhydrin test method, that is, a negative result proves that the condensation reaction is successful; remove the liquid in the peptide solid phase synthesis tube, and wash it repeatedly with DMF and DCM for 3 times to obtain the peptide resin after the second amino acid condensation.

[0058] (3) Repeat the "Fmoc deprotection-amino acid condensation" reaction steps of steps (1) and (2) on the obtained peptide resin until the last Boc-Arg(Pbf)-OH amino acid is reacted, wash it with DMF and DCM three times each, add a coupling solution containing photosensitizer Ce6, and react for 2 hours with nitrogen. After the reaction is completed, wash it with DMF and DCM three times each, wash it with methanol three times, and drain it.

[0059] (4) The synthesized peptide resin was taken out from the peptide solid phase synthesis tube and cleaved in an ice water bath for 2 h. After filtering the resin, it was blown dry with nitrogen, washed three times with anhydrous ether under ice bath conditions, and purified using preparative reverse phase HPLC to obtain an enzyme-responsive polypeptide with mitochondrial targeting function, which was stored at -20°C for future use.

[0060] The prepared enzyme-responsive polypeptide with mitochondrial targeting function was characterized by mass spectrometry using desorption electrospray ionization (mass spectrometry ionization) technology (the instrument model used was Waters, Micromass ZQ), such as Figure 1 As shown, the molecular weight of the enzyme-responsive polypeptide with mitochondrial targeting function is 4816.87, the highest corresponding peak is 804.10, and it is a 6-valent fragment.

[0061] Example 2

[0062] This embodiment provides an enzyme-responsive polypeptide nano-drug with mitochondrial targeting function encapsulated with siRNA, comprising a phospholipid molecule, a Glut1 siRNA molecule, and an enzyme-responsive polypeptide with mitochondrial targeting function prepared in Example 1. Among them, the Glut1 siRNA molecule is combined with the positively charged cationic polypeptide through the negatively charged phosphate group through electrostatic adsorption and is encapsulated in the nano-delivery system. The preparation method of the enzyme-responsive polypeptide nano-drug with mitochondrial targeting function encapsulated with siRNA is as follows:

[0063] The enzyme-responsive peptide with mitochondrial targeting function and phospholipid (lecithin, Aladdin, CAS number: 8002-43-5) were dissolved in the organic solvent DMSO, and then Glut1 siRNA was added to obtain a mixed solution (wherein the molar ratio of Glut1 siRNA, enzyme-responsive peptide and phospholipid was 1:87.8:175.6), and then the mixed solution was added to water (the volume ratio of the mixed solution to water was 1:20, v / v) for self-assembly to obtain an enzyme-responsive peptide nanodrug with mitochondrial targeting function encapsulating siRNA, named DCK@siRNA.

[0064] The sequence of Glut1 siRNA is as follows:

[0065] Sense strand: 5'-CUUCCUGCUCAUCAAUCGUTT-3' (SEQ ID NO. 2);

[0066] Antisense strand: 5'-ACGAUUGAUGAGCAGGAAGTT-3' (SEQ ID NO. 3).

[0067] like Figure 2 As shown, the DCK@siRNA prepared in this example was observed under a transmission electron microscope (model: Leica Microsystems GmbH. Ernst-Leitz-Strasse 17-37. D-35578), and the nanoparticle structure was visible.

[0068] Example 3

[0069] In this example, the formation of DCK@siRNA prepared in Example 2 under different N / P ratio conditions was investigated by gel retardation experiments. Glut1 siRNA (~0.3 μg) was added to the enzyme-responsive polypeptide with mitochondrial targeting function at different N / P ratios, and then mixed with 5X RNA electrophoresis loading buffer to obtain a mixture. The mixture was added to a 1% agarose gel using GelRed. Subsequently, the agarose gel was electrophoresed at 80V for 30 minutes in Tris acetate EDTA buffer, and imaging observation was performed using the Tanon 1600 fully automatic digital gel image analysis system.

[0070] The results are as follows Figure 3 As shown, the results show that when the N / P ratio is greater than or equal to 10, the siRNA and polypeptide in the nanomedicine exhibit strong binding ability.

[0071] Example 4

[0072] Cellular uptake assay:

[0073] The present invention selected 4T1 cells as model cells and cultured 4T1 cells at 2.0×10 5 Cells were seeded in 6-well plates at 100 μg / well and incubated at 37°C and 5% CO2 for 24 hours. The cells were washed with PBS and fresh medium containing DCK@siRNA was added and cultured for 0.5, 2, 4 and 8 hours. The cells were then trypsinized and washed with PBS. The cells were resuspended in PBS for flow cytometry analysis.

[0074] like Figure 4 As shown, the results of flow cytometry showed that the fluorescence intensity gradually increased with the extension of incubation time, indicating that the peptide nanomedicine was successfully taken up by 4T1 cells.

[0075] Example 5

[0076] In vitro transfection gene silencing experiment:

[0077] 4T1 cells were cultured at 1.0 × 10 5 The cells were inoculated in 12-well plates at 37°C and 5% CO2 for 12 hours, and then fresh medium containing DCK and DCK@siRNA was added (the amount of DCK and DCK@siRNA added in fresh medium was 5 μg / mL in terms of Ce6) and incubated for 48 hours. Fresh medium without DCK and DCK@siRNA was used as control. Among them, the two illumination groups (DCK+L and DCK@siRNA+L) were illuminated (658 nm, 200 mW / cm 2 , 1min), and continued to incubate for 24 hours. The cells were then washed with PBS, digested with trypsin, washed with PBS again, and collected. The efficiency of peptide nanomedicine in silencing the Glut1 gene at the siRNA level was detected by real-time fluorescence quantitative PCR (qPCR).

[0078] Among them, DCK in this embodiment and the following text is the abbreviation of enzyme-responsive polypeptide nanodrug with mitochondrial targeting function, and its preparation method is as follows:

[0079] The enzyme-responsive polypeptide with mitochondrial targeting function and phospholipid were dissolved in an organic solvent at a molar ratio of 1:2 to obtain a mixed solution, and then the mixed solution was added to water (1:20, v / v) for self-assembly to obtain an enzyme-responsive polypeptide nanodrug with mitochondrial targeting function, named DCK.

[0080] The results are as follows Figure 5 As shown in A, compared with the groups without siRNA (control group, DCK group and DCK+L group), both DCK@siRNA and DCK@siRNA+L groups can specifically induce Glut1 gene silencing.

[0081] Example 6

[0082] In vitro protein immunoblotting experiments:

[0083] 4T1 cells were cultured at 2.0×10 5The cells were inoculated in 6-well plates at 37°C and 5% CO2 for 12 hours, and then fresh medium containing DCK and DCK@siRNA was added (the amount of DCK and DCK@siRNA added in fresh medium was 5 μg / mL in terms of Ce6) and incubated for 48 hours. Fresh medium without DCK and DCK@siRNA was used as control. Among them, the two illumination groups (DCK+L and DCK@siRNA+L) were illuminated at 658 nm, 200 mW / cm 2 , 1min), and continued to incubate for 24 hours. The cells were then washed with PBS, digested with trypsin, washed with PBS again, and collected. Western blotting experiments were performed to explore the efficiency of peptide nanomedicine in silencing the Glut1 gene at the protein expression level.

[0084] like Figure 6 As shown, compared with the groups without siRNA (control group, DCK group and DCK+L group), both DCK@siRNA and DCK@siRNA+L groups could significantly down-regulate the specific protein expression of Glut1 gene.

[0085] Example 7

[0086] In vitro mitochondrial co-localization assay:

[0087] 4T1 cells were cultured at 2.0×10 5 / well of cells were inoculated in a confocal dish and incubated for 24 hours at 37°C and 5% CO2. Then, fresh culture medium containing DCK and DCK@siRNA was added thereto (the amount of DCK and DCK@siRNA added in fresh culture medium was 5 μg / mL in terms of Ce6), and the culture medium without DCK and DCK@siRNA was used as a control. The cells were then washed with PBS and incubated with Mitotracker Green fluorescent probe for 30 minutes. Finally, the cells were washed with PBS and the cell imaging was observed under a laser confocal microscope.

[0088] like Figure 7 As shown, the enzyme-responsive peptide nanomedicine with mitochondrial targeting function has a significant co-localization phenomenon with mitochondria.

[0089] Example 8

[0090] In vitro mitochondrial membrane potential detection experiment:

[0091] 4T1 cells were cultured at 2.0×10 5The cells were inoculated in a confocal dish at 37°C and 5% CO2 for 24 hours. Fresh culture medium containing DCK and DCK@siRNA was then added (the amount of DCK and DCK@siRNA added to the fresh culture medium was 5 μg / mL in terms of Ce6) and incubated for 24 hours. Fresh culture medium without DCK and DCK@siRNA was used as a control. The illumination group, DCK@siRNA+L group, was illuminated at 12 hours (658 nm, 200 mW / cm 2 , 1 min), and continue to incubate for 12 hours. Then the cells were washed with PBS, and incubated with JC-1 fluorescent probe for 30 minutes. Finally, the cells were washed with PBS and observed under a laser confocal microscope.

[0092] In the mitochondria of normal cells, JC-1 exists in the mitochondrial matrix in the form of polymers, emitting strong red fluorescence. In damaged mitochondria, due to the decrease or loss of membrane potential, JC-1 can only exist in the form of monomers, producing green fluorescence. Figure 8 As shown in the figure, the cells treated with the control group showed obvious red fluorescence, while the 4T1 cells treated with peptide nanomedicine showed a strong green fluorescence signal, and as the drug components increased, the intensity of green fluorescence gradually increased. The results showed that peptide nanomedicine can change the mitochondrial membrane potential and induce tumor cell apoptosis.

[0093] Example 9

[0094] In vitro Caspase 3 enzyme activity detection experiment:

[0095] 4T1 cells were cultured at 1.0 × 10 5 The cells were inoculated in 12-well plates at 37°C and 5% CO2 for 24 hours. Fresh culture medium containing DCK and DCK@siRNA was then added (the amount of DCK and DCK@siRNA added to the fresh culture medium was 5 μg / mL in terms of Ce6) and incubated for 24 hours. Fresh culture medium without DCK and DCK@siRNA was used as a control. The illumination group, DCK@siRNA+L group, was illuminated at 658 nm, 200 mW / cm 2 , 1 min), and continued to incubate for 12 hours. Then the cells were washed with PBS, and the cells were collected after trypsin digestion. The Caspase 3 activity of each group was detected using a Caspase 3 activity detection kit.

[0096] like Fig. 9 As shown, the peptide nanodrugs encapsulating siRNA can significantly activate intracellular Caspase 3 activity.

[0097] Example 10

[0098] Cytotoxicity assay:

[0099] 4T1 cells were cultured at 8.0×10 4 The cells were inoculated into 96-well plates at 37°C and 5% CO2 for 24 hours. Fresh culture medium containing different concentrations (in terms of Ce6 concentration) of DCK and DCK@siRNA was then added and incubated for 24 hours. Fresh culture medium without DCK and DCK@siRNA was used as control. The illumination group, DCK@siRNA+L group, was illuminated at 658nm, 200mW / cm 2 , 1min), and continue incubation for 12 hours. Then the CCK8 kit was used to evaluate the cytotoxicity of the drug, and the ultraviolet absorbance values ​​of all samples were measured at a wavelength of 450nm. The calculation formula for cell viability is as follows:

[0100] Cell survival rate (%) = (A sample -A blank ) / (A control -A blank )×100%;

[0101] Among them, A sample , A control , A blank Respectively represent the absorbance values ​​measured for the sample treatment group, fresh culture medium group and blank control group.

[0102] The results are as follows Fig.10 and Fig.11 As shown, compared with DCK, DCK@siRNA can induce 4T1 cell death to a greater extent, and has a stronger ability to induce apoptosis after illumination.

[0103] Embodiment 11

[0104] In vivo biodistribution imaging experiments:

[0105] In this example, 6-7 week old BALB / c female mice were injected subcutaneously with 2×10 6 4T1 tumor cells were used to establish an orthotopic breast cancer tumor model in mice. 3 The experiment was performed at 48 hours. Then the solution containing DCK@siRNA was injected into the mice through the tail vein (the injection dose of DCK@siRNA was 2.5 mg / kg in terms of Ce6). The fluorescence intensity was observed at different time points. The animals were killed at 48 hours, and the main organs (heart, liver, spleen, lung, kidney) and tumors were collected for ex vivo imaging.

[0106] Using Ce6 as a fluorescent tracer, it is possible to monitor the location of drugs in mice. Fig.12 and Fig.13 As shown, the tumor tissue of mice treated with DCK@siRNA showed obvious fluorescence signals, indicating its effective enrichment in tumor tissue. The fluorescence signal of DCK@siRNA in the tumor site had a high intensity within 48 hours, which means that DCK@siRNA was retained in the tumor site for a long time and had good tumor tissue retention ability.

[0107] Example 12

[0108] In vivo anti-tumor experiment:

[0109] In this example, 6-7 week old BALB / c female mice were injected subcutaneously with 2×10 6 4T1 tumor cells were used to establish an orthotopic breast cancer tumor model in mice. 3 The mice were randomly divided into 4 groups (n=5), namely PBS group, DCK group, DCK@siRNA group and DCK@siRNA+L group. The drug was injected into the tail vein once every two days (the injection dose of DCK group, DCK@siRNA group and DCK@siRNA+L group was 2.5 mg / kg in terms of Ce6; the PBS group was replaced with an equal amount of PBS), and the illumination group - DCK@siRNA+L was illuminated (658 nm, 200 mW / cm 2 The weight and tumor volume of mice were monitored every two days.

[0110] The tumor volume was calculated as follows:

[0111] V=a 2 ×b×(1 / 2)

[0112] Where a and b are the shortest and longest diameters of the tumor, respectively.

[0113] The results are as follows Fig.14 As shown in the figure, the weight of the mice in the experimental group and the control group was similar and constant, which proved that the peptide nanomedicine has good in vivo biosafety. Fig.15 As shown, compared with the PBS and DCK groups, the tumor growth of mice in the DCK@siRNA group was significantly inhibited, and the degree of inhibition was more obvious after illumination, indicating that the therapeutic efficacy of DCK@siRNA is obvious, and the anti-tumor effect is stronger after combined with photodynamic therapy.

[0114] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An enzyme-responsive polypeptide with mitochondrial targeting function, characterized in that: The amino acid sequence of the enzyme-responsive polypeptide is RGD-RRRRRRRRRR-DEVD-Kd(KLAKLAK)2-K; The 17th amino acid K at the N-terminus of the enzyme-responsive polypeptide is connected to the photosensitizer through an amide bond; the 32nd amino acid K at the N-terminus of the enzyme-responsive polypeptide is connected to stearic acid through an amide bond; The photosensitizer is dihydrochlorin e6.

2. A use of the enzyme-responsive polypeptide as claimed in claim 1 in a delivery system for preparing an enzyme-responsive polypeptide nanomedicine with mitochondrial targeting function encapsulating siRNA.

3. A method for preparing an enzyme-responsive polypeptide nanodrug with mitochondrial targeting function encapsulating siRNA, characterized in that: The following steps are involved: The carrier and the enzyme-responsive polypeptide according to claim 1 are dissolved in an organic solvent, and then siRNA is added and mixed evenly to obtain a mixed solution, and the mixed solution is added into water for self-assembly to obtain the enzyme-responsive polypeptide nanodrug with mitochondrial targeting function encapsulating siRNA; The carrier is phospholipid.

4. The preparation method according to claim 3, characterized in that: The volume ratio of the mixed solution to the water is 1:

20.

5. The preparation method according to claim 3, characterized in that: The molar ratio of the siRNA, the enzyme-responsive polypeptide and the phospholipid is 1:87.8:175.

6.

6. The preparation method according to claim 3, characterized in that: The siRNA is silencing Glut1 siRNA of the gene.

7. An enzyme-responsive polypeptide nanodrug with mitochondrial targeting function encapsulating siRNA, prepared according to the preparation method according to any one of claims 3 to 6.

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