Targeting tumor stem cell multifunctional nano-carrier as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological pharmacy, and particularly discloses a targeted tumor stem cell multifunctional nano-carrier as well as a preparation method and application thereof. The nano-carrier provided by the invention integrates functions of radiotherapy sensitization, up-regulation of MHC I on the surfaces of CSCs and targeting of PD-1 / PD-L1 immune checkpoints, and can act on multiple channels and multiple target points at the same time to play a synergistic effect. Firstly, the nano-carrier can enhance radiotherapy-mediated apoptosis, trigger stronger immunogenic cell death (ICD) and activate adaptive immune response; and secondly, the occurrence of the immune escape behavior of the CSCs can be restrained, and the sensitivity of the CSCs to the immunoreaction induced by radiotherapy is improved. Under the synergistic effect of the two aspects, the proportion of CSCs after radiotherapy is effectively reduced, so that residual CSCs in tumor tissues after radiotherapy are effectively removed, and a new treatment strategy is provided for inhibiting recurrence and metastasis of postoperative tumors.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to a multifunctional nanocarrier targeting tumor stem cells and a preparation method and application thereof. Background Art

[0002] Radiotherapy (RT) is one of the main means of cancer treatment. It has significantly improved the therapeutic effect of various malignant tumors such as breast cancer, colorectal cancer and esophageal cancer, and prolonged the survival of patients. Radiotherapy mainly damages the DNA of tumor cells through ionizing radiation, thereby inhibiting the growth of tumor cells. At the same time, radiotherapy can activate the body's anti-tumor immune response and further kill tumor cells. However, radiotherapy will increase the proportion of cancer stem cells (CSCs) in the tumor microenvironment. Studies have shown that CSCs have enhanced abilities such as DNA damage repair, reactive oxygen species (ROS) scavenging and apoptosis resistance, and can avoid direct damage caused by radiotherapy. At the same time, they can achieve immune escape by downregulating major histocompatibility complex (MHC) class I molecules and upregulating programmed death ligand 1 (PD-L1), thereby leading to drug resistance, recurrence and metastasis of various malignant tumors. Therefore, enhancing the effective killing of CSCs by radiotherapy and the immune system is crucial to improving the therapeutic effect of malignant tumors.

[0003] In order to achieve precise release of drugs in tumor sites with high expression of MMP2, Chinese patent CN116063389B (Guangzhou Medical University) discloses a polypeptide carrier for delivering nucleic acid drugs. Six arginine residues are synthesized at both ends of the MMP2 enzyme-cleavable peptide PLGLAG to obtain a cationic peptide: 6R-PLGLAG-6R, which can controllably load miRNA (such as miR-126-3pmimics) and release miRNA under environmental response conditions to achieve the purpose of controlled release and effectively induce cancer cell apoptosis. This patent uses MMP2 enzyme-responsive peptides to bind miRNAmimics through electrostatic adsorption, and uses RBCM (red blood cell membrane) to wrap nanoparticles for modification, giving them a stealth function. The REMAIN prepared in this way has the smallest particle size and the largest transfection efficiency, which is conducive to the accumulation of nanoparticles in tumor tissues, thereby ensuring the targeted delivery of miRNA mimics, and can also improve blood circulation life, immune escape ability and biosafety. The experimental results show that when MMP2 enzyme-responsive bionic nanoparticles are absorbed by lung cancer cells, 6R-PLGLAG-6R can be cleaved and decomposed by MMP2 due to the presence of a large amount of MMP2 enzyme in the cells, and miR-126-3pmimics are released, thereby effectively inducing apoptosis of cancer cells. However, there are no reports on the delivery of such nanoparticles for lung cancer gene drugs. Therefore, new cancer treatment strategies with great application potential need to be explored and discovered in the clinic. Summary of the invention

[0004] The main technical problem solved by the present invention is to provide a multifunctional nanocarrier targeting tumor stem cells, which integrates the functions of radiotherapy sensitization, upregulation of MHC I, and targeting of PD-1 / PD-L1 immune checkpoints. It can effectively eliminate residual CSCs in tumor tissue after radiotherapy, and provide a new treatment strategy for inhibiting the recurrence and metastasis of postoperative tumors.

[0005] Secondly, the present invention provides a method for preparing a multifunctional nanocarrier targeting tumor stem cells.

[0006] Again, the present invention provides an application of a multifunctional nanocarrier targeting tumor stem cells in the preparation of anti-tumor drugs.

[0007] Finally, the present invention provides an anti-tumor drug.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A multifunctional nanocarrier targeting tumor stem cells, wherein the nanocarrier is obtained by self-assembly using a bifunctional peptide and a hydrophobic drug that upregulates the expression of MHC I molecules as raw materials;

[0010] The bifunctional peptide is obtained by connecting a radiosensitizing peptide as a hydrophobic end and an anti-enzymatic PD-1 / PD-L1 blocking peptide as a hydrophilic end via a cleavable group.

[0011] As a preferred embodiment of the present invention, the size of the nanocarrier is 50-300 nm.

[0012] As a preferred embodiment of the present invention, the hydrophobic drugs that upregulate the expression of MHC I molecules include, but are not limited to, one or more of tazemetostat, docetaxel, decitabine, and the like.

[0013] Specifically, the English name of Tazemetostat is Tazemetostat (abbreviated as Taz hydrophobic drug), and its molecular formula is: C 34 H 44 N4O4, molecular weight: 572.74, manufacturer: MedChemExpress (MCE), origin: United States.

[0014] As a preferred embodiment of the present invention, the self-assembly is performed by hydrophobic interaction, wherein the hydrophobic properties of the hydrophobic drug that upregulates the expression of MHC I molecules enable it to be encapsulated in the hydrophobic core of the nanocarrier.

[0015] As a preferred embodiment of the present invention, the radiosensitizing peptide includes but is not limited to Smac polypeptide.

[0016] Specifically, the amino acid sequence of the Smac polypeptide is: AVPIAQK (SEQ ID NO: 2).

[0017] As a preferred embodiment of the present invention, the enzymatically resistant PD-1 / PD-L1 blocking peptide is a polypeptide composed of D-configuration amino acids, having excellent anti-protease activity, including but not limited to D One or more of PPA-1 polypeptide (i.e., D1 peptide), Pep-39 polypeptide (targeting PD-L1 and capable of blocking the interaction between PD-1 / PD-L1), etc.

[0018] Specifically, the D The amino acid sequence of the PPA-1 polypeptide is: NYSKPTDRQYHF (SEQ ID NO: 3), and the above amino acids are all in D configuration.

[0019] Specifically, the amino acid sequence of the Pep-39 polypeptide is: AMSDHHWTQRDK (SEQ ID NO: 4).

[0020] As a preferred embodiment of the present invention, the cleavable group is a group that can be cleaved by a substance highly expressed in the tumor site, including but not limited to an MMP2-responsive cleavable group (i.e., a group that can be recognized and cleaved by the MMP2 enzyme highly expressed in the tumor tissue), a pH-responsive cleavable group (i.e., a group that can respond to the acidic microenvironment of the tumor to achieve cleavage), a GSH-responsive cleavable group (i.e., a group that can react with excess glutathione (GSH) in tumor cells to achieve cleavage), and a ROS-responsive cleavable group (e.g., thioketal (TK) is a ROS-responsive group, and the chemical bond of thioketal breaks in a high-level ROS environment to achieve drug release).

[0021] Specifically, the MMP2-responsive cleavable group is an MMP2 enzyme-cleavable peptide (also called an MMP2 enzyme-cleaved fragment), and its amino acid sequence is: PLGLAG.

[0022] As a preferred embodiment of the present invention, the bifunctional peptide has Smac polypeptide as the hydrophobic end, D The PPA-1 polypeptide serves as the hydrophilic end and is connected by a peptide cleavable by the MMP2 enzyme.

[0023] Specifically, the amino acid sequence of the bifunctional peptide is: AVPIAQK-LL-PLGLAG-NYSKPTDRQYHF (SEQ ID NO: 1), referred to as Smac-D1 polypeptide.

[0024] A method for preparing a multifunctional nanocarrier targeting tumor stem cells comprises the following steps:

[0025] The bifunctional peptide and the hydrophobic drug for upregulating the expression of MHC I molecules are self-assembled through hydrophobic interaction to obtain a multifunctional nanocarrier targeting tumor stem cells.

[0026] As a preferred embodiment of the present invention, the mass ratio of the bifunctional peptide to the hydrophobic drug for upregulating the expression of MHC I molecules is 1-5:1.

[0027] As a preferred embodiment of the present invention, the preparation method comprises the following steps: uniformly mixing the bifunctional peptide, the hydrophobic drug for up-regulating the expression of MHC I molecules and DMSO, then dropping them into water for ultrasonic treatment, during which the hydrophobic properties of the hydrophobic drug for up-regulating the expression of MHC I molecules cause it to be encapsulated in the hydrophobic core of the nanocarrier, and standing to obtain the product.

[0028] Specifically, the conditions of the ultrasonic treatment are: temperature 20-30° C., ultrasonic power 450-500 W, frequency 30-50 kHz, and time 10-30 min.

[0029] Application of a multifunctional nanocarrier targeting tumor stem cells in the preparation of anti-tumor drugs.

[0030] An anti-tumor drug, wherein the drug efficacy component of the anti-tumor drug comprises a multifunctional nanocarrier targeting tumor stem cells.

[0031] As a preferred embodiment of the present invention, the active ingredients of the drug further include other auxiliary anti-tumor ingredients, including but not limited to RT radiotherapy, and the radiotherapy dose is 8-12Gy.

[0032] As a preferred embodiment of the present invention, the content of the multifunctional nanocarrier targeting tumor stem cells in the drug is an effective amount, which can be in the range of 0.01wt%-99.9wt%.

[0033] As a preferred embodiment of the present invention, the anti-tumor drug further comprises a pharmaceutically acceptable carrier or excipient, including but not limited to excipients, preservatives, stabilizers, wetting agents, emulsifiers, salts for adjusting osmotic pressure, and buffers.

[0034] As a preferred embodiment of the present invention, the dosage form of the anti-tumor drug is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection solution, dispersion solution, spray, capsule, tablet, and pill.

[0035] Beneficial effects of the present invention:

[0036] The present invention provides a nanocarrier that integrates the functions of radiosensitization, upregulation of MHC I on the surface of CSCs, and targeting of PD-1 / PD-L1 immune checkpoints, which can act on multiple pathways and multiple targets at the same time to exert a synergistic effect. First, the nanocarrier can enhance radiotherapy-mediated cell apoptosis, induce stronger immunogenic cell death (ICD), and activate adaptive immune responses, providing a basis for subsequent anti-tumor immunotherapy; at the same time, it can also curb the occurrence of CSCs immune escape behavior and increase the sensitivity of CSCs to radiotherapy-induced immune responses. Under the synergistic effect of the above two aspects, the proportion of CSCs after radiotherapy is effectively reduced, thereby effectively eliminating the residual CSCs in the tumor tissue after radiotherapy, providing a new treatment strategy for inhibiting the recurrence and metastasis of postoperative tumors.

[0037] The multifunctional nanocarrier targeting tumor stem cells provided by the present invention can use the radiosensitizing peptide Smac as the hydrophobic end, connect with the hydrophilic end anti-enzymatic PD-1 / PD-L1 blocking peptide (D1 peptide) through the MMP2 enzyme cleavage sequence (PLGLAG peptide segment), self-assemble to form a nanocarrier driven by hydrophobic interaction force, and the hydrophobic drug Taz is encapsulated in the hydrophobic core of the nanocarrier, thereby successfully constructing the full carrier Smac-D1@Taz. The multifunctional Smac-D1@Taz self-assembling nanocarrier can overcome the multi-factor treatment dilemma of cancer, and by integrating the three drugs into the same system for delivery, it can promote the synergistic effect of the drugs in vivo.

[0038] The MMP2-responsive polypeptide self-assembly nanocarrier Smac-D1@Taz prepared by the present invention has a spherical structure of uniform size and uniform dispersion, and can effectively load drugs. Among them, the D1 peptide in the outermost layer is a D configuration, which gives the carrier the ability to prolong the in vivo circulation time and enhance stability. Due to the presence of the MMP2 enzyme cleavage sequence in the carrier, the drug can be accurately released in tumor tissues with high expression of the MMP2 enzyme, providing a basis for the effective anti-tumor effect.

[0039] The multifunctional nanocarrier targeted to tumor stem cells provided by the present invention has multifunctional integration, a simple preparation process, and is easy to operate, and is particularly suitable for large-scale and standardized production. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the characterization of the polypeptide self-assembled nanocarrier in the examples.

[0041] Figure 2 This is the effect of nanocarriers on apoptosis and cell viability of 4T1 and 4T1 tumor sphere cells in the experimental example.

[0042] Figure 3 This is the effect of the nanocarrier on the expression of MHC I on the surface of 4T1 tumor spheres and tumor cells in the experimental example.

[0043] Figure 4 This is the effect of nanocarriers on T cell IL-2 secretion in the experimental example.

[0044] Figure 5 This is the in vivo distribution of the nanocarriers in the experimental example.

[0045] Figure 6 This is the inhibitory effect of nanocarriers combined with radiotherapy on 4T1 tumor growth in vivo in the experimental example.

[0046] In order to more clearly illustrate the technical solution to be protected by the present invention, the drawings in the embodiments and experimental examples are briefly introduced above. It should be understood that the above drawings should not be regarded as any limitation on the protection scope of the present invention. For ordinary technicians in this field, other relevant drawings can also be obtained based on these drawings without paying creative work. DETAILED DESCRIPTION

[0047] The technical solution of the present invention will be clearly and completely explained below in conjunction with specific embodiments and experimental examples.

[0048] Those skilled in the art should understand that the following embodiments and experimental examples are only used to illustrate the technical solutions and technical effects of the present invention and should not be regarded as any limitation on the protection scope of the present invention. Based on the following embodiments, other technical solutions obtained by those skilled in the art without creative work, such as technical solutions obtained after modification, deformation or simple replacement, all belong to the protection scope of the present invention.

[0049] Unless otherwise specified, the raw materials, reagents, and equipment used in the embodiments and experimental examples are all commercially available products.

[0050] The methods used in the embodiments and experimental examples are conventional methods unless otherwise specified.

[0051] The terms and abbreviations used in the embodiments and experimental examples have the conventional meanings in the art, such as PBS (pH 7.2) is phosphate buffered saline.

[0052] Example

[0053] This embodiment provides a multifunctional nanocarrier targeting tumor stem cells, which is obtained by self-assembly through hydrophobic interaction using a bifunctional peptide Smac-D1 and a hydrophobic drug Taz as raw materials. The amino acid sequence of the bifunctional peptide Smac-D1 is: AVPIAQK (radiotherapy sensitizing peptide Smac)-LL-PLGLAG (MMP2 enzyme cleavable peptide)-NYSKPTDRQYHF (anti-enzymatic PD-1 / PD-L1 blocking peptide) D PPA-1).

[0054] This example provides a method for preparing a multifunctional nanocarrier targeting tumor stem cells (see the schematic diagram of the synthesis scheme Figure 1 ), including the following steps:

[0055] (1) Peptide preparation: Smac-D1 (AVPIAQK-LL-PLGLAG-NYSKPTDRQYHF) was synthesized by the Fmoc solid phase method, and the peptide was separated and purified by a reverse HPLC system. The purified peptide was identified by mass spectrometry to confirm that the sequence of the synthesized peptide was correct and stored for future use.

[0056] (2) Construction of the whole vector: 7.5 mg of Smac-D1 peptide and 1.5 mg of Taz hydrophobic drug were weighed into a 4 mL EP tube and dissolved in 200 μL DMSO. The DMSO solution mixed with Smac-D1 and Taz was then added dropwise to 1.3 mL deionized water. The 4 mL EP tube was transferred to an ultrasonicator for ultrasonication (ultrasonic power 475.2 W, frequency 40 kHz, 25 °C, 20 min). During the ultrasonication, Smac-D1 and Taz self-assembled through hydrophobic interaction forces. Taz was encapsulated into the hydrophobic core of the nanocarrier due to its own hydrophobic properties. The nanocarrier was then allowed to stand at room temperature for 30 min to stabilize, thereby obtaining the multifunctional nanocarrier Smac-D1@Taz. The size is shown in the experimental example.

[0057] This embodiment provides an application of a multifunctional nanocarrier targeting tumor stem cells in the preparation of an anti-tumor drug.

[0058] This embodiment provides an anti-tumor drug, comprising a pharmaceutically effective amount of a multifunctional nanocarrier targeting tumor stem cells, and an appropriate amount of a pharmaceutically acceptable carrier and excipient, for preparing the drug into an injection dosage form.

[0059] In other embodiments of the present invention, the anti-enzymatic PD-1 / PD-L1 blocking peptide may be a Pep-39 polypeptide.

[0060] In other embodiments of the present invention, the cleavable group is a ROS-responsive cleavable group thiolketal (TK).

[0061] In other embodiments of the present invention, the hydrophobic drug that upregulates the expression of MHC I molecules on the surface of CSCs is docetaxel.

[0062] In other embodiments of the present invention, the mass ratio of the bifunctional peptide to the hydrophobic drug for upregulating the expression of MHC I molecules can be any value within the range of 1-5:1.

[0063] In other embodiments of the present invention, the conditions of the ultrasonic treatment can be arbitrarily set within a given range without substantially affecting the physicochemical properties and functions of the nanocarrier.

[0064] In other embodiments of the present invention, the anti-tumor drug can be prepared into other dosage forms that are easy to carry and take, and the content of the nanocarrier in the drug is the effective amount, and corresponding amounts of pharmaceutically acceptable carriers and excipients can be added.

[0065] Experimental example

[0066] 1. Experimental Methods

[0067] 1. Zeta potential and particle size distribution

[0068] 1 mL of the nanocarrier prepared in the example was taken and placed in a potential cup and a particle size cup respectively, and the particle size and Zeta potential of the nanocarrier were measured by a Malvern particle size potential instrument. The results are as follows: Figure 1 shown.

[0069] 2. TEM morphology characterization

[0070] The dried copper mesh was placed in a bacterial dish, and the nanocarriers prepared in the example (1 mg / mL) were dripped onto the surface of the copper mesh, and allowed to stand for 2 minutes. Excess liquid was removed with absorbent paper, and the sample was dripped again, and repeated 3 times. Negative staining was performed using uranyl acetate, specifically: the supernatant of uranyl acetate after centrifugation was taken, dripped onto the surface of the copper mesh, and allowed to stand for 2 minutes. Excess dye was removed after the staining was completed, and the prepared copper mesh was placed in a fume hood and dried naturally overnight. TEM images of the nanocarriers were then taken using TEM, and the results were as follows: Figure 1 shown.

[0071] 3. MMP2 enzyme digestion experiment

[0072] 1.8 mg of Smac-D1 polypeptide and 1.73 mg of Smac-cp-D1 polypeptide (to verify the responsiveness of Smac-D1 to MMP2, the restriction sequence PLGLAG was replaced with PGGGAG to synthesize the control peptide Smac-cp-D1 with no responsive effect: AVPIAQK-LL-PGGGAG-NYSKPTDRQYHF, molecular weight 2886.22 Da) were weighed and dissolved in 3 mL TCNB (50 mM Tris, 10 mM CaCl2, 150 mM NaCl, 160 μL 30% Brij, 100 mL triple distilled water) to a final concentration of 200 μM. The enzyme was activated before adding the MMP2 enzyme, 4.5 μL of the unactivated enzyme solution was taken, 0.5 μL APMA (10 mM) was added, and the mixture was incubated at 37°C for 1 h, and finally 1.125 mL of the prepared TCNB solution was added to dilute the activated MMP2 enzyme to 0.4 μg / mL. Then, 100 μL of Smac-D1 and Smac-cp-D1 solution was incubated with 100 μL of activated MMP2 enzyme at 37°C for 3 h. 50 μL of Smac-D1 and Smac-cp-D1 solution after MMP2 enzyme cleavage was taken, and the effect of peptide release in response to MMP2 enzyme cleavage was detected by mass spectrometry. The morphological changes of nanocarriers after MMP2 enzyme cleavage were observed by TEM. The results are as follows: Figure 1 shown.

[0073] 4. Study on the in vitro radiotherapy sensitization effect of multifunctional nanocarrier Smac-D1@Taz

[0074] (1) 4T1 tumor sphere plating: 4T1 tumor spheres induced to spheroidize for 14 days were collected and digested with 0.5% trypsin at 37°C for 5 min. The digestion was terminated with 1640 medium containing 10% FBS, washed twice with PBS (pH 7.2), resuspended in dry medium and the cell density was adjusted to 2×10 4 The cells were then seeded in ultra-low adsorption 6-well plates (2 mL system per well).

[0075] (2) Drug addition: After the cells were stable for 12 h, three replicate wells were set up in each group and different drugs were added for 12 h. The experimental groups were as follows: PBS (pH 7.2), Smac-D1@Taz, Smac-cp-D1@Taz and C16-D1@Taz.

[0076] To verify the responsiveness of Smac-D1 to MMP2, the restriction sequence PLGLAG was replaced with PGGGAG to synthesize the control peptide Smac-cp-D1 (AVPIAQK-LL-PGGGAG-NYSKPTDRQYHF, molecular weight 2886.22Da) with no responsiveness. At the same time, to verify the radiosensitization effect of Smac peptide, the Smac peptide segment was replaced with palmitic acid C16 to eliminate its radiosensitization effect, and C16-D1 (C16-PLGLAG-NYSKPTDRQYHF, molecular weight 2302.67Da) was synthesized as a control peptide. The preparation method of Smac-cp-D1@Taz and C16-D1@Taz groups is the same as that of Smac-D1@Taz.

[0077] (3) Irradiation: After 12 h of drug treatment, the cells were irradiated with a dose of 6 Gy, and then the well plate was placed in an incubator and cultured for a further 72 h.

[0078] (4) Sample collection: Collect cells into 1.5 mL EP tubes, centrifuge (3000 rpm, 5 min, 4°C), discard the supernatant, add 500 μL 0.5% trypsin to digest for 5 min, then add 1 mL 1640 medium containing 10% FBS to terminate digestion, centrifuge (3000 rpm, 5 min, 4°C), discard the supernatant, add 1 mL PBS (pH 7.2) to resuspend the cells, centrifuge (3000 rpm, 5 min, 4°C), discard the supernatant, and add 250 μL 1× Binding Buffer to each tube to resuspend the cells.

[0079] (5) Staining: Add 2.5 μL Annexin V-APC and 5 μL 7-AAD to each tube, vortex gently to mix, and incubate at room temperature in the dark for 5 min.

[0080] (6) Detection: The stained cells are transferred to the flow cytometry tube and tested on the machine. The results are as follows: Figure 2 shown.

[0081] The CCK-8 experimental method is as follows:

[0082] (1) 4T1 plating: 4T1 cells in the logarithmic growth phase were digested with 0.5% trypsin, resuspended into a single cell suspension using 1640 medium containing 10% FBS, and counted. 3 The cells were plated at a density of 10 cells / mL in a flat-bottom 96-well plate (200 μL system per well), and the plate was placed in a 37° C., 5% CO 2 cell culture incubator for culture.

[0083] (2) 4T1 tumor sphere plating: 4T1 tumor spheres induced to spheroidize for 14 days were collected and digested with 0.5% trypsin at 37°C for 5 min. The digestion was terminated with 1640 medium containing 10% FBS, washed twice with PBS (pH 7.2), resuspended in dry medium, and the cell density was adjusted to 5×10 3 The cells were seeded in a flat-bottom 96-well plate (200 μL system per well).

[0084] (3) Synchronization: On the second day, carefully remove the old culture medium, wash once with PBS, add 160 μL of serum-free 1640 culture medium, and starve the cells for 7 h (6-8 h is acceptable) to allow the cells to grow synchronously.

[0085] (4) Drug addition: Experimental groups were set up, and 3 replicate wells were set up in each group, divided into zero-adjustment wells: no cell and culture medium group; control group: PBS 7.2 group; experimental group: Smac-D1@Taz group, Smac-cp-D1@Taz group and C16-D1@Taz group. Drugs diluted with 20 μL serum-free culture medium were added to each well (try to avoid the generation of bubbles to avoid affecting the OD value reading). The drug concentration was quantified by Taz: 12 μg / mL, and the amount of carrier was substituted into the drug loading calculation formula. Then 20 μL serum was added (this step was omitted for tumor spheres), and the cells were irradiated with 6 Gy after culturing for 12 hours.

[0086] (5) Color development: 4 hours before the 72-h time point, add 10 μL of CCK-8 to each well, mix thoroughly, and place the plate in an incubator for a further 4 h.

[0087] (6) Reading: Use an ELISA reader to read the OD value of the sample at a wavelength of 450 nm. After reading, save and analyze the data. The results are as follows: Figure 2 shown.

[0088] 5. Effect of Smac-D1@Taz on cell surface MHC I expression

[0089] (1) 4T1 tumor sphere plating: 4T1 tumor spheres induced to sphere formation for 14 days were collected and digested with 0.5% trypsin at 37°C for 5 min. The digestion was terminated with 1640 medium containing 10% FBS, washed twice with PBS (pH 7.2), resuspended with tumor sphere culture medium and the cell density was adjusted to 5×10 4 cell / mL, and the cells were seeded in ultra-low adsorption 6-well plates (2 mL system per well).

[0090] (2) Drug addition: After the cells in each group were stable for 12 h, three replicate wells were set up in each group, and different drugs were added for treatment for 48 h. The well plates were then placed in a 37°C, 5% CO2 cell culture incubator for continued culture. The experimental groups were as follows: PBS (pH 7.2) group, Taz group, Smac-D1 group, and Smac-D1@Taz group.

[0091] (3) Sample collection: Collect cells into 1.5 mL EP tubes, centrifuge (3000 rpm, 5 min, 4°C), discard the supernatant, add 500 μL 0.5% trypsin to digest for 5 min, then add 1 mL 1640 medium containing 10% FBS to terminate digestion, centrifuge (3000 rpm, 5 min, 4°C), discard the supernatant, add 1 mL PBS (pH 7.2) to resuspend the cells, centrifuge (3000 rpm, 5 min, 4°C), discard the supernatant, and resuspend the cells in 50 μL PBS (pH 7.2) per tube.

[0092] (4) Flow cytometry antibody staining: Add anti-mouse MHC Class I (H-2Kd) APC antibody to each tube, vortex gently to mix, and incubate on ice in the dark for 30 min.

[0093] (5) Detection: After staining, add 1 mL of PBS (pH 7.2) to each tube to wash away unbound antibodies, centrifuge (3000 rpm, 5 min, 4°C), and then transfer the sample to a flow cytometer for detection. The results are as follows: Figure 3 shown.

[0094] 6. Co-culture experiment of Jurkat cells and CHO-K1-hPD-L1 cells

[0095] (1) CHO-K1-hPD-L1 cell plating: CHO-K1-hPD-L1 cells with good growth status were collected and the cell density was adjusted to 1×10 using 1640 medium containing 10% FBS. 5 cell / mL, 500 μL of cell suspension was inoculated into a 24-well plate, and the plate was placed in a 37°C, 5% CO2 cell culture incubator for 12 h.

[0096] (2) Co-culture: Jurkat cells stimulated with 25 ng / mL PMA and 1 μg / mL PHA for 24 h were collected and the cell density was adjusted to 4×10 5cell / mL, 500 μL of Jurkat cell suspension was extracted and the cells were inoculated into the 24-well plate of (1), and three replicate wells were set up in each group to add different drugs respectively. Then the well plate was placed in a cell culture incubator at 37°C and 5% CO2 for further culture for 48 h. The experimental groups were as follows: Jurkat group, Jurkat+CHO-K1-hPD-L1(J+C) group, J+C+D1 group, J+C+LAG-D1 group (LAG-D1 is one of the polypeptides of Smac-D1 after MMP2 cleavage, and the other is Smac-LL-PLG), J+C+Smac-D1@Taz group and J+C+Smac-cp-D1@Taz group.

[0097] (3) Addition of protein transport inhibitors: 4 h before the end of co-culture, add 1 μL of protein transport inhibitor to each well to make the final concentration 1 μL / mL (1 mL system per well), and continue culturing in the incubator for 4 h.

[0098] (4) Surface antibody staining: The cells were collected into a 1.5 mL EP tube, filled with PBS (pH 7.2) and centrifuged (3500 rpm, 5 min, 4°C). The supernatant was discarded and the cells were resuspended in 50 μL PBS (pH 7.2). Anti-human CD45 FITC antibody was added and the EP tube was placed on ice and incubated for 30 min in the dark.

[0099] (5) Intracellular antibody staining: After staining, add 1 mL of PBS (pH 7.2) to the tube for centrifugal washing (3500 rpm, 5 min, 4°C), discard the supernatant (keep a portion of the liquid), place the cells on a vortex to mix thoroughly, then add 200 μL of 4% paraformaldehyde fixative and fix at room temperature for 30 min. After fixation, add 800 μL of membrane permeabilization agent and centrifuge (3500 rpm, 5 min, 4°C), discard the supernatant, resuspend the cells with 50 μL of membrane permeabilization agent, add anti-human IL-2APC antibody, and then place the EP tube on ice and incubate for 30 min in the dark.

[0100] (6) Flow cytometry: After staining, add 1 mL of PBS (pH 7.2) and centrifuge (3500 rpm, 5 min, 4°C). Resuspend with 250 μL of PBS (pH 7.2) and transfer the cell suspension to a flow cytometer for detection. The results are as follows: Figure 4 shown.

[0101] 7. Study on the distribution of Smac-D1@Taz in vivo

[0102] (1) Construction of 4T1 subcutaneous tumor model: BALB / c female mice of appropriate age of 6-8 weeks were selected to bear tumors with a tumor load of 1×10 6Use a pipette to gently blow the cell suspension evenly, draw 200 μL of 4T1 cell suspension with a 1 mL syringe, and carefully inoculate the cell suspension into the subcutaneous area on the right side of the mouse's back.

[0103] (2) Monitor and record the growth of mouse tumors until the tumor volume grows to 150-200mm 3 The mice were randomly divided into three groups: control group, free DIR group and Smac-D1@DIR group. The drug concentration was quantitatively measured by DIR (0.5 mg / kg). The mice were anesthetized by gas at 1h, 4h, 8h, 12h, 24h and 48h after the injection of nanocarriers. After the mice were completely anesthetized, the in vivo distribution of nanocarriers was observed and recorded using a living imaging device. After 72h, the heart, liver, spleen, lung, kidney, brain and tumor of the mice were removed using surgical instruments for imaging. The results are shown in Figure 2. Figure 5 shown.

[0104] 8. Tumor-bearing mouse treatment model

[0105] (1) On the seventh day of tumor bearing, when the tumor grows to 40-80 mm 3 At the same time, the mice were treated with drugs by tail vein injection and were divided into the following groups: Control group, RT group (10 Gy radiotherapy was performed on the subcutaneous tumors on the back of mice), Taz group, Smac-D1@Taz group, Smac-D1@Taz+RT group and C16-D1@Taz+RT group.

[0106] (2) The drug injection dose for each group was calculated according to the amount of Taz in the nanocarrier (5 mg / kg). The injection solution was prepared with PBS (pH 7.2), and 200 μL of the injection solution was drawn using a 1 mL syringe and administered via tail vein injection. The control group was injected with the same volume of PBS (pH 7.2) as a control. The drug was administered once every 3 days for a total of 6 times. 24 hours after the first administration, the mouse back tumor was subjected to local radiotherapy at a dose of 10 Gy.

[0107] (3) During the drug administration period, the weight of the mice was weighed and recorded every two days so that the weight change curve could be drawn later to observe the effect of the drug on the weight of the mice. At the same time, a vernier caliper was used to measure the length (a) and width (b) of the tumor on the back of each mouse. According to the formula: tumor volume = 0.5 × a × b 2 Calculate the tumor volume and draw a tumor growth curve. The results are as follows Figure 6 shown.

[0108] 2. Experimental Results

[0109] 1. Characterization of Smac-D1@Taz

[0110] The physicochemical properties of the MMP2-responsive peptide self-assembled nanocarrier Smac-D1@Taz prepared in the example were characterized. Transmission electron microscopy (TEM) results showed that Smac-D1 and Smac-D1@Taz both had good dispersibility and presented a spherical structure of uniform size. Combined with the results of dynamic light scattering (DLS) measurement, the average hydrated particle sizes of Smac-D1 and Smac-D1@Taz were 132.27±1.54nm and 144.93±3.00nm, respectively. Figure 1 b, c), the particle size measured by DLS is slightly larger than that by TEM. This is because the size measured by TEM is the actual size of the sample in a dry state. After Smac-D1 was loaded with Taz, the Zeta potential dropped from 29.93±1.96mV to -3.79±0.28mV ( Figure 1 d). The above results all indicate that the multifunctional nanocarrier Smac-D1@Taz was successfully constructed.

[0111] In order to evaluate whether MMP2 can effectively cleave Smac-D1 and release two functional peptides (Smac-LL-PLG, LAG-D1), an experiment was designed to co-incubate Smac-D1 with MMP2 for 3 h. The LC-MS detection results showed the characteristic peaks of two functional peptides ( Figure 1 eg).

[0112] 2. Pro-apoptotic effect of Smac-D1@Taz

[0113] The effects of the multifunctional nanocarrier Smac-D1@Taz on apoptosis and cell viability of 4T1 and CSCs cells were studied by using the AnnexinV / 7-AAD cell apoptosis detection kit and CCK-8 assay. Figure 2 As shown in Figure 2, after being treated with Smac-D1@Taz, the sensitivity of both cells to radiotherapy increased significantly, and the total apoptosis rates of 4T1 and 4T1 tumor spheres reached about 36% and 26%, respectively, which was 3 times that of the PBS group ( Figure 2 A, B). Due to the lack of Smac peptide and limited release, C16-D1@Taz and Smac-cp-D1@Taz groups could not effectively induce apoptosis of 4T1 and 4T1 tumor sphere cells. In the CCK-8 experiment, after the cells were treated with Smac-D1@Taz for 12 hours, the OD value at 450nm was significantly reduced, and the cell viability was significantly inhibited ( Figure 2 C). The above results indicate that Smac-D1@Taz is an effective radiosensitizer that promotes apoptosis of 4T1 and 4T1 tumor sphere cells.

[0114] 3. Smac-D1@Taz upregulates MHC I on the surface of CSCs and 4T1 cells

[0115] In order to evaluate the ability of Smac-D1@Taz to upregulate MHC I, an experiment was designed to construct a 4T1 tumor sphere model enriched with CSCs, and the fluorescence intensity of MHC I (H-2Kd) on the surface of CSCs and 4T1 was detected by flow cytometry and immunofluorescence experiments. Figure 3 As shown in Figure 2, after 48 h of treatment with Taz and Smac-D1@Taz, flow cytometry results showed that the MHC I signal on the surface of CSCs and 4T1 was upregulated by 2-3 times compared with that in the PBS group ( Figure 3 A, B, D, E). DAPI was used to stain the nuclei of CSCs and 4T1 cells. Consistent with the flow cytometry results, a significantly upregulated red fluorescent signal was observed on the cell membrane surface ( Figure 3 C, F).

[0116] 4. Smac-D1@Taz blocks PD-1 / PD-L1 binding to CD8 + Effects on T cell function

[0117] Jurkat cells were co-cultured with CHO-K1-hPD-L1 in vitro and treated with different drug groups. The effect of Smac-D1@Taz on T cell function was detected by flow cytometry, and the D1 peptide alone was used as a positive control. Figure 4 As shown, co-culture of activated Jurkat cells with CHO-K1-hPD-L1 cells significantly reduced Jurkat's ability to secrete IL-2. When the multifunctional nanocarrier Smac-D1@Taz was added, Jurkat's ability to secrete IL-2 was restored, which to some extent alleviated the inhibitory effect of CHO-K1-hPD-L1 on Jurkat. In addition, compared with the Smac-cp-D1@Taz group, no significant upregulation of IL-2 was observed due to the limited release of the D1 peptide. There was no significant difference in IL-2 secretion between the LAG-D1 group and the D1 group, indicating that the presence of the three amino acids of LAG does not affect the blocking function of the D1 peptide ( Figure 4 A, B).

[0118] 5. Distribution of Smac-D1@Taz in vivo

[0119] The effective accumulation of nanocarriers in the tumor site is a prerequisite for their therapeutic effect. To this end, an experiment was designed to construct a 4T1 subcutaneous transplant tumor model to further investigate the distribution of the nanocarrier in vivo. Free DIR and nanocarrier-encapsulated DIR (Smac-D1@DIR) were set up to investigate the accumulation of nanocarriers in the tumor site. Free DIR and Smac-D1@DIR were injected into the tail vein, and mice were anesthetized and fluorescently imaged at 1, 4, 8, 12, 24, and 48 hours after administration. At 72 hours after administration, tumors and major tissues were taken for in vitro imaging analysis and fluorescence quantitative statistics of the images were performed to evaluate the ability of Smac-D1@DIR to accumulate in the tumor site.

[0120] The experimental results are as follows Figure 5 As shown in Figure 2, at 12 h, the accumulation of Smac-D1@DIR began to be observed in the tumor site, and the fluorescence gradually increased over time ( Figure 5 A). At 72 hours, the tumor, heart, liver, spleen, lung and kidney of the mice were taken for in vitro imaging, and the results were consistent with the in vivo results. Compared with free DIR, DIR carried by Smac-D1 showed higher accumulation in the tumor site, showing enhanced fluorescence signal ( Figure 5 B). The above results show that nanocarriers can carry drugs and effectively accumulate them in tumor sites, providing a basis for exerting anti-tumor effects.

[0121] 6. Inhibitory effect of Smac-D1@Taz combined with radiotherapy on 4T1 tumor growth

[0122] In order to evaluate the anti-tumor effect of Smac-D1@Taz in vivo, 4T1 cells were implanted subcutaneously in the back of 6-8-week-old BALB / c female mice to construct a 4T1 tumor model. On the 7th day after tumor inoculation, mice were treated with PBS (pH 7.2), RT, Taz, Smac-D1@Taz, Smac-D1@Taz and C16-D1@Taz drugs by tail vein injection. The drugs were administered every three days for a total of 6 times. After 24 hours, the tumors of mice in the RT group and the combined treatment group were treated with a single local radiotherapy of 10 Gy ( Figure 6 A) Starting from the first administration, the weight and tumor volume of the mice were recorded every other day. Figure 6 As shown in the figure, from the tumor growth curve and the weight of the in vitro tumor, compared with PBS, the other groups can inhibit tumor growth to a certain extent, among which the Smac-D1@Taz+RT combination group has the strongest anti-tumor activity. Although the anti-tumor effect of the C16-D1@Taz+RT group is better than that of the Smac-D1@Taz or RT alone group, since C16-D1@Taz+RT does not have a radiosensitizing effect, its ability to inhibit tumor growth is slightly lower than that of the Smac-D1@Taz+RT group ( Figure 6 BE). In order to evaluate the safety of nanocarriers in vivo, the body weight of mice was measured during the treatment. The statistical results showed that there was no significant change in the body weight of mice in each group ( Figure 6 F). Since Smac-D1@Taz can enhance radiotherapy-mediated cell apoptosis, TUNEL staining (yellow) was performed on the tumor tissues of each group, and immunohistochemical analysis of Ki67 expression was performed. The results showed that Smac-D1@Taz combined with radiotherapy can significantly induce cell apoptosis and inhibit tumor cell proliferation ( Figure 6 GH).

[0123] Although the technical scheme and technical effects of the present invention have been described in detail above with general descriptions, specific embodiments and experimental examples, for those skilled in the art, any modification, replacement or improvement made without departing from the spirit of the present invention shall fall within the scope of protection of the present invention.

Claims

1. A multifunctional nanocarrier targeting tumor stem cells, characterized in that: The nanocarrier is obtained by self-assembly using a bifunctional peptide and a hydrophobic drug that upregulates the expression of MHC I molecules as raw materials; The bifunctional peptide is obtained by connecting a radiosensitizing peptide as a hydrophobic end and an anti-enzymatic PD-1 / PD-L1 blocking peptide as a hydrophilic end via a cleavable group.

2. The multifunctional nanocarrier targeting tumor stem cells according to claim 1, characterized in that: The size of the nanocarrier is 50-300 nm.

3. The multifunctional nanocarrier targeting tumor stem cells according to claim 1, characterized in that: The hydrophobic drugs that upregulate the expression of MHC I molecules include, but are not limited to, one or more of tazemetostat, docetaxel, and decitabine; And / or, the radiosensitizing peptide includes but is not limited to Smac polypeptide; The amino acid sequence of the Smac polypeptide is: AVPIAQK; And / or, the anti-enzymatic PD-1 / PD-L1 blocking peptides include but are not limited to D One or both of PPA-1 polypeptide and Pep-39 polypeptide; Said D The amino acid sequence of the PPA-1 polypeptide is: NYSKPTDRQYHF, and the above amino acids are all in D configuration; The amino acid sequence of the Pep-39 polypeptide is: AMSDHHWTQRDK; and / or, the cleavable group includes but is not limited to a MMP2-responsive cleavable group, a pH-responsive cleavable group, a GSH-responsive cleavable group, a ROS-responsive cleavable group; The MMP2-responsive cleavable group is an MMP2 enzyme-cleavable peptide, and its amino acid sequence is: PLGLAG.

4. The multifunctional nanocarrier targeting tumor stem cells according to claim 1, characterized in that: The amino acid sequence of the bifunctional peptide is: AVPIAQK-LL-PLGLAG-NYSKPTDRQYHF.

5. A method for preparing a multifunctional nanocarrier targeting tumor stem cells according to any one of claims 1 to 4, characterized in that: The following steps are involved: The bifunctional peptide and the hydrophobic drug for upregulating the expression of MHC I molecules are self-assembled through hydrophobic interaction to obtain a multifunctional nanocarrier targeting tumor stem cells.

6. The preparation method according to claim 5, characterized in that: The mass ratio of the bifunctional peptide to the hydrophobic drug for upregulating the expression of MHC I molecules is 1-5:

1.

7. The preparation method according to claim 5, characterized in that: The method comprises the following steps: uniformly mixing the bifunctional peptide, the hydrophobic drug for up-regulating the expression of MHC I molecules and DMSO, then dripping them into water for ultrasonic treatment, and standing to obtain the product.

8. Use of the multifunctional nanocarrier targeting tumor stem cells according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.

9. An anti-tumor drug, characterized in that: The pharmaceutical ingredient of the anti-tumor drug comprises the multifunctional nanocarrier targeting tumor stem cells as described in any one of claims 1 to 4.

10. The anti-tumor drug according to claim 9, characterized in that: The active ingredients of the drug also include other auxiliary anti-tumor ingredients, including but not limited to RT radiotherapy, and the radiotherapy dose is 8-12Gy. And / or, the content of the multifunctional nanocarrier targeting tumor stem cells in the drug is 0.01wt%-99.9wt%; And / or, the anti-tumor drug further comprises a pharmaceutically acceptable carrier or excipient, including but not limited to an excipient, a preservative, a stabilizer, a wetting agent, an emulsifier, a salt for adjusting osmotic pressure, and a buffer; And / or, the dosage form of the anti-tumor drug is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection solution, dispersion solution, spray, capsule, tablet, and pill.

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