Tumor cell treatment method based on polypeptide nanotechnology

Abstract

The invention belongs to the technical field of nano medical treatment, and discloses a tumor cell treatment method based on a polypeptide nano technology, which comprises the following specific steps: step 1, material synthesis and characterization: synthesizing a polypeptide material by adopting a standard Fmoc-SPPS method, performing corresponding modification, taking a small amount of trifluoroacetic acid as a catalyst, and performing ultrasonic treatment on the polypeptide material; beta-CD-NH2 and cinnamyl aldehyde (CA) are connected through a Schiff base reaction, a material beta-CD-CA (CCA) is obtained through cold ether precipitation and drying, and the molecular weight is represented by mass spectrometry. Oxidative stress of tumor cells is amplified through polypeptide nanoparticles, oxidation-reduction balance of the tumor cells is destroyed, so that immunogen cell death (I CD) is induced, and in combination with a subject-object drug carrier system, polypeptide fibrosis deformation, NO gas generation and other mechanisms, cinnamyl aldehyde is gradually released in the cells, NO gas is generated, and the tumor cell death effect is improved. Further, the release of ROS is increased through the combination of the polypeptide and mitochondria, so that the function disorder of the mitochondria is aggravated, the redox imbalance in the cells is finally caused, the immunogenic death of the tumor cells is induced, and the tumor cells can be directly killed.

Description

Technical Field

[0001] The present invention belongs to the field of nanomedical technology, and specifically is a tumor cell treatment method based on polypeptide nanotechnology. Background Art

[0002] A tumor refers to a disease in which a space-occupying tumor is formed due to abnormal proliferation of local tissue cells. It is a neoplasm formed by the long-term abnormal proliferation of tissue cells in a part of the body. It is harmful to the body. Common symptoms include: lumps or swelling, persistent pain, digestive system problems, difficulty breathing or persistent cough, weight changes, persistent fatigue, skin changes, bone abnormalities, etc.

[0003] Cancer treatment is a major challenge in modern medicine. Although chemotherapy and radiotherapy have significant effects in inhibiting tumor growth, they are usually accompanied by serious side effects, such as hair loss, nausea, immunosuppression, etc., and inevitably damage normal cells. In addition, tumor cells often show drug resistance, resulting in reduced treatment efficacy. With the deepening of understanding of tumor biological characteristics, immunotherapy as an emerging treatment method has gradually become a research hotspot, especially the application of immune checkpoint inhibitors and cancer vaccines. However, immunotherapy also faces problems such as tumor immune escape and tumor heterogeneity. Therefore, finding more precise, effective and less side-effect treatment strategies has become an urgent need for today's tumor research and treatment. Summary of the invention

[0004] The purpose of the present invention is to provide a tumor cell treatment method based on polypeptide nanotechnology to solve the problems raised in the above background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solution: a tumor cell treatment method based on polypeptide nanotechnology, the specific steps are as follows:

[0006] Step 1: Material synthesis and characterization

[0007] The peptide material was synthesized by the standard Fmoc-SPPS method and modified accordingly. A small amount of trifluoroacetic acid was used as a catalyst to connect β-CD-NH2 and CA through Schiff base reaction. The material CCA was obtained by cold ether precipitation and drying, and the molecular weight was characterized by mass spectrometry.

[0008] Step 2: Preparation of spherical nanoparticles and detection of in vitro fibrosis deformation

[0009] Based on CCA and TPF NO Preparation of supramolecular nanoparticles TPF by host-guest interaction NO / CCA NPs, TPF NOThe Fc part of β-CD was inserted into the hydrophobic cavity to form a hydrophobic core, which served as the basis for self-assembled spherical nanoparticles. The TPF was detected by transmission electron microscopy and dynamic light scattering. NO / CCA NPs, and after oxidation, the host-guest dissociation of Fc / β-CD occurred, and the supramolecular nanoparticles TPF NO / CCA NPs transformed into nanofiber TPF NO / CCA NFs, the particle size increases accordingly, and then circular dichroism is used to detect whether β-sheet structure appears in the material;

[0010] Step 3: Disrupting cellular redox homeostasis

[0011] TPF NO After CCA is internalized into cancer cells, CA molecules are released in the acidic lysosomal environment, causing mitochondrial dysfunction, inducing increased ROS levels, and promoting the fibrillar deformation of nanoparticles. Subsequently, the fibrotic TPF NO The peptides will wrap around the mitochondrial surface to form a fibrous shell, which will lead to a decrease in mitochondrial membrane potential and further increase the mitochondrial ROS level, thereby amplifying the cellular oxidative stress caused by the material and destroying the cellular redox homeostasis;

[0012] Step 4: Mitochondrial targeting and mitochondrial surface fibrosis detection

[0013] TPF labeled with Ce6 NO After tracing with / CCe6NPs, the co-localization degree of the material and mitochondria was observed using a confocal microscope to detect the targeting of the material to mitochondria. Then, a scanning electron microscope was used to observe the morphological characteristics of the fibrous shell formed by the material on the surface of mitochondria. Then, the JC-1 fluorescent probe was used to detect the changes in mitochondrial membrane potential induced by the material, thereby confirming that the material has the ability to form a fibrous network on the surface of mitochondria.

[0014] Step 5: In vitro anti-tumor effect detection

[0015] CCK-8 reagent was used to detect the half-maximal inhibitory concentration of the material, and then Calcein / PI was used to perform live and dead staining on tumor cells to determine the effect of the material on the activity of tumor cells, and then to clarify the direct killing effect of oxidative stress amplification and mitochondrial damage on tumor cells;

[0016] Step 6: Evaluation of the material's anti-tumor ability and immune effects

[0017] A mouse subcutaneous tumor model was constructed, and the anti-tumor ability of the material in vivo was evaluated through tumor volume, tumor weight, survival rate and body weight indicators, as well as the activation effect of the material on immune cells and cytokines in the tumor microenvironment.

[0018] As a preferred technical solution of the present invention, the specific operation mode of the Fmoc-SPPS method described in step 1 is to use Rink Amide resin microbeads as a solid phase synthesis carrier, start the synthesis from the C-terminus of the polypeptide, and select Fmoc-Lys(Dde)-OH as the first amino acid at the C-terminus. The remaining polypeptide sequences and TPP groups are synthesized in sequence from the C-terminus to the N-terminus.

[0019] As a preferred technical solution of the present invention, the oxidation treatment described in step 2 uses H2O2 as an oxidant.

[0020] As a preferred technical solution of the present invention, the change in ROS level described in step three is detected by ROS fluorescent probe DCFH-DA.

[0021] As a preferred technical solution of the present invention, when the confocal microscope described in step 4 is used, Mito-tracker is used to mark mitochondria.

[0022] As a preferred technical solution of the present invention, when the tumor cells described in step 5 are subjected to live-dead staining, Calcein is used to label the cytoplasm of live cells, and PI is used to label the nuclei of dead cells, and a confocal microscope is used for observation at the same time.

[0023] As a preferred technical solution of the present invention, the anti-tumor effect described in step five can also be detected by detecting ICD markers released by cancer cells such as CRT, HMGB1 and ATP, and using AnnexinV-FITC / PI reagent to detect the induction of tumor cell apoptosis and necrosis by the material.

[0024] As a preferred technical solution of the present invention, the construction method of the mouse subcutaneous tumor model described in step six adopts the method of subcutaneous injection of tumor cells.

[0025] As a preferred technical solution of the present invention, the materials described in steps 1 to 6 also need to undergo tumor targeting evaluation and in vivo safety evaluation.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention amplifies the oxidative stress of tumor cells through polypeptide nanoparticles, destroys their redox balance, thereby inducing ICD, combines the host-guest drug carrier system, polypeptide fibrosis deformation and NO gas generation mechanisms, gradually releases drugs in cells and produces NO gas, further increases the release of ROS through the binding of polypeptides to mitochondria, thereby aggravating mitochondrial dysfunction, and ultimately leads to redox imbalance in cells, inducing immunogenic death of tumor cells, thereby being able to directly kill tumor cells and activate the anti-tumor response of the immune system, providing a new tumor treatment method with broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The structure and mass spectrum characterization diagram of TPF of the present invention;

[0029] Figure 2 The structure and mass spectrum characterization diagram of CCA of the present invention;

[0030] Figure 3 TPF of the present invention NO / Particle size change of CCA before and after fiberization deformation measured by dynamic light scattering in aqueous solution;

[0031] Figure 4 The present invention TPF NO / Transmission electron microscopy images of CCA before and after fiberization and deformation in aqueous solution;

[0032] Figure 5 The present invention TPF NO / Circular dichroism spectra of CCA before and after fiberization deformation in aqueous solution;

[0033] Figure 6 This is a confocal microscope image of the effect of the material on the intracellular ROS level after DCFH-DA labeled intracellular ROS in the present invention;

[0034] Figure 7 Semi-quantitative analysis of intracellular ROS for the effect of the material of the present invention on intracellular ROS levels;

[0035] Figure 8 After the Mito-tracker of the present invention labels mitochondria, TPF NO / Confocal microscopy images of mitochondrial targeting of CCe6;

[0036] Fig. 9 TPF of the present invention NO / Pearson colocalization coefficient plot of CCA and mitochondria;

[0037] Fig.10The TPF is a method for labeling the cytoplasm of living cells with Calcein and the nucleus of dead cells with PI. NO / Confocal microscopy images of CCA cytotoxicity;

[0038] Fig.11 This is a diagram of the CCK-8 cytotoxicity experiment of the present invention. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] like Figures 1 to 11 As shown, the embodiment of the present invention provides a tumor cell treatment method based on polypeptide nanotechnology, and the specific steps are as follows:

[0041] Step 1: Material synthesis and characterization

[0042] The peptide material was synthesized by the standard Fmoc-SPPS method and modified accordingly. A small amount of trifluoroacetic acid was used as a catalyst to connect β-CD-NH2 and CA through a Schiff base reaction. The material CCA was obtained by cold ether precipitation and drying, and the molecular weight was characterized by mass spectrometry.

[0043] Step 2: Preparation of spherical nanoparticles and detection of in vitro fibrosis deformation

[0044] Based on CCA and TPF NO Preparation of supramolecular nanoparticles TPF by host-guest interaction NO / CCA NPs, TPF NO The Fc part of β-CD was inserted into the hydrophobic cavity to form a hydrophobic core, which served as the basis for self-assembled spherical nanoparticles. The TPF was detected by transmission electron microscopy and dynamic light scattering. NO / CCA NPs, and after oxidation, the host-guest dissociation of Fc / β-CD occurred, and the supramolecular nanoparticles TPF NO / CCA NPs transformed into nanofiber TPF NO / CCA NFs, the particle size increases accordingly, and then circular dichroism is used to detect whether β-sheet structure appears in the material;

[0045] Step 3: Disrupting cellular redox homeostasis

[0046] TPF NOAfter CCA is internalized into cancer cells, CA molecules are released in the acidic lysosomal environment, causing mitochondrial dysfunction, inducing increased ROS levels, and promoting the fibrillar deformation of nanoparticles. Subsequently, the fibrotic TPF NO The peptides will wrap around the mitochondrial surface to form a fibrous shell, which will lead to a decrease in mitochondrial membrane potential and further increase the mitochondrial ROS level, thereby amplifying the cellular oxidative stress caused by the material and destroying the cellular redox homeostasis;

[0047] Step 4: Mitochondrial targeting and mitochondrial surface fibrosis detection

[0048] TPF labeled with Ce6 NO After tracing with / CCe6NPs, the co-localization degree of the material and mitochondria was observed using a confocal microscope to detect the targeting of the material to mitochondria. Then, a scanning electron microscope was used to observe the morphological characteristics of the fibrous shell formed by the material on the surface of mitochondria. Then, the JC-1 fluorescent probe was used to detect the changes in mitochondrial membrane potential induced by the material, thereby confirming that the material has the ability to form a fibrous network on the surface of mitochondria.

[0049] Step 5: In vitro anti-tumor effect detection

[0050] CCK-8 reagent was used to detect the half-maximal inhibitory concentration of the material, and then Calcein / PI was used to perform live and dead staining on tumor cells to determine the effect of the material on the activity of tumor cells, and then to clarify the direct killing effect of oxidative stress amplification and mitochondrial damage on tumor cells;

[0051] Step 6: Evaluation of the material's anti-tumor ability and immune effects

[0052] A mouse subcutaneous tumor model was constructed, and the anti-tumor ability of the material in vivo was evaluated through tumor volume, tumor weight, survival rate and body weight indicators, as well as the activation effect of the material on immune cells and cytokines in the tumor microenvironment.

[0053] Multifunctional polypeptide-based materials are constructed through host-guest interaction and supramolecular chemistry, so that they can respond to the acidic environment of lysosomes, release cinnamaldehyde, interfere with the electron transport chain of tumor cells, and increase the level of ROS in tumor cells; after targeting mitochondria, they respond to the oxidative environment around mitochondria through host-guest interaction and deform, forming a fibrous network on the surface of mitochondria, interfering with the normal function of mitochondria and triggering a surge in ROS; they consume residual GSH in cells and release NO, which reacts with ROS to generate RNS with stronger toxicity and longer half-life. Through this cascade amplification process, the nanomaterial will generate and amplify cellular oxidative stress, destroy redox homeostasis, induce ICD in tumor cells, release damage-associated molecular patterns, enhance the presentation of tumor antigens, promote T cell immune response, and thus enhance anti-tumor immunity.

[0054] Among them, the specific operation mode of the Fmoc-SPPS method in step 1 is to use Rink Amide resin microbeads as a solid phase synthesis carrier, start the synthesis from the C-terminus of the polypeptide, and use Fmoc-Lys(Dde)-OH as the first amino acid at the C-terminus. The remaining polypeptide sequences and TPP groups are synthesized in sequence from the C-terminus to the N-terminus.

[0055] The Dde protecting group of the first amino acid was removed by hydrazine hydrate to expose the active amino group, which was then connected to the Fc group. The resin microbeads were cut by trifluoroacetic acid cutting solution, and then the polypeptide monomer TPF was obtained by cold ether precipitation and drying. The molecular weight was characterized by mass spectrometry. Tert-butyl nitrite was used as an NO donor and reacted with the TPF polypeptide overnight in a light-proof environment to load NO. TPF was obtained after cold ether precipitation and drying. NO Peptide.

[0056] Among them, the oxidation treatment in step 2 uses H2O2 as an oxidant.

[0057] After H2O2 treatment, nanofiber TPF can be detected by circular dichroism. NO / CCA NFs have a positive peak at a particle size of 190 nm and a negative peak at 210 nm, indicating the presence of a β-folded structure in the material.

[0058] Among them, the change of ROS level in step three is detected by ROS fluorescent probe DCFH-DA.

[0059] The results showed that TPF NO / CCA can significantly increase the intracellular ROS level. Compared with CCA or TPF alone, NO / CD only resulted in a slight increase in intracellular ROS levels.

[0060] In step 4, when the confocal microscope is used, Mito-tracker is used to label mitochondria.

[0061] As the incubation time increased, the degree of co-localization between the material and mitochondria increased significantly, and the Pearson colocalization coefficient at 12 h was 0.85, proving that the material has the ability to target and form fibers.

[0062] Among them, when the tumor cells in step five are stained for live and dead, Calcein marks the cytoplasm of live cells and PI marks the nucleus of dead cells, and a confocal microscope is used for observation at the same time.

[0063] Calcein / PI tumor cell live-death staining results showed that TPF NOThe number of dead cells in the CCA group increased significantly, and the results of CCK-8 reagent test showed that TPF NO / CCA has the strongest toxicity to tumor cells, and its toxicity increases with increasing concentration. In contrast, the use of CCA or TPF alone NO The dead cells and cytotoxicity of / CD were less than TPF NO / CCA group, confirmed TPF NO / CCA can effectively increase the oxidative stress of cancer cells, interfere with the normal function of mitochondria, and thus induce cell death.

[0064] Among them, the anti-tumor effect in step five can also be detected by detecting ICD markers released by cancer cells such as CRT, HMGB1 and ATP, and using Annexin V-FITC / PI reagent to detect the induction of tumor cell apoptosis and necrosis by the material.

[0065] The in vitro anti-tumor effect can be further clarified by detecting ICD markers released by cancer cells such as CRT, HMGB1 and ATP, as well as the induction of apoptosis and necrosis of tumor cells by the material.

[0066] Among them, the mouse subcutaneous tumor model in step six is ​​constructed by subcutaneous injection of tumor cells.

[0067] Among them, the materials in steps one to six also need to undergo tumor targeting evaluation and in vivo safety evaluation.

[0068] After the construction of the mouse subcutaneous tumor model was completed, the in vivo imaging technology was used to evaluate the fibrosis deformation and retention of the material in the organism, as well as the distribution and accumulation of the material in the tumor tissue and major organs; the systemic toxicity of the material was evaluated by blood indicators (ALP, ALT, AST, BUN, CREA, etc.); and the tissue toxicity of the material on major organs was studied by histological analysis (H&E staining), so as to analyze the balance between the dosage and tumor targeting and safety.

[0069] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0070] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for treating tumor cells based on polypeptide nanotechnology, characterized in that: The specific steps are as follows: Step 1: Material synthesis and characterization The peptide material was synthesized by the standard Fmoc-SPPS method and modified accordingly. A small amount of trifluoroacetic acid was used as a catalyst to connect β-CD-NH2 and CA through Schiff base reaction. The material β-CD-CA (CCA) was obtained by cold ether precipitation and drying, and the molecular weight was characterized by mass spectrometry. Step 2: Preparation of spherical nanoparticles and detection of in vitro fibrosis deformation Based on CCA and TPF NO Preparation of supramolecular nanoparticles TPF by host-guest interaction NO / CCA NPs, TPF NO The Fc part of β-CD was inserted into the hydrophobic cavity to form a hydrophobic core, which served as the basis for self-assembled spherical nanoparticles. TPF was detected by transmission electron microscopy and dynamic light scattering. NO / CCA NPs particle size, after oxidation treatment, Fc / β-CD host-guest dissociation, supramolecular nanoparticles TPF NO / CCA NPs transformed into nanofiber TPF NO / CCA NFs, the particle size increases accordingly, and then circular dichroism is used to detect whether β-sheet structure appears in the material; Step 3: Disrupting cellular redox homeostasis TPF NO After CCA NPs enter cancer cells, CA molecules are released in the acidic lysosomal environment, causing mitochondrial dysfunction, inducing increased ROS levels, and promoting the fibrillar deformation of nanoparticles. Subsequently, the fibrotic TPF NO The peptides will wrap around the mitochondrial surface to form a fibrous shell, which will lead to a decrease in mitochondrial membrane potential and further increase the mitochondrial ROS level, thereby amplifying the cellular oxidative stress caused by the material and destroying the cellular redox homeostasis; Step 4: Mitochondrial targeting and mitochondrial surface fibrosis detection TPF labeled with Ce6 NO The path of the CCe6 NPs tracer particles in the cells was followed, and the co-localization degree of the material and mitochondria was observed by confocal microscopy to detect the targeting of the material to mitochondria. Then, the morphological characteristics of the fibrous shell formed by the material on the mitochondrial surface were observed by scanning electron microscopy. The changes in mitochondrial membrane potential induced by the material were detected by JC-1 fluorescent probe, thus confirming that the material has the ability to form a fibrous network on the mitochondrial surface. Step 5: In vitro anti-tumor effect detection CCK-8 reagent was used to detect the half-maximal inhibitory concentration of the material, and then Calcein / PI was used to perform live and dead staining on tumor cells to determine the effect of the material on the activity of tumor cells, and then to clarify the direct killing effect of oxidative stress amplification and mitochondrial damage on tumor cells; Step 6: Evaluation of the material's anti-tumor ability and immune effects A mouse subcutaneous tumor model was constructed, and the anti-tumor ability of the material in vivo was evaluated through tumor volume, tumor weight, survival rate and body weight indicators, as well as the activation effect of the material on immune cells and cytokines in the tumor microenvironment.

2. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: The specific operation mode of the Fmoc-SPPS method described in step 1 is to use Rink Amide resin microbeads as a solid phase synthesis carrier, start the synthesis from the C-terminus of the polypeptide, and select Fmoc-Lys(Dde)-OH as the first amino acid at the C-terminus. The remaining polypeptide sequences and TPP groups are synthesized in sequence from the C-terminus to the N-terminus.

3. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: The oxidation treatment described in step 2 uses H2O2 as an oxidant.

4. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: The changes in ROS levels described in step three were detected using the ROS fluorescent probe DCFH-DA.

5. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: When using a confocal microscope as described in step 4, label mitochondria using Mito-tracker.

6. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: When the tumor cells described in step 5 are subjected to live-dead staining, Calcein is used to label the cytoplasm of live cells, and PI is used to label the nuclei of dead cells, and the cells are observed simultaneously using a confocal microscope.

7. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: The anti-tumor effect described in step five can also be detected by detecting ICD markers released by cancer cells such as CRT, HMGB1 and ATP, and using Annexin V-FITC / PI reagent to detect the induction of apoptosis and necrosis of tumor cells by the material.

8. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: The mouse subcutaneous tumor model described in step six is ​​constructed by injecting tumor cells.

9. The method for treating tumor cells based on polypeptide nanotechnology according to claim 1, characterized in that: The materials described in steps 1 to 6 also need to undergo tumor targeting evaluation and in vivo safety evaluation.

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