Cyanobacterial nanogene carrier and preparation method thereof

Abstract

The application discloses a cyanobacterial nanogene carrier and a preparation method thereof. By irradiating cyanobacteria under appropriate wavelength light, toxic active oxygen is generated in the cyanobacteria, lysosomes are destroyed, and the gene carrier trapped in the lysosomes is released into the cytoplasm, so that the biological function of the gene can be better exerted, and the problems of the gene carrier in the prior art, such as cationic lipids, cationic polymers, difficult lysosome escape and low gene transfection efficiency, are overcome. Meanwhile, the preparation process is complex, other substances are easily introduced, and the safety is low, and the like are overcome, so that the application is more suitable for efficient delivery of the gene carrier, an effective treatment method is provided for treatment of tumors and the like, and the treatment of cancer and the like is beneficial.

Description

Technical Field

[0001] This application relates to the fields of nanomedicine and tumor therapy, specifically to a cyanobacterial nanogene carrier and its preparation method. Background Technology

[0002] In recent years, with in-depth research into the pathogenesis of tumors, targeting key kinases in tumor cell signal transduction pathways has become a new approach for anti-tumor drug research. This approach can yield novel targeted drugs that are highly effective, low in toxicity, and highly specific. Currently, novel targeted drugs under research or clinical application are generally inhibitors of disease genes or target genes. They effectively inhibit tumor growth by silencing highly expressed disease genes or target genes in tumor cells.

[0003] Small interfering RNA (siRNA), also known as silencing RNA, short interfering RNA, or non-coding RNA, can specifically silence the expression of drug resistance-related genes through RNA interference, thereby improving the anti-cancer effect of chemotherapy drugs. RNA interference can simply and efficiently silence the expression of target genes, thus achieving the same effect as targeted drugs. Targeted inhibitors in targeted drugs act directly on proteins, which may interfere with the expression of other proteins; while siRNA mainly acts on mRNA, specifically inhibiting the expression of target genes. Its site of action is specific, and it usually does not affect normal gene expression. Therefore, siRNA has better selectivity and specificity as a targeted drug. Theoretically, siRNA can inhibit the expression of almost any gene in the body, making it more promising for treatment and application than currently widely used small molecule targeted drugs.

[0004] However, small interfering RNA (sRNA) needs to be carried on gene vectors during treatment to transfect it into target cells. While nanogene vectors enter cells via endocytosis, aiming to reach the cytoplasm, most nanogene vectors remain trapped in lysosomes. This prevents the release of sRNA into the cytoplasm, hindering the drug's effectiveness and resulting in poor gene therapy efficacy. To overcome this lysosomal trapping problem and enable sRNA release to interfere with the target gene, the gene vector needs to effectively escape from the lysosome. This allows the drug to be released into the cytoplasm for interference. However, current gene vectors, such as cationic lipids and polymers, are difficult to escape from lysosomes, leading to low transfection efficiency and hindering drug efficacy. Furthermore, methods to promote lysosomal escape require the introduction of other substances, increasing experimental insecurity and adding complexity to the process. Summary of the Invention

[0005] The purpose of this application is to provide a highly efficient cyanobacterial nanogene vector that promotes escape from lysosomes, thereby solving the problems of gene vector retention in lysosomes and low transfection efficiency mentioned in the background art.

[0006] To achieve the above objectives, this application provides the following technical solution:

[0007] The first aspect of this application is to provide a method for preparing a cyanobacterial nanogene vector, comprising the following steps:

[0008] S1. Culturing cyanobacteria under light to obtain cyanobacterial culture medium;

[0009] S2. Prepare a polypeptide gene vector solution;

[0010] S3. Extract cyanobacteria and mix them with polyleucine nanogene particle solution to prepare cyanobacterial nanogene vector.

[0011] S4. Targeted transfection of macrophages with cyanobacterial nanogene vectors.

[0012] S5. Laser irradiation of macrophages.

[0013] Preferably, in step S5, the laser is a 660nm laser, the irradiation time is 20-60 minutes, and the laser intensity is 0.5W·cm. -2 .

[0014] Preferably, in step S3, the centrifugation speed for extracting cyanobacteria is 5000-8000 rpm, and the centrifugation time is 5 minutes.

[0015] Preferably, in step S3, the cyanobacteria need to be treated with N-hydroxysuccinimide for 20 minutes to attach thiol groups to the surface of the cyanobacteria; the cyanobacteria are incubated with the polypeptide gene carrier solution for 20 minutes to prepare the cyanobacterial nanogene carrier solution.

[0016] Preferably, in step S1, the cyanobacteria are cultured in an aerobic environment, and the density of the cyanobacteria is controlled to be 2 × 10⁻⁶ before the culture step in step S1. 8 Colonies / ml.

[0017] Preferably, in step S2, the polypeptide gene vector comprises nanospheres formed by the self-assembly of a triblock polypeptide, wherein the triblock polypeptide comprises polyethylene glycol, polylysine, and polyleucine connected in sequence; one end of the polylysine is connected to polyethylene glycol via an amide bond, and the other end of the polylysine is connected to polyleucine via a peptide bond. In addition, the N / P ratio of the polypeptide and the small interfering RNA is 1:2, and the polypeptide gene vector is coated with hyaluronic acid.

[0018] A second aspect of this application is to provide a cyanobacterial nanogene vector, which is prepared by the above-described method.

[0019] The third aspect of this application is to provide a method for preparing a drug for treating tumors, which includes two preparation methods. The first preparation method involves inserting a tumor drug gene into the cyanobacterial nanogene carrier prepared above.

[0020] The second preparation method involves inserting a tumor drug gene into the cyanobacterial nanogene carrier during the preparation process. The tumor drug gene is added to the cyanobacterial culture medium before laser irradiation.

[0021] Furthermore, after adding the tumor drug gene, hyaluronic acid was added to the cyanobacterial culture medium, and then the cyanobacterial culture medium was mixed.

[0022] Compared with the prior art, the beneficial effects of this application are:

[0023] This application provides a cyanobacterial nanogene carrier and its preparation method. By irradiating cyanobacteria with light of an appropriate wavelength, toxic reactive oxygen species (ROS) are generated within the cyanobacteria. These ROS, primarily singlet oxygen, can destroy lysosomes. This process helps release the gene carrier trapped in the lysosome into the cytoplasm, allowing the gene to better perform its biological function and thus improving gene transfection efficiency. This application overcomes the problems of existing gene carriers, such as cationic lipids and cationic polymers, which suffer from difficulties in lysosomal escape and low gene transfection efficiency. It also overcomes the disadvantages of complex preparation processes, easy introduction of other substances, and low safety. This makes this application more suitable for the efficient delivery of gene carriers, providing an effective treatment method for diseases such as tumors, and is beneficial for the treatment of cancer and other diseases. This application does not introduce other substances, has high experimental safety, and the steps are simple. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0025] Figure 1 The figure shows the results of a fluorescence colocalization experiment to detect the effect of cyanobacterial nanogene vectors on transfection efficiency using fluorescence microscopy.

[0026] Figure 2 A statistical bar chart showing the trend of lysosomal fluorescence values ​​under different light conditions;

[0027] Figure 3 A statistical bar chart showing the trend of siRNA fluorescence values ​​under different light conditions. Detailed Implementation

[0028] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] Macrophages (M) are a type of leukocyte located in peripheral blood and inflamed tissues, playing an immunomodulatory role in disease. In animals, macrophages participate in non-specific immune regulation by phagocytosing bacteria, dead cells, and cell debris. They also digest phagocytosed substances and present their characteristics to subsequent lymphocytes and other immune cells, participating in specific immune regulation. Macrophages are ideal targets for many diseases; therefore, developing drugs that activate macrophage function is an effective treatment approach.

[0030] Lysosomes are present in immune cells such as macrophages. Lysosomes function as digestive organs within the cell, and nanocarriers that enter the cell via endocytosis are ultimately degraded within the lysosome. However, most nanocarriers enter the cell via endocytosis and remain in the lysosome, leading to poor efficacy of gene therapy. Small interfering RNA (SRNA) is also carried in gene carriers; therefore, SRNA is also retained in the lysosome, resulting in low gene transfection efficiency and preventing SRNA drugs from functioning properly.

[0031] To address the aforementioned issues, this application discloses a cyanobacterial nanogene carrier that promotes lysosomal escape, thereby facilitating gene transfection and improving the therapeutic efficiency of gene drugs.

[0032] The cyanobacteria used in this application were formerly known as blue algae or blue-green algae. They are a type of Gram-negative bacteria with a long evolutionary history. Cyanobacteria are non-flagellated, contain chlorophyll a, but do not contain chloroplasts. Cyanobacteria are large single-celled prokaryotes capable of aerobic photosynthesis.

[0033] First, in this application, the cyanobacterial nanogene carrier is only phagocytosed by macrophages. Subsequently, the cyanobacterial photodynamic effect generates toxic reactive oxygen species (ROS) that disrupt the lysosomal structure of macrophages, thereby promoting the escape of the gene carrier from the lysosomes and ultimately enhancing the gene transfection efficiency of macrophages. Therefore, the cyanobacterial nanogene carrier disclosed in this application has the function of targeting and activating macrophages, providing an effective raw material for tumor therapeutic drugs.

[0034] The technical solution of this patent will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the methods used in the embodiments are conventional methods.

[0035] Example 1: Preparation method of cyanobacterial nanogene carrier

[0036] Cyanobacterial gene nanocarriers exhibit photodynamic effects under 660nm laser irradiation. The cyanobacterial gene nanocarriers prepared in this application are characterized by their ability to be massively amplified in vitro using cyanobacterial culture medium, and their ability to target tumors, thus improving the efficiency of nanocarrier entry into macrophages. Because the cyanobacterial surface is loaded with a large number of nanocarriers, a significant amount of nanocarriers are carried into the cells during phagocytosis by macrophages, resulting in a significantly improved efficiency compared to simple nanocarrier entry.

[0037] This embodiment provides a method for preparing a cyanobacterial nanogene vector. The cyanobacterial nanogene vector produced by this method targets macrophages. Through cyanobacterial photodynamic therapy, toxic ROS are generated, disrupting macrophage lysosomes and efficiently promoting the escape of siRNA drugs from lysosomes, thereby improving gene transfection efficiency and ultimately targeting and activating macrophages. The preparation process of this application is simple, and the resulting cyanobacterial nanogene vector is stable and biocompatible.

[0038] This embodiment details the preparation method of cyanobacterial nanogene vectors.

[0039] (1) Prepare a PBS solution of polypeptide nanomicelles;

[0040] (2) Add siRNA (N / P = 1:2) to polyleucine solution, and polypeptide nanomicelles and small interfering RNA form polypeptide nanogene carriers through electrostatic self-assembly.

[0041] (3) Hyaluronic acid (HA) containing maleimide groups is added to the polypeptide nanogene carrier solution and mixed well. Maleimide groups are introduced through electrostatic adsorption.

[0042] (4) After culturing cyanobacteria in an aerobic environment, N-hydroxysuccinimide (NHS) was added to the cyanobacterial solution and treated for 20 minutes, thiol groups were attached to the surface of the cyanobacteria.

[0043] (5) Add the cyanobacteria from step (4) to the solution from step (3) and place it on a shaker for 20 minutes to obtain the cyanobacterial nanogene vector.

[0044] Example 2: Detection of the photodynamic effect of cyanobacterial nanogene carriers

[0045] This embodiment details the triggering conditions for photodynamic generation of toxic reactive oxygen species (ROS) from cyanobacterial nanogene carriers, as well as the method for detecting ROS. The specific steps of this process are as follows:

[0046] (1) In a sterile operating table, the cyanobacterial nanogene vector was added to a culture dish, with a cyanobacterial density of 2 × 10⁻⁶. 8CFU / mL (colony count / mL);

[0047] (2) The solution in the petri dish was placed in an environment with a radiation exitance of 0.5 W / cm. -2 Irradiation with a 660nm wavelength laser for 20 minutes triggered photodynamic activity in cyanobacteria.

[0048] (3) In order to assess whether the irradiation in the second step is sufficient, the absorbance of the solution needs to be detected by an ELISA reader. A probe for detecting singlet oxygen (SOSG) is added to each well of the multi-well culture plate in advance to quantitatively detect the level of singlet oxygen production and assess the photodynamic effect of cyanobacteria in an in vitro hypoxic environment.

[0049] Please see the appendix Figure 1 , Figure 1 The image shows the results of a fluorescence colocalization experiment used to detect the effect of cyanobacterial nanogene vectors on transfection efficiency using fluorescence microscopy. Figure 1 The horizontal axis represents various substances in macrophages under different fluorescent labels, and the vertical axis represents the effects of light and time. Figure 1 The results showed that after cyanobacterial fluorescent nanocarriers in macrophages were treated with light, from the 12th to the 24th hour, the fluorescence intensity of lysosomes decreased with increasing time, while the fluorescence intensity of siRNA in the cytoplasm increased. This indicates that the light treatment of cyanobacterial fluorescent nanocarriers destroyed the lysosomes in macrophages and released siRNA into the cytoplasm, which is beneficial for siRNA to exert its function and thus improves the efficiency of gene transfection.

[0050] Example 3: Verification of the efficacy of cyanobacterial nanogene vectors in siRNA drugs.

[0051] This embodiment details a method for verifying the efficacy of cyanobacterial nanogene vectors.

[0052] (1) The siRNA was stained with DAPI, and then the cyanobacterial nanogene vector was prepared using the method in Example 1;

[0053] (2) Add cyanobacterial nanogene vectors to primary macrophages in a six-well plate and incubate them together for 2 hours;

[0054] (3) The culture medium of the above macrophages was placed in an environment with a radiation exitance of 0.5 W / cm. -2 Irradiated with a 660nm wavelength laser for 20 minutes;

[0055] (7) At 12 hours and 24 hours after irradiation, the lysosomes in the culture medium of the above macrophages were stained with lysosomal fluorescent probe (Lysotraker) dye for 30 minutes.

[0056] (8) Fix the above-stained macrophages with 4% paraformaldehyde for 30 minutes;

[0057] (9) Observe the fluorescence intensity of lysosomes and siRNA under a confocal microscope and analyze their colocalization.

[0058] (10) Collect macrophage samples at 24 hours for qPCR detection to detect the expression of siRNA target genes.

[0059] Please refer to the appendix for detailed experimental results. Figure 2 and attached Figure 3 , Figure 2 This is a statistical bar chart showing the fluorescence values ​​of lysosomes at different times under both light and no light conditions. Figure 3 This is a statistical bar chart showing the fluorescence values ​​of siRNA at different time points under both light and no light conditions. Figure 2 and Figure 3 The horizontal axis represents time, and the vertical axis represents the fluorescence values ​​of lysosomes and siRNA, respectively. Figure 2 The results showed that under no-light conditions, the fluorescence value of lysosomes decreased from the 12th to the 24th hour; while under light conditions, the fluorescence value of lysosomes decreased steadily. Figure 3 The results showed that under no-light conditions, the fluorescence value of siRNA remained at a low level, while under light conditions, from the 12th hour to the 24th hour, the fluorescence value of siRNA increased significantly over time. Figure 2 and Figure 3 This further reflects that light treatment of macrophages carrying cyanobacterial fluorescent nanocarriers significantly increased the proportion of lysosomes escaping from macrophages, while also significantly enhancing the transfection efficiency of siRNA.

[0060] This application discloses a cyanobacterial nanogene carrier and its preparation method. The cyanobacterial nanogene carrier produced by this method targets macrophages. Through cyanobacterial photodynamic therapy, toxic ROS are generated, disrupting the lysosomes of macrophages, thereby efficiently promoting the escape of genes loaded on the carrier from the lysosomes and improving gene transfection efficiency. This application overcomes the shortcomings of existing gene carriers, such as easy introduction of other substances, low safety, and complex preparation processes. The production cost of this application is low, the process is simple, and it does not introduce other impurities. This application provides a new carrier for the efficient delivery of targeted drugs in the treatment of diseases such as tumors.

[0061] The above description is merely a specific embodiment of this application, enabling those skilled in the art to understand or implement this application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of this application. Therefore, this application is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features claimed herein.

Claims

1. A method for preparing a cyanobacterial nanogene vector, characterized in that, Includes the following steps: S1. Prepare a cyanobacterial culture medium containing cyanobacteria; S2. Prepare a polypeptide gene vector solution; S3. Extract the cyanobacteria from the cyanobacterial culture medium, mix the cyanobacteria with the polypeptide gene carrier solution to prepare a cyanobacterial nanogene carrier. S4. Targeted transfection of macrophages with the cyanobacterial nanogene vector; S5. Irradiate the macrophages with a 660 nm laser for 20-60 minutes at a laser intensity of 0.5 W / cm². -2 ; In step S1, the cyanobacteria are cultured in an aerobic environment, and the density of the cyanobacteria is controlled to be 2 × 10⁻⁶ before the culture step in step S1. 8 Colonies / ml; In step S2, the polypeptide gene vector comprises nanospheres formed by the self-assembly of a triblock polypeptide. The triblock polypeptide comprises polyethylene glycol, polylysine, and polyleucine connected in sequence. One end of the polylysine is linked to polyethylene glycol via an amide bond, and the other end of the polylysine is linked to polyleucine via a peptide bond. Furthermore, the N / P ratio of the polypeptide and the small interfering RNA is 1:

2. The polypeptide gene vector is coated with hyaluronic acid, which has maleimide groups on it. In step S3, the cyanobacteria are treated with N-hydroxysuccinimide for 20 minutes to attach thiol groups to the surface of the cyanobacteria; the cyanobacteria are incubated with the polypeptide gene carrier solution for 20 minutes to prepare the cyanobacterial nanogene carrier solution.

2. The method according to claim 1, characterized in that, In step S3, the centrifugation speed for extracting the cyanobacteria is 5000-8000 rpm, and the centrifugation time is 5 minutes.

3. A cyanobacterial nanogene vector, characterized in that, The cyanobacterial nanogene vector is prepared by any of the methods in claims 1-2.

4. A method for preparing a drug for treating tumors, characterized in that, The tumor drug gene is inserted into the cyanobacterial nanogene vector prepared by any one of the methods of claims 1-2.

5. A method for preparing a drug for treating tumors, characterized in that, In the preparation process of any of the preparation methods in claims 1-2, a tumor drug gene is inserted, and the tumor drug gene is added to the cyanobacterial culture medium before the laser irradiation.

6. The preparation method according to claim 5, characterized in that, After adding the tumor drug gene, hyaluronic acid was added to the cyanobacterial culture medium, and then the cyanobacterial culture medium was mixed.

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