Preparation method and application of nano drug delivery system for inhibiting melanoma growth

Abstract

The present invention relates to the field of biomedicine technology, and discloses a method for preparing a nano drug delivery system for inhibiting melanoma growth, including constructing a shRNA-Ptpn2 plasmid, synthesizing HA-DOX, preparing an M1 macrophage membrane, and preparing a nano drug delivery system M1HD@RPR; the nano drug delivery system and its application are also disclosed. The present invention achieves targeted delivery of drugs to tumor sites in vivo, and is used for the inhibition and treatment of primary melanoma and lung metastases, providing a new direction for breaking through the current dilemma of no significantly effective treatment options for melanoma; it can efficiently transfect melanoma cells, has good stability, can self-replicate, and exerts an efficient melanoma treatment rate. The present invention converts shRNA into siRNA through intracellular cleavage, thereby exerting the effect of RNAi to silence genes, and utilizes shRNA, a small interfering RNA sequence, as a "short hairpin" cloned into a plasmid vector, so that it can exert a relatively stable RNAi effect in cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a preparation method and application of a nano drug delivery system for inhibiting melanoma growth. Background Art

[0002] Malignant melanoma is a highly aggressive malignant tumor that is prone to treatment resistance. Worldwide, the incidence of melanoma has been steadily increasing, leading to increasingly serious public health problems. It is currently becoming the leading cause of death from the skin.

[0003] Currently, chemotherapy is the mainstay of treatment for patients with advanced melanoma that has metastasized, either as a single agent or in combination. However, chemotherapy often suffers from significant side effects, unsatisfactory long-term effects, and the development of drug resistance. Advances in tumor immunology have revealed the high immunogenicity of melanoma. Immunotherapy has gained widespread application in the biological treatment of malignant melanoma and has become a research hotspot for personalized treatment. However, because tumor development and progression are the result of multiple genetic mutations and factors, single-agent treatment is unlikely to achieve optimal results. Therefore, combining immunotherapy with other therapeutic approaches has become a growing trend in melanoma treatment. For example, combining chemotherapy with immunotherapy can enhance the effectiveness of chemotherapy by activating and enhancing the immune response before antigen release, amplifying antigen recognition and thereby activating T cells. Furthermore, chemotherapy can enhance the immunogenicity of tumor cells by inducing these responses. Therefore, the combination of chemotherapy and immunotherapy offers a mutually reinforcing relationship.

[0004] Inspired by the above, the inventors plan to combine doxorubicin (DOX) with immunotherapy to treat mouse melanoma. In 2017, Nature reported that the loss of Ptpn2 can: ① enhance the sensitivity of tumor cells to immunotherapy; ② enhance the expression of cytotoxic T cells CD8 in tumor cells. +③ Enhance IFN-γ release and tumor sensitivity to IFN-γ; and ④ Promote polarization of M0 macrophages to M1 macrophages in the tumor microenvironment. Therefore, targeted downregulation of the Ptpn2 gene can exert tumor immunotherapy effects. Due to the significant side effects of doxorubicin, especially cardiotoxicity, it imposes a significant burden on patients in clinical applications. Nanoparticle drug delivery systems, due to their unique surface properties, such as large surface area and multifunctional modification based on different preparation processes and raw materials, can be used as therapeutic drug delivery vehicles, achieving targeted and controlled drug release. They can also simultaneously encapsulate multiple therapeutic drugs and biomacromolecules, thereby achieving the synergistic effect of multiple therapeutic approaches. With the development of nanoparticle drug delivery systems in the field of immunotherapy, inspired by the drug delivery capabilities of liposomes' phospholipid bilayers, many functional cell membranes, such as erythrocyte membranes, tumor cell membranes, neutrophil membranes, and macrophage membranes, have been incorporated into nanoparticle design for biotherapeutic purposes. These membranes naturally carry numerous biomarker molecules, enabling drug delivery systems to achieve long-term circulation or targeted delivery in vivo, and even exert immunotherapy effects. Studies have found that the M1 macrophage membrane can have an excellent targeting effect on tumor cells by forming nanotube tunnels between it and tumor cells and upregulating its own chemokine receptor 2 (Chemoattractant Cell Receptor 2, CCR2) and CCR4. Summary of the Invention

[0005] Based on the above problems, the present invention provides a preparation method and application of a nano-drug delivery system for inhibiting melanoma growth. The nano-drug delivery system of the present invention can efficiently transfect melanoma cells, has good stability, can self-replicate, and can achieve a high melanoma treatment rate, providing a new direction for breaking the current dilemma of no significantly effective treatment options for melanoma.

[0006] To solve the above technical problems, the present invention provides a method for preparing a nano drug delivery system for inhibiting melanoma growth, the specific steps of which are as follows:

[0007] S1: Construction of shRNA-Ptpn2 plasmid

[0008] Primers were designed to prepare the shRNA-Ptpn2 gene, which was then loaded into the pGpU6 / GFP / Neo plasmid to construct a shRNA-Ptpn2 plasmid that targeted downregulation of Ptpn2. The shRNA-Ptpn2 plasmid was then amplified in Escherichia coli.

[0009] S2: Synthesis of HA-DOX

[0010] The aromatic aldehyde formed by oxidation of hyaluronic acid HA reacts with the amino group of doxorubicin DOX through an imine bond to form a Schiff base HA-DOX, referred to as HD;

[0011] S3: Preparation of M1 macrophage membranes

[0012] S4: Preparation of Nanodrug Delivery System M1HD@RPR

[0013] 5 μg of the shRNA-Ptpn2 plasmid constructed in step S1 was mixed with 25 μg of the cell-penetrating peptide iRGD and allowed to stand for 5 minutes to form a blend of iRGD and shRNA-Ptpn2, iRGD-shRNA-Ptpn2, referred to as RR; 125 μg of PEI was added to the blend RR, and the mixture was allowed to stand for 5 minutes to obtain a complex of PEI and RR, iRGD-PEI-shRNA-Ptpn2, referred to as RPR; the mixture was allowed to stand for another 5 minutes, and HA-DOX containing 50 μg of DOX was added, and the mixture was mixed and self-assembled to obtain HD@RPR nanoparticles HA-DOX@iRGD-PEI-shRNA-Ptpn2; then a micro-extruder was used to repeatedly pass the M1 macrophage membrane prepared in step S3 and the HD@RPR nanoparticles through a 400 nm polycarbonate membrane to obtain the M1HD@RPR nanodrug delivery system, which was stored at 4°C.

[0014] Furthermore, the primers in step S1 include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2.

[0015] Furthermore, the synthesis method of HA-DOX in step S2 is as follows:

[0016] a. Oxidation of HA

[0017] 0.4 g of HA was dissolved in 10 mL of deionized water to prepare a HA solution, and then 0.053 g of NaIO4 was dissolved in 5 mL of deionized water to prepare a NaIO4 solution. The NaIO4 solution was added dropwise to the HA solution, stirred in the dark for 12 h, and then ethylene glycol was added and stirred for 0.5 h. The resulting solution was dialyzed against deionized water to remove NaIO4. After purification for 48 h, the sponge-like product OHA was freeze-dried.

[0018] b. Synthesis of HA-DOX

[0019] Take 0.2 g of OHA from step a, add 0.2 g of OHA and 40 mg of DOX·HCl to 30 mL of ultrasonically degassed ddH2O. React in the dark at 50°C under nitrogen protection for 48 h. Then dialyze with deionized water for 48 h to remove unreacted DOX·HCl. Freeze-dry to obtain a red spongy product HD.

[0020] Furthermore, the preparation method of the M1 macrophage membrane in step S3 is as follows: RAW264.7 cells in the logarithmic growth phase are taken, and when the cell density reaches 70%, 100 ng / mL lipopolysaccharide (LPS) and 10 ng / mL IFN-γ are added. After 24 hours of action, the M1 macrophages are collected; the cells are then resuspended in pre-cooled Tris-magnesium salt buffer to a cell concentration of 2×10 7 The cells were repeatedly squeezed out 20 times using a micro-extruder with only a gasket to destroy the cells. The cell membrane was then extracted using sucrose gradient centrifugation and then ultrasonicated at 100 W for 2 minutes to obtain the desired M1 macrophage membrane.

[0021] Furthermore, the ratio of M1 macrophage cell membrane to shRNA-Ptpn2 in step S4 is as follows: 0.034 mg M1 macrophage cell membrane is added for every 5 μg shRNA-Ptpn2.

[0022] Furthermore, in step S4, the M1 macrophage membrane and HD@RPR nanoparticles were passed through a 400 nm polycarbonate membrane 20 times.

[0023] In order to solve the above technical problems, the present invention also provides a nano drug delivery system.

[0024] To solve the above technical problems, the present invention also provides the use of a nano drug delivery system in the preparation of a drug for treating melanoma or lung metastasis melanoma.

[0025] Compared with the prior art, the present invention has the following beneficial effects: the present invention cleverly combines the chemotherapy drug doxorubicin with the silent immunosuppressive gene shRNA-Ptpn2, and uses M1 macrophage membrane to coat it, thereby achieving targeted delivery of the drug to the tumor site in the body, which is used for the inhibition and treatment of primary melanoma and lung metastases, and provides a new direction for breaking through the current dilemma of no significantly effective treatment options for melanoma; since shRNA-Ptpn2, as a nucleic acid biomacromolecule, is susceptible to nuclease degradation in the body, in order to enable it to efficiently transfect melanoma cells, have good stability, self-replicate, and exert a high melanoma treatment rate, the present invention cuts shRNA into siRNA in the cell, thereby exerting the effect of RNAi to silence the gene, and uses shRNA, a small interfering RNA sequence, as a "short hairpin" cloned into a plasmid vector, so that it can exert RNAi effects more stably in the cell. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Schematic diagram of the construction route of M1HD@RPR and its application mechanism in combined cancer therapy according to an embodiment of the present invention;

[0027] Figure 2Schematic diagram of the synthesis route of HA-DOX of the present invention;

[0028] Figure 3 This is a structural verification diagram of HA-DOX according to an embodiment of the present invention;

[0029] Figure 4 This is a characterization result diagram of the nano drug delivery system according to an embodiment of the present invention;

[0030] Figure 5 This is a diagram showing the distribution of mouse organs after tail vein injection of Free DOX, HD@RPR, and M1HD@RPR in mice according to an embodiment of the present invention;

[0031] Figure 6 These are confocal images of DOX fluorescence distribution in tumor tissue sections 24 hours after tail vein injection of Free DOX, HD@RPR, and M1HD@RPR in mice according to the examples of the present invention (scale bar: 200 μm);

[0032] Figure 7 and Figure 8 All of them are graphs showing the growth of mouse tumors according to the embodiments of the present invention;

[0033] Figure 9 These are representative images and H&E results of the lungs of B16F10 lung metastasis mice after treatment in different groups according to the examples of the present invention (scale bar is 2000 μm). DETAILED DESCRIPTION

[0034] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with examples and drawings. The exemplary embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0035] Example:

[0036] This embodiment provides a method for preparing a nano drug delivery system for inhibiting melanoma growth. The specific steps are as follows:

[0037] S1: Construction of shRNA-Ptpn2 plasmid

[0038] The shRNA-Ptpn2 gene was prepared using primer design. The shRNA-Ptpn2 gene was carried into the pGpU6 / GFP / Neo plasmid to construct a shRNA-Ptpn2 plasmid that targets downregulation of Ptpn2. The shRNA-Ptpn2 plasmid was then amplified by Escherichia coli. The primers used in this example include a forward primer and a reverse primer, and the sequences are as follows:

[0039] Forward primer 5′-3′: CACAAAGAAGTTACATCTT;

[0040] Reverse primer 3′-5′: AAGATGTAACTTCTT TGTG;

[0041] The nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.2;

[0042] S2: Synthesis of HA-DOX

[0043] The aromatic aldehyde formed by oxidation of hyaluronic acid HA and the amino group of doxorubicin (DOX) form a Schiff base HA-DOX (abbreviated as HD) through an imine bond. Because the Schiff base is pH sensitive, it can achieve the targeted release of doxorubicin in the weakly acidic tumor microenvironment; see the attached Figure 2 The synthesis method of HA-DOX in this embodiment is as follows:

[0044] Oxidation of HA: 0.4 g of HA was dissolved in 10 mL of deionized water to prepare a HA solution, and then 0.053 g of NaIO4 was dissolved in 5 mL of deionized water to prepare a NaIO4 solution. The NaIO4 solution was added dropwise to the HA solution and stirred in the dark for 12 h. Ethylene glycol was then added and stirred for 0.5 h to terminate the reaction. The resulting solution was dialyzed against deionized water (Mn = 1000 Da) to remove NaIO4. After purification for 48 h, the sponge-like product OHA was freeze-dried.

[0045] Synthesis of HA-DOX: Take 0.2 g of OHA from step a, add 0.2 g of OHA and 40 mg of DOX·HCl to 30 mL of ultrasonically degassed ddH2O, react at 50°C in the dark under nitrogen protection for 48 h, then dialyze with deionized water (Mw = 1000 Da) for 48 h to remove unreacted DOX·HCl, and freeze-dry to obtain a red sponge product HD; then use an ultraviolet spectrophotometer to determine the DOX content and calculate the DOX grafting rate, which is 12%. The product was then stored in the dark at 4°C for future use; HA-DOX structural characterization: using 1 The structure was confirmed by H-NMR nuclear magnetic resonance and FT-IR infrared spectroscopy. The results are shown in the attached Figure 3 , where: a, HA, b, OHA, c, DOX, d, HA-DOX; 1H-NMR results showed that, relative to HA, OHA had an aldehyde proton peak at a chemical shift of 8.3 ppm and a resonant hemiacetal proton peak between 5.0 and 5.25 ppm, indicating that HA was successfully oxidized to OHA. Compared with OHA and DOX, the aromatic ring proton peak of DOX at 6.85 to 7.25 ppm in HA-DOX was weakened and the amino proton peak at 7.85 disappeared, while the aldehyde proton peak at 8.3 ppm was weakened relative to OHA, indicating that HA-DOX was successfully synthesized. FT-IR infrared spectrum showed that, relative to HA, OHA had a 1731 cm -1 The typical aldehyde C=O stretching vibration band appears at 2750cm -1 The C(O)-H absorption peak at 1604 cm coincides with the CH absorption peak of HA itself, while the FT-IR spectrum of HA-DOX is at 1604 cm -1 The stretching band belonging to the aromatic ring of DOX appeared, so both FITR and 1H NMR indicated that HA-DOX was successfully synthesized;

[0046] S3: Preparation of M1 macrophage membranes

[0047] The preparation method of M1 macrophage membranes in this example is as follows: RAW264.7 cells in the logarithmic growth phase (M0) are taken. When the cell density reaches 70%, 100 ng / mL lipopolysaccharide (LPS) and 10 ng / mL IFN-γ are added. After 24 hours of treatment, the M1 macrophages are collected. The cells are then resuspended in pre-chilled Tris-magnesium buffer (TM buffer, pH 7.4, 0.01 M Tris and 0.001 M MgCl) to a cell concentration of 2×10 7 / mL, repeatedly squeeze out 20 times in a micro-extruder with only a gasket to destroy the cells, then extract the cell membrane by sucrose gradient centrifugation, and then use 100W power ultrasound for 2 minutes to obtain the desired M1 macrophage membrane; the specific operation method of extracting the cell membrane by sucrose gradient centrifugation in this embodiment is as follows: dilute the above-mentioned TM buffer to a sucrose concentration of 0.25M with 1M sucrose solution, centrifuge at 2000g and 4°C for 10 minutes, collect the supernatant, centrifuge at 4000g and 4°C for 30 minutes, resuspend and wash the precipitate with TM buffer containing 0.25M sucrose, centrifuge at 4000g and 4°C for 30 minutes, and collect the cell membrane;

[0048] The protein quantification of the extracted M1 macrophage membrane was performed using the BCA protein quantification method. The results showed that every 10 8 The protein content of each cell is about 0.17 mg;

[0049] S4: Preparation of Nanodrug Delivery System M1HD@RPR

[0050] M1HD@RPR was prepared by electrostatic adsorption method, and the nanoparticle size and transfection efficiency were finally determined. Figure 1 The specific method is as follows: 5 μg of the shRNA-Ptpn2 plasmid constructed in step S1 was mixed with 25 μg of a cell-penetrating peptide linear arginine-glycine-aspartic acid short peptide (iRGD), and the mixture was allowed to stand for 5 minutes to form a blend of iRGD and shRNA-Ptpn2 iRGD-shRNA-Ptpn2, referred to as RR; 125 μg of polyethyleneimine (PEI) was added to the blend RR, and the mixture was allowed to stand for 5 minutes to obtain a complex of PEI and RR iRGD-PEI-shRNA-Ptpn2, referred to as RPR; the mixture was allowed to stand for another 5 minutes, and 50 μg of HA-DOX of DOX was mixed and self-assembled to obtain HD@RPR nanoparticles HA-DOX@iRGD-PEI-shRNA-Ptpn2; then, the M1 macrophage membrane prepared in step S3 and the HD@RPR nanoparticles were repeatedly passed through a 400nm polycarbonate membrane using a micro-extruder to obtain the M1HD@RPR nanodrug delivery system, which was stored at 4°C and used as soon as possible; in this example, the amount of M1 macrophage membrane added was based on the following ratio of M1 macrophage membrane to shRNA-Ptpn2: 0.034mg M1 macrophage membrane was added for every 5μg shRNA-Ptpn2; in this example, the M1 macrophage membrane and HD@RPR nanoparticles were passed through a 400nm polycarbonate membrane 20 times;

[0051] In this step, blank nanoparticles (BlankNPs) were prepared by self-assembly using PEI, iRGD, and OHA in the same amount as M1HD@RPR. The particle size potential and appearance morphology of M1HD@RPR were characterized by Malvern particle size analyzer and transmission electron microscope. The results are shown in the attached Figure 4 , where A is the particle size, Zeta potential and appearance morphology characterization results of the HD@RPR nanodrug delivery system, and B is the particle size, Zeta potential and appearance morphology characterization results of the M1HD@RPR nanoparticles. Due to electrostatic self-assembly, HD@RPR self-assembles into a relatively uniform complex with a particle size of 123.6±14.4nm and an average surface charge of -13.5±6.8mV. Transmission electron microscopy (TEM) results show that the appearance is a uniform quasi-circular shape. Since the cell membrane is a phospholipid bilayer structure with a negative outer layer and a positive inner layer, it can be coated on the negatively charged HD@RPR surface to form M1HD@RPR. Compared with HD@RPR, the particle size of M1HD@RPR increased significantly, reaching 155.2±17.7nm, with a surface charge of -27.6±5.3mV. TEM results also showed that a relatively complete shell-core structure was formed.

[0052] This example verifies the effect of the M1HD@RPR nano-drug delivery system prepared above on the targeted sustained release of melanoma cells. In this example, Free DOX, HD@RPR (not wrapped with M1 cell membrane), and M1HD@RPR with a DOX content of 2.5 mg / kg were injected into mice through the tail vein. Blood was collected at 1 hour, 4 hours, 12 hours, and 24 hours, and the mice were killed. The DOX content in the blood and organs of the mice at different time points was measured to analyze the in vivo distribution of DOX of different preparations. The results are shown in the attached Figure 5 As can be seen, in the Free DOX group, 1 hour after injection, DOX rapidly distributed to the heart in addition to the blood, which explains its strong cardiotoxicity. 4 hours after injection, DOX levels in the heart, liver, spleen, and lungs increased rapidly, but decreased rapidly in the heart after 12 and 24 hours, indicating rapid metabolism. In the HD@RPR group, nanoparticle encapsulation enabled DOX to rapidly distribute to the liver, with less distribution in the heart. Furthermore, due to the nanoparticle size effect, DOX also distributed more in the blood. By 4 hours, DOX distribution further increased in the liver, spleen, and kidneys, while gradually decreasing in the blood. DOX also distributed more in the tumor than in the Free DOX group. This is due to the Schiff base-modified DOX's sensitive release to the tumor microenvironment. After 24 hours, DOX distribution also decreased, approaching that of Free DOX, but DOX distribution in the tumor was significantly higher than in the Free DOX group.

[0053] Attachment Figure 5 In the M1HD@RPR group, due to the long-circulating effect of M1 macrophage membrane encapsulation, DOX was still present in the blood at 1 and 4 hours, followed by the liver. This is due to the weak clearance of M1HD@RPR by macrophages. DOX distribution in the tumor reached its maximum at 4 hours, and even at 24 hours, a significant amount of DOX was still present in the tumor. Separate analysis of DOX distribution in each tumor site revealed that M1HD@RPR had significantly higher DOX distribution in the tumor site than the Free DOX group and even the HD@RPR group, demonstrating that M1 macrophage membrane encapsulation significantly enhances DOX distribution in the tumor site.

[0054] In this example, the distribution of Free DOX, HD@RPR, and M1HD@RPR in the tumor was observed visually 24 hours after administration. Paraffin-embedded sections were made and photographs were taken to observe the distribution of DOX. The results are shown in the attached figure. Figure 6 The results showed that the red fluorescence of DOX in the Free DOX group was significantly weaker than that in the nanoparticle group after 24 hours, and that in the M1HD@RPR group was significantly stronger than that in the HD@RPR group, further proving that M1 macrophage membrane encapsulation can significantly enhance the accumulation of DOX in the tumor site.

[0055] This example also verifies the inhibitory effect of the M1HD@RPR nanoparticle drug delivery system on primary melanoma tumors. Inspired by the in vitro anti-tumor effect and in vivo targeted anti-tumor effect of M1HD@RPR nanoparticles, this example uses B16F10 tumor-bearing mice as experimental subjects to evaluate the in vivo anti-tumor effect of nanoparticles. In this example, the tumor volume of each group of mice was recorded every other day, and a tumor volume growth curve was drawn. After the mice were sacrificed, the tumor tissue was removed, photographed, and weighed. The specific results are shown in the attached figure. Figure 7 and attached Figure 8 , attached Figure 7 A is the in vitro image of mouse tumors after treatment in each group, and B is the growth curve of tumor volume in each group after treatment (n=5); Figure 8 Figure A shows the comparison of tumor weights after treatment in each group, and Figure B shows the weight growth curve of mice in each group after treatment. As can be seen from the figure, the tumor volume of mice in the PBS group grew very quickly, reaching 1500mm on the 15th day after inoculation. 3 The blank nanoparticle group showed a growth curve close to that of the PBS group; the final tumor volume of the M1 group (only free M1 cell membrane was injected) and the OHA@RPR group (only shRNA-Ppn2 gene treatment group) was significantly lower than that of the PBS group, and the tumor volume growth of the HD@RPR and M1HD@RPR groups was significantly slower than that of the PBS group, and even the tumor disappeared completely, indicating that DOX chemotherapy combined with shRNA-Ptpn2 immunotherapy has a good effect, which is attributed to the sensitive release of DOX and the in vivo tumor microenvironment targeting effect of M1 macrophage membranes.

[0056] This example also verifies the inhibitory effect of the M1HD@RPR nano-drug delivery system on melanoma lung metastasis. In this example, melanoma cells were injected into the tail vein of mice to establish a melanoma lung metastasis model. Then, each treatment component was injected into the tail vein. The mice were killed 10 days after the end of treatment and the growth of melanoma in the mice's lungs was observed. The results are shown in the attached figure. Figure 9 As shown in the figure, mice in the PBS and Blank NPs groups were almost completely covered with melanoma tumors, and their lung tissue was severely damaged, indicating that melanoma is extremely invasive to the lungs. H&E histochemical analysis of the lungs of mice in each group revealed dense and clear tumor tissue, occupying more than 50% of the lung space. The significant improvement in the M1 group may be due to the fact that M1 macrophages are the first to settle in the lungs. Their surface proteins mediate the killing of lung tumor cells, reducing the occurrence of lung metastases. The OHA@RPR, HD@RPR, and M1HD@RPR groups were almost free of melanoma cells, and the lungs were intact. H&E results showed intact lung cells, no expansion of alveoli, and clear structure, indicating almost no lung metastasis. These results indicate that nanoparticles have an excellent therapeutic effect on lung metastatic melanoma by activating immunotherapy.

[0057] This example cleverly utilizes electrostatic self-assembly to construct a dual-gene and chemotherapy drug delivery system (M1HD@RPR) to achieve the effect of chemotherapy combined with immunotherapy for primary melanoma tumors and metastatic tumors in mice. This example is the first to use a non-viral vector to deliver a shRNA system that targets and downregulates the Ptpn2 gene into the tumor site, thereby downregulating the Ptpn2 gene and achieving the effect of gene immunotherapy. This provides an important basis for the application of pharmaceutical methods in tumor-targeted immunotherapy.

[0058] In summary, this embodiment successfully constructed an shRNA system shRNA-Ptpn2 targeting the silencing cell Ptpn2 gene to exert the effect of immunotherapy; and used Schiff base as a bridging arm of HA and DOX to prepare HA-DOX, and the negatively charged HA modified DOX so that it could wrap DOX through electrostatic action, and the pH-sensitive effect of Schiff base could make DOX sensitively released in the tumor microenvironment, thereby improving the targeted therapeutic effect of DOX and reducing its toxicity; M1 macrophage membrane was used to successfully encapsulate shRNA-Ptpn2 and HA-DOX to prepare M1HD@RPR nanoparticles, slowing down macrophage endocytosis and thus prolonging the circulation time in the body, and realizing targeted delivery to the tumor site; the M1HD@RPR nanodrug delivery system constructed in this embodiment can significantly kill B16F10 tumor cells in vitro, and significantly inhibit tumor volume growth in vivo, with significant tumor inhibition effects in vitro and in vivo, and M1HD@RPR nanoparticles can significantly enhance the treatment of melanoma metastatic lesions.

[0059] The above are embodiments of the present invention. The above embodiments and the specific parameters therein are only for the purpose of clearly describing the invention verification process and are not intended to limit the scope of patent protection of the present invention. The scope of patent protection of the present invention shall still be subject to the claims. Any equivalent structural changes made by using the contents of the description and drawings of the present invention shall also be included in the scope of protection of the present invention. Sequence Listing <110> Affiliated Hospital of Guizhou Medical University <120> Preparation method and application of nano drug delivery system for inhibiting melanoma growth <130> 2021.7.28 <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 19 <212> DNA <213> Artificial synthesis <400> 1 cacaaagaag ttacatctt 19 <210> 2 <211> 19 <212> DNA <213> Artificial synthesis <400> 2 aagatgtaac ttctttgtg 19

Claims

1. A method for preparing a nano drug delivery system for inhibiting melanoma growth, characterized in that: The specific steps are as follows: S1: Construction of shRNA-Ptpn2 plasmid Primers were designed to prepare the shRNA-Ptpn2 gene, which was then loaded into the pGpU6 / GFP / Neo plasmid to construct a shRNA-Ptpn2 plasmid that targeted downregulation of Ptpn2. The shRNA-Ptpn2 plasmid was then amplified in Escherichia coli. S2: Synthesis of HA-DOX The aromatic aldehyde formed by oxidation of hyaluronic acid HA reacts with the amino group of doxorubicin DOX through an imine bond to form a Schiff base HA-DOX, referred to as HD; S3: Preparation of M1 macrophage membranes S4: Preparation of Nanodrug Delivery System M1HD@RPR 5 μg of the shRNA-Ptpn2 plasmid constructed in step S1 was mixed with 25 μg of the cell-penetrating peptide iRGD and allowed to stand for 5 minutes to form a blend of iRGD and shRNA-Ptpn2, iRGD-shRNA-Ptpn2, referred to as RR; 125 μg of polyethyleneimine was added to the blend RR, and the mixture was allowed to stand for 5 minutes to obtain a complex of polyethyleneimine and RR, iRGD-polyethyleneimine-shRNA-Ptpn2, referred to as RPR; the mixture was allowed to stand for another 5 minutes, and HA-DOX containing 50 μg of DOX was added, and the mixture was mixed and self-assembled to obtain HD@RPR nanoparticles HA-DOX@iRGD-polyethyleneimine-shRNA-Ptpn2; the M1 macrophage membrane prepared in step S3 and the HD@RPR nanoparticles were then repeatedly passed through a 400 nm polycarbonate membrane using a micro-extruder to obtain the M1HD@RPR nanodrug delivery system, which was then stored at 4°C; The primers in step S1 include a forward primer and a reverse primer. The nucleotide sequence of the forward primer is shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

2.

2. The method for preparing a nano drug delivery system for inhibiting melanoma growth according to claim 1, characterized in that: The synthesis method of HA-DOX in step S2 is as follows: a. Oxidation of HA 0.4 g of HA was dissolved in 10 mL of deionized water to prepare a HA solution, and then 0.053 g of NaIO4 was dissolved in 5 mL of deionized water to prepare a NaIO4 solution. The NaIO4 solution was added dropwise to the HA solution, stirred in the dark for 12 h, and then ethylene glycol was added and stirred for 0.5 h. The resulting solution was dialyzed against deionized water to remove NaIO4. After purification for 48 h, the sponge-like product OHA was freeze-dried. b. Synthesis of HA-DOX Take 0.2 g of OHA from step a, add 0.2 g of OHA and 40 mg of DOX·HCl to 30 mL of ultrasonically degassed ddH2O. Incubate at 50°C in the dark under nitrogen for 48 h. Then dialyze against deionized water for 48 h to remove unreacted DOX·HCl. Freeze-dry to obtain a red spongy product, HD.

3. The method for preparing a nano drug delivery system for inhibiting melanoma growth according to claim 1, characterized in that: The preparation method of M1 macrophage membrane in step S3 is as follows: take RAW264.7 cells in the logarithmic growth phase, and when the cell density reaches 70%, add 100 ng / mL lipopolysaccharide (LPS) and 10 ng / mL IFN-γ. After 24 hours, collect M1 macrophages; then resuspend the cells in pre-cooled Tris-magnesium salt buffer to a cell concentration of 2×10 7 The cells were repeatedly squeezed out 20 times using a micro-extruder with only a gasket to destroy the cells. The cell membrane was then extracted using sucrose gradient centrifugation and then ultrasonicated at 100 W for 2 minutes to obtain the desired M1 macrophage membrane.

4. The method for preparing a nano drug delivery system for inhibiting melanoma growth according to claim 1, characterized in that: The ratio of M1 macrophage cell membrane to shRNA-Ptpn2 in step S4 is as follows: 0.034 mg of M1 macrophage cell membrane is added for every 5 μg of shRNA-Ptpn2.

5. The method for preparing a nano drug delivery system for inhibiting melanoma growth according to claim 1, characterized in that: In step S4, the M1 macrophage membrane and HD@RPR nanoparticles were passed through a 400 nm polycarbonate membrane 20 times.

6. The nano drug delivery system prepared by the preparation method according to any one of claims 1 to 5.

7. Use of the nano drug delivery system according to claim 6 in the preparation of a drug for treating melanoma or lung metastatic melanoma.

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