A fat cell carrier drug and its application

Abstract

The present invention provides a kind of fat cell carrier drug and its application, belonging to the field of biopharmaceutical technology. The fat cell carrier drug provided by the present invention is to transfect a vector containing IL‑15 / IL‑15Rα fusion gene into fat cells, screen and obtain IL‑15 / IL‑15Rα engineered fat cells; use the engineered fat cells to load anti-tumor drugs to obtain fat cell carrier drugs. The fat cell carrier drug constructed by the present invention integrates real-time monitoring, targeted delivery and synergistic treatment, and uses fat cells to express IL‑15 / IL‑15Rα complex protein to activate NK cells and produce immune killing effects on tumor cells. At the same time, dual active targeted delivery of tumor cells and mitochondria of amphiphilic cisplatin prodrugs is achieved, and the immunogenic cell death of tumor cells induced by cisplatin effectively enhances the immune response mediated by NK cells, thereby realizing chemotherapy-immunosynergistic treatment of tumor cells.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a fat cell carrier drug and application thereof. Background Art

[0002] With the development of nanotechnology, a large number of organic, inorganic and composite nanoparticles have been used as drug and / or gene delivery carriers in medical research and clinical trials for the treatment of malignant tumors. However, there are still many problems in the nano-delivery system that have not been properly solved in clinical applications and research, such as low drug loading rate (<10%, w / w), which can only be used as an adjuvant without direct therapeutic effect, and its metabolites can also cause short-term or long-term toxicity; even some carriers interact with cell surface receptors to cause adverse immune reactions such as interferon response, cytokine storm and / or lymphocyte activation, affecting the therapeutic effect. In order to overcome the above problems, the body's own "circulating cell" delivery carriers began to appear. Among various circulating cells, red blood cells, white blood cells, and stem cells have been hot spots in the field of cell-based drug delivery research in recent years, and have attracted widespread attention in the fields of biomedicine and materials science. Especially in recent years, with the deepening of research, many scholars have continuously tried to combine circulating cell carriers with different treatment methods such as chemotherapy, immunotherapy, and metabolic therapy to act on tumor cells in order to improve the therapeutic effect. Immunotherapy uses delivery vehicles to deliver therapeutic drugs to the treatment site and then activate or enhance the systemic immune function to kill tumor cells. This treatment method requires the delivery system to overcome multiple physiological and pathological barriers to achieve targeted and controlled release; at the same time, it can effectively solve the low response rate and immune-related side effects in solid tumor immunotherapy, and transform "cold tumors" into "hot tumors". Cell-based drug delivery systems just meet this condition. Although the chemotaxis of cell carriers to pathological environments provides a new therapeutic approach for targeted treatment of tumors, brain diseases, etc., several types of cell carriers are still There are urgent problems to be solved: the source, purification, storage, life cycle, drug loading rate, etc. of cell carriers limit their practical application; at the same time, the effect of integrated nanomedicine on the function of circulating cells is still unknown, and more pharmacokinetic and pharmacodynamic experiments are needed to confirm the anti-tumor advantages of cell carriers. Therefore, adipocytes are particularly outstanding in drug loading capacity, preparation methods and biosafety due to their unique hydrophobic structure, easy induction of transformation / isolation and purification in vitro, and widespread presence in the body. As a new drug delivery carrier, it has attracted widespread attention from experts and scholars in various fields.

[0003] In particular, tumor-associated adipocytes (TAA) are widely present in the microenvironment surrounding tumor tissues. They can not only indirectly act on tumor cells through inflammation, angiogenesis, fibrosis, etc., but also directly act on tumor cells through paracrine signals of adipose inflammatory factors, such as hormones, growth factors, cytokines, etc. In melanoma, white adipocytes can transfer lipids to tumor cells through fatty acid binding protein 4 (FABP4), inducing their metabolic reprogramming, growth, and invasion. In prostate cancer, adipocytes secrete chemokine ligand 7, which stimulates chemokine 3-positive tumor cells to migrate to the periprostatic adipose tissue (a common area of ​​prostate cancer recurrence). Adipose inflammatory factors can also indirectly regulate tumor insulin resistance and inflammation through autocrine effects, such as adiponectin acting locally on adipocytes to regulate adipocyte glucose absorption, lipogenesis, and lipid storage; leptin regulates adipocyte lipid decomposition through the central nervous system. In short, in the tumor microenvironment, stromal adipocytes drive tumor cell metabolic reprogramming and promote tumorigenesis; tumor cells promote the activation of adipocyte decomposition metabolic pathways and provide substrates for tumor composition metabolism. Therefore, adipocytes as drug delivery carriers can not only utilize their unique structure to increase the drug loading rate, but also use the metabolism-related signals of tumor cells themselves like a Trojan horse to achieve controllable delivery and release of drugs, thereby achieving specific killing of cancer cells. Although the research on adipocyte-based drug delivery systems in the field of tumor treatment has made certain progress, the current treatment system mainly uses fat-soluble drugs to transform adipocytes. While fat-soluble drugs transform adipocytes to increase the drug loading capacity, they also affect the physiological activity and tropism of the adipocytes themselves, thereby reducing the treatment effect and severely limiting the clinical application of adipocyte drug delivery carriers. Therefore, how to construct a new multifunctional drug efficient delivery carrier based on adipocytes has always been a technical problem that needs to be solved urgently in this field. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a fat cell carrier drug to achieve chemotherapy-immunotherapy of tumor cells.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides a fat cell carrier drug, comprising the following steps: transfecting a vector containing an IL-15 / IL-15Rα fusion gene into fat cells, and screening to obtain IL-15 / IL-15Rα engineered fat cells; using the IL-15 / IL-15Rα engineered fat cells to load an anti-tumor drug to obtain the fat cell carrier drug; the IL-15 / IL-15Rα fusion gene adopts a P2A sequence to connect IL-15 and IL-15Rα.

[0007] Preferably, the vector containing the IL-15 / IL-15Rα fusion gene includes a pcDNA3.1(+) vector or adenovirus.

[0008] Preferably, the adipocytes include mouse adipocytes 3T3-F442A or 3T3-L1.

[0009] Preferably, the anti-tumor drug includes cisplatin.

[0010] Preferably, when the IL-15 / IL-15Rα engineered adipocytes are used to load cisplatin, the following steps are included: chemically modifying cisplatin using long-chain fatty acids as axial ligands to obtain amphiphilic cisplatin; and loading the amphiphilic cisplatin into the IL-15 / IL-15Rα engineered adipocytes by a low-osmotic dialysis drug co-culture method.

[0011] Preferably, the long-chain fatty acids include ω-3:7, 10, 13, 16, 19-docosahexaenoic acid.

[0012] Preferably, the co-culture method is to co-culture the 10 μg / mL amphiphilic cisplatin with IL-15 / IL-15Rα engineered adipocytes in a maintenance culture medium.

[0013] The present invention also provides an application of the above-mentioned fat cell carrier drug in preparing a product for treating tumors.

[0014] Preferably, the tumor comprises melanoma.

[0015] Beneficial effects of the present invention:

[0016] The adipocyte carrier drug constructed by the present invention is a type of adipocyte carrier drug that integrates real-time monitoring, targeted delivery and synergistic treatment. The adipocyte carrier drug provided by the present invention utilizes adipocytes to express IL-15 / IL-15Rα complex protein to activate NK cells, produce immune killing effects on tumor cells, and then combined with the efficacy of the loaded anti-tumor drug itself, can achieve chemotherapy-immunosynergistic treatment of tumor cells.

[0017] In addition, when the tumor drug loaded by the fat cell carrier drug constructed by the present invention is cisplatin modified with long-chain anticancer fatty acids, dual active targeted delivery of the amphiphilic cisplatin prodrug to tumor cells and mitochondria can be achieved. At the same time, the immunogenic cell death of tumor cells induced by cisplatin effectively enhances the immune response mediated by NK cells, thereby achieving chemotherapy-immunotherapy of tumor cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is a schematic diagram of the pIRM structure;

[0019] Figure 2 3T3-F442A stably expressing IL-15 / IL-15Rα / mCherry fusion protein IRM Fluorescence detection results of cell lines;

[0020] Figure 3 A standard curve of concentration and absorbance determined using standards;

[0021] Figure 4 1×10 5 3T3-F442A IRM IL-15 / IL-15Rα concentration in cell supernatant;

[0022] Figure 5 This is a flow chart for constructing the adipocyte carrier drug of the present invention;

[0023] Figure 6 The synthesis and fragmentation diagram of cisplatin prodrug pro-Cisplatin, where A is the synthesis process of cisplatin prodrug pro-Cisplation; B is the spontaneous fragmentation diagram of pro-Cisplatin in the tumor microenvironment;

[0024] Figure 7 The results of inhibiting the growth of B16-F10 tumors in different groups, where A is the weight change of tumor-bearing mice in each group; B is the tumor growth curve; C is the tumor photo; D is the tumor weight;

[0025] Figure 8 Representative H&E images of various organs after drug administration;

[0026] Fig. 9 1H NMR spectra of cisplatin alone and amphiphilic cisplatin prodrugs, wherein A is the cisplatin alone group and B is the amphiphilic cisplatin prodrug group;

[0027] Fig.10 To verify the drug loading, release rate and toxicity in vitro, A is the 3T3-F442A induced differentiation and maturation; B is the UV-visible spectrum of cisplatin; C is the drug toxicity result. DETAILED DESCRIPTION

[0028] The present invention provides a fat cell carrier drug, comprising the following steps: transfecting a vector containing an IL-15 / IL-15Rα fusion gene into fat cells, and screening to obtain IL-15 / IL-15Rα engineered fat cells; using the IL-15 / IL-15Rα engineered fat cells to load an anti-tumor drug to obtain the fat cell carrier drug; the IL-15 / IL-15Rα fusion gene adopts a P2A sequence to connect IL-15 and IL-15Rα.

[0029] In the present invention, the amino acid sequence of the P2A sequence is ATNFSLLKQAGDVEENPGP (SEQ ID NO.1), and the cDNA sequence of the IL-15 / IL-15Rα fusion gene is preferably as shown in SEQ ID NO.2. Although the IL-15 signal is a means of effectively mediating NK cell immunotherapy based on improving the response of NK cells and CD8 T cells, the IL-15 protein has a short half-life and low biological activity in vivo, resulting in a low response rate in tumor patients, which makes its application have certain limitations. The present invention uses the P2A sequence to connect IL-15 and IL-15Rα, which overcomes the above defects. In the present invention, the IL-15 / IL-15Rα fusion gene is preferably an IL-15 / IL-15Rα fusion gene containing a fluorescent protein. The IL-15 / IL-15Rα fusion gene containing a fluorescent protein is preferably a fluorescent protein connected to the IL-15 / IL-15Rα fusion gene using a T2A sequence, and the amino acid sequence of the T2A sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO.3). The fluorescent protein preferably includes a green fluorescent protein and a red fluorescent protein; when the fluorescent protein is a red fluorescent protein, the cDNA sequence of the IL-15 / IL-15Rα fusion gene containing a red fluorescent protein is preferably as shown in SEQ ID NO.4; when the fluorescent protein is a green fluorescent protein, the cDNA sequence of the IL-15 / IL-15Rα fusion gene containing a green fluorescent protein is as shown in SEQID NO.5.

[0030] In the present invention, the vector containing the IL-15 / IL-15Rα fusion gene preferably includes a pcDNA3.1(+) vector or adenovirus, and the present invention has no special restrictions on the specific sources of the pcDNA3.1(+) vector and adenovirus. The present invention has no special restrictions on the specific method of transfection, and any conventional transfection method in the art can be used. In the present invention, the adipocytes preferably include mouse adipocytes 3T3-F442A or 3T3-L1, and the present invention has no special restrictions on the specific sources of mouse adipocytes 3T3-F442A and 3T3-L1. The vector containing the IL-15 / IL-15Rα fusion gene is transfected into the adipocytes, and IL-15 / IL-15Rα engineered adipocytes are screened and obtained, and the screening criterion is that the IL-15 / IL-15Rα engineered adipocytes can express the above-mentioned fusion gene or the above-mentioned fusion gene containing fluorescent protein. The present invention has no special restrictions on the specific method of screening. The IL-15 / IL-15Rα engineered adipocytes obtained by screening of the present invention realize the organic combination of the immune activation mechanism of the IL-15 / IL-15Rα complex protein secreted by adipocytes and the drug delivery ability of the engineered adipocytes, which greatly improves the tumor prevention and treatment effect of the IL-15 / IL-15Rα engineered adipocytes. Moreover, the present invention realizes the change of the biological properties and anti-tumor mechanism of adipocytes through IL-15 gene modification. In addition, the IL-15 / IL-15Rα engineered adipocytes obtained by screening of the present invention are an ideal drug delivery carrier, and their huge lipid droplet structure gives them excellent drug loading capacity, which can greatly increase the drug concentration at the target site and improve the therapeutic effect. Their superior biocompatibility can greatly reduce cytotoxicity and immunogenicity and increase the circulation time in the body. Their interaction signals with tumor cells can effectively control the delivery and release of drugs. They are easy to be genetically modified and transformed for multi-mode combined treatment.

[0031] After screening and obtaining IL-15 / IL-15Rα engineered adipocytes, the IL-15 / IL-15Rα engineered adipocytes are used to load anti-tumor drugs, and the anti-tumor drugs preferably include cisplatin. In the present invention, when IL-15 / IL-15Rα engineered adipocytes are used to load cisplatin, the following steps are preferably included: chemically modifying cisplatin with anti-cancer long-chain fatty acids as axial ligands to obtain amphiphilic cisplatin; and loading the amphiphilic cisplatin into IL-15 / IL-15Rα engineered adipocytes by a co-culture method. In the present invention, the long-chain fatty acids preferably include ω-3:7, 10, 13, 16, 19-docosahexaenoic acid. In the present invention, the process of obtaining amphiphilic cisplatin is preferably: in H 2 O 2Cisplatin is oxidized to a hydroxy coordination compound (DH-Cisplatin) in an aqueous solution, and then DH-Cisplatin and ω-3:7,10,13,16,19-docosahexaenoic acid (DHA) are catalyzed by EDC and NHS, and the hydroxyl group of cisplatin and the carboxyl group of DHA undergo an esterification reaction, and each cisplatin molecule can connect two fatty acid chains to form an amphiphilic cisplatin prodrug pro-Cisplatin; the amphiphilic cisplatin refers to cisplatin with one end being hydrophobic and the other end being hydrophilic. After obtaining the amphiphilic cisplatin, the amphiphilic cisplatin is loaded into IL-15 / IL-15Rα engineered adipocytes by a co-culture method. The co-culture method is preferably to co-incubate 10 μg / mL of amphiphilic cisplatin with IL-15 / IL-15Rα engineered adipocytes in a maintenance medium (DMEM / F12 [1:1], containing 10% fetal bovine serum and 1.5 μg / mL insulin), and the incubation time is preferably 48 hours.

[0032] The construction flow chart of the adipocyte carrier drug of the present invention is as follows Figure 5 As shown. According to the above content, the present invention comprehensively utilizes IL-15-mediated immune activation, long-chain fatty acid modification of chemotherapeutic drugs, adipocyte / tumor cell metabolic signals, fluorescent labeling and other functional units to achieve real-time monitoring of adipocyte carrier drugs, dual targeting and synergistic treatment of tumors. When the tumor drug loaded by the adipocyte carrier drug constructed by the present invention is cisplatin modified with long-chain anticancer fatty acids, under the mediation of fatty acid binding protein FABP4 and carnitine palmitoyltransferase (Carnitine palmitoyltransferase1, CPT1), dual active targeted delivery of tumor cells and mitochondria of amphipathic cisplatin prodrug is achieved. Wherein fatty acid binding protein family 4 (FABP4) is a kind of adipocyte type fatty acid binding protein, which is a partner molecule of intracellular free fatty acids in the transport process. FABP4 can bind to free fatty acids and transport them into cells. Carnitine palmitoyltransferase is a key enzyme for carnitine-dependent trans-mitochondrial inner membrane transport, mediating the transport of long-chain acylcarnitines. Since long-chain fatty acids cannot pass through the inner membrane of mitochondria by themselves, they need to shuttle into mitochondria through free carnitine, and carnitine needs high-affinity carnitine transporter to be transported into cells. Both are proteins that must be bound when cells or mitochondria obtain fatty acids.

[0033] The present invention also provides a use of the above-mentioned adipocyte carrier drug in the preparation of a product for treating tumors. In the present invention, the tumor preferably includes melanoma, and the product preferably includes a drug.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0036] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0037] Example 1

[0038] Using pcDNA3.1(+) as the vector, IL-15 cDNA (GenBank: NM_001254747.4) and IL-15Rα cDNA (GenBank: NM_001271497.1) as the expression target genes, red fluorescent protein (mCherry) as the reporter gene, the P2A sequence of porcine teschovirus (ATNFSLLKQAGDVEENPGP) and the T2A sequence of Thosea asigna virus (EGRGSLLTCGDVEENPGP) as the co-translational splicing sequence, the cDNA codon sequence of IL-15-P2A-IL-15Rα-T2A was optimized by SnapGene 5.0.5 software, and the cDNA synthesized by integrated DNA technology was cloned and expressed at the BamH I and EcoR I was inserted into the pcDNA3.1(+) vector to construct a CMV promoter-driven tricistronic IL-15 / IL-15Rα / mCherry complex protein co-expression vector (the cDNA sequence of the IL-15 / IL-15Rα / mCherry complex protein is shown in SEQ ID NO.4), denoted as pIRM (the schematic diagram is shown in Figure 1 shown).

[0039] After linearization of the pIRM plasmid with restriction endonuclease BgIII, Mouse adipocytes 3T3-F442A were transfected with 2000 DNA / pIRM, screened in RPMI 1640 (containing 10% fetal bovine serum) containing 600 μg / μL G418, and detected by fluorescence microscopy (the results are shown in Figure 2 IL-15 / IL-15Rα / mCherry complex protein stably expressed in mouse adipocytes 3T3-F442A IRM .

[0040] Detection of 3T3-F442A by ELISA IRM The expression level of IL-15 / IL-15Ra in the culture supernatant. 5 3T3-F442A or 3T3-F442A IRMInoculate into 6-well plates, 1.5 mL of culture medium per well. Collect 1 mL of cell culture supernatant at 48 h and 60 h, centrifuge, and follow the instructions of Mouse IL-15 / IL-15R ELISA Kit (Lianke Biotechnology, Cat: EK215R / 2-96) to detect OD450 and OD570 of each well with an ELISA reader. Determine the concentration and absorbance standard curve using the standard (see Figure 3 ), and the IL-15 / IL-15Rα concentration in the supernatant of each group was determined. Cells from each well were collected and counted by a hemocytometer, and the results were recorded in the table. The group numbers in the table represent parallel experiments of different groups, where the control group was tested twice in parallel and the experimental group was tested three times in parallel. The experimental results are shown in Tables 1 and Figure 4 As shown in Figure 2, after 48 h of stable transfection, the expression level of IL-15 / IL-15Ra in the culture supernatant was 31.446 pg / mL / 1×10 5 cells; at 60h, the expression level of IL-15 / IL-15Rα was 41.798pg / mL / 1×10 5 cells.

[0041] Table 1 Elisa method for determination of 3T3-F442A IRM IL-15 / IL-15Ra content in supernatant

[0042]

[0043] 48h 1x10 5 3T3-F442A IRM The content of IL-15 / IL-15Rα produced by cells was 31.446 pg / mL

[0044] 60h 1x105 3T3-F442A IRM The content of IL-15 / IL-15Rα produced by cells was 41.798 pg / mL

[0045] Example 2

[0046] In H 2 O 2 Cisplatin is oxidized to a hydroxyl coordination compound (DH-Cisplatin) in aqueous solution, and then DH-Cisplatin reacts with ω-3:7,10,13,16,19-docosahexaenoic acid (DHA) under the catalysis of EDC and NHS. The hydroxyl group of cisplatin and the carboxyl group of DHA undergo an esterification reaction, and each cisplatin molecule can be connected to two fatty acid chains, that is, the cisplatin prodrug pro-Cisplatin. The specific synthesis process is shown in Figure 6 A.

[0047] Compared with the microenvironment of normal tissues and cells, the tumor microenvironment (TME) has high expression of glutathione-related metabolic enzymes, resulting in an imbalance in the redox state. This specific redox state can break the ester bond and release cisplatin into the TME ( Figure 6 B).

[0048] The IL-15 / IL-15Rα engineered adipocytes prepared in Example 1 were induced to differentiate. Specifically, the IL-15 / IL-15Rα engineered adipocytes prepared in Example 1 were placed in a DMEM medium (containing a mixture of 10% FBS and 1% penicillin-streptomycin) for culture. When the cells were fully grown and contact-inhibited for 2 days, the culture medium was replaced with a differentiation medium (DMEM / F12[1:1]+10% FBS+10μM rosiglitazone+10μg / ml insulin, 1μM dexamethasone); after two days of differentiation, it was replaced with a maintenance medium (DMEM / F12[1:1]+10% fetal bovine serum+1.5μg / mL insulin+10μg / mL of cisplatin prodrug pro-Cisplatin (amphiphilic cisplatin)); after two days of maintenance, it was replaced with a complete culture medium (DMEM / F12[1:1]+10% FBS) to obtain the adipocyte carrier drug pro-Cisplatin@Adipocytes IL-15 / IL-15Rα .

[0049] Example 3

[0050] Tumor-bearing mice

[0051] 1×10 6 B16-F10 melanoma cells were subcutaneously injected into the right flank of C57BL6 mice, and the tumor formation was observed daily and the tumor volume was measured with a vernier caliper. 3 The drug injection was started at 1:00 p.m. The tumor volume was calculated as follows: tumor volume = length × width × width / 2.

[0052] Drug administration in mice

[0053] When the tumor size reaches 100 mm 3 The mice were randomly divided into 8 groups, each with 6 mice, and different drugs were injected intraperitoneally on days 1, 5, and 8, including (I) an equal amount of PBS as a control group (PBS group), (II) 1×10 9 differentiated 3T3-F442A adipocytes (Adipocytes group), (III) 0.2 mg / kg IL-15 / IL-15Rα fusion protein (purchased from medchemexpress, Cat: HY-P78558, denoted as IL-15 / IL-15Rα group), (IV) 1×10 9Differentiated 3T3-F442A adipocytes stably transfected with IL-15 / IL-15Rα (obtained in Example 1) IL-15 / IL-15Rα group), (V) 10 mg / kg DHA (DHA group), (VI) 5 mg / kg cisplatin (Cisplatin group), (VII) 5 mg / kg cisplatin prodrug (pro-Cisplatin group, obtained in Example 2), (VIII) 1×10 9 A stable IL-15 / IL-15Rα differentiated 3T3-F442A loaded with cisplatin prodrug (obtained in Example 2, pro-Cisplatin@Adipocytes IL-15 / IL-15Rα Group).

[0054] The body weight of mice was recorded every day. After 16 days of drug treatment, the animals were euthanized. The tumors of all tumor-bearing mice were removed and the tumor weights were recorded. The heart, liver, spleen, lung, and kidney were separated, fixed with 10% formalin buffer, stained with hematoxylin / eosin (H&E), and observed under a microscope.

[0055] The results are as follows Figure 7 and Figure 8 As shown. Figure 7 It can be seen that the weight of mice in each group is slowly increasing, and the difference is not obvious ( Figure 7 A) The tumor volume of PBS and empty vector Adipocytes group increased rapidly. IL-15 / IL-15Rα The tumor growth rate of the pro-Cisplatin group was slower than that of the IL-15 / IL-15Rα group; the tumor growth rate of the pro-Cisplatin group combined with DHA and cisplatin was slower than that of the single-drug DHA group and the Cisplatin group; the pro-Cisplatin@Adipocytes loaded with DHA and cisplatin IL-15 / IL-15Rα The tumor growth rate of the group was significantly slower than that of the single-drug DHA group, Cisplatin group, and pro-Cisplain group ( Figure 7 B) Tumor tissue was isolated from tumor-bearing mice and pro-Cisplatin@Adipocytes loaded with DHA and cisplatin IL-15 / IL-15Rα The tumors in the group were significantly reduced, and the anti-cancer effect was obvious ( Figure 7 C, D). The hearts, livers, spleens, lungs, and kidneys of the mice were stained, and it was observed that the drugs in each group had no obvious toxicity ( Figure 8 ).

[0056] Example 4

[0057] The pcDNA3.1(+) vector was used, IL-15 cDNA (GenBank: NM_001254747.4) and IL-15Rα cDNA (GenBank: NM_001271497.1) were used as the target genes, green fluorescent protein (GFP) was used as the reporter gene, the P2A sequence of porcine teschovirus (ATNFSLLKQAGDVEENPGP) and the T2A sequence of Thosea asignavirus (EGRGSLLTCGDVEENPGP) were used as the co-translational splicing sequence, and the SnapGene The cDNA codon sequence of IL-15-P2A-IL-15Rα-T2A was optimized using 5.0.5 software. The cDNA synthesized by integrated DNA technology was inserted into the pcDNA3.1(+) vector at BamHⅠ and EcoRⅠ to construct a tricistronic IL-15 / IL-15Rα / GFP complex protein co-expression vector driven by a CMV promoter (the cDNA sequence of the IL-15 / IL-15Rα / GFP complex protein is shown in SEQ ID NO.5), which was denoted as pIRG.

[0058] After linearization of the pIRG plasmid with restriction endonuclease BgⅢ, Mouse adipocytes 3T3-L1 were transfected with 2000DNA / pIRG, screened in RPMI 1640 (containing 10% fetal bovine serum) containing 600μg / μL G418, and detected by fluorescence microscopy to obtain mouse adipocytes 3T3-L1 stably expressing IL-15 / IL-15Rα / GFP complex protein. IRG .

[0059] In H 2 O 2 Cisplatin was oxidized to a hydroxyl coordination compound (DH-Cisplatin) in aqueous solution, and then DH-Cisplatin and ω-3:7,10,13,16,19-docosahexaenoic acid (DHA) were esterified under the catalysis of EDC and NHS. Each cisplatin molecule can connect two fatty acid chains to form an amphiphilic cisplatin prodrug pro-Cisplatin. The properties of cisplatin and amphiphilic cisplatin prodrug were analyzed by multinuclear NMR spectroscopy. 1 Spectral chemical migration characteristics of H elements ( Fig. 9). The nuclear magnetic resonance hydrogen spectrum of cisplatin alone shows three obvious peaks, namely the amino peak of cisplatin, the water peak and the peak of the solvent DMSO. In the synthesized amphiphilic cisplatin prodrug, the peaks of water and DMSO are relatively obvious, while the cisplatin amino peak is relatively weak and has undergone a significant position change, migrating from the original 3.93ppm to 4.17ppm, reflecting the interaction between the amino group in the amphiphilic cisplatin prodrug and the carboxyl portal of DHA.

[0060] 3T3-L1 IRG The cells were cultured in DMEM medium (containing a mixture of 10% FBS and 1% penicillin-streptomycin). After the cells were fully grown and contact inhibited for 2 days, the medium was replaced with differentiation medium (DMEM / F12[1:1]+10% FBS+10μM rosiglitazone+10μg / ml insulin, 1μM dexamethasone); after two days of differentiation, it was replaced with maintenance medium (DMEM / F12[1:1]+10% fetal bovine serum+1.5μg / mL insulin+10μg / mL cisplatin prodrug pro-Cisplatin (amphiphilic cisplatin)); after two days of maintenance, it was replaced with complete medium (DMEM / F12[1:1]+10% FBS) to obtain the adipocyte carrier drug pro-Cisplatin@Adipocytes IL-15 / IL-15Rα Oil red O fat staining method verified that 3T3-F442A preadipocytes had differentiated and matured ( Fig.10 A). By scanning cisplatin at 10-800 nm wavelength using UV-visible absorption spectroscopy, it was found that cisplatin has an obvious absorption peak at 301 nm ( Fig.10 B). Using this wavelength, the absorbance of different concentrations of cisplatin was measured, and the regression equation of the cisplatin standard curve was obtained. Then, the absorbance before and after culture in the maintenance medium was substituted into the regression equation, and the loading rate of the amphiphilic cisplatin prodrug was obtained to be 62.37%; the pro-Cisplatin@Adipocytes cultured for 2 days was measured. IL-15 / IL-15Rα Supernatant OD 301 The release rate of the amphiphilic cisplatin prodrug was 69.21%. Different concentrations of cisplatin alone, DHA alone, and amphiphilic cisplatin prodrug were mixed with 1×10 7 The results of MTT showed that with the increase of drug concentration, the cell viability decreased more obviously, and the amphiphilic cisplatin prodrug was more toxic and had better anticancer effect than cisplatin or DHA alone ( Fig.10 C). It was further confirmed that DHA and cisplatin have a synergistic effect.

[0061] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A fat cell carrier drug, characterized in that: The method comprises the following steps: transfecting a vector containing an IL-15 / IL-15Rα fusion gene into fat cells, and screening to obtain IL-15 / IL-15Rα engineered fat cells; using the IL-15 / IL-15Rα engineered fat cells to load an anti-tumor drug, and obtaining a fat cell carrier drug; the IL-15 / IL-15Rα fusion gene is a P2A sequence connecting IL-15 and IL-15Rα; the cDNA sequence of the IL-15 / IL-15Rα fusion gene is shown in SEQ ID NO.2; The anti-tumor drug includes cisplatin; When the IL-15 / IL-15Rα engineered adipocytes are used to load cisplatin, the method comprises the following steps: chemically modifying cisplatin using long-chain fatty acids as axial ligands to obtain amphiphilic cisplatin; and loading the amphiphilic cisplatin into the IL-15 / IL-15Rα engineered adipocytes by a co-culture method; The long-chain fatty acids include ω-3:7, 10, 13, 16, 19-docosahexaenoic acid; The co-culture is to culture the IL-15 / IL-15Rα engineered adipocytes for two days, dissolve the amphiphilic cisplatin in a maintenance medium, and co-culture with the IL-15 / IL-15Rα engineered adipocytes for 2 days.

2. The adipocyte carrier drug according to claim 1, characterized in that: The backbone vector containing the IL-15 / IL-15Rα fusion gene includes a pcDNA3.1(+) vector or adenovirus.

3. The adipocyte carrier drug according to claim 1, characterized in that: The adipocytes include mouse adipocytes 3T3-F442A or 3T3-L1.

4. The adipocyte carrier drug according to claim 1, characterized in that: The co-culture method is to dissolve the 10 μg / mL amphiphilic cisplatin in a maintenance culture medium and co-culture it with the IL-15 / IL-15Rα engineered adipocytes.

5. Use of the adipocyte carrier drug according to any one of claims 1 to 4 in the preparation of a product for treating melanoma.

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