CN117138053B

CN117138053B - A fat cell carrier drug and its application

Info

Publication number: CN117138053B
Application number: CN202311003894.5A
Authority: CN
Inventors: 张元新; 李佟; 张仁文; 劳军; 葛雅琨
Original assignee: Jilin Institute of Chemical Technology
Current assignee: JILIN UNIVERSITY OF CHEMICAL TECHNOLOGY
Priority date: 2023-08-10
Filing date: 2023-08-10
Publication date: 2025-02-14
Anticipated expiration: 2043-08-10
Also published as: CN117138053A

Abstract

The invention provides an adipocyte carrier drug and application thereof, belonging to the technical field of biopharmacology. The invention provides an adipocyte carrier drug, which is prepared by transfecting a carrier containing IL-15/IL-15 Ralpha fusion genes into adipocytes, screening to obtain IL-15/IL-15 Ralpha engineering adipocytes, and loading an antitumor drug with the engineering adipocytes. The adipocyte carrier drug constructed by the invention integrates real-time monitoring, targeted delivery and cooperative treatment, and utilizes adipocyte expression IL-15/IL-15 Ralpha composite protein to activate NK cells and generate immune killing effect on tumor cells. Meanwhile, double active targeting delivery of tumor cells and mitochondria of amphiphilic cisplatin precursor medicines is realized, and immunogenic cell death of the tumor cells induced by cisplatin effectively promotes NK cell-mediated immune response, so that chemotherapy-immune cooperative treatment of the tumor cells is realized.

Description

Adipocyte carrier drug and application thereof

Technical Field

The invention belongs to the technical field of biopharmacology, and particularly relates to an adipocyte carrier drug and application thereof.

Background

With the development of nanotechnology, a large number of organic, inorganic and composite nanoparticles are applied to medical research and clinical experiments of malignant tumor treatment as drugs and/or gene delivery vectors, however, in clinical application and research, various problems of a nano delivery system are not solved, such as low drug loading rate (< 10%, w/w), only can be used as an adjuvant, no direct treatment effect exists, and metabolites of the nano delivery system can cause short-term or long-term toxicity, and even the interaction of some vectors and cell surface receptors can cause adverse immune reactions such as interferon response, cytokine storm and/or lymphocyte activation, and the like, so that the treatment effect is influenced. To overcome the above problems, the human body's own "circulating cell" delivery vehicle has begun to appear, and among various circulating cells, erythrocytes, leukocytes and stem cells are hot spots in the field of recent cell-based drug delivery research, and have attracted a wide range of attention in the fields of biomedicine and material science. In particular, in recent years, as research proceeds, many scholars continuously try to combine a circulating cell carrier with different treatment modes such as chemotherapy, immunotherapy, metabolic treatment and the like to act on tumor cells in order to improve the treatment effect. The immune therapy is to utilize a delivery carrier to deliver therapeutic drugs to a treatment part and then activate or improve immune function of a system to kill tumor cells, the treatment mode requires the delivery system to overcome various physiological and pathological barriers to realize targeted and controllable release, and meanwhile, the immune therapy can effectively solve the problems of low response rate and immune related side effects in the immune therapy of solid tumors, and convert cold tumors into hot tumors, and the cell-based drug delivery system just meets the condition. Although chemotaxis of cell carriers to pathological environment provides a new therapeutic approach for targeted treatment of tumors, brain diseases and the like, the current several types of cell carriers still have the problems to be solved in the drug delivery process, namely the sources, purification, storage, life cycle, drug loading rate and the like of the cell carriers limit the practical application of the cell carriers, and meanwhile, the influence of the integrated nano-drugs on the functions of circulating cells is unknown, and more pharmacokinetic and pharmacodynamic experiments are needed to confirm the anti-tumor advantages of the cell carriers. Therefore, the fat cell (Adipocytes) is particularly superior in the aspects of drug carrying capacity, preparation method and biosafety due to the unique hydrophobic structure, easy in vitro induced transformation/separation and purification, wide in vivo existence and the like, and is widely focused by experts and scholars in various fields as a novel drug delivery carrier.

In particular, tumor-associated adipocytes (TAA) are widely present in the microenvironment surrounding tumor tissue, and can act directly on tumor cells not only indirectly through inflammation, angiogenesis, fibrosis, etc., but also through the paracrine signals of adiposities, such as hormones, growth factors, cytokines, etc. In melanoma, white adipocytes can transfer lipids into tumor cells through fatty acid binding protein 4 (FABP 4), inducing their metabolic reprogramming, growth, and invasion. In prostate cancer, adipocytes secrete chemokine ligand 7, stimulating migration of chemokine 3 positive tumor cells to periprostatic adipose tissue (a common area of recurrence of prostate cancer). The lipoinflammatory factors can also indirectly regulate insulin resistance and inflammatory properties of tumors through autocrine effects, such as adiponectin acting on adipocytes locally, regulating glucose absorption, adipogenesis, and lipid storage of adipocytes, and leptin regulating lipolysis of adipocytes through the central nervous system. In summary, in the tumor microenvironment, stromal adipocytes drive the metabolic reprogramming of tumor cells, promoting tumorigenesis, and tumor cells promote activation of adipocyte catabolic pathways, providing substrates for tumor anabolism. Therefore, the fat cell serving as a drug delivery carrier not only can utilize the unique structure of the fat cell to improve the drug loading rate, but also can utilize the metabolic related signals of tumor cells like Trojan horse to realize the controllable delivery and release of the drug so as to achieve the specific killing of cancer cells. Although research on the field of tumor treatment is advanced on the basis of a drug delivery system based on adipocytes, the current treatment system mainly utilizes fat-soluble drugs to modify adipocytes, and the fat-soluble drugs improve the drug loading capacity of the adipocytes and influence the physiological activity and the tendency of the adipocytes, so that the treatment effect is reduced, and the clinical application of the adipocyte drug delivery carrier is severely limited. Therefore, how to construct and obtain a novel multifunctional drug high-efficiency delivery carrier based on adipocytes is always a technical problem to be solved in the field.

Disclosure of Invention

In view of the above, the present invention aims to provide an adipocyte carrier drug for realizing chemotherapy-immune cooperative treatment of tumor cells.

In order to achieve the above object, the present invention provides the following technical solutions:

The invention provides an adipocyte carrier drug, which comprises the following steps of transfecting a carrier containing an IL-15/IL-15 Ralpha fusion gene into adipocytes, screening to obtain IL-15/IL-15 Ralpha engineering adipocytes, loading an antitumor drug into the IL-15/IL-15 Ralpha engineering adipocytes to obtain the adipocyte carrier drug, wherein the IL-15/IL-15 Ralpha fusion gene is formed by connecting IL-15 and IL-15 Ralpha by adopting a P2A sequence.

Preferably, the vector containing the IL-15/IL-15Rα fusion gene comprises a pcDNA3.1 (+) vector or an adenovirus.

Preferably, the adipocytes comprise mouse adipocytes 3T3-F442A or 3T3-L1.

Preferably, the antitumor drug comprises cisplatin.

Preferably, when the IL-15/IL-15Rα engineering adipocytes are used for loading cisplatin, the method comprises the steps of chemically modifying the cisplatin by using long-chain fatty acid as an axial ligand to obtain amphiphilic cisplatin, and loading the amphiphilic cisplatin into the IL-15/IL-15Rα engineering adipocytes by a low-permeability chromatography drug co-culture method.

Preferably, the long chain fatty acid comprises omega-3:7, 10,13,16, 19-docosahexaenoic acid.

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

The invention also provides application of the adipocyte carrier drug in preparing a tumor treatment product.

Preferably, the tumor comprises melanoma.

The invention has the beneficial effects that:

The adipocyte carrier medicine constructed by the invention is an adipocyte carrier medicine integrating real-time monitoring, targeted delivery and cooperative treatment. The adipocyte carrier drug provided by the invention activates NK cells by utilizing the IL-15/IL-15 Ralpha composite protein expressed by adipocytes, generates an immune killing effect on tumor cells, and combines the effect of the loaded antitumor drug, thereby realizing chemotherapy-immune cooperative treatment on the tumor cells.

In addition, when the tumor drug loaded by the fat cell carrier drug constructed by the invention is cisplatin modified by long-chain anticancer fatty acid, dual active targeting delivery of tumor cells and mitochondria of amphiphilic cisplatin precursor drug can be realized. Meanwhile, the immunogenic cell death of tumor cells induced by cisplatin effectively promotes the NK cell-mediated immune response, thereby realizing the chemotherapy-immune cooperative treatment of the tumor cells.

Drawings

FIG. 1 is a schematic diagram of pIRM;

FIG. 2 shows the fluorescence detection results of A3T 3-F442A ^IRM cell line stably expressing IL-15/IL-15Rα/mCherry fusion protein;

FIG. 3 is a concentration and absorbance standard curve determined using a standard;

FIG. 4 is a graph showing the concentration of IL-15/IL-15Rα in 1X10 ⁵ supernatants of 3T3-F442A ^IRM cells;

FIG. 5 is a flow chart of the construction of the adipocyte carrier drug of the present invention;

FIG. 6 is a diagram showing the synthesis and cleavage of the cisplatin prodrug pro-CISPLATIN, wherein A is the synthesis scheme of the cisplatin prodrug pro-Cisplation, and B is the spontaneous cleavage diagram of pro-CISPLATIN in the tumor microenvironment;

FIG. 7 shows the results of inhibiting the growth of B16-F10 tumors in different groups, wherein A is the weight change of each group of tumor-bearing mice, B is the tumor growth curve, C is the tumor photograph, and D is the tumor weight;

FIG. 8 is a representative H & E image of each organ after administration;

FIG. 9 is a nuclear magnetic resonance 1H spectrum of cisplatin alone and an amphiphilic cisplatin prodrug, wherein A is cisplatin alone group and B is amphiphilic cisplatin prodrug group;

FIG. 10 shows in vitro verification of drug loading, release rate and toxicity, wherein A is 3T3-F442A induced differentiation maturation, B is cisplatin UV visible spectrum, and C is drug toxicity result.

Detailed Description

In the 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 shown as SEQ ID NO. 2. Although IL-15 signaling is a means based on improving NK cell and CD 8T cell responses and can effectively mediate NK cell immunotherapy, IL-15 protein has short half-life and low in vivo biological activity, so that the response rate of the IL-15 protein in tumor patients is low, and the application of the IL-15 protein has a certain limit. The invention adopts P2A sequence to connect IL-15 and IL-15Rα, which overcomes the defects. In the invention, the IL-15/IL-15Rα fusion gene is preferably an IL-15/IL-15Rα fusion gene containing fluorescent protein, the IL-15/IL-15Rα fusion gene containing fluorescent protein is preferably formed by connecting fluorescent protein and the IL-15/IL-15Rα fusion gene by adopting a T2A sequence, the amino acid sequence of the T2A sequence is EGRGSLLTCGDVEENPGP (SEQ ID NO. 3), the fluorescent protein preferably comprises green fluorescent protein and red fluorescent protein, when the fluorescent protein is red fluorescent protein, the cDNA sequence of the IL-15/IL-15Rα fusion gene containing red fluorescent protein is preferably shown as SEQ ID NO.4, and when the fluorescent protein is green fluorescent protein, the cDNA sequence of the IL-15/IL-15Rα fusion gene containing green fluorescent protein is shown as SEQ ID NO. 5.

In the present invention, the vector containing the IL-15/IL-15Rα fusion gene preferably includes a pcDNA3.1 (+) vector or an adenovirus, and the specific sources of the pcDNA3.1 (+) vector and the adenovirus are not particularly limited. The specific mode of transfection is not particularly limited in the present invention, and any transfection method conventional in the art may be used. In the present invention, the adipocytes preferably include mouse adipocytes 3T3-F442A or 3T3-L1, and the specific sources of mouse adipocytes 3T3-F442A and 3T3-L1 are not particularly limited. Transfecting a vector containing an IL-15/IL-15Rα fusion gene into an adipocyte, and screening to obtain an IL-15/IL-15Rα engineered adipocyte, wherein the screening is based on the fact that the IL-15/IL-15Rα engineered adipocyte is capable of expressing the fusion gene or the fusion gene containing a fluorescent protein. The specific method of screening is not particularly limited in the present invention. The IL-15/IL-15Rα engineering adipocytes obtained by screening realize the organic combination of an IL-15/IL-15Rα composite protein immune activation mechanism secreted by adipocytes and the drug delivery capability of the engineering adipocytes, greatly improve the control effect of the IL-15/IL-15Rα engineering adipocytes on tumors, change the biological properties and the anti-tumor action mechanism of the adipocytes by modifying the IL-15 gene, and are ideal drug delivery carriers, the huge lipid drop structure of the IL-15/IL-15Rα engineering adipocytes obtained by screening can ensure that the IL-15/IL-15Rα engineering adipocytes have excellent drug loading capability, can greatly improve the drug concentration at an action target point, improve the treatment effect, the superior biocompatibility of the IL-15/IL-15Rα engineering adipocytes can greatly reduce cytotoxicity and immunogenicity, increase the internal circulation time, the interaction signals of the IL-15/IL-15Rα engineering adipocytes can effectively control the delivery and release of drugs, and are easy to be genetically modified and modified for carrying out various mode combined therapies.

After the IL-15/IL-15Rα engineering adipocytes are obtained by screening, the IL-15/IL-15Rα engineering adipocytes are used for loading an anti-tumor drug, wherein the anti-tumor drug preferably comprises cisplatin. In the invention, when the IL-15/IL-15Rα engineering adipocytes are used for loading cisplatin, the method preferably comprises the following steps of chemically modifying the cisplatin by using an anticancer long-chain fatty acid as an axial ligand to obtain amphiphilic cisplatin, and loading the amphiphilic cisplatin into the IL-15/IL-15Rα engineering adipocytes by a co-culture method. In the present invention, the long chain fatty acid preferably comprises omega-3:7, 10,13,16, 19-docosahexaenoic acid. In the invention, the process of obtaining the amphiphilic cisplatin is preferably that Cisplatin (CISPLATIN) is oxidized into a hydroxyl coordination compound (DH-CISPLATIN) in an H ₂O₂ aqueous solution, then DH-CISPLATIN and omega-3:7, 10,13,16, 19-docosahexaenoic acid (DHA) are subjected to esterification reaction with carboxyl groups of the DHA under the catalysis of EDC and NHS, each cisplatin molecule can be connected with two fatty acid chains to form an amphiphilic cisplatin precursor pro-CISPLATIN, and the amphiphilic cisplatin refers to cisplatin with one end being hydrophobic and the other end being hydrophilic. After the amphiphilic cisplatin is obtained, the amphiphilic cisplatin is loaded into IL-15/IL-15 Ralpha engineering adipocytes by a co-culture method. The co-culture method is preferably to co-incubate 10. Mu.g/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. Mu.g/mL of insulin), preferably for a period of 48 hours.

The construction flow chart of the adipocyte carrier drug of the present invention is shown in FIG. 5. According to the above, the invention comprehensively utilizes the IL-15 mediated immune activation, long-chain fatty acid modification of chemotherapeutics, fat cell/tumor cell metabolic signals, fluorescent markers and other functional units, thereby realizing the real-time monitoring, dual targeting and cooperative treatment of tumors of the fat cell carrier drugs. When the tumor drug loaded by the fat cell carrier drug constructed by the invention is cisplatin modified by long-chain anticancer fatty acid, double active targeting delivery of tumor cells and mitochondria of amphiphilic cisplatin precursor drug is realized under the mediation of fatty acid binding protein FABP4 and carnitine palmitoyl transferase (CARNITINE PALMITOYLTRANSFERASE, CPT 1). Wherein fatty acid binding protein family 4 (FABP 4) is an adipocyte-type fatty acid binding protein, which is a chaperone for free fatty acids in cells during transport, and FABP4 is capable of binding to free fatty acids and transporting them into cells. Carnitine palmitoyltransferase is a key enzyme for carnitine-dependent trans-granular endomembrane transport, mediating long chain acyl carnitine transport. Since long chain fatty acids cannot self-pass through the inner mitochondrial membrane, they need to shuttle into the mitochondria through free carnitine, which requires high affinity carnitine transporter enzymes to transport into the cell. Both are proteins that must be bound when the cell or mitochondria acquire fatty acids.

The invention also provides application of the adipocyte carrier drug in preparing a tumor treatment product. In the present invention, the tumor preferably comprises melanoma and the product preferably comprises a drug.

The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.

In the following examples, conventional methods are used unless otherwise specified.

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Example 1

The cDNA codon sequence of IL-15-P2A-IL-15Rα -T2A is optimized by SnapGene 5.0.5 software, and cDNA synthesized by an integrated DNA technology is inserted into the pcDNA3.1 (+) vector at BamH I and EcoR I to construct a CMV promoter-started polycistronic IL-15/IL-15Rα/mCherry complex protein coexpression vector (the cDNA sequence of IL-15/IL-15Rα/mCherry complex protein is shown as SEQ ID NO. 4) by using pcDNA3.1 (+) as a vector, IL-15cDNA (GenBank: NM-001254747.4) and IL-15Rα cDNA (GenBank: NM-001271497.1) as expression target genes, red fluorescent protein (mCherry) as a reporter gene, and P2A sequence (ATNFSLLKQAGDVEENPGP) of porcine teschovirus and T2A sequence (EGRGSLLTCGDVEENPGP) of Thosea asigna virus) as a cotranslational cleavage sequence.

Linearizing pIRM the plasmid with the restriction enzyme BgIII, then2000DNA/pIRM transfected mouse adipocytes 3T3-F442A, were screened in RPMI 1640 (containing 10% fetal bovine serum) containing 600. Mu.g/. Mu. L G418 and examined by fluorescence microscopy (results shown in FIG. 2) to obtain IL-15/IL-15Rα/mCherry complex protein stably expressed mouse adipocytes 3T3-F442A ^IRM.

IL-15/IL-15Ra expression levels in the supernatants of 3T3-F442A ^IRM media were assayed by ELISA. 3X 10 ⁵ 3T3-F442A or 3T3-F442A ^IRM were inoculated into 6-well plates with 1.5mL of culture broth per well. At 48h, 60h, 1mL of cell culture supernatant was collected, centrifuged, and the wells OD450 and OD570 were assayed by a microplate reader, following the instructions of Mouse IL-15/IL-15R ELISAKit (Union, cat: EK 215R/2-96). The concentration and absorbance standard curves (see FIG. 3) were determined by standard, and the IL-15/IL-15Rα concentration in each supernatant group was determined. The cells from each well were collected and counted by a hemocytometer and the results are recorded in the table. The group numbers in the tables represent the parallel runs of the different groups, respectively, wherein the control group was run in duplicate and the test group was run in triplicate, respectively. As shown in Table 1 and FIG. 4, the IL-15/IL-15Ra expression level in the culture medium supernatant after 48 hours of the stable transfection of the cells was 31.446 pg/mL/1X 10 ⁵ cells, and the IL-15/IL-15Rα expression level at 60 hours was 41.798 pg/mL/1X 10 ⁵ cells.

TABLE 1 Elisa determination of IL-15/IL-15Ra content in supernatants of 3T3-F442A ^IRM

48H 1X10 ⁵ 3T3-F442A ^IRM cells produced IL-15/IL-15Rα at a level of 31.446pg/mL

60H 1X105 cells producing IL-15/IL-15Rα at 41.798pg/mL of 3T3-F442A ^IRM

Example 2

Cisplatin (CISPLATIN) is oxidized into a hydroxyl coordination compound (DH-CISPLATIN) in an H ₂O₂ aqueous solution, DH-CISPLATIN and omega-3:7, 10,13,16, 19-docosahexaenoic acid (DHA) are subjected to esterification reaction with carboxyl groups of DHA under the catalysis of EDC and NHS, and each cisplatin molecule can be connected with two fatty acid chains, namely, a cisplatin prodrug pro-CISPLATIN, and the specific synthesis process is shown in figure 6A.

Compared with the microenvironment of normal tissues and cells, glutathione-related metabolic enzymes are highly expressed in Tumor Microenvironment (TME), and the redox state is unbalanced. This specific redox state can break the ester bond and cisplatin is released into the TME (fig. 6B).

The IL-15/IL-15Rα -engineered adipocytes prepared in example 1 were induced to differentiate. Specifically, IL-15/IL-15Rα -engineered adipocytes prepared in example 1 were cultured in DMEM medium (mixed solution containing 10% FBS and 1% penicillin streptomycin), after the cells were fully contacted and inhibited for 2 days, the medium was replaced with differentiation medium (DMEM/F12 [1:1] +10% FBS+10. Mu.M rosiglitazone+10. Mu.g/mL insulin, 1. Mu.M dexamethasone), after two days of differentiation, with maintenance medium (DMEM/F12 [1:1] +10% fetal bovine serum+1.5. Mu.g/mL insulin+10. Mu.g/mL cisplatin pro-CISPLATIN (amphiphilic cisplatin)), and after two days of maintenance, with complete medium (DMEM/F12 [1:1] +10% FBS), the adipocyte carrier drug pro-Cisplatin@Adipomes ^{IL-15/IL-15Rα} was obtained.

Example 3

Tumor-bearing of mice

1X 10 ⁶ B16-F10 melanoma cells were subcutaneously injected into the right flank of C57BL6 mice, the tumor formation was observed daily, and the tumor volume was measured with a vernier caliper. When the tumor reached 100mm ³, injection of the drug was started. The tumor volume calculation formula is tumor volume = length x width/2.

Administration to mice

When the tumor size reached 100mm ³, mice were randomly divided into 8 groups of 6, each, and different drugs were intraperitoneally injected on days 1,5, 8, respectively. An equivalent amount of PBS (I) was included as a control group (PBS group), (II) 1X 10 ⁹ differentiated 3T3-F442A adipocytes (Adipocytes group), (III) 0.2mg/kg IL-15/IL-15Rα fusion protein (purchased from medchemexpress company, cat: HY-P78558, designated IL-15/IL-15Rα group), (IV) 1X 10 ⁹ differentiated 3T3-F442A adipocytes (obtained in example 1) (Adipocytes ^{IL-15/IL-15Rα} group), (V) 10mg/kgDHA (DHA group), (VI) 5mg/kg cisplatin (CISPLATIN group), (VII) 5mg/kg cisplatin prodrug (pro-CISPLATIN group, obtained in example 2), (VIII) 1X 10 ⁹ stabilized cisplatin prodrug-loaded stabilized IL-15/IL-15Rα differentiated 3T3-F442A (obtained in example 2), (pro-Cisplatitin@Adsystem ^{IL-15/IL-15Rα} group).

The body weight of the mice was recorded daily. Animals were euthanized 16 days after drug treatment. Tumors were excised from all tumor-bearing mice and tumor weights were recorded. Centrifugation, liver, spleen, lung, kidney were performed, fixed with 10% formalin buffer, and hematoxylin/eosin (H & E) stained for microscopic observation.

The results are shown in fig. 7 and 8. As can be seen from fig. 7, the body weight of each group of mice was slowly increasing, and the difference was not obvious (fig. 7A). The tumor volumes of PBS and empty vector Adipocytes increased rapidly, the tumor growth rate of Adipocytes ^{IL-15/IL-15Rα} group was slower than that of IL-15/IL-15Rα group, the tumor rate of pro-CISPLATIN group in which DHA was combined with cisplatin was slower than that of DHA group alone, CISPLATIN group alone, and the tumor growth rate of pro-Cisplatin@adinocytes ^{IL-15/IL-15Rα} group loaded with DHA and cisplatin was significantly slower than that of DHA group alone, CISPLATIN group and pro-Cisplain group alone (FIG. 7B). Tumor tissues were isolated from tumor-bearing mice, and the pro-cispatatin@adicocytes ^{IL-15/IL-15Rα} group carrying DHA and cisplatin showed significantly reduced tumor and significant cancer inhibition effect (FIG. 7C, D). No apparent toxicity of each drug group was observed by staining the heart, liver, spleen, lung, kidney of the dosed mice (fig. 8).

Example 4

The cDNA sequence of IL-15-P2A-IL-15Rα -T2A is optimized by SnapGene 5.0.5 software, and cDNA synthesized by an integrated DNA technology is inserted into a pcDNA3.1 (+) vector at BamHI and EcoRI to construct a CMV promoter-started tricistronic IL-15/IL-15Rα/GFP composite protein coexpression vector (IL-15/IL-15 Rα/GFP composite protein cDNA sequence is shown as SEQ ID NO. 5) by using pcDNA3.1 (+) as a vector, IL-15cDNA (GenBank: NM-001254747.4), IL-15Rα cDNA (GenBank: NM-001271497.1) as an expression target gene, green Fluorescent Protein (GFP) as a reporter gene, P2A sequence (ATNFSLLKQAGDVEENPGP) of porcine teschovirus and T2A sequence (EGRGSLLTCGDVEENPGP) of Thosea asignavirus) as a cotranslational shearing sequence.

Linearizing pIRG the plasmid with the restriction enzyme BgIII, then2000DNA/pIRG transfected mouse adipocyte 3T3-L1, and the IL-15/IL-15Rα/GFP complex protein stably expressed mouse adipocyte 3T3-L1 ^IRG was obtained by screening in RPMI 1640 (containing 10% fetal bovine serum) containing 600 μg/μ L G418 and detecting by fluorescence microscopy.

Cisplatin (CISPLATIN) is oxidized to a hydroxyl coordination compound (DH-CISPLATIN) in an aqueous H ₂O₂ solution, DH-CISPLATIN and omega-3:7, 10,13,16, 19-docosahexaenoic acid (DHA) are subjected to esterification reaction with the carboxyl groups of DHA under the catalysis of EDC and NHS, and each cisplatin molecule can be connected with two fatty acid chains to form the amphiphilic cisplatin precursor pro-CISPLATIN. The ¹ H element spectrochemical migration profile of cisplatin and amphiphilic cisplatin prodrugs was analyzed by a polynuclear nuclear magnetic resonance spectrometer (Multinuclear NMR spectroscopy) (fig. 9). Three distinct peaks can be seen in the nuclear magnetic resonance hydrogen spectrum of cisplatin alone, respectively the amino peak, the water peak and the solvent DMSO peak of cisplatin. In the synthesized amphiphilic cisplatin prodrug, the peak value of water and DMSO is relatively obvious, the peak value of cisplatin amino is relatively weak, and obvious position change occurs, and the shift from original 3.93ppm to 4.17ppm reflects the interaction between amino in the amphiphilic cisplatin prodrug and the carboxyl portal of DHA.

3T3-L1 ^IRG cells were cultured in DMEM medium (mixed solution containing 10% FBS and 1% penicillin streptomycin), after 2 days of cell growth contact inhibition, the medium was changed to differentiation medium (DMEM/F12 [1:1] +10% FBS+10. Mu.M rosiglitazone+10. Mu.g/mL insulin, 1. Mu.M dexamethasone), after two days of differentiation, to maintenance medium (DMEM/F12 [1:1] +10% fetal bovine serum+1.5. Mu.g/mL insulin+10. Mu.g/mL cisplatin prodrug pro-CISPLATIN (amphipathic cisplatin)), and after two days of maintenance, to complete medium (DMEM/F12 [1:1] +10% FBS), the adipocyte carrier drug pro-Cisplatin adinocytes ^{IL-15/IL-15Rα} was obtained. The oil red O fat staining method verifies that 3T3-F442A preadipocytes have differentiated to maturation (FIG. 10A). Cisplatin was scanned at 10-800 wavelengths by UV-visible absorption spectroscopy, and was found to have a distinct absorption peak at 301nm (FIG. 10B). By using the wavelength, the absorbance of cisplatin with different concentrations is measured to obtain a regression equation of a cisplatin standard curve, the absorbance before and after the culture of a maintenance medium is brought into the regression equation to obtain the loading rate of the amphiphilic cisplatin prodrug of 62.37 percent, and the OD ₃₀₁ value of the supernatant of pro-Cisplatin@adinocytes ^{IL-15/IL-15Rα} after 2 days of culture is measured to obtain the release rate of the amphiphilic cisplatin prodrug of 69.21 percent. Co-culturing cisplatin single drug, DHA single drug and amphiphilic cisplatin prodrug with 1×10 ⁷ B16-F10 melanoma cells respectively, wherein MTT results show that the more obvious the cell viability decline trend is along with the increase of the drug concentration, and the more toxic and better anticancer effect of the amphiphilic cisplatin prodrug than that of cisplatin or DHA alone (figure 10C). It was further demonstrated that DHA has a synergistic effect with cisplatin.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. A fat cell carrier drug, characterized in that it comprises the following steps: transfecting a vector containing an IL-15/IL-15Rα fusion gene into fat cells, screening and obtaining IL-15/IL-15Rα engineered fat cells; using the IL-15/IL-15Rα engineered fat cells to load anti-tumor drugs to obtain fat cell carrier drugs; 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 comprises a pcDNA3.1(+) vector or adenovirus.

3 . The fat cell carrier drug according to claim 1 , wherein the fat cells comprise mouse fat cells 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 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.