A genetically engineered RGD peptide exosome drug delivery biological agent and its preparation and application

Abstract

The present invention provides a genetically engineered RGD peptide exosome drug delivery biological preparation and its preparation and application, which belongs to the technical field of anti-tumor drug preparation. The genetically engineered RGD peptide exosome provided by the present invention is to electroporate the RGD peptide and CD63 protein fusion plasmid into HEK293T cells by genetic manipulation, and then obtain the RGD peptide modified exosomes in the supernatant. The genetically engineered RGD peptide exosome drug delivery biological preparation provided by the present invention is to enrich the anticancer drug DOX into exosomes by electroporation technology, and obtain DOX-loaded RGD exosomes after washing. The DOX-loaded RGD exosomes provided by the present invention can target lung cancer cells, enhance the effect of drug delivery, reduce the side effects of drugs, and inhibit the growth of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drug preparation, and more specifically, the present invention relates to a genetically engineered RGD peptide exosome drug delivery biological preparation and its preparation and application. Background Art

[0002] Integrins are highly expressed on the surface of tumor cells, and they bind to extracellular matrix proteins to mediate tumor invasion and metastasis. Integrin αvβ3 is expressed at a low level on normal cells, normal vascular epithelial cells, and endothelial cells, but is highly expressed in a variety of tumor cells and neovascular endothelial cells. Therefore, integrin αvβ3 is an important target for the treatment of a variety of malignant tumors. The RGD tripeptide (Arg-Gly-Asp, arginine-glycine-aspartic acid peptide) sequence can specifically bind to integrin αvβ3, which can be used for tumor treatment itself, and can also be used as a tumor-targeted carrier for precise drug delivery.

[0003] In recent years, studies have found that exosomes with a lipid bilayer structure play a significant role in intercellular signal transduction and tumor suppression. Exosomes are an important paracrine form outside of cell-soluble cytokines. Their main characteristics include: (1) a diameter of 30-150nm; (2) having the cytoplasm and membrane components of parent cells; (3) a density of 1.13-1.19g / mL; (4) containing specific proteins of their source cells and exosome-related proteins such as CD9, CD81, Alix, etc. Exosome components mainly include: proteins, nucleotides (mRNA, miRNA and mtDNA, etc.), lipids and metabolites, etc.

[0004] Exosomes are gradually being regarded by researchers as a superior drug delivery carrier due to their many unique advantages. Compared with traditional liposome-based nanomaterials, their advantages include: (1) As a natural carrier produced by endogenous cells, they have better biocompatibility, stability and lower cytotoxicity. (2) They can not only efficiently escape macrophage phagocytosis, but also freely penetrate the blood-brain barrier. (3) The surface of the lipid bilayer can be endowed with disease-targeting molecules or therapeutic agents, and the interior can be loaded with functional molecules to avoid degradation. In recent years, engineered exosomes can promote the loading of tumor therapeutic molecules such as nucleic acids, proteins, nanoparticles, etc. into the interior or surface of exosomes, thereby achieving tumor-specific targeting and effective tissue penetration and precise drug delivery. With the increase in basic research on exosomes and the continuous development of modern biotechnology, engineered exosomes will have broader application prospects in future tumor treatment and regenerative medicine. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] The targeting of exosomes as drug carriers in tumor treatment still faces challenges. Although exosomes have a certain degree of natural targeting, relying solely on the natural targeting of exosomes cannot accurately deliver drugs to specific sites. In view of the shortcomings of the prior art, the present invention proposes and prepares a genetically engineered RGD peptide exosome for the first time, and uses it for chemotherapy drug delivery to achieve targeted treatment of tumor cells.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In one aspect, the present invention provides an RGD-CD63 fusion protein, which comprises a CD63 protein and a plurality of repeated RGD peptide sequences intermittently inserted in the extracellular macroloop region of the CD63 protein, wherein the coding region of the extracellular macroloop region is bp 309-609 of CD63. The fusion protein also comprises a fluorescent protein connected to CD63.

[0010] In one aspect, the present invention provides a fusion plasmid comprising a nucleotide sequence encoding the above-mentioned RGD-CD63 fusion protein.

[0011] In one aspect, the present invention provides a genetically engineered RGD peptide exosome, the surface of which is enriched with the above-mentioned RGD-CD63 fusion protein.

[0012] In one aspect, the present invention provides a method for preparing the above-mentioned genetically engineered RGD peptide exosomes, the preparation method comprising: electroporating a fusion plasmid containing a nucleotide sequence encoding the above-mentioned RGD-CD63 fusion protein into HEK293T cells, and then obtaining the RGD peptide-modified exosomes in the supernatant.

[0013] In one aspect, the present invention provides the use of the genetically engineered RGD peptide exosomes in the preparation of drug delivery biological preparations.

[0014] In one aspect, the present invention provides a genetically engineered RGD exosome drug delivery biological preparation, wherein the biological preparation is the genetically engineered RGD peptide exosomes loaded with drugs for preventing or treating tumors. Optionally, the drugs for preventing or treating tumors include chemotherapy drugs such as DOX, paclitaxel, and camptothecin.

[0015] In one aspect, the present invention provides the use of the above-mentioned genetically engineered RGD exosome drug delivery biological agent in the preparation of a drug for preventing or treating tumors.

[0016] In one embodiment, the amino acid sequence of CD63 is SEQ ID NO: 1, the encoding nucleotide sequence of CD63 is SEQ ID NO: 2, the amino acid of the RGD peptide is Arg-Gly-Asp (RGD), and the number of the RGD peptide sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9 or more. The fusion protein comprises the amino acid sequences shown in SEQ ID NO: 4 and 5.

[0017] In one embodiment, the fluorescent protein is selected from GFP, YFP, BFP, RFP or mCherry. The amino acid sequence of mCherry is SEQ ID NO:3.

[0018] In one embodiment, the steps of obtaining the genetically engineered RGD peptide exosomes for delivering drug biologics according to the present invention include:

[0019] (1) Multiple repeated RGD peptide sequences were intermittently inserted into the extracellular macroloop region of a highly enriched CD63 protein on the surface of the exosome membrane; the red fluorescent protein mCherry sequence was connected to the C-terminus of the CD63 protein. Subsequently, the artificially synthesized fusion protein expression sequence was cloned into a plasmid vector;

[0020] (2) HEK293T cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, and 2 mg of positive control CD63-mCherry and target RGD-CD63-mCherry plasmids were added to 1×10 6 HEK293T cells were mixed and added to the electrode cup for electroporation. Puromycin was added for screening to obtain CD63-mCherry and RGD-CD63-mCherry positive cells expressing red fluorescence. Positive expression cells were inoculated into high-glucose medium containing fetal bovine serum for the first amplification culture. When the cells were fused to 50%-60%, 10% fetal bovine high-glucose medium without serum exosomes was added. The second amplification culture was carried out using serum-free exosome medium. After 48 hours, the cell culture supernatant was collected, and the floating live cells were removed by centrifugation to obtain the cell supernatant of RGD peptide overexpressing exosomes.

[0021] (3) The supernatant of the RGD peptide-overexpressing exosomes obtained in step (2) was separated and purified using a kit method to obtain CD63-mCherry and RGD-CD63-mCherry-overexpressing HEK293T cell-derived exosomes.

[0022] (4) The exosomes of HEK293T cells overexpressing CD63-mCherry and RGD-CD63-mCherry and the blank control HEK293T cells were mixed with the chemotherapy drug doxorubicin (DOX) and then electroporated. 5 μL was taken each time and added to the electrode cup for electroporation.

[0023] The DOX-loaded exosomes obtained in step (4) were placed at 37°C for 1 hour to restore the integrity of the exosome membrane. Finally, DOX-loaded RGD-overexpressing exosomes were obtained; the three types of DOX-loaded exosomes obtained were added to an ultrafiltration tube with a molecular weight cutoff of 100 kDa and washed three times to remove excess unloaded drugs. After filtration and sterilization, pure DOX@RGD HEK293T cell exosomes were obtained.

[0024] In one embodiment, the genetically engineered RGD peptide exosome drug delivery biological preparations of the present invention can be used for tumor targeted therapy applications, wherein the tumors include various tumor diseases with high expression of αvβ3, such as lung cancer.

[0025] In one embodiment, the genetically engineered RGD peptide exosome drug delivery biological agent involved in the present invention can target and bind to integrin αvβ3 on the surface of lung cancer cells, efficiently deliver the chemotherapy drug DOX, and then target and inhibit the proliferation of lung cancer cells.

[0026] (III) Beneficial effects

[0027] The present invention proposes and prepares a genetically engineered RGD peptide exosome drug delivery biological preparation, and uses it for chemotherapy drug delivery to achieve targeted treatment of tumor cells. Compared with the prior art, it has the following beneficial effects:

[0028] The exosomes involved in the present invention are secreted by genetically engineered human embryonic kidney 293T cells (HEK293T) and extracted through certain steps. They can be stored in a refrigerator for a long time and remain active (more than 1 year). They can be used as an effective alternative to cell therapy and have excellent clinical application value. At the same time, the genetically engineered RGD peptide exosome drug delivery biological preparations are independent of HEK293T cells after separation and extraction, and do not contain any cellular components, which is convenient for transportation and storage. Secondly, RGD peptide exosomes target tumor cells by binding to integrin αvβ3 on the surface of tumor cells; a single CD63 protein can achieve effective expression of 4 RGD peptides (1:4), thereby significantly improving the targeted adhesion of exosomes to tumor cells. RGD peptide exosomes can efficiently deliver the chemotherapy drug doxorubicin (DOX); DOX@RGD peptide exosomes can efficiently inhibit tumor cell proliferation. In addition, mCherry protein can show excellent imaging efficiency in full imaging of living cells and animals, which further enables CD63-RGD engineered exosomes to have imaging characteristics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 is the expression of αvβ3 integrin in normal and tumor cells;

[0031] Figure 2 Construction and verification of RGD-CD63-mCherry fusion plasmid;

[0032] Figure 3 Inverted fluorescence microscopy was used to detect the fluorescence expression efficiency of HEK293T cells after electroporation of two fusion plasmids for 72 hours;

[0033] Figure 4 Western blot was used to detect the expression of CD63 fusion protein in HEK293T cells after electroporation of two fusion plasmids for 72 hours;

[0034] Figure 5 NTA was used to detect the particle size and concentration of three exosomes derived from HEK293T cells;

[0035] Figure 6 AFM was used to detect the morphology and height of three exosomes derived from HEK293T cells;

[0036] Figure 7 Western blot was used to detect the expression of CD63 protein in three exosomes derived from HEK293T cells;

[0037] Figure 8 A multifunctional microplate reader was used to detect the targeting binding ability of RGD-engineered exosomes in vitro;

[0038] Fig. 9 The RGD-engineered exosomes can better target human lung cancer cells A549;

[0039] Fig.10 Flow chart for the preparation of RGD-engineered exosomes loaded with DOX;

[0040] Fig.11 UV spectrophotometer was used to detect the spectrum of RGD-engineered exosome loaded with DOX;

[0041] Fig.12 The particle size and concentration of DOX loaded in RGD-engineered exosomes were tested for NTA;

[0042] Fig.13 The changes of Zeta potential of three exosomes before and after loading with DOX were detected by NTA;

[0043] Fig.14 Fluorescence microscopy was used to detect the uptake of DOX-loaded exosomes after incubation with human lung cancer cells A549 for 12 h;

[0044] Fig.15 The effect of DOX-loaded exosomes on the proliferation of A549 cells was detected by CCK8. DETAILED DESCRIPTION

[0045] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The present application embodiment provides a genetically engineered RGD peptide exosome for chemotherapy drug delivery to achieve targeted therapy of tumor cells, solving the targeting problem of exosome as a drug carrier in tumor treatment.

[0047] The technical solution in the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows: RGD (Arg-Gly-Asp, arginine-glycine-aspartic acid peptide) modified exosomes are obtained by electroporating the RGD peptide and CD63 protein fusion plasmid into HEK293T cells by genetic manipulation, and then obtaining the RGD peptide modified exosomes in the supernatant. Genetically engineered RGD peptide exosome drug delivery biological preparations use electroporation technology to enrich the anticancer drug DOX into exosomes, and obtain DOX-loaded RGD exosomes after washing. DOX-loaded RGD exosomes can target lung cancer cells, enhance the effect of drug delivery, reduce the side effects of drugs, and inhibit the growth of tumor cells.

[0048] The amino acid sequence of CD63 is shown in SEQ ID NO.1:

[0049] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFSLIMLVEVAAAIAGYVFRDKVMSEFNNNF RQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCINVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM;

[0050] The coding nucleotide sequence of CD63 is shown in SEQ ID NO.2:

[0051] ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTGCTGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCTTGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTGGTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGGGGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTATCATGTTGGTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGTGATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAACAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGGGCTGCTAACTACACAGATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTCCCCGACTCCTGCTGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGAAGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAAAATGTGCTGGTGGTAGCTGCAGCAGCCCTTGGAATTGCTTTTGTCGAGGTTTTGGGAATTGTCTTTGCCTGCTGCCTCGTGAAGAGTATCAGAAGTGGCTACGAGGTGATGTAG;

[0052] The amino acid sequence of mCherry is shown in SEQ ID NO. 3:

[0053] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK;

[0054] The amino acid sequence of RGD-CD63 is shown in SEQ ID NO.4:

[0055] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQRGDMENYPKNNHTASILDRMQRGDADFKCCGAANYTDWEKIPSMSRGDKNRVPDSCCINVTVGCGINFNRGDEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVM;

[0056] The sequence of the amino acid sequence of RGD-CD63-mCherry is shown in SEQ ID NO.5:

[0057] MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQ QRGDMENYPKNNHTASILDRMQRGDADFKCCGAANYTDWEKIPSMSRGDKNRVPDSCCINVTVGCGINFNRGDEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSI RSGYEVMMVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQD SSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK.

[0058] In order to better understand the above technical solution, the above technical solution will be described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0059] Example 1: Detection of integrin αVβ3 protein on the surface of tumor cells

[0060] The main materials and sources used in Example 1 are as follows:

[0061] Human keratinocytes (HaCaT), human embryonic kidney 293T cells (HEK293T), human umbilical vein endothelial cells (HUVEC), human non-small cell lung cancer cells (A549), human breast cancer cell lines (MCF-7 and MDA-231 cells), human gastric cancer cell lines (NGI-N87, SGC7901, HGC-27 and MGC803 cells) culture medium: high-glucose DMEM medium, 1640 medium, fetal bovine serum (Gibco), trypsin (Sigma), carbon dioxide incubator (Forma);

[0062] Biological microscope, ultra-sensitive chemiluminescence imager (GE, USA), clean bench, desktop centrifuge;

[0063] BCA protein quantification kit, horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG secondary antibody (Beijing Kangwei Century Company), premixed HRP chemiluminescent substrate, 100-kDa MWCO ultrafiltration centrifuge tube, 0.22 μm sterile filter membrane (Millipore Company, USA); atomic force microscope (FEI Tecnai 12, Philips Company);

[0064] rabbit anti-human integrin αV antibody, rabbit anti-human integrin β3 antibody, rabbit anti-human CD63 antibody, and rabbit anti-human β-actin antibody (CST);

[0065] The specific implementation steps are as follows:

[0066] (1) Culture of normal human cells and various tumor cells: HaCaT, HEK293T and HUVEC cells were cultured in 10% high glucose medium and maintained in an incubator at 37°C and 5% CO2 saturated humidity; A549, MCF-7, MDA-231, NGI-N87, SGC7901, HGC-27 and MGC803 cells were cultured in 10% high glucose medium and 10% 1640 medium, respectively, and maintained in an incubator at 37°C and 5% CO2 saturated humidity;

[0067] (2)① Add an appropriate proportion of cell lysis buffer containing protease inhibitors to samples such as cells and exosomes, and vortex to mix and lyse thoroughly. Vortex for 1 minute, place on ice for 5 minutes, and repeat 6 times. ② Centrifuge the lysed sample at 4℃ and 12000g for 15 minutes, carefully discard the cell pellet, and transfer the supernatant to a new 1.5mL EP tube. ③ Take 1.5μL and use the machine to detect the absorbance value of the sample protein at 280nm, and then further detect the protein concentration using the BCA kit method. ④ Add 1 / 3 volume of 4× protein loading buffer to the total protein extract, mix well, boil in a boiling water bath for 10 minutes, and then cool on ice. The prepared protein sample can be directly subjected to electrophoresis or frozen in a -20℃ refrigerator.

[0068] (3) Western blot detection of integrin αVβ3 protein expression in normal human cells and various tumor cells: 12% SDS-PAGE electrophoresis gel was prepared, and the extracted cells were fully lysed and added with 1 / 4 volume of 5×SDS loading buffer, boiled for 5 min, and 200 μg of protein was loaded. The protein was transferred to PVDF membrane by electrotransfer (350 mA, 120 min), blocked with TBS / T containing 50 g / L skim milk at room temperature for 1 h, and reacted with rabbit anti-human integrin αV, integrin β3 antibodies and β-actin antibodies (1:500) at 4°C overnight. The next day, the membrane was washed three times with TBS / 0.5% Tween 20, and incubated with HRP-labeled goat anti-rabbit IgG secondary antibody at 37°C for 1 h. After washing the membrane three times with TBS / 0.5% Tween 20, the premixed HRP chemiluminescent substrate was added, and the membrane was detected by chemiluminescent gel imaging system, as shown in the attached figure. Figure 1 As shown, αVβ3 protein was strongly positively expressed on the surface of HUVEC and various tumor cells.

[0069] Example 2: Preparation of genetically engineered RGD peptide exosomes for delivery of drug biologics

[0070] The steps for obtaining genetically engineered RGD peptide exosomes to deliver drug biologics include:

[0071] (1) Multiple repeated RGD peptide sequences were intermittently inserted into the extracellular macroloop region (bp 309-609 of SEQ ID NO:2) of a highly enriched CD63 protein (SEQ ID NO:1) on the surface of the exosome membrane (to obtain RGD-CD63 SEQ ID NO:4); the red fluorescent protein mCherry (SEQ ID NO:3) sequence was connected to the C-terminus of the CD63 protein (to obtain RGD-CD63-mCherry SEQ ID NO:5). Subsequently, the artificially synthesized fusion protein expression sequence was cloned into a plasmid vector;

[0072] (2) HEK293T cells were cultured in high-glucose DMEM medium containing 10% fetal bovine serum, and 2 mg of positive control CD63-mCherry and target RGD-CD63-mCherry (SEQ ID NO: 5) plasmids were added to 1×10 6HEK293T cells were mixed and added to the electrode cup for electroporation. The parameters for electroporation conditions were as follows: the decay mode was selected, and the perforation voltage (PpV) was adjusted to 135V, the perforation voltage duration (Pp on) was 3ms, and the rest time after perforation (Pp off) was 10ms; the driving voltage (Pd V) was 25V, the driving voltage duration (Pd on) was 50ms, and the rest time after driving (Pd off) was 50ms; the number of driving cycles (Pd Cycle N) was 10, and the capacitance was 940μF. Subsequently, after the detection resistance was in the appropriate range, electroporation was started; puromycin was added for screening to obtain CD63-mCherry and RGD-CD63-mCherry positive cells expressing red fluorescence. The positively expressing cells were inoculated into a high-glucose medium containing fetal bovine serum for the first amplification culture. When the cells were fused to 50%-60%, 10% fetal bovine high-glucose medium without serum exosomes was added. The second amplification culture was performed using serum-free exosome culture medium. After 48 hours, the cell culture supernatant was collected and centrifuged to remove floating live cells to obtain the cell supernatant of RGD peptide-overexpressing exosomes.

[0073] (3) The supernatant of the RGD peptide-overexpressing exosomes obtained in step (2) was separated and purified using a kit method to obtain CD63-mCherry and RGD-CD63-mCherry-overexpressing HEK293T cell-derived exosomes.

[0074] (4) The exosomes of HEK293T cells overexpressing CD63-mCherry and RGD-CD63-mCherry and the blank control HEK293T cells in step (3) were mixed with the chemotherapeutic drug doxorubicin (DOX) and then electroporated. 5 μL was taken each time and added to the electrode cup for electroporation. The parameters for setting the electroporation conditions were as follows: the decay mode was selected, and the perforation voltage (Pp V) was adjusted to 110 V, the perforation voltage duration (Pp on) was 3 ms, and the rest time after perforation (Pp off) was 10 ms; the driving voltage (Pd V) was 25 V, the driving voltage duration (Pd on) was 50 ms, and the rest time after driving (Pd off) was 50 ms; the number of driving cycles (Pd Cycle N) was 10, and the capacitance was 940 μF. Subsequently, after the detection resistance was in the appropriate range, electroporation was started.

[0075] The DOX-loaded exosomes obtained in step (4) were placed at 37°C for 1 hour to restore the integrity of the exosome membrane. Finally, DOX-loaded RGD-overexpressing exosomes were obtained; the three types of DOX-loaded exosomes obtained were added to an ultrafiltration tube with a molecular weight cutoff of 100 kDa and washed three times to remove excess unloaded drugs. After filtration and sterilization, pure DOX@RGD HEK293T cell exosomes were obtained.

[0076] Construction and verification of CD63-mCherry and RGD-CD63-mCherry (SEQ ID NO: 5) fusion expression plasmids: GenScript Biotech was commissioned to clone the chimeric sequence of RGD and CD63 extracellular macroloop into the plasmid vector and perform gel analysis ( Figure 2 A) and sequencing ( Figure 2 B) Verification, as attached Figure 2 As shown, CD63-mCherry and RGD-CD63-mCherry sequences were successfully cloned into the vector plasmid.

[0077] 2mg of overexpression plasmids CD63-mCherry and RGD-CD63-mCherry and 1x10 6 HEK293T cells were mixed and added to the electrode cup for electroporation. Puromycin was added for screening 72 hours after plasmid transfection, and the expression efficiency of CD63-mCherry and RGD-CD63-mCherry fusion proteins was detected by fluorescence microscopy and western blot, as shown in the attached Figure 3 and 4 As shown, CD63-mCherry and RGD-CD63-mCherry proteins were successfully expressed on the cell membrane surface.

[0078] Example 3: RGD-engineered HEK293T cells secrete exosomes to express CD63

[0079] (1) Isolation and purification of exosomes in the supernatant secreted by genetically engineered HEK293T cells (kit method): The collected HEK293T supernatant was centrifuged at 4°C and 2,000×g for 10 min to remove cell debris; after collecting the supernatant, it was centrifuged at 4°C and 10,000×g for 30 min to remove cell organelles; the supernatant was transferred to a 100-kDa MWCO ultrafiltration centrifuge tube and concentrated by centrifugation at 4°C and 1,000×g for 30 min; the concentrate was slowly transferred to a 5 ml 30% sucrose / heavy water density cushion (ρ=1.210 g / cm3) and incubated at 4°C and 100 000×g centrifugation for 3h; add EXquick-KIT reagent (1:5) and mix well, incubate at 4℃ overnight for more than 12h, centrifuge at 4℃ and 1500×g for 30min, wash with PBS three times; discard the supernatant, resuspend in PBS, and finally filter with a 0.22μm sterile filter membrane for sterilization, store at -70℃ after aliquoting, and perform protein quantitative detection using the BCA protein quantification kit method;

[0080] (2) Detection of exosome particle size by nanoparticle tracking analyzer: Take 10 μL of control exosome (CTR-exosome), CD63-exosome, and RGD-CD63-exosome, dilute them with PBS to a certain multiple, and inject the sample into the injection port with a 5 mL syringe. Figure 5 As shown, the peak values ​​of the three exosome diameters are around 130 nm;

[0081] (3) Observe the morphology and height of exosomes using atomic force microscopy: Pipette 10 μL of CTR-exosomes, CD63-exosomes, and RGD-CD63-exosomes derived from HEK293T cells and dilute them with deionized water in an appropriate ratio. Mix the diluted exosomes thoroughly and drop them on a clean cell slide, and dry them naturally at room temperature overnight. The next day, add deionized water to the center of the slide for 4-5 times to remove the interference of PBS salt crystals. Then, place the slide under AFM for observation and photography, as shown in the attached figure. Figure 6 As shown, all three exosomes present a “disc-shaped” structure;

[0082] (4) Western blot detection of exosome characteristic marker proteins: prepare 12% SDS-PAGE electrophoresis gel, fully lyse the exosomes extracted above, add 1 / 4 volume of 5×SDS loading buffer, boil for 5 min, load 200 μg of protein, transfer the protein to PVDF membrane by electrotransfer (350 mA, 120 min), block with TBS / T containing 50 g / L skim milk at room temperature for 1 h, react with rabbit anti-human CD63 antibody and β-actin antibody (1:500) at 4°C overnight, wash the membrane three times with TBS / 0.5% Tween 20 the next day, incubate with HRP-labeled goat anti-rabbit IgG secondary antibody at 37°C for 1 h, wash the membrane three times with TBS / 0.5% Tween 20, add premixed HRP chemiluminescent substrate, and detect with chemiluminescent gel imaging system, as shown in the attached Figure 7 As shown, the CD63 protein of the three exosomes derived from HEK293T cells was positively expressed.

[0083] Example 4: RGD-engineered HEK293T cells secrete exosomes that efficiently target integrin αVβ3 protein in vitro;

[0084] The main materials and sources used in Example 4 are as follows:

[0085] αVβ3 integrin, casein blocking agent, PKH67 dye (Invitrogen); human non-small cell lung cancer A549 cells (purchased from ATCC);

[0086] Cytation 5 multifunctional microplate reader (Biotek, USA); inverted microscope, biological microscope, clean bench, desktop centrifuge;

[0087] Detection of RGD-CD63-exosome targeting integrin αVβ3: The specific steps of RGD-CD63-exosome binding to αVβ3 integrin in vitro are as follows Figure 8 As shown in A; Figure 8 As shown in B, the detection results of the multifunctional microplate reader showed that compared with the CTR-exosome and CD63-exosome groups, RGD-CD63-exosome could bind to αVβ3 integrin more efficiently in vitro, indicating that RGD-CD63-exosome has a significant ability to target new blood vessels and tumor cells;

[0088] Detection of RGD-CD63-exosome targeting A549 cells: RGD-CD63-exosomes with the same number of particles labeled with PKH67 (green fluorescence) were co-incubated with human non-small cell lung cancer A549 cells for 12 hours, and the uptake efficiency of A549 cells for exosomes in different treatment groups was detected by Cytation 5 multifunctional microplate reader; as shown in the attached Fig. 9 As shown, all three exosomes can be taken up by A549 cells. The RGD-CD63-exosome incubation group has more significant tumor cell targeting because it can efficiently bind to the αVβ3 protein on the surface of A549 cells.

[0089] Example 5: Preparation and characterization of RGD-engineered HEK293T cell-secreted exosomes efficiently loaded with DOX biological agents;

[0090] The main materials and sources used in Example 5 are as follows:

[0091] Doxorubicin HCl (DOX); BEX new CFB16-HB full-function cell fusion instrument (Jiangsu University School of Medicine), UV spectrophotometer (Jiangsu University School of Materials Science and Engineering), Zetaview nanoparticle tracking analyzer (Germany, Particle Metrix, 220-Twin), clean bench, desktop centrifuge;

[0092] UV spectrophotometer was used to detect the spectral expression of DOX and RGD-CD63-exosome loaded with DOX: The specific steps of RGD-CD63-exosome loaded with DOX are shown in the attached Fig.10 As shown in the figure, the DOX group, CTR-exosome (unmodified and not loaded with DOX) and RGD-CD63-exosome@DOX group were added to the cuvette, and the emission spectrum intensity of DOX at 200nm to 1200nm was detected by UV spectrophotometer. Fig.11 As shown in Figure 2, the UV spectrophotometer detected strong spectral signals of DOX drug at 250nm and 480nm. Fig.11 dotted line in the middle), RGD-CD63-exosome@DOX (attached Fig.11 The two-point dashed line graph in the middle) and the DOX group (attached Fig.11 In the middle solid line graph), there are two obvious emission spectrum peaks at around 250 nm and 480 nm, which indicates that DOX is successfully loaded into RGD-CD63-exosome;

[0093] NTA detection of particle size and Zeta potential of RGD-CD63-exosome@DOX: Take 10 μL of control exosomes (CTR-exosome@DOX), CD63-exosome@DOX, and RGD-CD63-exosome@DOX, dilute them with PBS, and inject the samples into the injection port with a 5 mL syringe, as shown in the attached Fig.12 As shown in Figure 2, the peak values ​​of the diameters of the three exosomes are around 150 nm. NTA detection found that there was no significant difference in the particle size of the RGD-CD63-exosome@DOX group after drug loading. Both DOX and RGD peptides carry positive charges, such as Fig.13 As shown, NTA detection found that after loading the drug, the Zeta potential of the RGD-CD63-exosome@DOX group was reduced compared with the CTR-exosome group, indicating that the negative charge on the surface of the vesicles in the RGD-CD63-exosome@DOX group was neutralized after loading the drug, thus manifesting as a decrease in the negative value of the membrane potential, which further demonstrated that DOX was successfully loaded into the RGD-CD63-exosome.

[0094] Example 6: RGD-engineered HEK293T cells secrete exosomes that can deliver DOX to human lung cancer cells A549;

[0095] The uptake of RGD-CD63-exosome@DOX by A549 cells was detected by a multifunctional microplate reader: CD63-exosome@DOX and RGD-CD63-exosome@DOX with the same number of particles were co-incubated with A549 cells for 12 hours, and the distribution of DOX (DOX spontaneous red fluorescence) in the cell nucleus and cytoplasm was detected by a multifunctional microplate reader. Fig.14 As shown, after RGD-CD63-exosome@DOX was co-incubated with A549 cells for 12 hours, a large amount of DOX drug was enriched in the cell nucleus and cytoplasm, which further demonstrated that RGD-CD63-exosome can efficiently load DOX and deliver it to the cell membrane and cytoplasm of A549 cells.

[0096] Example 7: RGD-engineered HEK293T cells secrete exosomes loaded with DOX biological agents, which significantly inhibit the proliferation of A549 cells;

[0097] CCK8 was used to detect the inhibitory effect of RGD-CD63-exosome@DOX on the proliferation of A549 cells: A549 cells with the same number of cells were seeded into a 96-well plate. After the cells adhered to the plate for 12 hours, CD63-exosome@DOX and RGD-CD63-exosome@DOX with the same number of particles were added for 48 hours. PBS treatment was used as a control. Fig.15 As shown, both CD63-exosome@DOX and RGD-CD63-exosome@DOX intervention groups significantly inhibited the proliferation of A549 cells. Since RGD-CD63-exosome@DOX has better targeting, it exhibits a more significant proliferation inhibition effect.

[0098] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0099] 1. The surface of exosomes is enriched with a variety of characteristic protein molecules, among which the most enriched proteins are the tetraspanin family, including CD9, CD63 and CD81. However, studies have shown that CD63 is the most specific marker protein for exosomes, while CD9 and CD81 are not specific. Therefore, engineered exosomes constructed based on CD63 protein modification can achieve effective loading of more target genes;

[0100] 2. CD63 is a four-transmembrane protein. The second largest loop in the cell membrane extracellular region has a long sequence (309-609bp) and can therefore be used for specific modification. In the present invention, four repeated RGD peptide sequences are intermittently inserted into the second largest loop region of the cell membrane extracellular region in view of this characteristic of CD63 on the surface of the exosome membrane. After genetic modification, overexpression of a single CD63 protein can achieve the effective expression of four RGD peptides (1:4). Therefore, the highly enriched CD63 protein on the surface of exosomes significantly improves the efficiency of RGD peptide overexpression, thereby significantly improving the targeted adhesion of exosomes to tumor cells;

[0101] 3. In addition, the present invention also connects the red fluorescent protein mCherry sequence to the C-terminus of the CD63 protein cell membrane. The mCherry protein is a visual tracking protein with lower background and more stability, which can show excellent imaging efficiency in the whole imaging of living cells and animals. This further enables the CD63-RGD engineered exosomes to have imaging characteristics, so that the in vivo visualization distribution and dynamic detection of exosomes and the information of tumor sites can be actually detected.

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An RGD-CD63 fusion protein, characterized in that: The fusion protein is CD63 protein and multiple repeated RGD peptide sequences intermittently inserted in the extracellular macroloop region of the CD63 protein, the coding region of the extracellular macroloop region is 309-609bp of CD63, the coding nucleotide sequence of CD63 is SEQ ID NO: 2, the amino acid sequence of the RGD peptide is Arg-Gly-Asp, the number of the RGD peptide sequences is 4, and the fusion protein is the amino acid sequence shown in SEQ ID NO:

4.

2. An RGD-CD63 fusion protein, characterized in that: The fusion protein is composed of the fusion protein according to claim 1 and a fluorescent protein connected to CD63, and the fluorescent protein is selected from GFP, YFP, BFP, RFP or mCherry.

3. The fusion protein according to claim 2, characterized in that The fusion protein is the amino acid sequence shown in SEQ ID NO:

5.

4. A fusion plasmid, characterized in that: The plasmid comprises a nucleotide sequence encoding the RGD-CD63 fusion protein according to any one of claims 1 to 3.

5. A genetically engineered RGD peptide exosome, characterized in that: The surface of the exosomes is enriched with the RGD-CD63 fusion protein according to any one of claims 1 to 3.

6. The method for preparing genetically engineered RGD peptide exosomes according to claim 5, characterized in that: The preparation method comprises: electroporating the fusion plasmid according to claim 4 into HEK293T cells, and then obtaining the RGD peptide-modified exosomes in the supernatant.

7. Use of the genetically engineered RGD peptide exosomes as claimed in claim 5 or the genetically engineered RGD peptide exosomes prepared by the preparation method as claimed in claim 6 in the preparation of drug delivery biological preparations.

8. A genetically engineered RGD exosome drug delivery biological agent, characterized in that: The biological preparation is the genetically engineered RGD peptide exosomes as described in claim 5 or the genetically engineered RGD peptide exosomes prepared by the preparation method as described in claim 6 loaded with drugs for preventing or treating tumors.

9. The use of the genetically engineered RGD exosome drug delivery biological agent as claimed in claim 8 in the preparation of drugs for preventing or treating tumors, characterized in that: The tumor is non-small cell lung cancer expressing integrin αvβ3 or breast cancer expressing integrin αvβ3.

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