CN111514311B - Target exosome loaded with adriamycin and si-PVT1 together, preparation method thereof and anti-osteosarcoma application thereof - Google Patents

Abstract

The invention provides a targeting exosome loaded with adriamycin and si-PVT1 together, a preparation method thereof and an anti-osteosarcoma application thereof, belonging to the technical field of biological medicines. The targeted exosome comprises exosome particles with targeting effect in gene editing, a small molecule drug adriamycin and a gene drug si-PVT1. The invention carries out artificial modification on the exosome by a gene editing method, so that the exosome has a targeting effect on osteosarcoma cells, other normal cells cannot be damaged, and the safety to a human body is enhanced; then the small molecule drug adriamycin and the gene drug si-PVT1 are loaded on the medicine, and the two drugs are simultaneously delivered to osteosarcoma cells to play the role of killing the cells. Compared with the traditional administration mode, the targeted exosome particle provided by the invention has high targeting property on osteosarcoma cells, and reduces the toxic and side effects of the drug; simultaneously, the adriamycin and the si-PVT1 can play a synergistic role, the capability of killing osteosarcoma cells is improved, and the anti-osteosarcoma effect is enhanced.

Description

Targeted exosomes co-loaded with doxorubicin and si-PVT1 and its preparation and anti-osteosarcoma application

field of invention

The invention belongs to the technical field of biomedicine, and in particular relates to targeted exosomes co-loaded with doxorubicin and si-PVT1 and its preparation and anti-osteosarcoma application.

Background of the invention

Osteosarcoma is a highly malignant bone tumor that mostly occurs in the metaphysis of long bones and is most common in children and adolescents. It is characterized by rapid progression, easy recurrence and metastasis. At present, the main clinical treatment for osteosarcoma is "preoperative neoadjuvant chemotherapy + surgical resection + postoperative adjuvant chemotherapy", but this treatment method has problems such as difficult treatment, unsatisfactory prognosis, and low survival rate of patients. Osteosarcoma is very prone to lung metastases, and patients with lung metastases at the early stage of treatment have a worse prognosis, with a 5-year survival rate of less than 30%. Although increasing the dose of chemotherapy drugs, changing the time of drug use and using multiple drugs in combination with chemotherapy in clinical practice, the survival rate of osteosarcoma patients has not been substantially improved. In addition, the traditional first-line chemotherapeutic drug doxorubicin is effective in killing osteosarcoma cells, but there are problems of poor selectivity and strong side effects in conventional drug administration methods, which limit its clinical use.

Exosomes are membranous vesicles with a diameter of about 10-100 nm released outside the cell after the fusion of the intracellular multivesicular body and the cell membrane, which contain various intracellular substances such as RNA, protein, and DNA fragments. Exosomes can shuttle between cells, facilitate the exchange of substances and information between cells, and can be loaded with chemotherapy drugs and siRNA for targeted therapy. As a drug carrier, exosomes have natural advantages: (1) have nano-scale size, and can easily escape the capture of the reticuloendothelial system in vivo; (2) have a phospholipid bilayer structure, which is similar to the composition of the cell membrane, and has a strong impact on the cell membrane. Strong affinity, easy to enter cells; (3) Belonging to endogenous vesicles, entering the body as a carrier will not cause immune reactions, and will not be identified as "non-self substances" by the immune system in the body and swallowed; (4) Others It is derived from cells and contains many transmembrane proteins on the surface. Through genetic modification, a ligand short peptide that can target target cells can be added to the appropriate protein end to achieve targeted therapy in vivo.

Many reports and studies can show that exosomes are an ideal drug carrier, which can solve the common immunogenicity shortcomings of exogenous carriers. However, for the specific application of exosomes in the treatment of osteosarcoma, it needs to be further modified, and a new type of exosome drug particle should be redesigned so that it has the ability to target osteosarcoma cells and has the ability to kill bone and flesh cells. function of tumor cells. Based on the above research background, the present invention provides a targeted exosome particle loaded with the small molecule drug doxorubicin and the gene drug si-PVT1 and its application in the preparation of drugs for the treatment of osteosarcoma.

Contents of the invention

In order to solve the deficiencies of the prior art, the object of the present invention is to provide a novel targeting exosome co-carrying doxorubicin and si-PVT1 gene and its preparation and application in the treatment of osteosarcoma.

For realizing the object of the present invention, technical scheme of the present invention is as follows:

Firstly, the present invention provides exosomes co-loaded with doxorubicin and si-PVT1 targeting exosomes, which are composed of targeting exosome particles, small molecule drug doxorubicin and gene drug si-PVT1.

Further, the targeting exosome particle is a fusion protein formed by inserting a short peptide with the function of targeting osteosarcoma cells into the N-terminus of lysosome-associated membrane protein 2 (LAMP2) on the surface of exosomes. The peptide insertion is specifically located between the signal peptide and the mature sequence of LAMP2; the amino acid sequence of the short peptide with targeting function in the exosome particle is shown in SEQ ID NO: 1; the targeting function in the exosome particle The nucleic acid sequence of the short peptide is shown in SEQ ID NO:2. In one aspect, the present invention provides a method for preparing targeted exosomes co-loaded with doxorubicin and si-PVT1, which is prepared according to the following steps:

(1) Insert a short peptide with the amino acid sequence of SEQ ID NO: 1 into the N-terminus of lysosome-associated membrane protein 2 (LAMP2) on the surface of exosomes by gene editing, specifically at the signal peptide and mature Between the sequences, a fusion protein is formed; construct the pcDNA3.1(+)-hLAMP2b-Cys-CRGDKGPDC plasmid, mix the plasmid and liposomes within 5-8 minutes, place at room temperature for 20-25 minutes, and then add to 293T In the cells, the culture medium containing 10% FBS was replaced after 6-8 hours, and the supernatant was collected after 24h-30h.

(2) Centrifuge the supernatant in step (1) three times. The three centrifugation parameters are as follows: ①4°C, 300×g for 10 minutes; ②4°C, 20,000×g for 10 minutes; ③4°C, 10,000 ×g centrifugation for 30 min; then the supernatant was collected for ultracentrifugation, the ultracentrifugation parameters were 4°C, 100,000×g ultracentrifugation for 70 min, then the supernatant was discarded, and the precipitated pellet was collected to obtain osteosarcoma cells. Exosome particles. After resuspending the exosome particles in PBS, store them at -80~-70°C.

(3) Mix 0.2 μL of exosome particles targeting osteosarcoma cells obtained in step (2), 1200 ng of gene drug si-PVT, and 50 μg of doxorubicin with a buffer (200 μL system), select a voltage of 500 V at room temperature, and continue After 15 seconds, electroporation was performed to obtain a mixture.

(4) Remove free doxorubicin and si-PVT1 from the mixture obtained in step (3) by ultracentrifugation to obtain the targeted exosome drug co-loaded with doxorubicin and si-PVT1 according to the present invention .

Further, in step (3), the sequence of the si-PVT1 is 5'-GCU UGG AGG CUG AGG AGU UTT-3'.

In another aspect, the present invention provides targeted exosomes co-loaded with doxorubicin and si-PVT1 for the preparation of drugs for treating osteosarcoma.

Beneficial effect

In the present invention, the exosomes are artificially modified by gene editing, and a short peptide is inserted on the lysosome-associated protein 2 (LAMP2) on the surface of the exosomes, so that the exosomes have a targeting effect on osteosarcoma cells, and at the same time It will not damage other normal cells and enhance the safety of the human body; then, the gene-edited exosome particles are loaded with small molecule drug doxorubicin and gene drug si-PVT1, and these two drugs are delivered to osteosarcoma cells at the same time Play the role of killing cells. Compared with traditional administration methods, the targeted exosome particles provided by the present invention have a high degree of targeting to osteosarcoma cells, reduce the toxic and side effects of drugs, and improve the safety of drug use; at the same time, it can make doxorubicin The two drugs, si-PVT1, play a synergistic effect, enhance the ability to kill osteosarcoma cells, and enhance the anti-osteosarcoma effect.

Description of drawings

Figure 1 Distribution of iRGD-Exo/si-PVT1/DOX in mice.

Fig. 2 The uptake efficiency of osteosarcoma cells to iRGD-Exo/si-PVT1/DOX detected by flow cytometry.

Figure 3 The distribution of iRGD-Exo/si-PVT1/DOX in osteosarcoma cells.

Figure 4 The effect of iRGD-Exo/si-PVT1/DOX on the proliferation of osteosarcoma cells, where (A) is the effect of different doses of DOX, si-PVT1, iRGD-Exo/si-PVT1/DOX on the proliferation of osteosarcoma cells Effect; (B) Effect of DOX, si-PVT1, Exo/si-PVT1/DOX, iRGD-Exo/DOX, iRGD-Exo/si-PVT1, iRGD-Exo/si-PVT1/DOX on the proliferation of osteosarcoma cells . * p <0.05; ** p <0.01.

Figure 5 The therapeutic effect of iRGD-Exo/si-PVT1/DOX on osteosarcoma xenograft tumor-bearing mice, where (A) is the tumor growth curve (** p <0.01); (B) is the HE staining microscope of tumor tissue sections Observe the picture below.

Detailed ways

Example 1

The novel targeted exosomes co-loaded with doxorubicin and si-PVT1 were prepared according to the following steps:

(1) Insert a short peptide with the amino acid sequence of SEQ ID NO: 1 into the N-terminus of lysosome-associated membrane protein 2 (LAMP2) on the surface of exosomes by gene editing, specifically at the signal peptide and mature Between the sequences, a fusion protein is formed; construct the pcDNA3.1(+)-hLAMP2b-Cys-CRGDKGPDC plasmid, mix the plasmid and the liposome within 5 minutes, and place it at room temperature for 20 minutes before adding it to 293T cells, 6 After 1 hour, the culture medium containing 10% FBS was replaced, and the supernatant was collected after 24 hours.

(2) Centrifuge the supernatant in step (1) three times. The three centrifugation parameters are as follows: ①4°C, 300×g for 10 minutes; ②4°C, 20,000×g for 10 minutes; ③4°C, 10,000 ×g centrifugation for 30 min; then the supernatant was collected for ultracentrifugation, the ultracentrifugation parameters were 4°C, 100,000×g ultracentrifugation for 70 min, then the supernatant was discarded, and the precipitated pellet was collected to obtain osteosarcoma cells. Exosome particles. Exosome particles were resuspended in PBS and stored at -80°C.

(3) Add 0.2 μL of exosome particles targeting osteosarcoma cells obtained in step (2), and 200 ng of si-PVT1 gene drug (si-PVT1 sequence is 5'-GCU UGG AGG CUG AGG AGU UTT-3') , 50 μg doxorubicin was mixed with buffer (200 μL system), at room temperature, a voltage of 500 V was selected for 15 s, and electroporation was performed to obtain a mixture.

(4) The mixture obtained in step (3) was ultracentrifuged to remove free doxorubicin and si-PVT1, so as to obtain the targeted exosomes co-loaded with doxorubicin and si-PVT1 according to the present invention.

In order to further illustrate the role and effect of the co-loaded doxorubicin and si-PVT1 targeting exosomes provided by the present invention in the treatment of osteosarcoma, the present invention provides cell experiments and animal experiments as illustrations.

Drug involved in the test of the present invention and preparation method thereof:

iRGD-Exo/si-PVT1/DOX: targeted exosomes co-loaded with doxorubicin and si-PVT1 provided by the present invention; prepared according to the method in Example 1.

Exo/si-PVT1/DOX: common exosome particle co-carrying doxorubicin and si-PVT1 gene; the exosome particle is loaded with si-PVT1 gene drug and DOX drug, but has no targeting function; the exosome particle The specific preparation method of exocytic granules follows the steps below:

(1) After the 293T cells adhere to the wall, collect the culture supernatant.

(2) Centrifuge the supernatant in step (1) three times. The three centrifugation parameters are as follows: ①4°C, 300×g for 10 minutes; ②4°C, 20,000×g for 10 minutes; ③4°C, 10,000 ×g centrifugation for 30 min; then the supernatant was collected for ultracentrifugation, the ultracentrifugation parameters were 4°C, 100,000×g ultracentrifugation for 70 min, then the supernatant was discarded, and the pellet was collected to obtain exosome particles. Exosome particles were resuspended in PBS and stored at -80°C.

(3) Mix 0.2 μL exosome particles obtained in step (2), 200 ng si-PVT1 gene drug (si-PVT1 sequence is 5'-GCU UGG AGG CUG AGG AGU UTT-3'), 50 μg doxorubicin The buffer was mixed evenly (200 μL system), and a voltage of 500 V was selected at room temperature for 15 s, and electroporation was performed to obtain a mixture.

(4) The mixture obtained in step (3) was ultracentrifuged to remove free doxorubicin and si-PVT1, so as to obtain common exosome particles co-loaded with doxorubicin and si-PVT1.

iRGD-Exo/DOX: It is a targeted exosome particle loaded with DOX alone; the specific preparation method of the exosome particle follows the following steps:

(1) Insert a short peptide with the amino acid sequence of SEQ ID NO: 1 into the N-terminus of lysosome-associated membrane protein 2 (LAMP2) on the surface of exosomes by gene editing, specifically at the signal peptide and mature Between the sequences, a fusion protein is formed; construct the pcDNA3.1(+)-hLAMP2b-Cys-CRGDKGPDC plasmid, mix the plasmid and the liposome within 5 minutes, and place it at room temperature for 20 minutes before adding it to 293T cells, 6 After 1 hour, the culture medium containing 10% FBS was replaced, and the supernatant was collected after 24 hours.

(2) Centrifuge the supernatant in step (1) three times. The three centrifugation parameters are as follows: ①4°C, 300×g for 10 minutes; ②4°C, 20,000×g for 10 minutes; ③4°C, 10,000 ×g centrifugation for 30 min; then the supernatant was collected for ultracentrifugation, the ultracentrifugation parameters were 4°C, 100,000×g ultracentrifugation for 70 min, then the supernatant was discarded, and the precipitated pellet was collected to obtain osteosarcoma cells. Exosome particles. Exosome particles were resuspended in PBS and stored at -80°C.

(3) Mix 0.2 μL exosome particles obtained in step (2) and 50 μg doxorubicin with buffer (200 μL system), select a voltage of 500 V at room temperature for 15 seconds, and perform electroporation to obtain a mixture.

(4) The mixture obtained in step (3) was subjected to ultracentrifugation to remove free doxorubicin to obtain DOX-loaded targeted exosome particles.

iRGD-Exo/si-PVT1: It is a targeted exosome particle loaded with si-PVT1 gene alone; the specific preparation method of the exosome particle follows the following steps:

(1) Insert a short peptide with the amino acid sequence of SEQ ID NO: 1 into the N-terminus of lysosome-associated membrane protein 2 (LAMP2) on the surface of exosomes by gene editing, specifically at the signal peptide and mature Between the sequences, a fusion protein is formed; construct the pcDNA3.1(+)-hLAMP2b-Cys-CRGDKGPDC plasmid, mix the plasmid and the liposome within 5 minutes, and place it at room temperature for 20 minutes before adding it to 293T cells, 6 After 1 hour, the culture medium containing 10% FBS was replaced, and the supernatant was collected after 24 hours.

(2) Centrifuge the supernatant in step (1) three times. The three centrifugation parameters are as follows: ①4°C, 300×g for 10 minutes; ②4°C, 20,000×g for 10 minutes; ③4°C, 10,000 ×g centrifugation for 30 min; then the supernatant was collected for ultracentrifugation, the ultracentrifugation parameters were 4°C, 100,000×g ultracentrifugation for 70 min, then the supernatant was discarded, and the pellet was collected to obtain exosome particles. Exosome particles were resuspended in PBS and stored at -80°C.

(3) Mix 0.2 μL of exosome particles obtained in step (2) and 200 ng of si-PVT1 gene drug (si-PVT1 sequence is 5'-GCU UGG AGG CUG AGG AGU UTT-3') with buffer (200 μL system), select a voltage of 500V at room temperature for 15s, and perform electroporation to obtain a mixture.

(4) The mixture obtained in step (3) was removed by ultracentrifugation to remove free si-PVT1, so as to obtain ordinary exosome particles loaded with si-PVT1 alone.

DOX: Free DOX.

si-PVT1: free si-PVT1.

Establishment of osteosarcoma xenograft tumor-bearing mouse model: BALB/C mice were selected, and 100 μL of human osteosarcoma cell MNNG/HOS suspension (containing about 2.5× ¹⁰⁶ cells) was subcutaneously injected into the left axilla of the mice to prepare subcutaneous Tumor model.

Experiment 1: Distribution of iRGD-Exo/si-PVT1/DOX in vivo

iRGD-Exo/si-PVT1/DOX was stained with the hydrophobic infrared fluorescent dye DIR. The stained iRGD-Exo/si-PVT1/DOX was injected into the osteosarcoma tumor-bearing mice through the tail vein, respectively, with different doses at 10 minutes, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours after injection. The mice were anesthetized with halothane, and the fluorescence distribution in the osteosarcoma tumor-bearing mice was detected using a small animal in vivo imaging system, and the excitation wavelength of DIR was 748nm, and the emission wavelength was 780nm for detection. The result is shown in Figure 1. It can be seen from Figure 1 that after the mice were injected with iRGD-Exo/si-PVT1/DOX for 1 hour, iRGD-Exo/si-PVT1/DOX showed a strong fluorescent signal at the tumor site of the mouse, and the fluorescent signal could Continuous display for 24 hours. The above results can indicate that the iRGD-Exo/si-PVT1/DOX provided by the present invention can be effectively taken up by osteosarcoma cells in large quantities, and can stay in osteosarcoma tissues for a long time.

Experiment 2: Uptake rate of iRGD-Exo/si-PVT1/DOX by osteosarcoma cells

To prepare iRGD-Exo/si-PVT1/DOX, doxorubicin was stained with Nile Red, and si-PVT1 was labeled with green fluorescein FAM. Add stained and labeled iRGD-Exo/si-PVT1/DOX (test group) and Exo/si-PVT1/DOX (control group) to MNNG/HOS osteosarcoma cells under dark operation, incubate for 4 hours, and then Fluorescence in osteosarcoma cells was detected by flow cytometry. The result is shown in Figure 2. It can be seen from Figure 2 that the uptake efficiency of iRGD-Exo/si-PVT1/DOX by MNNG/HOS osteosarcoma cells was significantly higher than that of the control group; higher than the control group. The above results can indicate that osteosarcoma cells can efficiently uptake iRGD-Exo/si-PVT1/DOX provided by the present invention.

Experiment 3: Distribution of iRGD-Exo/si-PVT1/DOX in osteosarcoma cells

To prepare iRGD-Exo/si-PVT1/DOX, doxorubicin was stained with Nile Red, and si-PVT1 was labeled with green fluorescein FAM. Add stained and labeled iRGD-Exo/si-PVT1/DOX (test group) and Exo/si-PVT1/DOX (control group) to MNNG/HOS osteosarcoma cells under dark operation, incubate for 3 hours, and then The uptake of fluorescent markers by osteosarcoma cells and the distribution of intracellular fluorescence were observed by laser confocal microscopy (LSCM). The result is shown in Figure 3. It can be seen from Figure 3 that both the red fluorescence and green fluorescence of the test group were strong, and the yellow fluorescence produced by the superposition of the red fluorescence of Nile Red and the green fluorescence of FAM was observed. The above results can show that the targeted exosome particles provided by the present invention are used as carriers to facilitate the entry of doxorubicin and si-PVT1 genes into osteosarcoma cells.

Experiment 4: Inhibitory effect of iRGD-Exo/si-PVT1/DOX on proliferation of osteosarcoma cells

Prepare iRGD-Exo/si-PVT1/DOX, DOX and si-PVT1 respectively in solutions with different concentration gradients (0.01, 0.1, 1, 2 μg/mL) (use the medium as a solvent to make exosome particle suspension or drug solution), and then the above solution was co-cultured with MNNG/HOS osteosarcoma cells for 24 hours, and the inhibition of cell proliferation was detected by CCK-8. The results are shown in Figure 4A. It can be seen from Figure 4A that the three drugs all have obvious inhibitory effects on osteosarcoma cells, but compared with the two groups of DOX and si-PVT1, the inhibition of osteosarcoma cells after treatment with iRGD-Exo/si-PVT1/DOX The rate was about 86.92%, which was 2 times and 3.5 times of the inhibition rate of DOX and si-PVT1 respectively, with significant difference (P<0.01).

iRGD-Exo/si-PVT1/DOX (targeted exosome particles co-carrying doxorubicin and si-PVT1 gene), Exo/si-PVT1/DOX (common Exosome particles), iRGD-Exo/DOX (targeted exosome particles loaded with DOX alone), iRGD-Exo/si-PVT1 (targeted exosome particles loaded with si-PVT1 gene alone), free DOX, Free si-PVT1 was formulated into 2 μg/mL solutions, and then co-cultured with MNNG/HOS osteosarcoma cells for 48 hours, and the inhibition of cell proliferation was detected by CCK-8. The results are shown in Figure 4B. It can be seen from Figure 4B that the inhibition rate of iRGD-Exo/si-PVT1/DOX on osteosarcoma cell proliferation can be as high as 80.23%, and the inhibition rate of DOX, si-PVT1 and Exo/si-PVT1/DOX on cell proliferation is 12%. —22%, compared with iRGD-Exo/si-PVT1/DOX, the difference was extremely significant (P<0.01); the cell proliferation inhibition rate of iRGD-Exo/DOX was 50.62%, and the cell proliferation The inhibition rate was 31.47%, compared with iRGD-Exo/si-PVT1/DOX, there was a significant difference or an extremely significant difference (P<0.05, P<0.01); the above results can show that the combination of DOX and si-PVT1 The inhibitory effect on cells when the two drugs are used alone is significantly improved; secondly, the present invention performs gene editing on exosomes to obtain exosome particles with targeting effect, and its targeting effect can effectively improve the effect of DOX and si-PVT1 on bone and flesh The inhibitory effect on tumor cell proliferation; most importantly, the drug of the present invention obtained by loading DOX and si-PVT1 with exosome particles with targeting effect as a drug carrier has a more obvious inhibitory effect on the proliferation of osteosarcoma cells. The obtained drug can simultaneously deliver DOX and si-PVT1 to osteosarcoma cells, and the two drugs can have a synergistic effect, thereby enhancing their anti-tumor ability.

Experiment 5: The therapeutic effect of iRGD-Exo/si-PVT1/DOX on osteosarcoma transplanted tumor-bearing mice

Nine osteosarcoma transplanted tumor-bearing mice, weighing 20±1 g, were randomly divided into 3 groups, 3 mice in each group, which were blank control group, DOX group, and iRGD-Exo/si-PVT1/DOX group. Normal saline was administered to the blank control group, 20 mg/kg DOX to the DOX group, and 20 mg/kg iRGD-Exo/si-PVT1/DOX to the iRGD-Exo/si-PVT1/DOX group; administered via tail vein injection every 3 days 1 time, 21 consecutive days.

During the administration process, the longest and shortest diameters of the transplanted tumors were measured every three days to observe their general conditions, calculate the tumor volume, and draw a curve of tumor volume change. The results are shown in Figure 5A. It can be seen from Figure 5A that there was no significant difference in the volume of xenografted tumors in nude mice in each group before the start of administration. There were significant differences in the volumes, and the iRGD-Exo/si-PVT1/DOX group had a significant inhibitory effect on the volume growth of transplanted tumors, and the inhibitory effect was better than that of DOX.

After the administration, the nude mice were sacrificed, the tumor tissues were separated and the tissue sections were stained with HE, and the staining conditions were observed under a microscope. The results are shown in Figure 5B. It can be seen from Fig. 5B that the transplanted tumor tissues of nude mice in the three groups all have the morphological characteristics of tumor cells. The growth of tumor cells in the blank control group was very dense, with relatively large nuclei, high nuclear-cytoplasmic ratio, and obvious nuclear atypia; compared with the control group, tumors of different degrees appeared in the DOX group and the iRGD-Exo/si-PVT1/DOX group The cell density decreased, the tissue structure was scattered, and some cells showed nuclear pyknosis and nuclear lysis. The cells showed nuclear pyknosis, nuclear lysis and other phenomena, indicating that the iRGD-Exo/si-PVT1/DOX of the present invention can well inhibit the growth of osteosarcoma transplanted tumor tissue, and has a good therapeutic effect on the osteosarcoma tumor-bearing mouse model.

The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: it can still be applied to the foregoing embodiments The technical solutions described in the examples are modified, or some or all of the technical features are equivalently replaced; and these modifications or replacements do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

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Claims (3)

1. The target exosome carrying the adriamycin and the si-PVT1 together is characterized in that the exosome consists of target exosome particles, a small molecular drug adriamycin and a gene drug si-PVT 1;

the targeted exosome particle is a fusion protein comprising an N-terminus of a lysosomal associated membrane protein 2 (LAMP 2) inserted into the surface of exosome with a short peptide having a function of targeting osteosarcoma cells; the amino acid sequence of the short peptide with the targeting function in the targeting exosome particle is shown as SEQ ID NO:1 is shown in the specification; the nucleic acid sequence of the short peptide with the targeting function in the exosome particle is shown as SEQ ID NO:2 is shown in the specification;

the amino acid sequence is SEQ ID NO: the short peptide shown in 1 is inserted into the N end of a lysosome associated membrane protein 2 (LAMP 2) on the surface of an exosome, and is specifically positioned between a signal peptide and a mature sequence of the LAMP 2;

the sequence of the gene medicine si-PVT1 is 5'-GCU UGG AGG CUG AGG AGU UTT-3'.

2. The preparation method of targeted exosome loaded with adriamycin and si-PVT1 is characterized by comprising the following steps:

(1) The amino acid sequence is SEQ ID NO:1, the short peptide is inserted into the N end of a lysosome associated membrane protein 2 (LAMP 2) on the surface of an exosome, and is specifically positioned between a signal peptide and a mature sequence of the LAMP2 to form a fusion protein; constructing pcDNA3.1 (+) -hLAMP2b-Cys-CRGDKGPDC plasmid, mixing the plasmid and liposome uniformly within 5-8 minutes, placing at room temperature for 20-25 minutes, adding into 293T cell, replacing culture solution containing 10% FBS after 6-8 hours, and collecting supernatant after 24-30 hours;

(2) And (2) carrying out three times of centrifugation on the supernatant in the step (1), wherein the three times of centrifugation parameters are as follows in sequence: (1) centrifuging at 300 Xg for 10 min at 4 ℃; (2) centrifuging at 20,000 Xg for 10 min at 4 ℃; (3) centrifuging at 10,000 Xg for 30 min at 4 ℃; then collecting supernatant fluid for ultracentrifugation, and ultracentrifuging for 70 min at 100,000 Xg at 4 ℃, discarding the supernatant fluid, and collecting precipitated pellets to obtain exosome particles capable of targeting osteosarcoma cells; after the PBS re-suspended the exosome particles, storing at-80 to-70 ℃;

(3) Uniformly mixing 0.2 mu L of the exosome particles targeting osteosarcoma cells obtained in the step (2), 1 ng of gene drug si-PVT and 50 mu g of adriamycin by using buffer, selecting voltage of 500V at normal temperature for 15s, and performing electroporation to obtain a mixture;

(4) Removing free adriamycin and si-PVT1 from the mixture obtained in the step (3) through ultracentrifugation to obtain the targeted exosome drug co-loaded with the adriamycin and the si-PVT 1;

the sequence of si-PVT1 in the step (3) is 5'-GCU UGG AGG CUG AGG AGU UTT-3'.

3. Use of the targeted exosome co-loaded with doxorubicin and si-PVT1 as defined in claim 1 or the exosome prepared by the preparation method of the targeted exosome co-loaded with doxorubicin and si-PVT1 as defined in claim 2 in the preparation of a medicament for treating osteosarcoma.

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