CN111514311B - Target exosome loaded with adriamycin and si-PVT1 together, preparation method thereof and anti-osteosarcoma application thereof - Google Patents
Target exosome loaded with adriamycin and si-PVT1 together, preparation method thereof and anti-osteosarcoma application thereof Download PDFInfo
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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
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a targeting exosome loaded with doxorubicin and si-PVT1 together, a preparation method thereof and an anti-osteosarcoma application thereof.
Background
Osteosarcoma is a highly malignant bone tumor, which frequently occurs at the metaphysis of long bones, is most common in children and adolescents, and is characterized by rapid progression, easy recurrence and metastasis. At present, the main scheme for clinically treating osteosarcoma is 'new auxiliary chemotherapy before operation + surgical resection + auxiliary chemotherapy after operation', but the treatment method has the problems of high treatment difficulty, unsatisfactory prognosis, low survival rate of patients and the like. Osteosarcoma is easy to generate lung metastasis, the prognosis of patients with lung metastasis in early diagnosis is worse, and the 5-year survival rate is lower than 30%. Despite the clinical increase in the dose of chemotherapeutic drugs, the change in the duration of drug use, and the use of combination chemotherapy with multiple drugs, there was no substantial improvement in the survival of osteosarcoma patients. In addition, the killing effect of the traditional first-line chemotherapeutic drug adriamycin on osteosarcoma cells is effective, but the application of the conventional administration method has the problems of poor selectivity and strong toxic and side effects, which limits the clinical application of the adriamycin.
Exosome is a membrane vesicle with a diameter of about 10-100nm, which is released to the outside of the cell after the intracellular vesicle is fused with the cell membrane, and contains a plurality of intracellular substances such as RNA, protein, DNA fragments and the like. The exosome can shuttle among cells, is beneficial to exchange substances and information among the cells, and can be loaded with chemotherapeutic drugs and siRNA for targeted therapy. Exosomes have natural advantages as drug carriers: (1) The nano-scale size is provided, and the capture of a reticuloendothelial system can be easily escaped in vivo; (2) The phospholipid bilayer structure is similar to the composition of a cell membrane, has strong affinity to the cell membrane and is convenient to enter cells; (3) The vesicle belongs to an endogenous vesicle, can not cause immune response when entering a body as a carrier, and can not be phagocytized by being identified as 'non-self substance' by an immune system in the body; (4) The protein is derived from cells, contains a plurality of transmembrane proteins on the surface, and can realize in-vivo targeted therapy by adding a ligand short peptide capable of targeting target cells at the tail end of a proper protein in a gene modification mode.
Many reports and studies can show that the exosome is an ideal drug carrier and can solve the defect of immunogenicity universally existing in exogenous carriers. However, when the exosome is specifically applied to the treatment of osteosarcoma, the exosome needs to be further modified, and a novel exosome drug particle is designed again, so that the exosome drug particle has the capacity of targeting osteosarcoma cells and has the effect of killing the osteosarcoma cells. Based on the research background, the invention provides a targeting exosome particle simultaneously loaded with a small-molecule drug adriamycin and a gene drug si-PVT1 and application thereof in preparing a drug for treating osteosarcoma.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide a novel targeting exosome carrying doxorubicin and a si-PVT1 gene together, and a preparation method and application thereof in a medicament for treating osteosarcoma.
In order to realize the purpose of the invention, the technical scheme of the invention is as follows:
firstly, the invention provides an exosome carrying doxorubicin and si-PVT1 targeting, and the exosome consists of targeting exosome particles, small molecule drug doxorubicin and gene drug si-PVT1.
Further, the targeted exosome particle is a fusion protein comprising an N-terminus of 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 short peptide insertion being specifically located between the signal peptide and mature sequence of LAMP 2; the amino acid sequence of the short peptide with the targeting function in the 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, respectively. In one aspect, the invention provides a preparation method of targeted exosomes loaded with doxorubicin and si-PVT1 together, which is prepared according to 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) centrifuging the supernatant obtained in the step (1) for three times, wherein the three 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 ℃; and then collecting the supernatant for ultracentrifugation, wherein the ultracentrifugation parameters are 4 ℃ and 100,000 Xg for 70 min, discarding the supernatant, and collecting the pellet to obtain the exosome particles capable of targeting osteosarcoma cells. After the PBS resuspended exosome particles, the exosome particles are stored at-80 to-70 ℃.
(3) And (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 (200 mu L system), selecting voltage of 500V at normal temperature, lasting for 15s, and performing electroporation to obtain a mixture.
(4) And (4) carrying out ultracentrifugation on the mixture obtained in the step (3) to remove free adriamycin and si-PVT1, thus obtaining the target exosome drug co-loaded with adriamycin and si-PVT1.
Further, in the step (3), the sequence of the si-PVT1 is 5'-GCU UGG AGG CUG AGG AGU UTT-3'.
On the other hand, the invention provides a targeted exosome carrying doxorubicin and si-PVT1 together, which is applied to the preparation of a medicament for treating osteosarcoma.
Advantageous effects
According to the invention, firstly, an exosome is artificially modified by a gene editing method, and a short peptide is inserted into a lysosome related protein 2 (LAMP 2) on the surface of the exosome, 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, small molecular drug adriamycin and gene drug si-PVT1 are loaded on the exosome particles after gene editing, and the two drugs are simultaneously delivered to osteosarcoma cells to play a role in killing the cells. Compared with the traditional administration mode, the targeted exosome particle provided by the invention has high targeting property on osteosarcoma cells, reduces the toxic and side effects of the drug, and improves the safety 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.
Drawings
FIG. 1 in vivo distribution of iRGD-Exo/si-PVT1/DOX in mice.
FIG. 2 flow cytometry was used to examine the uptake efficiency of iRGD-Exo/si-PVT1/DOX by osteosarcoma cells.
FIG. 3 iRGD-Exo/si-PVT1/DOX distribution map in osteosarcoma cells.
FIG. 4 effect of iRGD-Exo/si-PVT1/DOX on proliferation of osteosarcoma cells, wherein (A) is the effect of different doses of DOX, si-PVT1, iRGD-Exo/si-PVT1/DOX on proliferation of osteosarcoma cells; (B) Effects of DOX, si-PVT1, exo/si-PVT1/DOX, iRGD-Exo/si-PVT1, and iRGD-Exo/si-PVT1/DOX on the proliferation of osteosarcoma cells. *p<0.05；**p<0.01。
FIG. 5 iRGD-Exo/si-PVT1/DOX vs. osteosarcomaTherapeutic effect of transplanted tumor-bearing mice, wherein (a) is tumor growth curve (. + -.) (ii)p<0.01 ); and (B) is an observation picture of the tumor tissue section under an HE staining microscope.
Detailed Description
Example 1
The novel targeting exosome loaded with doxorubicin and si-PVT1 together is prepared according to 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; pcDNA3.1 (+) -hLAMP2b-Cys-CRGDKGPDC plasmid was constructed, this plasmid and liposome were mixed well within 5 minutes, left at room temperature for 20 minutes, added to 293T cells, after 6 hours the culture solution containing 10% FBS was replaced, and after 24 hours the supernatant was collected.
(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 ℃; and then collecting the supernatant for ultracentrifugation, wherein the ultracentrifugation parameters are 4 ℃ and 100,000 Xg for 70 min, discarding the supernatant, and collecting the pellet to obtain the exosome particles capable of targeting osteosarcoma cells. PBS resuspended exosome particles and then stored at-80 ℃.
(3) And (3) mixing 0.2 mu L of the exosome particles of the targeted osteosarcoma cells obtained in the step (2), 200ng of the si-PVT1 gene drug (the si-PVT1 sequence is 5'-GCU UGG AGG CUG AGG AGU UTT-3'), 50 mu g of adriamycin uniformly by using buffer (200 mu L system), selecting voltage of 500V at normal temperature, lasting for 15s, and performing electroporation to obtain a mixture.
(4) And (4) removing free adriamycin and si-PVT1 from the mixture obtained in the step (3) through ultracentrifugation to obtain the targeted exosome co-loaded with adriamycin and si-PVT1.
To further illustrate the role and effect of the co-loaded doxorubicin and si-PVT1 targeting exosomes provided by the present invention in treating osteosarcoma, the present invention provides cell assays and animal assays as illustrations.
The invention relates to a medicament and a preparation method thereof, wherein the medicament comprises the following components in part by weight:
iRGD-Exo/si-PVT1/DOX: the target exosome of the co-loading adriamycin and si-PVT1 provided by the invention; prepared according to the method of example 1.
Exo/si-PVT1/DOX: is a common exosome particle carrying adriamycin and si-PVT1 genes; the exosome particle is loaded with si-PVT1 gene drugs and DOX drugs, but has no targeting function; the preparation method of the exosome particle comprises the following steps:
(1) After the 293T cells were attached to the wall, the culture supernatant was collected.
(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 ℃; and then collecting the supernatant for ultracentrifugation, wherein the ultracentrifugation parameters are 4 ℃ and 100,000 Xg for 70 min, discarding the supernatant, and collecting the pellet to obtain the exosome particles. PBS resuspended exosome particles and then stored at-80 ℃.
(3) And (3) mixing 0.2 mu L of the exosome particles obtained in the step (2), 200ng of the si-PVT1 gene drug (the si-PVT1 sequence is 5'-GCU UGG AGG CUG AGG AGU UTT-3') and 50 mu g of adriamycin uniformly by using a buffer (200 mu L system), selecting voltage of 500V at normal temperature, lasting for 15s, and carrying out electroporation to obtain a mixture.
(4) And (4) removing free adriamycin and si-PVT1 from the mixture obtained in the step (3) through ultracentrifugation to obtain the common exosome particles carrying both adriamycin and si-PVT1.
iRGD-Exo/DOX: targeted exosome particles loaded with DOX alone; the preparation method of the exosome particle comprises 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; pcDNA3.1 (+) -hLAMP2b-Cys-CRGDKGPDC plasmid was constructed, this plasmid and liposome were mixed well within 5 minutes, left at room temperature for 20 minutes, added to 293T cells, after 6 hours the culture solution containing 10% FBS was replaced, and after 24 hours the supernatant was collected.
(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 ℃; and then collecting the supernatant for ultracentrifugation, wherein the ultracentrifugation parameters are 4 ℃ and 100,000 Xg for 70 min, discarding the supernatant, and collecting the pellet to obtain the exosome particles capable of targeting osteosarcoma cells. PBS resuspended exosome particles and then stored at-80 ℃.
(3) And (3) uniformly mixing 0.2 mu L of the exosome particles obtained in the step (2) and 50 mu g of adriamycin by using a buffer (200 mu L system), selecting the voltage of 500V at normal temperature, lasting for 15s, and carrying out electroporation to obtain a mixture.
(4) And (4) removing free adriamycin from the mixture obtained in the step (3) through ultracentrifugation to obtain the single DOX-loaded targeted exosome particles.
iRGD-Exo/si-PVT1: targeting exosome particles for loading si-PVT1 genes alone; the preparation method of the exosome particle comprises 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; pcDNA3.1 (+) -hLAMP2b-Cys-CRGDKGPDC plasmid was constructed, this plasmid and liposome were mixed well within 5 minutes, left at room temperature for 20 minutes, added to 293T cells, after 6 hours the culture solution containing 10% FBS was replaced, and after 24 hours the supernatant was collected.
(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 ℃; and then collecting the supernatant for ultracentrifugation, wherein the ultracentrifugation parameters are 4 ℃ and 100,000 Xg for 70 min, discarding the supernatant, and collecting the pellet to obtain the exosome particles. PBS resuspended exosome particles and then stored at-80 ℃.
(3) And (3) uniformly mixing 0.2 mu L of the exosome particles obtained in the step (2) and 200ng of the si-PVT1 gene drug (the sequence of the si-PVT1 is 5'-GCU UGG AGG CUG AGG AGU UTT-3') by using a buffer (200 mu L system), selecting the voltage of 500V at normal temperature, lasting for 15s, and carrying out electroporation to obtain a mixture.
(4) And (4) removing free si-PVT1 from the mixture obtained in the step (3) through ultracentrifugation to obtain the si-PVT 1-loaded common exosome particles.
And (4) DOX: free DOX.
si-PVT1: free si-PVT1.
Establishing an osteosarcoma transplantation tumor-bearing mouse model: BALB/C mice were selected and injected subcutaneously into the left axilla of mice with 100. Mu.L of human osteosarcoma MNNG/HOS suspension (approximately containing 2.5X 10 cells) 6 One), a subcutaneous tumor model was prepared.
Test 1: iRGD-Exo/si-PVT1/DOX distribution in vivo
The iRGD-Exo/si-PVT1/DOX was stained with the hydrophobic infrared fluorescent dye DIR. Injecting the dyed iRGD-Exo/si-PVT1/DOX into an osteosarcoma tumor-bearing mouse through a tail vein, anesthetizing the osteosarcoma tumor-bearing mouse with isoflurane 10 minutes, 1 hour, 2 hours, 4 hours, 8 hours and 24 hours after injection, detecting the fluorescence distribution condition in the osteosarcoma tumor-bearing mouse by using a small animal living body imaging system, and detecting by adopting the excitation wavelength 748nm and the emission wavelength 780nm of DIR. The results are shown in FIG. 1. As can be seen from FIG. 1, the iRGD-Exo/si-PVT1/DOX showed strong fluorescence signals at the tumor sites of the mice after the mice were injected with iRGD-Exo/si-PVT1/DOX for 1 hour, and the fluorescence signals were able to be continuously displayed for 24 hours. The results show that the iRGD-Exo/si-PVT1/DOX provided by the invention can be effectively taken up by osteosarcoma cells in a large amount in cells and can be retained in osteosarcoma tissues for a long time.
Test 2: uptake rate of iRGD-Exo/si-PVT1/DOX by osteosarcoma cells
For the preparation of iRGD-Exo/si-PVT1/DOX, doxorubicin was stained with Nile Red (Nile Red) and si-PVT1 was labeled with green fluorescein FAM. Stained and labeled iRGD-Exo/si-PVT1/DOX (test group) and Exo/si-PVT1/DOX (control group) were added to MNNG/HOS osteosarcoma cells in the dark, incubated for 4 hours, and then fluorescence in the osteosarcoma cells was detected by flow cytometry. The results are shown in FIG. 2. As can be seen from FIG. 2, the uptake efficiency of iRGD-Exo/si-PVT1/DOX by MNNG/HOS osteosarcoma cells was significantly higher than that of the control group; in particular, the proportion of MNNG/HOS cells with double positive of two fluorescein in the test group is obviously higher than that of the control group. The above results indicate that osteosarcoma cells can efficiently take the iRGD-Exo/si-PVT1/DOX provided by the present invention.
Test 3: distribution of iRGD-Exo/si-PVT1/DOX in osteosarcoma cells
For the preparation of iRGD-Exo/si-PVT1/DOX, doxorubicin was stained with Nile Red (Nile Red) and si-PVT1 was labeled with green fluorescein FAM. The stained and labeled iRGD-Exo/si-PVT1/DOX (test group) and Exo/si-PVT1/DOX (control group) were added to MNNG/HOS osteosarcoma cells in the dark, incubated for 3 hours, and then the uptake of the fluorescent marker by the osteosarcoma cells and the distribution of the fluorescence in the cells were observed using a laser confocal microscope (LSCM). The results are shown in FIG. 3. As can be seen from fig. 3, the red fluorescence and the green fluorescence of the test group are both strong, and yellow fluorescence generated by superimposing the red fluorescence of nile red and the green fluorescence of FAM is observed. The above results can show that the targeting exosome particles provided by the invention are used as vectors to facilitate the entry of the adriamycin and si-PVT1 genes into osteosarcoma cells.
Test 4: inhibitory effect of iRGD-Exo/si-PVT1/DOX on osteosarcoma cell proliferation
Solutions (prepared into exosome particle suspensions or drug solutions by using culture media as solvents) with different concentration gradients (0.01, 0.1, 1 and 2 mu g/mL) are respectively prepared for iRGD-Exo/si-PVT1/DOX, DOX and si-PVT1, and after the solutions are co-cultured with MNNG/HOS osteosarcoma cells for 24 hours, the cell proliferation inhibition condition is detected by CCK-8. The results are shown in FIG. 4A. As can be seen from FIG. 4A, all three drugs have significant inhibitory effects on osteosarcoma cells, but compared with the two groups of DOX and si-PVT1, the inhibitory rate of osteosarcoma cells treated with iRGD-Exo/si-PVT1/DOX is about 86.92%, which is 2 times and 3.5 times that of the two groups of DOX and si-PVT1, respectively, and has significant difference (P < 0.01).
iRGD-Exo/si-PVT1/DOX (targeting exosome particles co-loaded with doxorubicin and si-PVT1 genes), exo/si-PVT1/DOX (common exosome particles co-loaded with doxorubicin and si-PVT1 genes), iRGD-Exo/DOX (targeting exosome particles loaded with DOX alone), iRGD-Exo/si-PVT1 (targeting exosome particles loaded with si-PVT1 genes alone), free DOX, and free si-PVT1 were prepared into solutions of 2. Mu.g/mL, respectively, and then were co-cultured with MNNG/HOS osteosarcoma cells for 48 hours, respectively, and then the inhibition of cell proliferation was detected by CCK-8. The results are shown in FIG. 4B. As can be seen from FIG. 4B, the inhibitory rate of iRGD-Exo/si-PVT1/DOX on osteosarcoma cell proliferation can reach as high as 80.23%, the inhibitory rate of DOX, si-PVT1 and Exo/si-PVT1/DOX on cell proliferation is 12% -22%, and the difference of iRGD-Exo/si-PVT1/DOX is very significant (P < 0.01); the cell proliferation inhibition rate of iRGD-Exo/DOX is 50.62%, the cell proliferation inhibition rate of iRGD-Exo/si-PVT1 is 31.47%, and iRGD-Exo/si-PVT1/DOX has significant difference or extremely significant difference compared with the same (P <0.05, P < -0.01); the results can show that, firstly, the combined use of DOX and si-PVT1 obviously improves the inhibition effect of the two drugs on cells when the two drugs are used independently; secondly, carrying out gene editing on exosomes to obtain exosome particles with a targeting effect, wherein the targeting effect can effectively improve the proliferation inhibition effect of DOX and si-PVT1 on osteosarcoma cells; most importantly, the medicine obtained by loading DOX and si-PVT1 on exosome particles with targeting effect as a medicine carrier has more obvious proliferation inhibition effect on osteosarcoma cells, the DOX and the si-PVT1 can be simultaneously delivered to the osteosarcoma cells by the medicine obtained by the invention, and the two medicines can generate synergistic effect, so that the anti-tumor capacity of the medicine is enhanced.
Test 5: therapeutic effect of iRGD-Exo/si-PVT1/DOX on osteosarcoma transplantation tumor-bearing mice
9 osteosarcoma transplantation tumor-bearing mice are taken, the body weight is 20 +/-1 g, the mice are randomly divided into 3 groups, and each group comprises 3 mice, namely a blank control group, a DOX group and an iRGD-Exo/si-PVT1/DOX group. The blank control group is administrated with physiological saline, the DOX group is administrated with 20mg/kg DOX, and the iRGD-Exo/si-PVT1/DOX group is administrated with 20mg/kg iRGD-Exo/si-PVT1/DOX; administered by tail vein injection 1 time every 3 days for 21 consecutive days.
In the administration process, the longest diameter and the shortest diameter of the transplanted tumor are measured every three days, the general condition of the transplanted tumor is observed, the tumor volume is calculated, and a tumor volume change curve graph is drawn. The results are shown in FIG. 5A. As can be seen from fig. 5A, there was no significant difference in the volume of the transplanted tumor in the nude mice of each group before the administration, and after the administration, compared with the placebo group and the DOX group, the volume of the transplanted tumor in the iRGD-Exo/si-PVT1/DOX group was significantly different, and the iRGD-Exo/si-PVT1/DOX group had a significant inhibitory effect on the volume increase of the transplanted tumor, which was superior to DOX.
After the administration, the nude mice were sacrificed, tumor tissues were isolated and tissue sections were HE stained, and the staining was observed under a microscope. The results are shown in FIG. 5B. As can be seen from FIG. 5B, the transplanted tumor tissues of three groups of nude mice all have the morphological characteristics of tumor cells. The tumor cells of the blank control group grow very compactly, the cell nucleus is relatively large, the nuclear plasma ratio is high, and the cell nucleus profile is obvious; compared with a control group, the DOX group and the iRGD-Exo/si-PVT1/DOX group have the phenomena of tumor cell density reduction, disorganized tissue structure, part of cells showing the phenomena of nuclear compaction, nuclear lysis and the like to different degrees; compared with the DOX group, the iRGD-Exo/si-PVT1/DOX group has the advantages that the tumor cell density is obviously reduced, the tissue structure is abnormal and disordered, and most cells show the phenomena of nuclear compaction, nuclear lysis and the like, so that the iRGD-Exo/si-PVT1/DOX can well inhibit the growth of osteosarcoma transplanted tumor tissues and has good treatment effect on osteosarcoma tumor-bearing mouse models.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions 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.
Sequence listing
<110> first subsidiary Hospital of Zhengzhou university
<120> target exosome co-loaded with adriamycin and si-PVT1, preparation and anti-osteosarcoma application thereof
<130> description, claims
<141> 2020-04-15
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> Artificial sequence (unknown)
<400> 1
Cys Arg Gly Asp Lys Gly Pro Asp Cys
1 5
<210> 2
<211> 27
<212> DNA
<213> Artificial sequence (unknown)
<400> 2
tgccgcggcg ataaaggccc ggattgc 27
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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