CN116676265A - Acid-sensitive fusion peptide targeted exosome and preparation method and application thereof - Google Patents
Acid-sensitive fusion peptide targeted exosome and preparation method and application thereof Download PDFInfo
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- CN116676265A CN116676265A CN202310414932.XA CN202310414932A CN116676265A CN 116676265 A CN116676265 A CN 116676265A CN 202310414932 A CN202310414932 A CN 202310414932A CN 116676265 A CN116676265 A CN 116676265A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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Abstract
The invention provides an acid-sensitive fusion peptide targeted exosome and a preparation method and application thereof, and relates to the technical field of biological medicines. According to the invention, pHLIP with a tumor targeting microenvironment is combined with a C1C2 structural domain of HEK 293T-derived exosome membrane surface protein Lactadherein for the first time, so that an exosome with a tumor targeting effect is prepared; and the targeted exosome is used as a carrier to load the traditional Chinese medicine monomer curcumenol with an anti-tumor effect, so that a medicine carrying system of CUR-ExosL2 with the targeted tumor effect is successfully constructed, and the medicine carrying system realizes high medicine carrying capacity of curcumenol while maintaining the complete exosome structure, property and function. The invention realizes targeted drug delivery by engineering modification of exosomes through genetic engineering, realizes specific drug delivery of gastric gland tumor, improves the bioavailability of the drug, and provides a new thought and reference for targeted treatment of gastric cancer by traditional Chinese medicine.
Description
Technical Field
The invention relates to the technical field of biological medicines, in particular to an acid-sensitive fusion peptide targeted exosome, a preparation method and application thereof.
Background
Gastric Cancer (GC) is one of five common cancers worldwide, has been identified as one of the major causes of Cancer-related deaths worldwide, and according to statistics of international Cancer institutions, 100 or more tens of thousands of new cases of Gastric Cancer worldwide and about 76.9 tens of thousands of deaths have occurred by 2020. Because early clinical symptoms of gastric cancer patients are not obvious and specific indexes are poor, more than 70% of patients are in the late stage when diagnosis is confirmed, and the optimal period of operation and radiotherapy is missed. Traditional chemotherapeutic drugs and targeted therapeutic drugs have gradually poor clinical treatment effects due to drug resistance and side effects of cytotoxicity. Therefore, the search for new anti-cancer drugs has great significance, and especially, low-toxicity and high-efficiency anti-tumor drugs are important and hot points of research in the world. Compared with synthetic chemical drugs, traditional Chinese medicines derived from natural plants have many advantages, such as: low toxicity, high safety, multiple routes, multiple targets, etc.
Because the traditional Chinese medicine has the synergistic and pharmacokinetic actions of various active compounds, the traditional Chinese medicine preparation plays an outstanding role in treating various diseases. At present, a plurality of traditional Chinese medicine formulas and traditional Chinese medicine monomers are successfully used for anti-tumor treatment. For example, the anti-tumor effect of the traditional Chinese medicine monomer curcumin has been widely studied, and can inhibit the occurrence, progress, invasion, metastasis and the like of tumors. Researchers found that hesperetin monomers are able to promote cisplatin-induced apoptosis of gastric cancer cells in vitro and in vivo by up-regulating PTEN expression in traditional Chinese medicine anti-gastric cancer therapies. Zhang et al found that luteolin induced apoptosis of gastric cancer cells SGC-7901 by affecting cell cycle progression to inhibit tumor cell activity. Although the traditional Chinese medicine shows good anti-tumor property in clinical research, the traditional Chinese medicine has the defects of low stability, poor water solubility, fast in-vivo metabolism, low bioavailability and the like, so that the traditional Chinese medicine is limited in clinical application, and therefore, a carrier with good stability and biocompatibility is urgently needed to improve the drug characteristics.
The exosomes are membrane vesicles with the diameter of about 30-150nm released outside cells after fusion of intracellular multivesicular bodies and cell membranes, and comprise a plurality of intracellular substances such as RNA, protein, DNA fragments and the like. The exosomes can shuttle among cells, are favorable for the exchange of intercellular substances and information, and can be loaded with chemotherapeutic drugs and siRNA for targeted treatment. Exosomes have natural advantages as drug carriers: (1) The nanometer size is provided, so that the capture of the reticuloendothelial system can be easily escaped in the body; (2) The phospholipid bilayer structure has a phospholipid bilayer structure, is similar to the cell membrane in composition, has stronger affinity to the cell membrane, and is convenient for entering cells; (3) Belongs to endogenous vesicles, can not cause immune response when entering the body as a carrier, and can not be identified as non-self substances by the immune system in the body to be phagocytized; (4) It is derived from cells, contains a plurality of transmembrane proteins on the surface, can be modified by gene, and adding ligand short peptide capable of targeting target cells at the tail end of a proper protein, so as to realize in vivo targeted treatment.
The low pH intercalating peptide (pH low insertion peptide, pHLIP) is derived from the C-helix of bacterial rhodopsin and is a water soluble molecule that interacts with the lipid membrane in a pH dependent manner. pHLIP has the ability to target acidic tissues and dual delivery, and various exogenous molecules can be delivered to the cell surface and within cells by coupling to the N-or C-terminus of pHLIP. pHLIP has been demonstrated to target tumor acidic microenvironment, and its targeting delivery system has strong specificity advantage compared with traditional targeting nanocarriers and transmembrane peptides, and has the following advantages in application: pHLIP is insensitive to the heterogeneous expression of receptors or antigens in tumor cells; pHLIP has specific markers that actively target the acidic tumor microenvironment; pHLIP not only can accurately target primary tumor focus and metastasis focus, but also can develop more application aspects by utilizing the characteristic that pHLIP is inserted into the surface of tumor cell membrane. The pHLIP technology can deliver different types of functional molecules into cells for treatment and diagnosis, has better application prospect in clinic, but still has the defect of needing to be optimized at present, wherein the improvement of the synthesis efficiency of pHLIP and exogenous molecules can obviously improve the clinical transformation capability of pHLIP and exogenous molecules. Although pHLIP has been shown to have specific targeting effect on acidic tumor microenvironment, there is still a need to further improve the targeting efficiency, and it is required to screen patients for inflammatory diseases in clinical application, thereby improving the specificity of pHLIP.
Disclosure of Invention
(one) solving the technical problems
Aiming at the defects of the prior art, the invention provides an acid-sensitive fusion peptide targeted exosome and a preparation method thereof, wherein the exosome is used for preparing a traditional Chinese medicine nano-carrier with a tumor targeting effect and is used for preparing an anti-gastric cancer drug.
(II) technical scheme
In order to achieve the above purpose, the invention is realized by the following technical scheme:
in one aspect, the invention provides an acid-sensitive fusion peptide targeted exosome, which is a fusion protein comprising a low pH insertion peptide and a domain C1C2 of the Lactadherein protein, which is bound to the exosome.
Further, the exosome is pHILP-C1C2-1 fusion protein formed by connecting a Lactadherein signal peptide-WT-pHLIP-Flag and C1C 2-HA; wherein the gene sequence of the fusion protein is shown as SEQ ID NO. 1; wherein the sequence of the Lactadherein signal peptide-WT-pHLIP-Flag is shown as SEQ ID NO. 2; the sequence of the C1C2-HA is shown as SEQ ID NO. 3.
Furthermore, the exosome also comprises a fusion protein pHILP-C1C2-2 with a glycosylation motif having a protective effect, and the gene sequence of the fusion protein is shown as SEQ ID NO. 4.
In yet another aspect, the invention also provides a method for preparing an acid-sensitive fusion peptide targeted exosome, wherein the exosome is prepared according to the following steps:
(1) The synthesized Lactadherein signal peptide pHLIP-Flag and C1C2-HA gene sequences are connected into fusion proteins pHLIP-C1C2-1 and pHLIP-C1C2-2 by utilizing an overlap extension PCR technology;
(2) Cloning the acid-sensitive fusion peptide gene to a multiple cloning site on a pCDH-CMV-MCS-EF1-GFP-Puro lentiviral vector to obtain a recombinant plasmid pCDH-pHLIP-C1C2-1-Puro and a recombinant plasmid pCDH-PHLIP-C1C2-2-Puro, which are respectively abbreviated as CT1 and CT2;
(3) Packaging recombinant plasmids pCDH-PHLIP-C1C2-1-Puro and pCDH-PHLIP-C1C2-2-Puro into lentivirus to transfect HEK293T cells, and collecting lentivirus liquid for concentration to infect the HEK293T cells; screening the monoclonal cells by combining puromycin and a fold dilution method to obtain HEK293T monoclonal cell strain HEK293TCT1/CT2 for stably expressing the acid-sensitive fusion protein;
(4) Extraction of exosomes
Normally passaging stable-rotation monoclonal HEK293T CT1/CT2 cells, and discarding the culture medium in the culture bottle when the cell fusion degree reaches 70% -80%; then adding PBS buffer solution to wash the cells, and discarding the PBS buffer solution after washing; then adding DMEM basal medium for hungerHunger culturing, placing in 37 deg.C, 5% CO 2 Culturing in a cell incubator for 24-48 hours; collecting cell supernatant, centrifuging at 4deg.C and 300 Xg for 10min, precipitating and removing living cells; collecting supernatant, centrifuging at 4deg.C and 2000 Xg for 20min, precipitating and removing dead cells; collecting supernatant, centrifuging at 4deg.C and 10000×g for 30min, precipitating and removing dead cell debris; collecting supernatant, filtering with 0.22 μm filter, removing vesicles larger than 220nm, and collecting filtrate; ultrafiltering the collected filtrate with a 15mL ultrafiltration tube to obtain a molecular weight cut-off of 100kDa; collecting ultrafiltrate, centrifuging at 100000 Xg at 4deg.C for 70min, and discarding supernatant; adding PBS buffer solution to resuspend the precipitate, separating from the precipitate for 70min at 4 ℃ and 100000 Xg, and discarding the supernatant to obtain the exosome.
In still another aspect, the invention also provides application of the acid-sensitive fusion peptide targeted exosome for preparing a traditional Chinese medicine nano-carrier with a tumor targeting effect; the Chinese medicinal monomer is a Chinese medicinal monomer with anti-tumor effect; the Chinese medicinal monomers include, but are not limited to, any one of curcumenol, curcumin, bisdemethoxycurcumin, quercetin, taxol, resveratrol, triptolide, luteolin, shikonin, farnesin, hederagenin and isomouse Li Suzhong.
Further, the acid-sensitive fusion peptide targeted exosome traditional Chinese medicine nano-carrier is prepared by adopting a room temperature co-incubation method, and specifically comprises the following steps:
1) Taking 7 parts of exosomes with the concentration of 1600 mug/mL, 800 mug/mL, 400 mug/mL, 200 mug/mL, 100 mug/mL, 50 mug/mL and 25 mug/mL respectively, and respectively incubating with 200 mug/mL curcumenol solution for 48 hours at room temperature;
2) And (3) respectively centrifuging the exosome and curcumenol mixed solution at the temperature of 4 ℃ for 70min, removing supernatant and free curcumenol, washing and resuspending by using 10ml PBS, centrifuging at the temperature of 120000 x g for 70min again, and re-suspending and precipitating by using a proper amount of PBS to obtain the pHLIP-Lamp2b-1/2 fusion protein exosome traditional Chinese medicine nano-carrier.
On the other hand, the application of the acid-sensitive fusion peptide targeted exosome provided by the invention in preparing targeted drugs for treating gastric cancer.
(III) beneficial effects
The invention provides an acid-sensitive fusion peptide targeted exosome, a preparation method and application thereof. According to the invention, pHLIP with a tumor targeting microenvironment is combined with HEK 293T-derived exosome membrane surface protein C1C2 for the first time, so that an exosome with a tumor targeting effect is prepared; and the targeted exosome is used as a carrier to load the traditional Chinese medicine monomer curcumenol with an anti-tumor effect, so that a medicine carrying system of CUR-ExosL2 with the targeted tumor effect is successfully constructed, and the medicine carrying system realizes high medicine carrying capacity of curcumenol while maintaining the complete exosome structure, property and function. The invention realizes targeted drug delivery by engineering modification of exosomes through genetic engineering, realizes specific drug delivery of gastric gland tumor, improves the bioavailability of the drug, and provides a new thought and reference for targeted treatment of gastric cancer by traditional Chinese medicine.
The invention adopts the combination of differential centrifugation, ultrafiltration and ultra-high speed centrifugation to extract the exosomes from HEK293T, CT1 and CT2 cells, firstly uses differential centrifugation to remove cell impurities, then uses an ultrafiltration tube to concentrate cell supernatant, enriches the exosomes, and then uses ultra-high speed centrifugation to extract the exosomes. The method can obviously improve the extraction quantity and purity of exosomes.
Drawings
FIG. 1 shows HEK293T cells, CT1 cells, CT2 cells, HEK293TExo, CT1-Exo and CT2-Exo marker protein expression.
FIG. 2 is a graph showing particle size distribution of HEK293T-Exos (FIG. 2A), CT1-Exos (FIG. 2B) and CT2-Exos (FIG. 2C).
FIG. 3 is a transmission electron microscope image of HEK293T-Exos (FIG. 3A), CT1-Exos (FIG. 3B), CT2-Exos (FIG. 3C).
FIG. 4 shows the detection of HEK293T-Exos, CT1-Exos and CT2-Exos acid-sensitive fusion proteins by Western Blot.
FIG. 5 shows the results of the observation of the fluorescent dye pKH 67-labeled exosomes under laser confocal.
FIG. 6 shows how gastric cancer cells HGC-27 can be taken into HEK293T-Exos and CT2 under laser confocal.
FIG. 7 is a transmission electron microscope image of CUR-HEK293Texos (FIG. 7A), CUR-CT2Exo (FIG. 7B).
FIG. 8 is a CUR-CT2Exo particle size distribution.
FIG. 9 is an HPLC chart of curcumenol (S3), CUR-CT2Exos (S2) and CUR-HEK293TExos (S1).
FIG. 10 is a standard graph of curcumenol.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described in the following in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Test 1
1. The preparation of the pHLIP-C1C2-1/2 acid-sensitive fusion peptide targeted exosome comprises the following steps:
(1) Amplification of the C1C2-HA Gene sequence of interest
Amplifying a C1C2 target gene sequence except the signal peptide by using a PCR amplification technology, and adding an HA tag sequence at the sequence end by using the PCR technology, wherein the obtained C1C2-HA sequence is shown as SEQ ID NO. 3;
(2) Amplification of the Lactadherein Signal peptide-WT-pHLIP-Flag Gene sequence
Amplifying the Lactadherein signal peptide-pHLIP-Flag by using a PCR technology, wherein the sequence of the obtained Lactadherein signal peptide-WT-pHLIP-Flag is shown as SEQ ID NO. 2;
(3) Construction of pHLIP-C1C2-1 and pHLIP-C1C2-2 fusion protein genes
The synthesized Lactadherein signal peptide pHLIP-Flag and C1C2-HA gene sequences are connected into fusion proteins pHLIP-C1C2-1 and pHLIP-C1C2-2 by utilizing an overlap extension PCR technology; wherein pHLIP-C1C2-2 contains glycosylation motif with protective effect, and the glycosylation protective site has a sequence shown in SEQ ID NO. 12; the sequences of pHLIP-C1C2-1 and pHLIP-C1C2-2 are shown in SEQ ID NO.1 and 4, respectively.
(4) Construction of recombinant plasmid of acid-sensitive fusion peptide
Cloning the acid-sensitive fusion peptide gene to a multiple cloning site on a pCDH-CMV-MCS-EF1-GFP-Puro lentiviral vector to obtain a recombinant plasmid pCDH-pHLIP-C1C2-1-Puro and a recombinant plasmid pCDH-pHLIP-C1C2-2-Puro, which are respectively abbreviated as CT1 and CT2;
(5) Establishing HEK293T monoclonal cell strain HEK293TCT1/CT2 for stably expressing acid-sensitive fusion protein
Packaging recombinant plasmids pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro into lentivirus transfected HEK293T cells, and collecting lentivirus liquid for concentration to infect the HEK293T cells; screening the monoclonal cells by combining puromycin and a fold dilution method to obtain HEK293T monoclonal cell strain HEK293TCT1/CT2 for stably expressing the acid-sensitive fusion protein;
(6) Extraction of acid sensitive fusion peptide exosomes (Exos)
Normally passaging stable-rotation monoclonal HEK293T CT1/CT2 cells, and discarding the culture medium in the culture bottle when the cell fusion degree reaches 70% -80%; then adding PBS buffer solution to wash the cells, and discarding the PBS buffer solution after washing; then adding DMEM basal medium for starvation culture, placing at 37deg.C, 5% CO 2 Culturing in a cell incubator for 24-48 hours; collecting cell supernatant, centrifuging at 4deg.C and 300 Xg for 10min, precipitating and removing living cells; collecting supernatant, centrifuging at 4deg.C and 2000 Xg for 20min, precipitating and removing dead cells; collecting supernatant, centrifuging at 4deg.C and 10000×g for 30min, precipitating and removing dead cell debris; collecting supernatant, filtering with 0.22 μm filter, removing vesicles larger than 220nm, and collecting filtrate; ultrafiltering the collected filtrate with a 15mL ultrafiltration tube to obtain a molecular weight cut-off of 100kDa; collecting ultrafiltrate, centrifuging at 100000 Xg at 4deg.C for 70min, and discarding supernatant; adding PBS buffer solution to resuspend the precipitate, centrifuging for 70min at the temperature of 4 ℃ and the speed of 100000 Xg, and discarding the supernatant to obtain the precipitate which is the exosome.
Exosomes derived from HEK293T, HEK293TCT1 and HEK293TCT2 cells were prepared according to the above methods, respectively designated HEK293T-Exos, CT1-Exos, CT2-Exos, and the prepared exosomes were identified.
2. Identification of pHLIP-C1C2-1/2 acid-sensitive fusion peptide targeted exosomes
(1) Western blot detection of exosome-specific proteins
The Western Blot method detects the expression of Exos specific marker proteins, including membrane protein CD81, and intein Alix, TSG101 and HSP70. As a result, as shown in FIG. 1, the protein extracted from the super-separation precipitation in this experiment expressed CD81 and Alix, TSG101, HSP70 positively, suggesting that the protein was isolated as Exos by super-high speed centrifugation.
(2) Dynamic Light Scattering (DLS) determination of particle size
As a result of measuring particle diameters of HEK293T-Exos, CT1-Exos and CT2-Exos in DLS, particles were concentrated at about 149.2nm, 151.5nm and 153.5nm, and the particle diameters were unimodal normal, and the polydispersity PDI was about 0.181, 0.168 and 0.178, as shown in FIG. 2. The particle dispersibility is better, the definition diameter of Exos is 30-150nm, and the particle sizes of HEK293T-Exos, CT1-Exos and CT2-Exos obtained by the experiment accord with the range of Exos.
(3) Observation of exosome form and size by transmission electron microscope
The result of a transmission electron microscope shows that HEK293T-Exos, CT1-Exos and CT2-Exos precipitates obtained by ultracentrifugation have obvious composition of a tea-tray-like double-layer membrane structure with a diameter ranging from 30 nm to 150nm, are in single distribution, have clear background and are typical in exosome forms, and are shown in figure 3.
The invention adopts the combination of differential centrifugation, ultrafiltration and ultra-high speed centrifugation to extract the exosomes derived from HEK293T, CT1 and CT2 cells, firstly uses an ultrafiltration tube to concentrate cell supernatant, enriches the exosomes, and then uses ultra-high speed centrifugation to extract the exosomes. By particle size analysis. The sediment obtained by ultra-high speed centrifugation is identified as an exosome by a transmission electron microscope and a Western blot method, and can be used for subsequent targeting verification experiments.
Test 2
Verifying targeting of pHLIP-C1C2-1/2 acid-sensitive fusion peptide exosomes
1. Western Blot detection of exosome marker proteins
The expression of CT1-Exos and CT2-Exos acid-sensitive fusion proteins is detected by a Western Blot method, and both CT1-Exos and CT2-Exos express the acid-sensitive fusion proteins. As shown in FIG. 4, compared with CT1-Exos, the expression level of pHLIP in CT2-Exos is higher, and pHLIP in CT1-Exos is obviously degraded, and the result shows that the glycosylation protection site is increased before pHLIP, so that the pHLIP can be effectively prevented from being degraded.
2. Dyeing marked exosomes
The exosomes derived from HEK293T, HEK293TCT1 and HEK293TCT2 cells were labeled with the fluorescent dye pKH67 and the labeling results of the exosomes were observed under laser confocal conditions, as shown in fig. 5.
3. Laser confocal observation of gastric cancer cell HGC-27 uptake condition of exosomes
The fluorescent dye pKH67 is used for marking exosomes derived from HEK293T and HEK293TCT2 cells, the exosomes are incubated with HGC-27 cells, and in order to verify the targeting of the exosomes containing the pHLIP-C1C2-2 fusion protein to tumor cells under different acid-base environments, the culture medium is adjusted to different pH values, and the pH values are set to 7.4 and 6.4. After 4h of incubation with gastric cancer cell HGC-27, uptake was observed using a laser confocal microscope, and the results are shown in FIG. 6. Under acidic conditions, the gastric cancer cells HGC-27 have obvious difference in uptake of HEK293T-Exos and HEK293TCT2-Exos, and the HGC-27 cells have stronger uptake capacity of HEK293TCT 2-Exos. The results prove that HEK293TCT2-Exos has targeting effect on gastric cancer cells.
Test 3
1. preparation of pHLIP-C1C2-2 acid-sensitive fusion peptide exosome traditional Chinese medicine nano-carrier
1) Taking 4 parts of purified exosomes (200 mug/mL), and respectively incubating with 600, 400, 200 and 100 mug/mL curcumenol solutions for 48 hours at room temperature;
2) And (3) respectively centrifuging the exosome and curcumenol mixed solution at the temperature of 4 ℃ for 70min, removing supernatant and free curcumenol, washing and resuspending by using 10ml PBS, centrifuging at the temperature of 120000 x g for 70min again, and re-suspending and precipitating by using a proper amount of PBS to obtain the traditional Chinese medicine nano-carrier of the pHLIP-C1C2-2 fusion protein exosome, which is called CUR-CT2Exos for short.
CUR-HEK293TExos were prepared simultaneously as described above.
2. Characterization of CUR-L2Exos
(1) Observation of CUR-CT2Exos morphology and size by transmission electron microscope
The transmission electron microscope results show that CUR-HEK293TExos and CUR-CT2Exo after drug loading have no larger change compared with Exos before drug loading, still have obvious tea-tray-like double-layer membrane structures, and the membrane recovery after ultrasonic incubation is good, which indicates that the drug loading method has no obvious influence on the morphology of exosomes, and the result is shown in figure 7.
(2) Dynamic Light Scattering (DLS) determination of CUR-CT2Exos particle size
The particle size of CUR-CT2Exos was measured by DLS, and as shown in FIG. 8, the particles were concentrated at about 170nm, and the particle size distribution was unimodal, and the polydispersity index (PDI) was 0.149, respectively. The particle dispersibility is better, and the particle size of the exosome after drug loading is slightly increased compared with that of the exosome before drug loading.
3. Determination of curcumenol content in CUR-CT2Exos by high-phase liquid chromatography
(1) Specificity of CUR-CT2Exos
The retention time of the chromatographic peak of the curcumenol and the CUR-Exos is 16.9min, the peak shape is good, the Exos has no chromatographic peak signal at the time point, and the detection of the curcumenol by exosomes is not interfered and the specificity is good. The results are shown in FIG. 10.
(2) Standard curve of curcumenol
The curcumenol reference solutions with the concentrations of 8, 16, 32, 64, 128 and 256 mug/mL are respectively prepared according to the following steps: YMC-triert C 18 Columns (250 mm. Times.4.6 mm,5 μm); mobile phase: acetonitrile-phosphoric acid water (60:40, v/v); flow rate: 1mL/min; column temperature: 30.0 ℃; sample injection amount: 10. Mu.L; detection wavelength: 200nm "chromatography conditions were used to draw standard curves.
The curcumenol standard graph is shown in fig. 10, and the linear regression equation is y=23.27x+8.246 (R 2 =0.999 9), curcumenol has good linear relation in the mass concentration range of 8-256 μg/mL.
(3) Determination of CUR-CT2Exos drug loading
100 mu L of CUR-CT2Exos demulsification solution is respectively taken and the mixture is subjected to column chromatography: YMC-triert C 18 Columns (250 mm. Times.4.6 mm,5 μm); mobile phase: acetonitrile-phosphoric acid water (60:40, v/v); flow rate: 1mL/min; column temperature: 30.0 ℃; sample injection amount: 10. Mu.L; detection wavelength: 200nm chromatographic condition sample injection for determining curcumenol content, and calculatingDrug Loading (LC). LC (%) =w 1 /W 2 X 100%, where W 1 Is the total amount of encapsulated curcumenol; w (W) 2 Protein content for CUR-CT2Exos.
The content of curcumenol in CUR-CT2Exos solution is calculated by a standard curve regression equation of curcumenol, and is taken as the medicine: the exosome is 1: the highest drug loading rate is as follows: the drug loading rate of 80.9ug, i.e., CUR-CT2Exos, was 20.225%.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.
Claims (7)
1. An acid-sensitive fusion peptide targeted exosome comprising a fusion protein of a low pH insertion peptide with domain C1C2 of the exosome-binding domain of the Lactadherin protein on the surface of the exosome.
2. The acid sensitive fusion peptide targeted exosome according to claim 1, wherein the exosome is a pHILP-C1C2-1 fusion protein comprising a Lactadherein signal peptide-WT-pHLIP-Flag and C1C2-HA linked together; wherein the gene sequence of the fusion protein is shown as SEQ ID NO. 1; wherein the sequence of the Lactadherein signal peptide-WT-pHLIP-Flag is shown as SEQ ID NO. 2; the sequence of the C1C2-HA is shown as SEQ ID NO. 3.
3. The acid-sensitive fusion peptide targeted exosome according to claim 1, wherein the exosome further comprises a fusion protein pHILP-C1C2-2 having a glycosylation motif with a protective effect, and the gene sequence of the fusion protein is shown in SEQ ID NO. 4.
4. The preparation method of the acid-sensitive fusion peptide targeted exosome is characterized by comprising the following steps of:
(1) The synthesized Lactadherein signal peptide pHLIP-Flag and C1C2-HA gene sequences are connected into fusion proteins pHLIP-C1C2-1 and pHLIP-C1C2-2 by utilizing an overlap extension PCR technology;
(2) Cloning the acid-sensitive fusion peptide to a multiple cloning site on a pCDH-CMV-MCS-EF1-GFP-Puro lentiviral vector to obtain a recombinant plasmid pCDH-pHLIP-C1C2-1-Puro and a recombinant plasmid pCDH-pHLIP-C1C2-2-Puro, which are respectively abbreviated as CT1 and CT2;
(3) Packaging recombinant plasmids pCDH-pHLIP-C1C2-1-Puro and pCDH-pHLIP-C1C2-2-Puro into lentivirus transfected HEK293T cells, and collecting lentivirus liquid for concentration to infect the HEK293T cells; screening the monoclonal cells by combining puromycin and a fold dilution method to obtain HEK293T monoclonal cell strains HEK293TCT1 and HEK293TCT2 for stably expressing the acid-sensitive fusion protein;
(4) Extraction of exosomes
Normally passaging stable-rotation monoclonal HEK293TCT1 and HEK293TCT2 cells, and discarding culture medium in the culture bottle when the cell fusion degree reaches 70% -80%; then adding PBS buffer solution to wash the cells, and discarding the PBS buffer solution after washing; then adding DMEM basal medium for starvation culture, placing at 37deg.C, 5% CO 2 Culturing in a cell incubator for 24-48 hours; collecting cell supernatant, centrifuging at 4deg.C and 300 Xg for 10min, precipitating and removing living cells; collecting supernatant, centrifuging at 4deg.C and 2000 Xg for 20min, precipitating and removing dead cells; collecting supernatant, centrifuging at 4deg.C and 10000×g for 30min, precipitating and removing dead cell debris; collecting supernatant, filtering with 0.22 μm filter, removing vesicles larger than 220nm, and collecting filtrate; ultrafiltering the collected filtrate with a 15mL ultrafiltration tube to obtain a molecular weight cut-off of 100kDa; collecting ultrafiltrate, centrifuging at 100000 Xg at 4deg.C for 70min, and discarding supernatant; adding PBS buffer solution to resuspend the precipitate, centrifuging for 70min at the temperature of 4 ℃ and the speed of 100000 Xg, and discarding the supernatant to obtain the precipitate which is the exosome.
5. The application of the acid-sensitive fusion peptide in targeting exosomes is characterized in that the exosomes are used for preparing traditional Chinese medicine nano-carriers with a tumor targeting effect; the Chinese medicinal monomer is a Chinese medicinal monomer with anti-tumor effect; the Chinese medicinal monomers include, but are not limited to, any one of curcumenol, curcumin, bisdemethoxycurcumin, quercetin, taxol, resveratrol, triptolide, luteolin, shikonin, farnesin, hederagenin and isomouse Li Suzhong.
6. The application of the acid-sensitive fusion peptide targeted exosome according to claim 3, wherein the targeted exosome traditional Chinese medicine nano-carrier is prepared by adopting a room temperature co-incubation method, and specifically comprises the following steps:
1) Taking 7 parts of exosomes with the concentration of 1600 mug/mL, 800 mug/mL, 400 mug/mL, 200 mug/mL, 100 mug/mL, 50 mug/mL and 25 mug/mL respectively, and respectively incubating with 200 mug/mL curcumenol solution for 48 hours at room temperature;
2) And (3) respectively centrifuging the exosome and curcumenol mixed solution at the temperature of 4 ℃ for 70min, removing supernatant and free curcumenol, washing and resuspension with 10ml PBS, centrifuging at the temperature of 120000×g for 70min again, and re-suspending and precipitating with a proper amount of anhydrous methanol to obtain the targeted exosome traditional Chinese medicine nano-carrier.
7. The application of the acid-sensitive fusion peptide targeted exosome is characterized in that the exosome is used for preparing targeted drugs for treating gastric cancer.
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