CN113908293A - Targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle and preparation method and application thereof - Google Patents

Targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle and preparation method and application thereof Download PDF

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CN113908293A
CN113908293A CN202111204735.2A CN202111204735A CN113908293A CN 113908293 A CN113908293 A CN 113908293A CN 202111204735 A CN202111204735 A CN 202111204735A CN 113908293 A CN113908293 A CN 113908293A
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exosome
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狄留庆
王若宁
赵华聪
王雪
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Nanjing University of Chinese Medicine
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Abstract

The invention discloses a preparation method and application of a targeting peptide modified traditional Chinese medicine multi-component 'exosome-like' fusion nanoparticle. The preparation method of the fusion nanoparticle comprises a polyethylene glycol induction method, a repeated freeze-thaw method, an extrusion method, an ultrasonic crushing method and an incubation method. The preparation method has the advantages of simple preparation process, mild conditions and low cost, and the prepared nanoparticles have various medicament-carrying varieties, high penetrability, biological target regulation, complementary treatment mechanism, biological safety and the like. The nano-drug delivery platform can realize multi-component co-delivery, combined drug delivery and cooperative and accurate treatment of traditional Chinese medicines, can realize cooperative target treatment of complex progressive diseases such as tumors, neurodegenerative diseases and the like, and has good application prospect.

Description

Targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle and preparation method and application thereof
Technical Field
The invention belongs to the technical field of nano preparations, and particularly relates to a targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle, and a preparation method and an application method thereof.
Background
Exosomes are endogenous, extracellular vesicles 40-100nm in diameter, and are secreted by various types of cells, which are ubiquitous in body fluids, including blood, urine, saliva, cerebrospinal fluid, and the like. The exosome contains protein and RNA related to cell sources, can transport protein and nucleic acid, participates in intercellular communication, and can be used for diagnosis and monitoring of diseases and screening of drug curative effects. Furthermore, the properties of exosomes in delivering active substances to diseased cells have prompted their research as therapeutic vectors. The membrane surface of the exosome has a plurality of characteristic transmembrane proteins, such as tetraspanin superfamily proteins CD9, CD81 and CD63, a tumor susceptibility gene TSG101, a lipid raft marker protein flotillin and the like. Delivery systems designed based on exosome-specific proteins are able to target tissue disease areas and exert disease therapeutic effects. Exosomes have good biocompatibility, can escape from the body for phagocytosis, and can penetrate biological barriers, especially the Blood Brain Barrier (BBB).
The liposome is an artificially synthesized nano material, has good biodegradability and compatibility, good size controllability, excellent drug sealing performance and easy modification, and is widely applied to the design of a nano drug delivery system. The liposome is used as a drug carrier, has lymphatic system tropism and passive targeting, and can be endowed with specific active targeting by modifying antibodies, sugar residues, receptor ligands and the like on a lipid bilayer of the liposome so as to deliver drugs to specific tissues, organs, cells or subcellular organelles. In recent years, the application of polypeptides in the pharmaceutical field has received attention. They are not only bioactive, but also adept at delivering cargo to target areas, and their application in targeted therapy has great promise. The RGD peptide (argine-glycine-aspartic acid) can target integrin alpha 5 beta 1 and mediate cell adhesion; luteinizing hormone releasing hormone GnRH (gonadotropin-releasing hormone) can be selectively combined with seven-transmembrane G protein coupled receptor GnRH-R; vascular endothelin 2(angiopep-2) is a 19-mer peptide that binds to low density lipoprotein receptor-related protein 1 on brain endothelial cells and is able to cross the BBB; the tLyp-1 peptide can be combined with neuropilin receptor 1 (NRP-1) with high affinity and specificity shown by tumor cells and blood vessels, and has the function of penetrating through tumor blood vessels and tumor stroma.
Cancer is one of the leading causes of death, second only to death caused by heart and circulatory diseases. At present, the combination of surgery, radiotherapy and chemotherapy is mainly adopted for treating cancer, but the clinical treatment effect is poor, the prognosis is poor, serious side effects can be caused, and the life quality of patients is greatly influenced. Among them, the anticancer drugs used lack tumor cell targeting, thereby inevitably bringing about normal cytotoxicity. In addition, clinically used anticancer drugs are often limited by problems of poor water solubility, low oral bioavailability, and the like. Conventional chemotherapeutic drugs such as gemcitabine, paclitaxel, doxorubicin, etc. cannot selectively accumulate at tumor sites and distribute throughout the body, and the clinical therapeutic effect is not good.
The traditional Chinese medicine has a long history of treating tumor-related diseases, tumors belong to 'abdominal mass accumulation' in the traditional Chinese medicine syndrome, and more than 70 medicines for treating the abdominal mass accumulation are recorded in 'Shen nong Ben Cao Jing'. The traditional Chinese medicines are various in types and rich in resources, and various traditional Chinese medicines show antitumor activity. The traditional Chinese medicine plays an important role in clinically relieving the side effects of radiotherapy and chemotherapy of patients, improving the life quality and the survival time of the patients and the like. The traditional Chinese medicine plays a role in drug effect depending on multiple components, multiple targets and overall regulation. However, most of the active ingredients of the traditional Chinese medicine have the problems of low solubility, low oral bioavailability, toxic and side effects and the like, and the active ingredients of the traditional Chinese medicine are difficult to permeate through BBB for central nervous system diseases. Therefore, a delivery system and a technical means for realizing the multi-component co-delivery of the traditional Chinese medicine, accurately delivering the traditional Chinese medicine to a diseased region, and performing the overall effect of the traditional Chinese medicine and targeted treatment of diseases need to be developed.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems, the invention aims to provide a targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle, and the invention aims to provide a preparation process of a targeting peptide modified co-delivery multi-component exosome-like fusion nanoparticle.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a targeting peptide modified traditional Chinese medicine multi-component 'exosome-like' fusion nanoparticle is mainly formed by fusing a functionalized liposome and an exosome, wherein the functionalized liposome mainly comprises targeting peptide, phospholipid and a traditional Chinese medicine liposoluble active drug, and the exosome is loaded with a traditional Chinese medicine water-soluble active drug.
Preferably, the traditional Chinese medicine fat-soluble active medicine is selected from one or more of traditional Chinese medicines of red sage root (cryptotanshinone, tanshinone IIA, dihydrotanshinone I, other tanshinone), triptolide, tripdiolide, other terpenes and alkaloids, coptis root (berberine, coptisine, other alkaloids and flavonoids) or fat-soluble active components in other traditional Chinese medicines, or is selected from one or more fat-soluble active components in a plurality of traditional Chinese medicines, but is not limited to the medicines; the traditional Chinese medicine water-soluble active medicine is selected from one or more of traditional Chinese medicines of salvia miltiorrhiza (salvianolic acid B, tanshinol and other phenolic acids), ginseng (ginsenoside Rg3, ginsenoside Rh2 and other saponins), angelica (polysaccharides) or water-soluble active components in other traditional Chinese medicines, or is selected from one or more of water-soluble active components in a plurality of traditional Chinese medicines, but is not limited to the medicines.
When the active medicine is applied to anti-tumor, the active medicine is distinguished from a mechanism angle, and the traditional Chinese medicine fat-soluble active medicine is one or more of a medicine for inhibiting tumor cell proliferation, a medicine for inducing tumor cell apoptosis, a medicine for causing tumor cell autophagy and a medicine for inhibiting tumor cell invasion and migration; the Chinese medicinal water-soluble active medicine is one or more of medicine for inhibiting tumor neovascularization, medicine for resisting tumor cell proliferation, medicine for retarding tumor cell growth cycle and medicine for inducing tumor cell apoptosis.
Preferably, the targeting peptide is selected from one or more of homing peptide tLyp-1, RGD, iRGD and angiopep-2, but is not limited to the substances; the phospholipid is selected from one or more of DOPC, DOPS, DOPE, DMPC and DMPE, but is not limited to the substances; the exosome is selected from one or more of blood source exosome, cell source exosome and urine source exosome, but is not limited to these sources.
Preferably, the particle size of the targeting peptide modified traditional Chinese medicine multi-component "exosome-like" fusion nanoparticle is 120-140 nm.
The invention also provides a preparation process of the targeting peptide modified multi-component exosome-like fusion nanoparticle of the traditional Chinese medicine, which mainly comprises the following steps: polyethylene glycol methods, ultrasonication methods, freeze-thaw methods, extrusion methods, and incubation methods. Comprises preparing target peptide modified liposome by using target peptide, phospholipid and traditional Chinese medicine fat-soluble active medicine as raw materials; loading the exosome with a traditional Chinese medicine water-soluble active drug, and then fusing the exosome with the targeting peptide modified liposome to obtain the target peptide modified liposome.
Preferably, the preparation method of the targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle comprises the following steps:
(1) preparing organic solution A of targeting peptide, phospholipid and traditional Chinese medicine fat-soluble active medicine and phosphate buffer solution B;
(2) dropwise adding the solution A into the solution B, emulsifying, removing the organic solvent by rotary evaporation after the emulsification is finished, and carrying out ultrasonic crushing to obtain the drug-loaded functionalized liposome;
(3) extracting and separating to obtain exosome;
(4) adding the traditional Chinese medicine water-soluble active drug and the electroporation medium solution into the exosome obtained in the step (3), performing electric shock, and after the electric shock is finished, incubating to obtain a drug-loaded exosome;
(5) mixing the drug-loaded functionalized liposome obtained in the step (2) with the drug-loaded exosome obtained in the step (4), and performing polyethylene glycol induction, ultrasonic crushing, repeated freeze thawing, extrusion or incubation;
(6) removing free drug by membrane.
Further preferred is:
in the step (1), when the mass of the solid is mg, the mass of the liquid is counted by mL, and the target peptide is 1-2 parts, the phospholipid is 3-7 parts, and the traditional Chinese medicine fat-soluble active medicine is 1 part.
In the step (2), the emulsifying time is 30-40 min, and the ultrasonic crushing time is 5-15 min.
In the step (3), the exosome is extracted and separated by adopting a mode of combining an iodixanol density gradient centrifugation method and an ultracentrifugation method.
In the step (4), the mass ratio of the exosome to the traditional Chinese medicine water-soluble active drug is 5 (2-5).
In the step (5), the liposome is calculated by the total amount of phospholipid, the exosome is calculated by the content of protein, and the mass ratio of the drug-loaded functionalized liposome to the drug-loaded exosome is 1: (1-4), wherein the ultrasonication time is 5-10 min.
The invention finally provides application of the targeting peptide modified traditional Chinese medicine multi-component 'exosome-like' fusion nanoparticle in preparation of medicines for treating tumors or neurodegenerative diseases. When the nanoparticle is applied, the nanoparticle can be dissolved by adding normal saline, phosphate buffer solution or 5% glucose solution and is administered by intravenous injection, intramuscular injection or oral administration, the bioavailability of the loaded active drug can be remarkably improved, combined administration can be realized, particularly multi-component combined delivery of the traditional Chinese medicine can be realized, the effects of multiple components, multiple targets and overall regulation of the traditional Chinese medicine can be fully exerted, and the nanoparticle has the curative effect of resisting tumors or treating neurodegenerative diseases.
The invention utilizes exosome to load traditional Chinese medicine water-soluble active components, utilizes liposome to load traditional Chinese medicine fat-soluble active components, constructs the fusion nanoparticles loading two types of medicine active components in a membrane fusion mode, and fully utilizes the hydrophilic and hydrophobic space of the 'exosome-like' fusion nanoparticles.
The targeted peptide modified traditional Chinese medicine multi-component 'exosome-like' fusion nanoparticle is prepared by an ultrasonic crushing method, and the nanoparticles successfully load drugs to permeate a biological barrier by utilizing endogenous exosomes and characteristic protein receptors on the surfaces of the endogenous exosomes; the drug-loaded fusion nanoparticle lesion site is endowed with specific targeting by utilizing the targeting peptide with surface modification; the passive targeting of the nanoparticles also improves the permeability of the active drug in the affected area. The obtained targeting peptide modified Chinese medicinal multi-component exosome-like fusion nanoparticle can increase the accumulation of medicaments in pathological cells, improve the targeting property of the medicaments, effectively reduce the toxicity of the medicaments and improve the treatment effect of diseases.
The invention utilizes the targeting peptide modified functionalized liposome, the exosome rich in specific protein and the traditional Chinese medicine active multi-component, and prepares the targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle by methods such as an ultrasonic crushing method, and the like, thereby effectively improving the problems of low drug loading capacity, weak targeting penetration, limited drug bioavailability, single treatment mechanism and the like of a single carrier. It has the following advantages:
(1) high endogenous: the exosome from natural sources is fused with the functionalized liposome, and the obtained 'exosome-like' fusion nanoparticle reserves the endogenous physiological characteristics of the exosome and has high endogenous property.
(2) High penetrability: the nano-grade particle size has passive targeting property and is easy to diffuse from the inside of a blood vessel to the outside of the blood vessel; endogenous exosomes and specific protein receptors they possess enable penetration of physiological barriers; the modification of the targeting peptide further enhances the penetration ability of cells and tissues thereof;
(3) multi-component co-delivery: the exosome is used for loading the active ingredients of the traditional Chinese medicine hydrophilic medicine, the liposome is used for loading the active ingredients of the traditional Chinese medicine fat-soluble medicine, and the fusion nanoparticles which are used for loading the two active ingredients of the traditional Chinese medicine are constructed in a membrane fusion mode, so that the multi-component combined delivery of the traditional Chinese medicine can be realized, and the overall effect of the traditional Chinese medicine can be favorably exerted.
(4) Biological target regulation: the exosome-like fusion nanoparticle reserves a specific protein receptor of an exosome and can be combined with a pathological cell surface receptor; the modified targeting peptide targets cells at a pathological part and other high-expression receptors, and has biological targeting regulation and control capability. Meanwhile, after the exosome and the functionalized liposome are fused, the exosome and the functionalized liposome have synergistic effect, and the targeting property can be further remarkably improved.
(5) High biological safety: the obtained 'exosome-like' fusion nanoparticle has good biocompatibility, biodegradability and low toxic and side effects.
(6) The treatment mechanism is complementary: the co-supported multi-component fusion nanoparticle constructed in a mode of fusing exosomes and liposome membranes can realize the complementation of combined administration and treatment mechanisms.
(7) Diversification of treatment means: the capacity of treating diseases or diagnosing diseases by the 'exosome-like' fusion nanoparticle is realized by modifying different targeting peptides, loading different active pharmaceutical ingredients or other diagnosis and monitoring ingredients.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the preparation method adopts methods such as an ultrasonic crushing method, a polyethylene glycol induction method, a freeze-thaw method and the like to prepare the exosome-like fusion nanoparticles, and has the advantages of simple method and low cost;
(2) the targeting peptide modified 'exosome-like' fusion nanoparticle provided by the invention can penetrate a physiological barrier, is specifically combined with tumor cells and other high-expression receptors, and has active targeting property; the nanoparticles retain the physiological characteristics of endogenous exosomes and have high endogenous and biological safety;
(3) the targeting peptide modified traditional Chinese medicine multi-component 'exosome-like' fusion nanoparticle provided by the invention can achieve the purposes of combined administration and synergistic accurate treatment by loading active drugs with different treatment mechanisms, and provides a new design idea for multi-drug delivery and multi-path disease treatment.
(4) The targeting peptide modified traditional Chinese medicine multi-component 'exosome-like' fusion nanoparticle provided by the invention has the advantages that traditional Chinese medicine hydrophilic active components are loaded through endogenous exosomes, traditional Chinese medicine lipophilic active components are loaded through functionalized liposomes, the traditional Chinese medicine multi-component co-delivery can be realized, the effects of multiple components, multiple targets and overall regulation of traditional Chinese medicines are favorably exerted, and design ideas and help are provided for the traditional Chinese medicine multi-component co-delivery and the modernization and internationalization of traditional Chinese medicines.
The targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle provided by the invention can complete in-vivo co-delivery of single or multiple traditional Chinese medicine anti-tumor active ingredients (such as cryptotanshinone, triptolide, berberine and the like) or tumor drugs comprising traditional Chinese medicine active multi-components and a diagnosis monitoring reagent, and the nano preparation has the characteristics of high endogenesis, high penetrability, various drug-carrying types, traditional Chinese medicine multi-component co-delivery, biological targeting regulation, high biological safety, complementary treatment mechanism, diversified treatment means and the like, successfully constructs a bionic exosome-like fusion nano delivery system for multi-drug co-delivery, provides a paradigm for efficient targeting treatment of tumors and other neurodegenerative diseases, traditional Chinese medicine multi-component co-delivery and construction of the multi-drug co-delivery system, and has wide application prospects and clinical transformation potentials.
Drawings
FIG. 1 is a "exosome-like" fusion nanoparticle membrane fusion study of targeted peptide modifications in example four 1.1, including fluorescence energy resonance transfer (FRET) fluorescence spectra (A); cellular uptake of FRET nanoparticles pattern (B); nanoparticle potential map (C);
fig. 2 is the form of targeting peptide modified multicomponent "exosome-like" fusion nanoparticles of the traditional Chinese medicine in example four 1.2;
FIG. 3 shows the expression of the surface characteristic protein of targeting peptide-modified multicomponent exosome-like fusion nanoparticle of the traditional Chinese medicine in example four 1.3
Fig. 4 is a co-localization situation of targeting peptide modified multicomponent of traditional Chinese medicine "exosome-like" fusion nanoparticles and cells in example four 1.4;
FIG. 5 is a study of apoptosis of multi-component "exosome-like" fusion nanoparticles of the targeting peptide-modified traditional Chinese medicine in example four 1.5;
fig. 6 is a cytotoxicity investigation of targeting peptide modified traditional Chinese medicine multicomponent "exosome-like" fusion nanoparticles in example four 1.6.
Fig. 7 is a tumor sphere penetration capability examination of targeting peptide modified multi-component "exosome-like" fusion nanoparticles of the traditional Chinese medicine in example four 1.8.
Detailed Description
The invention is further illustrated by the following examples. These examples are purely illustrative and they are intended to describe the invention in detail only and should not be interpreted as limiting the invention. The invention is further described with reference to the following figures and examples:
the first embodiment is as follows: extracting and separating in serum to obtain exosome (Exo)
Obtaining blood of a rat by taking blood from an abdominal aorta, standing for 30min, and centrifuging for 12min at 1600 Xg to obtain serum; centrifuging serum at 2000 Xg for 12min, and collecting supernatant; centrifuging the supernatant at 12000 Xg for 30min, collecting supernatant, and filtering with 0.22 μm water system microporous membrane to obtain fresh serum. Adding 50% iodixanol solution, 30% iodixanol solution, 10% iodixanol solution and serum into a centrifugal tube in sequence, centrifuging at 27000rpm and 4 ℃ for 150min, wherein an exosome density layer is positioned between 10% and 30% iodixanol solution layers, sucking out the exosome density layer to an ultracentrifugal tube, adding PBS solution into the ultracentrifugal tube, centrifuging at 40000rpm and 4 ℃ for 80min, and adding the PBS solution into the obtained precipitate for heavy suspension to obtain an exosome (Exo) solution.
Example two: preparation process of Cryptotanshinone (CPT) -entrapped functionalized liposome (tLipo/CPT)
Collecting 670 μ g (850nmol) of DOPC phospholipid, 300 μ g (370nmol) of DOPS phospholipid, and DSPE-PEG2000230 mu g (61nmol) of tLyp-1 targeting peptide and 100 mu g (337nmol) of CPT powder are dissolved in 1mL of chloroform, the chloroform is fully dissolved, the solution is dropwise added into 2mLPBS buffer solution and stirred while being dropwise added, the stirring is carried out for 30min, the organic solvent is removed by rotary evaporation, the solution is transferred into a penicillin bottle, 195W is carried out, and the ice water bath is carried out ultrasonic crushing for 5min, thus obtaining tLipo/CPT.
Example three: preparation process of exosome (Exo/SAB) containing Salvianolic Acid B (SAB)
1mg of SAB powder is taken and added with 21 percent iodixanol PBS solution to prepare 1mg/mL of SAB solution. 100 mu g of exosome Exo calculated by the amount of protein is taken, 40 mu L (40 mu g) of SAB solution is added, the mixture is fully mixed, and 21% iodixanol PBS solution is added to make up the total volume to 200 mu L. Electroporation was performed at 1000V, 0.5ms, 2 pulses. And after the electroporation is finished, incubating at the constant temperature of 37 ℃ for 1h to promote the closure of an Exo membrane, thus obtaining the Exo/SAB.
Example four: target peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle property investigation
1.1 Targeted peptide-modified multicomponent Chinese medicine exosome-like fusion nanoparticle membrane fusion investigation
The membrane fusion of Exo/SAB and tLipo/CPT is induced by adopting an ultrasonic disruption method, and the specific operation is that Exo/SAB (obtained in the third embodiment) with the protein content of 100 mu g is mixed with tLipo/CPT (obtained in the second embodiment) with the phospholipid total content of 100 mu g, the power is 195W, the mixture is ultrasonically disrupted for 5min on an ice water bath, the mixture is incubated for 1h at the constant temperature of 37 ℃ after the ultrasonic disruption is finished, the closure of a fusion nanoparticle membrane is promoted, and the mixture passes through a 0.8 mu m filter membrane after the incubation is finished, so that the 'exosome-like' fusion nanoparticle Hybrid/SAB-CPT with the CPT and the SAB being carried together is obtained.
1.1.1FRET fluorescence Spectroscopy scanning to verify fusion occurred
And (2) dissolving 670 mu g of DOPC phospholipid, 300 mu g of DOPS phospholipid, 400 mu g of NBD-DMPE phospholipid and 400 mu g of Rho-DMPE phospholipid in 1mL of chloroform, fully dissolving, dropwise adding into 2mL of PBS buffer solution while stirring, stirring for 30min, rotationally evaporating to remove the organic solvent, transferring the solution into a penicillin bottle, 195W, and ultrasonically crushing in an ice water bath for 5min to obtain NBD/Rho-Lipo.
Exo and NBD/Rho-Lipo were induced to undergo membrane fusion by ultrasonication. Respectively taking 100 mu g, 50 mu g and 25 mu g of Exo based on the amount of protein, taking 100 mu g of NBD/Rho-Lipo based on the total amount of phospholipid, mixing, performing ultrasonic crushing on an ice water bath for 5min, performing constant temperature incubation for 1h at 37 ℃ after the ultrasonic crushing is finished, and filtering through a 0.8 mu m filter membrane after the incubation is finished to obtain the fusion nanoparticle NBD/Rho-Hybrid fused with Exo and NBD/Rho-Lipo in different proportions.
The fluorescence spectrophotometer is used for setting the excitation wavelength of the NBD fluorescent molecule to be 460nm, and the fluorescence curve of the fused nanoparticle NBD/Rho-Hybrid in the wavelength range of 450-700nm is scanned. As a result, as shown in the fluorescence spectrum curve of NBD/Rho-Lipo in FIG. 1A, when the excitation wavelength of the donor molecule NBD is set to 460nm to excite the NBD molecule, which generates fluorescence at 530nm and is capable of exciting Rhodamin B to generate an emission wavelength of 580nm, a FRET phenomenon exists. When membrane fusion occurs with different ratios of Exo and NBD/Rho-Lipo, because the phospholipid bilayer of NBD/Rho-Lipo is diluted by Exo membrane insertion, the spatial distance between the donor fluorescent molecule NBD and the acceptor fluorescent molecule Rhodamine B in the phospholipid bilayer is increased, and the FRET efficiency is weakened, so that the peak intensity of the fused nanoparticle at 530nm is increased and the peak intensity at 580nm is reduced after fusion of Exo and NBD/Rho-Lipo with different ratios, which indicates the occurrence of Exo and NBD/Rho-Lipo membrane fusion. When the ratio of Exo to NBD/Rho-Lipo is 1:1, the amount of the added Exo is the largest, and the NBD/Rho-Lipo phospholipid membrane is more diluted, so that the peak intensity of the fluorescence curve of Exo: NBD/Rho-Lipo ═ 1:1 is the largest at 530nm and the peak intensity is the largest at 580nm, which indicates that the fusion of Exo and NBD/Rho-Lipo membranes occurs the highest efficiency.
1.1.2 cellular uptake of FRET fusion nanoparticles
Dissolving 670 mu g of DOPC phospholipid and 300 mu g of DOPS phospholipid in 1mL of chloroform, adding 10 mu L of 10mM DiO dye solution and 10 mu L of 10mM DiI dye solution, fully mixing uniformly, dropwise adding into 2mL of PBS buffer solution while stirring, stirring for 30min, removing the organic solvent by rotary evaporation, transferring the solution into a penicillin bottle, 195W, and ultrasonically crushing in ice water bath for 5min to obtain Lipo/DiI-DiO.
Exo and Lipo/DiI-DiO were induced to undergo membrane fusion by ultrasonication. Taking 100 mu g of Exo based on the amount of protein, taking 100 mu g of Lipo/DiI-DiO based on the total amount of phospholipid, mixing, performing ultrasonic crushing on an ice water bath for 5min at the power of 195W, performing constant-temperature incubation for 1h at 37 ℃, and after the incubation is finished, passing through a 0.8 mu m filter membrane to obtain the fusion nanoparticle Hybrid/DiI-DiO fused with Exo and Lipo/DiI-DiO.
Taking logarithmic growth glioma U87 cell, 1 × 104Inoculating the cells in 12-well plate at 37 deg.C and 5% CO2Culturing until the cells adhere to the wall. After the cells are attached to the wall, single culture solution containing Hybrid/DiI-DiO and Lipo/DiI-DiO is respectively added for culturing for 12 h. After 12h, taking out the pore plate, sucking out the culture solution, adding 1mLPBS for cleaning twice, adding 4% paraformaldehyde solution for fixing cells for 20min, sucking out paraformaldehyde after fixing, adding 1mLPBS solution for cleaning twice, dropwise adding an anti-fluorescence quenching sealing tablet containing DAPI for sealing, and then measuring the FRET efficiency of the fusion nanoparticle Hybrid/DiI-DiO under a confocal microscope.
After the cells take the FRET fluorescent pair labeled nanoparticles, yellow fluorescent distribution after superposition of DiO green and DiI red fluorescent signals is displayed in cytoplasm and is distribution of Hybrid/DiI-DiO or Lipo/DiI-DiO. Adjusting a confocal shooting mode to a FRET mode, bleaching DiI fluorescence during shooting, and obtaining FRET efficiency E according to fluorescence intensity of DiO before and after bleaching, wherein the calculation formula of E is as follows:
E=(Ipost-Ipre)/Ipost
wherein, IpostRefers to the fluorescence intensity of DiO after bleaching, IpreRefers to the fluorescence intensity of DiO before bleaching.
Results as shown in fig. 1B, Lipo/DiI-DiO, which was not membrane-fused with Exo, had a FRET efficiency of 30.86% due to FRET fluorescence in the presence of DiO and DiI; when the fusion nanoparticle Hybrid/DiI-DiO is subjected to membrane fusion with Exo, the FRET efficiency of the fusion nanoparticle Hybrid/DiI-DiO is 3.24 percent and is greatly reduced, which indicates that the Exo and Hybrid/DiI-DiO membrane fusion occurs.
1.1.3 potential Change of fused nanoparticles
The fused nanoparticles NBD/Rho-Hybrid obtained by fusing different Exo obtained in example four 1.1.1 with NBD/Rho-Lipo proportional membranes were subjected to nanoparticle potential measurement, and the results are shown in fig. 1C, where the absolute value of the potential of the fused nanoparticles is between Exo alone and NBD/Rho-Lipo alone, and the occurrence of Exo fusion with NBD/Rho-Lipo membranes was also laterally verified to some extent.
1.2Hybrid/SAB-CPT morphology
The Hybrid/SAB-CPT nanoparticles obtained in example four 1.1 were taken and placed under a transmission electron microscope by a negative staining method to observe the surface morphology of the fused nanoparticles, as shown in FIG. 2.
1.3 expression of Hybrid/SAB-CPT surface characteristic protein
Total protein content was determined by BCA protein concentration assay using the Hybrid/SAB-CPT nanoparticles obtained in example four 1.1. Mixing Hybrid/SAB-CPT solution and RIPA lysate with equal volume, reacting on ice for 15min, taking 10 μ L of reaction solution, adding 10 μ L of LPBS solution, mixing, adding 200 μ L of LBCA working solution, oscillating at 37 ℃ for 30min, measuring absorbance value at 562nm wavelength, and calculating protein concentration according to standard.
Taking 1 mu g of Hybrid/SAB-CPT based on the amount of protein, sequentially adding primary anti-FITC-CD 9, PE-CD81 and PE/Cy7-CD71(TfR) solutions with fluorescent labels, adding 500 mu L of binding solution, and incubating at room temperature for 20 min; and another part of Hybrid/SAB-CPT is taken, corresponding fluorescence labeled isotype control antibody solutions FITC-ctl, PE-ctl and PE/Cy7-ctl are sequentially added, after uniform mixing, 500 mu L of binding solution is added, incubation is carried out for 20min at room temperature, and the protein expression condition is detected by using a flow cytometer.
The results are shown in FIG. 3, CD9, CD81 and CD71 in the Hybrid/SAB-CPT are all expressed positively, which indicates that the fusion nanoparticle Hybrid/SAB-CPT retains the characteristic membrane protein on the surface of Exo.
1.4Hybrid/SAB-CPT and cell Co-localization Studies
Taking DSPE-PEG2000Adding 1mg of-tLyp-1 targeting peptide into chloroform to prepare a 1mg/mL targeting peptide solution, adding 5 mu L of Cy5 dye solution with the concentration of 10mM, mixing uniformly, incubating overnight at 4 ℃ in a dark place, and filtering with a 0.8 mu m filter membrane to obtain Cy5 labeled targeting peptide Cy5-DSPE-PEG2000-tLyp-1。
Collecting 670 μ g DOPC phospholipid, 300 μ g DOPS phospholipid, and Cy5-DSPE-PEG2000Dissolving 230 mu g of-tLyp-1 targeting peptide in 1mL of chloroform, adding 10 mu L of 1mg/mL coumarin 6(C6) solution, fully and uniformly mixing, dropwise adding into 2mLPBS buffer solution while stirring, stirring for 30min, rotationally evaporating to remove the organic solvent, transferring the solution into a penicillin bottle, 195W, and ultrasonically crushing in an ice-water bath for 5min to obtain Cy 5-tLipo/C6.
And taking 100 mu g of Exo based on the amount of the protein, adding 10 mu L of DiI dye solution with the concentration of 10mM, uniformly mixing, incubating at the constant temperature of 37 ℃ for 1h, and filtering through a 0.8 mu m filter membrane after the incubation is finished to obtain the DiI-Exo.
And (3) taking 100 mu g of DiI-Exo in terms of the amount of protein, taking 100 mu g of Cy5-tLipo/C6 in terms of the amount of total phospholipid, uniformly mixing, performing ultrasonic crushing on an ice water bath for 5min at a power of 195W, performing constant-temperature incubation for 1h at 37 ℃ after the ultrasonic crushing is finished, and filtering through a 0.8 mu m filter membrane after the incubation is finished to obtain the fluorescence labeled fusion nanoparticle DiI/Cy 5-Hybrid/C6.
Taking glioma U87 cell of logarithmic growth, 1 × 104Inoculating the cells in 12-well plate at 37 deg.C and 5% CO2Culturing until the cells adhere to the wall. After the cells were attached, a single culture solution containing DiI/Cy5-Hybrid/C6 was added for culture for 4 h. Taking out the pore plate, removing the culture solution by suction, adding 1mLPBS for cleaning twice, adding 4% paraformaldehyde solution for fixing cells for 20min, removing paraformaldehyde by suction after the fixation is finished, adding 1mLPBS solution for cleaning twice, dropwise adding an anti-fluorescence quenching sealing tablet containing DAPI for sealing, and observing under a confocal microscope.
The results are shown in fig. 4, and the fluorescence signals of purple Cy5, red DiI and green C6 of the fusion nanoparticle DiI/Cy5-Hybrid/C6 are highly shown in the cytoplasm of U87, indicating that U87 cells can efficiently take up the fusion nanoparticle. Also, the high coincidence of the three fluorescence signals indicates the occurrence of fusion of Exo with tLipo/C6.
1.5Hybrid/SAB-CPT induced apoptosis Studies
Taking glioma U87 cell of logarithmic growth, 1 × 105The cells were seeded at a density of 12-well plates at 37 ℃ in 5% CO2Culturing until the cells adhere to the wall.
The contents of the SAB and CPT in the tLipo/CPT and Hybrid/SAB-CPT samples obtained in example four 1.1 were measured by High Performance Liquid Chromatography (HPLC). 1mg of SAB powder was added with PBS to prepare a 1mg/mL (1.4mM) solution of SAB, and 1.8mg of CPT powder was added with anhydrous DMSO to prepare a 2mg/mL (8mM) solution of CPT. A blank control group, a Free drug Free CPT group, a two-drug combined Free SAB + CPT group, a CPT liposome tLipo/CPT group and a co-carried two-drug fusion nanoparticle Hybrid/SAB-CPT group are arranged, and according to the HPLC measurement result, single culture is respectively added to prepare each drug-containing culture medium with the SAB concentration of 20 mu M and the CPT concentration of 16 mu M.
After the U87 cells adhered to the wall, the well plate was removed, the culture medium was aspirated, 1mL of drug-containing culture medium containing different drug groups was administered to each well at 37 ℃ with 5% CO2And culturing for 24 h. After 24h, taking out the pore plate, sucking out liquid medicine of each pore, storing in a centrifuge tube, adding PBS for washing once per pore, adding 200 mu L of pancreatin without EDTA for digestion, adding 1mL of full culture to stop digestion, uniformly mixing with the sucked corresponding liquid medicine, 1500rpm, centrifuging for 5min, discarding supernatant, adding PBS for heavy suspension, washing once, 1500rpm, centrifuging for 5min, discarding supernatant, taking a tube of blank cells, adding 0.5mL of positive liquid medicine for uniform mixing, reacting at room temperature for 30min, taking another tube of blank cells, adding 0.5mL of binding liquid for heavy suspension, mixing with positive drug tube cells, passing through a 300-mesh nylon net, uniformly dividing to two tubes, performing AnnexinV FITC single staining on one tube, and performing PI single staining on one tube. And adding 0.5mL of binding solution into the other tubes for resuspension, sieving by a 300-mesh nylon net, adding annexin V FITC and PI for double staining, and performing flow cytometry detection.
As shown in FIG. 5, the apoptosis rate of the Hybrid/SAB-CPT group was 13.09. + -. 0.75% compared to that of the other groups, which was 3.67-fold higher than that of the Free CPT group, 2.71-fold higher than that of the Free SAB + CPT group, and 3.53-fold higher than that of the tLipo/CPT group, and it was confirmed that the Hybrid/SAB-CPT group had the strongest ability to induce apoptosis in U87 cells.
1.6 cytotoxic Effect of Hybrid/SAB-CPT on cells
Taking glioma U87 cell of logarithmic growth, 1 × 104The cells were seeded at a density of 5% CO at 37 ℃ in 96-well plates2Culturing until the cells adhere to the wall.
The content of SAB and CPT was measured by HPLC using tLipo/CPT and Hybrid/SAB-CPT obtained in example IV 1.1, and SAB powder (1 mg) was prepared as a 1mg/mL (1.4mM) SAB solution in PBS, and CPT powder (1.8 mg) was prepared as a 2mg/mL (8mM) CPT solution in anhydrous DMSO. A blank control group, a Free drug Free CPT group, a two-drug combined Free SAB + CPT group, a CPT liposome tLipo/CPT group and a co-carried two-drug fusion nanoparticle Hybrid/SAB-CPT group are arranged, and according to the HPLC measurement result, single culture is respectively added to prepare each drug-containing culture medium with the SAB concentration of 20 mu M and the CPT concentration of 16 mu M.
After the U87 cells adhere to the wall, the well plate is taken out, the culture solution is aspirated, 100 mu L of drug-containing culture solution containing different drug groups is given to each well, the temperature is 37 ℃, and the CO content is 5 percent2Culturing for 12 h. After 12h, taking out the pore plate, absorbing the liquid medicine in each pore, adding 100 mu L of Calcein AM/EthD-1 fluorescence working solution (10 mu LEthD-1 and 2.5 mu LCalcein AM staining solution are added into 5mLPBS for mixing, and then the mixture is ready for use), incubating for 30min at room temperature, and observing the survival condition of each group of cells under an inverted fluorescence microscope.
The result is shown in fig. 6, the Hybrid/SAB-CPT group can obviously inhibit the survival of U87 cells compared with other groups, and the survival cells are spherical in shape and lose star antenna shape, thus proving that the fusion nanoparticle Hybrid/SAB-CPT has the strongest effect of resisting glioma U87.
1.7 investigation of cell proliferation inhibitory Capacity of Hybrid/SAB-CPT
The content of SAB and CPT was measured by HPLC using tLipo/CPT and Hybrid/SAB-CPT obtained in example IV 1.1, and 1.5mg of SAB powder was prepared as a 1mg/mL (1.4mM) SAB solution in PBS, 2.7mg of CPT powder was prepared as a 2mg/mL (8mM) CPT solution in anhydrous DMSO. Free drug Free CPT, two-drug combination Free SAB + CPT, CPT liposome tLipo/CPT and co-carried two-drug fusion nanoparticle Hybrid/SAB-CPT groups are arranged, and according to HPLC determination results, single culture is respectively added to prepare gradient concentration drug-containing culture media containing different concentrations of SAB and CPT.
Taking glioma U87 cell of logarithmic growth, 5X 103The cells were seeded at a density of 5% CO at 37 ℃ in 96-well plates2Culturing until the cells adhere to the wall. Taking out the well plate, removing culture medium by suction, adding 100 μ L of gradient concentration drug-containing culture medium into each well, 37 deg.C, and 5% CO2And culturing for 24 h. After 24h, the well plates were removed and 10 μ LCCK8 solution was added to each well at 37 ℃ with 5% CO2After incubation for 1.5h, the cells were removedTaking out of the well plate, measuring absorbance of each well at 450nm wavelength, and calculating half inhibitory concentration IC of each group of drugs50The value is obtained.
Results are shown in Table 1, IC for Free CPT5021.76 mu M, after CPT is loaded by utilizing liposome and fusion nanoparticles, the IC of the drug CPT is reduced by both tLipo/CPT and Hybrid/SAB-CPT50Respectively reduced to 17.92. mu.M and 8.36. mu.M, wherein IC of CPT of Hybrid/SAB-CPT group50The value is reduced by half compared with the tLipo/CPT group, which indicates that Hybrid/SAB-CPT has stronger capability of inhibiting the proliferation of U87 cells. In addition, IC of Free SAB + CPT from mixed Free CPT and SAB treated U87 cells50IC at 21.83. mu.M, SAB50IC value of 54.56. mu.M, and Hybrid/SAB-CPT group CPT50IC at 8.36. mu.M, SAB50The value is 20.91 mu M, the IC50 of the two drugs is obviously reduced by the fused nanoparticles, which shows that the Hybrid/SAB-CPT can obviously inhibit the proliferation of U87 cells and shows stronger tumor cell proliferation inhibition capability.
TABLE 1 IC of Free CPT, Free SAB + CPT, tLipo/CPT and Hybrid/SAB-CPT on U87 cells50
Figure BDA0003306396070000121
1.8Hybrid/SAB-CPT tumor sphere penetration Capacity study
Labeling Exo with a fluorescent dye DiI to obtain DiI-labeled exosome DiI-Exo: and taking 100 mu g of Exo based on the amount of the protein, adding 10 mu L of DiI dye solution with the concentration of 10mM, uniformly mixing, incubating at the constant temperature of 37 ℃ for 1h, and filtering through a 0.8 mu m filter membrane after the incubation is finished to obtain the DiI-Exo. Labeling tLipo with fluorescent dye DiD to obtain DiD labeled liposome tLipo/DiD: collecting 670 μ g DOPC phospholipid, 300 μ g DOPS phospholipid, and DSPE-PEG2000230 mu g of-tLyp-1 targeting peptide is dissolved in 1mL of chloroform, 10 mu L of DiD dye solution with the concentration of 5mM is added, the mixture is fully and uniformly mixed, the mixture is dropwise added into 2mLPBS buffer solution and stirred while being dropwise added, the stirring is carried out for 30min, the organic solvent is removed by rotary evaporation, the solution is transferred into a penicillin bottle, 195W is carried out, and the mixture is ultrasonically crushed for 5min in ice water bath, thus obtaining tLipo/DiD. Taking 100 mu g of DiI-Exo in terms of the amount of protein, and taking tLipo/DiD in terms of total phosphorusMeasuring the amount of the lipid by 100 mu g, uniformly mixing, carrying out ultrasonic crushing on an ice water bath for 5min at the power of 195W, carrying out constant-temperature incubation for 1h at the temperature of 37 ℃ after the ultrasonic crushing is finished, and filtering through a 0.8 mu m filter membrane after the incubation is finished to obtain the fluorescence labeled fusion nanoparticle DiI-Hybrid/DiD.
Taking U87 cells in logarithmic growth phase, 1X 104The cells were seeded on 2% agarose gel at 37 ℃ in 5% CO2Culturing until the cells are spherical. After the formation of U87 tumor cells, the well plate was removed, the tumor cells were aspirated, and 100. mu.L of each of the drug-containing culture solutions containing DiI-Exo, tLipo/DiD and DiI-Hybrid/DiD was administered at 37 ℃ with 5% CO2Culturing for 12 h. After 12h, the well plate was removed, the culture medium was aspirated, PBS was added for washing, and U87 tumor balls were aspirated and placed in confocal imaging.
The results are shown in fig. 7, compared with DiI-Exo and tLipo/DiD, the fused nanoparticle DiI-Hybrid/DiD shows stronger green DiI signal and red DiD signal with the increase of the depth of the U87 tumor sphere, indicating that the fused nanoparticle DiI-Hybrid/DiD has the strongest penetration depth, which indicates that the Hybrid/SAB-CPT can have stronger U87 tumor sphere penetration capability, and the fused nanoparticle after Exo and tLipo fusion can carry the drug to penetrate to the deep part of the tumor.

Claims (10)

1. A targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle is characterized in that the exosome-like fusion nanoparticle is mainly formed by fusing a functionalized liposome and an exosome, the functionalized liposome mainly comprises targeting peptide, phospholipid and a traditional Chinese medicine fat-soluble active drug, and the exosome is loaded with a traditional Chinese medicine water-soluble active drug.
2. The targeted peptide modified traditional Chinese medicine multi-component "exosome-like" fusion nanoparticle according to claim 1, wherein the targeted peptide is selected from one or more of homing peptide tllp-1, RGD, iRGD and angiopep-2; the phospholipid is selected from one or more of DOPC, DOPS, DOPE, DMPC and DMPE; the exosome is selected from one or more of exosomes derived from blood, cells and urine.
3. The targeted peptide modified traditional Chinese medicine multicomponent exosome-like fusion nanoparticle according to claim 1, wherein the traditional Chinese medicine liposoluble active drug is selected from one or more of tanshinone, tripterygium wilfordii and coptis chinensis; the Chinese medicinal water soluble active medicine is selected from one or more of salvianolic acids, ginsenosides, and polysaccharides.
4. The preparation method of the targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle as claimed in any one of claims 1 to 3, which is characterized by comprising the steps of preparing targeting peptide modified liposome by taking targeting peptide, phospholipid and traditional Chinese medicine liposoluble active medicine as raw materials; loading the exosome with a traditional Chinese medicine water-soluble active drug, and then fusing the exosome with the targeting peptide modified liposome to obtain the target peptide modified liposome.
5. The preparation method of the targeting peptide modified traditional Chinese medicine multicomponent exosome-like fusion nanoparticle according to claim 4, which is characterized by comprising the following steps:
(1) preparing organic solution A of targeting peptide, phospholipid and traditional Chinese medicine fat-soluble active medicine and phosphate buffer solution B;
(2) dropwise adding the solution A into the solution B, emulsifying, removing the organic solvent by rotary evaporation after the emulsification is finished, and carrying out ultrasonic crushing to obtain the drug-loaded functionalized liposome;
(3) extracting and separating to obtain exosome;
(4) adding the traditional Chinese medicine water-soluble active drug and the electroporation medium solution into the exosome obtained in the step (3), performing electric shock, and after the electric shock is finished, incubating to obtain a drug-loaded exosome;
(5) mixing the drug-loaded functionalized liposome obtained in the step (2) with the drug-loaded exosome obtained in the step (4), and performing polyethylene glycol induction, ultrasonic crushing, repeated freeze thawing, extrusion or incubation;
(6) removing free drug by membrane.
6. The preparation method of the targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle according to claim 5, wherein in the step (1), when the mass of the solid is mg, the mass of the liquid is mL, and the targeted peptide is 1-2 parts, the phospholipid is 3-7 parts, and the traditional Chinese medicine fat-soluble active drug is 1 part.
7. The preparation method of the targeting peptide modified traditional Chinese medicine multi-component exosome-like fusion nanoparticle as claimed in claim 5, wherein in the step (2), the emulsifying time is 30-40 min, and the ultrasonication time is 5-15 min.
8. The preparation method of the targeting peptide modified multi-component traditional Chinese medicine exosomal-like fusion nanoparticle according to claim 5, wherein in the step (3), the exosomal is obtained by extraction and separation in a manner of combining iodixanol density gradient centrifugation and ultracentrifugation; in the step (4), the mass ratio of the exosome to the traditional Chinese medicine water-soluble active drug is 5 (2-5).
9. The preparation method of the targeting peptide modified traditional Chinese medicine multicomponent exosome-like fusion nanoparticle according to claim 5, wherein in the step (5), the liposome is calculated by the total amount of phospholipid, the exosome is calculated by the content of protein, and the mass ratio of the drug-carrying functionalized liposome to the drug-carrying exosome is 1: (1-4), wherein the ultrasonication time is 5-10 min.
10. The use of the targeted peptide-modified traditional Chinese medicine multicomponent "exosome-like" fusion nanoparticle according to any one of claims 1 to 3 in the preparation of anti-tumor or neurodegenerative disease treatment drugs.
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