CN112569362B - Method for inhibiting tumor metastasis by breaking up CTC cell mass - Google Patents
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Abstract
The invention discloses a method for inhibiting tumor metastasis by scattering CTC cell masses, which relates to the technical field of medical treatment and comprises the steps of constructing a nano preparation based on exosome loaded ouabain, evaluating the effect of scattering CTC clusters by the nano preparation based on exosome loaded ouabain through an in-vitro experiment, and evaluating the effect of scattering CTC clusters and inhibiting tumor metastasis by the nano preparation based on exosome loaded ouabain through an in-vivo experiment; according to the invention, the tumor specific polypeptide iRGD is modified on the surface of the exosome, so that the exosome can be specifically combined with the alpha v beta 3 integrin on the surface of CTCs (CTCs), and the capacity of targeting the exosome to the CTCs is improved; the ouabain is introduced into exosomes by utilizing an electroporation method, so that the solubility of the ouabain in water is improved, the dosage form of the ouabain is improved, and the toxic and side effects of the ouabain are reduced; tumor metastasis is inhibited by using an exosome-based ouabain-loaded nano-formulation by scattering CTC cell masses in blood.
Description
Technical Field
The invention relates to the technical field of medical treatment, in particular to a method for inhibiting tumor metastasis by scattering CTC cell masses.
Background
In recent ten years, the incidence of tumors is rapidly rising, and the tumors become female malignant tumors with the first incidence worldwide, which pose serious threats to the life health of women worldwide. Tumor metastasis is one of the leading causes of death in tumor patients. It has been reported that 90% of cases of tumor death are attributable to tumor metastasis. Tumor metastasis mainly occurs in lung, bone, brain, lymph node and other parts, and lung metastasis occurs when 60-70% of tumor patients die. To date, surgical resection, chemotherapy, radiation therapy, and targeted therapy are considered the primary means of treating tumors. Early stage tumors can be treated by means of surgical resection, however, no effective treatment strategy for tumors with metastasis can obviously prolong the life of patients at present. Traditional chemotherapy drugs can inhibit the proliferation of cancer cells, but have relatively serious toxic and side effects and are easy to induce tumor resistance. Therefore, how to effectively inhibit tumor metastasis is a key problem to be solved urgently in tumor treatment.
The Daniel a. haber team at the general hospital cancer center in ma, usa found through in situ tumor bearing model experiments: lung metastases from about half of the tumors are initiated by CTC clusters. The metastatic potential of CTC clusters is 25-50 fold higher than that of single CTCs, and thus CTC clusters are key to tumor metastasis. The CTC clusters are broken up, so that the CTC clusters can lose the transfer capacity, and further the transfer and diffusion of tumor cells are inhibited, therefore, the invention provides a method for inhibiting the tumor transfer by breaking up CTC cell clusters.
Disclosure of Invention
The invention aims to provide a method for inhibiting tumor metastasis by scattering CTC cell masses, which is characterized in that tumor specific polypeptide iRGD is modified on the surface of an exosome, so that the exosome can be specifically combined with alpha v beta 3 integrin on the surface of CTCs cells, and the capacity of targeting CTCs by the exosome is improved; the ouabain is introduced into exosomes by utilizing an electroporation method, so that the solubility of the ouabain in water is improved, the dosage form of the ouabain is improved, and the toxic and side effects of the ouabain are reduced; tumor metastasis is inhibited by using an exosome-based ouabain-loaded nano-formulation by scattering CTC cell masses in blood.
In order to achieve the purpose, the invention provides the following technical scheme: a method of inhibiting tumor metastasis by breaking up a mass of CTC cells, comprising: the method comprises the steps of constructing a nano preparation based on exosome loaded ouabain, evaluating the effect of scattering CTC clusters by the nano preparation based on exosome loaded ouabain through an in vitro experiment, and evaluating the effect of scattering CTC clusters and inhibiting tumor metastasis by the nano preparation based on exosome loaded ouabain through an in vivo experiment; the construction of the exosome-loaded ouabain-based nano preparation comprises the following steps:
s1: extracting exosome from milk by adopting an exosome separation technology based on anion exchange magnetic beads;
s2: identifying and characterizing exosomes;
s3: modifying iRGD on the surface of the exosome to obtain a targeting modified exosome;
s4: evaluating the tumor targeting property of the iRGD modified exosome;
s5: electroporation introduces ouabain into exosomes.
Preferably, the in vitro experiment evaluation of the effect of scattering CTC clusters by the exosome-loaded ouabain-based nano-preparation comprises the following steps:
s1, forming a CTC cluster in vitro and representing the CTC cluster;
s2, evaluating the effect of scattering CTC clusters by the exosome-loaded ouabain-based nano preparation through an in vitro experiment.
Preferably, the in vivo experiment evaluation of the effect of the exosome-loaded ouabain-based nano-preparation in breaking up CTC clusters and inhibiting tumor metastasis comprises the following steps:
s1: constructing an in-situ tumor bearing model;
s2, evaluating the effect of the exosome-loaded ouabain-based nano preparation on scattering CTC clusters in blood;
s3, evaluating the effect of the nano preparation based on the exosome loading ouabain on inhibiting tumor metastasis.
Preferably, the tumor comprises a tumor of various parts of the human body.
Preferably, the nano-preparation comprises a nano-preparation based on exosomes, liposomes, gold nanoparticles and magnetic nanoparticles.
Preferably, the drug that breaks up the CTC cell mass comprises a Na +/K + -atpase inhibitor and a tubulin-binding agent.
Preferably, the Na +/K + -ATPase inhibitor comprises digitoxin ouabain.
Preferably, the tubulin binding agents include the group consisting of rigosertib, pradafelone, colchicine and vincristine sulfate.
Compared with the prior art, the invention has the beneficial effects that:
1. according to the invention, the tumor specific polypeptide iRGD is modified on the surface of the exosome, so that the exosome can be specifically combined with the alpha v beta 3 integrin on the surface of CTCs (CTCs), and the capacity of targeting the exosome to the CTCs is improved;
2. the invention introduces the ouabain into exosomes by utilizing an electroporation method, which is beneficial to improving the solubility of the ouabain in water, improving the dosage form of the ouabain and reducing the toxic and side effects of the ouabain;
3. the invention inhibits tumor metastasis by scattering CTC cell mass in blood by applying the exosome-based ouabain-loaded nano preparation.
Drawings
FIG. 1 is a flow chart of a method of the present invention for inhibiting tumor metastasis by breaking up CTC cell masses;
FIG. 2 is a diagram showing the steps of anion exchange magnetic beads separating exosomes by a method of inhibiting tumor metastasis by breaking up CTC cell masses according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Referring to fig. 1-2, an embodiment of the present invention is shown: a method for inhibiting tumor metastasis by scattering CTC cell masses comprises constructing a nano preparation based on exosome-loaded ouabain, evaluating the effect of scattering CTC clusters by the nano preparation based on exosome-loaded ouabain through in vitro experiments, and evaluating the effect of scattering CTC clusters and inhibiting tumor metastasis by the nano preparation based on exosome-loaded ouabain through in vivo experiments; the construction of the exosome-loaded ouabain-based nano preparation comprises the following steps:
s1: extracting exosomes from a cell culture medium by using an exosome separation technology based on anion exchange magnetic beads;
the invention adopts the independently developed exosome separation technology based on anion exchange magnetic beads to separate exosomes from a cell culture medium. According to the difference of the charge property and the charge quantity of the surface of the exosome and impurities such as protein, nucleic acid, lipid and the like, the exosome is separated from the impurities such as the protein, the nucleic acid, the lipid and cell debris, and thus the exosome with high purity is obtained. The specific operation is shown in fig. 2: firstly, incubating the anion exchange magnetic beads and a fresh cell culture medium to ensure that the anion exchange magnetic beads with positive electricity are fully combined with the exosomes with negative electricity. The positively or uncharged impurities such as proteins, nucleic acids, lipids and cell debris are then washed away with a low concentration of saline solution. And finally, eluting with a high-concentration NaCl solution, wherein the exosome can be replaced from the surface of the anion exchange magnetic bead by high-concentration Cl-, and the exosome is obtained by collecting eluent.
S2: identifying and characterizing exosomes;
after obtaining the exosomes, the morphology and size of the exosomes were first observed using a transmission electron microscope FEI Tecnai Spirit TEM D1266. And then detecting the particle size distribution and the particle concentration of the exosome by using a nanoparticle tracking analysis method, and calculating the concentration and the recovery rate of the exosome. Next, the total protein concentration of exosomes was measured with the BCA kit, and the purity of exosomes was calculated (the purity of exosomes ═ particle concentration of exosomes/total protein concentration). Next, markers CD81, TSG101 and Alix of exosomes were detected using western blotting. Finally, the influence of the conditions of the anion exchange magnetic beads on the concentration, purity and uniformity of the prepared exosome, such as reaction concentration, reaction time, salt solubility of cleaning solution, pH of eluent, salt concentration and the like, is researched, and the parameters are optimized to obtain the exosome with high yield and high purity.
S3: modifying iRGD on the surface of the exosome to obtain a targeting modified exosome;
the invention uses Sulfo-EMCS to perform surface modification on exosome. Sulfo-EMCS is a bifunctional crosslinker containing an N-hydroxysuccinimide ester and a maleimide group. The modification process is as follows: firstly, a Sulfo-EMCS cross-linking agent is added into an exosome solution with a proper concentration, the pH value is adjusted to 7.0, and the reaction is carried out for 2 hours at room temperature, so that N-hydroxysuccinimide ester and exosome surface protein react to form an amido bond through primary amine. The unreacted EMCS was then removed by ultrafiltration 3 times (4000g,20 min) using a 50kD ultrafiltration tube. Finally, exosome-EMCS was mixed with iRGD polypeptide (sequence: CRGDRGPDC) to give a final concentration of 0.5mM of iRGD, pH was adjusted to 7.0, and the reaction was carried out at room temperature for 2 hours to allow maleimide to react with cysteine of iRGD via thiol group to form a stable thioether bond. And ultrafiltered 3 times (4000g,20 min) using a 50kD ultrafilter tube to remove free iRGD, thereby obtaining an iRGD-modified exosome (iRGD-EXO).
S4: evaluating the tumor targeting property of the iRGD modified exosome;
after targeted modification, we evaluated iRGD-EXO targeting to tumor cells LM2, MCF7, HCC1954 below. LM2, MCF7, HCC1954 and human fibroblast cell line (HSF) were first plated in 96-well plates and cultured overnight at 37 ℃ to allow the cells to adhere. EXO and iRGD-EXO were then incubated with red fluorescent dye Dil in PBS buffer, respectively, free dye was removed by ultrafiltration, and Dil-labeled EXO and iRGD-EXO were added to 96-well plates for incubation with LM2, MCF7, HCC1954, and HSF cells. Then, the cell culture solution was aspirated, washed three times with PBS, and fixed by adding 4% paraformaldehyde to each well of a 96-well plate. Finally, staining was performed with DAPI solution at room temperature, and uptake of EXO and iRGD-EXO by LM2, MCF7, HCC1954 and HSF cells was observed using a laser confocal microscope. The targeting of iRGD-EXO to LM2, MCF7, HCC1954 was evaluated.
S5: introducing ouabain into exosomes by electroporation;
after the targeting property of the iRGD-EXO is evaluated, the following exosomes modified by the iRGD are required to load drugs. The invention introduces ouabain into exosome by electroporation. The specific operation is as follows: firstly, adding an ouabain solution with a proper concentration into iRGD-EXO, and adding an electrotransfer buffer solution to adjust the resistance to 30-50 ohms. The mixture was then added to an electroporation cuvette and an electroporation signal (spring: 200V/5ms, length: 5ms, interval: 2 x 10%/+) was first applied using a NEPA21 instrument to perforate the exosome membrane. An electrical transfer signal (transfer: 20V/5ms, length: 50ms, interval: 5 x 40%/+) was then applied to cause ouabain to flow into the exosomes along the potential difference. Then, the mixture was incubated at 37 ℃ for 30 minutes to sufficiently repair the membrane of the exosome. And finally, carrying out ultrafiltration for 3 times (4000g for 20 minutes) at 4 ℃ by using a 50kD ultrafiltration tube to remove free ouabain, thereby obtaining the iRGD-EXO (i.e. iRGD-EXO-Ob) loaded with ouabain.
In this example, the in vitro experiment to evaluate the effect of the exosome-loaded ouabain-based nano-formulation in breaking up CTC clusters comprises the following steps:
s1, forming a CTC cluster in vitro and representing the CTC cluster;
the present invention selects LM2, MCF7 and HCC1954 cells to simulate tumor CTCs. The GFP plasmid was first transferred into LM2, MCF7 and HCC1954 cells to obtain LM2-GFP, MCF7-GFP and HCC 1954-GFP. Then LM2 in the logarithmic phase with the concentration of 0.1, 0.2, 0.4, 0.8, 1.6, 3.2 and 6.4 x 104/well is inoculated in an ultra-low adsorption 96-well plate, LM2-GFP, MCF7-GFP and HCC1954-GFP in the logarithmic phase are inoculated in the ultra-low adsorption 96-well plate, the culture medium is contained, the culture is cultured in a constant temperature incubator at 37 ℃ and 5% CO2, the culture solution is periodically replaced according to the growth speed of the cells and the color change of the culture solution, and the CTC clusters are obtained after 1-week culture; and finally, characterizing and analyzing the CTC cluster by using a direct observation method under an optical microscope.
S2, evaluating the effect of scattering CTC clusters by the exosome-loaded ouabain-based nano preparation through an in vitro experiment;
the experiment was divided into four groups: negative Control (NC) group, ouabain (Ob) group, iRGD-EXO group and iRGD-EXO-Ob group, the iRGD-EXO and iRGD-EXO-Ob groups were first labeled with red fluorescent dye Dil, and the above groups were incubated with CTC clusters, respectively, for 24, 48 and 72 hours at 37 ℃. And (3) observing the condition that the nano preparation is taken up by tumor cells, and the distribution and penetration effect in CTC clusters by using a laser confocal microscope. The fluorescent signal can be converted into a digital signal by combining Image J data processing software, and the penetration amount of the drug in the CTC cluster is compared. And (4) observing and counting the diameter change of the CTC cluster under an optical microscope, and evaluating the effect of the nano preparation on scattering the CTC cluster. And finally, detecting the methylation level of the single CTC and the transcription factor binding sites such as OCT4, SOX2, NANOG and SIN3A in the CTC cluster by using a methylation kit, and evaluating the methylation level change of key transcription factor binding sites before and after breaking up the CTC cluster.
In this example, the in vivo experiment to evaluate the effect of the exosome-loaded ouabain-based nano-formulation in breaking up CTC clusters and inhibiting tumor metastasis includes the following steps:
s1: constructing an in-situ tumor bearing model;
LM2-GFP, MCF7-GFP and HCC1954-GFP cells were first cultured to logarithmic phase, trypsinized, centrifuged to remove the cell culture medium, and cell culture medium was added to prepare suspensions of LM2-GFP, MCF7-GFP and HCC1954-GFP at a cell concentration of 5X 106 cells/mL. 96 female NSG mice, 4-6 weeks old, were then divided into 3 groups of 32 mice each. Finally, a second pair of mammary fat pads of NSG mice were inoculated with LM2-GFP, MCF7-GFP and HCC1954-GFP cells (5X 105 cells/mouse), and two weeks were raised until the tumor volume reached 100-150mm3, thereby obtaining LM2, MCF7 and HCC1954 tumor-bearing mice, i.e., tumor-bearing models in situ.
S2, evaluating the effect of the exosome-loaded ouabain-based nano preparation on scattering CTC clusters in blood;
taking LM2, MCF7 and HCC1954 tumor-bearing mice (32 mice each), respectively randomly dividing into 4 groups, 8 mice each group, and setting an NC group, an Ob group, an iRGD-EXO group and an iRGD-EXO-Ob group; the mice of each group were orally administered with PBS, Ob, iRGD-EXO and iRGD-EXO-Ob, respectively, twice a week for three consecutive weeks. Subsequently, the effect of the nano-formulations on breaking up CTC clusters was evaluated. Approximately 1mL of blood was collected by mouse cardiac puncture into EDTA anticoagulation tubes, CTCs were isolated from mouse blood using a CTC separator, and the isolated CTCs were stained with anti-EpCAM-AF488 antibody, anti-HER2-AF488 antibody, anti-EGFR-FITC antibody, and anti-CD45-BV605 antibody. And then analyzing the number and the proportion of the CTC clusters and single CTC by using a fluorescence microscope, and evaluating the effect of scattering the CTC clusters by the exosome-loaded ouabain-based nano preparation. And detecting the methylation level of the binding sites of transcription factors such as OCT4, SOX2, NANOG and SIN3A in the CTC cluster and single CTC by using a methylation detection kit.
S3, evaluating the effect of the nano preparation based on the exosome loading ouabain on inhibiting tumor metastasis;
the number and fluorescence values of spontaneous metastases of the lung, the liver and the brain of NC group, Ob group, iRGD-EXO group and iRGD-EXO-Ob group LM2, MCF7 and HCC1954 tumor-bearing mice are observed by a high-resolution fluorescence imaging technology, the tumor metastasis index (Ph/sec/cm2/sr) is calculated, the difference detected by each group is analyzed, and whether the exosome-supported ouabain-based nano preparation can reduce the metastasis risk of the tumor is evaluated. Finally, the effects of the nano preparation in inhibiting tumor metastasis and prolonging survival time are evaluated by measuring the tumor volume after treatment, detecting the survival time of mice, analyzing pathological sections and the like.
In this embodiment: the tumor includes tumors of various parts of the human body.
In this embodiment: the nano preparation comprises a nano preparation based on exosome, liposome, gold nanoparticle and magnetic nanoparticle.
In this embodiment: the drug for breaking up the CTC cell mass comprises a Na +/K + -ATPase inhibitor and a tubulin binding agent.
In this embodiment: the Na +/K + -ATPase inhibitor comprises digitoxin ouabain.
In this embodiment: the tubulin binding agents include rigosertib, pradafelone, colchicine and vincristine sulfate.
In this embodiment: the animal experiment scheme related by the invention passes the examination and verification of the biological and medical ethics committee of Beijing aerospace university; abbreviations and key term definitions to which the present invention relates: CTC: circulating tumor cells; iRGD: a tumor targeting peptide; EXO: an exosome; ob is ouabain.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (2)
1. A nanosystem for inhibiting tumor metastasis by breaking up CTC cell masses, characterized in that: the nano system is an exosome-loaded ouabain-based nano preparation constructed by drug ouabain for breaking up CTC cell aggregates and exosomes for loading drugs, and the exosome-loaded ouabain-based nano preparation is prepared by the following steps:
s1: extracting exosome from milk by adopting an exosome separation technology based on anion exchange magnetic beads;
s2: identifying and characterizing exosomes;
s3: modifying iRGD on the surface of the exosome to obtain a targeting modified exosome;
s4: evaluating the tumor targeting property of the iRGD modified exosome;
s5: introducing ouabain into exosomes by electroporation;
the method for extracting the exosomes from the milk by adopting the exosome separation technology based on the anion exchange magnetic beads comprises the following steps: incubating the anion exchange magnetic beads with fresh milk to ensure that the anion exchange magnetic beads with positive electricity are fully combined with the exosomes with negative electricity; then washing with low concentration salt solution to remove positively or uncharged protein, nucleic acid, lipid and cell debris impurities; finally, eluting with high-concentration NaCl solution to obtain high-concentration Cl-The exosome can be replaced from the surface of anion exchange magnetic beads, and the exosome is obtained by collecting eluent;
the exosome surface modified iRGD to obtain the targeting modified exosome comprises: adding a Sulfo-EMCS cross-linking agent into an exosome solution, adjusting the pH value to 7.0, reacting for 2 hours at room temperature to enable N-hydroxysuccinimide ester to react with exosome surface protein through primary amine to form an amido bond, then carrying out ultrafiltration for 3 times by using a 50kD ultrafiltration tube to remove unreacted EMCS, finally mixing exosome-EMCS with iRGD polypeptide to enable the final concentration of iRGD to be 0.5mM, adjusting the pH value to 7.0, reacting for 2 hours at room temperature to enable maleimide to react with cysteine of iRGD through sulfydryl to form stable thioether bond, and carrying out ultrafiltration for 3 times by using the 50kD ultrafiltration tube to remove free iRGD, thereby obtaining the iRGD modified exosome iRGD-EXO;
the electroporation of ouabain into exosomes comprises: adding an ouabain solution into iRGD-EXO, adding an electrotransfer buffer solution to adjust the resistance to 30-50 ohms, then adding the mixed solution into an electrotransfer cup, applying an electroporation signal, punching a hole on an exosome membrane, then applying an electrotransfer signal to enable ouabain to flow into an exosome along a potential difference, incubating the obtained mixed solution at 37 ℃ for 30 minutes to fully repair the membrane of the exosome, and finally performing ultrafiltration for 3 times at 4 ℃ by using a 50kD ultrafiltration tube to remove free ouabain, thereby obtaining the iRGD-EXO loaded with ouabain.
2. The nanosystem of claim 1 for inhibiting tumor metastasis by breaking up a mass of CTC cells, wherein: the nano preparation is any one of liposome, gold nanoparticles and magnetic nanoparticles.
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