CN114504655A - Drug-loaded microsphere for targeted inhibition of TNBC cell activity, preparation method and application thereof - Google Patents
Drug-loaded microsphere for targeted inhibition of TNBC cell activity, preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, a preparation method and application thereof, and relates to the technical field of molecular biology. The microsphere comprises a CD24 aptamer and a carrier, wherein the CD24 aptamer is positioned on the surface of the carrier, the carrier is a composite microsphere, the composite microsphere comprises a microsphere body and a loading agent, and the microsphere body is a PC-cobP microsphere, so that the technical problem of low inhibition efficiency for inhibiting the activity of TNBC cells can be mainly solved; the invention also provides a preparation method of the drug-loaded microsphere for the targeted inhibition of the activity of the TNBC cells, which is simple to operate and is convenient for the batch production of the drug-loaded microsphere for the targeted inhibition of the activity of the TNBC cells.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a drug-loaded microsphere for targeted inhibition of TNBC cell activity, a preparation method and application thereof.
Background
Breast cancer is the cancer with the highest morbidity and mortality among women, and in recent years, the morbidity and mortality of breast cancer among women are about 24.2% and 15%, respectively. With the use of various targeted anticancer drugs, although the 5-year relative survival rate of breast cancer has been greatly improved, the prognosis is still not satisfactory, especially for Triple Negative Breast Cancer (TNBC). TNBC accounts for about 15-20% of breast cancers, and is more aggressive, more recurrent and more mortality than other types of breast cancers. Due to the lack of specific receptors for TNBC, most TNBC patients do not benefit from targeted therapy. Standard chemotherapy is the only approved treatment, but has poor therapeutic effect and large toxic and side effects, and thus, targeted therapy is very essential for TNBC.
However, the existing TNBC targeted therapy has poor curative effect, and is mainly related to that targeted therapy medicaments are not only easily phagocytosed by lysosomes in human cells, but also inaccurate in targeting, low in inhibition efficiency for inhibiting TNBC cell activity and the like.
Disclosure of Invention
The first purpose of the invention is to provide a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which can mainly solve the technical problem of low inhibition efficiency for inhibiting TNBC cell activity.
The second purpose of the invention is to provide a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, the method discloses a carrier and a preparation process of the finally prepared drug-loaded microsphere in detail so as to facilitate preparation by operators, and the preparation method is simple in operation and facilitates batch production of the drug-loaded microsphere for targeted inhibition of TNBC cell activity.
The third purpose of the invention is to provide an application of the drug-loaded microspheres for targeted inhibition of TNBC cell activity in preparation of drugs for treating triple negative breast cancer.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
In one aspect, the application provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises a CD24 aptamer and a carrier, wherein the CD24 aptamer is located on the surface of the carrier, the carrier is a composite microsphere, the composite microsphere comprises a microsphere body and a loading agent, and the microsphere body is a PC-coBP microsphere. The TNBC mainly comprises CD24 positive cells, the sensitivity to chemotherapy is poor, an iron death pathway can be activated through targeted intervention of NF2-YAP signals, the expression conditions of CD24 and CD47 on the surface of a membrane can be reduced, the phagocytosis of a phagocytosis system is promoted, the activity of TNBC cells is inhibited from two aspects, the aim of comprehensively inhibiting the activity of the TNBC cells is fulfilled, in addition, the structure of the microsphere is stable, the phagocytosis of the drug-loaded nano microsphere by immune cells in blood can be effectively avoided, the stable transportation is realized, the CD24 on the surface of the microsphere can accurately target TNBC cells with high expression of CD24 aptamer, a loading agent is fed into the TNBC cells with high expression of CD24, the iron death of the TNBC cells with high expression of the CD24 is promoted, the expression conditions of CD24 protein and CD47 protein on the surface of the TNBC cells are reduced, and the phagocytosis of the cells is promoted.
In a second aspect, the application also provides a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises the following steps of obtaining an intermediate product by using CPADB, EDC and sulfo-NHS reaction, and using NH2reacting-PEG-COOH with intermediate product and adding hydrogen chloride to obtain COOH-PEG-CPADB, dissolving COOH-PEG-CPADB in anhydrous dioxane, adding BMA and V501, sealing with membrane, and introducing N2Reacting at 65-75 ℃ for 16-20h to obtain COOH-PEG-CPADB- [ co-BMA]To COOH-PEG-CPADB- [ co-BMA]Middle addingAdding PDSMA and V70, sealing with diaphragm and introducing N2Reacting at 65-75 deg.C for 16-20h to obtain COOH-PEG-CPADB- [ co-BMA-co-PDSMA]Obtaining PC-cobP microspheres; dissolving and standing the PC-cobP microspheres, mixing supernatant with a loading agent to obtain a mixture, adding water into the mixture, performing vortex treatment and ultrasonic treatment in sequence to obtain a crude product, adding DTT (draw texturing yarn) into the crude product for crosslinking, performing dialysis to obtain a purified product, and crosslinking the purified product with a CD24 aptamer to obtain the drug-loaded microspheres for targeted inhibition of TNBC cell activity. The method fully discloses the preparation process of the carrier in the microsphere and the synthesis route of the microsphere, so that operators can synthesize the microsphere by using the most basic raw materials, and further improve the synthesis efficiency of the microsphere.
In a third aspect, the application also provides application of the drug-loaded microsphere for targeted inhibition of TNBC cell activity in preparation of a drug for treating triple negative breast cancer.
Compared with the prior art, the invention has at least the following advantages or beneficial effects:
the invention provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which can activate an iron death pathway, reduce the expression conditions of CD24 and CD47 on the surface of a membrane, promote phagocytosis of a phagocytosis system, inhibit the activity of TNBC cells from two aspects, and further achieve the aim of comprehensively inhibiting the activity of the TNBC cells, and in addition, the microsphere has a stable structure, can effectively avoid immune cells in blood from phagocytosing the drug-loaded nanoparticle, thereby realizing stable transportation, and a CD24 aptamer on the surface of the microsphere can accurately target the TNBC cells with high expression of CD24, so that a loading agent is fed into the TNBC cells with high expression of CD24, thereby promoting the TNBC cells with high expression of CD24 to have iron death and reducing the expression conditions of CD24 protein and CD47 protein on the surface of the TNBC cells, and further promoting phagocytosis of the cells.
The invention also provides a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, which fully discloses a preparation process of a carrier in the microsphere and a synthesis route of the microsphere, so that an operator can synthesize the microsphere by using the most basic raw materials, and further the synthesis efficiency of the microsphere is improved.
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In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 shows the ionization equation of 1-2M2ge in accordance with one embodiment of the present invention;
fig. 2 is a synthesis route chart and a chromatogram of a drug-loaded microsphere for targeted inhibition of TNBC cell activity provided in an embodiment of the present invention;
FIG. 3 is a schematic view of the observation of the drug-loaded microsphere in test example 1 of the present invention under a transmission electron microscope;
FIG. 4 is a zeta potential diagram of the drug-loaded microspheres in test example 1 of the present invention;
FIG. 5 is a graph showing the results of DLS analysis of the drug-loaded microspheres in test example 2 of the present invention;
FIG. 6 is a schematic view of PC-coBP microspheres in experimental example 3 of the present invention observed under a transmission electron microscope;
FIG. 7 is a zeta potential diagram of PC-coBP microspheres in test example 3 of the present invention;
FIG. 8 is a DLS analysis result chart of PC-coBP microspheres in test example 3 of the present invention;
FIG. 9 is a graph showing encapsulation efficiency detection of YAP overexpression plasmid and NF2 knock-out plasmid in Experimental example 4 of the present invention;
FIG. 10 is a schematic view showing the observation of the drug-loaded microspheres in a solution with a high GSH level in Experimental example 5 of the present invention;
FIG. 11 is a schematic view showing the observation of the drug-loaded microspheres in an acidic solution in test example 5 of the present invention;
FIG. 12 is a graph showing the release rate detection of YAP overexpression plasmid and NF2 knock-out plasmid in the drug-loaded microspheres of Experimental example 5 of the present invention;
fig. 13 is a schematic amino acid modification diagram of a CD24 aptamer targeted to a drug-loaded microsphere that inhibits TNBC cell activity, provided in an example of the invention;
FIG. 14 is a graph showing the phagocytic effect of macrophages in an example of the effect of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments in the present application may be combined with each other without conflict. The present invention will be described in detail below with reference to specific examples.
In one aspect, the application provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises a CD24 aptamer and a carrier, wherein the CD24 aptamer is located on the surface of the carrier, the carrier is a composite microsphere, the composite microsphere comprises a microsphere body and a loading agent, and the microsphere body is a PC-coBP microsphere. The TNBC cells are mainly CD24 positive cells and are poor in chemotherapy sensitivity, the iron death pathway can be activated through targeted intervention of NF2-YAP signals, the expression conditions of CD24 and CD47 on the surface of a membrane can be reduced, phagocytosis of a phagocytosis system is promoted, the activity of the TNBC cells is inhibited from two aspects, the aim of comprehensively inhibiting the activity of the TNBC cells is fulfilled, in addition, the structure of the microsphere is stable, phagocytosis of the drug-loaded nano microsphere by immune cells in blood can be effectively avoided, stable transportation is achieved, the CD24 on the surface of the microsphere can accurately target the TNBC cells highly expressed by CD24 aptamers, a loading agent is fed into the TNBC cells highly expressed by the CD24, the iron death of the TNBC cells highly expressed by the CD24 is promoted, the expression conditions of the CD24 proteins and the CD47 proteins on the surfaces of the TNBC cells are reduced, and the phagocytosis of the cells is promoted. Studies currently suggest that CD24 and CD47 are highly expressed on the surface of tumor cells and act as "don't eat me" signals by interacting in cis with the signal receptor protein alpha (sirp α) on the surface of macrophages. Meanwhile, researches show that phagocytosis of tumor cells by macrophages can be promoted by blocking interaction between the tumor cells and the macrophages, in addition, iron death of the tumor cells in solid tumors can be determined by an NF2-YAP signal axis, NF2 or over-expressed YAP can be specifically knocked out, and the sensitivity of tumors to iron death pathways can be effectively improved, so that the drug-loaded microspheres provided by the invention can effectively regulate TNBC cells from the two aspects, and can activate phagocytic cells to phagocytize TNBC cells while promoting iron death of the TNBC cells so as to improve the efficiency of reducing the activity of the TNBC cells, the PC-cobP microspheres have a simple structure of COOH-PEG-CPADB- [ co-BMA-co-PDSMA ], and the drug-loaded microspheres have high GSH expression content and acidic pH value in the TNBC cells, so that the microspheres can react based on GSH after entering the TNBC cells, further leading the carrier to be dissociated, and releasing the loading agent into TNBC cells so as to realize the function of immediately releasing the medicament in the medicament-carrying microsphere.
The above-mentioned loading agents include NF2 knock-out plasmid, YAP overexpression plasmid, iron death agonist and 1-2M2 ge. The NF2 knockout plasmid and the YAP overexpression plasmid can enter the TNBC cell to play the effect of specifically knocking out the NF2 gene or specifically expressing the YAP gene, thereby activating the iron death of the TNBC cell; the iron death agonist can kill MAD-MB-231 cells so as to inhibit the growth of TNBC cells, can induce the death of TNBC related cell lines by regulating MUC1-C/XCT signal channels, has a structural formula of 1-2M2ge and a dissociation equation under an acidic condition as shown in figure 1, wherein the pH value in the TNBC cells is acidic, 1-2M2ge dissociates under the acidic condition after entering the TNBC cells to generate carbon dioxide, and the generated carbon dioxide can further push the NF2 knockout plasmid, the YAP overexpression plasmid and the iron death agonist to further reach corresponding positions in the TNBC cells so as to play a regulating role, and can also enable the NF2 knockout plasmid, the YAP overexpression and the iron death agonist to escape from the phagocytosis of lysosomes due to the pushing effect of the carbon dioxide, both the NF2 knockout plasmid and the YAP overexpression plasmid were induced by CRISPR-Cas 9.
The weight ratio of the NF2 knockout plasmid, the YAP overexpression plasmid and the PC-cobP microspheres is (1-2) to (3-5). The weight ratio interval can play a good role in coating, when the ratio is higher than the interval, the PC-cobP microspheres have poor coating effect, so that the prepared drug-loaded microspheres are unstable in structure and possibly cannot reach TNBC cells, and when the ratio is lower than the interval, NF2 knockout plasmids and YAP overexpression plasmids in the drug-loaded microspheres are difficult to enter the cells, so that the loading agent in the drug-loaded microspheres cannot be immediately released into the TNBC cells, and the release efficiency of the drug is reduced; in addition, the encapsulation rate of the PC-cobP microspheres in the proportion interval to the iron death agonist is (23.34 +/-2.45) wt%, and the encapsulation rate interval can simultaneously ensure the stability of the iron death agonist and the release efficiency of the iron death agonist to TNBC cells.
The iron death agonist is Erastin or RSL 3. Erastin has the chemical name of 2- [ 1- {4- [2- (4-chlorophenoxy) acetyl ] -1-piperazinyl } ethyl ] -3- (2-ethoxyphenyl) -4- (3H) quinazolinone, can inhibit the growth of TNBC tumors by killing MDA-MB-231, and can inhibit the MUC1-C/XCT signal pathway to induce the generation of mitochondrial reactive oxygen species and further promote the iron death of TNBC cells; RSL3 is a small molecule compound RAS-selective reactive small molecule 3, which can directly bind to GPX4 and inhibit the enzymatic activity in TNBC cells, thereby interfering the capacity of the TNBC to prevent lipid peroxidation, or inducing GPX4 protein degradation to cause the accumulation of ROS in TNBC cells, and finally inducing the cells to generate pig death.
In a second aspect, the application also provides a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises the following steps of obtaining an intermediate product by using CPADB, EDC and sulfo-NHS reaction, and using NH2Reacting-1000 DA-COOH with an intermediate product and adding hydrogen chloride to obtain COOH-PEG-CPADB, dissolving COOH-PEG-CPADB in anhydrous dioxane, adding BMA and V501, sealing with a diaphragm, and introducing N2Reacting at 65-75 ℃ for 16-20h to obtain COOH-PEG-CPADB- [ co-BMA]To COOH-PEG-CPADB- [ co-BMA]Adding PDSMA and V70, sealing with a diaphragm and then introducing N2Reacting at 65-75 deg.C for 16-20h to obtain COOH-PEG-CPADB- [ co-BMA-co-PDSMA]Obtaining PC-cobP microspheres; dissolving and standing PC-cobP microspheres, mixing supernate with a loading agent to obtain a mixture, adding water into the mixture, performing vortex and ultrasonic treatment in sequence to obtain a crude product, adding DTT (diethylenetriamine pentaacetic acid) into the crude product for crosslinking, and performing dialysis to obtain a purified product, and crosslinking the purified product with a CD24 aptamer to obtain the drug-loaded microspheres for targeted inhibition of TNBC cell activity. The method fully discloses the preparation process of the carrier in the microsphere and the synthesis route of the microsphere, thereby facilitating the synthesis of the microsphere by using the most basic raw materials by operators, further improving the synthesis efficiency of the microsphere, in addition, the preparation process of the microsphere is simple and convenient, and easy to operate, thereby facilitating the large-scale popularization of the microsphere, wherein the chemical reaction equation and the structural formula of the preparation process of the PC-coBP microsphere are shown in figure 2, wherein figure 2 also discloses COOH-PEG-CAPDB, COOH-PEG-CAPDB- [ co-BMA]And COOH-PEG-CAPDB- [ co-BMA-co-PDSMA]1hnmr spectrum and gel permeation chromatogram. The volume ratio of the mixture to the water used for the vortex is preferably 1 (7-9), the water in the proportion range can sufficiently remove residual impurities such as ethanol, when the proportion of the mixture to the water is lower than the range, the impurities cannot be sufficiently removed, and when the proportion of the mixture to the water is higher than the range, the concentration of a crude product is low.
The mixture also comprises primary centrifugation before swirling, wherein the primary centrifugation comprises the following steps of centrifuging the mixture for 3-7min, standing and taking supernatant to obtain a pretreatment substance. The insoluble impurities in the solution are sufficiently removed by primary centrifugation.
The pre-treatment substance also comprises a second centrifugation before swirling, the second centrifugation comprises the following steps of adding ethanol into the pre-treatment substance, centrifuging for 3-7min, and taking supernatant to obtain a crude extract, wherein the volume ratio of the pre-treatment substance to the ethanol is 1 (2.5-3.5). The proportion interval can ensure that the pretreatment substance is completely dissolved in the ethanol, and the pretreatment substance and the ethanol completely react without generating other impurities. When the volume ratio of the pretreatment substance to the ethanol is lower than the range, the residual ethanol impurities after the reaction of the pretreatment substance and the ethanol are excessive, and when the volume ratio of the pretreatment substance to the ethanol is higher than the range, the pretreatment substance cannot completely react with the ethanol.
The temperature of the ultrasonic treatment is 35-40 ℃. The temperature interval can ensure the stability of ultrasonic treatment, and when the temperature of ultrasonic treatment is higher or lower than the interval, the efficiency of ultrasonic treatment is reduced.
The CD24 aptamer further comprises an amino acid modification prior to crosslinking with the purified product. The CD24 aptamer modified by amino acid is crosslinked on a purified product, so that the finally obtained drug-loaded microsphere has a stable structure, and the amino acid modification schematic diagram of the CD24 aptamer is shown in FIG. 13.
In a third aspect, the application also provides application of the drug-loaded microsphere for targeted inhibition of TNBC cell activity in preparation of a drug for treating triple negative breast cancer.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises a CD24 aptamer and a carrier, wherein the CD24 aptamer is located on the surface of the carrier, the carrier is a composite microsphere, the composite microsphere comprises a microsphere body and a negative carrier, the microsphere body is a PC-coBP microsphere, wherein the load agent comprises NF2 knockout plasmid, YAP overexpression plasmid, iron death agonist Erastin and 1-2M2ge, and the weight ratio of the NF2 knockout plasmid, the YAP overexpression plasmid and the PC-coBP microsphere is 1.5:1.5: 4.
The embodiment also provides a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises the following steps of using CPADB, EDC and sulfo-NHS to react to obtain an intermediate product, and using NH2reacting-PEG-COOH with intermediate product and adding hydrogen chloride to obtain COOH-PEG-CPADB, dissolving COOH-PEG-CPADB in anhydrous dioxane, adding BMA and V501, sealing with membrane, and introducing N2Reacting at 65 ℃ for 16h to obtain COOH-PEG-CPADB- [ co-BMA]To COOH-PEG-CPADB- [ co-BMA]Adding PDSMA and V70, sealing with a diaphragm and then introducing N2Reacting for 16h at 65 ℃ to obtain COOH-PEG-CPADB- [ co-BMA-co-PDSMA]To obtain PC-cobP microA ball; dissolving PC-cobP microspheres in ethanol, standing, mixing with a loading agent to obtain a mixture, centrifuging the mixture for 3min, standing to obtain a pre-treatment substance, adding 2.5 times of ethanol into the pre-treatment substance, centrifuging for 3min to obtain a crude extract, adding 7 times of water into the crude extract, performing vortex treatment and ultrasonic treatment at 35 ℃ in sequence to obtain a crude product, adding DTT into the crude product, performing crosslinking and dialysis to obtain a purified product, and crosslinking the purified product with a CD24 aptamer modified by amino acid to obtain the drug-loaded microspheres for targeted inhibition of TNBC cell activity.
Example 2
The embodiment provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises a CD24 aptamer and a carrier, wherein the CD24 aptamer is located on the surface of the carrier, the carrier is a composite microsphere, the composite microsphere comprises a microsphere body and a negative carrier, the microsphere body is a PC-cobP microsphere, wherein a loading agent comprises an NF2 knockout plasmid, a YAP overexpression plasmid, an iron death agonist Erastin and 1-2M2ge, and the weight ratio of the NF2 knockout plasmid to the YAP overexpression plasmid to the PC-cobP microsphere is 2:2: 5.
The embodiment also provides a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises the following steps of using CPADB, EDC and sulfo-NHS to react to obtain an intermediate product, and using NH2reacting-PEG-COOH with intermediate product and adding hydrogen chloride to obtain COOH-PEG-CPADB, dissolving COOH-PEG-CPADB in anhydrous dioxane, adding BMA and V501, sealing with membrane, and introducing N2Reacting at 75 ℃ for 20h to obtain COOH-PEG-CPADB- [ co-BMA]To COOH-PEG-CPADB- [ co-BMA]Adding PDSMA and V70, sealing with a diaphragm and then introducing N2Reacting at 75 ℃ for 20h to obtain COOH-PEG-CPADB- [ co-BMA-co-PDSMA]Obtaining PC-cobP microspheres; dissolving PC-cobP microspheres in ethanol, standing, mixing with a loading agent to obtain a mixture, centrifuging the mixture for 7min, standing to obtain a pre-treated substance, adding 3.5 times of ethanol to the pre-treated substance, centrifuging for 7min to obtain a crude extract, adding 9 times of water to the crude extract, performing vortex treatment and ultrasonic treatment at 40 deg.C to obtain a crude product, adding DTT to the crude product, crosslinking, dialyzing to obtain a purified product, and mixing the purified product with the obtained productAnd (3) crosslinking the amino acid modified CD24 aptamer to obtain the drug-loaded microsphere for targeted inhibition of TNBC cell activity.
Example 3
The embodiment provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises a CD24 aptamer and a carrier, wherein the CD24 aptamer is located on the surface of the carrier, the carrier is a composite microsphere, the composite microsphere comprises a microsphere body and a negative carrier, the microsphere body is a PC-coBP microsphere, wherein the load agent comprises NF2 knockout plasmid, YAP overexpression plasmid, iron death agonist Erastin and 1-2M2ge, and the weight ratio of the NF2 knockout plasmid, the YAP overexpression plasmid and the PC-coBP microsphere is 1:1: 3.
The embodiment also provides a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, which comprises the following steps of using CPADB, EDC and sulfo-NHS to react to obtain an intermediate product, and using NH2reacting-PEG-COOH with intermediate product and adding hydrogen chloride to obtain COOH-PEG-CPADB, dissolving COOH-PEG-CPADB in anhydrous dioxane, adding BMA and V501, sealing with membrane, and introducing N2Reacting at 70 ℃ for 18h to obtain COOH-PEG-CPADB- [ co-BMA]To COOH-PEG-CPADB- [ co-BMA]Adding PDSMA and V70, sealing with a diaphragm and then introducing N2Reacting at 70 ℃ for 28h to obtain COOH-PEG-CPADB- [ co-BMA-co-PDSMA]Obtaining PC-cobP microspheres; dissolving PC-cobP microspheres in ethanol, standing, mixing with a loading agent to obtain a mixture, centrifuging the mixture for 5min, standing to obtain a pre-treatment substance, adding 3 times of ethanol into the pre-treatment substance, centrifuging for 5min to obtain a crude extract, adding 8 times of water into the crude extract, performing vortex treatment and ultrasonic treatment at 37 ℃ in sequence to obtain a crude product, adding DTT into the crude product, performing crosslinking, dialyzing to obtain a purified product, and crosslinking the purified product with a CD24 aptamer modified by amino acid to obtain the drug-loaded microspheres for targeted inhibition of TNBC cell activity.
Test example 1
Taking the drug-loaded microspheres for targeted inhibition of TNBC cell activity prepared in examples 1-3, and performing morphology observation and zeta potential analysis under a transmission electron microscope, wherein the observation result of the transmission electron microscope of the drug-loaded microspheres prepared in example 3 is shown in figure 3, the zeta potential analysis result of the drug-loaded microspheres prepared in example 3 is shown in figure 4, and the drug-loaded microspheres are regular and uniform spherical structures and have fuzzy flocculent appearance at the edges according to the result of figure 3, which indicates that CD24 aptamer is successfully modified on the surfaces of the drug-loaded nanospheres; the results in fig. 4 show that the drug-loaded microspheres have good dispersity and uniform distribution on the whole.
Test example 2
The drug-loaded microspheres for targeted inhibition of TNBC cell activity prepared in examples 1 to 3 were examined by DLS (dynamic light scattering), wherein the examination results of the drug-loaded microspheres prepared in example 3 are shown in fig. 5, and it can be seen from the examination results in fig. 5 that the drug-loaded microspheres prepared in example 3 are 141.11 ± 13.43nm on average, the polydispersity index (PDI) thereof is 0.371 ± 0.054, and the dispersion effect is good.
Test example 3
Taking the uncrosslinked CD24 aptamer and the Pc (cobP) microspheres loaded with the carrier in examples 1-3, and performing morphology observation under a transmission electron microscope, and then performing DLS analysis and zeta potential analysis, wherein the observation results of the Pc (cobP) microspheres prepared in example 3 are shown in FIG. 6, the zeta potential analysis results of the Pc (cobP) microspheres prepared in example 3 are shown in FIG. 7, the DLS analysis results of the Pc (cobP) microspheres prepared in example 3 are shown in FIG. 8, the results of FIG. 6 show that the PC (cobP) microspheres have smooth surfaces, the zeta potential of the PC (cobP) microspheres shows negative potential, the results of FIG. 8 show that the particle size of the PC (cobP) microspheres is about 127.67 + -17.52, and the PDI is 0.353 + -0.074.
Test example 4
The drug-loaded microspheres for targeted inhibition of TNBC cell activity prepared in examples 1-3 were taken, agarose gel electrophoresis was used to detect the plasmid encapsulation efficiency of YAP _ OV and NF2_ KO, and the results shown in FIG. 9 indicate that the drug-loaded microspheres for targeted inhibition of TNBC cell activity prepared in example 3 have the best encapsulation efficiency when the ratio of YAP _ OV, NF2_ KO, and PC (cobP) is 1:1: 3.
Test example 5
When the drug-loaded microspheres for targeted inhibition of TNBC cell activity prepared in examples 1-3 are placed in a high-level GSH solution, TEM images show that the structure of the nanoparticles is irregular and the nanoparticles aggregate, wherein the detection results of example 3 are shown in fig. 10. Meanwhile, PC (cobP) Ca is under the condition of acidic PH (< 6.8), the compound 1-2M2ge is subjected to ionization equilibrium reaction to induce a large amount of carbon dioxide to generate, TEM images show that the structure of the drug-loaded microsphere drug-loaded nanospheres which can inhibit the activity of TNBC cells in a targeted manner is damaged, irregular and porous, wherein the detection result of example 3 is shown in figure 11, the release speed of YAP overexpression plasmids and NF2 knockout plasmids is also observed to be obviously accelerated, and the detection result of example 3 is shown in figure 12.
Examples of effects
CD24high cells were isolated from TNBC and labelled with FITC or GFP, anti-CD 11b labelled with A647 or rhodamine, respectively, to label macrophages. Pre-treating CD24high cells with pc (cobp) Ca (1 × 104 cells/mg/ml) or anti-CD 24 antibody (1 × 105 cells/mg/ml) at 37 ℃ for 2 hours, then co-culturing with macrophages, (a) detecting the phagocytosis rate of the CD24high cells by macrophages using fluorescence activated cell sorting, (B) obtaining statistical results of the phagocytosis rate (n ═ 5 per group), (C) representative fluorescence microscopy images (bar graph, 50 μm), (D) of CD24high cell in vitro phagocytosis, and performing statistical analysis (n ═ 5 per group) P <0.05, the obtained results are shown in fig. 14, and according to the results in fig. 14, the drug-loaded microspheres can effectively inhibit the activity of TNBC cells, promote the iron death of TNBC cells and phagocytosis of TNBC cells by phagocytes.
In conclusion, the invention provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, a preparation method and application thereof:
the invention provides a drug-loaded microsphere for targeted inhibition of TNBC cell activity, which can activate an iron death pathway, reduce the expression conditions of CD24 and CD47 on the surface of a membrane, promote phagocytosis of a phagocytosis system, inhibit the activity of TNBC cells from two aspects, and further achieve the aim of comprehensively inhibiting the activity of the TNBC cells, and in addition, the microsphere has a stable structure, can effectively avoid immune cells in blood from phagocytosing the drug-loaded nanoparticle, thereby realizing stable transportation, and a CD24 aptamer on the surface of the microsphere can accurately target the TNBC cells with high expression of CD24, so that a loading agent is fed into the TNBC cells with high expression of CD24, thereby promoting the TNBC cells with high expression of CD24 to have iron death and reducing the expression conditions of CD24 protein and CD47 protein on the surface of the TNBC cells, and further promoting phagocytosis of the cells.
The invention also provides a preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity, which fully discloses a preparation process of a carrier in the microsphere and a synthesis route of the microsphere, so that an operator can synthesize the microsphere by using the most basic raw materials, and further the synthesis efficiency of the microsphere is improved.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (10)
1. The drug-loaded microsphere is characterized by comprising a CD24 aptamer and a carrier, wherein the CD24 aptamer is located on the surface of the carrier, the carrier is a composite microsphere, the composite microsphere comprises a microsphere body and a loading agent, and the microsphere body is a PC-coBP microsphere.
2. The drug-loaded microsphere for targeted inhibition of TNBC cell activity according to claim 1, wherein the drug-loaded microsphere comprises an NF2 knockout plasmid, a YAP overexpression plasmid, an iron death agonist and 1-2M2 ge.
3. The drug-loaded microsphere for targeted inhibition of TNBC cell activity according to claim 2, wherein the weight ratio of the NF2 knockout plasmid, the YAP overexpression plasmid and the PC-coBP microsphere is (1-2): (1-2): (3-5).
4. The drug-loaded microsphere for targeted inhibition of TNBC cell activity according to claim 2, wherein the iron death agonist is Erastin or RSL 3.
5. A method for preparing drug-loaded microspheres for targeted inhibition of TNBC cell activity according to any one of claims 1-4, comprising the steps of using CPADB, EDC and sulfo-NHS reaction to obtain intermediate product, using NH2Reacting PEG-COOH with the intermediate product and adding hydrogen chloride to obtain COOH-PEG-CPADB, dissolving COOH-PEG-CPADB in anhydrous dioxane, adding BMA and V501, sealing with a diaphragm, and introducing N2Reacting at 65-75 ℃ for 16-20h to obtain COOH-PEG-CPADB- [ co-BMA]To COOH-PEG-CPADB- [ co-BMA]Adding PDSMA and V70, sealing with a diaphragm and then introducing N2Reacting at 65-75 deg.C for 16-20h to obtain COOH-PEG-CPADB- [ co-BMA-co-PDSMA]Obtaining the PC-cobP microspheres;
dissolving the PC-cobP microspheres and standing, mixing supernatant with the loading agent to obtain a mixture, adding water into the mixture, performing vortex treatment and ultrasonic treatment in sequence to obtain a crude product, adding DTT (draw texturing yarn) into the crude product for crosslinking, performing dialysis to obtain a purified product, and crosslinking the purified product and the CD24 aptamer to obtain the drug-loaded microspheres for targeted inhibition of TNBC cell activity.
6. The preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity according to claim 5, wherein the mixture further comprises a primary centrifugation before the vortexing, the primary centrifugation comprises the following steps of centrifuging the mixture for 3-7min, and standing to obtain a supernatant to obtain a pretreatment.
7. The preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity according to claim 6, wherein the pre-treatment substance further comprises a second centrifugation step before the vortex, the second centrifugation step comprises the following steps of adding ethanol into the pre-treatment substance, then centrifuging for 3-7min, and taking supernatant to obtain a crude extract, wherein the volume ratio of the pre-treatment substance to the ethanol is 1 (2.5-3.5).
8. The preparation method of the drug-loaded microsphere for targeted inhibition of TNBC cell activity according to claim 5, wherein the temperature of the ultrasonic treatment is 35-40 ℃.
9. The method for preparing drug-loaded microspheres for targeted inhibition of TNBC cell activity according to claim 5, wherein the CD24 aptamer further comprises amino acid modification before cross-linking with the purified product.
10. Use of the drug-loaded microsphere for targeted inhibition of TNBC cell activity according to any one of claims 1-4 in the preparation of a medicament for treating triple negative breast cancer.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116478999A (en) * | 2023-01-16 | 2023-07-25 | 广西师范大学 | Screening and application of immune suppression molecule CD24 aptamer |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016179189A1 (en) * | 2015-05-04 | 2016-11-10 | University Of Massachusetts | Polymers and polymeric assemblies for peptide and protein encaosulation and release, and methods thereof |
| CN111743861A (en) * | 2020-07-15 | 2020-10-09 | 南通大学 | Targeted triple-negative breast cancer hypoxia response chiral drug micelle and preparation method thereof |
| AU2021100957A4 (en) * | 2021-02-20 | 2021-04-29 | Dwivedi, Sumeet DR | A formulation for treatment of cancer and method of preparation thereof |
-
2022
- 2022-01-19 CN CN202210061371.5A patent/CN114504655B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2016179189A1 (en) * | 2015-05-04 | 2016-11-10 | University Of Massachusetts | Polymers and polymeric assemblies for peptide and protein encaosulation and release, and methods thereof |
| CN111743861A (en) * | 2020-07-15 | 2020-10-09 | 南通大学 | Targeted triple-negative breast cancer hypoxia response chiral drug micelle and preparation method thereof |
| AU2021100957A4 (en) * | 2021-02-20 | 2021-04-29 | Dwivedi, Sumeet DR | A formulation for treatment of cancer and method of preparation thereof |
Non-Patent Citations (5)
| Title |
|---|
| JIAO WU等: "Intercellular interaction dictates cancer cell ferroptosis via NF2-YAP signalling", 《NATURE》 * |
| JOANNA FAFIŃSKA等: "DNA Aptamers for the Maligant Transformation Marker CD24", 《NUCLEIC ACID THERAPEUTICS》 * |
| REN-MAN ZHU等: "Synchronous Synthesis of Polymeric Vesicles with Controllable Size and Low-Polydispersity by Polymerization-Induced Self-Assembly", 《CHINESE JOURNAL OF CHEMISTRY》 * |
| SHIH-HSUAN CHAN等: "Identification of the Novel Role of CD24 as an Oncogenesis Regulator and Therapeutic Target for Triple-Negative Breast Cancer", 《MOLECULAR CANCER THERAPEUTICS》 * |
| XIAOTIAN JI等: "Enzyme-polymer hybrid nanogels fabricated by thiol-disulfide exchange reaction", 《COLLOIDS AND SURFACES B: BIOINTERFACES》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116478999A (en) * | 2023-01-16 | 2023-07-25 | 广西师范大学 | Screening and application of immune suppression molecule CD24 aptamer |
| CN116478999B (en) * | 2023-01-16 | 2024-04-05 | 广西师范大学 | Screening and application of immune suppression molecule CD24 aptamer |
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