CN113546058A - Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application - Google Patents

Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application Download PDF

Info

Publication number
CN113546058A
CN113546058A CN202110762977.7A CN202110762977A CN113546058A CN 113546058 A CN113546058 A CN 113546058A CN 202110762977 A CN202110762977 A CN 202110762977A CN 113546058 A CN113546058 A CN 113546058A
Authority
CN
China
Prior art keywords
msn
dox
nanoparticles
lmb
mesoporous silicon
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202110762977.7A
Other languages
Chinese (zh)
Inventor
秦洪双
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Luliang University
Original Assignee
Luliang University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Luliang University filed Critical Luliang University
Priority to CN202110762977.7A priority Critical patent/CN113546058A/en
Publication of CN113546058A publication Critical patent/CN113546058A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/5115—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5192—Processes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Abstract

The invention belongs to the technical field of biological medicines, and discloses nanoparticles for inhibiting TERT (TERT-like transition nucleus), a preparation method and application thereof. The preparation method of the nanoparticle for inhibiting TERT nuclear evolution comprises the following steps: loading the compound leptin B (LMB) for inhibiting TERT from generating nucleus and the chemotherapeutic drug Dox into the mesoporous silicon nano particles at the same time, and wrapping hyaluronic acid of the targeted tumor stem cells outside the drug-loaded mesoporous silicon. The nanoparticle Dox-LMB @ MSN-HA can improve the sensitivity of tumor stem cells to chemotherapeutic drugs and enhance the damage of Dox to mitochondria by inhibiting TERT (telomerase reverse transcriptase) to nucleate, thereby effectively removing the tumor stem cells.

Description

Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to nanoparticles for inhibiting TERT (TERT-electron transfer) nucleation, a preparation method and application.
Background
At present: tumor stem cells are the source of tumor metastasis and recurrence. The traditional antitumor drugs can kill most of tumor cells to reduce the tumor volume, but cannot eliminate tumor stem cells. The surviving tumor stem cells are rapidly proliferated and differentiated to form new tumor cells, so that the patient finally dies from the metastasis or recurrence of the tumor. With the rapid development of targeted chemotherapy, many anti-tumor stem cell compounds directed to different targets have been discovered, such as compounds directed to multiple kinases, mitochondria, or the tumor microenvironment. However, the compounds (such as oligomycin A, piperacillin and repitaxin) have weak selectivity on tumor stem cells and strong toxic and side effects on normal somatic cells. Therefore, it is an urgent problem to find a more effective target according to the biological characteristics of tumor stem cells, and thus to develop an antitumor stem cell compound with low toxicity.
Telomerase has attracted considerable attention in recent years in the treatment of cancer. Telomerase is highly expressed in tumor stem cells and most malignant cells, but is not or is poorly expressed in normal cells. Therefore, the telomerase is a good target for developing antitumor stem cell medicines with low toxicity and obvious curative effect. Telomerase includes an RNA Template (TERC) and a protein catalytic subunit (TERT). Its classical function is to add DNA repetitive sequence 5 '-TTAGGG-3' to telomere end by TERT catalytic function using its RNA as template. In addition to classical functions, in a cell stress state, TERT can translocate from a nucleus to mitochondria, protect mitochondrial DNA from DNA damage, enhance respiratory chain activity and mitochondrial membrane potential, inhibit ROS production, further enhance mitochondrial function, promote cell survival and inhibit apoptosis, and finally cause serious resistance to chemotherapeutic drugs, including etoposide, cisplatin, 5-fluorouracil, adriamycin (Dox) and the like. Therefore, inhibiting TERT transfer from the nucleus to the mitochondria can enhance the killing effect of chemotherapeutic drugs on tumor stem cells.
Through the above analysis, the problems and defects of the prior art are as follows:
(1) the existing drugs for inhibiting tumor growth or resisting tumor can kill most of tumor cells, reduce the volume of the tumor, but can not eliminate tumor stem cells.
(2) The existing antitumor stem cell medicines have weak selectivity on tumor stem cells and strong toxic and side effects on normal somatic cells.
The difficulty in solving the above problems and defects is: effective targets are searched according to the biological characteristics of the tumor stem cells, so that the anti-tumor stem cell compound which specifically targets the tumor stem cells and has small toxicity to normal body cells is developed.
The significance of solving the problems and the defects is as follows: the target of the specific targeting tumor stem cells is found, so that the antitumor stem cell compound with low toxicity is developed, a new thought can be provided for the design of antitumor stem cell medicines, and a new opportunity is provided for the treatment of the tumor stem cells.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a nanoparticle for inhibiting TERT nucleation, a preparation method and application thereof.
The invention is realized by the nanoparticles for inhibiting TERT nucleus emergence, namely the nanoparticles Dox-LMB @ MSN-HA. The materials of silicon dioxide and hyaluronic acid used by the nano platform have better biocompatibility in vitro and in vivo, so that the nano platform has good potential of being developed into clinical medicines.
Another object of the present invention is to provide a method for preparing TERT-nucleation inhibiting nanoparticles from the nanoparticles, the method comprising:
loading the compound leptin B (LMB) for inhibiting TERT from generating nucleus and the chemotherapeutic drug Dox into the mesoporous silicon nano particles at the same time, and wrapping hyaluronic acid of the targeted tumor stem cells outside the drug-loaded mesoporous silicon.
Further, the preparation method of the nanoparticles for inhibiting TERT nuclear evolution comprises the following steps:
synthesizing mesoporous silicon nano-particle MSP by using N-cethyltetramethonium bromide (CTAB) and NaOH;
stirring, ultrasonic treatment, dispersion, reflux and other treatments are carried out on the prepared mesoporous silicon nano particles to aminate the mesoporous silicon;
thirdly, carrying out drug loading on the aminated mesoporous silicon by using Dox and LMB to obtain drug-loaded nanoparticles LMB @ MSN, Dox @ MSN and Dox-LMB @ MSN;
and fourthly, performing hyaluronic acid coating on the obtained drug-loaded nano particles to obtain hyaluronic acid-coated mesoporous silicon nano particles MSN-HA, and hyaluronic acid-coated drug-loaded mesoporous silicon nano particles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA.
Further, in the step one, the synthesizing of the mesoporous silicon nanoparticles using N-cetyltrimethylammonioide, namely CTAB, and NaOH includes:
dissolving 0.50g of CTAB in 240mL of ultrapure water to obtain a CTAB solution; adding 1.75mL of 2MNaOH into the CTAB solution, and heating to 80 ℃ to obtain a mixed solution;
dropwise adding 2.5ml TEOS into the mixed solution, stirring for 2h until a white precipitate appears, filtering, and keeping the white precipitate; and washing the white precipitate by using deionized water and ethanol, and drying the washed white precipitate to obtain the mesoporous silicon nano particles.
Further, in the second step, the amination of the mesoporous silicon by stirring, ultrasonic treatment, dispersion, reflux treatment and other treatments of the prepared mesoporous silicon nanoparticles comprises:
adding 500mg MSP into 50ml HMMe, stirring, performing ultrasonic treatment, and fully dispersing to obtain a mixed solution; 250 μ L of 0.2 μmol of 3- (2-aminoethylamino) -propyl-trimethoxysilane was added to the above mixed solution under N2Refluxing for 24h under the condition; the product was filtered and washed sequentially with PhMe, THF and EtOH; after cleaning, the product is dried in vacuum for 24h to obtain aminated mesoporous silicon amino-functionalized MSP, namely MSN.
Further, in the third step, the loading of the drug on the aminated mesoporous silicon by Dox and LMB to obtain drug-loaded nanoparticles LMB @ MSN, Dox @ MSN and Dox-LMB @ MSN comprises:
adding 4mg of Dox into ultrapure water, fully stirring, adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing the nanoparticles with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain drug-loaded nanoparticles LMB @ MSN;
adding 0.16mg of LMB into ultrapure water, fully stirring, then adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing the nanoparticles with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain drug-loaded nanoparticles Dox @ MSN;
adding 4mg of Dox and 0.16mg of LMB into ultrapure water, fully stirring, then adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain the drug-loaded nanoparticles Dox-LMB @ MSN.
Further, in the fourth step, the hyaluronic acid-coated drug-loaded nanoparticles to obtain hyaluronic acid-coated mesoporous silicon nanoparticles MSN-HA, and hyaluronic acid-coated drug-loaded mesoporous silicon nanoparticles LMB @ MSN-HA, Dox @ MSN-HA, and Dox-LMB @ MSN-HA include:
adding 30mg Hyaluronic Acid (HA) into ultrapure water, stirring overnight, and fully hydrating; hydrated hyaluronic acid HA was added to a solution containing 2mg mL-1EDC、2mg mL-1Stirring for 30min in 2- (N-morpholino) ethane-sulfonic acid pH 6.0buffer of NHS;
then 20 mul of PBS buffer with 100mM and pH 7.4 is added, 12mg of MSN or MSN loaded with medicine is added, and the mixture is stirred for 20 hours at room temperature;
and centrifuging to remove unreacted hyaluronic acid to obtain the mesoporous silicon nano particles MSN-HA wrapped by the hyaluronic acid, and the mesoporous silicon nano particles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA which are wrapped by the hyaluronic acid and loaded with the drugs.
The invention also aims to provide application of the nanoparticles for inhibiting TERT nuclear generation in preparing medicines for preventing and/or treating cancers.
The invention also aims to provide application of the nanoparticles for inhibiting TERT nuclear generation in preparing medicines for eliminating or killing tumor stem cells.
The invention also aims to provide application of the nanoparticles for inhibiting TERT nucleation in preparing medicines for treating and/or preventing tumor diseases which are caused by drug resistance and relapse due to tumor stem cells.
By combining all the technical schemes, the invention has the advantages and positive effects that: the invention provides a nanoparticle Dox-LMB @ MSN-HA, which can improve the sensitivity of tumor stem cells to chemotherapeutic drugs by inhibiting TERT (telomerase reverse transcriptase) nucleation. The hyaluronic acid HA on the surface of the nanoparticle is combined with a CD44 receptor on a cell membrane of a tumor stem cell, LMB and Dox are simultaneously loaded into the tumor stem cell, after entering the cell, the LMB enhances the damage of the Dox to mitochondria by inhibiting TERT from generating nucleus, thereby effectively removing the tumor stem cell.
Drawings
FIG. 1 is a schematic diagram of the synthesis of nanoparticles Dox-LMB @ MSN-HA provided in the embodiment of the present invention.
FIG. 2 is a flow chart of a method for preparing nanoparticles for inhibiting TERT nucleation according to an embodiment of the present invention.
Fig. 3 is a TEM image of the aminated mesoporous silicon nanoparticle MSN provided in the embodiments of the present invention.
FIG. 4 is a TEM image of the nanoparticles coated with HA according to the present invention.
Fig. 5 is a schematic diagram illustrating the effect of the nanoparticles on the integrity of the mitochondrial DNA of breast cancer stem cells provided by the embodiments of the present invention.
Fig. 6 is a schematic diagram illustrating the effect of the nanoparticles on ATP levels of breast cancer stem cells according to the embodiment of the present invention.
Fig. 7 is a schematic diagram illustrating the effect of nanoparticles on the viability of breast cancer stem cells, according to an embodiment of the present invention.
Fig. 8 is a schematic diagram illustrating the effect of nanoparticles on the viability of breast cancer stem cells, according to an embodiment of the present invention.
Fig. 9 is a schematic diagram illustrating the effect of nanoparticles on the viability of breast cancer stem cells according to an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In view of the problems of the prior art, the present invention provides a nanoparticle for inhibiting TERT nucleation, which is described in detail below with reference to the accompanying drawings.
The nanoparticle for inhibiting TERT nuclear generation, namely the nanoparticle Dox-LMB @ MSN-HA, provided by the embodiment of the invention.
As shown in fig. 1, the method for preparing nanoparticles for inhibiting TERT nucleation according to the embodiment of the present invention includes:
loading the compound leptin B (LMB) for inhibiting TERT from generating nucleus and the chemotherapeutic drug Dox into the mesoporous silicon nano particles at the same time, and wrapping hyaluronic acid of the targeted tumor stem cells outside the drug-loaded mesoporous silicon.
As shown in fig. 2, the method for preparing nanoparticles for inhibiting TERT nucleation according to the embodiment of the present invention includes the following steps:
s101, synthesizing mesoporous silicon nano-particle MSP by using N-cethyltetramethonium bromide (CTAB) and NaOH;
s102, stirring, ultrasonic treatment, dispersion, reflux and other treatment are carried out on the prepared mesoporous silicon nano particles to aminate the mesoporous silicon;
s103, carrying out drug loading on the aminated mesoporous silicon by using Dox and LMB to obtain drug-loaded nanoparticles LMB @ MSN, Dox @ MSN and Dox-LMB @ MSN;
s104, performing hyaluronic acid coating on the obtained drug-loaded nanoparticles to obtain hyaluronic acid-coated mesoporous silicon nanoparticles MSN-HA, and hyaluronic acid-coated drug-loaded mesoporous silicon nanoparticles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA.
The mesoporous silicon nano particle synthesized by N-cethyltetramethonium bromide (CTAB) and NaOH provided by the embodiment of the invention comprises the following components:
dissolving 0.50g of CTAB in 240mL of ultrapure water to obtain a CTAB solution; adding 1.75mL of 2MNaOH into the CTAB solution, and heating to 80 ℃ to obtain a mixed solution;
dropwise adding 2.5ml TEOS into the mixed solution, stirring for 2h until a white precipitate appears, filtering, and keeping the white precipitate; and washing the white precipitate by using deionized water and ethanol, and drying the washed white precipitate to obtain the mesoporous silicon nano particles.
The method for amination of mesoporous silicon by stirring, ultrasonic treatment, dispersion, reflux treatment and other treatment of the prepared mesoporous silicon nanoparticles comprises the following steps:
adding 500mg MSP into 50ml HMMe, stirring, performing ultrasonic treatment, and fully dispersing to obtain a mixed solution; 250 μ L of 0.2 μmol of 3- (2-aminoethylamino) -propyl-trimethoxysilane was added to the above mixed solution under N2Refluxing for 24h under the condition; the product was filtered and washed sequentially with PhMe, THF and EtOH; after cleaning, the product is dried in vacuum for 24h to obtain aminated mesoporous silicon amino-functionalized MSP, namely MSN.
The method for carrying out drug loading on aminated mesoporous silicon by using Dox and LMB to obtain drug-loaded nanoparticles LMB @ MSN, Dox @ MSN and Dox-LMB @ MSN provided by the embodiment of the invention comprises the following steps:
adding 4mg of Dox into ultrapure water, fully stirring, adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing the nanoparticles with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain drug-loaded nanoparticles LMB @ MSN;
adding 0.16mg of LMB into ultrapure water, fully stirring, then adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing the nanoparticles with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain drug-loaded nanoparticles Dox @ MSN;
adding 4mg of Dox and 0.16mg of LMB into ultrapure water, fully stirring, then adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain the drug-loaded nanoparticles Dox-LMB @ MSN.
The method for coating the obtained drug-loaded nanoparticles with hyaluronic acid to obtain the hyaluronic acid-coated mesoporous silicon nanoparticles MSN-HA, and the hyaluronic acid-coated drug-loaded mesoporous silicon nanoparticles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA provided by the embodiment of the invention comprise the following steps:
adding hyaluronic acid HA 30mg into ultrapure water, stirring overnight, and fillingHydration; hydrated hyaluronic acid HA was added to a solution containing 2mg mL-1EDC、2mg mL-1Stirring for 30min in 2- (N-morpholino) ethane-sulfonic acid pH 6.0buffer of NHS;
then 20 mul of PBS buffer with 100mM and pH 7.4 is added, 12mg of MSN or MSN loaded with medicine is added, and the mixture is stirred for 20 hours at room temperature;
and centrifuging to remove unreacted hyaluronic acid to obtain the mesoporous silicon nano particles MSN-HA wrapped by the hyaluronic acid, and the mesoporous silicon nano particles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA which are wrapped by the hyaluronic acid and loaded with the drugs.
The technical solution of the present invention is further described with reference to the following specific embodiments.
Example 1 enrichment culture and identification of breast cancer Stem cells
MCF-7 cells were seeded at a density of 5000/ml into DMEM/F12 medium, to which 20ng/ml basic fibroblast growth factor bFGF, 20ng/ml human recombinant epithelial growth factor EGF, 4. mu.g/ml heparin, 1% streptomycin and penicillin were added, and cultured in 6-well ultra-low-adhesion culture plates, which were then placed at 37 ℃ and 5% CO2The microspheres were incubated in a humidified incubator, after 7d, the formed microspheres were digested into single cells with 0.25% trypsin, and cultured for a second time at a cell density of 5000 cells/ml, after 7d, the microspheres were collected and similarly digested into single cells.
Will be 1 × 106The individual cells were dispersed in 100. mu.l of PBS buffer, to which PE-labeled CD24 antibody and APC-labeled CD44 antibody were added, incubated at room temperature for 30min, then washed twice with PBS, resuspended in 500. mu.l of PBS, and finally tested for CD44 by flow cytometry+/CD24-/lowThe proportion of cells.
At 1X 1061mL of Trizol lysate was added to each cell, and the cells were blown up and transferred to a 1.5mL EP tube. Add 200. mu.L chloroform to Trizol solution, mix vigorously for 15-20s, after standing for 2min at room temperature, centrifuge for 15min (4 ℃, 12000 rpm). The aqueous phase was transferred to another EP tube, an equal volume of precooled isopropanol was added, the mixture was inverted and mixed several times and allowed to stand at room temperature for 10 min. Centrifuging for 10min (4 ℃, 12000rpm), discardingThe precipitate was washed by adding 1mL of 75% ethanol to the clear solution, centrifuged for 5min (4 ℃ C., 7500rpm), and the procedure was repeated once. The supernatant was discarded, dried naturally, and dissolved in 15 to 20. mu.L of 0.1% DEPC water at 37 ℃ for 30min to obtain an RNA solution. Carrying out reverse transcription on the RNA solution, wherein the reaction system comprises: mRNA template 1 μ g; 1. mu.L of adsorbed Oligo (dT)18 (0.5. mu.g/mL); 2 × TS Reaction Mix 10 μ L; TransScript RT/RI Enzyme Mix 1. mu.L; RNase-free Water was added to 20. mu.L. The reaction process is as follows: the reagents of the system are sequentially added into an EP tube, mixed evenly and incubated for 30min at 42 ℃. TransScript RT was inactivated by heating at 85 ℃ for 5 min. And finally, carrying out qRT-PCR reaction on the reverse transcription product, and detecting the expression quantity of ALDH 1. Reaction system: brilliant II SYBR Green QPCR mastermix 1 ×; 1 μ g of cDNA; primers were mixed at 0.8. mu.M. Reaction procedure: 10min at 95 ℃; 10sec at 95 ℃; 1min at 60 ℃; 60 ℃ for 30sec, 40 cycles. ALDH1 primer sequence: upstream 5'-TCG TCT GCT GCT GGC GAC AAT G-3', downstream 5'-CCC AAC CTG CAC AGT AGC GCA A-3'.
Culturing breast cancer stem cells at the density of 5000/ml, and simultaneously adding nanoparticles MSP, MSN-HA, LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA. Then, the influence of the nanoparticles on the mitochondria and the cell activity of the breast cancer stem cells is detected by using a PCR amplification technology, an ATP detection technology, a flow cytometry technology and a microsphere formation experiment.
Example 2 nanoparticle Synthesis
Mesoporous silicon nanoparticles (MSP) were synthesized. 0.50g of N-Cethylthrimethyllamoniumbromide (CTAB) was dissolved in 240mL of ultrapure water, 1.75mL of 2M NaOH was added to the CTAB solution, and heated to 80 ℃. Then 2.5mL of TEOS was added dropwise to the above solution and stirred for 2h until a white precipitate appeared, which was mesoporous silicon. The product was filtered, washed with deionized water and ethanol, and dried.
Amination of mesoporous silicon. Add 500mg MSP to 50mL PhMe, stir, sonicate to disperse it well. 250 μ L of 3- (2-aminoethylamino) -propyl-trimethyoxysilane (0.2 μmol) was added to the above solution under N2Refluxing for 24h under the condition. The product was then filtered and washed sequentially with PhMe, THF and EtOH. After cleaning, cleaningAnd drying the product in vacuum for 24h to obtain aminated mesoporous silicon amino-functionalized MSP (MSN).
Drug loading. 4mg of Dox, 0.16mg of LMB, or 4mg of Dox and 0.16mg of LMB were added to ultrapure water, and the mixture was stirred well. 20mg of MSN was added to each solution and stirred for 24 hours. Centrifugally separating the nano particles, fully washing with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain drug-loaded nano particles LMB @ MSN, Dox @ MSN and Dox-LMB @ MSN.
And (4) coating with hyaluronic acid. Hyaluronic Acid (HA) 30mg was added to ultrapure water, and stirred overnight to fully hydrate it. Then adding hydrated hyaluronic acid HA into the solution containing 2mg mL-1EDC、2mg mL-1NHS 2- (N-morpholino) ethane-sulfonic acid (MES) buffer (pH 6.0), stirred for 30 min. To the above solution was added 20. mu.L of PBS buffer (100mM, pH 7.4), and 12mg of MSN or drug-loaded MSN was added, followed by stirring at room temperature for 20 hours. And centrifuging to remove unreacted hyaluronic acid to obtain the mesoporous silicon nano particles MSN-HA wrapped by the hyaluronic acid, and the mesoporous silicon nano particles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA which are wrapped by the hyaluronic acid and loaded with the drugs.
FIG. 1 is a schematic of nanoparticle construction. FIG. 3 is a TEM image of the amino mesoporous silicon nanoparticle MSN, and the image shows that the MSN has a pore channel capable of loading a drug. FIG. 4 is a TEM image of the nanoparticles after being coated with HA, and the image shows that the periphery of MSN is successfully coated with HA and the drug-carrying pore canal is successfully sealed.
Example 3 Effect of nanoparticles on mitochondria
Mitochondrial DNA detection: after the breast cancer stem cells and the nanoparticles are incubated for 3d, mitochondrial DNA is extracted. Mitochondrial DNA integrity was tested by long-chain DNA PCR. An upstream primer 5'-ATA CCC ATG GCC AAC CTC CTA CTC CTC ATT-3' and a downstream primer 5'-CTA GAA GTG TGA AAA CGT AGG CTT GGA TTA AGG C-3'. Reaction system: buffe 1 ×; dNTP 0.75 mM; 150ng of template DNA; primer mixture 0.4. mu.M each; long DNA polymerase 2.5U/50. mu.l. Reaction procedure: 3min at 95 ℃; 30sec at 95 ℃; 30sec at 55 ℃; 5min at 68 ℃; 15min at 68 ℃; 30 cycles. The PCR product was mixed with a DNA dye, and the mixture was subjected to electrophoresis using 1% agarose gel, and the electrophoresis result was observed in a gel imager. FIG. 5 is a graph of the effect of nanoparticles on mitochondrial DNA integrity. FIG. 5 shows that Dox-LMB @ MSN-HA damages mitochondrial DNA more significantly than Dox @ MSN-HA,
the fact that Dox-LMB @ MSN-HA can remarkably enhance the damage of Dox to the mitochondrial DNA of breast cancer stem cells is shown.
ATP detection: after the breast cancer stem cells and the nanoparticles are incubated for 3d, the cells are collected by centrifugation. 1X 106Cells were lysed by adding 200. mu.L of lysis solution to each cell. After lysis, the cells were centrifuged at 12000rpm/min at 4 ℃ for 5min, and the supernatant was collected and used for measurement. Add 100. mu.L of ATP detection working solution to the detection well. The mixture is left at room temperature for 3-5min to consume all background ATP, thereby reducing the background. Add 20. mu.L of sample to the wells and mix them quickly with a micropipette, after 2sec intervals, measure the RLU value with a chemiluminescence apparatus (luminometer). Figure 6 is the effect of nanoparticles on ATP levels of breast cancer stem cells. In FIG. 6, it is shown that Dox-LMB @ MSN-HA significantly inhibited ATP production compared to Dox @ MSN-HA, indicating that Dox-LMB @ MSN-HA significantly enhanced Dox impairment of mitochondrial function in breast cancer stem cells.
And (3) ROS detection: the breast cancer stem cells and the nanoparticles are incubated for 3d, and are incubated with DCFH-DA (10 mu M) for 30min, and the intracellular ROS content is detected by using a flow cytometry. Figure 7 is the effect of nanoparticles on breast cancer stem cell ROS levels. In FIG. 7, it is shown that Dox-LMB @ MSN-HA significantly enhances ROS production compared to Dox @ MSN-HA, again demonstrating that Dox-LMB @ MSN-HA significantly enhances Dox's impairment of breast cancer stem cell mitochondrial function.
Example 4 Effect of nanoparticles on Breast cancer Stem cell viability
Inoculating 5000 breast cancer stem cells into a 6-hole ultralow-adhesion culture plate, adding nanoparticles, observing the number and size of microspheres under a microscope after 3 days, and taking a picture; the microspheres were then digested with trypsin into single cells and counted. Fig. 8 is a photograph showing the effect of nanoparticles on breast cancer tumor stem cell viability, and fig. 9 is statistical data showing the effect of nanoparticles on breast cancer tumor stem cell viability. As can be seen from FIGS. 8 and 9, Dox-LMB @ MSN-HA HAs a significantly stronger effect on cell viability than Dox @ MSN-HA, indicating that the nanoparticle Dox-LMB @ MSN-HA can significantly enhance the sensitivity of breast cancer stem cells to Dox by inhibiting TERT nucleation.
The above description is only for the purpose of illustrating the present invention and the appended claims are not to be construed as limiting the scope of the invention, which is intended to cover all modifications, equivalents and improvements that are within the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. Nanoparticles for inhibiting TERT nuclear export, namely nanoparticles Dox-LMB @ MSN-HA.
2. The method of claim 1, wherein the method comprises the steps of:
loading the compound leptin B (LMB) for inhibiting TERT from generating nucleus and the chemotherapeutic drug Dox into the mesoporous silicon nano particles at the same time, and wrapping hyaluronic acid of the targeted tumor stem cells outside the drug-loaded mesoporous silicon.
3. The method of claim 2, wherein the method comprises the steps of:
synthesizing mesoporous silicon nano-particle MSP by using N-cethyltetramethonium bromide (CTAB) and NaOH;
stirring, ultrasonic treatment, dispersion, reflux and other treatments are carried out on the prepared mesoporous silicon nano particles to aminate the mesoporous silicon;
thirdly, carrying out drug loading on the aminated mesoporous silicon by using Dox and LMB to obtain drug-loaded nanoparticles LMB @ MSN, Dox @ MSN and Dox-LMB @ MSN;
and fourthly, performing hyaluronic acid coating on the obtained drug-loaded nano particles to obtain hyaluronic acid-coated mesoporous silicon nano particles MSN-HA, and hyaluronic acid-coated drug-loaded mesoporous silicon nano particles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA.
4. The method as claimed in claim 3, wherein the step one, the step of synthesizing mesoporous silica nanoparticles with N-Cethyltetramethyionitumbromide (CTAB) and NaOH comprises:
dissolving 0.50g of CTAB in 240mL of ultrapure water to obtain a CTAB solution; adding 1.75mL of 2M NaOH into the CTAB solution, and heating to 80 ℃ to obtain a mixed solution;
dropwise adding 2.5mL of TEOS into the mixed solution, stirring for 2 hours until a white precipitate appears, filtering, and keeping the white precipitate; and washing the white precipitate by using deionized water and ethanol, and drying the washed white precipitate to obtain the mesoporous silicon nano particles.
5. The method for preparing nanoparticles for inhibiting TERT nucleation according to claim 3, wherein in the second step, the step of subjecting the prepared mesoporous silicon nanoparticles to stirring, ultrasonic treatment, dispersion, reflux treatment and other treatments to aminate mesoporous silicon comprises:
adding 500mg MSP into 50ml HMMe, stirring, performing ultrasonic treatment, and fully dispersing to obtain a mixed solution; 250 μ L of 0.2 μmol of 3- (2-aminoethylamino) -propyl-trimethoxysilane was added to the above mixed solution under N2Refluxing for 24h under the condition; the product was filtered and washed sequentially with PhMe, THF and EtOH; after cleaning, the product is dried in vacuum for 24 hours to obtain aminated mesoporous silicon amino-functionalized MSP (MSN).
6. The method for preparing nanoparticles for inhibiting TERT nucleation according to claim 3, wherein in the third step, the loading of the aminated mesoporous silicon with drugs by Dox and LMB to obtain drug-loaded nanoparticles LMB @ MSN, Dox @ MSN and Dox-LMB @ MSN comprises:
adding 4mg of Dox into ultrapure water, fully stirring, adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing the nanoparticles with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain drug-loaded nanoparticles LMB @ MSN;
adding 0.16mg of LMB into ultrapure water, fully stirring, then adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing the nanoparticles with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain drug-loaded nanoparticles Dox @ MSN;
adding 4mg of Dox and 0.16mg of LMB into ultrapure water, fully stirring, then adding 20mg of MSN, and stirring for 24 hours; centrifugally separating the nanoparticles, fully washing with ultrapure water, and removing unloaded and adsorbed drug molecules to obtain the drug-loaded nanoparticles Dox-LMB @ MSN.
7. The method for preparing nanoparticles for inhibiting TERT coring according to claim 3, wherein in step four, the hyaluronic acid-coated drug-loaded nanoparticles are obtained to obtain hyaluronic acid-coated mesoporous silicon nanoparticles MSN-HA, and the hyaluronic acid-coated drug-loaded mesoporous silicon nanoparticles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA comprise:
adding 30mg Hyaluronic Acid (HA) into ultrapure water, stirring overnight, and fully hydrating; hydrated hyaluronic acid HA was added to a solution containing 2mg mL-1 EDC、2mg mL-1Stirring for 30min in 2- (N-morpholino) ethane-sulfonic acid pH 6.0buffer of NHS;
then 20 mul of PBS buffer with 100mM and pH 7.4 is added, 12mg of MSN or MSN loaded with medicine is added, and the mixture is stirred for 20 hours at room temperature;
and centrifuging to remove unreacted hyaluronic acid to obtain the mesoporous silicon nano particles MSN-HA wrapped by the hyaluronic acid, and the mesoporous silicon nano particles LMB @ MSN-HA, Dox @ MSN-HA and Dox-LMB @ MSN-HA which are wrapped by the hyaluronic acid and loaded with the drugs.
8. Use of the TERT nuclear-outgrowth-inhibiting nanoparticle of claim 1 for the preparation of a medicament for the prevention and/or treatment of cancer.
9. Use of the TERT nucleation inhibiting nanoparticle of claim 1 for the preparation of a medicament for the elimination or killing of tumor stem cells.
10. Use of the TERT nuclear-outgrowth-inhibiting nanoparticle of claim 1 in the preparation of a medicament for treating and/or preventing tumor diseases which are resistant and recurrent due to tumor stem cells.
CN202110762977.7A 2021-07-06 2021-07-06 Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application Pending CN113546058A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202110762977.7A CN113546058A (en) 2021-07-06 2021-07-06 Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202110762977.7A CN113546058A (en) 2021-07-06 2021-07-06 Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application

Publications (1)

Publication Number Publication Date
CN113546058A true CN113546058A (en) 2021-10-26

Family

ID=78102755

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202110762977.7A Pending CN113546058A (en) 2021-07-06 2021-07-06 Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application

Country Status (1)

Country Link
CN (1) CN113546058A (en)

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101732308A (en) * 2008-11-17 2010-06-16 中国人民解放军军事医学科学院毒物药物研究所 New application of leptomycin B, pharmaceutical compositions and products thereof
CN102557051A (en) * 2012-01-04 2012-07-11 复旦大学 Method for preparing hollow mesocellular silica nanometer particles based on polymer formwork
US20130157959A1 (en) * 2010-04-21 2013-06-20 Konkuk University Industrial Cooperation Corp Use of hades as tumor suppressor target
CN105997881A (en) * 2016-07-26 2016-10-12 上海应用技术学院 Tumor cell targeting mesoporous silicon nanometer assembly and preparation method for same
CN106177982A (en) * 2016-07-26 2016-12-07 上海应用技术学院 A kind of hyaluronic acid decorated lipid mesoporous silicon core-shell nano assembly and preparation method thereof
CN107375298A (en) * 2017-06-30 2017-11-24 中国科学院长春应用化学研究所 Application of the chiral metal super molecular compound in resisting tumour stem cells medicine is prepared
US20180326078A1 (en) * 2017-05-10 2018-11-15 Graybug Vision, Inc. Extended release microparticles and suspensions thereof for medical therapy
CN109464421A (en) * 2018-10-25 2019-03-15 湖北大学 Functional mesoporous silicon substrate cancer target transport controlled release system and preparation method thereof
CN109939238A (en) * 2019-04-10 2019-06-28 大连理工大学 Hyaluronic acid decorated load medicine composite nano materials and preparation method thereof

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101732308A (en) * 2008-11-17 2010-06-16 中国人民解放军军事医学科学院毒物药物研究所 New application of leptomycin B, pharmaceutical compositions and products thereof
US20130157959A1 (en) * 2010-04-21 2013-06-20 Konkuk University Industrial Cooperation Corp Use of hades as tumor suppressor target
CN102557051A (en) * 2012-01-04 2012-07-11 复旦大学 Method for preparing hollow mesocellular silica nanometer particles based on polymer formwork
CN105997881A (en) * 2016-07-26 2016-10-12 上海应用技术学院 Tumor cell targeting mesoporous silicon nanometer assembly and preparation method for same
CN106177982A (en) * 2016-07-26 2016-12-07 上海应用技术学院 A kind of hyaluronic acid decorated lipid mesoporous silicon core-shell nano assembly and preparation method thereof
US20180326078A1 (en) * 2017-05-10 2018-11-15 Graybug Vision, Inc. Extended release microparticles and suspensions thereof for medical therapy
CN107375298A (en) * 2017-06-30 2017-11-24 中国科学院长春应用化学研究所 Application of the chiral metal super molecular compound in resisting tumour stem cells medicine is prepared
CN109464421A (en) * 2018-10-25 2019-03-15 湖北大学 Functional mesoporous silicon substrate cancer target transport controlled release system and preparation method thereof
CN109939238A (en) * 2019-04-10 2019-06-28 大连理工大学 Hyaluronic acid decorated load medicine composite nano materials and preparation method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHUANWEN LU ET AL: "Chemotherapeutic Sensitization of Leptomycin B Resistant Lung Cancer Cells by Pretreatment with Doxorubicin", 《PLOS ONE》 *
HONGSHUANG QIN ET AL: ""Developing Enzyme-Responsive Nanomedicine for Inhibition of hTERT Mitochondrial Translocation", 《ADVANCED THERAPEUTICS》 *

Similar Documents

Publication Publication Date Title
Frede et al. Colonic gene silencing using siRNA-loaded calcium phosphate/PLGA nanoparticles ameliorates intestinal inflammation in vivo
Zhou et al. Ru (II)-modified TiO2 nanoparticles for hypoxia-adaptive photo-immunotherapy of oral squamous cell carcinoma
CN111150853B (en) Preparation and application of drug carrier for combined treatment of tumors
CN106929508B (en) SaRNA for activating PTPRO gene expression and transport vector thereof
CN108201533A (en) A kind of N- succinyl-chitosan nanoparticle pharmaceutical delivery systems of load siRNA and its preparation method and application
CN101332301A (en) Antineoplastic composition and use thereof
Cong et al. How to exploit different endocytosis pathways to allow selective delivery of anticancer drugs to cancer cells over healthy cells
CN106860427B (en) Cerium oxide/iron oxide/mesoporous silicon nano composite material and preparation method and application thereof
WO2022143010A1 (en) Use of pyridoxal in preparation of drugs for treating ovarian cancer
CN112426437B (en) DNA functionalized copper oxide nano enzyme and preparation method and application thereof
CN113546058A (en) Nanoparticles for inhibiting TERT (TERT-terminal electron transfer) nucleation, preparation method and application
Xu et al. Development of fullerene nanospherical miRNA and application in overcoming resistant breast cancer
Huang et al. Effects of biofilm nano-composite drugs OMVs-MSN-5-FU on cervical lymph node metastases from oral squamous cell carcinoma
CN111888482A (en) Liver cancer cell targeted graphene oxide drug-loaded compound and preparation method thereof
CN108969479B (en) Method for constructing reduction response type anticancer nano-drug by polypeptide-drug co-assembly
CN110921669A (en) Preparation and application of hollow tungsten carbide nano material
CN113577086B (en) Application of isovaleryl spiramycin compound or composition thereof in preparation of medicines for treating immune disorder
CN113476606B (en) Application of UPK1A-AS1 inhibitor in preparation of antitumor drugs
CN114887061A (en) Preparation method and application of photo-thermal gene combination therapy nano system for targeting tumors
CN114939165A (en) Bimetallic nanoparticle capable of reversing multidrug resistance and preparation method and application thereof
CN108619528A (en) A kind of cyclodextrin-mesoporous silicon multifunctional nano load medicine particle
CN111330020B (en) Preparation and application of silver nano targeted tumor treatment preparation
EP3384918B1 (en) Bacteriophages for use in diseases associated with microbiota deficiency
CN112791083A (en) Application of small molecule compound in preparation of medicine for preventing and treating silkworm microparticle
CN110898035B (en) Tumor chemotherapy pharmaceutical preparation and preparation method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20211026

RJ01 Rejection of invention patent application after publication