CN113082226A - Preparation method of drug-loaded nano cage and application of drug-loaded nano cage in targeted circulating tumor cell release of triptolide - Google Patents

Preparation method of drug-loaded nano cage and application of drug-loaded nano cage in targeted circulating tumor cell release of triptolide Download PDF

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CN113082226A
CN113082226A CN202110459985.4A CN202110459985A CN113082226A CN 113082226 A CN113082226 A CN 113082226A CN 202110459985 A CN202110459985 A CN 202110459985A CN 113082226 A CN113082226 A CN 113082226A
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CN113082226B (en
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张振
陈子超
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Shandong University of Traditional Chinese Medicine
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Abstract

The invention discloses a preparation method of a mesoporous structure drug-loaded nano cage, which specifically comprises the following steps: preparing an amino mesoporous silicon nanocage, continuously stirring a functionalized DNA sequence, the amino mesoporous silicon nanocage and a medicine triptolide in a dispersion liquid, precipitating, centrifuging and cleaning twice, realizing the mesoporous encapsulation and the embedding of an anticancer medicine triptolide in the amino mesoporous silicon nanocage, and obtaining the medicine-carrying nanocage, and simultaneously providing the application of the medicine in releasing triptolide by targeting circulating tumor cells. The invention belongs to the technical field of medicine, and particularly relates to a preparation method of a drug-loaded nano cage and application of the drug-loaded nano cage in targeted circulating tumor cell release of triptolide.

Description

Preparation method of drug-loaded nano cage and application of drug-loaded nano cage in targeted circulating tumor cell release of triptolide
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to a preparation method of a drug-loaded nano cage and application of the drug-loaded nano cage in targeted circulating tumor cell release of triptolide.
Background
Reduced Glutathione (GSH) is a tripeptide condensed from glutamic acid, cysteine and glycine and containing gamma-amido bond and sulfhydryl, is also a main non-protein sulfhydryl compound found in cells, and is an antioxidant factor with the most abundant content in cells. Reduced Glutathione (GSH) plays an important role in cellular metabolism, cell proliferation, intracellular homeostasis, and disease resistance. Glutathione is present in the redox balance between disulfide compounds (GSSG oxidized) and sulfhydryl compounds (GSH reduced), and the levels of reduced glutathione in living cells significantly alter the corresponding oxidative stress that has been implicated in many diseases and accelerated aging processes. During oxidative stress, reduced glutathione can be converted to oxidized form (GSSG) to protect cells from oxidative stress and to help capture free radicals that damage RNA or DNA. As a sensitive indicator, any change in the optimal ratio of intracellular GSH to GSSG can lead to human diseases such as: cancer, heart disease, stroke, and many neurological disorders. Especially in some cancer cells, the concentration of reduced glutathione in the cytoplasm is generally higher compared to normal cells. Therefore, detection of glutathione in vivo plays a very important role in disease diagnosis.
Currently, fluorescence detection, electrochemistry, high performance liquid chromatography, chemiluminescence, and spectrophotometry are widely used to study GSH. However, these methods have derivatization, complicated experimental procedures, time-consuming, limited in vivo applications, which are disadvantageous for development of practical procedures, and have difficulty in achieving effective targeted drug delivery. Due to high sensitivity of Quartz Crystal Microbalance (QCM) to mass change, the sensor has the advantages of good specificity, high sensitivity, low cost and simple operation, is more and more concerned by the scientific community, and has good application potential in the field of disease analysis and detection. In recent years, mesoporous silicon nanocages as nanocarriers have attracted considerable attention, and the nanocages have a surface that can be chemically modified, a high specific surface area, a characteristic nanostructure, biocompatibility, and a high loading function. As a molecule at the nanometer level, DNA is actively used in nanoscience, bioscience, and material science with many advantages such as its own programmability, structural diversity, and controllability of changes.
Therefore, it is necessary to design a DNA functionalized nanocage by using the precise pairing of bases and sequence designable characteristics to realize the detection of high specificity and high sensitivity of the circulating tumor cells and the release of anticancer drugs to the targeted reduced glutathione in the circulating tumor cells.
Disclosure of Invention
Aiming at the situation and in order to make up for the existing defects, the invention prepares a mesoporous structure drug-loaded nano-cage based on a DNA functionalized nano-cage and takes the nano-cage as an anticancer drug carrier; based on the chip for modifying the circulating tumor cell aptamer, the high-specificity and high-sensitivity detection of the circulating tumor cells is realized through a quartz crystal microbalance technology, meanwhile, the targeted reduced glutathione in the cells releases anti-cancer drugs, and unnecessary damage to normal cells is reduced.
The invention provides the following technical scheme:
a preparation method of a drug-loaded nano cage specifically comprises the following steps: preparing an amino mesoporous silicon nanocage, continuously stirring a functionalized DNA sequence, the amino mesoporous silicon nanocage and a medicine tripterygium wilfordii in a dispersion liquid, precipitating, centrifuging and cleaning twice, and then realizing the mesoporous encapsulation of the amino mesoporous silicon nanocage and embedding an anticancer medicine triptolide, thus obtaining the medicine-carrying nanocage.
Further, the functionalized DNA sequence is a chain hybridization product containing disulfide bonds and generated by chain hybridization reaction of three DNA sequences.
Further, the three DNA sequences are sequence one, sequence two and sequence three respectively, wherein the sequence one is: 5 '-ATC AGA CTG ATG TTG A CAA AGT-/HS-SH/-T CAA CAT CAG TCT- (BHQ) -GAT AAG CTA-3'; the second sequence is: 5 '-ACT TTG TCA ACA TCA GTC TGA T TAG CT-/HS-SH/-T A TCA GAC TGA TGT TGA-3', the third sequence being: 5 '-COOH-TTT TTT TTT TTT TAG CTT ATC AGA CTG ATG TTG A-3'.
Preferably, the drug-loaded nano cage is a mesoporous structure drug-loaded nano cage.
An application of triptolide released by drug-loaded nanocages targeted circulating tumor cells specifically comprises the following processes:
(1) the method is characterized in that a drug-loaded nano cage is used as a functional probe, functional nucleic acid is modified on a quartz crystal microbalance chip, the mesoporous-structure drug-loaded nano cage is incubated to enter circulating tumor cells, the cells are captured based on an aptamer recognition principle, the circulating tumor cells are captured by the quartz crystal microbalance chip, the sensitivity of the quartz crystal microbalance for detecting the circulating tumor cells is improved by the mesoporous-structure drug-loaded nano cage, and the high-sensitivity detection of the circulating tumor cells is realized;
(2) adding incision endonuclease into the quartz crystal microbalance chip combined with the circulating tumor cells, reacting the mesoporous structure drug-loaded nano cage with reduced glutathione in the circulating tumor cells of a detected object based on thiol exchange reaction, and releasing anticancer drug triptolide in the circulating tumor cells in a targeted manner.
The invention with the structure has the following beneficial effects: compared with the prior art, the drug-loaded nano-cage, the preparation method thereof and the application of the drug-loaded nano-cage in targeted circulating tumor cell release of triptolide have the following main innovations and advantages:
1. the mesoporous structure drug-loaded nano cage is prepared by a two-step method, and the preparation method is simple to operate;
2. according to the invention, the mesoporous structure drug-loaded nano cage is used as a probe, so that the sensitivity of a Quartz Crystal Microbalance (QCM) for detecting circulating tumor cells is improved, and the high-sensitivity detection of the circulating tumor cells is realized;
3. the invention takes the mesoporous structure drug-loaded nano cage as the drug-loaded probe, realizes the release of the anti-cancer drug in the circulating tumor cells with high specificity and selectivity, and reduces unnecessary damage to normal cells.
Drawings
FIG. 1 is a scanning electron microscope image of the mesoporous structure drug-loaded nanocage of the present invention;
FIG. 2 is a graph of the relationship between the number of circulating tumor cells and the Quartz Crystal Microbalance (QCM) signal response according to the present invention;
FIG. 3 is a graph of the linear relationship between the number of circulating tumor cells and the frequency intensity of a Quartz Crystal Microbalance (QCM) according to the present invention;
FIG. 4 is a graph comparing fluorescence intensity of drug release amount of mesoporous structure drug-loaded nanocages (a), normal cells (b) and circulating tumor cells (c) according to the present invention;
FIG. 5 is an imaging diagram of the mesoporous structure drug-loaded nanocage of the present invention used for targeted circulating tumor cell release of drugs.
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 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.
1. Preparing a mesoporous structure drug-loaded nano cage:
(1) 70 g of triethanolamine, 390 g of hexadecyl trimethyl benzene sulfonic acid, 200mL of ultrapure water, organic amine micromolecule solution and 2200g of tetraethoxysilane are mixed and stirred for 3 hours at 80 ℃;
(2) filtering and washing the mixture obtained in the step (1), and baking the mixture in an oven at 95 ℃ overnight for 24 hours to synthesize mesoporous silicon;
(3) weighing 10 g of the synthesized mesoporous silicon, dissolving the mesoporous silicon in 1000 ml of absolute ethyl alcohol, adding 3-aminopropyltriethoxysilane, and stirring for 6 hours at 37 ℃;
(4) filtering the product prepared in the step (3), washing with absolute ethyl alcohol, and drying at 60 ℃ to obtain an amino mesoporous silicon nanocage;
(5) the DNA sequence generates chain hybridization, the hybridization product is continuously stirred with amino mesoporous silicon nano cage (30mg/ml) and drug triptolide at 37 ℃ for 3 hours in dispersion liquid, and the suspension of mesoporous structure drug-loaded nano cage is obtained after twice sedimentation and centrifugal washing.
2. Quartz Crystal Microbalance (QCM) detects circulating tumor cells:
the aptamer is modified on the QCM chip, the suspension (20mg/ml) of the mesoporous structure drug-loaded nano cage is incubated to enter circulating tumor cells MDA-MB-231, the circulating tumor cells MDA-MB-231 are captured by the QCM chip based on the aptamer recognition principle in a QCM reaction pool at 37 ℃, and QCM tests are carried out, wherein the experimental results are shown in fig. 2 and 3.
As shown in FIG. 2, the intensity of QCM frequency increased with increasing numbers of cells added to circulating tumor MDA-MB-231.
As shown in fig. 3, when the number of circulating tumor MDA-MB-231 cells is in the range of 10 to 9000, the QCM intensity response is linearly related to the number of circulating tumor MDA-MB-231 cells, and the linear regression equation is Δ F-137.18 lgN-122.19, where N is the number of circulating tumor MDA-MB-231 cells, Δ F-F0, F is the QCM frequency intensity after the circulating tumor MDA-MB-231 cells act, F0 is the frequency intensity before the circulating tumor cells act, and R is 0.992.
3. Fluorescence intensity comparison of released drugs:
through a fluorescence spectrophotometer detection technology, the fluorescence intensity of triptolide released by the mesoporous structure drug-loaded nano-cage itself is used as a reference sample, and the fluorescence intensity of triptolide released by the mesoporous structure drug-loaded nano-cage (20mg/ml) in normal cells and circulating tumor cells MDA-MB-231 is matched and compared, the mesoporous structure drug-loaded nano-cage can target reduced glutathione in the circulating tumor cells to trigger thiol exchange reaction, so that the release of anticancer drug triptolide in the circulating tumor cells is realized, and the experimental result is shown in figure 4.
The data in fig. 4 show the comparison of the fluorescence intensity of three drug release amounts of the mesoporous structure drug-loaded nanocage (a), the mesoporous structure drug-loaded nanocage in normal cells (b) and the mesoporous structure drug-loaded nanocage in circulating tumor cells MDA-MB-231(c), and the comparative data show that: the mesoporous structure drug-loaded nano cage can target reduced glutathione in circulating tumor cells to trigger thiol exchange reaction, so that the anticancer drug triptolide can be effectively released in the circulating tumor cells, the drug release amount to normal cells is less, and the damage to the normal cells is reduced.
4. Targeting circulating tumor cells to release drugs:
3 mu L of incision enzyme is added into the QCM chip reaction pool combined with the circulating tumor cells MDA-MB-231, the QCM chip is separated from the liquid to obtain a product, and the product is subjected to laser confocal microscope imaging test, and the experimental result is shown in figure 5.
As shown in figure 5, the fluorescence imaging diagram of the drug released by the mesoporous structure drug-loaded nanocage targeted circulating tumor cell MDA-MB-231 shows that a large amount of anticancer drug triptolide is released in the circulating tumor cell through the visualization imaging technology.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Moreover, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Sequence listing
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Claims (5)

1. The preparation method of the drug-loaded nano cage is characterized by comprising the following steps:
preparing an amino mesoporous silicon nanocage, continuously stirring a functionalized DNA sequence, the amino mesoporous silicon nanocage and a medicine tripterygium wilfordii in a dispersion liquid, precipitating, centrifuging and cleaning twice, and then realizing the mesoporous encapsulation of the amino mesoporous silicon nanocage and embedding an anticancer medicine triptolide, thus obtaining the medicine-carrying nanocage.
2. The method for preparing a drug-loaded nanocage according to claim 1, wherein the functionalized DNA sequence is a disulfide bond-containing chain hybridization product of three DNA sequences undergoing a chain hybridization reaction.
3. The method for preparing the drug-loaded nanocage according to claim 1, wherein the three DNA sequences are sequence one, sequence two and sequence three, respectively, and the sequence one is: 5 '-ATC AGA CTG ATG TTG A CAAAGT-/HS-SH/-T CAA CAT CAG TCT- (BHQ) -GAT AAG CTA-3'; the second sequence is: 5 '-ACT TTG TCA ACA TCA GTC TGA T TAG CT-/HS-SH/-T A TCA GAC TGA TGT TGA-3', the third sequence being: 5 '-COOH-TTT TTT TTT TTT TAG CTT ATC AGA CTG ATG TTG A-3'.
4. The method for preparing the drug-loaded nanocage according to claim 1, wherein the drug-loaded nanocage is a mesoporous drug-loaded nanocage.
5. An application of triptolide released by drug-loaded nanocages targeted circulating tumor cells is characterized by comprising the following steps:
(1) the method is characterized in that a drug-loaded nano cage is used as a functional probe, functional nucleic acid is modified on a quartz crystal microbalance chip, the mesoporous-structure drug-loaded nano cage is incubated to enter circulating tumor cells, the cells are captured based on an aptamer recognition principle, the circulating tumor cells are captured by the quartz crystal microbalance chip, the sensitivity of the quartz crystal microbalance for detecting the circulating tumor cells is improved by the mesoporous-structure drug-loaded nano cage, and the high-sensitivity detection of the circulating tumor cells is realized;
(2) adding incision endonuclease into the quartz crystal microbalance chip combined with the circulating tumor cells, reacting the mesoporous structure drug-loaded nano cage with reduced glutathione in the circulating tumor cells of a detected object based on thiol exchange reaction, and releasing anticancer drug triptolide in the circulating tumor cells in a targeted manner.
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