CN113082226B - Preparation method of drug-loaded nanocage and application of drug-loaded nanocage in targeting circulating tumor cells to release triptolide - Google Patents
Preparation method of drug-loaded nanocage and application of drug-loaded nanocage in targeting circulating tumor cells to release triptolide Download PDFInfo
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/58—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin
- A61K31/585—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids containing heterocyclic rings, e.g. danazol, stanozolol, pancuronium or digitogenin containing lactone rings, e.g. oxandrolone, bufalin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
- A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
- G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
- G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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Abstract
The invention discloses a preparation method of a medicine carrying nano cage with a mesoporous structure, which comprises the following steps: the preparation method comprises the steps of preparing an amino mesoporous silicon nanocage, continuously stirring a functionalized DNA sequence, the amino mesoporous silicon nanocage and the triptolide in a dispersion liquid, precipitating, centrifugally cleaning for two times, and realizing the embedding of the anti-cancer triptolide in the mesoporous encapsulation of the amino mesoporous silicon nanocage, so that the drug-loaded nanocage can be obtained, and the application of the drug-loaded nanocage for targeting circulating tumor cells to release the triptolide is provided. The invention belongs to the technical field of medicine, and particularly relates to a preparation method of a medicine-carrying nanocage and application of the medicine-carrying nanocage in targeting circulating tumor cells to release triptolide.
Description
Technical Field
The invention belongs to the technical field of medicine, and particularly relates to a preparation method of a medicine-carrying nanocage and application of the medicine-carrying nanocage in targeting circulating tumor cells to release triptolide.
Background
Reduced Glutathione (GSH) is a tripeptide containing gamma-amide bonds and sulfhydryl groups, which is formed by condensing glutamic acid, cysteine and glycine, and is also a main non-protein sulfhydryl compound found in cells, and is the most abundant antioxidant factor in cells. Reduced Glutathione (GSH) plays an important role in cellular metabolism, cellular proliferation, intracellular environmental stability, and disease resistance. Glutathione is present in the redox balance prior to disulfide (GSSG oxidized) and sulfhydryl (GSH reduced) compounds, and the levels of reduced glutathione within living cells significantly alter the corresponding oxidative stress that has been implicated in many diseases and accelerated aging processes. Upon oxidative stress, reduced glutathione may be converted to oxidized form (GSSG) to protect cells from oxidative stress and to assist in capturing that radical that damages RNA or DNA. As a sensitive indicator, any change in the optimal ratio of intracellular GSH and GSSG can lead to diseases in humans such as: cancer, heart disease, stroke, and many neurological disorders. In particular, in some cancer cells, the concentration of intracellular reduced glutathione is generally higher than in 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, complex experimental procedures, time consuming, limited in vivo applications, which are detrimental to the development of practical procedures, and difficult to achieve for effective targeted drug delivery. Quartz Crystal Microbalance (QCM) has been receiving more and more attention from the scientific community due to its high sensitivity to mass changes, and the sensor has advantages of good specificity, high sensitivity, low cost and simple operation, and has a very good application potential in the field of disease analysis and detection. In addition, in research in recent years, mesoporous silicon nanocages as nanocarriers are attracting attention, and the surfaces of the nanocages can be chemically modified, have a high specific surface area, a characteristic nanostructure, biocompatibility and a high-load function. As a nano-scale molecule, DNA is active in nano science, bioscience, and material science with various advantages of its own programmability, structural diversity, and controllability of variation.
Therefore, it is necessary to design a DNA functionalized nanocage by utilizing the precise pairing of bases and the designable sequence characteristics, so as to realize the detection of high specificity and high sensitivity of the circulating tumor cells and the release of anticancer drugs into the circulating tumor cells by targeting reduced glutathione.
Disclosure of Invention
Aiming at the situation, in order to make up the existing defects, the invention prepares a medicine carrying nano-cage with a mesoporous structure based on a DNA functionalized nano-cage and taking the nano-cage as an anticancer medicine carrier; the invention provides a mesoporous structure drug-loaded nano cage which is simple in preparation method, high in sensitivity and high in drug-targeted performance for detecting the circulating tumor cells, and is suitable for rapid detection and targeted drug release of the circulating tumor cells in various human bodies.
The invention provides the following technical scheme:
the preparation method of the drug-loaded nano cage specifically comprises the following steps: preparing an amino mesoporous silicon nanocage, continuously stirring the functionalized DNA sequence, the amino mesoporous silicon nanocage and the medicine triptolide in a dispersion liquid, and performing precipitation and centrifugal cleaning twice to realize mesoporous encapsulation of the amino mesoporous silicon nanocage and embedding of the anticancer medicine triptolide, thus obtaining the medicine carrying nanocage.
Further, the functionalized DNA sequence is a disulfide bond-containing chain hybridization product of three DNA sequences subjected to chain hybridization reaction.
Further, the three DNA sequences are sequence one, sequence two and sequence three, respectively, 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 ATCA GAC TGA TGT TGA-3', the sequence three is: 5'-COOH-TTT TTT TTT TTT TAG CTT ATC AGA CTG ATG TTG A-3'.
Preferably, the drug-carrying nanocages are mesoporous structure drug-carrying nanocages.
The application of the drug-loaded nanocage to release triptolide from circulating tumor cells specifically comprises the following steps:
(1) The medicine carrying nano-cage is used as a functional probe, the medicine carrying nano-cage with a mesoporous structure is used for modifying functional nucleic acid on the quartz crystal microbalance chip, incubating the medicine carrying nano-cage with a mesoporous structure into circulating tumor cells, capturing the cells based on an aptamer recognition principle, capturing the circulating tumor cells by the quartz crystal microbalance chip, improving the sensitivity of detecting the circulating tumor cells by the quartz crystal microbalance, and realizing high-sensitivity detection of the circulating tumor cells;
(2) And adding a quartz crystal microbalance chip combined with the circulating tumor cells into the incision enzyme, and carrying out reaction between the medicine carrying nano cage with a mesoporous structure and reduced glutathione in the circulating tumor cells of the tested object based on thiol exchange reaction, so as to release the anticancer medicine triptolide in the circulating tumor cells in a targeted manner.
The beneficial effects obtained by the invention by adopting the structure are as follows: compared with the prior art, the invention has the main innovation and superiority as follows:
1. the invention adopts a two-step method to prepare the medicine carrying nano cage with the mesoporous structure, and the preparation method is simple to operate;
2. the invention takes the medicine carrying nano cage with a mesoporous structure as a probe, improves the sensitivity of Quartz Crystal Microbalance (QCM) for detecting the circulating tumor cells, and realizes the high-sensitivity detection of the circulating tumor cells;
3. the invention takes the medicine carrying nano-cage with mesoporous structure as the medicine carrying probe, realizes the release of the anticancer medicine in the circulating tumor cells with high specific selectivity, and reduces the unnecessary damage to the normal cells.
Drawings
FIG. 1 is a scanning electron microscope image of a drug-loaded nano cage with a mesoporous structure;
FIG. 2 is a graph showing the response of the number of circulating tumor cells to a Quartz Crystal Microbalance (QCM) signal in accordance with the present invention;
FIG. 3 is a graph showing 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 showing the comparison of fluorescence intensity of drug release amounts of drug-loaded nanocages (a), normal cells (b) and circulating tumor cells (c) with mesoporous structures according to the present invention;
FIG. 5 is an imaging view of the mesoporous structured drug-loaded nanocages of the present invention for targeted delivery of drugs to circulating tumor cells.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention; all other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
1. Preparation of a medicine carrying nano cage with a mesoporous structure:
(1) 70 g of triethanolamine, 390 g of hexadecyl trimethyl benzene sulfonic acid, 200mL of ultrapure water, organic amine small molecule 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 synthesized mesoporous silicon, dissolving the mesoporous silicon in 1000 ml of absolute ethyl alcohol, adding 3-aminopropyl triethoxysilane, 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 nano cage;
(5) The DNA sequence is hybridized in chain, the hybridized product is continuously stirred with amino mesoporous silicon nano cage (30 mg/ml) and medicine triptolide at 37 ℃ for 3 hours in dispersion liquid, and the suspension of medicine carrying nano cage with mesoporous structure is obtained after precipitation and centrifugal washing twice.
2. Quartz Crystal Microbalance (QCM) detection of circulating tumor cells:
modifying an aptamer on a QCM chip, incubating a suspension (20 mg/ml) of a medicine carrying nano cage with a mesoporous structure into a circulating tumor cell MDA-MB-231, capturing the circulating tumor cell MDA-MB-231 by the QCM chip based on an aptamer recognition principle in a QCM reaction tank at 37 ℃, and carrying out QCM test, wherein the experimental result is shown in figures 2 and 3.
As shown in FIG. 2, the QCM frequency intensity increased with increasing cell number of the circulating tumor MDA-MB-231.
As shown in fig. 3, when the number of the MDA-MB-231 cells of the circulating tumor is in the range of 10 to 9000, the QCM intensity response is in a linear relationship with the number of the MDA-MB-231 cells of the circulating tumor, the linear regression equation is Δf= 137.18lgN-122.19, where N is the number of the MDA-MB-231 cells of the circulating tumor, Δf=f-f0, F is the QCM frequency intensity after the MDA-MB-231 cells of the circulating tumor act, F0 is the frequency intensity before the MDA-MB-231 cells of the circulating tumor act, and r=0.992.
3. Fluorescence intensity contrast of released drug:
the fluorescence intensity of triptolide released by the medicine carrying nano-cage with a mesoporous structure (20 mg/ml) is used as a reference sample by a fluorescence spectrophotometer detection technology, the fluorescence intensity of triptolide released by the medicine carrying nano-cage with a mesoporous structure in normal cells and circulating tumor cells MDA-MB-231 is matched and compared, the medicine carrying nano-cage with the mesoporous structure can target reduced glutathione in the circulating tumor cells, a thiol exchange reaction is initiated, the release of the anti-cancer medicine triptolide in the circulating tumor cells is realized, and the experimental result is shown in figure 4.
The data in fig. 4 shows fluorescence intensity contrast of three drug release amounts of mesoporous structure drug-loaded nanocages (a), mesoporous structure drug-loaded nanocages in normal cells (b) and mesoporous structure drug-loaded nanocages in circulating tumor cells MDA-MB-231 (c), and the contrast data shows that: the mesoporous structure drug-loaded nano cage can target reduced glutathione in circulating tumor cells to trigger thiol exchange reaction, so that the anti-cancer drug triptolide in the circulating tumor cells can be effectively released, the drug release amount to normal cells is small, and the damage to the normal cells is reduced.
4. Targeting circulating tumor cells releases drugs:
and 3 mu L of nicking endonuclease is added into a QCM chip reaction tank combined with the circulating tumor cells MDA-MB-231, the QCM chip is separated from the liquid to obtain a product, and a laser confocal microscope imaging test is carried out, wherein the experimental result is shown in figure 5.
Fig. 5 shows a fluorescence imaging diagram of drug-loaded nanocage targeting circulating tumor cells MDA-MB-231 with mesoporous structure to release drugs, and the visual imaging technology shows that a large amount of anticancer drugs triptolide are released in the circulating tumor cells.
It is noted that relational terms such as first and second, and the like are 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 understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made therein 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
<110> university of Shandong traditional Chinese medicine
<120> preparation method of drug-loaded nanocage and application of drug-loaded nanocage in targeting circulating tumor cells to release triptolide
<130> university of Shandong traditional Chinese medicine
<160> 3
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<210> 3
<211> 34
<212> DNA
<213> sequence 3
<400> 3
tttttttttt tttagcttat cagactgatg ttga 34
Claims (2)
1. The preparation method of the drug-loaded nano cage is characterized by comprising the following steps of: preparing an amino mesoporous silicon nanocage, continuously stirring the functionalized DNA sequence, the amino mesoporous silicon nanocage and the medicine triptolide in a dispersion liquid, and performing precipitation and centrifugal cleaning twice to realize mesoporous encapsulation of the amino mesoporous silicon nanocage and embedding of the anticancer medicine triptolide, thus obtaining the medicine carrying nanocage;
the functionalized DNA sequence is a chain hybridization product containing disulfide bonds, wherein the chain hybridization reaction of the three DNA sequences is carried out;
the three DNA sequences are respectively a sequence I, a sequence II and a sequence III, wherein the sequence I is as follows: 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 sequence three is: 5'-COOH-TTT TTT TTT TTT TAG CTT ATC AGA CTG ATG TTG A-3'.
2. The method for preparing the drug-loaded nanocages according to claim 1, wherein the drug-loaded nanocages are mesoporous drug-loaded nanocages.
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