CN111467323A - Synthesis method and application of VB12 combined nano-composite carrying miRNA - Google Patents
Synthesis method and application of VB12 combined nano-composite carrying miRNA Download PDFInfo
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- CN111467323A CN111467323A CN202010271802.1A CN202010271802A CN111467323A CN 111467323 A CN111467323 A CN 111467323A CN 202010271802 A CN202010271802 A CN 202010271802A CN 111467323 A CN111467323 A CN 111467323A
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Classifications
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- 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
- A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
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- 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
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
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- 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
- A61K9/5123—Organic compounds, e.g. fats, sugars
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- 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 discloses a synthesis method and application of a VB12 combined nano compound carrying miRNA, wherein the synthesis method comprises the steps of mixing and dissolving a P L GA-PEG nano compound, vitamin B12, EDC and DMAP in an anhydrous solvent A, stirring and reacting at 15-40 ℃, dialyzing and removing the solvent after the reaction is finished to obtain the vitamin B12 combined nano compound.
Description
Technical Field
The invention relates to a method for synthesizing a nano-drug, in particular to a method for synthesizing a nano-drug with better targeting property to tumor cells.
Background
MicroRNAs (miRNAs) are small single-stranded non-coding RNAs of about 22 nucleotides in length, which are bound to the 3' untranslated regions (UTRs) of a target gene by means of base complementary pairing, thereby cleaving the transcript of the target gene or continuing the translation of the transcript, and finally playing a role in inhibiting the post-transcriptional regulation of target gene expression. However, miRNAs are small molecular nucleic acids, and the nucleic acids are easily degraded by nuclease in blood in a free state to be ineffective, so that how to realize the efficient and targeted delivery of the miRNAs to tumor focuses is the key for realizing tumor treatment.
In recent years, a nano Drug Delivery System (DDS) is concerned by more and more researchers and widely applied to medical intervention such as disease prevention, diagnosis and treatment, and the like, and has outstanding effects in the fields of Drug performance improvement, Drug delivery and the like, wherein a polylactic acid-glycolic acid copolymer (P L) has wide application prospects in the clinical and biological fields due to the characteristics of low immunogenicity, biocompatibility, biodegradability and the like of the polylactic acid-glycolic acid copolymer, and is a water-soluble polyethylene glycol material (GA) approved by the United states Food and Drug Administration (FDA), and PEG 6335-PEG L-PEG-linked to form a PEG-L-PEG-6335-PEG-L.
The nano carrier is a drug delivery system with excellent performance, can be passively targeted to a tumor focus through permeability enhancement and retention (EPR) of a tumor in blood circulation, but lacks active targeting, and has certain limitation in clinical use. Therefore, there is a need to explore novel nanocomplexes with tumor targeting.
Vitamin B12(VB12), also known as mecobalamin, is a water-soluble vitamin essential to human body, and is involved in methylation of human cell metabolism, synthesis of deoxyribonucleic acid (DNA), synthesis of nucleic acid and protein, and other important links. During the proliferation and development of tumor cells, the synthesis of nucleic acid and protein is accelerated obviously, so VB12 has certain uptake targeting property in tumor tissues. However, VB12 has poor stability and is sensitive to both light and heat, and how to couple VB12 to nanocarriers under mild reaction conditions is a challenging task.
Disclosure of Invention
The invention aims to overcome at least one defect of the prior art and provides a synthesis method of vitamin B12-binding nano-complex carrying miRNA.
The technical scheme adopted by the invention is as follows:
in some examples, the synthesis method of the P L GA-PEG nanocarrier comprises:
1) mixing P L GA, NHS and EDC in an anhydrous solvent B, activating, fully reacting, washing by using an anhydrous solvent C to remove unreacted micromolecules, and drying to obtain a P L GA-NHS pellet;
2) dissolving P L GA-NHS pellets in an anhydrous solvent D, and adding bifunctional modified NH2-PEG-COOH and diisopropylethylamine, and the reaction was continued for 24 hours;
3) after the reaction is finished, washing the reaction product by using an anhydrous solvent C, and finally drying the reaction product in vacuum to obtain the P L GA-PEG nano carrier.
In some examples, the method for synthesizing the vitamin B12-binding nano-carrier comprises the following steps:
1) mixing and dissolving P L GA-PEG nano-carrier, vitamin B12, EDC and DMAP in an anhydrous solvent A, and stirring for reaction at 15-40 ℃;
2) after the reaction is finished, dialyzing and removing the solvent to obtain the vitamin B12 combined nano-carrier which is recorded as VB12-P L GA-PEG nano-carrier.
In some examples, the molar ratio of the P L GA-PEG nano-carrier to the vitamin B12 is 1 (1-1.2).
In some examples, the molar ratio of P L GA-PEG nanocarrier to EDC to DMAP is 1 (1-1.2) to (0.05-0.12).
In some examples, the anhydrous solvent a is selected from butylene oxide.
In some examples, the P L GA-PEG nanocarrier is loaded with miRNA.
In some examples, the synthesis of miRNA-loaded P L GA-PEG nanocomplexes was as follows:
1) dispersing P L GA-PEG nano-carrier and cholestrol 3 β - [ N- (N, N-dimethyllaminoethane) in an anhydrous solvent D, and stirring until no precipitate exists to obtain a P L GA-PEG dispersion liquid;
2) adding a mixed solution of polyvinyl alcohol and miRNA mimics into the P L GA-PEG dispersion solution, and stirring for reacting for 6 hours;
3) and after the reaction is finished, washing with deionized water, and filtering to obtain the miRNA-loaded P L GA-PEG nano composite, which is marked as miRNA @ P L GA-PEG nano composite.
In some embodiments, the method for further loading miRNA on VB12-P L GA-PEG nanocarriers is as follows:
1) dispersing VB12-P L GA-PEG nano carrier and cholestrol 3 β - [ N- (N, N-dimethyllaminoethane) in an anhydrous solvent D, and stirring until no precipitate exists to obtain P L GA-PEG-VB12 dispersion liquid;
2) adding a mixed solution of polyvinyl alcohol and miRNA mimics into the P L GA-PEG-VB12 dispersion liquid, and stirring for reaction for 6 hours;
3) after the reaction is finished, washing with deionized water, and filtering to obtain the miRNA-loaded P L GA-PEG-VB12 nano complex which is marked as miRNA @ P L GA-PEG-VB12 nano complex.
In some examples, the miRNA mimics are miR-532-3p mimics.
In some examples, the anhydrous solvent B and the anhydrous solvent D are independently selected from dichloromethane and chloroform.
In some examples, the anhydrous solvent C is a mixture of methanol and diethyl ether.
The invention has the beneficial effects that:
the synthesis method has mild preparation conditions, cheap required raw materials, convenient operation, simple process, high efficiency and practicability. The prepared nano-composite optimizes the nucleic acid medicament in three aspects of water solubility, stability and tumor targeting, endows the nucleic acid medicament with high-efficiency and low-toxicity anti-tumor effect, and has wide application prospect in the field of tumor treatment.
The vitamin B12 combined nano-drug carrying the nucleic acid drug can prevent the nano-compound from being phagocytized by a reticuloendothelial system, and has good stability, excellent biocompatibility, biodegradability and water solubility.
VB12 is used for modifying miR-532-3P @ P L GA-PEG nanometer material, so that the nanometer compound is endowed with active targeting, and can enter tumor cells more in a targeted and efficient manner under the mediation of CD320 receptors on the surfaces of cell membranes.
Drawings
FIG. 1 is the physical and chemical characterization of P L GA-PEG and VB12-P L GA-PEG nano-carrier, A. transmission electron microscope picture, B. infrared absorption spectrum, C. particle size and electric potential;
FIG. 2 is an experimental result of the safety of the miR-532-3P @ P L GA-PEG-VB12 nano-complex;
FIG. 3 shows the targeting experiment results of the miR-532-3P @ P L GA-PEG-VB12 nano-composite;
FIG. 4 shows the experimental results of the antitumor effect of the miR-532-3P @ P L GA-PEG-VB12 nano-composite, the A.CCK-8 cell proliferation experiment and the B.cell apoptosis level detection.
FIG. 5 is the experimental results of the interaction of miR-532-3p with ARC; A. the generation information analysis predicts the possible combining part of the miR-532-3p and the ARC; B. dual luciferase gene reporter experiments.
FIG. 6 is an experimental result of an anti-tumor action mechanism of the miR-532-3P @ P L GA-PEG-VB12 nano-complex, A. mitochondrial membrane potential (MTPs), B. mitochondrial ROS (mitoROS) generation level, C. intracellular ATP generation level, D. mitochondrial membrane permeability (mPTP), E. expression level of apoptosis pathway related protein.
Detailed Description
In some examples, the synthesis method of the P L GA-PEG nanocarrier comprises:
1) mixing P L GA, NHS and EDC in an anhydrous solvent B, activating, fully reacting, washing by using an anhydrous solvent C to remove unreacted micromolecules, and drying to obtain a P L GA-NHS pellet;
2) dissolving P L GA-NHS pellets in an anhydrous solvent D, and adding bifunctional modified NH2-PEG-COOH and diisopropylethylamine, and the reaction was continued for 24 hours;
3) after the reaction is finished, washing the reaction product by using an anhydrous solvent C, and finally drying the reaction product in vacuum to obtain the P L GA-PEG nano carrier.
In some embodiments, a method for synthesizing a vitamin B12-binding nano-carrier includes:
1) mixing and dissolving P L GA-PEG nano-carrier, vitamin B12, EDC and DMAP in an anhydrous solvent A, and stirring for reaction at 15-40 ℃;
2) after the reaction is finished, dialyzing and removing the solvent to obtain the vitamin B12 combined nano-carrier which is recorded as VB12-P L GA-PEG nano-carrier.
The reaction temperature is preferably room temperature (20-25 ℃), and additional temperature control is avoided.
In some examples, the molar ratio of the P L GA-PEG nano-carrier to the vitamin B12 is 1 (1-1.2), so that the reaction of the P L GA-PEG nano-carrier can be more complete, wherein the molar molecular weight of the P L GA-PEG is 6000.
In some examples, the molar ratio of the P L GA-PEG nano-carrier to EDC to DMAP is 1 (1-1.2) to (0.05-0.12), so that the activation effect is better, and the full utilization of reactants is facilitated.
In some examples, the anhydrous solvent a is selected from butylene oxide.
In some examples, the P L GA-PEG nanocarrier is loaded with miRNA.
In some examples, the synthesis of miRNA-loaded P L GA-PEG nanocomplexes was as follows:
1) dispersing P L GA-PEG and cholestrol 3 β - [ N- (N, N-dimethylxanthoethane) in an anhydrous solvent D, and stirring until no precipitate exists to obtain a P L GA-PEG dispersion liquid;
2) adding a mixed solution of polyvinyl alcohol (PVA) and miRNA into the P L GA-PEG dispersion, and stirring for reaction;
3) and after the reaction is finished, washing with deionized water, and filtering to obtain the miRNA-loaded P L GA-PEG nano composite, which is marked as miRNA @ P L GA-PEG nano composite.
In some examples, the concentration of PVA is 1-2% (w/v), preferably 1% (w/v).
In some examples, filtration is performed using a 0.22 μm filter.
In some embodiments, the VB12-P L GA-PEG nanocomplexes loaded with miRNA were synthesized as follows:
1) dispersing VB12-P L GA-PEG and cholestrol 3 β - [ N- (N, N-dimethyllaminoethane) in an anhydrous solvent D, and stirring until no precipitate exists to obtain a P L GA-PEG-VB12 dispersion liquid;
2) adding a mixed solution of 1 percent polyvinyl alcohol (PVA) and mirnaamimics into the P L GA-PEG-VB12 dispersion, and stirring for reaction for 6 hours;
3) after the reaction is finished, washing with deionized water, and filtering (a filter with the diameter of 0.22 mu m) to obtain the miRNA-loaded P L GA-PEG-VB12 nano complex which is marked as miRNA @ P L GA-PEG-VB12 nano complex.
In some examples, the miRNA mimics are miR-532-3p mimics. Of course, the miRNA may also be other mirnas having a therapeutic effect on tumors.
For convenience of use and cost considerations, in some examples, the anhydrous solvents B, D are independently selected from dichloromethane, trichloromethane. Other solvents may also be selected.
In some examples, the anhydrous solvent C is a mixture of methanol and diethyl ether.
The noun abbreviations of the present invention are as follows:
p L GA polylactic acid-glycolic acid copolymer
PEG: polyethylene glycol
EDC: 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride
NHS: n-hydroxysuccinimide
DMAP: the Chinese name is 4-dimethylaminopyridine, the English name: 4-dimethylaminopyrdine
PVA: polyvinyl alcohol
Butene oxide: (S) - (-) -1,2-Epoxybutane, CAS No.: 30608-62-9, structural formula
Other abbreviations not indicated have the well-known meaning.
The technical scheme of the invention is further explained by combining the embodiment and the experimental data.
The inventor researches and discovers that microRNA-532-3p (miR-532-3p) is a gene closely related to tumorigenesis and development, and the up-regulation of the expression of miR-532-3p can inhibit the malignant progression of tumor cells; meanwhile, miR-532-3p can induce mitochondrial dysfunction in cells and promote apoptosis of the cells by inhibiting the action of caspase recruitment domain apoptosis inhibitor (ARC). Based on the above, the inventor guesses that miR-532-3p can induce more tumor cells to undergo apoptosis by regulating mitochondrial-mediated apoptosis signal pathways, so as to achieve the purpose of tumor treatment, and therefore, the miR-532-3p is expected to be an effective nucleic acid medicament for treating tumors. In the following examples and experiments, the technical scheme is further described by taking miR-532-3p as an example of miRNA.
In order to break through the technical barrier, the invention uses a cationic adsorbent polyvinyl alcohol (PVA) to mediate VB12-P L GA-PEG nano carrier and miR-532-3P to carry out electron adsorption to form a miR-532-3P @ P L GA-PEG-VB12 nano compound.
Example 1 Synthesis and physicochemical characterization of miR-532-3P @ P L GA-PEG-VB12 nanocomposite
(1) Material synthesis:
1) synthesis of P L GA-PEG-COOH derivatives A250 mg solution of P L GA was first reacted with NHS and EDC in dry dichloromethane (CH)2Cl2) Mixing under the condition; after 4 hours, the mixture was collected with precooled MeOH/Et2Washing with O (1:1) twice, drying in vacuum, dissolving dried pellets of P L GA-NHS in dry chloroform, and adding bifunctional modified NH2-PEG-COOH and diisopropylethylamine; after 24 hours, with MeOH/Et2Washing with O (1:1), and vacuum drying to obtain P L GA-PEG-COOH compound;
2) preparing VB 12-labeled P L GA-PEG nano-complex, namely adding the P L GA-PEG nano-complex (1mol) obtained in the step ①, vitamin B12(1mol) and EDC (1.1mol) into a butylene oxide solution containing or not containing DMAP (0.1mol), stirring for 4 hours at room temperature, and finally obtaining VB12 binding nano-carrier (P L GA-PEG-VB12) carrying nucleic acid after dialysis and freeze drying;
3) synthesis of miR-532-3P @ P L GA-PEG-VB12 nanocomposite, namely, P L GA-PEG-VB12(25mg), chloroform (0.8m L) and DC-cholesterol (choleestenol 3 β - [ N- (N, N-dimethyllaminoethane)) (1mg) are mixed in an ultrasonic stirrer until no precipitate exists, then, 1% of polyvinyl alcohol (PVA) (w/v,3m L) and miR-532-3P micics are added and stirred continuously for 6 hours, and finally, the miR-532-3P @ P L GA-PEG-VB12 nanocomposite is obtained through DEPC water washing and filtering (a filter with the size of 0.22 mu m) and stored at the temperature of-20 ℃.
(2) Infrared absorption spectroscopy inspection:
prior to analysis, the samples were mixed well with potassium bromide (KBr) and pressed into a granular mixture. It was then dissolved in DMSO-D6 and finally analyzed by Nicolet/Nexus 670FTIR analyzer to obtain an infrared spectrum of the material.
(3) Transmission electron microscopy:
and (3) dropwise adding the nano composite on a Cu net, plating a conductive gold film by using an ion sputtering instrument after the solution is evaporated at normal temperature, and observing the original particle size and shape of the nano particles by adopting a TEM (transmission electron microscope) under the accelerating voltage of 200 kV.
(4) Particle size and potential analysis:
the P L GA-PEG, miR-532-3P @ P L GA-PEG and miR-532-3P @ P L GA-PEG-VB12 nano-composite synthesized in the research institute is dispersed by using sufficient deionized water, and then the particle size and the potential of the nano-composite are detected and analyzed by a Marvin dynamic light scattering instrument.
FIG. 1 shows the physicochemical characteristics of P L GA-PEG-VB12 nanocomposite, A. transmission electron microscopy, B. infrared absorption spectrum, C. particle size and potential. from the transmission electron microscopy results of FIG. 1A, it can be seen that the miR-532-3P @ P L GA-PEG-VB12 nanocomposite, which is a spherical nanoparticle having a diameter of about 100nm, was successfully synthesized, and in the near infrared spectroscopy (FIG. 1B), the absorption peak of the C-O-C bond (1093.84 cm) was seen after DMAP modification, as compared with the DMAP-unmodified group-1And 1399.92cm-1) And absorption peak of C ═ O bond (1742.60 cm)-1) And specific ester group absorption peaks are obtained, which further indicates that vitamin B12 activated by DMAP can be subjected to esterification reaction with P L GA-PEG-COOH to successfully construct VB12-P L GA-PEG nano-carrier, and in addition, the particle size result of FIG. 1C reveals that even though P L GA-PEG nano-carrier is modified by VB12 and miR-532-3P, the particle size of the miR-532-3P @ P L GA-PEG-VB12 nano-complex is maintained at 107nm to ensure the high permeability and retention effect (EPR effect) of the nano-complex, and meanwhile, the potential of the miR-532-3P @ P L GA-PEG-VB12 nano-complex is-28 +/-1.5 mV, and the nano-complex of miR-532-3P carrying vitamin B12 binding type nano-complex (miR-532-3P @ P L-PEG-VB 12) is more easily transported in blood circulation.
Example 2 safety of VB12-P L GA-PEG nanocarriers
After co-incubation of P L GA-PEG and P L GA-PEG-VB12 nanocarriers (2-500ug/m L) at different concentrations with gastric normal mucosal epithelial cells (GES-1) for 24 hours, Cell Counting Kit (CCK-8) experiments were used to examine Cell survival rates.
FIG. 2 shows the safety results of the miR-532-3P @ P L GA-PEG-VB12 nano-composite. the CCK-8 results show that the cell survival rate of GES-1 cells is over 90 percent (FIG. 2) under the treatment of P L GA-PEG and VB12-P L GA-PEG nano-carriers at various concentrations ranging from 2 to 500 mu g/m L, so that the P L GA-PEG and VB12-P L GA-PEG nano-carriers are relatively safe nano-materials.
Example 3 targeting of the miR-532-3P @ P L GA-PEG-VB12 nanocomplex
(1) And laser confocal experiments, namely inoculating gastric cancer cells into a confocal dish, adding FITC-labeled miR-532-3P @ L ipo3000, miR-532-3P @ P L GA-PEG and miR-532-3P @ P L GA-PEG-VB12 for co-incubation, carrying out DAPI staining after 4 hours, and finally carrying out photographic analysis under a laser inverted microscope.
(2) An RT-qPCR experiment, namely inoculating gastric cancer cells into a six-well plate, adding miR-532-3P @ L ipo3000, miR-532-3P @ P L GA-PEG and miR-532-3P @ P L GA-PEG-VB12 for co-incubation, collecting the cells after 24 hours, carrying out the experimental steps of total RNA extraction, reverse transcription, fluorescent quantitative PCR and the like, and collecting data for analysis.
FIG. 3 is a laser confocal map, B.RT-qPCR experiment from the laser confocal experiment result (FIG. 3A), it can be known that the green fluorescence intensity in the cells of the miR-532-3P @ P L GA-PEG and miR-532-3P @ P L GA-PEG-VB12 treated groups is obviously higher than that in the cells of the traditional plasmid transfection group (miR-532-3P @ L ipo3000) at the same time point, and the fluorescence intensity in the cells of the miR-532-3P @ P L GA-PEG-VB12 treated group is highest, in addition, the RT-qPCR experiment (FIG. 3B) also shows the same trend, the miR-532-3P expression level in the cells of the miR-532-3P @ P5 GA-VB 12 treated group is highest, and the potential delivery value of drugs to gastric cancer cells is improved, and the VB12-P L-PEG-3P @ PEG-VB12 nano-carrier can carry more drugs and is delivered to gastric cancer cells.
Example 4 antitumor Effect of miR-532-3P @ P L GA-PEG-VB12 nanocomposite
(1) And CCK-8 cell proliferation experiments, namely inoculating gastric cancer cells into a 96-well plate, adding miR-532-3P @ L ipo3000, miR-532-3P @ P L GA-PEG and miR-532-3P @ P L GA-PEG-VB12 after the cells are attached to the wall, co-incubating for a period of time (24h,48h,72h and 96h), adding 10% of CCK-8 reagent into each group of wells, continuously incubating at 37 ℃ for 2h, and measuring the absorbance (A) value of each well with the 490nm wavelength in an enzyme labeling instrument.
(2) And detecting the apoptosis level, namely inoculating gastric cancer cells into a six-hole plate, adding miR-532-3P @ L ipo3000, miR-532-3P @ P L GA-PEG and miR-532-3P @ P L GA-PEG-VB12 for co-incubation, collecting cell precipitates after 24 hours, adding an apoptosis detection reagent, incubating for 15 minutes in a dark place, and finally detecting on a flow cytometer.
FIG. 4 shows the antitumor effect of the miR-532-3P @ P L GA-PEG-VB12 nano-composite, a. CCK-8 cell proliferation experiment, and B. cell apoptosis level detection, the results show that miR-532-3P @ P L GA-PEG and miR-532-3P @ P L GA-PEG-VB12 can more remarkably inhibit the proliferation capability of gastric cancer cells (FIG. 4A) and induce more gastric cancer cells to undergo apoptosis (FIG. 4B) compared with a control group and a plasmid transfection group, wherein the antitumor effect of miR-532-3P @ P L GA-PEG-VB12 is more remarkable.
Example 5: interaction relation of miR-532-3p and ARC
(1) Bioinformatic analysis: based on a miRBase (http:// www.mirbase.org Starbase) database, according to the secondary structure and free energy of RNA, the Starbase database data is utilized to predict the correlation coefficient and the possible binding site of miR-532-3p and ARC through biological information analysis.
(2) A dual-luciferase gene reporter experiment comprises the steps of inoculating 293T cells into a 96-well plate, transfecting the 293T cells with pmirG L O-ARC-WT or pmirG L O-ARC-MUT and miR-532-3p mimics or miR-532-3p NC under the assistance of L ipofectamine 3000 after the cells are attached to the wall, adding L uciferase Reagent II (L AR II) solution after incubating for 6 hours, detecting the Firefly luciferase value in a fluorescence instrument, and then adding Stop&
Reagent, detecting the value of Renilla luciferase in a fluorescence instrument; finally, taking Fireflyluciferase value as an internal reference, and cotransfecting 3' UTR-Wt and miR-532-3PNCThe group luciferase activities were normalized to 1 and the relative luciferase activities were calculated.
FIG. 5 is an experimental result of the interaction relationship between miR-532-3P and ARC to clarify the anti-tumor action mechanism of the miR-532-3P @ P L GA-PEG-VB12 nano-complex, the inventors first predict the binding site of miR-532-3P and downstream key gene ARC through a public database (FIG. 5A), and at the same time, the dual-luciferase gene report experimental result shows (FIG. 5B) that luciferase activity in the ARC-WT cell strain after miR-532-3P mimics is transfected is obviously reduced.
Example 6 anti-tumor action mechanism of miR-532-3P @ P L GA-PEG-VB12 nano-complex:
(1) and mitochondrial function detection, namely inoculating gastric cancer cells into a 6-well plate, respectively treating the cells by miR-532-3pNC @ P L GA-PEG-VB12 and miR-532-3P @ P L GA-PEG-VB12, collecting the cells after 24 hours, respectively adding a JC-1 detection kit, a mitoROS detection kit, an ATP detection kit and a mPTP detection kit into the cells to respectively detect the intracellular mitochondrial membrane potential (MTP), the mitoROS generation level, the ATP production amount and the mPTP openness degree, constructing an ARC over-expressed gastric cancer cell line by a plasmid transfection technology, carrying out a recovery experiment, and observing whether the damage of miR-532-3P @ P L GA-PEG-VB12 to the mitochondrial function can be reduced after the ARC expression level in the gastric cancer cells is adjusted upwards.
(2) And detecting an apoptosis pathway, namely inoculating gastric cancer cells into a 6-well plate, respectively treating the cells by miR-532-3P NC @ P L GA-PEG-VB12 and miR-532-3P @ P L GA-PEG-VB12, collecting the cells after 24 hours, extracting, electrophoresing, transferring membranes, closing and the like of total proteins, respectively incubating with antibodies such as cytoC, caspase 9, cleared caspase 9, caspase 3, cleared caspase 3 and the like, then incubating with an HRP-labeled goat anti-rabbit IgG secondary antibody, finally carrying out photographic analysis under the action of a developing solution, and comparing the expression levels of related proteins in the cells.
FIG. 6 is the experimental results of the anti-tumor mechanism of action of miR-532-3P @ P L GA-PEG-VB12 nanocomplex A. mitochondrial membrane potential (MTPs), B. mitochondrial ROS (mitoROS) production level, C. intracellular ATP production level, D. mitochondrial membrane permeability (mPTP), E. apoptosis through the expression level of the associated proteins the results show that treatment of miR-532-3P @ P L GA-PEG-VB12 can down-regulate mitochondrial membrane potential in gastric cancer cells (FIG. 6A), induce mitochondrial ROS miteros (mROS) production (FIG. 6B), inhibit intracellular ATP synthesis (FIG. 6C), increase opening of mitochondrial permeability transition pore (mPTP) (FIG. 6C), thereby promoting cytochrome C (cytoc) release, activating caspase-dependent apoptosis pathway (FIG. 6D), furthermore, after up-regulating intracellular ARC 532 expression level, miR-3P @ P can reduce the down-dependent mitochondrial apoptosis of miR-3 GA-VB 32, thereby increasing the intracellular mitochondrial ROS-dependent mitochondrial ROS-GG-GCA 12.
Claims (10)
1. A method for synthesizing vitamin B12 conjugated nano-carrier comprises the following steps:
1) mixing and dissolving P L GA-PEG nano-carrier, vitamin B12, EDC and DMAP in an anhydrous solvent A, and stirring for reaction at 15-40 ℃;
2) after the reaction is finished, dialyzing and removing the solvent to obtain the vitamin B12 combined nano-carrier which is recorded as VB12-P L GA-PEG nano-carrier.
2. The synthesis method of claim 1, wherein the molar ratio of the P L GA-PEG nanocarrier to the vitamin B12 is 1 (1-1.2).
3. The synthesis method of claim 1, wherein the molar ratio of P L GA-PEG nano-carrier to EDC to DMAP is 1 (1-1.2) to (0.05-0.12).
4. The method for synthesizing the P L GA-PEG nano-carrier according to claim 1, wherein the method for synthesizing the P L GA-PEG nano-carrier comprises the following steps:
1) mixing P L GA, NHS and EDC in an anhydrous solvent B, activating, fully reacting, washing by using an anhydrous solvent C to remove unreacted micromolecules, and drying to obtain P L GA-NHS pellets, wherein the molar ratio of P L GA to NHS to EDC is 1 (1-1.2) to 1-1.2;
2) dissolving P L GA-NHS pellets in an anhydrous solvent D, and adding bifunctional modified NH2-PEG-COOH and diisopropylethylamine;
3) after the reaction is finished, washing and drying to obtain the P L GA-PEG nano carrier.
5. The synthesis method according to any one of claims 1 to 4, characterized in that: the anhydrous solvent A is selected from butylene oxide.
6. The synthesis method of claim 4, wherein the P L GA-PEG nanocarrier is loaded with miRNA, and the synthesis method is as follows:
1) dispersing P L GA-PEG and cholestrol 3 β - [ N- (N, N-dimethylxanthoethane) in an anhydrous solvent D, and stirring until no precipitate exists to obtain a P L GA-PEG dispersion liquid;
2) adding a mixed solution of polyvinyl alcohol and miRNA mimics into the P L GA-PEG dispersion solution, and stirring for reaction;
3) and after the reaction is finished, washing with deionized water, and filtering to obtain the P L GA-PEG nano composite loaded with miRNA mics, which is marked as miRNA @ P L GA-PEG nano composite.
7. The synthesis method of claim 1, wherein the method for further loading miRNA in VB12-P L GA-PEG nanocarrier comprises:
1) dispersing VB12-P L GA-PEG and cholestrol 3 β - [ N- (N, N-dimethyllaminoethane) in an anhydrous solvent D, and stirring until no precipitate exists to obtain a VB12-P L GA-PEG dispersion liquid;
2) adding a mixed solution of polyvinyl alcohol and miRNA mimics into VB12-P L GA-PEG dispersion liquid, and stirring for reaction;
3) after the reaction is finished, washing with deionized water, and filtering to obtain a VB12-P L GA-PEG nano compound loaded with miRNA mimics, which is marked as miRNA @ P L GA-PEG-VB12 nano compound.
8. The synthesis method according to claim 6 or 7, characterized in that: the miRNA mimics is miR-532-3 pmimics.
9. The method of synthesis according to claim 4, 6 or 7, characterized in that: the anhydrous solvent B and the anhydrous solvent D are independently selected from dichloromethane and trichloromethane.
10. The method of synthesis according to claim 4, characterized in that: the anhydrous solvent C is a mixed solution of methanol and diethyl ether.
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Application publication date: 20200731 Assignee: Guangdong Bonn Life Sciences Co.,Ltd. Assignor: NANFANG HOSPITAL, SOUTHERN MEDICAL University Contract record no.: X2024980002452 Denomination of invention: Synthesis method and application of VB12 binding nanocomposites carrying miRNA Granted publication date: 20220104 License type: Common License Record date: 20240304 |