CN110964816A - Solution containing blood-stable nanoparticles, preparation method thereof and detection method of miRNA marker - Google Patents

Solution containing blood-stable nanoparticles, preparation method thereof and detection method of miRNA marker Download PDF

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CN110964816A
CN110964816A CN201911144488.4A CN201911144488A CN110964816A CN 110964816 A CN110964816 A CN 110964816A CN 201911144488 A CN201911144488 A CN 201911144488A CN 110964816 A CN110964816 A CN 110964816A
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mirna
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nanoparticle solution
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赵鹏飞
林泉
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Shenzhen Kunpeng Future Technology Co Ltd
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    • C08J2367/00—Characterised by the use of polyesters obtained by reactions forming a carboxylic ester link in the main chain; Derivatives of such polymers
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Abstract

The present invention provides a nanoparticle solution comprising polymeric micellar nanoparticles having blood stability, in particular nanoparticles modified with a functional molecule such as a nucleotide sequence. The invention also provides a method for preparing the nanoparticle solution and a method for detecting a micro RNA (miRNA) marker by using the nanoparticle solution.

Description

Solution containing blood-stable nanoparticles, preparation method thereof and detection method of miRNA marker
Technical Field
The invention relates to the field of nano materials and medical nanoprobes, in particular to a nano particle solution which comprises polymer micelle nano particles with blood stability, particularly nano particles modified with functional molecules such as nucleotide sequences. The invention also relates to a method for preparing the nanoparticle solution and a method for detecting a micro RNA (miRNA) marker by using the nanoparticle solution.
Background
Due to the unique physical and chemical properties of the nano material, the nano material has good application prospects in the medical field, particularly in the aspects of drug delivery, medical detection, biosensing, medical material modification and the like.
In the biomedical diagnosis application of nano materials, nano particles are adopted to detect disease markers, and diseases are diagnosed by comparing changes of fluorescence, color, aggregation state and the like of the nano particles before and after detection. Among them, nanoparticles using biodegradable polymers and natural phospholipids as raw materials have been widely used in drug delivery and biosensing applications due to their good biocompatibility and cycling stability.
The early screening, early diagnosis and treatment of cancer patients can obviously improve the treatment survival rate of the patients, and has important significance for the clinical treatment of cancer. Monoclonal antibodies against characteristic secretions and specific antigens of cancer cells have been used for clinical screening of early cancers due to their advantages of convenient detection method, high detection sensitivity, and the like. However, the detection of tumor secretion is greatly influenced by individual difference, and the detection of cancer cell monoclonal antibody has the disadvantages of high detection cost, more interference factors, poor detection stability and the like, and also limits the clinical application of the monoclonal antibody.
mirnas are non-coding single-stranded RNA molecules 20-24 nucleotides in length that regulate gene expression by regulating gene translation processes. Among them, mirnas capable of regulating processes such as cancer cell immortalization, immune escape, cell migration, and angiogenesis are becoming specific for cancer detection and treatment. In addition, miRNA has the characteristics of high specificity, conservative sequence and the like, and is a potential choice for realizing ultra-early and ultra-sensitive in-vitro diagnosis of cancer.
The existing cancer early diagnosis technology has the defects of poor detection specificity, unstable detection result, high detection cost and the like, and is difficult to popularize in cancer early screening. On the other hand, the traditional miRNA detection means mainly comprises Northern hybridization, microarray analysis and fluorescent quantitative PCR, and the detection means is time-consuming and labor-consuming, complex in process, high in processing technical requirement and high in cost, and cannot meet the requirement of clinical cancer early screening.
The rapid and efficient miRNA detection is realized by means of the high-stability and high-safety nano material, and the method has important application and research significance.
Disclosure of Invention
The invention aims to provide a polymer micelle nanoparticle solution which is simple in preparation method, high in blood stability and good in biological safety. The nanoparticle solution and the detection method can promote the miRNA detection technology to be more convenient and efficient, and are suitable for the ultra-early and ultra-sensitive in-vitro detection of various diseases, particularly cancer markers.
The present invention includes the following aspects:
according to a first aspect of the present invention, there is provided a nanoparticle solution comprising polymer micelle nanoparticles having blood stability, the nanoparticles containing polyglycolide-lactide-polyethylene glycol block copolymer (PLGA-PEG-COOH) and distearoylphosphatidylethanolamine-polyethylene glycol block copolymer (DSPE-PEG-COOH) in a mass ratio of 20:1 to 5: 1; in another embodiment, the nanoparticles consist of polyglycolide-lactide-polyethylene glycol block copolymer (PLGA-PEG-COOH) and distearoylphosphatidylethanolamine-polyethylene glycol block copolymer (DSPE-PEG-COOH) in a mass ratio of 20:1 to 5: 1.
Preferably, the nanoparticle solution of the present invention comprises nanoparticles having functional molecules bound to the surface thereof, such as nucleotide sequences complementary to specific miRNA markers, targeting molecules capable of targeting tumor cells, antibodies capable of binding to specific antigens, and the like.
Preferably, the nanoparticle solution of the present invention contains nanoparticles having a particle size of 30 to 200nm and a surface charge of-20 to-50 mV.
Preferably, in the nanoparticle solution of the present invention, the functional molecule bound to the surface of the nanoparticle is a nucleotide sequence that can be complementarily paired with a specific miRNA marker. After the nucleotide sequence is complementary and paired with the specific miRNA marker to form a nucleotide double strand, the adriamycin molecule can be embedded into the nucleotide double strand, so that the embedded adriamycin molecule is subjected to fluorescence quenching in fluorescence intensity detection. More preferably, the nucleotide sequence is a single-stranded DNA with 5' terminal amination which can be combined with the carboxyl on the surface of the nanoparticle through a chemical crosslinking method.
In an embodiment of the invention, the miRNA markers include: cancer miRNA markers, such as miR-96-5p, miRNA-23b, miRNA-31, miRNA-21, miRNA-152, miRNA-451; characteristic miRNAs of rheumatoid arthritis, such as miRNA-16, miRNA-155; a miRNA characteristic of tuberculosis, such as miRNA-150.
According to a second aspect of the present invention there is provided a method of detecting a specific miRNA marker in a sample using a nanoparticle solution according to the first aspect of the present invention, the method comprising the steps of:
(1) adding a micro sample (<0.1mL) into a nanoparticle solution, uniformly mixing, and standing at normal temperature for 10-60 min, wherein a nucleotide sequence which can be complementarily paired with the specific miRNA marker is combined on the surface of a nanoparticle in the nanoparticle solution;
(2) adding 10-100 mu M of adriamycin molecules into the mixed solution obtained in the step (1), and uniformly mixing;
(3) and (3) detecting the fluorescence intensity of the adriamycin in the mixed solution obtained in the step (2), wherein the detection result is positive if the fluorescence intensity of the adriamycin molecules is obviously reduced or disappeared compared with that of a negative control group, and the detection result is negative if the fluorescence intensity is not changed. In vitro cell experiments, a mixed solution can be prepared as a negative control using an equal amount of PBS instead of the sample. In human blood detection experiments, a healthy human blood sample can be used as a negative control.
In an embodiment of the invention, the miRNA markers include: cancer miRNA markers, such as miR-96-5p, miRNA-23b, miRNA-31, miRNA-21, miRNA-152, miRNA-451; characteristic miRNAs of rheumatoid arthritis, such as miRNA-16, miRNA-155; a miRNA characteristic of tuberculosis, such as miRNA-150.
According to a third aspect of the present invention, there is provided use of the nanoparticle solution according to the first aspect of the present invention for the preparation of a test agent for the detection of diseases including cancer, rheumatoid arthritis, tuberculosis and the like.
According to a fourth aspect of the present invention, there is provided a method for preparing a nanoparticle solution, comprising the steps of:
(1) mixing a polyglycolide-lactide-polyethylene glycol block copolymer (PLGA-PEG-COOH) and a distearoyl phosphatidyl ethanolamine-polyethylene glycol block copolymer (DSPE-PEG-COOH) in a mass ratio of 20: 1-5: 1 in a first solution;
(2) dropwise adding the mixture obtained in the step (1) into a 4% ethanol water solution, and simultaneously carrying out ultrasonic mixing at the power of 60-150W to promote the self-assembly of PLGA-PEG-COOH and DSPE-PEG-COOH to obtain a polymer micelle solution;
(3) adding 1- (3-bis 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxythiosuccinimide (NHS) into the polymer micelle solution in sequence, and binding a 5' -end aminated DNA single strand which can be complementarily paired with a specific miRNA marker to form a nucleotide double strand onto the surface of the micelle through a chemical crosslinking method, wherein the molar ratio of the added EDC and NHS to the polymer in the solution is in the range of 5: 1-30: 1 respectively;
(4) and removing free molecular impurities by using a dialysis bag with the molecular weight cutoff of 10-100 kD through a dialysis method to obtain a purified nanoparticle solution.
In further embodiments, the first solution is chloroform, acetonitrile or dimethyl sulfoxide.
The term "polymeric micelle" as used herein has the usual meaning in the polymer and nanomaterial arts and generally refers to a micelle structure formed by polymers having amphiphilic segments in a solution system. In the context of the present invention, the terms "polymeric micelle," "polymeric nanomicelle," "polymeric micellar nanoparticle," "nanoparticle," and the like are used interchangeably.
The invention also includes any combination of the above embodiments. Equivalent substitutions of various components, elements, steps in the embodiments are also encompassed by the invention.
Compared with the prior art, the invention has the advantages that:
1. the nano-particle solution disclosed by the invention is low in preparation cost, simple in preparation method, excellent in system blood stability and extremely high in detection specificity and sensitivity.
2. The fluorescence quantitative detection method for miRNA has the advantages of low detection cost, simple clinical operation, accurate and sensitive detection result, realization of batch and automatic detection, and the like.
3. The invention can be used for personalized design and preparation of miRNA markers of different diseases, particularly cancers, and is beneficial to high sensitivity and ultra-early accurate in-vitro diagnosis of cancers.
4. The polymer micelle nano-particle containing the polyglycolide-lactide-polyethylene glycol block copolymer (PLGA-PEG-COOH) and the distearoyl phosphatidyl ethanolamine-polyethylene glycol block copolymer (DSPE-PEG-COOH) can be modified by various functional molecules (such as a nucleotide sequence which can be complementarily matched with a specific miRNA marker, a targeting molecule which can target tumor cells, an antibody which can be combined with a specific antigen and the like) as required, so that various medical effects are realized.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings needed to be used in the description of the embodiments are briefly introduced below, and it is obvious that the drawings in the following description are some embodiments of the present application, and it is obvious for those skilled in the art to obtain other drawings based on these drawings without creative efforts.
FIG. 1 is a graph of the particle size distribution of nanoparticles of the present invention;
FIG. 2 is a transmission electron micrograph of a nanoparticle of the present invention;
FIG. 3 is a graph showing the effect of different PLGA-PEG-COOH and DSPE-PEG-COOH mass ratios on nanoparticle particle size and polydispersity;
FIG. 4 is a graph of the effect of DSPE-PEG-COOH on the particle size stability of nanoparticles;
FIG. 5 is a comparison of fluorescence intensity values after detection of negative control and colorectal cancer supernatant;
FIG. 6 shows fluorescence spectra of doxorubicin.
Detailed Description
Inventive preparation example 1
10mg of PLGA-PEG-COOH (produced by Creative PEGworks, USA, the product number DLG-5k20k21) and 1mg of DSPE-PEG-COOH (produced by Avanti Polar lipids, USA, the product number 880135) were dissolved in 2mL of chloroform and mixed uniformly; dropwise adding the chloroform solution into a 4% ethanol aqueous solution, and continuously performing ultrasonic treatment for 5min at the frequency of 20kHz and the power of 90W by using a cell ultrasonic crusher while dropwise adding, so as to promote the self-assembly of the two block copolymers and promote the volatilization of the chloroform solution, thereby obtaining the polymer micelle solution.
1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (Merck sigma, Germany, cat # E6383) and N-hydroxysuccinimide (NHS) (Merck sigma, Germany, cat # 130672) were added sequentially (please add the amounts of EDC and NHS), and a single strand of 5 ' aminated complementary DNA (purchased from Shanghai Biotechnology Co., Ltd.) corresponding to cDNA (SEQ ID NO: 5'-GGGAGCTCATAGGCCGGCCCCTGGCTGCAGATG-3') as a custom-synthesized colorectal cancer marker miR-96-5p was chemically crosslinked to carboxyl groups on the micelle surface via EDC/NHS. And dialyzing the mixed solution for 8h by using a dialysis bag with the molecular weight cutoff of 10kD, and replacing the dialyzate for 3 times in the period to obtain the purified nano-particles. The particle size of the nano-particles detected by a particle size analyzer is 98.6nm (figure 1), the surface potential is-28.4 mV, and the result of a transmission electron microscope shows that the nano-particles are in spherical monodispersion (figure 2).
Inventive preparation example 2
Nanoparticle solutions were prepared according to preparative example 1 of the present invention, except that: the mass ratio of PLGA-PEG-COOH to DSPE-PEG-COOH is 5: 1; carrying out ultrasonic treatment at 60W power in the self-assembly process; the coupling was performed using a dialysis bag with a molecular weight cut-off of 20 kD.
Inventive preparation example 3
Nanoparticle solutions were prepared according to preparative example 1 of the present invention, except that: the mass ratio of PLGA-PEG-COOH to DSPE-PEG-COOH is 20: 1; carrying out ultrasonic treatment at 130W power in the self-assembly process; the coupling was performed using dialysis bags with a molecular weight cut-off of 50 kD.
Comparative preparation example 1
Nanoparticle solutions were prepared according to preparative example 1 of the present invention, except that: the mass ratio of PLGA-PEG-COOH to DSPE-PEG-COOH is 1: 1.
Comparative preparation example 2
Nanoparticle solutions were prepared according to preparative example 1 of the present invention, except that: the mass ratio of PLGA-PEG-COOH to DSPE-PEG-COOH was 30: 1.
As can be seen from FIG. 3, the mass ratio of PLGA-PEG-COOH to DSPE-PEG-COOH is in the range of 20: 1-5: 1, the size distribution of the nanoparticles is more uniform, and the particle size is smaller.
Comparative preparation example 3
A nanoparticle solution was prepared according to preparation example 1 of the present invention, but nanoparticles were prepared using PLGA-PEG-COOH alone without adding DSPE-PEG-COOH.
As can be seen from fig. 4, the single PLGA-PEG-COOH prepared nanoparticles have low size uniformity and system stability, and the particles spontaneously aggregate, which is not favorable for long-term storage of the reagent and may lead to unreliable detection results. Nanoparticles formed by using PLGA-PEG-COOH and DSPE-PEG-COOH together (mass ratio of 10:1) have high size uniformity and can be stable for a long time.
Detection of miRNA markers
Detection 1: 100 μ L of the nanoparticle solution obtained in the preparation example of the present invention was added to a 96-well plate, 100 μ L of the supernatant of Caco-2 human colorectal cancer cell culture fluid (negative control group was 100 μ L of PBS) was added to the nanoparticle solution, and after standing at room temperature for 30min, 5 μ M of doxorubicin molecule (purchased from Merck sigma, germany, under the product number D1515) was added and mixed well, and the fluorescence intensity of doxorubicin at 597nm was measured using a multifunctional microplate reader (fig. 5). It can be seen that in the mixed solution added with the Caco-2 human colorectal cancer cell culture supernatant, the fluorescence intensity of the adriamycin is remarkably reduced, which indicates that the adriamycin is subjected to fluorescence quenching, and indicates that the detection result of the sample is positive.
And (3) detection 2: the difference of the preparation of the nanoparticle solution according to the method of the preparation example of the present invention is that a DNA sequence which can be complementarily paired with the bladder cancer marker miRNA-23b is bonded to the surface of the nanoparticle. And (3) detecting by referring to the method of detection 1, adding the supernatant of the J82 human bladder cancer cell culture solution into the nanoparticle solution, mixing, adding trace adriamycin into the mixed sample, and detecting the fluorescence intensity of the adriamycin of the sample by using an enzyme-labeling instrument, wherein the fluorescence intensity is obviously reduced.
And (3) detection: the difference of the preparation of the nanoparticle solution by referring to the method of the preparation embodiment of the invention is that the surface of the nanoparticle is bonded with a DNA sequence which can be complementarily paired with the lung cancer marker miRNA-31. And (3) detecting by referring to the method of detection 1, adding the culture solution supernatant of the NCI-H226 human lung cancer cell into the nanoparticle solution for mixing, then adding trace adriamycin into the mixed sample, and detecting the fluorescence intensity of the adriamycin of the sample by using an enzyme-labeling instrument, wherein the fluorescence intensity is obviously reduced.
And (4) detection: the difference of the preparation of the nanoparticle solution by referring to the method of the preparation embodiment of the invention is that the surface of the nanoparticle is bonded with a DNA sequence which can be complementarily paired with the liver cancer marker miRNA-21. And (3) detecting according to the method of detection 1, adding the supernatant of the HepG2 human liver cancer cell culture solution into the nanoparticle solution, mixing, adding trace adriamycin into the mixed sample, and detecting the fluorescence intensity of the adriamycin of the sample by using an enzyme-labeling instrument, wherein the fluorescence intensity is obviously reduced.
And (5) detection: the method of the preparation example of the present invention is referred to prepare nanoparticle solution, and the difference is that the surface of the nanoparticle is bonded with a DNA sequence which can be complementarily paired with the cervical cancer marker miRNA-152. And (3) detecting according to the method of detection 1, adding the supernatant of the HeLa human cervical carcinoma cell culture solution into the nanoparticle solution for mixing, then adding trace adriamycin into the mixed sample, and detecting the adriamycin fluorescence intensity of the sample by using an enzyme-labeling instrument, wherein the fluorescence intensity is obviously reduced.
And (6) detection: the method of the preparation example of the present invention is referred to prepare nanoparticle solution, except that a DNA sequence that can be complementarily paired with the gastric cancer marker miRNA-451 is bonded to the surface of the nanoparticle. And (3) detecting according to the method of detection 1, adding the supernatant of the MKN45 human gastric cancer cell culture solution into the nanoparticle solution, mixing, adding trace adriamycin into the mixed sample, and detecting the adriamycin fluorescence intensity of the sample by using an enzyme-labeling instrument, wherein the fluorescence intensity is obviously reduced.
And (7) detection: the difference of the preparation of the nanoparticle solution by referring to the method of the preparation embodiment of the invention is that a DNA sequence which can be complementarily paired with the glioma marker miRNA-21 is bonded on the surface of the nanoparticle. The detection is carried out according to the method of detection 1, the culture solution supernatant of the U87MG human glioblastoma cell is added into the nanoparticle solution for mixing, then trace doxorubicin is added into the mixed sample, and the fluorescence intensity of the doxorubicin of the sample is detected by using a microplate reader, so that the fluorescence intensity is obviously reduced.
And (8) detection: a nanoparticle solution was prepared according to the method of the preparation example of the present invention, except that a DNA sequence complementary-paired with miRNA-16 characteristic of rheumatoid arthritis was bonded to the surface of the nanoparticle. Mixing the nanoparticle solution with hydrarthrosis of patients with rheumatoid arthritis. The detection was carried out by referring to the method of detection 1, in which a trace amount of doxorubicin was added to the mixed sample of this example, and the fluorescence intensity of the sample was detected by a microplate reader, and the fluorescence intensity was significantly decreased.
And (9) detection: the nanoparticle solution was prepared according to the method of the preparation example of the present invention, except that a DNA sequence that can be complementarily paired with miRNA-150 characteristic of tuberculosis was bonded to the surface of the nanoparticle. The nanoparticle solution is mixed with a peripheral blood sample of a patient with tuberculosis. The detection was carried out by referring to the method of detection 1, in which a trace amount of doxorubicin was added to the mixed sample of this example, and the fluorescence intensity of the sample was detected by a microplate reader, and the fluorescence intensity was significantly decreased.

Claims (11)

1. A nanoparticle solution, characterized by: the nanoparticle solution comprises polymer micelle nanoparticles with blood stability, wherein the nanoparticles contain polyglycolide-lactide-polyethylene glycol block copolymer (PLGA-PEG-COOH) and distearoyl phosphatidyl ethanolamine-polyethylene glycol block copolymer (DSPE-PEG-COOH) in a mass ratio of 20: 1-5: 1.
2. The nanoparticle solution of claim 1, wherein the surface of the nanoparticles is bound with functional molecules, such as nucleotide sequences that can be complementarily paired with specific miRNA markers, targeting molecules that can target tumor cells, antibodies that can bind to specific antigens, and the like.
3. The nanoparticle solution as set forth in claim 2, wherein the nanoparticles have a particle size of 30 to 200nm and a surface charge of-20 to-50 mV.
4. The nanoparticle solution of claim 2 or 3 wherein the functional molecule is a nucleotide sequence that can be complementarily paired to a specific miRNA marker; and after the nucleotide sequence is complementary and paired with the specific miRNA marker to form a nucleotide double strand, the adriamycin molecule can be embedded into the nucleotide double strand, so that the embedded adriamycin molecule is subjected to fluorescence quenching in fluorescence intensity detection.
5. The nanoparticle solution as claimed in claim 4, wherein the nucleotide sequence is a single-stranded DNA with 5' terminal amination capable of binding to the carboxyl group on the surface of the nanoparticle by a chemical cross-linking method.
6. The nanoparticle solution of claim 4, wherein the miRNA markers comprise: cancer miRNA markers, such as miR-96-5p, miRNA-23b, miRNA-31, miRNA-21, miRNA-152, miRNA-451; characteristic miRNAs of rheumatoid arthritis, such as miRNA-16, miRNA-155; a miRNA characteristic of tuberculosis, such as miRNA-150.
7. A method for detecting specific miRNA markers in a sample using the nanoparticle solution of any one of claims 4-6, the method comprising the steps of:
(1) adding a trace sample into a nanoparticle solution, uniformly mixing, and standing at normal temperature for 10-60 min, wherein a nucleotide sequence which can be complementarily paired with the specific miRNA marker is combined on the surface of a nanoparticle in the nanoparticle solution;
(2) adding 10-100 mu M of adriamycin molecules into the mixed solution obtained in the step (1), and uniformly mixing;
(3) and (3) detecting the fluorescence intensity of the adriamycin in the mixed solution obtained in the step (2), wherein the detection result is positive if the fluorescence intensity of the adriamycin molecules is obviously reduced or disappeared compared with that of a negative control group, and the detection result is negative if the fluorescence intensity is not changed.
8. The method of claim 7, wherein the miRNA markers comprise: cancer miRNA markers, such as miR-96-5p, miRNA-23b, miRNA-31, miRNA-21, miRNA-152, miRNA-451; characteristic miRNAs of rheumatoid arthritis, such as miRNA-16, miRNA-155; a miRNA characteristic of tuberculosis, such as miRNA-150.
9. Use of the nanoparticle solution of any one of claims 4-6 in the preparation of a test agent for the detection of diseases including cancer, rheumatoid arthritis, tuberculosis, and the like.
10. A method for preparing a nanoparticle solution comprising the steps of:
(1) mixing a polyglycolide-lactide-polyethylene glycol block copolymer (PLGA-PEG-COOH) and a distearoyl phosphatidyl ethanolamine-polyethylene glycol block copolymer (DSPE-PEG-COOH) in a mass ratio of 20: 1-5: 1 in a first solution;
(2) dropwise adding the mixture obtained in the step (1) into a 4% ethanol water solution, and simultaneously carrying out ultrasonic mixing at the power of 60-150W to promote the self-assembly of PLGA-PEG-COOH and DSPE-PEG-COOH to obtain a polymer micelle solution;
(3) adding 1- (3-bis 1- (3-dimethylaminopropyl) -3-Ethylcarbodiimide (EDC) and N-hydroxythiosuccinimide (NHS) into the polymer micelle solution in sequence, and binding a 5' -end aminated DNA single strand which can be complementarily paired with a specific miRNA marker to form a nucleotide double strand onto the surface of the micelle through a chemical crosslinking method, wherein the molar ratio of the added EDC and NHS to the polymer in the solution is in the range of 5: 1-30: 1 respectively;
(4) and removing free molecular impurities by using a dialysis bag with the molecular weight cutoff of 10-100 kD through a dialysis method to obtain a purified nanoparticle solution.
11. The method of claim 10, wherein the first solution is chloroform, acetonitrile or dimethyl sulfoxide.
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