CN112852920A - DNA zipper molecule modified liposome vesicle and preparation method and application thereof - Google Patents
DNA zipper molecule modified liposome vesicle and preparation method and application thereof Download PDFInfo
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Abstract
The invention provides a DNA zipper molecule modified liposome vesicle and a preparation method and application thereof, wherein the liposome vesicle is composed of a DNA zipper molecule modified bilayer membrane and a molecular probe wrapped in the bilayer membrane, wherein the DNA zipper molecule has a DNA zipper sequence for promoting liposome fusion, and the DNA zipper molecule and an exosome modified by the DNA zipper molecule are subjected to membrane fusion rapidly under the action of a DNA zipper, so that the molecular probe contained in the vesicle is delivered into the exosome, and the in-situ, accurate and rapid detection of a nucleic acid-containing biomarker in the exosome is realized; the preparation method of the liposome vesicle comprises the following steps: mixing the bilayer membrane with a molecular probe, extruding to obtain liposome vesicles, and incubating with the lipophilic group-modified DNA zipper molecules; according to the invention, the DNA zipper molecules modified on the surface of the liposome vesicle are actively fused with the exosomes, so that the efficiency is higher, the nucleic acid marker to be detected does not need to be separated from the exosomes in advance, and the detection effect is better.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to a DNA zipper molecule modified liposome vesicle and a preparation method and application thereof.
Background
Exosomes are receiving more and more attention because they contain important intercellular information exchange molecules in the organism, and they can be used in a variety of fields such as biological detection and diagnostic treatment. Exosomes originally refer to 40-100nm vesicular structures formed by fusion of vesicles with plasma membranes secreted during reticulocyte differentiation. The outer layer of exosomes is phospholipidsThe bilayer, which contains proteins, mRNA, miRNA, DNA fragments, etc. associated with the source cell, is an important basis for the exosomes to participate in the communication of intercellular information and substances. Exosomes are widely present in blood, saliva, urine, tears, cerebrospinal fluid, and the like, and have a high abundance. Particularly, it is noted that miRNA contained in exosome plays a very important role in intercellular communication, and becomes an important influencing factor for regulating biological activity of receptor cells and phenotypic change of cells. For example, tumor-derived exosomes usually contain a large amount of tumor-associated bioactive substances, such as tumor-associated mRNA, miRNA, etc., and the content of miRNA is very similar to that of cancer cells. The existing research shows that miRNA in exosome actively participates in the processes of tumor growth, metastasis, tumor immune escape, formation of tumor microenvironment and the like, for example, miR-214 secreted by tumor is transmitted to receptor CD4 through exosome+T cells, leading to CD4+The down-regulation of PTEN protein in T cells, the expansion of regulatory T cells, ultimately leads to immune escape of the host and rapid growth of the tumor. Although the function and related molecular mechanism of miRNA contained in exosome in tumor environment are still to be further improved, a great deal of research has proved that miRNA in exosome can be used as an important tumor marker for early diagnosis of cancer.
The existing clinical tumor diagnosis is mainly divided into: image diagnosis, tissue biopsy, and fluid biopsy. Among them, the most mature of image diagnosis and tissue biopsy are difficult to be applied to early diagnosis of tumor and to obtain more comprehensive tumor-related information due to its hysteresis and invasiveness. Therefore, the later developed liquid biopsy, a non-invasive one, is expected to obtain more comprehensive information, and a method with simple sample source is very promising. Fluid biopsies can be further classified into circulating tumor cells, circulating tumor nucleic acids, and exosome assays. According to statistics, only 1-10 tumor cells can be detected in each milliliter of blood, which brings great difficulty to the separation and detection of the tumor cells. The amount of circulating tumor nucleic acid that can be detected in the blood by the presence of nucleases is also minimalThis also negatively affects the accuracy of the detection. The exosome has high abundance in blood, and about 10 can be detected per milliliter9And protects the internal nucleic acid molecules from nuclease degradation during blood circulation due to its protection with a phospholipid bilayer membrane. Therefore, the analysis and detection of the nucleic acid molecules in the exosome are beneficial to the diagnosis of diseases and the analysis of curative effects, and have extremely important scientific research and clinical significance.
The detection of nucleic acid molecules in the existing exosomes mainly depends on a mode of crushing and collecting internal nucleic acid after separation. The common exosome separation methods include differential centrifugation, density gradient centrifugation, size exclusion chromatography, and the like. These methods solve the problem of exosome collection, but have certain limitations. Differential centrifugation separates each component in a sample by using different rotating speeds, but the finally obtained exosome is mixed with more other impurities due to the existence of various vesicles and protein aggregates in plasma, and in addition, the step of final ultra-high speed centrifugation easily causes the damage of the exosome; density gradient centrifugation is the combination of ultracentrifugation and sucrose density gradient to separate exosomes from non-vesicular particles, but impurities with similar densities can still be found in the extracted product; size exclusion is based on the size of the molecule rather than the molecular weight, but this method is tedious and not suitable for the processing of large numbers of samples. However, to detect a specific nucleic acid in an exosome, not only is the exosome isolated and collected, but also the exosome is subsequently disrupted to detect the nucleic acid therein. The exposure of nucleic acid is easy to cause nuclease degradation, and the accuracy of the detection result is influenced.
The molecular probe for detecting nucleic acid molecules includes molecular beacon, nucleic acid fluorescent probe, fluorescent flare probe based on nanometer particle, etc. Taking a molecular beacon as an example, the molecular beacon is a fluorescent probe with a special hairpin structure, one end of the fluorescent probe is provided with a fluorescent group, and the other end of the fluorescent probe is provided with a corresponding quenching group. After the detection molecule binds to the molecular probe, the quenched fluorescent signal is released to detect the corresponding nucleic acid sequence. The method is widely applied to aspects of gene screening, biosensor development, nucleotide detection and the like. The molecular beacon is endowed with excellent sensitivity and selectivity by the thermal stability of the hairpin structure, the high efficiency of internal signal transfer and the designability of the hairpin structure sequence, can detect a target sequence under the condition of not separating an unbound probe, and provides possibility for real-time detection of nucleic acid. Through the incubation of the molecular beacon and the exosome, the molecular beacon passively diffuses into the exosome, and the detection of a tumor marker miR-21 in the exosome can be realized. However, the single molecular beacon has low transmembrane efficiency, and may not ensure the detection accuracy. The diffusion efficiency of the molecular beacon can be improved by adding the enzyme for increasing the membrane permeability of the exosome, but the leakage of the biomarker in the exosome can be brought, and errors are brought to detection. Meanwhile, free miRNA may exist in the extracted exosome solution, interference on actual miRNA content determination in exosomes may be caused by direct coculture of the molecular beacon with the hairpin structure and exosomes, and the naked molecular beacon may be degraded by nucleic acid in body fluid to influence detection accuracy. Based on the above analysis, the existing technology for detecting nucleic acid molecules in exosomes by using molecular probes has the following problems (1) exosomes need to be separated in advance, the separation steps are long, the types of extracellular vesicles are various, the separated products often contain other types of vesicles and protein aggregates, and the separation steps can also cause damage to exosomes; (2) the efficiency of the molecular probe entering the exosome through diffusion is low, and the molecular probe is easily decomposed by nuclease in body fluid and possibly generates non-specific signals, so that the accurate and efficient detection of nucleic acid molecules in the exosome is difficult to realize.
Disclosure of Invention
In view of the defects in the prior art, one of the purposes of the invention is to provide a DNA zipper molecule modified liposome vesicle with a bilayer membrane and a carrier molecule probe.
The invention also aims to provide a preparation method of the liposome vesicle modified by the DNA zipper molecules. Obtaining the vesicles with controllable quality and uniform size, simultaneously realizing the modification of the lipid groups of the DNA fragments, and introducing the modified DNA fragments into the surface of the vesicles after complementary pairing.
The invention also aims to provide the application of the liposome vesicle modified by the DNA zipper molecules, which can be used for analyzing and detecting nucleic acid molecules in exosomes and sorting disease-related exosomes in a body fluid sample. The detection result can be used for early diagnosis, curative effect evaluation and prognosis analysis of diseases. Specifically, the vesicle can be rapidly fused with exosome modified by a DNA zipper under the assistance of the DNA zipper. Thereby delivering the molecular probe in the vesicle to the exosome and realizing the in-situ, accurate and rapid detection of the nucleic acid molecules in the exosome. The vesicle can protect the molecular probe wrapped in the vesicle and prevent the molecular probe from being degraded by enzyme in body fluid; in addition, the separation and crushing process of the exosome can be avoided.
In order to achieve one of the above purposes, the solution of the invention is as follows:
a liposome vesicle modified by DNA zipper molecules is composed of a bilayer membrane and a molecular probe wrapped in the bilayer membrane, wherein the surface of the bilayer membrane is modified with DNA zipper molecules capable of promoting membrane fusion.
The DNA zipper molecule consists of short-chain DNA and long-chain DNA which are modified with lipophilic groups, wherein a section of the short-chain DNA and a section of the long-chain DNA close to the lipophilic groups are completely complementarily assembled to form the DNA zipper molecule.
The lipophilic group is selected from more than one of cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylcholine and phosphatidylcholine.
According to one embodiment of the present invention, the DNA zipper molecule is pretreated before modifying the lipophilic group by a method selected from the group consisting of:
A. the DNA zipper molecule with the modification of the sulfhydryl group reacts with lipid with maleimide to obtain the DNA zipper molecule with the modification of the lipid group.
B. Reacting the DNA zipper molecule modified by maleimide with lipid with sulfhydryl group to obtain the DNA zipper molecule modified by lipid group.
C. The DNA zipper molecule with the azide modification is reacted with the lipid with the alkynyl to obtain the DNA zipper molecule with the lipid group modification.
D. And reacting the DNA zipper molecule modified by alkynyl with the lipid modified by azide to obtain the DNA zipper molecule modified by the lipid group.
E. The DNA zipper molecule with the amino group modification reacts with the lipid with the carboxylic acid to obtain the DNA zipper molecule with the lipid group modification.
F. And (3) connecting the phosphoramidite monomer with the lipid group to one end of the DNA zipper molecule by utilizing a DNA solid-phase synthesis reaction to obtain the DNA zipper molecule modified by the lipid group.
Preferably, the bilayer membrane is selected from more than one of a bilayer membrane synthesized by a natural biological membrane and a bilayer membrane synthesized by an amphiphilic molecule.
More preferably, the natural biofilm is selected from one or more of a cell membrane, a bacterial membrane and a viral membrane.
Preferably, the amphiphilic molecule is selected from one or more of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylcholine.
Preferably, the molecular probe is selected from more than one of a nucleic acid molecular beacon, a nucleic acid fluorescent probe and a nano flare probe. That is, the inner portion of the bilayer membrane may simultaneously encapsulate one or more molecular probes.
In order to achieve the two objects, the solution of the invention is:
a preparation method of the DNA zipper molecule modified liposome vesicle is selected from one of the following methods:
A. when the bilayer membrane is a natural biological membrane, firstly crushing the bilayer membrane, mixing the crushed bilayer membrane with a molecular probe, extruding to obtain vesicles, and then incubating with a DNA zipper molecule (DNA fragment) with one end modified by a lipophilic group to obtain liposome vesicles modified by the DNA zipper molecule; alternatively, the first and second electrodes may be,
B. when the bilayer membrane is synthesized by amphiphilic molecules, the bilayer membrane is directly mixed with a molecular probe, vesicles are obtained by extrusion, and then the vesicles are incubated with DNA zipper molecules (DNA fragments) with lipophilic group modification at one end to obtain liposome vesicles modified by the DNA zipper molecules; alternatively, the first and second electrodes may be,
conventional thin film hydration: dissolving amphiphilic molecules in an organic solvent, carrying out rotary evaporation to obtain a solid film, adding water, carrying out ultrasonic treatment, extruding by a liposome extruder, and obtaining liposome vesicles with specific sizes by a polycarbonate film with specific pore diameters.
Preferably, the natural biological membrane is selected from one or more of a cell membrane, a bacterial membrane and a viral membrane.
Preferably, the amphiphilic molecule is selected from one or more of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylcholine.
Preferably, the lipophilic group is selected from one or more of cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylcholine and phosphatidylcholine.
Preferably, to obtain a DNA fragment with lipophilic group modification, the DNA fragment is pre-treated by a method selected from one of the following:
A. and reacting the DNA fragment with the sulfhydryl modification with lipid with maleimide to obtain the DNA fragment with the lipid group modification.
B. Reacting the DNA fragment with maleimide modification with lipid with sulfhydryl group to obtain the DNA fragment with lipid group modification.
C. And reacting the DNA fragment with the azide modification with the lipid with the alkynyl to obtain the DNA fragment with the lipid group modification.
D. And reacting the DNA fragment modified by alkynyl with the lipid modified by azide to obtain the DNA fragment modified by the lipid group.
E. Reacting the DNA fragment with the amino modification with the lipid with the carboxylic acid to obtain the DNA fragment with the lipid group modification.
F. And (3) utilizing a DNA solid phase synthesis reaction to connect a phosphoramidite monomer with a lipid group to one end of the DNA fragment to obtain the DNA fragment modified by the lipid group.
Preferably, the particle size of the liposome vesicle modified by the DNA zipper molecule is 50nm-1.5 μm.
In order to achieve the three objects, the solution of the invention is:
the application of the liposome vesicle modified by the DNA zipper molecule in detecting the nucleic acid molecular marker in the exosome can efficiently and quickly detect the nucleic acid biomarker in the target exosome by using the vesicle.
Preferably, the nucleic acid molecule is selected from more than one of mRNA, miRNA, lncRNA, circRNA and DNA.
The detection target exosome needs to be incubated with a sequence complementarily matched with a vesicle surface DNA sequence, so that the target exosome can be smoothly fused with the vesicle.
Due to the adoption of the scheme, the invention has the beneficial effects that:
the DNA zipper molecule modified liposome vesicle comprises a bilayer membrane and a molecular probe wrapped in the bilayer membrane, wherein a DNA sequence is inserted into the surface of the bilayer membrane, and the DNA sequence inserted into the surface of an exosome undergo base complementary pairing 'DNA zipper opening reaction', so that the distance between the exosome and the liposome vesicle carrying the molecular probe is shortened as much as possible, a membrane fusion process is carried out between the exosome and the liposome vesicle, the molecular probe in the liposome vesicle is delivered into the exosome, and then a nucleic acid biomarker in the exosome is detected, and in-situ, accurate and rapid detection of nucleic acid molecules in the exosome is realized. The method can avoid separating exosome in advance, avoid the molecular probe from being exposed in body fluid and degraded by nuclease in the body fluid, and has the advantages of higher detection speed, shorter time consumption and more accurate detection result. The process for performing membrane fusion detection by using exosome and liposome vesicle surface modified DNA zipper molecules belongs to active fusion, and has higher fusion efficiency and better detection effect.
Secondly, the molecular probe entrapped in the liposome vesicle can be designed according to a specific target nucleic acid sequence to be detected, so that the detection of specific nucleic acid molecules of various diseases can be realized, and corresponding diagnostic information can be obtained.
Thirdly, the method adopts a simple and feasible strategy of inserting DNA into a lipid membrane to promote membrane fusion, and can finish liposome vesicle surface modification with the efficiency close to 100% only by incubation at normal temperature, so the method has the potential of large-scale production and application.
Drawings
Fig. 1 is a schematic diagram of the fusion process of liposome vesicles and exosomes of the present invention.
Fig. 2 is a transmission electron micrograph of a liposome vesicle in an example of the present invention.
Fig. 3 is a schematic diagram of particle size and concentration of liposome vesicles in an embodiment of the invention.
FIG. 4 is an agarose gel electrophoresis image of a liposome vesicle in an example of the present invention.
FIG. 5 is a graph showing the variation of particle size of liposome vesicles in PBS and 50% fetal calf serum in the examples of the present invention.
FIG. 6 is a transmission electron micrograph of exosomes in an example of the invention.
FIG. 7 is a graph of particle size and concentration of exosomes in an example of the invention.
Fig. 8 is an expression diagram of exosome surface markers CD63, CD9, CD81 in an experiment of fusion of liposome vesicles and exosomes in the example of the present invention.
FIG. 9 is a graph of fluorescence intensity versus time in an assay of liposome vesicle fusion with exosome in accordance with an embodiment of the present invention.
FIG. 10 is a flow analysis scattergram of the fusion products of liposome vesicles and normal human serum in the example of the present invention.
Fig. 11 is a flow analysis scattergram of liposome vesicles and human tumor patient serum fusion products in an embodiment of the invention.
FIG. 12 is a flow analysis histogram of the fusion products of liposome vesicles with normal human serum in an example of the present invention.
FIG. 13 is a histogram of flow analysis of liposome vesicles and human tumor patient serum fusion products in an embodiment of the invention.
Detailed Description
The invention provides a DNA zipper molecule modified liposome vesicle and a preparation method and application thereof.
The biological membrane fusion is strictly regulated and controlled by SNARE protein (soluble N-ethylmaleimide sensitive factor attachment protein receptor); molecular recognition between proteins located in the transport vesicle and the target cell membrane results in the formation of SNARE protein complexes that catalyze the necessary lipid rearrangement to incorporate adjacent lipid bilayers. Based on the inspiration, the invention adopts a method for realizing the fusion of lipid vesicles through membrane-anchored DNA chain hybridization (namely DNA zipper molecules), and efficiently delivers molecular probes through a method for fusing a liposome vesicle membrane and an exosome membrane, thereby detecting nucleic acid molecules in exosome. The detection means does not need an acid environment, enzyme or ion for changing membrane permeability, and membrane fusion protein in a complex biosynthesis process, and has the potential of simple, high-efficiency and large-scale production and application.
< DNA zipper molecule-modified Liposomal vesicle >
The DNA zipper molecule modified liposome vesicle comprises a bilayer membrane and a molecular probe wrapped in the bilayer membrane, wherein the surface of the bilayer membrane is modified with a DNA zipper molecule capable of promoting a membrane fusion process.
As shown in figure 1, the DNA zipper molecules (DNA fragments) are subjected to base complementary pairing with the DNA sequence modified on the surface of the exosome membrane, so that the liposome vesicle and the exosome are efficiently fused, the molecular probe in the vesicle is delivered into the exosome, the nucleic acid molecules in the exosome are further detected, and the in-situ, accurate and rapid detection of the nucleic acid molecules in the exosome is further realized. The DNA inserted into the exosome membrane and the liposome membrane needs to be modified with a lipophilic group at one end of the DNA in advance, if a double-stranded DNA zipper mode is adopted, annealing treatment is needed to obtain double-stranded DNA, and the DNA is modified on the surfaces of the exosome and the liposome membrane by utilizing excellent compatibility between the lipophilic group and the membrane. The exosome detection means can avoid separating exosomes in advance, prevent the molecular probes from being exposed in body fluid and degraded by nuclease in the body fluid, and has the advantages of higher detection speed, shorter time consumption and more accurate detection result. The DNA sequence on the surface of the liposome vesicle provided by the invention can be actively fused with the DNA modified exosome, so that the fusion efficiency is higher, and the detection effect is better.
Among them, one end of the DNA zipper molecule may be modified with various lipid groups so as to be inserted into the membrane, i.e., a lipid group having good compatibility with lipids constituting the cell membrane. The DNA zipper molecule is composed of a short-chain DNA and a long-chain DNA modified with lipophilic groups, and specifically, the sequence of the short-chain DNA may be designed as: 3 ' -X-A ' -5 ', the sequence of the long-chain DNA can be designed as follows: 5 ' -X-A-B-3 ', wherein the part A ' in the short-chain DNA and the part A in the long-chain DNA are complementary sequences; when the DNA zipper molecule is used for exosome detection, the DNA zipper molecule embedded in exosome is designed to be a long-chain sequence as follows: 3 '-X-A' -B '-5', the short chain sequence is designed as follows: 5 '-X-a-3'; in the above sequence design, A and A 'are complementary sequences, and B' are complementary sequences. Wherein, the sequence of the short-chain DNA includes but is not limited to: 5 '-AGGCAGCACGGA-X-3', the sequence of the long-chain DNA includes but is not limited to the sequences: 5 '-X-TCCGTCGTGCCTTATTTCTGATGTCCA-3', X is a lipophilic group, and the modified lipophilic group includes, but is not limited to, lipids such as cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylcholine, and phosphatidylcholine. The DNA zipper molecule is not particularly limited as long as it satisfies the purpose of zipper, that is, the DNA zipper sequence on the vesicle can be complementarily paired with the zipper on the exosome.
In addition, the DNA zipper molecule is pre-treated before modification of the lipophilic group by a method selected from one of the following:
A. the DNA zipper molecule with the modification of the sulfhydryl group reacts with lipid with maleimide to obtain the DNA zipper molecule with the modification of the lipid group.
B. Reacting the DNA zipper molecule modified by maleimide with lipid with sulfhydryl group to obtain the DNA zipper molecule modified by lipid group.
C. The DNA zipper molecule with the azide modification is reacted with the lipid with the alkynyl to obtain the DNA zipper molecule with the lipid group modification.
D. And reacting the DNA zipper molecule modified by alkynyl with the lipid modified by azide to obtain the DNA zipper molecule modified by the lipid group.
E. The DNA zipper molecule with the amino group modification reacts with the lipid with the carboxylic acid to obtain the DNA zipper molecule with the lipid group modification.
F. And (3) connecting the phosphoramidite monomer with the lipid group to one end of the DNA zipper molecule by utilizing a DNA solid-phase synthesis reaction to obtain the DNA zipper molecule modified by the lipid group.
The bilayer membrane is selected from more than one of natural biological membrane synthesized bilayer membrane and amphiphilic molecule synthesized bilayer membrane. In fact, natural biological membranes include, but are not limited to, cell membranes, bacterial membranes, and viral membranes, among others. Amphiphilic molecules include, but are not limited to dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylethanolamine, phosphatidylcholine, and the like.
The molecular probe includes but is not limited to nucleic acid molecular beacon, nucleic acid fluorescent probe, nanometer flare probe, etc. One or more molecular probes can be wrapped in the bilayer membrane at the same time, so that the simultaneous detection of specific nucleic acid molecules of various diseases can be realized, and richer diagnosis information can be obtained.
< preparation method of liposome vesicle modified with DNA zipper molecule >
The preparation method of the DNA zipper molecule modified liposome vesicle is selected from one of the following methods:
A. when the bilayer membrane is a natural biological membrane, firstly, a large number of biological membranes are obtained by utilizing cell culture, cells are ground and crushed, membrane fragments are obtained by centrifugation, the membrane fragments are mixed with a molecular probe, then, vesicles are obtained by extrusion, and then, the surfaces of the vesicles are incubated with DNA fragments with lipophilic group modification at one end by adopting a physical combination method to obtain the DNA zipper molecule modified liposome vesicles; alternatively, the first and second electrodes may be,
B. when the bilayer membrane is synthesized by amphiphilic molecules, the bilayer membrane is directly mixed with a molecular probe, vesicles are obtained by extrusion, and then the surfaces of the vesicles are incubated with DNA fragments with lipophilic group modification at one end by adopting a physical combination method to obtain DNA zipper molecule modified liposome vesicles; alternatively, the first and second electrodes may be,
conventional thin film hydration: dissolving amphiphilic molecules in an organic solvent, carrying out rotary evaporation to obtain a solid film, adding water, carrying out ultrasonic treatment, extruding by a liposome extruder, and obtaining liposome vesicles with specific sizes by a polycarbonate film with specific pore diameters.
Among them, natural biological membranes include, but are not limited to, cell membranes, bacterial membranes, and viral membranes.
Amphiphilic molecules include, but are not limited to dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylethanolamine, phosphatidylcholine, and the like.
Lipophilic groups include, but are not limited to, cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylcholine, phosphatidylcholine, and the like.
To obtain a DNA fragment with lipophilic group modification, the DNA fragment is pre-treated by a method selected from one of the following:
A. and reacting the DNA fragment with the sulfhydryl modification with lipid with maleimide to obtain the DNA fragment with the lipid group modification.
B. Reacting the DNA fragment with maleimide modification with lipid with sulfhydryl group to obtain the DNA fragment with lipid group modification.
C. And reacting the DNA fragment with the azide modification with the lipid with the alkynyl to obtain the DNA fragment with the lipid group modification.
D. And reacting the DNA fragment modified by alkynyl with the lipid modified by azide to obtain the DNA fragment modified by the lipid group.
E. Reacting the DNA fragment with the amino modification with the lipid with the carboxylic acid to obtain the DNA fragment with the lipid group modification.
F. And (3) utilizing a DNA solid phase synthesis reaction to connect a phosphoramidite monomer with a lipid group to one end of the DNA fragment to obtain the DNA fragment modified by the lipid group.
Specifically, when the liposome vesicle is obtained by extrusion, a polycarbonate membrane can be adopted, and the pore diameter of the polycarbonate membrane is 50nm-1.5 μm, so that the particle diameter of the vesicle is 50nm-1.5 μm, and the uniform size and controllable quality of the vesicle can be realized.
< use of liposome vesicle modified with DNA zipper molecule >
The DNA zipper molecule modified liposome vesicle can be applied to detection of nucleic acid molecular markers in exosomes, and detection results can be used for early diagnosis, curative effect evaluation and prognosis analysis of diseases.
Wherein the nucleic acid molecule is any one of mRNA, miRNA, lncRNA, circRNA and DNA.
The present invention will be further described with reference to the following examples and the accompanying drawings.
Example (b):
the preparation of liposome vesicles using amphiphilic molecules and performance characterization are described in detail.
1. Preparation of liposome vesicles encapsulating molecular probes
Dissolving the selected artificial lipid with good compatibility with the exosome membrane into a proper amount of chloroform to form a uniform and transparent solution system, and performing rotary evaporation to obtain a multi-component mixed uniform lipid membrane solid tablet. Thereafter, the mixture was put into a freeze-dryer overnight to further remove the solvent. Mixing the lipid sheet and the molecular probe in a buffer solution (10mM Tris/HCl, 150mM NaCl, 1mM EDTA) with the pH value of 7.5, emulsifying the system by ultrasonic waves, sequentially passing polycarbonate membranes with the diameters of 800nm, 400nm and 200nm through a liposome extruder, and repeatedly extruding the polycarbonate membranes with different pore diameters to obtain the liposome vesicle loaded with the molecular probe.
2. Solid phase synthesis of lipid molecule modified DNA
The lipophilic phosphoramidite compounds are combined at the 5' end of the oligonucleotide to form the final "base", and the lipophilic phosphoramidite is dissolved in dichloromethane and coupled with the oligomer by using a syringe synthesis technology. Briefly, lipophilic phosphoramide (200. mu.L) was mixed with activator (200. mu.L acetonitrile, 0.2mM 5-ethylthiotetrazole) and the mixture was pushed back and forth through the CpG column 10 times using two syringes. Alternatively, the lipophilic phosphoramidite can also be coupled using a DNA synthesizer (15min coupling time). After synthesis, the DNA was cleaved from CpG using a C4 column (Biobasic-4, 200 mM. times.4.6 mM, Thermo-Scientific), 100mM triethylamine-acetic acid buffer (TEAA, pH 7.5) -acetonitrile (0-30min, 10-100%) as eluent and deprotected and purified by reverse phase high performance liquid chromatography.
3. Preparing liposome vesicles with surface modified DNA zipper molecules, wherein the DNA zipper molecules are formed by pairing two DNA single chains with different lengths, and the specific sequence of the example is as follows: 5 '-X-TCCGTCGTGCCTTATTTCTGATGTCCA-3', 5 '-AGGCAGCACGGA-X-3', X is dioleoylphosphatidylcholine (lipophilic group), but the sequence of the DNA zipper is not limited to this sequence.
And (2) mixing the DNA fragment modified by the lipophilic group with the prepared liposome vesicle according to the molar ratio of 100: 1 and incubating for 30min at 37 ℃ after mixing to obtain the liposome vesicle with the surface modified by the DNA zipper molecules.
4. Characterization of Liposomal vesicles
Dropping the liposome vesicle on a sealing membrane, floating a copper net on the drop to adsorb for 20min, dyeing with 2% phosphotungstic acid for 1min, and detecting the size and shape of the liposome by using a transmission electron microscope. As shown in FIG. 2, the liposome vesicles were uniformly dispersed and were of a saucer type or a hemispherical shape with one side concave.
The concentration of the liposome vesicles was determined using a Malvern nanoparticle tracking analyzer, and the concentration of the liposome vesicles was about 1.62X 10 as shown in FIG. 310particle/mL, particle size about 100 nm.
In order to examine the situation that the DNA is inserted into the liposome vesicle, different fluorescent dyes are respectively adopted to mark the DNA and the liposome vesicle, as shown in FIG. 4, after the DNA is inserted, a free DNA band disappears, the efficiency is close to 100%, agarose gel electrophoresis shows that two fluorescent signals are well overlapped, and the originally dispersed DNA chain is retained in an upper sample hole, which indicates that the DNA is modified on the liposome vesicle.
To investigate the stability of this liposome vesicle in a simulated physiological environment, i.e., 0.01M PBS and 50% fetal calf serum. The particle size change of the liposome vesicle in two liquid environments within 72h is monitored by a dynamic light scattering instrument, as shown in fig. 5, no obvious particle size change is seen in the liposome vesicle within 72h, which indicates that the liposome vesicle has good stability in a simulated physiological environment.
5. Experiment for fusion of liposome vesicle and exosome modified by DNA zipper molecules and carrying nucleic acid molecular beacon
If liposome vesicles are used for detecting nucleic acid molecules in exosomes, exosomes do not need to be extracted in advance, but the conditions in a laboratory are limited, and the exosomes are extracted first and then the environment of the exosomes in a human body is simulated. This demonstrates the fusion of liposome vesicles with exosomes.
5.1 extraction, separation and identification of exosomes
Human breast cancer MCF-7 cell exosome and human normal breast MCF-10A cell are used as research objects. When MCF-7 or MCF-10A cells in the culture dish grow to 70%, washing with PBS for 2 times, removing the culture medium except exosome, culturing for 48h, collecting the supernatant, centrifuging at 2000 Xg for 10min at 4 ℃, and removing cells and cell debris in the culture solution. The supernatant was centrifuged at 20000 Xg for 30min to remove the large-size vesicles from the culture. The supernatant was centrifuged at 110000 Xg for 70min, the supernatant was removed, the bottom pellet was resuspended in PBS and centrifuged at 110000 Xg for 70 min. And discarding the supernatant, and carrying out PBS heavy suspension precipitation to obtain the tumor or normal cell exosomes. And (4) measuring the concentration of the exosome by a nanoparticle tracking analyzer, and preparing an exosome standard solution.
And detecting the size and the form of the exosome by adopting a transmission electron microscope. As shown in FIG. 6, the exosome is of a saucer type or a hemisphere with a concave side, and the particle size is 30-150 nm.
The concentration of exosomes was determined using a malvern nanoparticle trace analyzer, and the results are shown in fig. 7, where the liposome vesicles are at a concentration of about 1.66 x 1011one/mL.
The expression of markers such as CD63, CD9, CD81 and the like on the surface of the exosome is examined by an immunoblotting method. As shown in FIG. 8, significant bands of markers such as CD63, CD9, CD81 and the like can be found in exosomes and MCF-7 cell membranes producing the exosomes, which indicates that the obtained vesicles are indeed exosomes.
5.2 in-situ detection of target nucleic acid molecules in exosomes by DNA zipper molecule modified liposome vesicles carrying nucleic acid molecule beacons
miR-21 is used as a detection target, and a molecular probe capable of detecting miR-21 is loaded in the liposome vesicle. The DNA zipper molecule modified liposome vesicle encapsulating the nucleic acid molecular beacon is respectively incubated with the exosome extracted from the normal cell and the exosome extracted from the tumor cell at room temperature according to a proper proportion, and the fluorescence intensity of the fusion product is measured by a steady-state transient fluorescence spectrometer (FLS 1000). The result is shown in fig. 9, the fluorescence intensity of the fusion product of the tumor exosome is obviously higher than that of the normal exosome group, and the quantitative result shows that the fluorescence intensity of the fusion product of the tumor exosome is increased by about 40%; the fluorescence intensity of the normal exosome fusion product is essentially unchanged; the fluorescence intensity of the naked molecular beacon detected by passive diffusion increased only by 10%. The results can be explained that the content of miR-21 in the tumor exosomes is obviously higher than that of normal exosomes, and the effect of exosome detection through passive diffusion is obviously better than that of simple molecular beacon detection.
6. Detection of nucleic acid molecules in tumor exosomes by DNA zipper molecule modified liposome vesicles carrying nucleic acid molecule beacons
In order to further investigate the effect of liposome vesicle in-situ detection of nucleic acid molecules in exosomes of clinical samples, blood samples of breast cancer patients and normal persons were collected. Removing redundant protein and large extracellular vesicles through primary ultrafiltration and filtration, adding a DNA fragment with lipid groups for incubation, adding liposome vesicles which are modified by DNA zipper molecules and carry nucleic acid molecular beacons into plasma of a patient and a volunteer, incubating for a certain time, and measuring fluorescence intensity through a flow cytometer. As shown in fig. 10 to 13, only one colony of the normal group exists in the region with weak fluorescence, indicating that it is a normal-source exosome, while one colony of the tumor patient group exists in each of the region with weak fluorescence and the region with strong fluorescence, indicating that it is a normal-source exosome and a tumor-source exosome. The results show that the liposome vesicle is expected to diagnose tumor patients by detecting nucleic acid molecules in exosomes.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.
Claims (9)
1. A DNA zipper molecule modified liposome vesicle, which is characterized in that: the liposome vesicle is composed of a bilayer membrane and a molecular probe wrapped in the bilayer membrane, and a DNA zipper molecule for promoting membrane fusion is modified on the surface of the bilayer membrane.
2. The DNA zipper molecule-modified liposome vesicle of claim 1, wherein: the DNA zipper molecule consists of short-chain DNA and long-chain DNA which are modified with lipophilic groups;
the lipophilic group is selected from more than one of cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylcholine and phosphatidylcholine.
3. The DNA zipper molecule-modified liposome vesicle of claim 1, wherein: the bilayer membrane is selected from more than one of a bilayer membrane synthesized by a natural biological membrane and a bilayer membrane synthesized by amphiphilic molecules;
preferably, the natural biological membrane is selected from one or more of a cell membrane, a bacterial membrane and a viral membrane;
preferably, the amphiphilic molecule is selected from one or more of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylcholine.
4. The DNA zipper molecule-modified liposome vesicle of claim 1, wherein: the molecular probe is selected from more than one of a nucleic acid molecular beacon, a nucleic acid fluorescent probe and a nano flare probe.
5. A method for preparing the DNA zipper molecule-modified liposome vesicle of claim 1, wherein: it includes:
firstly, mixing the bilayer membrane with a molecular probe, extruding to obtain a liposome vesicle, and then incubating the liposome vesicle with a DNA zipper molecule with one end modified by a lipophilic group to obtain the liposome vesicle modified by the DNA zipper molecule.
6. The method of claim 5, wherein: the bilayer membrane is selected from more than one of a bilayer membrane synthesized by a natural biological membrane and a bilayer membrane synthesized by amphiphilic molecules; if the bilayer membrane is synthesized by a natural biological membrane, the bilayer membrane needs to be firstly crushed and then mixed with a molecular probe;
preferably, the natural biological membrane is selected from one or more of a cell membrane, a bacterial membrane and a viral membrane;
preferably, the amphiphilic molecule is selected from one or more of dioleoylphosphatidylcholine, dioleoylphosphatidylethanolamine, phosphatidylethanolamine and phosphatidylcholine.
7. The method of claim 5, wherein: the lipophilic group is selected from more than one of cholesterol, distearoylphosphatidylethanolamine, dioleoylphosphatidylcholine and phosphatidylcholine.
8. Use of a DNA zipper molecule modified liposome vesicle of claim 1 for detecting an in-vitro nucleic acid molecular marker.
9. Use according to claim 8, characterized in that: the nucleic acid molecule is selected from more than one of mRNA, miRNA, lncRNA, circRNA and DNA.
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