CN113144211B - RNA packaging method - Google Patents
RNA packaging method Download PDFInfo
- Publication number
- CN113144211B CN113144211B CN202110318427.6A CN202110318427A CN113144211B CN 113144211 B CN113144211 B CN 113144211B CN 202110318427 A CN202110318427 A CN 202110318427A CN 113144211 B CN113144211 B CN 113144211B
- Authority
- CN
- China
- Prior art keywords
- rna
- nanoparticles
- loading
- encapsulating
- packaging method
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
- A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
- A61K47/6923—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being an inorganic particle, e.g. ceramic particles, silica particles, ferrite or synsorb
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/52—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an inorganic compound, e.g. an inorganic ion that is complexed with the active ingredient
-
- 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
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
- A61K9/143—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with inorganic compounds
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Inorganic Chemistry (AREA)
- Molecular Biology (AREA)
- Nanotechnology (AREA)
- Ceramic Engineering (AREA)
- General Engineering & Computer Science (AREA)
- Medical Informatics (AREA)
- Biotechnology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Biophysics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Saccharide Compounds (AREA)
Abstract
The invention discloses an RNA packaging method, which comprises the following steps: mixing and incubating an RNA aqueous solution and a Zn ion aqueous solution, performing one-step self-assembly under the driving of coordination crosslinking between zinc ions and RNA nucleotide, simply synthesizing a Zn-RNA nanostructure, and washing with water after centrifugation to obtain a solid. The packaging method successfully realizes the protective loading of RNA, has simple process and stronger universality, is not influenced by the type, the composition, the shape and the length of loaded RNA, and the assembled nanosphere has adjustable size, excellent RNA loading efficiency, better biocompatibility, enzymolysis and biodegradation resistance and has original innovation and application prospect.
Description
Technical Field
The invention belongs to the technical field of gene medicines, and relates to a nano structure constructed based on RNA self-assembly.
Background
With the development of biotechnology, RNA vaccines and RNA intervention medicine have become a hot tide, and especially functional RNA is often used for disease treatment, for example, BNT162b2 mRNA may encode SARS-CoV-2 spike protein, the expression of which causes immune response against antigen in a receptor, BNT162b2 mRNA is formulated with lipid nanoparticles, modified by nucleoside, prepared into mRNA vaccine, which may be used for preventing new coronavirus diseases 2019 (COVID-19), the vaccine is proved to be effective by clinical evaluation, and a series of approval or authorization for emergency use has been obtained at present. RNA interference (RNAi) technologies based on small interfering RNA (siRNA) or microrna (miRNA) show great potential in cancer therapy due to their unique sequence-specific gene silencing effects. However, the conversion of RNA into drugs still faces serious challenges due to the poor pharmacokinetic properties of RNA, inability to penetrate cell membranes, and susceptibility to degradation by rnases in body fluids.
At present, the packaging carrier of RNA mainly comprises virus, inorganic nano material, organic polymer material and bionic nano carrier. The most commonly used viral vectors Are Adenovirus (AAV) and lentiviral vectors, for example, studies by Yu Miyazaki et al have shown that the use of adenovirus to deliver miR-196a into mice can effectively inhibit androgen receptor decay, confirming the use of disease-specific miRNA in neurodegenerative diseases. Trang et al achieved good results by delivering let-7 for inhibition of mouse non-small cell lung cancer growth via lentivirus and lipid mimics, respectively. However, since the procedures for loading RNA into viral vectors are complicated and viral vectors have safety problems, applying this method to clinical treatment is very challenging. The inorganic and organic nano-carriers can improve the capacity of miRNAs to penetrate cell membranes, jae-Hong Kim and the like carry DNA thiolate, combine the thiolated carrying DNA on the surface of gold nanoparticles through Jin Qiu bonds, and combine the carried DNA with a part of AMO-miR-29b through base complementary pairing, so as to load the AMO-miR-29b to enter tumor cells, thereby inhibiting the growth and proliferation of the tumor cells. Gibori et al developed a biodegradable amphiphilic polyglutamic acid polymer nanocarrier (APA) whose electropositivity could well adsorb miR-34a and siRNA to make it more easily taken up by cells and more easily taken up by cells. However, the nano-carrier also suffers from low loading efficiency, toxic side effects, and the like. Aiming at the problem of poor biocompatibility of inorganic materials and organic materials, researchers further develop bionic nano materials, such as liposomes, exosomes, chitosan, N-acetyl-D-galactosamine, polypeptide nano carriers and the like, wherein the carriers have good biocompatibility and no side effect in cells, and can better realize in vivo delivery of microRNAs. Derrick Gibbings et al, which integrates siRNA sequences into the stem-loop backbone of the miRNA-451 precursor, achieve endogenous expression and packaging of therapeutic siRNAs in extracellular vesicles. Yuan Lijun (an exosome loaded with ABCA1 mRNA and its construction method and use, chinese patent, publication No. CN109234237 a): constructing an ABCA1 fusion expression vector and a CD9 fusion expression vector, co-transfecting the ABCA1 fusion expression vector and the CD9 fusion expression vector to hepatocytes, culturing, and collecting exosomes to obtain the exosomes loaded with ABCA1 mRNA. In addition, a plurality of DNA sequences can be used to construct a stable nano structure through the design of DNA sequences, and can be assembled together through the complementary pairing with partial basic groups of RNA to carry RNA into cells, katherine E.Bujold et al utilize 6 reasonably designed DNA sequences to form a cuboid DNA nano box through the complementary pairing and self-assembly of the basic groups, and then two ends of another nucleic acid loaded with siRNA are respectively complementary with the ridges of a pair of diagonal lines of the cuboid, so that the siRNA is coated in the middle of the nano box. However, the existing scheme has the problems of limitation on the length and type of the sequence, complex design and synthesis and the like, so that the exploration of an RNA packaging method with high loading preparation efficiency, simple operation steps and good universality has important significance on the biomedical application of RNA.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the prior art and provide a simple and universal RNA encapsulation method.
In order to solve the technical problem, the invention adopts the following technical scheme:
a method for encapsulating RNA, comprising the steps of:
mixing and incubating an RNA aqueous solution and a Zn ion aqueous solution, synthesizing a Zn-RNA nanostructure by zinc ion and RNA nucleotide coordination cross-linking driven self-assembly, centrifuging, and washing with water to obtain a solid.
In the method for encapsulating RNA, preferably, the ratio of RNA to zinc ions in the aqueous solution is 10 μ M to 100mM:1 mM-10M.
In the method for encapsulating RNA, the reaction temperature is preferably 60 ℃ to 100 ℃.
In the method for encapsulating RNA, the reaction time is preferably 0.5 to 3 hours.
In the method for encapsulating RNA, the reaction pH is preferably 5.0 to 7.0.
In the method for encapsulating RNA, preferably, the RNA includes microRNA, siRNA, long non-coding RNA, circular RNA and messenger RNA fragments.
In the method for encapsulating RNA, the centrifugation rate is preferably not lower than 15,000r.p.m. and the time is preferably 20min.
Compared with the prior art, the invention has the advantages that:
1. according to the RNA packaging method, the RNA is loaded through zinc ion coordination triggering assembly for the first time, and then the RNA nano material is constructed.
2. According to the RNA packaging method, the loading RNA type, composition, shape and length have little influence on the loading result, successful loading can be realized, and spherical nanoparticles with different sizes are formed.
3. In the method for encapsulating RNA of the present invention, there may be a plurality of RNA oligonucleotides loaded at one time.
4. The packaging method of RNA of the invention has simple process, adjustable size of the assembled nanospheres, excellent RNA loading efficiency, better biocompatibility, enzymolysis and biodegradation resistance, and original innovation and application prospect.
Drawings
FIG. 1 is a representation of Zn-RNA nanoparticles prepared in example 1.
FIG. 2 is a quantification of RNA loading efficiency in example 1.
FIG. 3 is a graph of RNA integrity before and after loading in example 1.
FIG. 4 is a transmission electron microscope image of Zn-RNA nanoparticles prepared in example 2.
FIG. 5 is a TEM image of Zn-RNA nanoparticles prepared in example 3.
FIG. 6 is a transmission electron micrograph, a scanning electron micrograph, and a confocal scanning microscope image of Hela cells after transfection of GFP mRNA of Zn-RNA nanoparticles prepared in example 4.
FIG. 7 is a scanning electron microscope image and a transmission electron microscope image of Zn-RNA nanoparticles prepared in example 5.
FIG. 8 is a verification of the simultaneous loading of two RNAs in example 5.
Fig. 9 is an abstract drawing.
Detailed Description
The embodiments of the present invention will be described in detail below with reference to the accompanying drawings: the embodiment is implemented on the premise of the technical scheme of the invention, and a detailed implementation manner and a process are given, so that the technical scheme features of the invention are easy to understand, and the protection scope of the invention is not limited at all. All the technical solutions formed by equivalent transformation or equivalent replacement fall within the protection scope of the present invention.
EXAMPLE 1 encapsulation of random sequence RNA
(1) Encapsulation of random sequence RNA
First, 5. Mu.L of Zn (NO) was added to a 1.5mL centrifuge tube containing 55. Mu.L of sterile water 3 ) 2 ·6H 2 O (20 mM), and 40. Mu.L of 28-base random strand RNA (miRNA-205) with a concentration of 50. Mu.M is added, i.e., the final concentration of RNA in the system is 20. Mu.M, and the final concentration of zinc ions is 1mM. After thoroughly mixing, the mixture was reacted at 95 ℃ for 1 hour. After natural cooling, centrifuging for about 20 minutes at the rotating speed of 15,000r.p.m by a high-speed refrigerated centrifuge, washing with sterile water, finally re-dispersing the collected Zn-RNA nanoparticles in the sterile water, and then storing at 4 ℃ for later use.
Dropwise adding the Zn-RNA nanoparticles prepared in the embodiment onto a copper net, and determining the morphology and the main components of the Zn-RNA nanoparticles by using a transmission electron microscope and a scanning electron microscope; finally, preparing a sample through a silicon chip, and analyzing elements and valence states of the sample by utilizing X-ray photoelectron spectroscopy.
(2) Quantification of RNA Loading efficiency
After synthesis of Zn-RNA nanoparticles by the one-pot method, all supernatants were collected during centrifugation and washing for subsequent calculation of the amount of unencapsulated RNA in the Zn-RNA nanoparticles. Then, a series of concentrations (10. Mu.M, 20. Mu.M, 30. Mu.M, 40. Mu.M and 50. Mu.M) of single-stranded miRNA-205 are prepared, and the absorbance of the RNA is measured by UV-visible spectroscopy at 260nm to draw a standard curve of the UV absorption of the RNA, wherein parameters are adjusted to make the absorbance value as small as 1 as possible. The absorbance of all supernatants collected during the above washing of Zn-RNA nanoparticles was determined under the same conditions, and then the RNA loading efficiency was calculated using the following formula: load efficiency = (M) i -M u )/M i ) X 100% where M u Is the total amount of unencapsulated RNA, M, in the Zn-RNA nanoparticle i Is the total amount of RNA initially added during synthesis.
(3) Verification of integrity before and after RNA Loading
And (3) gel electrophoresis characterization: preparing DNAmarker, fRNA (miRNA-205), synthesized Zn-RNA nanoparticles and a mixed sample obtained by processing the Zn-RNA nanoparticles with disodium salt solution of EDTA in advance, sequentially taking out 5 mu L of the four samples, dripping the samples on a clean disposable transparent plastic glove, respectively adding 1 mu L of 5 × loading buffer and 2 mu L of 100 × SYBR Gold, and fully and uniformly mixing the samples for later use by repeatedly blowing and beating. After the gel is completely solidified, carefully taking out the comb, keeping the process vertical to avoid the aperture deflection, cleaning the gel sampling hole with purified water for 2 times, and then cleaning with 1 × TBE electrophoresis working solution for 2 times. Then, about 800mL of the 1 XTBE electrophoresis working solution was poured into the electrophoresis tank to ensure that the liquid surface was not over the gel inlet, and pre-electrophoresis was performed for 2-3min with the voltage set at 90V. Finally, carefully injecting the prepared sample into a sample inlet hole of the gel through a microsyringe, adding 1 × TBE electrophoresis buffer solution to submerge the glass groove, adjusting the set voltage to 90V for 150min, starting electrophoresis, and keeping the whole process away from light as much as possible. During the experiment, the electrophoresis time was adjusted by observing the position of the bromophenol blue band, then the gel block of the electrophoresis tank was carefully removed, and finally the removed gel was imaged using a gel imager.
And (3) fluorescence characterization: set up 3 sets of parallel experiments, the first set was 2 μ L of Zn-RNA nanoparticles (containing miRNA-205 at about 200 nM) added in 98 μ LPBS (10 mm pbs, ph = 7.4) buffer; the second group was the addition of molecular beacon MB1 (loop containing sequences that can be complementarily paired with miRNA-205, final concentration 100 nM) in PBS (10 mm PBS, ph = 7.4) buffer; the third group was performed in PBS (10mM PBS,5mM MgCl) 2 pH = 7.4) buffer was added to molecular beacon MB1 (final concentration 100 nM) for about 30min for sufficient assembly, and then the degraded Zn-RNA nanoparticles (i.e. particles were degraded by treatment with disodium EDTA salt solution, containing miRNA-205 at about 200 nM) were added and mixed well. The system of three parallel experiments is 100 mu L, then after reacting for 2h at 37 ℃, the fluorescence intensity of each group of reaction samples is measured by F-7000, and each group of experiment samples is repeated in parallel for three times. Wherein the excitation wavelength of F-7000 is set to 480nm, the collected emission range is 505 nm-650 nm, and the slit width is fullThe cell was set to 10nm, the voltage was 600V, and a quartz cuvette with an optical length of 1.0cm was used during the experiment.
And (3) mass spectrum characterization: after Zn-RNA nano-particles are prepared by a simple one-pot method, the Zn-RNA nano-particles are concentrated after centrifugal washing, and then sterile EDTA disodium salt solution is added to degrade the nano-particles, even if RNA single chains are released, the whole process needs strict sterility, so that the RNA concentration of the finally obtained degraded Zn-RNA nano-particles is about 100 mu M. Finally, the degraded Zn-RNA nanoparticles obtained above and 100 μ M of free single-stranded miRNA-205 were characterized using mass spectrometry.
Fig. 1 is a representation of Zn-RNA nanoparticles prepared in this example, (a) scanning electron microscopy images of Zn-RNA nanoparticles, (B) transmission electron microscopy images of Zn-RNA nanoparticles, (C) large angle circular dark field scanning transmission electron microscopy images of Zn-RNA nanoparticles and corresponding elemental mapping images, (D) energy dispersive X-ray spectroscopy of Zn-RNA nanoparticles, (E) X-ray photoelectron spectroscopy of Zn-RNA nanoparticles. As shown in FIGS. 1A-B, the Zn-RNA nanoparticles prepared in this example have a discrete spherical morphology with a particle size of 60-120 nm. As shown in fig. 1C-E, the nanoparticles contain phosphorus, nitrogen (from RNA) and zinc elements, further demonstrating the formation of Zn-RNA nanoparticles with these elements uniformly distributed in the nanoparticles.
FIG. 2 shows quantification of RNA loading efficiency in this example, which was calculated to determine that the RNA loading efficiency was about 61.53%.
FIG. 3 shows the verification of RNA integrity before and after loading in this example, (A) gel electrophoresis test of Zn-RNA nanoparticles or free RNA (28 nt) by different treatments, (B) fluorescence spectra of Molecular Beacons (MB), zn-RNA Nanoparticles (NPs) and a mixture of molecular beacons and degraded nanoparticles (MB + degraded NPs), (C) ESI mass spectrum of degraded Zn-RNA nanoparticles, and (D) ESI mass spectrum of free RNA. As shown in FIG. 3A, due to the zinc coordination-driven RNA self-assembly, zn-RNA nanoparticles with higher molecular weight are trapped in the loading hole, the electrophoretic shift is not obvious, the formation of the nanoparticles is illustrated, and then the disodium salt of EDTA is adopted to compete with the zinc ions for binding, and the disodium salt of EDTA induces the nanoparticle to dropAfter the lysis, a fast migration electrophoretic band was observed, and in addition, the migration position of the sample and the free RNA was similar, indicating that the RNA sequence of the nanoparticles remained intact before and after loading. It can be seen from the fluorescence spectrum 3B that when only molecular beacons or Zn-RNA nanoparticles exist in the system, the fluorescence values of both are low and negligible, and after the Zn-RNA nanoparticles are treated with the disodium salt of chelating agent EDTA, the fluorescence of MB is significantly recovered, which indicates that RNA still retains its hybridization function in the synthesis process of Zn-RNA nanoparticles. The molecular weights were determined by tandem mass spectrometry in FIGS. 3C-D, and the molecular weight of the disassembled RNA was found to be 9043 g-mol -1 Consistent with the molecular weight of free RNA. These results indicate that the RNA sequence is well preserved during loading, and that the RNA maintains its integrity before and after loading.
EXAMPLE 2 encapsulation of different base RNAs
A method for encapsulating RNA, comprising the steps of:
first, 5. Mu.L of Zn (NO) was added to a 1.5mL centrifuge tube containing 55. Mu.L of sterile water 3 ) 2 ·6H 2 O (20 mM), and then 40. Mu.L of rA20 (A20) with a concentration of 50. Mu.M was added, i.e., the final concentration of RNA in the system was 20. Mu.M, and the final concentration of zinc ions was 1mM. After thoroughly mixing, the mixture was reacted at 95 ℃ for 1 hour. After natural cooling, centrifuging for about 20 minutes at the rotating speed of 15,000r.p.m by a high-speed refrigerated centrifuge, washing with sterile water, finally re-dispersing the collected Zn-RNA nanoparticles in the sterile water, and then storing at 4 ℃ for later use. rU20 (U20), rC20 (C20) and rG20 (G20) were packaged as above.
The Zn-RNA nanoparticles prepared in this example were dropped onto a copper mesh for transmission electron microscopy characterization experiments.
FIG. 4 is a TEM image of Zn-RNA nanoparticles prepared in this example, wherein (A) is A20, (B) is C20, (C) is U20, and (D) is G20. As shown in the figure, the nanoparticles synthesized by a20, C20 and U20 in this example are all spherical nanoparticles, while the nanostructure synthesized by G20 is gel-like structure. This nucleic acid sequence dependent assembly is likely due to the different nucleotides and Zn 2+ Has different coordination affinity, zn 2+ Higher affinity with the guanine N7 position results in the attachment of more RNA molecules to form a hydrogel, and in addition, strong guanine-guanine interactions may also lead to gelation.
EXAMPLE 3 encapsulation of RNA at different solution pH
A method for encapsulating RNA, comprising the steps of:
first, 5. Mu.L of Zn (NO) was added to a 1.5mL centrifuge tube containing 55. Mu.L of sterile water 3 ) 2 ·6H 2 O (20 mM), and adding 40 μ L of miRNA-205 with a concentration of 50 μ M, wherein the final concentration of RNA in the system is 20 μ M, and the final concentration of zinc ions is 1mM. After thoroughly mixing them, the mixture was reacted at 95 ℃ for 1 hour, and synthesized under conditions of pH =5.0, pH =7.0, and pH =9.0, respectively. After natural cooling, centrifuging for about 20 minutes at the rotating speed of 15,000r.p.m by a high-speed refrigerated centrifuge, washing with sterile water, finally re-dispersing the collected Zn-RNA nanoparticles in the sterile water, and then storing at 4 ℃ for later use.
The Zn-RNA nanoparticles prepared in the embodiment are dripped on a copper net for a transmission electron microscope characterization experiment.
Fig. 5 is a transmission electron microscope image of Zn-RNA nanoparticles prepared in this example, (a) pH =5.0, (B) pH =7.0, and (C) pH =9.0. As shown, the nanoparticles synthesized under acidic (pH = 5.0) and neutral conditions (pH = 7.0) have spherical nanostructures, but are not substantially formed under conditions of pH 9.0. The reason for this may be Zn 2+ Hydrolysis is favored at higher pH values, which in turn may lead to competition with the coordination reaction between the nucleotides of the RNA.
Example 4 encapsulation of different types, compositions, shapes and lengths of RNA
RNA plays an important role in gene expression and regulation, such as siRNA, microRNA, mRNA, circular RNA, long non-coding RNA and the like can be used as a therapeutic agent or vaccine, but the functional RNA secondary structure has uncertainty, and the RNA secondary structure can be eliminated due to high-temperature reaction in the assembly process.
(1) Encapsulation of different RNAs
First, 5. Mu.L of Zn (NO) was added to a 1.5mL centrifuge tube containing 55. Mu.L of sterile water 3 ) 2 ·6H 2 O (20 mM), and adding 40 μ L of long-chain mRNA (996-nucleotide GFP mRNA) with the concentration of 50 μ M, wherein the final concentration of RNA in the system is 20 μ M, and the final concentration of zinc ions is 1mM. After thoroughly mixing, the mixture was reacted at 95 ℃ for 1 hour. After natural cooling, centrifuging for about 20 minutes at the rotating speed of 15,000r.p.m by a high-speed refrigerated centrifuge, washing with sterile water, finally re-dispersing the collected Zn-RNA nanoparticles in the sterile water, and then storing at 4 ℃ for later use. Circular RNA (214-nucleotide circular) was encapsulated as described above.
The Zn-RNA nanoparticles prepared in this example were dropped onto a copper mesh for transmission electron microscopy characterization experiments.
(2) Verification of functional integrity before and after RNA Loading
Hela cells were transfected with Zn-RNA nanoparticles loaded with GFP mRNA, electroporation transfection and free GFP mRNA, respectively, and GFP expression in Hela cells was observed under a confocal scanning microscope.
FIG. 6 is a transmission electron micrograph, a scanning electron micrograph, and a confocal scanning electron micrograph of Hela cells after transfection of GFP mRNA of Zn-RNA nanoparticles prepared in this example, (A-D) transmission electron micrograph and scanning electron micrograph of Zn-RNA nanoparticles, (A, C) Long-chain RNA, (B, D) circular RNA, (E-G) confocal scanning microscope image of Hela cells after transfection of GFP mRNA, (E) Zn-RNA nanoparticles, (F) electroporation transformation, (G) free GFPmRNA, and (H) statistical analysis of mRNA transfer efficiency. As shown in fig. 6A-D, both RNAs successfully synthesized spherical nanoparticles, and since RNA contained more bases and the chain length was also longer, the size of both spherical nanoparticles exceeded 400nm, further confirming the versatility of the encapsulation method. As can be seen from FIGS. 6E-H, the transfection efficiency of Zn-RNA nanoparticles was higher than that of free RNA, and the apparent GFP fluorescence indicates that the Zn-RNA nanoparticles retain the function of GFP mRNA.
Example 5 Simultaneous encapsulation of two RNAs
(1) Simultaneous encapsulation of two RNAs
First, 5. Mu.L of Zn (NO) was added to a 1.5mL centrifuge tube containing 55. Mu.L of sterile water 3 ) 2 ·6H 2 O (20 mM), and then 20 μ L of miRNA-205 and antagomiR-221 with the concentration of 50 μ M are respectively added, namely the final concentration of the two RNAs in the system is 10 μ M, and the final concentration of zinc ions is 1mM. After thoroughly mixing, the mixture was reacted at 95 ℃ for 1 hour. After natural cooling, centrifuging for about 20 minutes at the rotating speed of 15,000r.p.m by a high-speed refrigerated centrifuge, washing with sterile water, finally re-dispersing the collected Zn-RNA nanoparticles in the sterile water, and then storing at 4 ℃ for later use.
The Zn-RNA nanoparticles prepared in this example were dropped onto a copper mesh for characterization experiments by scanning electron microscopy and transmission electron microscopy.
(2) Verification of Simultaneous Loading of two RNAs
Set up 6 parallel experiments, wherein the purpose of the first three groups was to verify the presence of the first RNA, namely miRNA-205, and the purpose of the latter three groups was to verify the presence of the second RNA, namely antagomiR-221. First, set up the first set of experiments to add 5 μ L of Zn-RNA nanoparticles (containing miRNA-205 at about 200 nM) in 95 μ L PBS (10 mm PBS, ph = 7.4) buffer; the second group was the addition of molecular beacon MB1 (loop containing sequences that can be complementarily paired with miRNA-205, final concentration 100 nM) in PBS (10 mm PBS, ph = 7.4) buffer; the third group was in PBS (10mM PBS,5mM MgCl 2 pH = 7.4) buffer was added to molecular beacon MB1 (final concentration 100 nM) for about 30min for sufficient assembly, and then the disassembled Zn-RNA nanoparticles (i.e., the particles were degraded by treatment with disodium salt solution of EDTA to release encapsulated miRNA, containing miRNA-205 at about 200 nM) were added and mixed well. The latter three sets of experiments were set up identically, the fourth set being identical to the first set, wherein the antagomiR-221 packaged with Zn-RNA nanoparticles was also about 200nM; the fifth group was prepared by adding molecular beacon MB2 (loop containing a sequence that can complementarily pair with antagomiR-221, final concentration 100 nM) in PBS (10mM PBS, ph = 7.4) buffer; the sixth group was in PBS (10mM PBS,5mM MgCl 2 pH = 7.4) buffer solution was sufficiently assembled by adding molecular beacon MB2 (final concentration 100 nM) for about 30min, and then adding disodium salt solution of the same EDTAThe treated Zn-RNA nanoparticles (containing antagomiR-221 of about 200 nM) were mixed well. The system of each parallel experiment is 100 mu L, then after reacting for 2h at 37 ℃, the fluorescence intensity of each group of reaction samples is measured by F-7000, and each group of experiment samples is repeated in parallel for three times. Wherein the excitation wavelength of F-7000 in the first three groups of experimental processes is set to 480nm, the collected emission range is 505 nm-650 nm, the slit width is set to 10nm, and the voltage is 600V; the excitation wavelength of the latter three groups was set to 625nm, the collected emission range was 650 nm-720 nm, the slit width was all set to 10nm, and the voltage was 600V.
FIG. 7 is a scanning electron microscope and a transmission electron microscope image of Zn-RNA nanoparticles prepared in this example, (A) a scanning electron microscope image of Zn-RNA nanoparticles, and (B) a transmission electron microscope image of Zn-RNA nanoparticles. As shown in FIGS. 7A-B, the Zn-RNA nanoparticles prepared in this example have discrete spherical morphology with a size of about 100 nm.
FIG. 8 shows the verification of the simultaneous loading of two RNAs in this example, (A) the fluorescence spectra of MB1 (molecular beacon-miR-205), zn-RNA Nanoparticles (NPs) and a mixture of MB1 and nanoparticles (MB + NPs) in PBS buffer (pH 7.4), (B) the fluorescence spectra of MB2 (molecular beacon-antmiR-221), zn-RNA Nanoparticles (NPs) and a mixture of MB2 and nanoparticles (MB + NPs) in PBS buffer (pH 7.4). As shown in FIGS. 8A-B, in the system, when only molecular beacons or Zn-RNA nanoparticles exist, the generated fluorescence values are low and can be ignored, and after the Zn-RNA nanoparticles are treated by a chelating agent EDTA disodium salt, the fluorescence of MB1 and MB2 is obviously recovered, which indicates that miRNA-205 and antagomiR-221 successfully synthesize Zn-RNA nanoparticles, and the hybridization function of the Zn-RNA nanoparticles is still maintained in the synthesis process, thereby indicating that Zn 2+ Coordination-driven RNA self-assembly is not limited to loading only one RNA, multiple RNAs can be loaded simultaneously, and this property makes Zn-RNA nanoparticles have great potential for the synergistic delivery of various therapeutic RNAs.
The above description is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-described embodiments. All technical schemes belonging to the idea of the invention belong to the protection scope of the invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention, and such modifications and embellishments should also be considered as within the scope of the invention.
Claims (5)
1. A method for encapsulating RNA, comprising the steps of:
mixing an RNA aqueous solution and a Zn ion aqueous solution, incubating in a high-temperature water bath, performing one-step self-assembly to synthesize a Zn-RNA nanostructure under the drive of coordination crosslinking between zinc ions and RNA nucleotide, centrifuging, and washing with water to obtain a solid;
the method is used for loading miRNA-205 and antagomiR-221 simultaneously;
in the method, the pH is 5.0-7.0.
2. The method for encapsulating RNA according to claim 1, wherein the concentration of RNA in the aqueous RNA solution is 10. Mu.M to 100mM.
3. The method for encapsulating RNA according to claim 1, wherein the concentration of Zn ions in the aqueous Zn ion solution is 1 mM-10M.
4. The method for encapsulating RNA according to claim 1, wherein the temperature in the method is 60 ℃ to 100 ℃.
5. The method for encapsulating RNA according to claim 1, wherein the time in the method is 0.5 to 3 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110318427.6A CN113144211B (en) | 2021-03-25 | 2021-03-25 | RNA packaging method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110318427.6A CN113144211B (en) | 2021-03-25 | 2021-03-25 | RNA packaging method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN113144211A CN113144211A (en) | 2021-07-23 |
| CN113144211B true CN113144211B (en) | 2023-01-24 |
Family
ID=76885098
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110318427.6A Active CN113144211B (en) | 2021-03-25 | 2021-03-25 | RNA packaging method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113144211B (en) |
Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1354958A1 (en) * | 2002-04-18 | 2003-10-22 | Max-Delbrück-Centrum Für Molekulare Medizin | Preparation and use of DNA-polyelectrolyte nanoparticles for gene transfer |
| US9732337B2 (en) * | 2009-06-16 | 2017-08-15 | The United Stated of America, as represented by the Secretary, Department of Health & Human Services | RNA nanoparticles and nanotubes |
| CN108578694A (en) * | 2018-04-24 | 2018-09-28 | 武汉科技大学 | The hollow gold nanometer ball of CpG oligodeoxynucleotides modification and preparation method, application |
| CN111110846B (en) * | 2018-10-30 | 2021-07-23 | 国家纳米科学中心 | Metal-nucleic acid nano-particle and preparation method and application thereof |
| CN109504739A (en) * | 2018-11-05 | 2019-03-22 | 桂林理工大学 | A kind of method that microRNA is quickly detected |
| CN111956809B (en) * | 2020-08-21 | 2022-11-01 | 湖南大学 | Nucleic acid biological medicine, preparation method thereof and application thereof in tumor treatment |
| CN112403411B (en) * | 2020-10-27 | 2022-02-01 | 中国科学院长春应用化学研究所 | Self-assembled nano-particles, self-assembled composite nano-particles, preparation method and application |
-
2021
- 2021-03-25 CN CN202110318427.6A patent/CN113144211B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN113144211A (en) | 2021-07-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ding et al. | A self-assembled RNA-triple helix hydrogel drug delivery system targeting triple-negative breast cancer | |
| Ren et al. | Co-delivery of as-miR-21 and 5-FU by poly (amidoamine) dendrimer attenuates human glioma cell growth in vitro | |
| Epple et al. | Application of calcium phosphate nanoparticles in biomedicine | |
| Liu et al. | Capturing intracellular oncogenic microRNAs with self-assembled DNA nanostructures for microRNA-based cancer therapy | |
| Shaat et al. | Modified gold nanoparticles for intracellular delivery of anti-liver cancer siRNA | |
| CN109355310B (en) | ROS (reactive oxygen species) -responsive gene delivery vector as well as preparation method and application thereof | |
| US10335491B2 (en) | Catalytic delivery nanosubstrates (CDNS) for highly efficient delivery of biomolecules | |
| CN106890343B (en) | Targeting polypeptide nano gene vector compound | |
| CN113999846B (en) | Interference RNA for inhibiting AFAP1-AS1 expression and application thereof in increasing breast cancer radiotherapy sensitivity | |
| CN112353950A (en) | Preparation method of siRNA nano delivery system and application of siRNA nano delivery system in prostatic cancer | |
| CN108201533A (en) | A kind of N- succinyl-chitosan nanoparticle pharmaceutical delivery systems of load siRNA and its preparation method and application | |
| CN113144211B (en) | RNA packaging method | |
| CN109045304B (en) | Nucleolar targeting nano-carrier carrying Polymerase I inhibitor and preparation method and application thereof | |
| CN103849652B (en) | A kind of nano-carrier complex for microRNA targeted delivery and preparation method thereof and application | |
| CN104974343A (en) | Modified polyethyleneimine and application thereof in the preparation of gene transfection vector reagent | |
| CN110403915B (en) | DNA and polymer hybrid nucleic acid drug carrier, and preparation method and application thereof | |
| CN116769837A (en) | Nanometer delivery system and preparation method and application thereof | |
| CN108619528A (en) | A kind of cyclodextrin-mesoporous silicon multifunctional nano load medicine particle | |
| CN108531514B (en) | Endogenous hyperbranched polyspermine cationic gene vector and preparation method and application thereof | |
| CN110448539B (en) | Mesoporous silicon sphere with surface modified amino group carrying microRNA and wrapped by mesenchymal stem cell membrane | |
| CN105267983B (en) | iNGR modified brain glioma targeted self-assembly RNAi nano drug delivery system and preparation method thereof | |
| CN113633779A (en) | Gene delivery vector based on surface burr structure nano particle and preparation method thereof | |
| Guo et al. | Multifunctional DNA nanosphere platform for real-time monitoring and fluorescence imaging-guided photodynamic therapy | |
| CN108403665B (en) | Prostate cancer targeted drug delivery carrier modified by EpDT3 aptamer, delivery system, preparation method and application thereof | |
| Sun et al. | The combined plasma membrane coating and cluster bombing strategy for improved tumor-targeting gene delivery of silicon nanoclusters |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |