CN113368261A - Non-viral vector and preparation method and application thereof - Google Patents

Non-viral vector and preparation method and application thereof Download PDF

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CN113368261A
CN113368261A CN202110673040.2A CN202110673040A CN113368261A CN 113368261 A CN113368261 A CN 113368261A CN 202110673040 A CN202110673040 A CN 202110673040A CN 113368261 A CN113368261 A CN 113368261A
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plasmid
protamine
viral vector
lip
cationic liposome
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张学农
王钰
黄归
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Suzhou University
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Abstract

The invention discloses a non-viral vector and a preparation method and application thereof, wherein the non-viral vector comprises protamine of a compressed plasmid medicament, a cationic liposome coated on the surface of the protamine of the compressed nucleic acid medicament, and a distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid (DSPE-PEG-HA) polymer modified on the surface of the cationic liposome. The non-viral vector of the invention can efficiently accumulate in tumor tissues by virtue of passive targeting and active targeting effects, increase the uptake of tumor cells and promote nucleic acid drugs to enter cell nucleus, accelerate the subsequent transcription and translation processes, and provide a new candidate system for the delivery of gene editing plasmids.

Description

Non-viral vector and preparation method and application thereof
Technical Field
The invention belongs to the technical field of gene editing, and particularly relates to a non-viral vector, and a preparation method and application thereof.
Background
The CRISPR/Cas9 has strong gene editing capability as a third-generation gene editing technology, can realize gene silencing or correction at the DNA level, has permanent action effect once being successfully edited, makes certain progress in clinical tests for treating blood diseases, hereditary retinal diseases and tumor diseases by applying the CRISPR/Cas9, and has wide application prospect in tumor-targeted gene therapy.
The CRISPR/Cas9 system has three common implementations, such as delivery of plasmids encoding Cas9 and sgRNA simultaneously, delivery of Cas9 mRNA and sgRNA, or delivery of Cas9 protein and sgRNA complexes, i.e., ribonucleoprotein complexes. Gene editing occurs in the nucleus, delivering plasmid and ribonucleoprotein complexes, the nuclear targeting ability of the vector is of crucial importance.
CRISPR/Cas9 system delivery methods include physical methods, viral vectors and non-viral vectors. Physical methods include microinjection, electroporation, nuclear infection and membrane deformation, mainly used for gene editing of cells and tissues in vitro or in vitro, which are poorly biocompatible in vivo; viral vectors, such as: related viruses such as lentivirus, adenovirus and the like are widely applied to in vivo and in vitro administration of a CRISPR/Cas9 system at present, but have the safety problems of carcinogenicity, insertion mutation, immunogenicity and the like, and the loading capacity is limited, so that the clinical transformation of a virus vector is seriously influenced; the non-viral vector has the advantages of strong entrapment capability, low immunogenicity and easy assembly, and is a more ideal delivery method.
The non-viral vector loads a CRISPR/Cas9 system mainly comprising a liposome (liposome), a polymer, a polypeptide or protein, a vesicle, a DNA (deoxyribonucleic acid) nanowire group, an inorganic nanoparticle and the like through electrostatic interaction, van der Waals force, hydrogen bonds, covalent bonds and the like. In order to play a gene editing role, the non-viral vector needs to efficiently deliver the CRISPR/Cas9 system to the cytoplasm of a target cell, and for the CRISPR/Cas9 plasmid and ribonucleoprotein complex, the delivery of the CRISPR/Cas9 plasmid and the ribonucleoprotein complex to the nucleus is more favorable for promoting the plasmid to express Cas9 protein and sgRNA, so that the editing efficiency is improved. However, the existing non-viral vectors have the problems of low targeting and stability in the delivery process.
Disclosure of Invention
In order to solve the technical problems, the non-viral vector for gene editing provided by the invention can be efficiently accumulated in tumor tissues by virtue of passive targeting and active targeting effects, increase the uptake of tumor cells and promote nucleic acid drugs to enter cell nuclei, accelerate subsequent transcription and translation processes, and provides a new candidate system for delivery of gene editing plasmids.
The first purpose of the invention is to provide a non-viral vector, which comprises protamine of a compressed plasmid medicament, cationic liposome coated on the surface of the protamine of the compressed nucleic acid medicament, and distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid (DSPE-PEG-HA) polymer modified on the surface of the cationic liposome.
Further, the cationic liposome is DOPE/DOTAP/Chol.
Further, the plasmid drug is a gene editing plasmid.
Furthermore, the gene editing plasmid is one or more of pCas9/sgMTH1, pCas9/sgKRAS, pCas9/sgPLK1 and pCas9/sgMETTL 3.
The second objective of the invention is to provide a preparation method of the non-viral vector, which comprises the following steps:
s1, preparing a distearoyl phosphatidyl ethanolamine-polyethylene glycol-hyaluronic acid polymer by using hyaluronic acid and distearoyl phosphatidyl ethanolamine-polyethylene glycol as raw materials;
s2, preparing cationic liposome by using DOTAP, DOPE and cholesterol as raw materials;
s3, mixing and incubating the protamine solution and the plasmid medicine solution to prepare a protamine/plasmid medicine compound;
s4, mixing and incubating the protamine/plasmid drug compound with the cationic liposome to obtain a protamine/plasmid drug compound coated by the cationic liposome;
s5, mixing and incubating the protamine/plasmid drug compound coated by the cationic liposome and the aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer to obtain the non-viral vector.
In the present invention, in step S1, Hyaluronic Acid (HA) is activated by carboxyl group through activator and then reacted with distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG-NH)2) The reaction takes place. In the step S3, the protamine solution and the plasmid drug solution form a complex through electrostatic interaction. In step S4, the cationic liposome is coated on the surface of the complex by electrostatic interaction. In the step of S5, distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer (DSPE-PEG-HA) is inserted into liposome through hydrophobic interaction, and is modified on the surface of non-viral vector.
Further, in the step S3, the incubation is carried out for 4-6min at 20-30 ℃, and the mass ratio of the protamine to the plasmid drug is 1.8-2.2: 1.
Further, in the step S4, the incubation is carried out at 20-30 ℃ for 15-25min, and the mass ratio of the protamine/plasmid drug complex to the cationic liposome is 3: 11-11.5.
Further, in the step S5, the incubation is performed at 50-60 deg.C for 15-25min, and the concentration of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer in the aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer is 19-21 mg/mL-1。
Further, in the step S1, the hyaluronic acid is activated by an activating agent, and then reacted with distearoylphosphatidylethanolamine-polyethylene glycol, wherein the activating agent is one or more of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC. HCl), N-hydroxysuccinimide (NHS), and Dicyclohexylcarbodiimide (DCC).
The third purpose of the invention is to provide the application of the non-viral vector in a CRISPR/Cas9 delivery system.
The protamine adopted by the invention HAs the functions of compressing plasmids and targeting cell nucleus, but can not protect the stable circulation of nucleic acid drugs in blood and deliver the drugs to tumor tissues when being used alone, in order to solve the problem, the invention coats cationic liposome on the outer layer after the protamine compresses the nucleic acid drugs to form a negative electricity compound, and modifies DSPE-PEG-HA on the surface, thereby protecting the nucleic acid drugs from being degraded by nuclease in the blood circulation, reducing the interaction between the vector and blood components, and endowing the vector with active targeting, for the tumor cells with high expression of CD44 receptors, the invention of the vector can increase the accumulation of the vector at the tumor site and mediate the vector to enter the cells. After the carrier is taken by cells, the escape of endosome is realized depending on the phase change of liposome components, the protamine/nucleic acid drug compound is released into cytoplasm, and a Nuclear Localization Signal (NLS) sequence in the protamine can mediate the compound to enter cell nucleus through nuclear pores, thereby promoting the function of nucleic acid drugs to be exerted. The vector formed by the method can realize nuclear delivery of some nucleic acid drugs, and provides a novel vector for plasmid drugs such as CRISPR/Cas9 plasmid.
By the scheme, the invention at least has the following advantages:
the non-viral vector of the invention can efficiently accumulate in tumor tissues by virtue of passive targeting and active targeting effects, increase the uptake of tumor cells and promote nucleic acid drugs to enter cell nucleus, accelerate the subsequent transcription and translation processes, and provide a new candidate system for the delivery of gene editing plasmids.
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a preferred embodiment of the present invention and is described in detail below.
Drawings
FIG. 1 shows HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in example 1 of the present invention1An H-NMR spectrum;
FIG. 2 shows IR spectra of HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in example 1 of the present invention;
FIG. 3 is the plasmid-coating ability of the vector in example 1 of the present invention, wherein a: marker; b: bare pMTH 1; c: PS/pMTH 1; d: PS @ HA-Lip/pMTH 1;
fig. 4 is a graph showing the particle size of the carrier measured by a dynamic light scattering particle size analyzer DLS in example 1 of the present invention, wherein a: the particle size distribution of different carriers; b: zeta potential of different supports;
FIG. 5 is an electron micrograph of PS, PS @ Lip and PS @ HA-Lip in example 1 of the present invention;
FIG. 6 is the serum stability of the vector in example 1 of the present invention;
FIG. 7 shows the in vitro safety of the drug-free carrier in CCK-8 experiment in example 1 of the present invention A: HUVEC cells, B: a549 cells;
FIG. 8 shows the cellular uptake of the vector by confocal laser microscopy as described in example 1 of the present invention;
FIG. 9 shows the cellular uptake of the vector for flow cytometry quantitative assessment A in example 1 of the present invention: a549 cells, B: MDA-MB-231 cells, C: HepG2 cells;
FIG. 10 is a confocal laser microscopy study of vector delivery into the nucleus of a cell in example 1 of the present invention;
FIG. 11 is a schematic diagram of a non-viral vector of the present invention.
Detailed Description
The following detailed description of the preferred embodiments of the present invention, taken in conjunction with the accompanying drawings, will make the advantages and features of the invention easier to understand by those skilled in the art, and thus will clearly and clearly define the scope of the invention.
Example 1:
preparation of a non-viral vector comprising the steps of:
(1) synthesis of DSPE-PEG-HA
Hyaluronic Acid (HA)64.0mg was weighed out and dissolved in 5mL deionized water, 49.0mg EDC.HCl and 29.0mg NHS were added with stirring, and activated at 45 ℃ for 2 h. 60mg of DSPE-PEG-NH is weighed2Dissolving in 5mL DMSO, dissolving completely, and mixing DSPE-PEG-NH2Dropwise adding activated HA, magnetically stirring for 24h, adding reaction product into dialysis bag (MWCO 3500), adding into deionized water, magnetically stirring, dialyzing for 48h, filtering dialysate, and freezing filtrateDrying to obtain white solid DSPE-PEG-HA.
(2) Preparation of liposomes
Precisely weighing DOTAP, DOPE and cholesterol (Chol), dissolving in chloroform and methanol (2:1, v/v) mixed organic solvent, and preparing mother liquor (20 mg.mL)-1). Adding 200 μ L of DOTAP mother liquor, 200 μ L of DOPE mother liquor, and 100 μ L of cholesterol mother liquor into a penicillin bottle, adding 2mL of mixed organic reagent and 10mL of deionized water, performing probe ultrasonic treatment (200W, 5min), transferring to a eggplant-shaped bottle, performing rotary evaporation at 45 deg.C to remove organic reagent, and filtering with 0.45 μm filter membrane to obtain liposome Lip (1 mg.mL)-1)。
(3) Mixing the protamine solution and pMTH1 plasmid solution, vortexing for 30s, and incubating at room temperature for 5min to obtain protamine/plasmid complex.
(4) And (4) mixing the compound obtained in the step (3) with the liposome according to the ratio determined by the experiment, vortexing for 30s, and incubating at room temperature for 20min to obtain the protamine/plasmid compound coated by the liposome.
(5) And (3) mixing the liposome-coated protamine/plasmid complex obtained in the step (4) with the aqueous solution of DSPE-PEG-HA obtained in the step (1), and incubating for 20min at 55 ℃ to obtain PS @ HA-Lip/pMTH 1.
The compounds in this example were characterized and performance of the non-viral vectors was determined as follows:
1. nuclear magnetic characterization
Dissolving HA in heavy Water (D)2O), DSPE-PEG-HA dissolved in deuterated dimethyl sulfoxide (d6-DMSO), 400MHz NMR instrument test,1the H-NMR spectrum is shown in FIG. 1: peaks at 5.91ppm and 4.50ppm are characteristic peaks of last methyl hydrogen on HA sugar ring, peaks at 1.24ppm are characteristic peaks of methylene hydrogen on distearoyl carbon chain, peaks at 3.50 ppm are characteristic peaks of methylene hydrogen on PEG, and characteristic peaks of HA and DSPE-PEG appear in DSPE-PEG-HA hydrogen spectrum, which indicates that DSPE-PEG-HA is successfully synthesized.
2. Infrared characterization
Taking appropriate amount of dried powder of HA, DSPE-PEG and DSPE-PEG-HA, and performing computer analysis to obtain Fourier transform infrared spectra of the three compounds.
The DSPE-PEG-HA infrared spectrum is shown in figure 2: 2916cm-1、2868cm-1Is the stretching vibration peak of PEG methylene; 3329cm in the infrared spectrum of DSPE-PEG-HA due to the formation of a new amide bond by the reaction-1The intensity of the stretching vibration peak of the primary amide is increased; 1733cm-1The peak of stretching vibration of carbonyl group of distearyl ester is shown at 1637cm-1、1573cm-1Enhancement of stretching vibration peak of carbonyl group of amide bond, 843cm-1The out-of-plane bending vibration of the N-H disappears, which indicates that the DSPE-PEG-HA is successfully synthesized.
3. Agarose electrophoresis investigation of plasmid coating by vector
According to the mass ratio of 2:1, preparing a plasmid/protamine compound, preparing PS @ HA-Lip/pMTH1 according to the N/P ratio of 2/1, fully and uniformly mixing the PS @ HA-Lip/pMTH1 with 6 XDNA loading buffer solution in proportion, loading, setting the voltage to be 150V, observing the result by using a gel imager after 30min, and obtaining the result shown in figure 3.
A complex of PS and pMTH1 is prepared, mixed with Lip at a ratio of 0.5/1(w/w), when the complex is negatively charged, coated with Lip, and surface-modified with HA by "post-insertion" to obtain PS @ HA-Lip/pMTH 1. The agarose electrophoresis results showed that pMTH1 can be completely coated with PS @ HA-Lip.
4. Particle size potential of the support
Preparation of Lip, P-Lip, HA-Lip, PS @ HA-Lip, Nano ZS90 nanometer particle size and zeta potential analyzer determination of carrier particle size, potential, Polydispersity (PDI), the results are shown in FIG. 4.
The particle sizes and the potentials of the pMTH1 loaded carriers Lip, P-Lip, HA-Lip and PS @ HA-Lip are measured, and the results show that the particle sizes of the four carriers are proper and the distribution is uniform; lip HAs stronger positive charge, the potential is 37.2mV when the carrier is prepared according to the N/P of 2/1, the positive charge can be reduced by the modification of PEG and HA, the PS @ HA-Lip is slightly increased compared with HA-Lip, and the whole potential of the PS @ HA-Lip prepared by the PSp is slightly increased due to the fact that the partial negative charge of the plasmid is combined by the PS. PEG and HA can reduce the surface charge of the carrier, reduce the interaction with plasma proteins, and contribute to the long circulation of the carrier in the blood.
5. Morphology of the Carrier under Electron microscope
The plasmid-loaded PS, PS @ Lip and PS @ HA-Lip were dropped on a carbon-supported membrane, dried overnight at 55 ℃, and a picture was taken using a Transmission Electron Microscope (TEM) to observe the morphology of the carrier, as shown in FIG. 5.
PS, PS @ Lip and PS @ HA-Lip can be observed to be in a round spherical shape under TEM; after the liposome is coated, the particle sizes of PS @ Lip and PS @ HA-Lip are larger than that of PS, and a layer of shell is uniformly distributed on the surface of the PS, so that the successful preparation of the carrier is proved and the carrier HAs a proper particle size.
6. Serum stability of the vehicle
The PBS solution, Lip, P-Lip, HA-Lip, PS @ HA-Lip in a volume of 100. mu.L were co-incubated with 100. mu.L of fetal bovine serum at 37 ℃ with the PBS group as a control, and the absorbance at 630nm of each group of samples was measured at predetermined time points to determine the relative turbidity (A)At a certain point in time/A0h) The incubation times were plotted and the serum stability of the vehicle was assessed as a change in turbidity of the mixture, the results being shown in FIG. 6.
The vector was incubated with fetal bovine serum, and the relative turbidity at predetermined time points of Lip, P-Lip, HA-Lip, PS @ HA-Lip was recorded to evaluate the serum stability of the vector. As can be seen from the above figure, Lip is easy to interact with negatively charged proteins in serum to generate precipitates due to the high positive charge, so that the relative turbidity is increased; modification of PEG is beneficial to improving the serum stability of Lip and obviously reducing the relative turbidity of a mixed system; although the HA-Lip and the PS @ HA-Lip have different charges, the relative turbidity is not greatly different, so that the HA-Lip and the PS @ HA-Lip can stably exist in serum, and the modification of HA can further reduce the interaction between a carrier and serum protein, improve the stability of the carrier and be beneficial to long circulation in vivo.
7. CCK-8 experiment for investigating safety of carrier
Taking logarithmically grown A549 cells and HUVEC cells, culturing in a 96-well plate (3000/well), co-culturing for 12h with a culture medium containing carriers with different concentrations for 24h, adding 20 mu L of CCK-8 solution, continuously incubating for 4h, and measuring the absorbance value OD by using a multifunctional enzyme-labeling instrument450. Cell viability was calculated according to the following formula and the safety of the vectors against normal and tumor cells was evaluatedAnd (4) completeness. Different concentrations of Lip, P-Lip, HA-Lip and PS @ HA-Lip loaded with negative control plasmid P-null without drug effect were administered.
Figure BDA0003119517750000061
OD450(treated): absorbance of cells, plus carrier and CCK-8 solution wells; OD0: cell-free, absorbance of medium plus CCK-8 solution wells; OD450(nontreated): with cells, CCK-8 was added, and the absorbance of the wells was not loaded.
The cell survival rate of A549 cells and HUVEC cells after the cells grow for 24 hours in culture media containing different carriers is determined, and the safety of the carriers is evaluated. As shown in FIG. 7, it is understood that Lip has significant cytotoxicity to both cells due to high positive surface charge, and the carrier concentration is 250. mu.g.mL-1Both cell viability rates were about 20%; the P-Lip presents certain cytotoxicity to tumor cells and normal cells, the cell survival rate is about 60% when the concentration of the carrier is the highest, and the cell survival rate is slightly increased to about 80% when the concentration is low, so that the PEG modification can reduce the cytotoxicity brought by overhigh surface charge; due to the further shielding of the HA on the carrier charge, the cell survival rate of the HA-Lip and the PS @ HA-Lip is higher than 95% at a higher concentration, and the existence of the HA-Lip and the PS @ HA-Lip can be regarded as having no influence on various life activities of cells and having higher safety.
8. Laser confocal microscope investigation of uptake and intracellular distribution of multifunctional non-viral vectors
It is contemplated that pMTH1 itself is not fluorescently labeled, and is replaced with FAM-labeled siRNA. Taking logarithmic growth A549 cells, inoculating a proper amount of the cells to a confocal dish, culturing for 12h, and reacting with P-Lip/siRNAFAM、HA-Lip/siRNAFAM、 PS@HA-Lip/siRNAFAM、HA+PS@HA-Lip/siRNAFAM(siRNAFAMConcentration: 50 nM). After 2h, washing with PBS 3 times, incubating with lysosome red dye solution (60nM) at 37 deg.C for 30min, washing with PBS 3 times, fixing with 4% formaldehyde solution for 10min, and Hoechst33258(10 μ g. mL)-1) Staining nuclei, incubation at room temperature for 10min, then PBS washing for 3 times, dripping a proper amount of fluorescence quenching mounting solution on the edge of the circular cover glass, slowly covering the circular cover glass into a confocal cuvette, and observing the distribution in cells and the escape condition of inclusion bodies by using a laser confocal microscope, wherein the result is shown in figure 8.
When the incubation time is 2h, the green fluorescence signal of the P-Lip is mainly distributed in the outer area of the cell, while the HA-modified two groups have uniform fluorescence distribution in the cell, when the PS @ HA-Lip/siRNA is usedFAMAfter co-incubation with free hyaluronic acid, vector distribution and P-Lip/siRNAFAMThe similarity is shown, which indicates that the free hyaluronic acid can compete with the carrier for the CD44 receptor on the cell surface, and once the CD44 receptor is saturated, the amount of the carrier entering the cell is obviously reduced, and indicates that the interaction of the hyaluronic acid-CD 44 receptor can promote the cellular uptake of the carrier. PS @ HA-Lip/siRNAFAMThe panel observed a separation of the red signal from the green fluorescence signal of the vector for lysosomes, suggesting that our vector was successfully evaded from the endosome into the cytoplasm.
9. Flow cytometry quantitative investigation of cellular uptake of multifunctional non-viral vectors
Taking logarithmic growth A549 cells, inoculating a proper amount of A549 cells to a 6-well plate (about 30 ten thousand per well), culturing for 12h, and reacting with P-Lip/siRNAFAM、HA-Lip/siRNAFAM、PS@HA-Lip/siRNAFAM、HA+PS@HA-Lip/siRNAFAM (siRNAFAMConcentration: 50 nM). After 2h the cells were washed 3 times with PBS, trypsinized, washed 3 times with PBS and finally resuspended in 0.3mL PBS and the uptake was analyzed using flow cytometry. Uptake experiments were performed with MDA-MB-231 cells and HepG2 cells, and the effect of cellular CD44 receptor expression levels on vector cell uptake was examined, and the results are shown in FIG. 9.
The cellular uptake of the vector was quantitatively analyzed using a flow cytometer. For A549 cells, the uptake of HA-Lip and PS @ HA-Lip cells is obviously higher than that of P-Lip, and the cell uptake of the vector is similar to that of a P-Lip group when HA exists at the same time, which indicates that HA modification can promote the vector to enter the A549 cells and is consistent with the result of a confocal picture; similar results were shown on MDA-MB-231 cells with high expression of CD44 receptor, but not HepG2 cells with low expression of CD44 receptor, indicating that the promotion of cellular uptake by hyaluronic acid depends on the expression of cellular CD44 receptor, further demonstrating that HA-Lip and PS @ HA-Lip can promote vector entry into cells through HA-CD44 receptor interaction.
10. Laser confocal microscope investigation of nuclear entry process of multifunctional non-viral vector
Taking a proper amount of logarithmic growth A549 cells to inoculate a confocal small dish (15 ten thousand per dish), culturing for 12h, and mixing with HA-Lip/siRNAFAM、PS@HA-Lip/siRNAFAMAnd (4) co-incubation. After 4h and 8h, the cells were washed 3 times with PBS, lysosome red stain (60nM) was added, incubation was carried out at 37 ℃ for 30min, PBS was washed 3 times, 4% formaldehyde solution was added for fixation for 10min, and then PBS was washed 3 times, Hoechst33258 (10. mu.g.mL. mu.L.)-1) Staining cell nucleus, incubating for 10min at room temperature, washing with PBS for 3 times, adding appropriate amount of anti-fluorescence quenching sealing liquid, sealing, storing in a refrigerator at 4 deg.C in dark place, and observing the distribution of carrier cells with confocal microscope, with the result shown in FIG. 10.
The confocal microscope examines the intracellular distribution of the vector in A549 cells after 4h and 8h of administration, and the result shows that the green fluorescent signals of HA-Lip and PS @ HA-Lip are uniformly distributed in cytoplasm after 4h, and the red fluorescent signal and the green fluorescent signal are separated to a certain extent to prompt part of the vector to escape from an endosome; after 8h, PS @ HA-Lip green fluorescence is also distributed in cell nuclei, so that the carrier is prompted to enter the cell nuclei, and no obvious green fluorescence signal exists in the cell nuclei of the HA-Lip group. In conclusion, the PS can promote the plasmid to enter the cell nucleus of the tumor cell through the NLS in the sequence, which is beneficial to the subsequent transcription and translation processes of the plasmid and improves the gene editing efficiency.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.

Claims (10)

1. A non-viral vector comprising protamine having a compressed plasmid drug, a cationic liposome coated on the surface of the protamine having a compressed nucleic acid drug, and a distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer modified on the surface of the cationic liposome.
2. The non-viral vector according to claim 1, wherein the cationic liposome is DOPE/DOTAP/Chol.
3. The non-viral vector according to claim 1, wherein the plasmid drug is a gene editing plasmid.
4. The non-viral vector according to claim 3, wherein the gene editing plasmid is one or more of pCas9/sgMTH1, pCas9/sgKRAS, pCas9/sgPLK1, pCas9/sgMETTL 3.
5. A method for producing the non-viral vector according to any one of claims 1 to 4, comprising the steps of:
s1, preparing a distearoyl phosphatidyl ethanolamine-polyethylene glycol-hyaluronic acid polymer by using hyaluronic acid and distearoyl phosphatidyl ethanolamine-polyethylene glycol as raw materials;
s2, preparing cationic liposome by using DOTAP, DOPE and cholesterol as raw materials;
s3, mixing and incubating the protamine solution and the plasmid medicine solution to prepare a protamine/plasmid medicine compound;
s4, mixing and incubating the protamine/plasmid drug compound with the cationic liposome to obtain a protamine/plasmid drug compound coated by the cationic liposome;
s5, mixing and incubating the protamine/plasmid drug compound coated by the cationic liposome and the aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer to obtain the non-viral vector.
6. The method according to claim 5, wherein in the step S3, the incubation is performed at 20-30 ℃ for 4-6min, and the mass ratio of the protamine to the plasmid drug is 1.8-2.2: 1.
7. The method according to claim 5, wherein in the step of S4, the incubation is performed at 20-30 ℃ for 15-25min, and the mass ratio of the protamine/plasmid drug complex to the cationic liposome is 3: 11-11.5.
8. The method according to claim 5, wherein the incubation in the step S5 is performed at 50-60 ℃ for 15-25min, and the concentration of the distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer in the aqueous solution of the distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer is 19-21 mg-mL-1。
9. The method according to claim 5, wherein in the step of S1, the hyaluronic acid is activated by an activating agent and then reacted with distearoylphosphatidylethanolamine-polyethylene glycol, wherein the activating agent is one or more selected from the group consisting of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and dicyclohexylcarbodiimide.
10. Use of the non-viral vector of any one of claims 1 to 4 in a system for delivering CRISPR/Cas 9.
CN202110673040.2A 2021-06-17 2021-06-17 Non-viral vector and preparation method and application thereof Pending CN113368261A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112143831A (en) * 2020-09-29 2020-12-29 重庆大学 Bionic cell-like structure sensor and preparation method and application thereof
CN114699538A (en) * 2022-04-02 2022-07-05 中国药科大学 Core-shell type efficient gene drug delivery system and preparation method thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103550783A (en) * 2013-04-27 2014-02-05 中国人民解放军军事医学科学院放射与辐射医学研究所 Nucleic acid drug targeting delivery system and preparation method thereof
CN108743970A (en) * 2018-06-07 2018-11-06 上海应用技术大学 A kind of hyaluronic acid decorated Mitochondrially targeted liposome and preparation method thereof
CA3069821A1 (en) * 2017-07-14 2019-01-17 Oncorus, Inc. Encapsulated polynucleotides and methods of use
CN110882383A (en) * 2019-11-26 2020-03-17 宁夏医科大学 Cationic liposome-protamine-mRNA tumor vaccine and preparation method and application method thereof

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103255174B (en) * 2013-05-02 2015-10-28 天津大学 With liquid and application that the hyaluronic acid of polyoxyethylene glycol grafting is outer field ternary complex and ternary complex
WO2018237369A2 (en) * 2017-06-23 2018-12-27 Vical Incorporated Lipid nanoparticle (lnp)-mediated delivery of a crispr-expressing plasmid dna for treating chronic hepatitis b virus infection

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103550783A (en) * 2013-04-27 2014-02-05 中国人民解放军军事医学科学院放射与辐射医学研究所 Nucleic acid drug targeting delivery system and preparation method thereof
CA3069821A1 (en) * 2017-07-14 2019-01-17 Oncorus, Inc. Encapsulated polynucleotides and methods of use
CN111212914A (en) * 2017-07-14 2020-05-29 昂克诺斯公司 Encapsulated polynucleotides and methods of use
CN108743970A (en) * 2018-06-07 2018-11-06 上海应用技术大学 A kind of hyaluronic acid decorated Mitochondrially targeted liposome and preparation method thereof
CN110882383A (en) * 2019-11-26 2020-03-17 宁夏医科大学 Cationic liposome-protamine-mRNA tumor vaccine and preparation method and application method thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIN HONG KIM等: "Hyaluronic acid-based nanomaterials for cancer therapy", 《POLYMERS》 *
SHIVANI RAI PALIWAL等: "Hyaluronic acid modified pH-sensitive liposomes for targeted intracellular delivery of doxorubicin", 《JOURNAL OF LIPOSOME RESEARCH》 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112143831A (en) * 2020-09-29 2020-12-29 重庆大学 Bionic cell-like structure sensor and preparation method and application thereof
CN112143831B (en) * 2020-09-29 2024-03-12 重庆大学 Bionic cell structure sensor and preparation method and application thereof
CN114699538A (en) * 2022-04-02 2022-07-05 中国药科大学 Core-shell type efficient gene drug delivery system and preparation method thereof

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