WO2022262050A1 - Non-viral vector, and preparation method therefor and use thereof - Google Patents
Non-viral vector, and preparation method therefor and use thereof Download PDFInfo
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- WO2022262050A1 WO2022262050A1 PCT/CN2021/106991 CN2021106991W WO2022262050A1 WO 2022262050 A1 WO2022262050 A1 WO 2022262050A1 CN 2021106991 W CN2021106991 W CN 2021106991W WO 2022262050 A1 WO2022262050 A1 WO 2022262050A1
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- protamine
- plasmid
- viral vector
- lip
- distearoylphosphatidylethanolamine
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Definitions
- the invention belongs to the technical field of gene editing, and in particular relates to a non-viral vector and its preparation method and application.
- CRISPR/Cas9 As the third-generation gene editing technology, CRISPR/Cas9 has powerful gene editing capabilities, and can realize gene silencing or correction at the DNA level. Once edited successfully, the effect will be permanent. It can be used to treat blood diseases, hereditary retinal diseases and tumors Some progress has been made in clinical trials of the disease, and it has broad application prospects in tumor-targeted gene therapy.
- CRISPR/Cas9 system There are three implementation forms of CRISPR/Cas9 system, such as delivery of plasmids encoding both Cas9 and sgRNA, delivery of Cas9 mRNA and sgRNA, or delivery of Cas9 protein and sgRNA complex, that is, ribonucleoprotein complex.
- Gene editing occurs in the nucleus, and the ability of the vector to target the nucleus is critical when delivering a plasmid and ribonucleoprotein complex.
- CRISPR/Cas9 system delivery methods include physical methods, viral vectors and non-viral vectors.
- Physical methods include microinjection, electroporation, nuclear infection, and membrane deformation, which are mainly used for gene editing of cells and tissues in vitro or in vitro, which have poor biocompatibility in vivo; viral vectors, such as: lentivirus, adenovirus, Adenovirus and other related viruses have been widely used in the in vivo and in vitro drug delivery of the CRISPR/Cas9 system, but there are safety issues such as carcinogenicity, insertion mutation, and immunogenicity, and the loading capacity is limited, which seriously affects the clinical application of viral vectors. Transformation: Non-viral vectors have the advantages of strong loading capacity, low immunogenicity, and easy assembly, and are ideal delivery methods.
- Non-viral vectors load CRISPR/Cas9 systems through electrostatic interactions, van der Waals forces, hydrogen bonds and covalent bonds, etc., mainly liposomes (lipsomes), polymers, polypeptides or proteins, vesicles, DNA nanocoils, inorganic nano Granules etc.
- liposomes liposomes
- non-viral vectors need to efficiently deliver the CRISPR/Cas9 system to the cytoplasm of target cells.
- delivering it to the nucleus is more conducive to promoting the expression of the plasmid Cas9 protein and sgRNA to improve editing efficiency.
- the existing non-viral vectors still have problems of low targeting and stability during the delivery process.
- the present invention provides a non-viral vector for gene editing.
- the non-viral vector of the present invention relies on passive targeting and active targeting effects, can efficiently accumulate in tumor tissues, increase the uptake of tumor cells and promote Nucleic acid drugs enter the nucleus to accelerate the subsequent transcription and translation processes, providing a new candidate system for the delivery of gene editing plasmids.
- the first object of the present invention is to provide a kind of non-viral vector, and described non-viral vector comprises the protamine that compresses plasmid drug, the cationic liposome coated on the surface of protamine that has compressed nucleic acid drug, and modification Distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid (DSPE-PEG-HA) polymer on the surface of cationic liposomes.
- DSPE-PEG-HA Distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid
- the cationic liposome is DOPE/DOTAP/Chol.
- the plasmid drug is a gene editing plasmid.
- the gene editing plasmid is one or more of pCas9/sgMTH1, pCas9/sgKRAS, pCas9/sgPLK1, pCas9/sgMETTL3.
- the second object of the present invention is to provide the preparation method of said non-viral vector, comprising the following steps:
- step S1 hyaluronic acid (HA) reacts with distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG-NH 2 ) after the carboxyl group is activated by an activator.
- step S3 the protamine solution and the plasmid drug solution form a complex through electrostatic interaction.
- step S4 cationic liposomes are coated on the surface of the complex through electrostatic interaction.
- the distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer (DSPE-PEG-HA) is inserted into the liposome through hydrophobic interaction and modified on the surface of the non-viral carrier.
- step S3 incubation is carried out at 20-30° C. for 4-6 minutes, and the mass ratio of protamine to plasmid drug is 1.8-2.2:1.
- step S4 the incubation is carried out at 20-30° C. for 15-25 min, and the mass ratio of the protamine/plasmid drug complex to the cationic liposome is 3:11-11.5.
- step S5 the incubation is at 50-60°C for 15-25min, in the aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer, distearoylphosphatidylethanolamine-polyethylene glycol
- concentration of ethylene glycol-hyaluronic acid polymer was 19-21 mg ⁇ mL -1 .
- step S1 hyaluronic acid is activated by an activator and then reacted with distearoylphosphatidylethanolamine-polyethylene glycol, and the activator is 1-(3-dimethylaminopropyl)-3- One or more of ethylcarbodiimide hydrochloride (EDC ⁇ HCl), N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC).
- EDC ⁇ HCl ethylcarbodiimide hydrochloride
- NHS N-hydroxysuccinimide
- DCC dicyclohexylcarbodiimide
- the third object of the present invention is to provide the application of the non-viral vector in delivering CRISPR/Cas9 system.
- the protamine used in the present invention has the functions of compressing plasmids and targeting the nucleus, but it cannot protect the stable circulation of nucleic acid drugs in the blood and deliver the drugs to tumor tissues when used alone.
- the present invention compresses protamine After the nucleic acid drug forms a negatively charged complex, the cationic liposome is coated on the outer layer, and the surface is modified with DSPE-PEG-HA, thereby protecting the nucleic acid drug from nuclease degradation in the blood circulation, reducing the interaction between the carrier and blood components, and at the same time The carrier is endowed with active targeting.
- the invented carrier can increase the accumulation of the carrier in the tumor site and mediate the carrier into the cell. After the carrier is taken up by the cell, it depends on the phase transition of the liposome components to escape from the endosome and release the protamine/nucleic acid drug complex into the cytoplasm.
- the nuclear localization signal (NLS) sequence in protamine can mediate the complex Drugs enter the nucleus through nuclear pores, thereby promoting the function of nucleic acid drugs.
- the carrier constructed in this way can realize the nuclear delivery of some nucleic acid drugs, and provide a new type of carrier for plasmid drugs such as CRISPR/Cas9 plasmids.
- the present invention has at least the following advantages:
- the non-viral vector of the present invention can efficiently accumulate in tumor tissue, increase the uptake of tumor cells and promote the entry of nucleic acid drugs into the nucleus, accelerate the subsequent transcription and translation processes, and provide a new way for the delivery of gene editing plasmids.
- a new candidate system was developed.
- Fig. 1 is the 1 H-NMR spectrum of HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in Example 1 of the present invention
- Fig. 2 is the infrared spectrum of HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in Example 1 of the present invention
- Figure 3 is the ability of the vector to coat the plasmid in Example 1 of the present invention, wherein, a: Marker; b: naked pMTH1; c: PS/pMTH1; d: PS@HA-Lip/pMTH1;
- Fig. 4 is the particle size of the carrier detected by the dynamic light scattering particle size analyzer DLS in Example 1 of the present invention, wherein, A: the particle size distribution of different carriers; B: the zeta potential of different carriers;
- Fig. 5 is an electron micrograph of PS, PS@Lip and PS@HA-Lip in Example 1 of the present invention
- Figure 6 is the serum stability of the carrier in Example 1 of the present invention.
- Figure 7 is the in vitro safety of the CCK-8 experiment in Example 1 of the present invention to investigate the non-pharmaceutical carrier A: HUVEC cells, B: A549 cells;
- Fig. 8 is the laser confocal microscopy investigation of the cellular uptake of the carrier in Example 1 of the present invention.
- Figure 9 is the quantitative investigation of the cellular uptake of the carrier by flow cytometry in Example 1 of the present invention
- Figure 10 is a laser confocal microscope in Example 1 of the present invention to investigate the carrier delivery plasmid into the nucleus;
- Figure 11 is a schematic diagram of a non-viral vector of the present invention.
- the preparation of non-viral vector comprises the following steps:
- HA hyaluronic acid
- DOTAP, DOPE and cholesterol (Chol) were precisely weighed, dissolved in a mixed organic solvent of chloroform and methanol (2:1, v/v) to prepare a mother solution (20 mg.mL -1 ).
- a mother solution (20 mg.mL -1 ).
- DOTAP mother solution 200 ⁇ L of DOPE mother solution, and 100 ⁇ L of cholesterol mother solution, and add them to a vial, then add 2 mL of mixed organic reagent and 10 mL of deionized water, ultrasonically (200W, 5min), transfer to an eggplant-shaped bottle, and remove by rotary evaporation at 45°C.
- Organic reagents were filtered through a 0.45 ⁇ m filter membrane to obtain liposome Lip (1 mg.mL -1 ).
- step (3) Mix the complex obtained in step (3) with the liposome according to the ratio determined in the experiment, vortex for 30 s, and incubate at room temperature for 20 min to obtain the liposome-coated protamine/plasmid complex.
- step (4) Mix the liposome-coated protamine/plasmid complex obtained in step (4) with the aqueous solution of DSPE-PEG-HA obtained in step (1), and incubate at 55°C for 20min to obtain PS@ HA-Lip/pMTH1.
- the 1 H-NMR spectrum is shown in the figure Shown in 1: the peaks at 5.91ppm and 4.50ppm are the characteristic peaks of methine hydrogen on the HA sugar ring, the peak at 1.24ppm is the characteristic peak of methylene hydrogen on the distearoyl carbon chain, and the peak at 3.50ppm is The characteristic peaks of methylene hydrogen on PEG, the characteristic peaks of HA and DSPE-PEG appeared in the hydrogen spectrum of DSPE-PEG-HA, indicating that DSPE-PEG-HA has been successfully synthesized.
- the infrared spectrum of DSPE-PEG-HA is shown in Figure 2: 2916cm -1 and 2868cm -1 are the stretching vibration peaks of PEG methylene; due to the reaction to generate new amide bonds, in the infrared spectrum of DSPE-PEG-HA, 3329cm
- the intensity of the stretching vibration peak of the primary amide at -1 increased; the carbonyl stretching vibration peak of distearyl ester appeared at 1733cm -1 , the stretching vibration peak of the carbonyl group of the amide bond at 1637cm -1 and 1573cm -1 strengthened, and the stretching vibration peak of the NH at 843cm -1
- PS, PS@Lip and PS@HA-Lip are round and spherical; after coating liposomes, the particle size of PS@Lip and PS@HA-Lip becomes larger than that of PS, and the surface of PS can be seen A layer of shell is uniformly distributed, which proves that the carrier is successfully prepared and has a suitable particle size.
- the carrier was co-incubated with fetal bovine serum, and the relative turbidity of Lip, P-Lip, HA-Lip, and PS@HA-Lip at predetermined time points was recorded to evaluate the serum stability of the carrier.
- Lip has a high positive charge, it is easy to interact with negatively charged proteins in serum to produce precipitation, resulting in an increase in relative turbidity; PEG modification is beneficial to improve the serum stability of Lip, significantly reducing The relative turbidity of the mixed system; although there is a difference in charge between HA-Lip and PS@HA-Lip, the relative turbidity is not much different, indicating that both can exist stably in serum, and the modification of HA can further reduce the carrier and serum protein interaction, improve the stability of the carrier, and facilitate long-term circulation in the body.
- A549 cells and HUVEC cells with logarithmic growth were taken and cultured in 96-well plates (3000 cells/well). After 12 hours, they were co-cultured with media containing different concentrations of carriers for 24 hours. 20 ⁇ L of CCK-8 solution was added and incubated for 4 hours. The absorbance value OD 450 was measured using a multifunctional microplate reader. The cell viability was calculated according to the following formula, and the safety of the vector to normal cells and tumor cells was evaluated. Lip, P-Lip, HA-Lip, PS@HA-Lip loaded with non-drug-effective negative control plasmid p-null at different concentrations were administered.
- OD 450(treated) Absorbance of wells with cells, loading body and CCK-8 solution
- OD 0 Absorbance of wells without cells, medium and CCK-8 solution
- OD 450(nontreated) cells, plus CCK-8 8. Absorbance of unloaded volumetric wells.
- the cell survival rate of HA-Lip and PS@HA-Lip is also higher than 95% at higher concentrations, which can be regarded as the presence of the two on the cells. There is no impact on various life activities, and it has high safety.
- pMTH1 itself does not have a fluorescent label
- FAM-labeled siRNA Take A549 cells with logarithmic growth, inoculate an appropriate amount into a confocal small dish, culture for 12 hours, and mix with P-Lip/siRNA FAM , HA-Lip/siRNA FAM , PS@HA-Lip/siRNA FAM , HA+PS@HA-Lip/ siRNA FAM (siRNA FAM concentration: 50nM) co-incubation.
- A549 cells with logarithmic growth were inoculated into 6-well plates (about 300,000 per well), cultured for 12 hours, and mixed with P-Lip/siRNA FAM , HA-Lip/siRNA FAM , PS@HA-Lip/siRNA FAM , HA +PS@HA-Lip/siRNA FAM (siRNA FAM concentration: 50nM) co-incubation. After 2 hours, the cells were washed 3 times with PBS, digested with trypsin, washed 3 times with PBS, and finally the cells were resuspended with 0.3 mL of PBS, and the uptake was analyzed by flow cytometry. The uptake experiment was carried out with MDA-MB-231 cells and HepG2 cells to investigate the effect of the expression level of CD44 receptor on the uptake of the vector cells, and the results are shown in FIG. 9 .
- a proper amount of A549 cells with logarithmic growth was inoculated into confocal small dishes (150,000 per dish), cultured for 12 hours, and co-incubated with HA-Lip/siRNA FAM and PS@HA-Lip/siRNA FAM .
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Abstract
Provided are a non-viral vector, and a preparation method therefor and the use thereof. The non-viral vector comprises protamine with a compressed plasmid drug, a cationic liposome coating the surface of the protamine with the compressed nucleic acid drug, and a distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid (DSPE-PEG-HA) polymer modified on the surface of the cationic liposome. The non-viral vector provides a candidate system for the delivery of gene editing plasmids.
Description
本发明属于基因编辑技术领域,尤其涉及一种非病毒载体及其制备方法与应用。The invention belongs to the technical field of gene editing, and in particular relates to a non-viral vector and its preparation method and application.
CRISPR/Cas9作为第三代基因编辑技术,具有强大的基因编辑能力,可在DNA水平实现基因沉默或修正,一旦编辑成功,作用效果永久,应用其治疗血液性疾病、遗传性视网膜疾病和肿瘤性疾病的临床试验取得一定进展,在肿瘤靶向基因治疗中具有广阔的应用前景。As the third-generation gene editing technology, CRISPR/Cas9 has powerful gene editing capabilities, and can realize gene silencing or correction at the DNA level. Once edited successfully, the effect will be permanent. It can be used to treat blood diseases, hereditary retinal diseases and tumors Some progress has been made in clinical trials of the disease, and it has broad application prospects in tumor-targeted gene therapy.
CRISPR/Cas9系统共有三种实现形式,如递送同时编码Cas9和sgRNA的质粒、递送Cas9mRNA和sgRNA或者递送Cas9蛋白和sgRNA复合物,即核糖核蛋白复合体。基因编辑发生在细胞核,递送质粒和核糖核蛋白复合物时,载体的细胞核靶向能力至关重要。There are three implementation forms of CRISPR/Cas9 system, such as delivery of plasmids encoding both Cas9 and sgRNA, delivery of Cas9 mRNA and sgRNA, or delivery of Cas9 protein and sgRNA complex, that is, ribonucleoprotein complex. Gene editing occurs in the nucleus, and the ability of the vector to target the nucleus is critical when delivering a plasmid and ribonucleoprotein complex.
CRISPR/Cas9系统递送方法包括物理方法、病毒载体和非病毒载体。物理方法包括微注射、电穿孔、核感染和膜变形,主要用于体外或离体细胞和组织的基因编辑,其在体内生物相容性较差;病毒载体,如:慢病毒、腺病毒、腺病毒等相关病毒等,目前已广泛应用于CRISPR/Cas9系统的体内外给药,但存在致癌性、插入突变、免疫原性等安全问题,且装载能力有限,这些严重影响了病毒载体的临床转化;非病毒载体具有包载能力强、免疫原性低、易于组装的优点,是较为理想的递送方法。CRISPR/Cas9 system delivery methods include physical methods, viral vectors and non-viral vectors. Physical methods include microinjection, electroporation, nuclear infection, and membrane deformation, which are mainly used for gene editing of cells and tissues in vitro or in vitro, which have poor biocompatibility in vivo; viral vectors, such as: lentivirus, adenovirus, Adenovirus and other related viruses have been widely used in the in vivo and in vitro drug delivery of the CRISPR/Cas9 system, but there are safety issues such as carcinogenicity, insertion mutation, and immunogenicity, and the loading capacity is limited, which seriously affects the clinical application of viral vectors. Transformation: Non-viral vectors have the advantages of strong loading capacity, low immunogenicity, and easy assembly, and are ideal delivery methods.
非病毒载体通过静电相互作用、范德华力、氢键和共价键等装载CRISPR/Cas9系统,主要为脂质体(lipsome)、聚合物、多肽或蛋白、囊泡、DNA纳米线团、无机纳米粒等。为了发挥基因编辑作用,非病毒载体需要将CRISPR/Cas9系统高效递送到靶细胞的胞浆,对于CRISPR/Cas9质粒和核糖核蛋白复合物,将其递送到细胞核更有利于促进质粒表达Cas9蛋白和sgRNA,提高编辑效率。但是现有的非病毒载体在递送过程中,还存在靶向性和稳定性不高的问题。Non-viral vectors load CRISPR/Cas9 systems through electrostatic interactions, van der Waals forces, hydrogen bonds and covalent bonds, etc., mainly liposomes (lipsomes), polymers, polypeptides or proteins, vesicles, DNA nanocoils, inorganic nano Granules etc. In order to play a role in gene editing, non-viral vectors need to efficiently deliver the CRISPR/Cas9 system to the cytoplasm of target cells. For the CRISPR/Cas9 plasmid and ribonucleoprotein complex, delivering it to the nucleus is more conducive to promoting the expression of the plasmid Cas9 protein and sgRNA to improve editing efficiency. However, the existing non-viral vectors still have problems of low targeting and stability during the delivery process.
发明内容Contents of the invention
为解决上述技术问题,本发明提供一种用于基因编辑的非病毒载体,本发明的非病毒载体依靠被动靶向和主动靶向效应,可以在肿瘤组织高效蓄积,增加肿瘤细胞的摄取并且促进核酸药物进入细胞核,加速后续的转录、翻译过程,为基因编辑质粒的递送提供了一种新的候选体系。In order to solve the above technical problems, the present invention provides a non-viral vector for gene editing. The non-viral vector of the present invention relies on passive targeting and active targeting effects, can efficiently accumulate in tumor tissues, increase the uptake of tumor cells and promote Nucleic acid drugs enter the nucleus to accelerate the subsequent transcription and translation processes, providing a new candidate system for the delivery of gene editing plasmids.
本发明的第一个目的是提供一种非病毒载体,所述的非病毒载体包括压缩质粒药物的鱼精蛋白,包覆在压缩了核酸药物的鱼精蛋白表面的阳离子脂质体,以及修饰在阳离子脂质体表面的二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸(DSPE-PEG-HA)聚合物。The first object of the present invention is to provide a kind of non-viral vector, and described non-viral vector comprises the protamine that compresses plasmid drug, the cationic liposome coated on the surface of protamine that has compressed nucleic acid drug, and modification Distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid (DSPE-PEG-HA) polymer on the surface of cationic liposomes.
进一步地,所述的阳离子脂质体为DOPE/DOTAP/Chol。Further, the cationic liposome is DOPE/DOTAP/Chol.
进一步地,所述的质粒药物为基因编辑质粒。Further, the plasmid drug is a gene editing plasmid.
进一步地,所述的基因编辑质粒为pCas9/sgMTH1、pCas9/sgKRAS、pCas9/sgPLK1、pCas9/sgMETTL3中的一种或几种。Further, the gene editing plasmid is one or more of pCas9/sgMTH1, pCas9/sgKRAS, pCas9/sgPLK1, pCas9/sgMETTL3.
本发明的第二个目的是提供所述的非病毒载体的制备方法,包括如下步骤:The second object of the present invention is to provide the preparation method of said non-viral vector, comprising the following steps:
S1、采用透明质酸和二硬脂酰基磷脂酰乙醇胺-聚乙二醇为原料制备二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物;S1. Using hyaluronic acid and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials to prepare distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer;
S2、采用DOTAP、DOPE和胆固醇为原料制备阳离子脂质体;S2, using DOTAP, DOPE and cholesterol as raw materials to prepare cationic liposomes;
S3、将鱼精蛋白溶液与质粒药物溶液混合孵育,制备鱼精蛋白/质粒药物复合物;S3. Mixing and incubating the protamine solution and the plasmid drug solution to prepare a protamine/plasmid drug complex;
S4、将鱼精蛋白/质粒药物复合物与阳离子脂质体混合孵育,得到阳离子脂质体包覆的鱼精蛋白/质粒药物复合物;S4. Mixing and incubating the protamine/plasmid drug complex with cationic liposomes to obtain cationic liposome-coated protamine/plasmid drug complexes;
S5、将阳离子脂质体包覆的鱼精蛋白/质粒药物复合物与二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物的水溶液混合孵育,得到所述的非病毒载体。S5. Mix and incubate the cationic liposome-coated protamine/plasmid drug complex with an aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer to obtain the non-viral vector.
在本发明中,S1步骤中,透明质酸(HA)经活化剂活化羧基后与二硬脂酰基磷脂酰乙醇胺-聚乙二醇(DSPE-PEG-NH
2)发生反应。S3步骤中,鱼精蛋白溶液与质粒药物溶液通过静电相互作用形成复合物。S4步骤中,阳离子脂质体通过静电相互作用包覆在复合物表面。S5步骤中,二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物(DSPE-PEG-HA)通过疏水作用插入脂质体,修饰在非病毒载体表面。
In the present invention, in step S1, hyaluronic acid (HA) reacts with distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG-NH 2 ) after the carboxyl group is activated by an activator. In step S3, the protamine solution and the plasmid drug solution form a complex through electrostatic interaction. In step S4, cationic liposomes are coated on the surface of the complex through electrostatic interaction. In step S5, the distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer (DSPE-PEG-HA) is inserted into the liposome through hydrophobic interaction and modified on the surface of the non-viral carrier.
进一步地,S3步骤中,孵育是在20~30℃孵育4-6min,鱼精蛋白与质粒药物的质量比为1.8-2.2:1。Further, in step S3, incubation is carried out at 20-30° C. for 4-6 minutes, and the mass ratio of protamine to plasmid drug is 1.8-2.2:1.
进一步地,S4步骤中,孵育是在20~30℃孵育15-25min,鱼精蛋白/质粒药物复合物与阳离子脂质体的质量比为3:11-11.5。Further, in step S4, the incubation is carried out at 20-30° C. for 15-25 min, and the mass ratio of the protamine/plasmid drug complex to the cationic liposome is 3:11-11.5.
进一步地,S5步骤中,孵育是在50-60℃孵育15-25min,二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物的水溶液中,二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物的浓度为19-21mg·mL
-1。
Further, in step S5, the incubation is at 50-60°C for 15-25min, in the aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer, distearoylphosphatidylethanolamine-polyethylene glycol The concentration of ethylene glycol-hyaluronic acid polymer was 19-21 mg·mL -1 .
进一步地,S1步骤中,透明质酸通过活化剂活化后与二硬脂酰基磷脂酰乙醇胺-聚乙二醇进行反应,所述活化剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC·HCl)、N-羟基琥珀酰亚胺(NHS)和二环己基碳二亚胺(DCC)中的一种或几种。Further, in step S1, hyaluronic acid is activated by an activator and then reacted with distearoylphosphatidylethanolamine-polyethylene glycol, and the activator is 1-(3-dimethylaminopropyl)-3- One or more of ethylcarbodiimide hydrochloride (EDC·HCl), N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC).
本发明的第三个目的是提供所述的非病毒载体在递送CRISPR/Cas9系统中的应用。The third object of the present invention is to provide the application of the non-viral vector in delivering CRISPR/Cas9 system.
本发明所采用的鱼精蛋白具有压缩质粒和细胞核靶向功能,但单独使用不能保护核酸药物在血液中稳定循环并把药物递送到肿瘤组织,为了解决这一问题,本发明在鱼精蛋白压缩核酸药物形成负电复合物后在外层包覆阳离子脂质体,并在表面修饰DSPE-PEG-HA,从而保护核酸药物在血液循环中免受核酸酶降解,减少载体与血液成分的相互作用,同时赋予载体主动靶向性,对于高表达CD44受体的肿瘤细胞,发明所述载体可以增加载体在肿瘤部位的蓄积并介导载体进入细胞。载体被细胞摄取后,依赖脂质体成分的相变实现内涵体逃逸,将鱼精蛋白/核酸药物复合物释放到胞浆中,鱼精蛋白中的核定位信号(NLS)序列可以介导复合物通过核孔进入细胞核,从而促进核酸药物功能的发挥。以此构成的载体可实现一些核酸药物的核递送,为质粒药物如CRISPR/Cas9质粒,提供一种新型载体。The protamine used in the present invention has the functions of compressing plasmids and targeting the nucleus, but it cannot protect the stable circulation of nucleic acid drugs in the blood and deliver the drugs to tumor tissues when used alone. In order to solve this problem, the present invention compresses protamine After the nucleic acid drug forms a negatively charged complex, the cationic liposome is coated on the outer layer, and the surface is modified with DSPE-PEG-HA, thereby protecting the nucleic acid drug from nuclease degradation in the blood circulation, reducing the interaction between the carrier and blood components, and at the same time The carrier is endowed with active targeting. For tumor cells with high expression of CD44 receptors, the invented carrier can increase the accumulation of the carrier in the tumor site and mediate the carrier into the cell. After the carrier is taken up by the cell, it depends on the phase transition of the liposome components to escape from the endosome and release the protamine/nucleic acid drug complex into the cytoplasm. The nuclear localization signal (NLS) sequence in protamine can mediate the complex Drugs enter the nucleus through nuclear pores, thereby promoting the function of nucleic acid drugs. The carrier constructed in this way can realize the nuclear delivery of some nucleic acid drugs, and provide a new type of carrier for plasmid drugs such as CRISPR/Cas9 plasmids.
借由上述方案,本发明至少具有以下优点:By means of the above solution, the present invention has at least the following advantages:
本发明的非病毒载体依靠被动靶向和主动靶向效应,可以在肿瘤组织高效蓄积,增加肿瘤细胞的摄取并且促进核酸药物进入细胞核,加速后续的转录、翻译过程,为基因编辑质粒的递送提供了一种新的候选体系。Relying on passive targeting and active targeting effects, the non-viral vector of the present invention can efficiently accumulate in tumor tissue, increase the uptake of tumor cells and promote the entry of nucleic acid drugs into the nucleus, accelerate the subsequent transcription and translation processes, and provide a new way for the delivery of gene editing plasmids. A new candidate system was developed.
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细说明如后。The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention are described in detail below.
图1是本发明的实施例1中HA(a)、DSPE-PEG(b)和DSPE-PEG-HA(c)的
1H-NMR图谱;
Fig. 1 is the 1 H-NMR spectrum of HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in Example 1 of the present invention;
图2是本发明的实施例1中HA(a)、DSPE-PEG(b)和DSPE-PEG-HA(c)的红外光谱;Fig. 2 is the infrared spectrum of HA (a), DSPE-PEG (b) and DSPE-PEG-HA (c) in Example 1 of the present invention;
图3是本发明的实施例1中载体对质粒的包覆能力,其中,a:Marker;b:裸露pMTH1;c:PS/pMTH1;d:PS@HA-Lip/pMTH1;Figure 3 is the ability of the vector to coat the plasmid in Example 1 of the present invention, wherein, a: Marker; b: naked pMTH1; c: PS/pMTH1; d: PS@HA-Lip/pMTH1;
图4是本发明的实施例1中动态光散射粒度分析仪DLS检测的载体粒径大小,其中,A:不同载体的粒径分布情况;B:不同载体的zeta电位;Fig. 4 is the particle size of the carrier detected by the dynamic light scattering particle size analyzer DLS in Example 1 of the present invention, wherein, A: the particle size distribution of different carriers; B: the zeta potential of different carriers;
图5是本发明的实施例1中PS、PS@Lip和PS@HA-Lip的电镜图;Fig. 5 is an electron micrograph of PS, PS@Lip and PS@HA-Lip in Example 1 of the present invention;
图6是本发明的实施例1中载体的血清稳定性;Figure 6 is the serum stability of the carrier in Example 1 of the present invention;
图7是本发明的实施例1中CCK-8实验考察无药效载体的体外安全性A:HUVEC细胞,B:A549细胞;Figure 7 is the in vitro safety of the CCK-8 experiment in Example 1 of the present invention to investigate the non-pharmaceutical carrier A: HUVEC cells, B: A549 cells;
图8是本发明的实施例1中激光共聚焦显微镜考察载体的细胞摄取;Fig. 8 is the laser confocal microscopy investigation of the cellular uptake of the carrier in Example 1 of the present invention;
图9是本发明的实施例1中流式细胞术定量考察载体的细胞摄取A:A549细胞,B:MDA-MB-231细胞,C:HepG2细胞;Figure 9 is the quantitative investigation of the cellular uptake of the carrier by flow cytometry in Example 1 of the present invention A: A549 cells, B: MDA-MB-231 cells, C: HepG2 cells;
图10是本发明的实施例1中激光共聚焦显微镜考察载体递送质粒进入细胞核;Figure 10 is a laser confocal microscope in Example 1 of the present invention to investigate the carrier delivery plasmid into the nucleus;
图11是本发明的非病毒载体的示意图。Figure 11 is a schematic diagram of a non-viral vector of the present invention.
下面结合附图对本发明的较佳实施例进行详细阐述,以使本发明的优势和特征能更易于被本领域技术人员理解,从而对本发明的保护范围做出更为清楚明确的界定。The preferred embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings, so that the advantages and features of the present invention can be more easily understood by those skilled in the art, so as to define the protection scope of the present invention more clearly.
实施例1:Example 1:
非病毒载体的制备,包括以下步骤:The preparation of non-viral vector comprises the following steps:
(1)DSPE-PEG-HA的合成(1) Synthesis of DSPE-PEG-HA
称取透明质酸(HA)64.0mg溶于5mL去离子水中,在搅拌条件下加入49.0mg EDC.HCl和29.0mg NHS,45℃活化2h。称取60mg DSPE-PEG-NH
2溶于5mL DMSO,充分溶解后,将DSPE-PEG-NH
2逐滴加入活化后的HA,磁力搅拌反应24h,将反应产物装进透析袋(MWCO=3500)置于去离子水中,磁力搅拌,透析48h,透析液过滤,滤液冷冻干燥得到白色固体DSPE-PEG-HA。
Weigh 64.0 mg of hyaluronic acid (HA) and dissolve it in 5 mL of deionized water, add 49.0 mg of EDC.HCl and 29.0 mg of NHS under stirring conditions, and activate at 45°C for 2 hours. Weigh 60mg DSPE-PEG-NH 2 and dissolve it in 5mL DMSO. After fully dissolving, add DSPE-PEG-NH 2 dropwise to the activated HA, stir and react for 24 hours, and put the reaction product into a dialysis bag (MWCO=3500) Place in deionized water, stir magnetically, dialyze for 48 hours, filter the dialysate, and freeze-dry the filtrate to obtain DSPE-PEG-HA as a white solid.
(2)脂质体的制备(2) Preparation of liposomes
精密称取DOTAP、DOPE和胆固醇(Chol),溶于三氯甲烷和甲醇(2:1,v/v)混合有机溶剂,配制母液(20mg.mL
-1)。取200μL DOTAP母液,200μL DOPE母液、100μL胆固醇母液,加入于西林瓶中,再加入2mL混合有机试剂和10mL去离子水,探头超声(200W,5min),转移至茄形瓶,45℃旋转蒸发除去有机试剂,0.45μm滤膜过滤,得脂质体Lip(1mg.mL
-1)。
DOTAP, DOPE and cholesterol (Chol) were precisely weighed, dissolved in a mixed organic solvent of chloroform and methanol (2:1, v/v) to prepare a mother solution (20 mg.mL -1 ). Take 200 μL of DOTAP mother solution, 200 μL of DOPE mother solution, and 100 μL of cholesterol mother solution, and add them to a vial, then add 2 mL of mixed organic reagent and 10 mL of deionized water, ultrasonically (200W, 5min), transfer to an eggplant-shaped bottle, and remove by rotary evaporation at 45°C. Organic reagents were filtered through a 0.45 μm filter membrane to obtain liposome Lip (1 mg.mL -1 ).
(3)按照鱼精蛋白溶液与pMTH1质粒溶液混合,涡旋30s,室温孵育5min,得到鱼精蛋白/质粒复合物。(3) Mix the protamine solution with the pMTH1 plasmid solution, vortex for 30 seconds, and incubate at room temperature for 5 minutes to obtain a protamine/plasmid complex.
(4)将步骤(3)中得到的复合物与脂质体按照实验确定的比例混合,涡旋30s,室温孵育20min,得到脂质体包覆的鱼精蛋白/质粒复合物。(4) Mix the complex obtained in step (3) with the liposome according to the ratio determined in the experiment, vortex for 30 s, and incubate at room temperature for 20 min to obtain the liposome-coated protamine/plasmid complex.
(5)将步骤(4)中得到的脂质体包覆的鱼精蛋白/质粒复合物与步骤(1)中的到的DSPE-PEG-HA的水溶液混合,55℃孵育20min,得到PS@HA-Lip/pMTH1。(5) Mix the liposome-coated protamine/plasmid complex obtained in step (4) with the aqueous solution of DSPE-PEG-HA obtained in step (1), and incubate at 55°C for 20min to obtain PS@ HA-Lip/pMTH1.
下面对该实施例中的化合物进行表征以及非病毒载体的性能测定:The compound in this embodiment is characterized and the performance measurement of non-viral vector below:
1、核磁表征1. NMR characterization
将HA溶于重水(D
2O),将DSPE-PEG、DSPE-PEG-HA溶于氘代二甲亚砜(d6-DMSO), 在400MHz核磁共振仪上测试,
1H-NMR图谱如图1所示:5.91ppm和4.50ppm处的峰为HA糖环上次甲基氢的特征峰,1.24ppm处的峰为二硬脂酰基碳链上亚甲基氢的特征峰,3.50ppm处为PEG上亚甲基氢的特征峰,DSPE-PEG-HA氢谱中出现了HA、DSPE-PEG的特征峰,说明已经成功合成DSPE-PEG-HA。
Dissolve HA in heavy water (D 2 O), dissolve DSPE-PEG and DSPE-PEG-HA in deuterated dimethyl sulfoxide (d6-DMSO), and test it on a 400MHz nuclear magnetic resonance instrument. The 1 H-NMR spectrum is shown in the figure Shown in 1: the peaks at 5.91ppm and 4.50ppm are the characteristic peaks of methine hydrogen on the HA sugar ring, the peak at 1.24ppm is the characteristic peak of methylene hydrogen on the distearoyl carbon chain, and the peak at 3.50ppm is The characteristic peaks of methylene hydrogen on PEG, the characteristic peaks of HA and DSPE-PEG appeared in the hydrogen spectrum of DSPE-PEG-HA, indicating that DSPE-PEG-HA has been successfully synthesized.
2、红外表征2. Infrared Characterization
取适量HA、DSPE-PEG、DSPE-PEG-HA的干燥粉末,上机分析,获取三种化合物的傅立叶变换红外光谱。Take an appropriate amount of dry powder of HA, DSPE-PEG, and DSPE-PEG-HA, analyze it on the machine, and obtain the Fourier transform infrared spectra of the three compounds.
DSPE-PEG-HA红外谱图如图2所示:2916cm
-1、2868cm
-1为PEG亚甲基的伸缩振动峰;由于反应生成新的酰胺键,DSPE-PEG-HA红外谱图中,3329cm
-1处伯酰胺的伸缩振动峰强度增大;1733cm
-1处出现二硬脂酯的羰基伸缩振动峰,1637cm
-1、1573cm
-1处酰胺键羰基伸缩振动峰增强,843cm
-1处N-H的面外弯曲振动消失,说明DSPE-PEG-HA已成功合成。
The infrared spectrum of DSPE-PEG-HA is shown in Figure 2: 2916cm -1 and 2868cm -1 are the stretching vibration peaks of PEG methylene; due to the reaction to generate new amide bonds, in the infrared spectrum of DSPE-PEG-HA, 3329cm The intensity of the stretching vibration peak of the primary amide at -1 increased; the carbonyl stretching vibration peak of distearyl ester appeared at 1733cm -1 , the stretching vibration peak of the carbonyl group of the amide bond at 1637cm -1 and 1573cm -1 strengthened, and the stretching vibration peak of the NH at 843cm -1 The out-of-plane bending vibration disappeared, indicating that DSPE-PEG-HA had been successfully synthesized.
3、琼脂糖电泳考察载体对质粒的包覆3. Agarose electrophoresis to investigate the coating of the vector on the plasmid
按照质量比2:1的制备质粒/鱼精蛋白复合物,按照N/P比2/1制备PS@HA-Lip/pMTH1,与6×DNA上样缓冲液按比例充分混匀并上样,设置电压150V,30min后使用凝胶成像仪观察结果,结果如图3所示。Prepare the plasmid/protamine complex according to the mass ratio of 2:1, prepare PS@HA-Lip/pMTH1 according to the N/P ratio of 2/1, mix well with 6×DNA loading buffer in proportion and load the sample, Set the voltage to 150V, and use a gel imager to observe the results after 30 minutes. The results are shown in Figure 3.
制备PS与pMTH1的复合物,将其与Lip以0.5/1(w/w)比例混合,此时复合物呈负电性,外层包覆Lip,通过“后插入法”表面修饰HA,得到PS@HA-Lip/pMTH1。琼脂糖电泳结果表明PS@HA-Lip可以完全包覆pMTH1。Prepare the complex of PS and pMTH1, mix it with Lip at a ratio of 0.5/1 (w/w), at this time the complex is negatively charged, the outer layer is coated with Lip, and the surface is modified with HA by the "post-insertion method" to obtain PS @HA-Lip/pMTH1. The results of agarose electrophoresis showed that PS@HA-Lip could completely coat pMTH1.
4、载体的粒径电位4. The particle size potential of the carrier
制备Lip、P-Lip、HA-Lip、PS@HA-Lip,Nano ZS90纳米粒度及zeta电位分析仪测定载体粒径、电位、多分散系数(polydispersity,PDI),结果如图4所示。Preparation of Lip, P-Lip, HA-Lip, PS@HA-Lip, Nano ZS90 nanometer particle size and zeta potential analyzer to measure carrier particle size, potential, polydispersity (polydispersity, PDI), the results are shown in Figure 4.
通过对装载pMTH1载体Lip、P-Lip、HA-Lip、PS@HA-Lip的粒径、电位进行测定,由结果可知四种载体粒径适宜,分布均匀;Lip具有较强的正电荷,按照N/P为2/1制备载体时电位为37.2mV,PEG和HA的修饰可以降低其正电荷,PS@HA-Lip相较HA-Lip略有升高,归因于PS结合掉质粒的部分负电荷,PSp制备成PS@HA-Lip整体电位略变大。PEG和HA可以降低载体表面电荷、减少与血浆蛋白的相互作用,有助于载体在血液中长循环。By measuring the particle size and potential of pMTH1-loaded carriers Lip, P-Lip, HA-Lip, and PS@HA-Lip, it can be seen from the results that the particle sizes of the four carriers are suitable and evenly distributed; Lip has a strong positive charge, according to When N/P is 2/1, the potential is 37.2mV. The modification of PEG and HA can reduce its positive charge. Compared with HA-Lip, PS@HA-Lip is slightly higher, which is due to the combination of PS and the part of the plasmid. Negative charge, the overall potential of PSp prepared into PS@HA-Lip becomes slightly larger. PEG and HA can reduce the surface charge of the carrier, reduce the interaction with plasma proteins, and help the carrier circulate in the blood for a long time.
5、载体的电镜下形态5. The shape of the carrier under the electron microscope
将装载质粒的PS、PS@Lip和PS@HA-Lip滴加到碳支持膜上,55℃烘干过夜,使用透射电镜(Transmission electron microscopy,TEM)获取图片,观察载体形态,结果如图5所示。Add the plasmid-loaded PS, PS@Lip and PS@HA-Lip dropwise onto the carbon support membrane, and dry it overnight at 55°C. Use a transmission electron microscope (TEM) to take pictures and observe the shape of the carrier. The results are shown in Figure 5 shown.
TEM下可以观察到PS、PS@Lip和PS@HA-Lip呈圆整的球形;包覆脂质体后,PS@Lip 和PS@HA-Lip较PS粒径变大,可以看到PS表面均匀分布一层外壳,证明载体制备成功且具有适宜粒径。Under TEM, it can be observed that PS, PS@Lip and PS@HA-Lip are round and spherical; after coating liposomes, the particle size of PS@Lip and PS@HA-Lip becomes larger than that of PS, and the surface of PS can be seen A layer of shell is uniformly distributed, which proves that the carrier is successfully prepared and has a suitable particle size.
6、载体的血清稳定性6. Serum stability of the carrier
将体积为100μL的PBS溶液、Lip、P-Lip、HA-Lip、PS@HA-Lip与100μL胎牛血清在37℃下共孵育,以PBS组做对照,在预定时间点,测定各组样品在630nm处的吸光度,以相对浊度(A
某一时间点/A
0h)对孵育时间作图,以混合物浊度变化评价载体血清稳定性,结果如图6所示。
Incubate 100 μL of PBS solution, Lip, P-Lip, HA-Lip, PS@HA-Lip and 100 μL fetal bovine serum at 37°C, and use the PBS group as a control, and measure the samples of each group at a predetermined time point The absorbance at 630nm was plotted against the incubation time by relative turbidity (A certain time point /A 0h ), and the carrier serum stability was evaluated by the change of turbidity of the mixture. The results are shown in FIG. 6 .
载体与胎牛血清共孵育,记录Lip、P-Lip、HA-Lip、PS@HA-Lip在预定时间点的相对浊度,评价载体的血清稳定性。由上图可以看出,Lip由于具有较高的正电荷,易与血清中的负电荷蛋白相互作用产生沉淀,导致相对浊度升高;PEG的修饰有利于提高Lip的血清稳定性,显著降低混合体系的相对浊度;虽然HA-Lip和PS@HA-Lip电荷有差异,但相对浊度相差不大,说明二者在血清中均可稳定存在,HA的修饰可以进一步减少载体与血清蛋白的相互作用,提高载体稳定性,有利于体内长循环。The carrier was co-incubated with fetal bovine serum, and the relative turbidity of Lip, P-Lip, HA-Lip, and PS@HA-Lip at predetermined time points was recorded to evaluate the serum stability of the carrier. As can be seen from the figure above, because Lip has a high positive charge, it is easy to interact with negatively charged proteins in serum to produce precipitation, resulting in an increase in relative turbidity; PEG modification is beneficial to improve the serum stability of Lip, significantly reducing The relative turbidity of the mixed system; although there is a difference in charge between HA-Lip and PS@HA-Lip, the relative turbidity is not much different, indicating that both can exist stably in serum, and the modification of HA can further reduce the carrier and serum protein interaction, improve the stability of the carrier, and facilitate long-term circulation in the body.
7、CCK-8实验考察载体的安全性7. CCK-8 experiment investigates the safety of the carrier
取对数生长的A549细胞和HUVEC细胞,培养于96孔板(3000个/孔),12h后,与含不同浓度载体的培养基共培养24h,加入20μL CCK-8溶液,继续孵育4h后,使用多功能酶标仪测定吸光度值OD
450。根据下式计算细胞活力,评价载体对正常细胞和肿瘤细胞的安全性。给药不同浓度装载无药效的阴性对照质粒p-null的Lip、P-Lip、HA-Lip、PS@HA-Lip。
A549 cells and HUVEC cells with logarithmic growth were taken and cultured in 96-well plates (3000 cells/well). After 12 hours, they were co-cultured with media containing different concentrations of carriers for 24 hours. 20 μL of CCK-8 solution was added and incubated for 4 hours. The absorbance value OD 450 was measured using a multifunctional microplate reader. The cell viability was calculated according to the following formula, and the safety of the vector to normal cells and tumor cells was evaluated. Lip, P-Lip, HA-Lip, PS@HA-Lip loaded with non-drug-effective negative control plasmid p-null at different concentrations were administered.
OD
450(treated):有细胞,加载体和CCK-8溶液孔的吸光度;OD
0:无细胞,加培养基和CCK-8溶液孔的吸光度;OD
450(nontreated):有细胞,加CCK-8,不加载体孔的吸光度。
OD 450(treated) : Absorbance of wells with cells, loading body and CCK-8 solution; OD 0 : Absorbance of wells without cells, medium and CCK-8 solution; OD 450(nontreated) : cells, plus CCK-8 8. Absorbance of unloaded volumetric wells.
测定A549细胞、HUVEC细胞在含有不同载体的培养基中生长24h后细胞存活率,评价载体的安全性。结果如图7所示,可知,由于表面正电荷较高,Lip对两种细胞具有明显的细胞毒性,载体浓度250μg.mL
-1时,两种细胞存活率约为20%;P-Lip对于肿瘤细胞和正常细胞均呈现一定的细胞毒性,载体浓度最高时,细胞存活率约为60%,低浓度时,细胞存活率略有升高,约为80%,说明PEG修饰可以减轻表面电荷过高带来的细胞毒性;得益于HA对载体电荷的进一步屏蔽,HA-Lip和PS@HA-Lip在较高浓度时细胞生存率也高于95%,可以视作二者的存在对细胞各项生命活动没有影响,具有较高的安全性。
The cell viability of A549 cells and HUVEC cells after 24 hours of growth in media containing different vectors was measured to evaluate the safety of the vectors. The results are shown in Figure 7. It can be seen that due to the high positive surface charge, Lip has obvious cytotoxicity to the two kinds of cells. When the carrier concentration is 250 μg.mL -1 , the survival rate of the two kinds of cells is about 20%. Both tumor cells and normal cells exhibit certain cytotoxicity. When the carrier concentration is the highest, the cell survival rate is about 60%, and when the carrier concentration is low, the cell survival rate is slightly increased, about 80%. High cytotoxicity; thanks to the further shielding of the carrier charge by HA, the cell survival rate of HA-Lip and PS@HA-Lip is also higher than 95% at higher concentrations, which can be regarded as the presence of the two on the cells. There is no impact on various life activities, and it has high safety.
8、激光共聚焦显微镜考察多功能非病毒载体的摄取与胞内分布8. Laser confocal microscopy to investigate the uptake and intracellular distribution of multifunctional non-viral vectors
考虑到pMTH1本身不带荧光标记,以FAM标记的siRNA代替。取对数生长的A549 细胞,适量接种至共聚焦小皿,培养12h,与P-Lip/siRNA
FAM、HA-Lip/siRNA
FAM、PS@HA-Lip/siRNA
FAM、HA+PS@HA-Lip/siRNA
FAM(siRNA
FAM浓度:50nM)共孵育。2h后用PBS洗3次,与溶酶体红染液(60nM)37℃孵育30min,PBS洗3次,4%甲醛溶液固定10min,Hoechst33258(10μg□mL
-1)染细胞核,室温孵育10min,然后PBS洗3次,在圆形盖玻片边缘滴加适量荧光淬灭封片液,缓慢将盖玻片盖到共聚焦小皿里,激光共聚焦显微镜观察细胞内分布和内含体逃逸情况,结果如图8所示。
Considering that pMTH1 itself does not have a fluorescent label, it was replaced by FAM-labeled siRNA. Take A549 cells with logarithmic growth, inoculate an appropriate amount into a confocal small dish, culture for 12 hours, and mix with P-Lip/siRNA FAM , HA-Lip/siRNA FAM , PS@HA-Lip/siRNA FAM , HA+PS@HA-Lip/ siRNA FAM (siRNA FAM concentration: 50nM) co-incubation. After 2 hours, wash 3 times with PBS, incubate with lysosome red staining solution (60nM) at 37°C for 30min, wash 3 times with PBS, fix with 4% formaldehyde solution for 10min, stain cell nuclei with Hoechst33258 (10μg mL -1 ), incubate at room temperature for 10min, Then wash with PBS for 3 times, drop an appropriate amount of fluorescent quenching mounting solution on the edge of the circular cover glass, slowly cover the cover glass into a confocal small dish, and observe the intracellular distribution and inclusion body escape under the laser confocal microscope. The result is shown in Figure 8.
孵育时间为2h时,P-Lip的绿色荧光信号主要分布在细胞的外部区域,而HA修饰的两组,胞内有均匀的荧光分布,当PS@HA-Lip/siRNA
FAM与游离透明质酸共孵育后,载体分布和P-Lip/siRNA
FAM呈现相近的情况,说明游离透明质酸可以与载体竞争细胞表面的CD44受体,而CD44受体一旦饱和,载体进入细胞的量会显著减少,说明透明质酸-CD44受体相互作用可以促进载体的细胞摄取。PS@HA-Lip/siRNA
FAM组可以观察到溶酶体的红色信号和载体的绿色荧光信号出现分离,提示我们载体成功从内涵体逃逸到胞浆中。
When the incubation time was 2h, the green fluorescence signal of P-Lip was mainly distributed in the outer area of the cells, while the two groups modified by HA had uniform fluorescence distribution in the cells. When PS@HA-Lip/siRNA FAM and free hyaluronic acid After co-incubation, the carrier distribution and P-Lip/siRNA FAM showed a similar situation, indicating that 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 will be significantly reduced. It shows that the interaction of hyaluronic acid-CD44 receptor can promote the cellular uptake of the carrier. PS@HA-Lip/siRNA FAM group can observe the separation of the red signal of lysosome and the green fluorescence signal of the carrier, suggesting that the carrier has successfully escaped from the endosome to the cytoplasm.
9、流式细胞术定量考察多功能非病毒载体的细胞摄取9. Quantitative investigation of cellular uptake of multifunctional non-viral vectors by flow cytometry
取对数生长的A549细胞,适量接种至6孔板(约30万每孔),培养12h,与P-Lip/siRNA
FAM、HA-Lip/siRNA
FAM、PS@HA-Lip/siRNA
FAM、HA+PS@HA-Lip/siRNA
FAM(siRNA
FAM浓度:50nM)共孵育。2h后用PBS清洗细胞3次,胰蛋白酶消化,再用PBS清洗3遍,最后用0.3mL的PBS重悬细胞,使用流式细胞仪分析摄取情况。用MDA-MB-231细胞和HepG2细胞进行摄取实验,考察细胞CD44受体表达水平对载体细胞摄取的影响,结果如图9所示。
A549 cells with logarithmic growth were inoculated into 6-well plates (about 300,000 per well), cultured for 12 hours, and mixed with P-Lip/siRNA FAM , HA-Lip/siRNA FAM , PS@HA-Lip/siRNA FAM , HA +PS@HA-Lip/siRNA FAM (siRNA FAM concentration: 50nM) co-incubation. After 2 hours, the cells were washed 3 times with PBS, digested with trypsin, washed 3 times with PBS, and finally the cells were resuspended with 0.3 mL of PBS, and the uptake was analyzed by flow cytometry. The uptake experiment was carried out with MDA-MB-231 cells and HepG2 cells to investigate the effect of the expression level of CD44 receptor on the uptake of the vector cells, and the results are shown in FIG. 9 .
使用流式细胞仪对载体的细胞摄取进行定量分析。对于A549细胞,HA-Lip和PS@HA-Lip细胞摄取显著高于P-Lip,HA同时存在时载体的细胞摄取与P-Lip组相近,说明HA修饰可以促进载体进入A549细胞,与共聚焦图片结果一致;在高表达CD44受体的MDA-MB-231细胞上出现了相似的结果,而低表达CD44受体的HepG2细胞没有此现象,说明透明质酸促进细胞摄取依赖于细胞CD44受体的表达,进一步证明HA-Lip和PS@HA-Lip可以通过HA-CD44受体相互作用促进载体进入细胞。Cellular uptake of the vector was quantified using flow cytometry. For A549 cells, the cellular uptake of HA-Lip and PS@HA-Lip was significantly higher than that of P-Lip, and the cellular uptake of the carrier was similar to that of the P-Lip group when HA coexisted, indicating that HA modification can promote the carrier into A549 cells, and confocal images The results were consistent; similar results appeared on MDA-MB-231 cells with high expression of CD44 receptors, but not in HepG2 cells with low expression of CD44 receptors, indicating that hyaluronic acid promotes cellular uptake depends on the cellular CD44 receptors expression, further proving that HA-Lip and PS@HA-Lip can promote the vector entry into cells through the HA-CD44 receptor interaction.
10、激光共聚焦显微镜考察多功能非病毒载体的入核过程10. Laser confocal microscopy to investigate the nuclear entry process of multifunctional non-viral vectors
取对数生长的A549细胞适量接种于共聚焦小皿(15万每皿),培养12h,与HA-Lip/siRNA
FAM、PS@HA-Lip/siRNA
FAM共孵育。4h、8h后,用PBS清洗细胞3次,加入溶酶体红染液(60nM),37℃孵育30min,PBS洗3次,加入4%甲醛溶液固定10min,再用PBS洗3次,Hoechst33258(10μg·mL
-1)染色细胞核,室温孵育10min,PBS洗3次,滴加适量抗荧光淬灭封片液封片,置于4℃冰箱避光保存,使用共聚焦显微镜观察载体细胞 内分布情况,结果如图10所示。
A proper amount of A549 cells with logarithmic growth was inoculated into confocal small dishes (150,000 per dish), cultured for 12 hours, and co-incubated with HA-Lip/siRNA FAM and PS@HA-Lip/siRNA FAM . After 4h and 8h, the cells were washed 3 times with PBS, added with lysosome red staining solution (60nM), incubated at 37°C for 30min, washed 3 times with PBS, fixed with 4% formaldehyde solution for 10min, washed 3 times with PBS, Hoechst33258( 10 μg·mL -1 ) to stain cell nuclei, incubate at room temperature for 10 min, wash 3 times with PBS, add an appropriate amount of anti-fluorescence quenching mounting solution dropwise to seal, store in a 4°C refrigerator in the dark, and use a confocal microscope to observe the distribution of the carrier cells , the result is shown in Figure 10.
共聚焦显微镜考察给药4h和8h后,载体在A549细胞的胞内分布,由结果可以看出4h后HA-Lip和PS@HA-Lip的绿色荧光信号均匀分布在胞浆中,红色荧光信号和绿色荧光信号呈现一定的分离,提示部分载体从内涵体逃逸;8h后,PS@HA-Lip绿色荧光在细胞核内也有分布,提示载体进入细胞核,HA-Lip组细胞核内没有明显的绿色荧光信号。综上可知,PS可以通过序列中的NLS促进质粒进入肿瘤细胞的细胞核,这有利于质粒后续的转录、翻译过程,提高基因编辑效率。Confocal microscopy was used to investigate the intracellular distribution of the carrier in A549 cells after 4 hours and 8 hours of administration. It can be seen from the results that after 4 hours, the green fluorescence signals of HA-Lip and PS@HA-Lip were evenly distributed in the cytoplasm, and the red fluorescence signals There was a certain separation from the green fluorescence signal, suggesting that some carriers escaped from endosomes; after 8 hours, the green fluorescence of PS@HA-Lip was also distributed in the nucleus, suggesting that the carrier entered the nucleus, and there was no obvious green fluorescence signal in the nucleus of the HA-Lip group . In summary, PS can promote the entry of plasmids into the nucleus of tumor cells through the NLS in the sequence, which is beneficial to the subsequent transcription and translation of the plasmids and improves the efficiency of gene editing.
以上仅是本发明的优选实施方式,并不用于限制本发明,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变型,这些改进和变型也应视为本发明的保护范围。The above are only preferred embodiments of the present invention, and are not intended to limit the present invention. It should be pointed out that for those of ordinary skill in the art, some improvements and modifications can be made without departing from the technical principles of the present invention. , these improvements and modifications should also be regarded as the protection scope of the present invention.
Claims (10)
- 一种非病毒载体,其特征在于,所述的非病毒载体包括压缩质粒药物的鱼精蛋白,包覆在压缩了核酸药物的鱼精蛋白表面的阳离子脂质体,以及修饰在阳离子脂质体表面的二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物。A kind of non-viral vector, it is characterized in that, described non-viral vector comprises the protamine that compresses plasmid drug, the cationic liposome that is coated on the surface of the protamine that has compressed nucleic acid drug, and is modified in cationic liposome Surface distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer.
- 根据权利要求1所述的非病毒载体,其特征在于,所述的阳离子脂质体为DOPE/DOTAP/Chol。The non-viral vector according to claim 1, wherein the cationic liposome is DOPE/DOTAP/Chol.
- 根据权利要求1所述的非病毒载体,其特征在于,所述的质粒药物为基因编辑质粒。The non-viral vector according to claim 1, wherein the plasmid drug is a gene editing plasmid.
- 根据权利要求3所述的非病毒载体,其特征在于,所述的基因编辑质粒为pCas9/sgMTH1、pCas9/sgKRAS、pCas9/sgPLK1、pCas9/sgMETTL3中的一种或几种。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/sgMETTL3.
- 一种权利要求1~4任一项所述的非病毒载体的制备方法,其特征在于,包括如下步骤:A method for preparing the non-viral vector according to any one of claims 1 to 4, characterized in that it comprises the steps of:S1、采用透明质酸和二硬脂酰基磷脂酰乙醇胺-聚乙二醇为原料制备二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物;S1. Using hyaluronic acid and distearoylphosphatidylethanolamine-polyethylene glycol as raw materials to prepare distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer;S2、采用DOTAP、DOPE和胆固醇为原料制备阳离子脂质体;S2, using DOTAP, DOPE and cholesterol as raw materials to prepare cationic liposomes;S3、将鱼精蛋白溶液与质粒药物溶液混合孵育,制备鱼精蛋白/质粒药物复合物;S3. Mixing and incubating the protamine solution and the plasmid drug solution to prepare a protamine/plasmid drug complex;S4、将鱼精蛋白/质粒药物复合物与阳离子脂质体混合孵育,得到阳离子脂质体包覆的鱼精蛋白/质粒药物复合物;S4. Mixing and incubating the protamine/plasmid drug complex with cationic liposomes to obtain cationic liposome-coated protamine/plasmid drug complexes;S5、将阳离子脂质体包覆的鱼精蛋白/质粒药物复合物与二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物的水溶液混合孵育,得到所述的非病毒载体。S5. Mix and incubate the cationic liposome-coated protamine/plasmid drug complex with an aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer to obtain the non-viral vector.
- 根据权利要求5所述的方法,其特征在于,S3步骤中,孵育是在20~30℃孵育4-6min,鱼精蛋白与质粒药物的质量比为1.8-2.2:1。The method according to claim 5, characterized in that in step S3, the incubation is at 20-30° C. for 4-6 minutes, and the mass ratio of protamine to plasmid drug is 1.8-2.2:1.
- 根据权利要求5所述的方法,其特征在于,S4步骤中,孵育是在20~30℃孵育15-25min,鱼精蛋白/质粒药物复合物与阳离子脂质体的质量比为3:11-11.5。The method according to claim 5, characterized in that, in step S4, incubation is at 20-30°C for 15-25min, and the mass ratio of protamine/plasmid drug complex to cationic liposome is 3:11- 11.5.
- 根据权利要求5所述的方法,其特征在于,S5步骤中,孵育是在50-60℃孵育15-25min,二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物的水溶液中,二硬脂酰基磷脂酰乙醇胺-聚乙二醇-透明质酸聚合物的浓度为19-21mg·mL -1。 The method according to claim 5, characterized in that, in step S5, the incubation is at 50-60°C for 15-25min, in the aqueous solution of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer , the concentration of distearoylphosphatidylethanolamine-polyethylene glycol-hyaluronic acid polymer is 19-21 mg·mL -1 .
- 根据权利要求5所述的方法,其特征在于,S1步骤中,透明质酸通过活化剂活化后与二硬脂酰基磷脂酰乙醇胺-聚乙二醇进行反应,所述活化剂为1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐、N-羟基琥珀酰亚胺和二环己基碳二亚胺中的一种或几种。The method according to claim 5, characterized in that, in step S1, hyaluronic acid reacts with distearoylphosphatidylethanolamine-polyethylene glycol after being activated by an activator, and the activator is 1-(3 -One or more of dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and dicyclohexylcarbodiimide.
- 权利要求1~4任一项所述的非病毒载体在递送CRISPR/Cas9系统中的应用。The application of the non-viral vector according to any one of claims 1 to 4 in delivering CRISPR/Cas9 system.
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