CN111440824B

CN111440824B - Liver-targeted cationic gene vector constructed based on lactose through amino-epoxy ring-opening reaction and preparation method thereof

Info

Publication number: CN111440824B
Application number: CN202010452576.7A
Authority: CN
Inventors: 徐福建; 祁宇; 俞丙然
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2020-05-26
Filing date: 2020-05-26
Publication date: 2022-07-12
Anticipated expiration: 2040-05-26
Also published as: CN111440824A

Abstract

The invention discloses a preparation method for constructing a liver-targeted cationic gene vector based on lactose through amino-epoxy ring-opening reaction, which comprises the following steps: 1) reacting aminated lactose and triglycidyl isocyanurate in dimethyl sulfoxide under the protection of inert gas at the reaction temperature of 40-60 ℃ for 24-48 hours; 2) and (2) after the reaction in the step 1), adding ethylenediamine, heating to 50-70 ℃, reacting for 1-3 hours, and dialyzing after the reaction is finished to obtain a white flocculent polymer. The cationic gene vector with low toxicity, high efficiency and liver targeting effect is prepared by the green amino-epoxy ring-opening reaction with mild reaction conditions based on lactose, not only can effectively realize gene transfection, but also can realize targeted delivery of nucleic acid, and can mediate CRIPSR/Cas9 gene editing system to realize effective gene editing.

Description

Liver-targeting cationic gene vector constructed based on lactose through amino-epoxy ring-opening reaction and preparation method thereof

Technical Field

The invention belongs to the field of non-viral gene vectors, and relates to a liver-targeted cationic gene vector constructed by amino-epoxy ring-opening reaction based on lactose and a preparation method thereof.

Background

The gene vector, which is a tool for introducing a foreign gene into a cell, should itself be low in toxicity and not cause an immune response; secondly, it should be able to form a complex with a stable structure with the gene, and at the same time, it should not cause the change of the gene structure; finally, the carrier should preferably have a targeting and degradation effect that allows treatment to be directed to specific cells and reduces the side effects of retention. The gene vectors widely used by people at present include two types: viral vectors (viral vector) and non-viral vectors (non-viral vector). Viral vectors include primarily retroviruses, adenoviruses, adeno-associated viruses, and herpes simplex viruses. Such viral vectors are generally capable of easily overcoming cellular barriers and immune defense mechanisms, and thus have high transfection efficiency. However, this vector lacks safety, is liable to cause carcinogenesis and autoimmune reaction (unimmune response) and viral changes in leukocytes, and in severe cases, causes organ failure and death. Meanwhile, the viral vector also causes insertion mutation, which may cause malignant transformation of host cells, and the viral vector has limited gene-carrying ability, thus being not suitable for large-scale industrial production. In response to the above drawbacks of viral vectors, more attention has been directed to non-viral vectors. The self safety performance is high, and many gene vectors with biocompatibility or biodegradability have low toxicity and very low immunotoxicity. Compared with viral vectors, the transfection efficiency of the non-viral vector is not high, and various barriers inside and outside cells are difficult to overcome sometimes, but the non-viral vector can carry more genes, can be produced industrially on a large scale, and has high commercial potential. Therefore, the development potential thereof promotes the research and development of non-viral vectors. Cationic polymers are currently the most frequently used non-viral vectors. The cationic non-viral vector can effectively complex genes through charge interaction, so that a positively charged nanoscale complex (complex) is formed, the genes can be protected from degradation by nuclease after passing through a cell membrane, and smooth expression of the genes is guaranteed.

Natural carbohydrates are a widely occurring natural product in nature and include monosaccharides, oligosaccharides, and polysaccharides. Natural carbohydrates have been extensively studied worldwide as excellent genetic carrier materials due to their excellent biocompatibility, biodegradability and unique bioactive properties. Lactose is a disaccharide, and lactose molecules are linked by a glycosidic bond between a molecule of glucose and a molecule of galactose, and can be further classified into alpha-lactose and beta-lactose depending on the glucose unit in lactose. According to research, lactose molecules can be specifically identified with asialoglycoprotein receptor (ASGPR) by virtue of galactose units in the molecules, and the ASGPR receptor exists on the surface of parenchymal liver cells in a large quantity, so that the lactose is a natural carbohydrate compound with liver targeting property. In addition, reports that disulfide bond groups are introduced into cationic gene vectors and that the reduction response of disulfide bonds and glutathione in organisms is utilized to realize the degradation of the vectors in organisms are frequently reported, and the method not only can reduce the toxicity of the vectors, but also is beneficial to the release of nucleic acid.

The amino-epoxy ring opening reaction is a novel step-wise polymerization process. It relies primarily on the chemical reaction of primary amine groups with epoxide groups. Secondary amine groups and hydroxyl groups will appear as the epoxy ring is opened. Secondary amine groups can complex negatively charged nucleic acids, while hydroxyl groups can provide hydrophilicity. The amino-epoxy ring-opening reaction does not depend on harsh reaction conditions, and the reaction process is mild and green.

Cell targeting technology is a technology for biological behavior research at the cellular and molecular level, and is the research method of life science and pharmacology which develops the fastest in recent years. In recent years, with the improvement of molecular biology techniques and the further understanding of tumor pathogenesis from the molecular level of cellular receptors, many new advances have been made in targeted gene therapy. Targeting means that the expression of a target gene is restricted to a specific target cell, tissue or organ, and does not affect the function of other normal cells, tissues or organs. For the targeted gene therapy, the key to the success is to construct a safe, efficient and targeted gene vector. The traditional method for constructing the targeting gene vector is to carry out post-modification on the constructed vector by means of esterification, amidation and the like, the modification strategy is relatively single, the preparation process is relatively complex, and the characterization difficulty is relatively high. These deficiencies have greatly limited the development of targeted nucleic acid delivery systems. How to break through the traditional synthesis mode, and the preparation method with simple, convenient and controllable establishment and wide universality becomes the key point of the research on the targeting vector. According to previous research, tumor cells over-express receptors on the cell surface that specifically recognize and bind to specific natural monosaccharides or oligosaccharides.

With the continuous progress of polymer science, how to better mutually blend and infiltrate the polymer with the subjects of modern medicine, biology, engineering and the like becomes a problem to be mainly solved by people at present. In the aspect of gene therapy, the high molecular material already shows high utilization value. Currently, a series of non-viral cationic gene vectors are reported in the literature, including Poly-L-Lysine (Poly (L-Lysine), PLL), polyethylene diamine dendrimer (Poly (amido amine), PAMAM), Poly (N, N-dimethylaminoethyl methacrylate) (Poly (2-dimethylethylene methacrylate), PDMAEMA), Polyethyleneimine (PEI), etc. Wherein PEI is the recognized "gold label" in cationic non-viral vectors. However, the cationic gene vector such as PEI cannot completely show the process of entering the material into the cell, and can only indirectly show the material through the characterization means such as transfection and the like. Therefore, the integration of gene vectors with cellular imaging technology is a hot spot for research today.

In recent years, researchers have devoted to the research work of the amino-epoxy ring opening theory and application, and can proficiently utilize the polymerization means to obtain a series of polymer materials which can be used in modern biomedicine, and simultaneously promote the application of the reaction in medical biopolymers. Ethylenediamine is a colorless liquid at room temperature and has an amine odor. Are widely used as chemical agents, agricultural chemicals, medicines, solvents, dye intermediates, rubber accelerators, surfactants, and the like. The use of amino-epoxy ring opening reactions to obtain cationic gene vectors has presented a number of problems in the research process, such as: the cationic vector has higher corresponding transfection efficiency along with the increase of molecular weight, but has higher toxicity, and how to improve the transfection efficiency and ensure the targeting effect to the greatest extent under the condition of ensuring moderate toxicity becomes the key point of attention of people; different monomers have different characteristics, some monomers have higher transfection efficiency and lower effective toxicity, and the problem that how to screen out high-efficiency and high-performance monomers is also needed to be considered by people.

Disclosure of Invention

In view of the above, the invention provides a liver-targeted cationic gene vector constructed by amino-epoxy ring-opening reaction based on lactose and a preparation method thereof. The invention specifically provides the following technical scheme:

1. a preparation method for constructing a liver-targeted cationic gene vector based on lactose through amino-epoxy ring-opening reaction comprises the following steps:

1) reacting aminated lactose and triglycidyl isocyanurate in dimethyl sulfoxide under the protection of inert gas at the reaction temperature of 40-60 ℃ for 24-48 hours;

2) and (2) after the reaction in the step 1), adding ethylenediamine, heating to 50-70 ℃, reacting for 1-3 hours, and dialyzing after the reaction is finished to obtain a white flocculent polymer.

Further, the cut-off number average molecular weight of the dialysis is 1000-7500.

Further, the cut-off number average molecular weight of the dialysis was 3000-5000.

Further, the molecular weight distribution index of the white flocculent polymer is 1.8-2.0.

Further, by mass, 0.4-0.6 part of aminated lactose, 0.1-0.4 part of triglycidyl isocyanurate and 0.1-0.3 part of ethylenediamine.

Further, by weight, 0.5-0.6 part of aminated lactose and 0.2-0.3 part of triglycidyl isocyanurate

0.2-0.3 part of ethylenediamine.

Further, by mass, 0.57 part of aminated lactose, 0.22 part of triglycidyl isocyanurate and 0.21 part of ethylenediamine.

Further, the reaction temperature in the step 1) is 50-55 ℃, and the reaction time is 24-36 hours.

Further, the reaction temperature in the step 2) is 55-60 ℃, and the reaction time is 1-2 hours.

2. The liver targeting cationic gene vector is constructed based on lactose through amino-epoxy ring opening reaction.

The invention has the beneficial effects that: the cationic gene vector with low toxicity, high efficiency and liver targeting effect is prepared by a green amino-epoxy ring-opening reaction with mild reaction conditions based on lactose, not only can gene transfection be effectively realized, but also the targeted delivery of nucleic acid can be realized, and the CRIPSR/Cas9 gene editing system can be mediated to realize effective gene editing. Not only has research significance, but also has wide application prospect and potential commercial value.

Drawings

In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:

FIG. 1 is a graph of transfection efficiency of the prepared cationic vectors in HEK293 cells.

Fig. 2 is a graph of intracellular toxicity of the prepared cationic carriers in HEK293 cells.

FIG. 3 is a diagram of the effect of gene editing produced by the prepared cationic vector mediated CRISPR/Cas9 gene editing system.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Example 1

513mg of beta-lactose was placed in a 50mL round bottom flask, dissolved completely by adding 5mL of anhydrous dimethyl sulfoxide, and sealed for further use after continuously venting with nitrogen for 15 min. Subsequently, 500mg of carbonyldiimidazole was dissolved in 5mL of anhydrous dimethyl sulfoxide, the dissolved solution was aspirated with a syringe, and slowly injected into the previously prepared β -lactose solution while rapidly stirring, and after the complete injection, the mixed solution was allowed to react at room temperature for 24 hours. After reacting for 24h, 4g of cystamine is dissolved in 3mL of anhydrous dimethyl sulfoxide and slowly injected into the reaction solution by using an injector to react for 24h at normal temperature. After the reaction is finished, the reaction solution is dripped into 100-200mL acetone for precipitation, and white powdery solid aminated lactose is obtained after repeated washing, centrifugation and vacuum drying.

Example 2

349mg of aminated lactose and 100mg of triglycidyl isocyanurate are placed in a 50mL round-bottom flask, 10mL of dimethyl sulfoxide is added to be fully dissolved, then nitrogen is used for exhausting for 15min, and the reaction is carried out for 24h at 50 ℃. After the reaction, 0.2mL of ethylenediamine was added, and the temperature was raised to 60 ℃ to react for 2 hours. Finally adding the reaction solution into 50mL of water, dialyzing for 24h by using a dialysis bag with the molecular weight cutoff of 3500, and finally freeze-drying to obtain white flocculent polymer which is recorded as LBP²Polymer (LBP)²) Number average molecular weight (M)_n) 17500 molecular weight distribution index (M)_w/M_n) Is 1.85.

Example 3

438mg of aminated lactose and 150mg of triglycidyl isocyanurate were charged into a 50mL round-bottom flask, 10mL of dimethyl sulfoxide was added thereto and dissolved sufficiently, and then, nitrogen gas was used to evacuate for 15min, followed by reaction at 50 ℃ for 24 hours. After the reaction, 0.2mL of ethylenediamine was added, and the temperature was raised to 60 ℃ to react for 2 hours. Finally, the reaction solution was added to 50mL of water, and the solution was partitioned with a cut-offDialyzing in a dialysis bag with a molecular weight of 3500 for 24h, and freeze-drying to obtain white flocculent polymer LBP³. Polymer (LBP)³) Number average molecular weight (M)_n) 19600, molecular weight distribution index (M)_w/M_n) Was 1.91.

Example 4

527mg of aminated lactose and 200mg of triglycidyl isocyanurate are put into a 50mL round-bottom flask, 10mL of dimethyl sulfoxide is added to be fully dissolved, then, nitrogen is used for exhausting for 15min, and the reaction is carried out for 24h at 50 ℃. After the reaction, 0.2mL of ethylenediamine was added, and the temperature was raised to 60 ℃ to react for 2 hours. Finally adding the reaction solution into 50mL of water, dialyzing for 24h by using a dialysis bag with the molecular weight cutoff of 3500, and finally freeze-drying to obtain white flocculent polymer LBP⁴. Polymer (LBP)⁴) Number average molecular weight (M) of_n) 20000, molecular weight distribution index (M)_w/M_n) Is 1.83.

Example 5

HEK293 cells at 6X 10⁴The cells were cultured in 24-well plates at a density per well for 24 h. Subsequently, 20. mu.L of LBP/pDNA complex was added to each well at a mass ratio of 10 to 60, the mass of pDNA in each well being 1.0. mu.g. After 4h the old medium was discarded and replaced with new medium, 500. mu.L per well. After another 20h, the old medium was discarded, each well was washed once with 500 μ L PBS, then 70 μ L of cell lysate was added, after lysis for 2h, the cells were observed under a microscope, after no apparent cell morphology was observed, cell debris in each well was scraped off with a cell scraper, then 10 μ L was added to 25 μ L of substrate, and the chemiluminescence intensity was measured with a luminometer. The results are shown in FIG. 1.

As can be seen from FIG. 1, the transfection efficiency of the three LBP cationic polymers tends to increase with increasing mass ratio, and then the transfection efficiency of LBP slightly decreases. In addition, at the same mass ratio, LBP⁴The transfection efficiency in both cells was significantly higher than that of LBP²And LBP³. This is due to LBP⁴The cationic polymer has more secondary amine groups, and can complex pDNA more effectively, thereby promoting the transfection efficiency, and the LBP cationic polymer can realize higher groupsDue to transfection efficiency.

Example 6

The MTT method was used to characterize the cytotoxicity of LBP/pDNA complexes in HEK293 cells. Cells were cultured in 96-well plates at a density of 2 × 104 cells per well. After 24h of culture, LBP/pDNA prepared in advance according to the mass ratio of 10-60 is added into each well, and after 4h, old culture medium is removed and MTT solution is added. And continuing culturing for 4h, taking out the 96-well plate, sucking out the MTT solution, washing for 1-2 times by using PBS, adding 100 mu L of dimethyl sulfoxide into each well, shaking for 10min, and placing on a microplate reader to read the absorbance. The results are shown in FIG. 2.

As can be seen from fig. 2, LBP did not exhibit significant cytotoxicity as the mass ratio increased. Whereas at the same mass ratio the cytotoxicity of PEI was significantly higher than LBP. This indicates that the LBP cationic polymer has lower intracellular toxicity.

Example 7

To characterize the gene editing effect produced by the prepared cationic vector-mediated CRISPR/Cas9 gene editing system, BEL7402 cells were cultured at 2X 10⁵Density of individual cells per well in 6-well plates, after 24h incubation, 80. mu.L of LBP per well was added⁴the/pCas 9-surv complex. After 48h, the cells were harvested and total DNA was extracted therefrom. The sequence with the editing site was amplified by PCR instrument and cleaved with T7E1 enzyme. Gold labeled PEI was used as a negative control.

The results are shown in FIG. 3. From FIG. 3, it can be seen that LBP was determined by the treatment with T7E1 enzyme compared to the control group and gold-labeled PEI⁴The experimental group shows a hybrid band after electrophoresis, and the prepared cationic vector mediated CRISPR/Cas9 gene editing system is proved to generate an effective gene editing effect. Therefore, the LBP cationic polymer can mediate the CRIPSR/Cas9 gene editing system to realize effective gene editing.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1. A preparation method for constructing a liver-targeted cationic gene vector based on lactose through amino-epoxy ring-opening reaction is characterized by comprising the following steps:

2. The preparation method for constructing the liver-targeting cationic gene vector based on lactose through amino-epoxy ring opening reaction according to claim 1, wherein the cut-off number average molecular weight of dialysis is 1000-7500.

3. The method for preparing the liver-targeting cationic gene vector by amino-epoxy ring-opening reaction based on lactose as claimed in claim 2, wherein the cut-off number average molecular weight of the dialysis is 3000-5000.

4. The preparation method for constructing the liver-targeting cationic gene vector based on lactose through amino-epoxy ring-opening reaction according to claim 1, wherein the molecular weight distribution index of the white flocculent polymer is 1.8-2.0.

5. The preparation method of the liver-targeting cationic gene vector based on lactose through amino-epoxy ring-opening reaction according to claim 1, wherein the preparation method comprises 0.4-0.6 part of aminated lactose, 0.1-0.4 part of triglycidyl isocyanurate and 0.1-0.3 part of ethylenediamine by mass.

6. The preparation method of the liver-targeting cationic gene vector based on lactose through amino-epoxy ring-opening reaction according to claim 5, wherein the preparation method comprises, by mass, 0.5-0.6 part of aminated lactose, 0.2-0.3 part of triglycidyl isocyanurate, and 0.2-0.3 part of ethylenediamine.

7. The preparation method of claim 6, wherein the aminated lactose is 0.57 parts, triglycidyl isocyanurate is 0.22 parts, and ethylenediamine is 0.21 parts by weight.

8. The preparation method for constructing the liver-targeted cationic gene vector based on lactose through amino-epoxy ring-opening reaction according to claim 1, wherein the reaction temperature in the step 1) is 50-55 ℃, and the reaction time is 24-36 hours.

9. The preparation method for constructing the liver-targeted cationic gene vector based on lactose through amino-epoxy ring-opening reaction according to claim 1, wherein the reaction temperature in the step 2) is 55-60 ℃, and the reaction time is 1-2 hours.

10. The gene vector prepared by the preparation method for constructing the liver-targeting cationic gene vector based on lactose through amino-epoxy ring opening reaction according to any one of claims 1 to 9.