CN112843246B

CN112843246B - Preparation method and application of CBX 3-containing L-argininated polyamine polymer gene vector

Info

Publication number: CN112843246B
Application number: CN202110062639.2A
Authority: CN
Inventors: 余涧坤; 秦孟孟
Original assignee: China Medical University
Current assignee: China Medical University
Priority date: 2021-01-18
Filing date: 2021-01-18
Publication date: 2022-10-11
Anticipated expiration: 2041-01-18
Also published as: CN112843246A

Abstract

The invention belongs to the technical field of new auxiliary materials of pharmaceutical preparations, new formulations of biological preparations and gene vectors, and particularly relates to a polyaminoamine polymer compound gene vector containing a target protein CBX3, and a preparation method and application thereof. The CBX3 target noggin is introduced into the compound gene vector, so that the compound gene vector has good membrane permeability and intracellular environment reduction sensitivity, and the CBX3 target noggin can be specifically positioned in the target effect of euchromatin. The disulfide bond and guanidino of the carrier main chain have reduction sensitivity in an intracellular environment and have better membrane permeability, meanwhile, CBX3 target protein is introduced and can be specifically positioned in an euchromatin area to up-regulate the expression of genes, and the CBX3 target protein is combined with a target gene, so that the compound carrier has good intracellular reduction sensitivity and has a special targeting effect of being positioned in euchromatin, the expression of the genes is promoted, and a new direction is opened up for gene therapy.

Description

Preparation method and application of CBX 3-containing L-argininated polyamine polymer gene vector

Technical Field

The invention belongs to the technical field of new auxiliary materials of pharmaceutical preparations, new formulations of biological preparations and gene vectors, and particularly relates to a polyamine polymer compound gene vector containing a target protein CBX3, and a preparation method and application thereof.

Background

As a new tumor treatment means, gene therapy has gained wide favor of experts in the medical field in recent 20 years and becomes an important part for research and development of antitumor drugs. The gene therapy technology has entered the growth stage from the introduction stage, and has the following obvious advantages: (1) Can treat the disease according to the occurrence and development reasons, and is expected to realize the radical cure of the disease; (2) Based on the complementary pairing principle of nucleotide, the natural targeting property is realized; (3) Compared with traditional chemical medicine, has lower toxic and side effect.

As an application technology in the growth stage, gene therapy has a plurality of implementation technical routes, and the technical routes have different principles, which means that it is difficult to realize in vivo delivery of gene therapy drugs by using a universal vector.

The gene therapy means that high-expression exogenous genes are introduced into the organism, and the original defective genes of target cells are corrected or compensated to ensure that the cells normally express, thereby achieving the purpose of treating diseases. In recent years, gene therapy has been achieved with considerable success, but many problems still remain to be solved as a therapeutic means in the growth stage. In terms of its embodiments, there is still a lack of exogenous gene delivery vectors that are efficient in performance and stable in quality.

The gene vector is divided into two main types of virus vector and non-virus vector. Although the viral vector has the advantage of high transfection efficiency, the viral vector has great potential safety hazard, and high immunogenicity can cause organism immunogenicity and has the risk of activating protooncogenes to cause tumors. The advantages of non-viral vectors are low immunogenicity, simple production and preparation, less restriction on genetic materials, etc.

The non-viral vectors are divided into two categories, namely polymers and liposomes, wherein the polymers comprise polyethyleneimine, chitosan, polyamino acid, dendritic macromolecules and the like; the latter are represented by cationic liposomes. These vectors have a common feature that they all have a large amount of positive charges and carry genetic materials by electrostatic adsorption. Polyethyleneimine is a gene vector material which has high transfection efficiency and is widely used, but the application of polyethyleneimine in vivo is inhibited due to the undegradability, high toxicity and the like. In recent years, biodegradable polycationic materials have attracted the attention of researchers. Compared with non-degradable polymer materials, the polypeptide and derivative materials thereof have the characteristics of low toxicity, low enrichment and good biocompatibility due to the composition of the protein, and are expected to become ideal gene vectors. Therefore, the development of non-viral gene vectors which have controllable quality, can carry exogenous genes into a nucleus and a transmembrane region, can efficiently express target genes, have no immunogenicity, and are easy to industrially produce has become a research hotspot in the field of gene therapy.

With the rapid development of molecular biology and the continuous and deep understanding of tumor pathogenesis by human beings, gene therapy is becoming an important component in tumor biological therapy. Gene transfection into specific cell types in vivo is a key factor in the success of gene therapy. In recent years, non-tumor cell components in tumor microenvironment, such as tumor-associated fibroblasts, vascular endothelial cells, and tumor-associated macrophages, have attracted attention. Aiming at the gene therapy of tumor interstitial cells, the method prevents the interstitial cells from promoting the tumorigenesis and development, thereby achieving the means of assisting the conventional treatment method and being a strategy with wide application prospect.

Therefore, there is a need to develop a gene vector complex with high efficiency, low toxicity, simple preparation and targeting effect.

Disclosure of Invention

In view of the problems in the prior art, the invention aims to provide a polyamine polymer compound gene vector containing a target protein CBX3, wherein the compound gene vector introduces the CBX3 target protein, has good membrane permeability and intracellular environment reduction sensitivity, and has a targeting effect that the CBX3 target protein can be specifically positioned in euchromatin. The disulfide bond and the guanidino group of the carrier main chain have reduction sensitivity in an intracellular environment and better membrane permeability, and simultaneously, CBX3 target noggin (heterochromatin proteinum 3) is introduced, is a paralog in a eukaryotic cell, can be specially positioned in an euchromatin area, can up-regulate the expression of genes, and combines the CBX3 target noggin with target genes, so that the compound carrier has good intracellular reduction sensitivity and a special targeting effect of positioning in euchromatin, promotes the expression of the genes, and opens up a new direction for gene therapy.

In order to achieve the above purpose, the invention adopts the following technical scheme.

A gene vector compound with euchromatin targeting function contains guanidinated polyamino amine basic vector of target noggin and disulfide bond, and the target noggin is CBX3.

The preparation method of the gene vector complex with the euchromatin targeting function specifically comprises the following steps.

Step 1, preparing guanidination disulfide bond-containing polyaminoamine carrier polymer ARG-CBA: the polymer is prepared by Michael addition polymerization of CBA with a diacryloyl structure and a guanidyl reagent L-Arginine (ARG) containing primary amino, wherein the Michael addition polymerization reaction equation of the CBA and the guanidyl reagent L-arginine is as follows:

n is 18-30, the molecular weight of the polymer is 7.8-15.1KDa, and the polymer is purified and collected by a freeze drying method.

Step 2, preparing a CBX3 target noggin and target gene compound: the CBX 3/target gene compound is prepared by adopting a self-assembly room temperature incubation method, and the specific steps are that the CBX 3/target gene compound is prepared according to different proportions of CBX 3/target gene in a buffer solution under simulated physiological conditions, the pH range is 6.8-8.2, and the CBX 3/target gene compound can be obtained by standing and incubating for 20-30 minutes at room temperature.

Step 3, preparing a CBX 3/target gene/ARG-CBA compound gene vector: preparing a gene carrier compound by adopting a self-assembly room temperature incubation method, specifically, incubating the polymer ARG-CBA prepared in the step (1) and the CBX 3/target gene prepared in the step (2) in a buffer solution at room temperature and still for 5-30 minutes according to different mass ratios (N/P ratio) at room temperature to obtain the CBX 3/target gene/ARG-CBA.

Further, the polymer in step 1 was purified by dialysis with a cut-off Molecular Weight (MWCO) of 1000 in dialysis bag.

Further, the mass ratio of the CBX3 to the target gene in the step 2 is 1:50-1:10000.

furthermore, the target gene comprises one or more of pDNA plasmid, siRNA segment and microRNA segment in any combination.

Further, the mass ratio of the polymer ARG-CBA prepared in the step 1 in the step 3 to the CBX3 prepared in the step 2 to the target gene is 12:1-48:1.

further, the buffer solution in step 3 is mainly 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution, and also includes other buffer solutions simulating physiological conditions, such as phosphate buffer solution, citric acid buffer solution, and serum-free cell culture solution, with pH range of 6.8-8.2.

The application of the gene vector compound with the euchromatin targeting function in preparing gene therapy medicines.

The gene vector complex with the euchromatin targeting function is mixed with a pharmaceutically acceptable excipient to prepare clinically acceptable injections, including local injections and intravenous injections.

Compared with the prior art, the invention has the following beneficial effects.

The invention provides a guanidinated polyamino amine gene carrier compound CBX 3/target gene segment/ARG-CBA with euchromatin targeting function, wherein the guanidinated polyamino amine polymer can form a compound with a target gene segment (cDNA, microRNA, siRNA, plasmid DNA, antisense oligonucleotide and the like) containing CBX3 under physiological conditions, and the main chain of the guanidinated polyamino amine polymer is rich in amino and guanidino functional groups and can efficiently permeate a biological membrane, so that the guanidinated polyamino amine polymer has intracellular reduction sensitivity due to the existence of disulfide bonds in the main chain, and in addition, CBX3 target head protein can be specifically positioned in a euchromatin region, so the polymer has intracellular reduction sensitivity and good nuclear positioning effect, becomes a new generation of compound carrier with accurate targeting nuclear positioning and is used for gene therapy process.

The invention provides a guanidinylated polyaminoamine gene vector compound CBX 3/target gene fragment/ARG-CBA with an euchromatin targeting function, wherein the CBX 3/target gene/ARG-CBA not only contains a disulfide bond capable of responding to a reduction environment in a cell, but also retains the reduction sensitivity of the guanidinylated reagent, and a CBX3 target protein is introduced, and the CBX3 target protein can be specifically positioned in an euchromatin area to promote the expression of genes, so that the finally prepared polymer gene vector has a good and accurate nuclear localization effect and has great significance for the application of gene therapy in the future.

Drawings

FIG. 1 is a structural representation of the ARG-CBA polymer prepared.

FIG. 2 shows the in vitro transfection efficiency of ARG-CBA polymer loaded pcDNA3.1-EGFP-cDNA plasmid containing CBX3.

FIG. 3 is a graph of the in vitro gene inhibition efficiency of ARG-CBA polymer loaded with pSliencer TM 4.1CMV-FANCF-shRNA plasmid containing CBX3.

FIG. 4 shows the in vitro gene inhibition efficiency of ARG-CBA polymer carrying a fragment of FACCF-shRNA containing CBX3.

FIG. 5 shows the in vitro transfection efficiency of ARG-CBA polymer carrying 328-microRNA fragment containing CBX3.

FIG. 6 shows the effect of the ARG-CBA polymer on cell viability of a complex formed by pcDNA3.1-EGFP-cDNA containing CBX3 and pSliencer TM 4.1CMV-FANCF-shRNA plasmid.

FIG. 7 shows the effect of the complex formed by ARG-CBA polymer carrying FANCF-shRNA containing CBX3 and 328-microRNA fragment on the cell viability.

FIG. 8 shows the intracellular nuclear localization effect of the complex formed by the ARG-CBA polymer carrying the pcDNA3.1-EGFP-cDNA plasmid containing CBX3.

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to examples and the accompanying drawings. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 preparation of ARG-CBA Polymer.

Placing 0.11g of L-arginine guanidino reagent and 0.105g of CBA into a 50ml round-bottom flask, adding 2.5ml of water and 6ml of DMF as solvents, keeping out of the sun at 45-60 ℃, and reacting for 3 days under the conditions of nitrogen protection and magnetic stirring; the progress of the reaction was monitored by TLC, and after 72 hours, an excess of about 10% L-arginine guanidino reagent (0.013 g) was added and the reaction was continued for 24 hours; stopping the reaction, adjusting the pH of the system to 2-5 by using 4mol/L hydrochloric acid, collecting the solution of the whole reaction system, transferring the solution into a dialysis bag, dialyzing and purifying the reaction product, wherein the dialysis medium is distilled water, the cut-off Molecular Weight (MWCO) of the dialysis bag is 1000, dialyzing for three days, and replacing the dialysis medium at least once every day; collecting the solution in the dialysis bag, placing in a dry culture dish, lyophilizing, and collecting the polymerProduct, structural characterization by NMR ¹ HNMR) measurements, the results are shown in fig. 1. The number average molecular weight and the mass average molecular weight of the final polymer product were determined by Gel Permeation Chromatography (GPC) and were: 7975,21123. The polydispersity index (PDI) is: 1.51.

example 2 preparation of CBX 3/Gene of interest/ARG-CBA Gene vector Complex.

(1) Preparing CBX 3/target gene complex.

pcDNA3.1-EGFP-cDNA plasmid, pSliencer ^TM 4.1 CMV-FACCF-shRNA plasmid, FACCF-shRNA, 328-microRNA, diluted to 0.016mg/ml with 50mM HEPES buffer solution with pH 7.4, CBX3 powder prepared with deionized water to a concentration of 5X 10 ^-3 Mu.g/. Mu.l stock solution, at a concentration of 5X 10 ^-3 Diluting the CBX3 stock solution of mu g/mu l to different concentrations by HEPES, mixing according to the mass ratio of CBX3 to plasmid gene as 1.

(2) Preparing a CBX 3/target gene/ARG-CBA gene vector compound.

The polymer gene vector of example 1, dissolved at 0.9mg/ml in HEPES buffer, and mixed at 1:1 was diluted with HEPES buffer, 0.5ml of ARG-CBA polymer carrier solution was taken and added to the solution in the ratio 1:500 of CBX 3/target gene compound solution, the mixed solution is vortexed in an EP tube for 5 seconds, the pH range is 6.8-8.2, and the mixed solution is incubated for 10-30min at room temperature, thus obtaining the CBX 3/target gene/ARG-CBA carrier compound with the ARG-CBA/target gene mass ratio of 48/1, 24/1 and 12/1. The particle size and Zeta potential of the compound are measured by a Dynamic Light Scattering (DLS) method, and are shown in table 1, and the table 1 is the average value of the particle size and Zeta potential measured in three times of experiments.

Table 1 shows the particle size and Zeta potential of the complex formed by ARG-CBA, the target gene and CBX3.

The particle size and Zeta potential measurements described in example 2 for the complexes formed between the different gene fragments and the polymer carrier show that: the particle sizes of the compounds are all in the nanometer level, the Zeta potential is positive, and the compounds have the physicochemical properties of common cationic polymer gene vectors.

Example 3 evaluation of transfection efficiency of CBX 3/Gene of interest/ARG-CBA Gene vectors in vitro.

(1) The vector comprises a research on the in vitro transfection efficiency of pcDNA3.1-EGFP-cDNA plasmid containing CBX3.

After preparing complexes with different N/P values (1/12, 1/24, 1/48) according to the method described in example 2, the three complexes were added to a six-well plate normally cultured with MCF10A cells at a concentration of 500. Mu.l per well, the medium containing serum (BSA); 48 hours after transfection, the expression of green fluorescent protein was detected by flow cytometry and the transfection efficiency was determined by flow cytometry software. Commercial transfection reagents Lipofectamine2000 and PEI were selected as controls and the results are shown in FIG. 2.

(2) In vitro transfection efficiency study of vector-containing pSliencer TM 4.1CMV-FANCF-shRNA plasmid containing CBX3.

After preparing complexes with different N/P values (1/12, 1/24, 1/48) according to the method described in example 2, the three complexes were added to a six-well plate normally cultured with MCF10A cells at a concentration of 500. Mu.l per well, the medium containing serum (BSA); at 48 hours after transfection, the expression of the FANCF gene was detected by immunofluorescence, and the silencing efficiency of FANCF was indirectly evaluated by evaluating the green fluorescence intensity by flow cytometry. Commercial transfection reagents Lipofectamine2000 and PEI were selected as controls and the results are shown in FIG. 3.

(3) The vector comprises in vitro transfection efficiency research of a FANCF-shRNA fragment containing CBX3.

After preparing complexes with different N/P values (1/12, 1/24, 1/48) according to the method described in example 2, the three complexes were added to a six-well plate normally cultured with MCF10A cells at a concentration of 500. Mu.l per well, the medium containing serum (BSA); at 48 hours after transfection, the expression of the FANCF gene was detected by immunofluorescence, and the silencing efficiency of FANCF was indirectly evaluated by evaluating the green fluorescence intensity by flow cytometry. Commercial transfection reagents Lipofectamine2000 and PEI were selected as controls and the results are shown in FIG. 4.

(4) In vitro transfection efficiency study of 328-microRNA fragment carrying CBX3.

After preparing complexes with different N/P values (1/12, 1/24, 1/48) according to the method described in example 2, the three complexes were added to a six-well plate normally culturing MCF10A cells at a concentration of 500. Mu.l per well, the medium containing serum (BSA); 48 hours after transfection, 328-microRNA expression was detected by Realtime-PCR. Commercial transfection reagents Lipofectamine2000 and PEI were selected as controls and the results are shown in FIG. 5.

Example 4 in vitro cytotoxicity assessment of CBX 3/Gene of interest/ARG-CBA gene vector.

After preparing complexes having different N/P values (1/12, 1/24, 1/48) as described in example 2, the three complexes were added to a six-well plate in which MCF10A cells were normally cultured at a concentration of 500. Mu.l per well, and the culture was continued for 24 hours while serum (BSA) was contained in the medium; the medium containing the complexes was then discarded and cell viability was determined using the classical MTT method with cell viability without complex treatment as 100% and with Lipo2000 and PEI as control vectors. The results are shown in FIGS. 6-7.

FIG. 6 shows the effect of a complex of three polymers carrying the plasmid pcDNA3.1-EGFP-cDNA containing CBX3, pSliencer TM 4.1CMV-FANCF-shRNA on cell viability, as described in example 2; FIG. 7 shows the effect of complexes formed by three polymers carrying CBX 3-containing FANCF-shRNA, 328-microRNA fragments on cell viability.

The result shows that the CBX 3/target gene/ARG-CBA gene vector has higher in vitro transfection efficiency and lower toxicity on pDNA plasmids, siRNA fragments and micro-RNA fragments.

Example 5 Targeted Nuclear localization Effect of CBX3/pDNA/ARG-CBA vectors.

Mu.g of pcDNA3.1-EGFP was incubated with 1. Mu.l of DAPI (5 mg/mL) in a water bath at 37 ℃ for 1h, and 1mL of a solution containing 1 XPBS and absolute ethanol, at a final concentration of 70%, at 12000 rpm before transfection, was addedAfter centrifugation for 10min, the supernatant was discarded and 100. Mu.l of 1 XPBS buffer was added to dissolve the pellet for further use. The stained pDNA was then incubated with CBX3 in HEPES buffer for 20-30 minutes at room temperature to prepare a CBX3/pDNA complex for use. MCF10A cells at 2X 10 ⁵ One/well density was inoculated in a dish dedicated for confocal imaging, cultured in RPMI1640 complete medium at 37 ℃ with 5% CO2 and saturation humidity for about 24 hours, washed once with 1 XPBS until the cell fusion rate reached about 90%, and replaced with 1ml of the foreign gene/vector complex containing DAPI blue fluorescent label prepared as described above (ARG-CBA, PEI; the N/P ratio was still 1/48), the complex prepared according to example 2 above was further cultured in complete medium at 4h, washed once with 1 XPBS, 1ml of complete medium containing 1 μ l of Nuclear-ID Green nucleolar dye was added, washed three times with 37 ℃ water bath 15min and 1 XPBS, imaged by a fluorescence microscope, automatically exposed, magnified 400X, green and blue fluorescence images were collected separately, and finally the images were superimposed by fluorescence microscopy software. Commercial transfection reagents Lipofectamine2000 and PEI were selected as controls and the results are shown in FIG. 8. FIG. 8 shows that N/P of CBX3/pDNA/ARG-CBA is 1/24; lipo and PEI were used at the recommended concentrations. The scale bar is 50 μm. NC: only pDNA was added, and no polymer carrier. pDNA was stained blue with DAPI and nucleoli were stained green with a specific dye. The superimposed image is displayed in the last line. The result shows that the gene vector containing CBX3/pDNA/ARG-CBA has good targeted nuclear localization effect on the pDNA, and the goal of expecting and constructing a more ideal gene vector is achieved.

Claims

1. A gene vector complex with an euchromatin targeting function is characterized by comprising a guanidinated polyaminoamine basic vector of target protein and disulfide bonds, wherein the target protein is CBX3;

the preparation method of the gene vector compound comprises the following steps:

n is 18-30, the molecular weight of the polymer is 7.8-15.1KDa, and the polymer is purified and collected by a freeze drying method;

step 2, preparing a CBX3 target noggin and target gene compound: preparing a CBX 3/target gene compound by adopting a self-assembly room temperature incubation method, preparing the CBX 3/target gene compound according to different ratios of CBX 3/target gene in a buffer solution under simulated physiological conditions, keeping the pH value within the range of 6.8-8.2, and standing and incubating for 20-30 minutes at room temperature to obtain the CBX 3/target gene compound;

step 3, preparing a CBX 3/target gene/ARG-CBA compound gene vector: and (2) preparing a gene vector compound by adopting a self-assembly room-temperature incubation method, specifically, performing room-temperature static incubation on the polymer ARG-CBA prepared in the step (1) and the CBX 3/target gene prepared in the step (2) for 5-30 minutes in a buffer solution according to different mass ratios to obtain the CBX 3/target gene/ARG-CBA.

2. The gene vector complex of claim 1, wherein the purification of the polymer in step 1 is performed by dialysis, and the dialysis bag has a molecular weight cut-off (MWCO) of 1000.

3. The gene vector complex of claim 1, wherein the mass ratio of CBX3 to the target gene in step 2 is 1:50-1:10000.

4. the gene vector complex of claim 1, wherein the target gene comprises one or any combination of more than two of pDNA plasmid, siRNA segment and microRNA segment.

5. The gene vector complex of claim 1, wherein the mass ratio of polymer ARG-CBA produced in step 1 to CBX3 produced in step 2/gene of interest in step 3 is 12:1-48:1.

6. the gene vector complex of claim 1, wherein the buffer solution is 4-hydroxyethylpiperazine ethanesulfonic acid, phosphate buffer solution, citric acid buffer solution, or serum-free cell culture solution, and the pH is 6.8-8.2.

7. Use of the gene vector complex of claim 1 for the preparation of a gene therapy medicament.

8. The injection of the gene vector complex of claim 1, which comprises mixing the gene vector complex of claim 1 with a pharmaceutically acceptable excipient to prepare a clinically acceptable injection, and the injection comprises a local injection and an intravenous injection.