WO2016101405A1 - Safe and efficient carrier for transfecting protein into cell - Google Patents

Abstract

Disclosed in the present invention are a carrier for transfecting a protein into a cell, the main component of the carrier being a low-molecular chitosan and/or a low-molecular chitosan derivative, the low-molecular chitosan having a polymerization degree of 2-120 and a molecular weight of 320-20,000 and obtained from chitosan through a biodegradation method, a chemical degradation method or a physical degradation method, or through a chemical synthesis method or an enzymatic synthesis method.

Description

A safe and efficient protein transfection into cells Technical field

The invention relates to a protein transfected cell carrier and belongs to the field of biological materials.

Background technique

Protein transfection is a process of directly transferring proteins into cells. Generally, a vector is used to help the target protein to be transfected into the cell membrane. Protein transfection into cells has very important scientific research value and application prospects. The main researches are: 1 protein-protein interaction; 2 intracellular signal transduction, cell cycle regulation, apoptosis, tumorigenesis and transcriptional regulation; 3 unknown proteins Function; 4 cell reprogramming; 5 functional transcription factor delivery and cancer treatment.

However, there are few people working on protein carriers entering cells. The main reason is that people in the field are generally accustomed to accept gene transfection, because the gene will be translated into protein after transfection into the cell, so it is considered unnecessary to directly transfect the protein into the cell. In addition, the technology of gene transfection is now mature, and there are a large number of stable gene transfection vectors, such as viruses, liposomes, and nanocarriers such as PEI. Among them, the virus is the most efficient, but there is an uncontrollable risk. Liposomes are generally considered to be more cytotoxic, and nanocarriers are generally considered to have a potential to become safer and more reliable gene carriers because of their size effect and non-virality. People usually focus on modification of nanocarriers, such as research to improve the efficiency of gene transfection without increasing the cytotoxicity of the vector.

In addition, those skilled in the art generally believe that the direct transfer of proteins into cells has the following problems: 1 the amount of protein transferred is limited, and may not reach an effective concentration; 2 proteins cannot be greatly compressed as image genes, so The formed carrier-protein complex has a relatively large size, the nano-effect is not obvious, and is easy to produce cytotoxicity; 3 the protein to be transferred is generally a prokaryotic expression protein, has no protein function, and is processed by the endoplasmic reticulum after being inserted into the cell. Only after the function, whether the cells will actively modify the transfected protein is unknown; 4 existing protein carriers can not meet the two basic requirements of safe low toxicity and high efficiency transfection, such as PEI, cationic liposome transfection efficiency is high However, the toxicity is large, and the microsphere-grafted penetrating peptide is less toxic, but the transfection efficiency is low, and the preparation is complicated; 5 the protein obtained by artificial recombination or the naturally isolated and purified protein is difficult to obtain, and the cost is high. In short, there are not many literatures and patents that directly transfer proteins into cells, and the research and development of gene vectors has been hot.

To sum up, it is known that there are fewer peers in the research of protein transfected cells. In addition, developed There are few protein carriers with reliable performance, especially the important requirements of high transfection efficiency, safety, low toxicity and wide adaptability. The existing protein carrier is basically a direct use of the currently more mature non-viral gene vector. In fact, the experience of borrowing genetic vectors to some extent is basically feasible. At present, at home and abroad, the following protein transfection reagents have been reported:

(1) Polystyrene microspheres: It has been reported that bifunctional groups are synthesized on the surface of polystyrene microspheres, one for linking proteins and one for intracellular tracking. Results Microspheres: (a) can stably link proteins, (b) have a certain protein transfection efficiency, and (c) after entering the cells, the released proteins are functional and traceable. (d) Microspheres can enter a variety of cells such as primary immune cells, embryonic stem cells, human neural stem cells, differentiated mouse neural stem cells, and several non-phagocytic cell lines. This is mainly due to the use of polystyrene microspheres with good hydrophilicity.

(2) Cationic peptide or cationic liposome reagent: The cationic peptide is called a protein transduction domain (PTD) and has been shown to be effective in permeating the cell membrane. Cationic liposome, such as LipofectamineTM2000, has become the most widely used non-viral vector due to its simple operation, high biosafety, good repeatability and high transfection efficiency, but it still has a large Toxicity limits its application. Some researchers have also mixed the above cationic peptides and cationic liposomes to improve transfection efficiency.

(3) Polyarginine: It is very toxic. It is generally considered to have no clinical application prospects, but it is suitable for a wide range of cells, and can effectively transfect proteins into various cells, including various primary cultured cells and suspension cells. Some researchers have designed and produced oligoarginine to reduce its toxicity, but the toxicity is still very large.

(4) PEI: Transfection efficiency is very high, but because it is very toxic, it is generally not used alone, and it is often used in combination with other substances to reduce its toxicity. For example, many researchers have grafted small molecule PEI onto low toxicity polymer carriers such as hyaluronic acid. Some researchers have also used small molecule PEI to transfect proteins, but the toxicity is still very large.

In short, the existing protein carrier can not have two basic requirements of safe, low toxicity and high efficiency transfection, and has certain selectivity to cells. Although a small number of peers have carried out research on protein carriers, chitosan, low molecular chitosan and its derivatives have not been used as protein transfection vectors.

Chitosan (chitosan), also known as chitosan, is obtained by deacetylation of chitin, which is widely found in nature. The chemical name is polyglucosamine (1-4)-2-amino-B-D glucose. It is worth noting that chitosan is not a natural product, it is a deacetylated product of the natural substance chitin. This high Molecular biocompatibility, blood compatibility, safety, microbial degradability, etc. are widely concerned in various industries, in medicine, food, chemical, cosmetics, water treatment, metal extraction and recycling, biochemical and biomedical engineering, etc. Significant progress has been made in applied research in the field. For example, chitosan has been reported to reduce blood fat and lower blood sugar. Chitosan has been used as a thickener and a filming agent in the national food additive standard GB-2760. The degradation of chitosan is also very safe. The literature shows that more than 30 enzymes can work on it, so there are quite a few enzymes in the body and in the cells that can degrade it, such as lysozyme. In general, chitosan is a very safe substance.

However, chitosan as a gene carrier has low transfection efficiency. Numerous literatures indicate that chitosan and its derivatives can be used as gene carriers to transfect genetic material into cells, but the transfection efficiency is still generally lower than that of cationic liposomes and viral vectors. Therefore, people in the field generally pay attention to modifying chitosan and improving its transfection efficiency. There have been a few documents and patents in this respect. The researchers mainly modify the following methods: 1. Graft or blend cationic carriers such as PEI or arginine, mainly because of the high efficiency of the cationic carrier; 2. Grafting or blending PEG or acid such as amber Acid, etc., mainly by increasing the hydrophilicity of the carrier to increase the transmembrane ability of the carrier; 3. Adding calcium phosphate, mainly because calcium phosphate and DNA are combined into a complex, which is easy to transfect into cells; 4. Grafting TAT peptide (penetrating peptide) in chitosan, thereby improving transmembrane efficiency; 5 compound drugs, improve the ability to target cells, such as complex galactose-targeted hepatocytes, such as complex LHRH peptide, targeting liver cancer cells.

In addition, chitosan as a gene carrier has certain selectivity to cells, which is another disadvantage of chitosan as a gene carrier. The literature shows that chitosan/DNA nanoparticles can easily enter tumor cells, macrophages, etc., and it is difficult to enter stem cells and fibroblasts.

In conclusion, although chitosan is safe, it has low transfection efficiency, is suitable for cell types, and is not suitable as a gene carrier, and has not been generally recognized by those in the field. Low molecular weight chitosan and its derivatives with smaller molecular weight than chitosan are less suitable as gene carriers. Because low molecular weight chitosan carries too little positive charge, it is difficult to encapsulate and compress the target gene. task.

Chitosan is also used in the field of pharmaceutical carriers. As a safe drug carrier, chitosan has been used in the research of drug carriers such as antibodies, such as controlled release and sustained release of protein drugs, and belongs to the field of pharmacy. In the field of controlled release, since chitosan and its derivatives have spongy special structure and solubility, and the degradation products do not contain any harmful substances, they have been used to control the sustained release of drugs and improve the solubility of drugs. Absorption, etc., such as by changing the cross-linking process of chitosan particles The amount of protein and the amount of protein loaded achieves the goal of optimizing drug encapsulation efficiency, particle size and release.

In the field of sustained release, chitosan as a sustained release agent can control the release of the drug, the blood concentration is stable, and it remains within the effective concentration range, prolonging the effective time without toxicity. Moreover, the sustained-release preparation made of chitosan can prolong the residence time in the gastrointestinal tract and improve the bioavailability of the drug. The sustained-release dosage forms currently studied include granules, tablets and capsules. For example, if a compound of chitosan, sodium alginate and protein is compounded, it is administered orally to study the enteric sustained-release effect of protein drugs.

However, the research and application of low molecular weight chitosan and its derivatives as carriers for transfecting proteins into cells have not yet been carried out.

In fact, the chitosan (molecular weight greater than 20,000) and its modified chitosan derivative purchased in general research cannot be used as a carrier to transfect proteins into cells. The main reason is that this molecular weight grade of chitosan is insoluble in water and can only be dissolved in acidic solutions. Even in an acidic environment, chitosan and protein can form a soluble complex. Once applied to a human or cell culture environment with a pH of 7.4, precipitation occurs immediately, so that proteins cannot be transfected into cells, even if there is a small amount. Entry, transfection efficiency is also low. As a gene carrier, the effect is small, because the gene has greater compressibility, and the chitosan and the gene can form a nano-scale complex by charge, which has a nano-size effect, so it can be dissolved in water and can be worn. Permeabilized cell membrane (although not efficient, it has a certain selectivity to cells). As a drug carrier, because the purpose of the research is not to mediate the entry of proteins into cells, but to control release and sustained release of protein drugs, it is required that the molecular weight of chitosan is large, which is favorable for sustained release, and is advantageous for wrapping more proteins. Therefore, in this field, the conventionally used chitosan has a molecular weight of usually 50-100 KD or even 200-500 KD.

Low-molecular chitosan has this ability because it is still soluble in water at neutral pH and does not undergo precipitation, which can efficiently mediate protein entry into cells.

Low-molecular chitosan can be divided into two types, one is chitosan oligosaccharide, generally chitosan with a degree of polymerization between 2 and 10, and a molecular weight of between 320 and 1700. Many properties of this chitosan oligosaccharide The properties of chitosan, which are generally applied to gene carriers and drug carriers, vary greatly; one is between chitosan oligosaccharides and chitosan, generally having a degree of polymerization between 10 and 60, and a molecular weight of 1700-10000. Between chitosan, the nature of this substance is between chitosan oligosaccharide and polymeric chitosan. In the present invention, Applicants have found that as long as chitosan (including derivative modification or addition of additives) is soluble in a neutral aqueous solution, the efficiency of transfection of the protein is high, and the efficiency without dissolving is low.

Summary of the invention

It is an object of the present invention to provide a vector which is safely and efficiently adapted to a wide range of proteins for transfection into cells.

To achieve the above object, the present invention provides a safe and efficient carrier for transfecting a protein into a cell, characterized in that the main component of the carrier is a low molecular chitosan and/or a low molecular chitosan derivative.

The low molecular weight chitosan of the present invention refers to the degradation of chitosan by biodegradation, chemical degradation or physical degradation, or the degree of polymerization obtained by chemical synthesis or enzymatic synthesis is 2 to 120, and the molecular weight is Low molecular chitosan of 320 to 20000.

The low molecular weight chitosan derivative of the present invention refers to a compound formed by substituting a low molecular chitosan as a precursor for replacing atoms or radicals with other atoms or groups of atoms, including acylation, carboxylation, hydroxylation, cyanation. Derivatives prepared by etherification, alkylation, esterification, aldimineation, azidation, salt formation, chelation, hydrolysis, oxidation, halogenation, grafting and crosslinking. Low molecular chitosan derivatives are sulfonated chitin, sulfonated carboxymethyl chitin, N-hydroxyethyl chitosan, N-acetylated chitosan, O-carboxymethyl chitooligosaccharide, N-carboxyl Methyl chitosan, N-trimethyl chlorinated chitosan oligosaccharide, N-hydroxypropyl chitosan acetate, cyanoethyl or phenyl cyanoethyl chitosan, N-propyl (butyl, hexyl) Acylated chitosan, N-methyl(phenyl)sulfonyl chitosan, N-phthaloyl chitosan, N-butyl (octyl, hexadecane)-based chitosan, N-carboxybutyl Chitosan, N-sulfate chitosan, 6-O-hydroxyethyl chitosan, ethylene glycol chitosan, glycerol (3-chloropropane-1,2-diol) chitosan, BD half-milk One or more of glycosidic branched chitosan, 6-deoxy chitosan, 6-O-monosulfate (3,6-O-disulfate) chitosan, sodium dodecyl sulfate chitosan Kind.

Further, the components of the carrier of the present invention further include a secondary component, which is PEG (polyethylene glycol), PEI (polyethyleneimine), liposome, calcium phosphate, metaphosphoric acid. One or more of calcium, sodium alginate, HIV-1 TAT short peptide, polyarginine, oligoarginine, oligolysine, polystyrene, carbon nanotubes.

The molar ratio of the main component to the minor component of the carrier of the present invention is from 1:0 to 100.

The vector of the present invention can bind to one or more types of proteins to form a complex, and since the protein and the carrier are non-covalently bound, the biological activity of the protein is not destroyed. In the complex, the molar ratio of the main component of the carrier to the protein is from 1:0.1 to 100.

The vector provided by the invention has the characteristics of safety, high efficiency and wide adaptability, wherein the safety refers to the growth period of the carrier itself after the vector itself and the complex formed by the carrier and the protein are transfected into the cell. Period, proliferation, apoptosis, necrosis have no obvious effect, the main components of the carrier have been approved by the FDA, with little or no cytotoxicity; high efficiency means that the proportion of the vector itself and / or complex transfected into the cell can exceed 80%, The vector can transfect high-concentration (up to 1-10mg/L) protein into the cell, and the vector transfects the protein into the cell for a fast time (generally 0.1-6h); the broad adaptation refers to the combination of the carrier itself and the carrier and protein. The substance can be transfected into all animal cells, and the application procedure of the vector transfected protein into the cell is simple, and can be protected from degradation of related enzymes outside the cell. The entry of the complex into the cell means that the formed complex enters the cell membrane, and the transfection of the protein into the cell refers to the cytoplasm or nucleus, endoplasmic reticulum, mitochondria and the like organelles of the protein entering the cell membrane.

Compared with the prior art, the vector for transfecting a protein provided by the present invention has the following technical effects:

(1) After binding of the carrier to the protein, the protein can be effectively protected from degradation by the enzyme. Low-molecular chitosan and protein are mainly combined by charge interaction, which is covalently bound and does not destroy the biological activity of the protein. However, this combination can mask the active groups of the protein and cannot be recognized by the enzyme in the medium, so that the protein can be effectively protected from degradation by the enzyme. Of course, the medium also contains an enzyme that degrades chitosan, but because the enzyme specificity and efficiency of degrading low molecular weight chitosan are poor, the low molecular chitosan carrier cannot be completely degraded in a short time, so Remove the protective effect of low molecular chitosan on proteins. Therefore, if applied to animals, low molecular weight chitosan will not (in a small amount) be degraded by enzymes in the extracellular matrix in a short time, but will mediate protein entry into cells.

(2) The vector mediates protein transfection into cells with high efficiency, quantity and time. In neutral aqueous solutions, low molecular chitosan is positively charged, while most proteins are negatively charged. Even if a small amount of protein is positively charged, the value of positive charge is much smaller than that of low molecular chitosan. After the two solutions are mixed, the protein and the low molecular chitosan will agglomerate, and in general, an irregular cationic substance, that is, the complex mentioned in the present invention, is formed. In the case of ultrasonic dispersion, if the ratio of protein to chitosan is appropriate, a positively charged dispersed sphere of uniform size will appear. Since the complex has cationic characteristics and is easy to enter cells, the efficiency of transfection into cells is high, the number is large, and the time is fast.

(3) The carrier has no obvious toxicity to cells. Carrier Materials Low molecular weight chitosan has proven to be non-toxic in the food and genetic carrier fields. Since there are many enzymes in the body and in the cells that can degrade chitosan, the low molecular chitosan will be gradually degraded and the protein will be released from the complex. Since the molecular weight of the low molecular chitosan is not large, the degradation process is not long, and there are not many degradation products, so the toxicity is little or no. The released protein will enter different cells according to its nature and cell type. To complete the next step of regulation. Therefore, the influence of the carrier on the cells is extremely small, and the influence of the protein on the cells is revealed, so it is very suitable for the study of the action of the protein.

(4) The carrier is widely adaptable and can be applied to different types of animal cells. Since different cells have different phagocytic abilities, the efficiency of some vectors entering cells varies widely. The complex provided by the present invention is capable of entering all types of cells for the first reason that it has cationic properties, the second is that the particles are relatively small, and the third is a glycosyl moiety.

(5) Simple operation. It is only necessary to simply blend low molecular weight chitosan and protein to prepare a complex that can be transfected into cells. In the case of ultrasonic dispersion, if the ratio of protein to chitosan is appropriate, a positively charged dispersed sphere of uniform size will appear. This has the potential to further reduce the toxicity of the carrier.

DRAWINGS

1 is a TEM top view of the composite formed in Example 3;

2 is an agarose gel electrophoresis pattern of the low molecular weight chitosan-Dnase I enzyme complex prepared in Application Example 1 of the present invention; A: 15000 kb DNA marker; B: 2 ul 1600 ng/ml Oct4 plasmid DNA; C: B + 7.5 ul 100 ug / ml Dnase I; DI: B + 7.5 ul low molecular chitosan-protein complex (D: 200 ug / ml low molecular chitosan - 5000 + 100 ug / ml DNase I; E: 50 ug /ml low molecular chitosan-5000+100ug/ml DNase I; F: 12.5ug/ml low molecular chitosan-5000+100ug/ml DNase I; G: 3.125ug/ml low molecular chitosan-5000+ 100ug/ml DNase I; H: D: 0.78ug/ml low molecular chitosan-5000+100ug/ml DNase I; I: 0.19ug/ml low molecular chitosan 5000+100ug/ml DNase I);

3 is a fluorescence micrograph showing the three vectors mediated by GFP protein entering cells in the application example 2 of the present invention;

4 is a top view of a light microscope after induction of hBMSC by a composite in Example 3 of the present invention;

5 is an immunofluorescence pattern of hMSCs induced by four myocardial regulatory proteins in Application Example 3 of the present invention;

Figure 6 is a diagram showing the heart EF after cell transplantation by echocardiography in Example 3 of the present invention; Sham: normal control group; PBS: PBS control group; iCPC: iCPC cell transplantation group, N=6, *P< 0.01 vs PBS group;

Figure 7 is a diagram showing the HE staining of the infarcted area 4 weeks after cell transplantation in the application example 3 of the present invention; In the figure, A: normal control group; B: PBS control group; C: iCPC cell transplantation group.

detailed description

Example 1

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, and the component thereof is a low molecular weight chitosan having a molecular weight of 500.

The above carrier was formulated into a 200 ug/ml aqueous solution; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube and sonicated for 15 seconds to obtain a mixed solution, that is, A complex was formed; the mixed solution was directly added to the cell culture medium of the cultured hMSC cells in a volume of 2 ml. The morphology of the complex was observed by TEM. The effect of protein transfection was observed by immunofluorescence. The results confirmed that the above vector could transfect the protein into hMSC cells.

Example 2

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, and the component thereof is a low molecular weight chitosan having a molecular weight of 1300.

The above carrier was formulated into a 200 ug/ml aqueous solution; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube and sonicated for 15 seconds to obtain a mixed solution, that is, A complex was formed; the mixed solution was directly added to the cell culture medium of the cultured hMSC cells in a volume of 2 ml. The morphology of the complex was observed by TEM. The effect of protein transfection was observed by immunofluorescence. The results confirmed that the above vector could transfect the protein into hMSC cells.

Example 3

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, and the component thereof is a low molecular weight chitosan having a molecular weight of 5000.

The above carrier was formulated into a 200 ug/ml aqueous solution; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube and sonicated for 15 seconds to obtain a mixed solution, that is, A complex was formed; the mixed solution was directly added to the cell culture medium of the cultured hMSC cells in a volume of 2 ml. The morphology of the complex was observed by TEM (as shown in Figure 3); the effect of protein transfection was observed by immunofluorescence, and it was confirmed that the above vector could transfect the protein into hMSC cells.

Example 4

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, and its component is molecular weight. Low molecular chitosan of 20000.

The above carrier was formulated into a 200 ug/ml aqueous solution; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube and sonicated for 15 seconds to obtain a mixed solution, that is, A complex was formed; the mixed solution was directly added to the cell culture medium of the cultured hMSC cells in a volume of 2 ml. The morphology of the complex was observed by TEM. The effect of protein transfection was observed by immunofluorescence. The results confirmed that the above vector could transfect the protein into hMSC cells.

Example 5

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, the component of which is a low molecular chitosan derivative, and the low molecular chitosan derivative is N-hydroxyethyl chitosan, N-acetylated shell Glycan, O-carboxymethylchitooligosaccharide.

The above low molecular chitosan derivative was prepared into a 200 ug/ml aqueous solution at a mass ratio of 1:1:1; Dnase I protein was taken to prepare a 100 ug/ml PBS solution; and the above two solutions were each taken at 7.5 ul. In the EP tube, ultrasonication was carried out for 15 seconds to obtain a mixed solution, that is, a complex was formed; the mixed solution was directly added to the cell culture medium of the cultured hMSC cells, and the medium volume was 2 ml. The effect of protein transfection was observed by immunofluorescence, and it was confirmed that the above vector was soluble in a neutral aqueous solution, and the protein could be transfected into hMSC cells.

Example 6

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, and the component thereof is composed of a low molecular weight chitosan having a molecular weight of 1300 and a low molecular chitosan derivative, wherein the low molecular chitosan derivative is an N-carboxyl group. Butyl chitosan and N-sulfate chitosan (both molecular weights are 20,000, both soluble in neutral water), and the molar ratio of low molecular chitosan to low molecular chitosan derivative is 1:1.

The above carrier was formulated into a 200 ug/ml aqueous solution; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube and sonicated for 15 seconds to obtain a mixed solution, that is, A complex was formed; the mixed solution was directly added to the cell culture medium of the cultured hMSC cells in a volume of 2 ml. The effect of protein transfection was observed by immunofluorescence, and it was confirmed that the above vector was soluble in a neutral aqueous solution, and the protein could be transfected into hMSC cells.

Example 7

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, and the component thereof is composed of a low molecular weight chitosan having a molecular weight of 1300 and a secondary component, wherein the secondary component is a PEG having a molecular weight of 5000. The molar ratio of the low molecular chitosan to the minor component is 1:0.2. The addition of PEG is mainly to improve the hydrophilic properties.

The above carrier was formulated into an aqueous solution of 200 ug/ml; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube, and ultrasonicated for 15 seconds to obtain a mixed solution. That is, a complex was formed; the mixed solution was directly added to the cell culture medium in which the hMSC cells were cultured, and the medium volume was 2 ml. The effect of protein transfection was observed by immunofluorescence, and it was confirmed that the above vector could transfect the protein into hMSC cells.

Example 8

The safe and efficient protein of the present embodiment is transfected into a carrier of a cell, the component of which is composed of a low molecular chitosan derivative and a secondary component, wherein the low molecular chitosan derivative is N-carboxymethyl shell polycondensation. The sugar, the minor component is a cationic liposome (LIPO2000), and the molar ratio of the low molecular chitosan derivative to the cationic liposome is 1:0.2.

The above carrier was formulated into an aqueous solution of 200 ug/ml; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube, and ultrasonicated for 15 seconds to obtain a mixed solution. That is, a complex was formed; the mixed solution was directly added to the cell culture medium in which the hMSC cells were cultured, and the medium volume was 2 ml. The effect of protein transfection was observed by immunofluorescence, and it was confirmed that the above vector could transfect the protein into hMSC cells.

Example 9

The safe and efficient protein provided by the present embodiment is transfected into a carrier of a cell, and the component thereof is composed of a low molecular weight chitosan having a molecular weight of 5000, a low molecular chitosan derivative and a secondary component, wherein the low molecular chitosan is derived. The substance is O-carboxymethyl chitosan, the secondary component is PEI (molecular weight: 20,000), and the molar ratio of low molecular chitosan, O-carboxymethyl chitosan and PEI is 1:1:0.2.

The above carrier was formulated into an aqueous solution of 200 ug/ml; Dnase I protein was taken and formulated into a 100 ug/ml PBS solution; 7.5 ul of each of the above two solutions was mixed in an EP tube, and ultrasonicated for 15 seconds to obtain a mixed solution. That is, a complex was formed; the mixed solution was directly added to the cell culture medium in which the hMSC cells were cultured, and the medium volume was 2 ml. The effect of protein transfection was observed by immunofluorescence, and it was confirmed that the above vector could transfect the protein into hMSC cells.

The invention provides a carrier for protein transfecting cells, which is simple to operate, comprising the steps of: preparing or purchasing low molecular chitosan, or preparing a derivative of low molecular chitosan, and formulating it into a certain concentration of aqueous solution; Or purchase one or more proteins and formulate them into a certain concentration of aqueous solution; The two solutions are mixed and oscillated in a certain ratio; the mixed solution is directly added to the cell culture medium of the cultured cells, or directly injected into an animal such as a vein.

Application Example 1

The vector prepared in Example 2 was used to form a complex with DNase I enzyme, and whether the complex could degrade OCT4 plasmid was determined, and the ratio of low molecular chitosan to DNase I enzyme was determined. Includes the following steps:

(1) The carrier was quantified into an aqueous solution having a concentration of 10 mg/ml, and a 0.1 mol/L sodium hydroxide solution was added to adjust the pH to 7.4. The above aqueous solution was diluted to a concentration of 200.00, 50.00, 12.50, 3.13, 0.78, 0.19 ug/ml.

(2) Take DNase I enzyme and prepare 100 ug/ml PBS solution.

(3) The above diluted low molecular chitosan solution was mixed with 7.5 ul of 100 ug/ml DNase I enzyme, and shaken overnight at 4 ° C, which was a series of "low molecular chitosan-DNase I enzyme" mixed solution.

(4) The mixed solution and the control group were subjected to agarose gel electrophoresis.

The results are shown in Figure 2. The concentration of DNase I was 100 ug/ml, and the concentration of low molecular chitosan was 200.00, 50.00, 12.50, 3.13, and 0.78 ug/ml, and the concentration of chitosan was 200.00. At 50.00 ug/ml, because the concentration is too large, the electrophoresis effect is poor and effective separation cannot be performed. 12.50, 3.13, 0.78ug/ml three concentrations of DNase I enzyme are suitable.

Application Example 2

Using the vectors prepared in Examples 3, 7 and 9, the GFP protein was transfected into hBMSC cells, and the transfection ability and experimental method of the above vectors were investigated. Includes the following steps:

(1) The carriers prepared in Examples 3, 7 and 9 were prepared and each formulated into a solution having a concentration of 200 ug/ml.

(2) Take GFP protein and prepare 100 ug/ml PBS solution

(3) The above carrier solution was separately mixed with 7.5 ul of 100 ug/ml GFP protein solution, and shaken overnight at 4 ° C to form a complex solution.

(4) A 1 ug/ml complex solution was added to 2 ml of the medium, and after 3 hours, the transfection status was observed under a fluorescence microscope.

As shown in Fig. 3, all of the above three vectors were able to efficiently transfect GFP protein into hBMSC cells. Because hBMSC cells are difficult to transfect into proteins. This indicates that the transfection ability of the above three vectors is very excellent. And as time increases, its transfection rate gradually increases.

Application Example 3

Using the vector prepared in Example 3, four recombinant proteins such as Tbx5, Hand2, Mef2c and Gata4 were mediated into hBMSC cells to observe whether the cells were transformed into myocardial progenitor cells. Includes the following steps:

(1) The carrier was quantified into an aqueous solution having a concentration of 10 mg/ml, and a 0.1 mol/L sodium hydroxide solution was added to adjust the pH to 7.4, and each was formulated into a solution having a concentration of 200 ug/ml.

(2) The above four proteins were separately dissolved in a PBS solution at a concentration of 100 ug/ml.

(3) 7ul of each of the above five solutions, blended and shaken to obtain a composite solution.

(4) The surface of the hBMSC cells was added to the medium, and the surface changes were observed at different times. The changes of the cells were observed by immunofluorescence.

(5) The 12d cells were directly injected into the myocardial infarcted myocardium of the myocardial infarction model. The superparamagnetic iron oxide particles (SPIO)-labeled piCPC was injected at the same time as the model, and the same amount of PBS was injected as a control.

(6) The cardiac function of each group was detected by echocardiography at 1 week, 2 weeks, 3 weeks and 4 weeks. The repair effect of transplanted cells on myocardial infarction was detected by HE staining.

The results of Fig. 4 and Fig. 5 show that the expression of myocardial specific protein such as isl-1 and myl2 is up-regulated after hMSCs are induced by four kinds of myocardial regulatory proteins, and hMSCs are present to the myocardium whether they are aggregated or digested after digestion. The tendency of progenitor cells to differentiate, expressing markers of their three lineages. This indicates that it is expected that the differentiation of hMSCs into myocardial progenitor cells can be achieved by protein transfection. The results of Figures 6 and 7 show that the use of transformed cells has a distinct advantage over the PBS group.

Claims (8)

1. A safe and efficient carrier for transfecting proteins into cells, characterized in that the main component of the carrier is a low molecular chitosan and/or a low molecular chitosan derivative.
2. The carrier for transfecting a safe and efficient protein into a cell according to claim 1, wherein the low molecular chitosan refers to degradation of chitosan by biodegradation, chemical degradation or physical degradation, or A low molecular weight chitosan having a degree of polymerization of from 2 to 120 and a molecular weight of from 320 to 20,000 by chemical synthesis or enzymatic synthesis.
3. The carrier for transfecting a safe and efficient protein into a cell according to claim 1, wherein the low molecular chitosan derivative refers to a low molecular chitosan as a parent, and an atom or a group of atoms thereof is used for other atoms. Or a group of substituted compounds, including acylation, carboxylation, hydroxylation, cyanation, etherification, alkylation, esterification, aldimineation, azidation, salt formation, chelation, hydrolysis, oxidation, halogenation , a derivative prepared by grafting and crosslinking reaction.
4. The safe and efficient protein transfection into a cell according to claim 3, wherein the low molecular chitosan derivative is sulfonated chitin, sulfonated carboxymethyl chitin, N-hydroxyethyl Chitosan, N-acetylated chitosan, O-carboxymethylchitosan oligosaccharide, N-carboxymethyl chitosan, N-trimethyl chlorinated chitosan oligosaccharide, N-hydroxypropyl chitosan B Acid ester, cyanoethyl or phenyl cyanoethyl chitosan, N-propyl (butyl, hexyl) acylated chitosan, N-methyl (phenyl) sulfonyl chitosan, N-phthaloyl Chitosan, N-butyl (octyl, hexadecane)-based chitosan, N-carboxybutyl chitosan, N-sulfate chitosan, 6-O-hydroxyethyl chitosan, ethylene glycol shell Glycan, glycerol (3-chloropropane-1,2-diol) chitosan, BD galactoside branched chitosan, 6-deoxy chitosan, 6-O-monosulphuric acid (3,6-O - Disulfate) One or more of chitosan, sodium lauryl sulfonate chitosan.
5. A carrier for transfecting a safe and efficient protein into a cell according to claim 1 or 2 or 3 or 4, wherein the component of the carrier further comprises a secondary component, and the secondary component is polyethylene. Glycol, polyethyleneimine, liposome, calcium phosphate, calcium metaphosphate, sodium alginate, HIV-1 TAT short peptide, polyarginine, oligoarginine, oligolysine, polystyrene, carbon One or more of the nanotubes.
6. A carrier for safe and efficient protein transfection into a cell according to claim 5, wherein the carrier has a molar ratio of a major component to a minor component of from 00 to 100.
7. A safe and efficient protein-transfecting vector into a cell according to claim 6, wherein the carrier is capable of binding to one or more types of proteins to form a complex.
8. The carrier for transfecting a safe and highly efficient protein into a cell according to claim 7, wherein the molar ratio of the main component of the carrier to the protein in the complex is from 1:0.1 to 100.

Images