CN112813104B - Preparation method and application of protein-polymer composite nano material gene vector - Google Patents
Preparation method and application of protein-polymer composite nano material gene vector Download PDFInfo
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- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
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- A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
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- A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
- A61K47/6933—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained by reactions only involving carbon to carbon, e.g. poly(meth)acrylate, polystyrene, polyvinylpyrrolidone or polyvinylalcohol
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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Abstract
The invention discloses a protein-polymer composite nano material gene vector, a preparation method and application thereof. The carrier is obtained by synthesizing a core-shell structure nano material through in-situ polymerization of a high-molecular shell on the surface of protein. The vector can effectively compound and compress nucleic acid, can efficiently release the nucleic acid into cytoplasm through degradation of cationic groups in a macromolecular shell while protecting the nucleic acid from degradation of nuclease, achieves extremely high gene transfection efficiency, has high biocompatibility, low cytotoxicity and designable functionality, is simple in preparation process and purification program, and has wide application prospect in related fields of gene therapy.
Description
Technical Field
The invention belongs to the field of nano materials, and particularly relates to construction and application of a gene presentation vector combining protein and a polymer.
Background
Nucleic acids are considered to be an excellent gene therapy agent, but are not themselves able to enter cells directly and are susceptible to nuclease degradation. Vectors commonly used for nucleic acid presentation are divided into two categories, viral vectors and non-viral vectors. Non-viral gene vectors have been extensively studied due to their properties of good safety, low immunogenicity, and high gene loading capacity, such as cationic liposomes and macromolecules. Despite significant advances in the design of polymeric carriers, research in this area has been plagued by complex synthetic processes, low transfection efficiency, poor biocompatibility and biodegradability.
Based on the technical problems of the existing carrier, the invention aims to graft a polymerizable monomer and a cross-linking agent molecular system on the surface of protein and initiate polymerization to obtain a protein-polymer composite nano material, further provides a gene presentation carrier taking the protein-polymer composite nano material as a tool, efficiently delivers target nucleic acid to cells through extremely strong nucleic acid adsorption capacity and gene encapsulation effect, and realizes the release and expression of the nucleic acid in the cells through the degradation of cationic groups on the surface of the composite nano material, thereby enhancing the gene transfection efficiency.
Disclosure of Invention
Based on the above purpose, the present invention firstly provides a protein-polymer composite nanomaterial gene vector, which is formed by a protein as an inner core and a high molecular polymer as an outer shell, wherein the high molecular polymer is formed by modifying a primary amino group of lysine in the protein to introduce a double bond, and then performing cross-linking polymerization on a neutral monomer, a cationic monomer and a cross-linking agent with a protein surface double bond in the presence of a free radical initiator.
In a preferred technical scheme, the cationic monomer is 2- (dimethylamino) ethyl methacrylate, the crosslinking agent is N, N' -methylene bisacrylamide, the electrically neutral monomer is acrylamide, the free radical initiator is ammonium persulfate and tetramethylethylenediamine, and modification of a primary amino group of lysine in the protein is acrylation modification.
More preferably, the modification of the primary amino group of lysine in the protein is performed using N-hydroxysuccinimide acrylate.
In a preferred embodiment, the protein is any polypeptide having more than two lysine residues.
In a particular embodiment of the invention, the protein is bovine serum albumin. Chemically modified (e.g., fluorescent molecular labeling) polypeptides are also included in embodiments of the invention, as long as the modifications do not interfere with the synthesis of the nanocomposite, as is well known to those skilled in the art.
More preferably, the bovine serum albumin: acrylamide: 2- (dimethylamino) ethyl methacrylate: n, N' -methylenebisacrylamide: ammonium persulfate: the mole ratio of the tetramethyl ethylene diamine is 1: 3000: 3000: 400: 250: 1000.
secondly, the invention also provides a preparation method of the protein-polymer composite nano material gene vector, which comprises the following steps:
(1) modifying a protein serving as a carrier inner core to enable primary amine groups of lysine in the protein to be modified to introduce double bonds;
(2) the method comprises the step of carrying out cross-linking polymerization on a neutral monomer, a cationic monomer and a cross-linking agent with double bonds on the surface of protein in the presence of a free radical initiator to grow a high molecular polymer shell wrapping a protein core in situ.
In a preferred embodiment, the protein in step (1) is any polypeptide having two or more lysine residues, and the modification is an acrylation modification using N-hydroxysuccinimide acrylate.
In another preferred embodiment, the electrically neutral monomer of step (2) is acrylamide, the cationic monomer is 2- (dimethylamino) ethyl methacrylate, the crosslinking agent is N, N' -methylenebisacrylamide, and the radical initiator is ammonium persulfate and tetramethylethylenediamine.
Thirdly, the invention also provides the protein-polymer composite nano material presentation carrier loaded with the drug molecules.
In a preferred embodiment, the drug molecule is a nucleic acid molecule.
More preferably, the mass ratio of the protein-polymer composite nanomaterial presentation carrier to the nucleic acid molecule is 9: 1.
Fourthly, the invention also provides a preparation method of the protein-polymer composite nano material gene presentation vector, and the method comprises the step of mixing the vector and the nucleic acid molecule.
Finally, the invention provides the use of the above vector in the preparation of a gene therapy or prophylactic agent.
Compared with the existing gene vector, the protein-polymer composite nano material provided by the invention has the following outstanding advantages as a novel gene presentation vector:
the invention cross-links and polymerizes the macromolecule from the surface of protein in situ, the preparation method of the material is simple and green, and the cost is low. The design idea of taking protein as the inner core ensures that the material has good biocompatibility and low cytotoxicity. The material and the nucleic acid form a non-covalent self-assembly compound, the influence on the gene is small, and the non-covalent self-assembly compound has good practical operability when used for gene transfection;
the cross-linking agent of the polymer shell is selected to be N, N' -methylene bisacrylamide after screening and optimization, so that the compression capacity of the polymer shell on DNA can be enhanced, the disturbance of a cell membrane double-layer structure is easily induced, and the DNA uptake capacity of cells is improved;
the cationic monomer of the high molecular shell is selected into 2- (dimethylamino) ethyl methacrylate with tertiary amino and ester bond after screening and optimization, the tertiary amino structure generates a proton sponge effect to facilitate endosome escape, and the ester bond structure connected with the tertiary amino is decomposed at a physiological temperature to degrade the cationic group so as to promote the dissociation of DNA from the self-assembly complex. Therefore, the vector can realize high-level nucleic acid release and achieve excellent gene transfection effect;
the gene vector achieves the highest transfection efficiency at 4 days after transfection, and the continuity of gene expression is superior to that of the commercialized liposome. And the release rate of the nucleic acid is controllable, so that the method has obvious advantages in some special applications that the gene expression rate needs to be regulated and controlled.
Drawings
FIG. 1 shows the preparation and the shape and size of a protein-polymer composite nano material. (a) A schematic diagram of the synthesis of composite nanomaterials; (b) transmission electron microscopy images; (c) atomic force microscopy images; (d) dynamic light scattering pattern.
FIG. 2 is a gel electrophoresis diagram of a complex formed by mixing a composite nano-material carrier and DNA according to different proportions. Wherein, M is DL2000 DNA marker, 0 is plasmid DNA, and 1-3 respectively represent the mass ratio of the vector to the plasmid DNA is 1:1, 3:1 and 9: 1.
FIG. 3 is a co-localization map of complexes formed by FITC-labeled composite nanomaterials and TOTO 3-labeled plasmids after internalization into HEK293T cells.
FIG. 4 shows the rate of GFP positive cells measured by flow cytometry after the composite nanomaterial and Lipo2000 transfection of pmaxGFP plasmid into HEK293T cells and continued culture for 1-4 days.
FIG. 5 is a fluorescent microscope image of composite nanomaterials transfected with pmaxGFP plasmid into HEK293T cells and cultured for 4 days.
FIG. 6 shows the properties and transfection efficiencies of material systems prepared with different cross-linking agents. (a) The chemical structure of the cross-linking agent used in the preparation of each material system; (b) testing the contact angle of each material system with water to characterize the hydrophobicity of the material; (c) internalization efficiency of HEK293T cells to each material system measured by flow cytometry; (d) internalization efficiency of HEK293T cells by flow cytometry for complexes formed by each material system with TOTO 3-labeled plasmids; (e) the flow cytometry results show that the rate of GFP positive cells in each material system after transfection of pmaxGFP plasmid into HEK293T cells continued to be cultured for 4 days.
FIG. 7 shows the gene transfection efficiency of material systems prepared with different cationic monomers. (a) Chemical structure of the cationic monomer used in preparing each material system; (b) the flow cytometry results show that the rate of GFP positive cells in each material system after transfection of pmaxGFP plasmid into HEK293T cells continued to be cultured for 4 days.
FIG. 8 shows the Zeta potential change of the composite nanomaterial at 37 ℃. Wherein, B 0 As a raw composite nanomaterial, B 37 Is a composite nano material after being placed for 4 days at 37 ℃.
FIG. 9 is B 0 And B 37 Gel electrophoresis image after complexing with DNA.
FIG. 10 is B 0 And B 37 Atomic force microscopy images after complexing with DNA.
FIG. 11 shows plasmid DNA stained with TOTO-3 dye and then combined with B 0 And B 37 Flow pattern 4h after transfection of the formed complexes into cells.
Detailed Description
The invention is further described below in conjunction with specific embodiments, and the advantages and features of the invention will become more apparent as the description proceeds. These examples are only illustrative and do not limit the scope of protection defined by the claims of the present invention.
Example 1: preparation and characterization of protein-polymer composite nanomaterials
1. Preparation method
1) Bovine serum albumin (Sigma-Aldrich # SRE0096) was dissolved in 100mM borate buffer at pH 8.5 and centrifuged four times through a 30kD ultrafiltration tube (Amicon # UFC5030BK) in this buffer to remove any small amount of NH4 that may be present in the protein + The bicinchoninic acid (BCA) method measures the concentration and dilutes the protein with this buffer to 10 mg/ml.
2) N-hydroxysuccinimide acrylate (Sigma-Aldrich # A8060) was dissolved in dimethyl sulfoxide (Sigma-Aldrich #276855) to prepare a 20mg/ml solution, and 5.1. mu.l of the solution was mixed with 100. mu.l of 10mg/ml bovine serum albumin solution (wherein N-hydroxysuccinimide acrylate: bovine serum albumin ═ 40: 1 molar ratio), reacting at room temperature of 25 ℃ for 6h to obtain a protein solution A with surface double bond modification.
3) Acrylamide (Sigma-Aldrich # A8887) is dissolved in deionized water to prepare a solution B of 200 mg/ml; dissolving N, N' -methylene bisacrylamide (Sigma-Aldrich # M7279) in dimethyl sulfoxide to prepare a solution C with the concentration of 100 mg/ml; ammonium persulfate (Sigma-Aldrich # A3678) is dissolved in deionized water to prepare a solution D of 100 mg/ml; both ethyl 2- (dimethylamino) methacrylate (Sigma-Aldrich #234907) and tetramethylethylenediamine (Sigma-Aldrich # T9281) were liquid reagents and were used directly in the reaction.
4) After adding 300. mu.l of pH 7.4, 100mM phosphate buffer to the solution A system, 16. mu.l of solution B, 7.6. mu.l of 2- (dimethylamino) ethyl methacrylate, 9.3. mu.l of solution C, 8.6. mu.l of solution D, and 2.1. mu.l of tetramethylethylenediamine were added and mixed uniformly (wherein, bovine serum albumin: acrylamide: 2- (dimethylamino) ethyl methacrylate: n, N' -methylenebisacrylamide: ammonium persulfate: tetramethylethylenediamine ═ 1: 3000: 3000: 400: 250: 1000 molar ratio), at room temperature 25 ℃ for 4 h.
5) See FIG. 1a for a schematic of the reaction. After the polymerization reaction, components of the reaction product which are not intended generally remain in the system, and therefore, purification is required. And centrifuging the product solution by using a Phosphate Buffer Solution (PBS) with the pH value of 7.4 and the concentration of 10mM in a 30kD ultrafiltration tube for 4 times to obtain a protein-polymer composite nano material solution. After the BCA assay, the concentration was diluted to 1.5mg/ml with PBS.
2. Characterization of
When the protein-polymer composite nanomaterial is observed by a transmission electron microscope (HT7700, Hitachi) and an atomic force microscope (Dimension Icon, Bruker), it is known that the particles are spherical and have a particle size distribution of 10-20nm, as shown in FIGS. 1b and 1 c. Dynamic light scattering (Zetasizer Nano, Malvern) determined an average particle size of 13.8nm (FIG. 1 d). The ability of the composite nanomaterial to concentrate plasmid DNA at different mass ratios was then investigated by gel retardation experiments (FIG. 2). Mu.l, 2.6. mu.l, 7.8. mu.l of 1.5mg/ml composite nanomaterial solution and 2.6. mu.l of 500. mu.g/ml plasmid solution (Lonza pmaxGFP) TM ) Mixing (mass ratio of 1:1, 3:1, 9:1) respectively, and adding deionized water to make the total volume 20 μ l. Placing the mixture at room temperature for 20min, adding a sample into 1% agarose gel, and carrying out electrophoresis under the conditions of 160V and 400mA for 20min to observe that the DNA of the unmixed composite nano material migrates to a positive electrode; at a recombination ratio of 1:1 of the composite nanomaterial to DNA, the DNA no longer migrates to the positive electrode but remains in the pores; at higher complexing ratios (3:1 or 9:1), however, the DNA begins to migrate toward the negative electrode. This demonstrates that the composite nanomaterial can concentrate DNA molecules that originally exhibit negative charges into positively charged particles, and that the higher the composite nanomaterial concentration, the stronger the ability of the concentrated DNA to complex. And performing subsequent cell experiments according to the mass ratio of the composite nano material to the nucleic acid of 9: 1. In FIG. 2, lane M is DL2000 DNA molecular marker, lane 0 is plasmid DNA, and the mass ratio of vector to plasmid DNA in lanes 1-3 is: 1:1,3:1,9:1.
Example 2: application of protein-polymer composite nano material presentation gene
The composite nanomaterial prepared in example 1 can be applied to gene transfection. The process is as follows:
1) and mixing the composite nano material with nucleic acid, and standing at room temperature for 20min to obtain the carrier/nucleic acid composite.
2) The vector/nucleic acid complex was added to serum-free medium of HEK293T cells in exponential growth phase, the medium was changed to complete medium 4h after transfection, and the culture was continued for 4 days.
3) The gene transfection effect of the vector in the cell is observed by means of a flow cytometer, a fluorescence microscope, or the like.
The following specific experimental procedures were used:
1. cellular internalization of composite nanomaterial carrier/nucleic acid
Before the internalization experiment of the composite nanomaterial vector/nucleic acid, the composite nanomaterial and pmaxGFP plasmid (Lonza) were modified separately. After incubating the composite nanomaterial with FITC fluorescent labeling reagent (Sigma-Aldrich # F3651) at a molar ratio of 1:4 in the dark for 1h, dialyzing the mixture with PBS to remove free FITC to obtain FITC labeled composite nanomaterial; plasmids were labeled with TOTO-3 fluorescent dye (Molecular Probes # T-3604) at a dye/base pair molar ratio of 1: 20. This mixture, after incubation in the dark for 2 hours, yielded TOTO 3-labeled nucleic acids. 18 mu g of FITC-labeled composite nanomaterial and 2 mu g of TOTO 3-labeled nucleic acid are mixed and kept stand for 20min, 1ml of serum-free DMEM medium is used for mixing uniformly, the mixture is added into HEK293T cells (ATCC source) growing to the exponential growth phase in a glass bottom culture dish with the diameter of 20mm for incubation for 4h, then DAPI dye (Invitrogen # D1306) is used for labeling cell nuclei, a high-resolution fluorescence microscope (Ti-E, Nikon) is used for observation (figure 3), the green fluorescence of FITC and the magenta fluorescence of TOTO3 have consistent subcellular localization, the excellent intracellular co-transfer efficiency of the composite nanomaterial and the nucleic acid is shown, and the composite nanomaterial can be used as a carrier to efficiently internalize DNA into the cells.
2. Gene expression after transfection of cells with composite nanomaterial vectors/nucleic acids
6 mu.l of 1.5mg/ml composite nano material and 2 mu.l of 500 mu g/ml pmaxGFP plasmid capable of expressing super-strong green fluorescent protein are mixed and kept stand for 20min, 500 mu.l of serum-free DMEM medium is used for mixing uniformly, the mixture is added into HEK293T cells growing to the exponential growth phase in a 24-well plate for incubation for 4h, then the DMEM medium containing 10% serum is used for replacing the culture medium, and the culture is continued for 1-4 days. The positive control was commercial liposome Lipo2000(Invitrogen # 11668). FIG. 4 shows the proportion of GFP positive cells after transfection with two vectors characterized by flow cytometry (Guava easy Cyte HT, Merck Millipore). 1 day after transfection, the ratio of Lipo2000 positive cells (75.2%) is nearly 3 times that of composite nano material positive cells (27.0%); 2 days after transfection, the difference between the positive cell rates of both decreased to 8.6% (79.1% and 70.5%); after 3 days of transfection, the positive cell rate of the composite nano material is over 80%, while the positive cell rate of Lipo2000 is gradually reduced, and the positive cell rate of the composite nano material is 18.7% higher than that of Lipo2000 after 4 days of transfection. In contrast, composite nanomaterials achieve gene expression for longer durations. Meanwhile, the gene release expression rate is low, so that the method is expected to be applied to some applications in which the gene expression rate needs to be regulated. Cells 4 days after transfection were labeled with DAPI dye, and very high expression rate of GFP in cells was also observed by fluorescence microscopy (rotation 5, BioTek) (fig. 5), further indicating that the protein-polymer composite nanomaterial is a very good gene-presenting vector.
Example 3: optimization of protein-polymer composite nano material preparation material
1. Optimized selection of cross-linking agents
Several other material systems were prepared by the same preparation method as example 1 (the crosslinker of B4 was N, N' -methylenebisacrylamide) only with the exchange of the crosslinker, wherein B5 was prepared without the addition of a crosslinker, the crosslinker of B6 was poly (DL-lactide) -B-poly (ethylene glycol) -B-poly (DL-lactide) -diacrylate triblock copolymer (polyticech # AI102), and the crosslinker of B7 was glycerol dimethacrylate (Sigma-Aldrich # 436895). The molecular structure of each crosslinker is shown in FIG. 6 a.
Fig. 6b shows the water contact angle of each material system measured by a surface interfacial tension meter (DCAT21, Dataphysics), and the larger the contact angle, the higher the hydrophobicity of the material. The result shows that N, N' -methylene bisacrylamide increases the hydrophobicity of the material system. In general, the more hydrophobic the polymer, the more strongly the polymer interacts with the lipid bilayer of the cell membrane, and the more cellular uptake of the polymer can be promoted. To demonstrate this correlation, each material system was labeled with FITC, 7.5 μ g of FITC-labeled each material system was mixed with 500 μ l serum-free DMEM medium, added to HEK293T cells in a 24-well plate for incubation for 4h, and then the cells were washed, digested, resuspended in PBS, and the average FITC fluorescence intensity of the uptake of each material system by the cells was characterized by flow cytometry. As shown in FIG. 6c, when N, N' -methylene bisacrylamide is used as a cross-linking agent, the degree of cellular internalization of the material is the highest, which is 2-3 times that of other material systems.
The ability of each material system to complex nucleic acids into cells was then characterized. Mu.l of each material system of 1.5mg/ml and 1. mu.g of pmaxGFP plasmid marked by TOTO3 were mixed, left to stand for 20min, mixed with 500. mu.l of serum-free DMEM medium, added into HEK293T cells grown to exponential phase in a 24-well plate, incubated for 4h, and then the cells were washed, digested, resuspended in PBS, and the average TOTO3 fluorescence intensity of the plasmid DNA taken up by the cells when different material systems were used as carriers was characterized by a flow cytometer. FIG. 6d shows that B4 has the highest efficiency of internalization for delivering nucleic acid into cells, which indicates that when N, N' -methylene bisacrylamide is used as a cross-linking agent, the material can enhance the ability of compressing nucleic acid and improve the ability of uptake nucleic acid by cells. And then, the expression condition of the gene of the composite nucleic acid of each material system after entering the cell is characterized. Mu.l of each material system at 1.5mg/ml was mixed with 2. mu.l of 500. mu.g/ml pmaxGFP plasmid, left to stand for 20min, mixed with 500. mu.l of serum-free DMEM medium, added to HEK293T cells grown to exponential growth phase in a 24-well plate and incubated for 4h, and then the medium was changed to 10% serum-containing DMEM medium and cultured for 4 days. Fig. 6e is the proportion of GFP positive cells after 4 days of transfection of the composite plasmids of each material system characterized by flow cytometry, and the results show that the transfection efficiency of B4 is the highest, the GFP positive cell rate reaches 73.6%, and the GFP positive cell rates of other vectors are all below 35%. The results show that the N, N' -methylene bisacrylamide is superior to the protein-polymer composite nano material in the application of serving as a gene vector when being used as a cross-linking agent.
2. Optimized selection of cationic monomers
Several other material systems were prepared using the same preparation method as in example 1 (the cationic monomer of B4 was 2- (dimethylamino) ethyl methacrylate) with the cationic monomer of B1 being N- (3-aminopropyl) methacrylamide (Sigma-Aldrich #731099), the cationic monomer of B9 being N- [ (3- (dimethylamino) propyl ] methacrylamide (Sigma-Aldrich #409472), the cationic monomer of B11 being 2-aminoethyl methacrylate (Sigma-Aldrich #516155) with only the cationic monomer replaced, and the molecular structure of each cationic monomer is shown in fig. 7 a.
And (3) characterizing the gene expression condition of the composite nucleic acid of each material system after entering cells. Mu.l of each material system at 1.5mg/ml was mixed with 2. mu.l of 500. mu.g/ml pmaxGFP plasmid, left to stand for 20min, mixed with 500. mu.l of serum-free DMEM medium, added to HEK293T cells grown to exponential growth phase in a 24-well plate and incubated for 4h, and then the medium was changed to 10% serum-containing DMEM medium and cultured for 4 days. FIG. 7B shows the GFP positive cell rate 4 days after transfection of the composite plasmids of each material system characterized by flow cytometry, and the results show that the transfection efficiency of B4 is the highest, which is about 9.3 times that of B1, 5.5 times that of B9 and 3 times that of B11. The results show that the 2- (dimethylamino) ethyl methacrylate as the cationic monomer has superiority in the application of the protein-polymer composite nano material as a gene vector.
Example 4: mechanism research of protein-polymer composite nano material presentation gene
The 4 days at 37 ℃ used in cell transfection was selected as the experimental conditions for evaluating the stability of the composite nanomaterial itself. B is 0 As a raw composite nanomaterial, B 37 Is a composite nano material after being placed for 4 days at 37 ℃.
As measured by a potentiometer (Zetasizer Nano, Malvern), the Zeta potential of the composite Nano-material at 37 ℃ is remarkably reduced from 13mV to 3mV (figure 8). The chemical composition of the bond can be attributed to the hydrolysis of ester bonds in the 2- (dimethylamino) ethyl methacrylate, resulting in the loss of tertiary amine groups over time, just as the degradation of the cationic groups results in the reduction of the surface charge of the composite nanomaterial. The gel retardation experiment showsAfter 4 days of incubation at 37 ℃, the complex of the carrier and DNA migrated toward the positive electrode (fig. 9), indicating a decrease in the ability of the composite nanomaterial to condense DNA. At the same time, with B 0 In contrast, B 37 The complex formed with the DNA was less tight, showing a partially relaxed free DNA linear structure (fig. 10). With B 0 Or B 37 After complexing with the TOTO 3-labeled plasmid at a mass ratio of 9:1, cells were incubated with HEK293T for 4 h. The average TOTO3 fluorescence intensity of the cells was measured by flow cytometry, as shown in B 37 The effective DNA amount loaded into the cells is only B 0 33% (fig. 11).
It follows that when the carrier/DNA complex enters the cell, at physiological temperature, the reduction of charge caused by the degradation of the composite nanomaterial carrier leads to the reduction of the condensation capacity of the carrier to DNA, which leads to the gradual release of DNA from the complex in the cell, and thus the gene can be expressed continuously. The self-degradation characteristic of the composite nano material promotes the successful expression of DNA internalized into cells, which is an important reason for good transfection effect of the composite nano material as a gene presentation vector.
Claims (1)
1. An application of protein-polymer composite nano material carrier in preparing gene carrier, the protein-polymer composite nano material carrier is formed by protein as an inner core and high molecular polymer as an outer shell, characterized in that the high molecular polymer is prepared by modifying a primary amino group of lysine in the protein and introducing a double bond, is prepared by crosslinking and polymerizing an electrically neutral monomer, a cationic monomer and a crosslinking agent with double bonds on the surface of protein in the presence of a free radical initiator, the cationic monomer is 2- (dimethylamino) ethyl methacrylate, the cross-linking agent is N, N' -methylene bisacrylamide, the charge neutral monomer is acrylamide, the free radical initiator is ammonium persulfate and tetramethyl ethylene diamine, the modification of the primary amino group of lysine in the protein is an acrylation modification by using N-hydroxysuccinimide acrylate; the protein is bovine serum albumin; when the protein-polymer composite nano-material carrier is prepared, the ratio of bovine serum albumin: acrylamide: 2- (dimethylamino) ethyl methacrylate: n, N' -methylenebisacrylamide: ammonium persulfate: the mole ratio of the tetramethylethylenediamine is 1: 3000: 3000: 400: 250: 1000.
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