CN118085201A - Protein-macromolecule conjugate with precise sequence structure, and preparation method and application thereof - Google Patents
Protein-macromolecule conjugate with precise sequence structure, and preparation method and application thereof Download PDFInfo
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- CN118085201A CN118085201A CN202410220772.XA CN202410220772A CN118085201A CN 118085201 A CN118085201 A CN 118085201A CN 202410220772 A CN202410220772 A CN 202410220772A CN 118085201 A CN118085201 A CN 118085201A
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Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a protein-macromolecule conjugate with a precise sequence structure, and a preparation method and application thereof, wherein the protein-macromolecule conjugate comprises the following components: the initiator is covalently connected with the protein to obtain a protein macromolecular initiator, the protein macromolecular initiator is subjected to in-situ polymerization reaction with the monomer of the first sequence, after the monomer of the first sequence is completely reacted, the monomer of the second sequence is put into the reaction to carry out in-situ polymerization reaction, and the operation is repeated until the protein-macromolecule conjugate with the accurate sequence structure is obtained. The method is simple, efficient and easy for mass production, and the obtained protein-macromolecule conjugate has good biocompatibility, biostability, controllable in-vivo organ distribution behavior and remarkably improved in-vivo circulation capacity, and has good application prospect in the fields of personalized protein medicines and accurate medical treatment.
Description
Technical Field
The invention relates to the field of biomedical materials, in particular to a protein-macromolecule conjugate with a precise sequence structure, and a preparation method and application thereof.
Background
The protein is a biological macromolecule with a complex structure formed by folding one or more linear peptide chains, is involved in almost all biological processes, is not only a specific and efficient catalyst and a main structural component of cells, but also can be used as an active drug for diagnosing and treating diseases. In recent years, protein drugs have received widespread attention. The protein medicine is a protein product which has biological functions and can be used for preventing, diagnosing and treating diseases, and compared with the common chemical medicine, the protein medicine has the advantages of clear biological functions, strong specificity, high activity, low toxicity and the like, and is easier to play a role in clinic. In addition, protein medicines gradually become an important component of biological medicines due to high biological safety and clinical curative effect and low cost.
Proteins usually need to maintain their own conformations to express biological activity, but due to the complexity and accuracy of their own spatial structures, they are easily affected by external environments such as temperature, pH, solvents, high-energy rays, heavy metal salts, etc., to cause denaturation and inactivation, and the application of the proteins in high-temperature oxidation-resistant protection is beneficial to the high-temperature environment. The protein-macromolecule conjugate has both bioactivity of protein and versatility and flexibility of macromolecule, and has increasingly wide application in biomedical field. In the field of natural biomacromolecules, both nucleic acids and proteins have precise sequences and complex functions, and the precise sequence structure in these biomacromolecules plays a key role in their functional expression. In the field of synthesizing polymers, the precise positioning of monomer units plays an important role in the structure of the polymers, and provides the polymers with information storage, molecular recognition, biocatalysis and other properties.
In the prior art, the sequence structure of the macromolecule in the protein-macromolecule conjugate obviously influences the biological function and clinical curative effect of the protein drug. However, the conventional protein-polymer conjugate cannot realize precise control over the structure of the polymer sequence, and the biological function and clinical efficacy of the conjugate are greatly limited. Therefore, there is a need to develop a controllable synthesis method for precisely controlling the sequence structure of the polymer in the protein-polymer conjugate.
Disclosure of Invention
Aiming at the problems that the protein medicine in the prior art has poor stability, short half-life period and poor targeting property, and the existing protein-polymer conjugate has single polymer chain structure and uncontrollable monomer sequence, and is difficult to further optimize and improve the biological performance of the protein medicine, the main purpose of the invention is to provide a preparation method of the protein-polymer conjugate with an accurate sequence structure, which is carried out by adopting a one-pot in-situ active polymerization method.
Another object of the present invention is to provide a protein-polymer conjugate having a precise sequence structure, which has a remarkable sequence dependence, high biosafety and high bioactivity.
The invention also aims to provide the application of the protein-macromolecule conjugate with the precise sequence structure in the preparation of protein medicines.
In order to achieve the above purpose, the invention adopts the following technical scheme:
the invention provides a preparation method of a protein-macromolecule conjugate with a precise sequence structure, which adopts a one-pot in-situ active polymerization method and comprises the following steps:
(1) Covalently connecting an initiator dissolved in an organic solvent with protein dissolved in a buffer solution to obtain a protein macromolecular initiator;
(2) The protein macromolecular initiator is subjected to in-situ polymerization reaction with the monomer of the first sequence; after the monomer of the first sequence is completely reacted, adding the monomer of the second sequence to perform in-situ polymerization reaction; repeating the above operation until the protein-macromolecule conjugate with accurate sequence structure is obtained.
In the preparation method, in-situ polymerization is realized by utilizing atom transfer radical polymerization, and in-situ polymerization reaction is always carried out in a mixed solution system of aqueous solution and organic solvent in a specific proportion, so that the activity of protein and the solubility of monomers participating in the polymerization reaction are ensured, and the efficiency of the polymerization reaction is ensured. In addition, the protein-macromolecule conjugates with various different precise sequence structures are successfully prepared by adopting the preparation method, and the protein-macromolecule conjugates with different sequence structures have sequence-dependent in-vivo and in-vitro biological behaviors.
Preferably, in step (1), the organic solvent a is dimethyl sulfoxide;
And/or the volume ratio of the protein macroinitiator to the monomer of the first sequence in dimethylformamide and ultrapure water is 1:4, carrying out in-situ polymerization reaction in the mixed solution.
Preferably, in the step (1), the initiator is selected from one or more of halides, halogenated esters, halogenated ketones and halogenated nitriles; in some embodiments, the initiator is a compound represented by formula (1):
preferably, in step (1), the monomer is an acrylic monomer selected from the chemical structures shown in any one of the following (2) to (4):
the monomer can be completely converted in the in-situ polymerization process, so that the accuracy of the sequence is ensured.
Preferably, in step (1), the protein comprises a protein and/or an antibody, in particular a therapeutic protein, selected from one or more of an interferon, insulin, monoclonal antibody, colony stimulating factor, growth hormone, therapeutic vaccine and enzyme; further such proteins include, but are not limited to, asparaginase, glutamate, arginase, arginine deaminase, adenosine deaminase ribonuclease, cytosine deaminase, trypsin, chymotrypsin, papain, epidermal Growth Factor (EGF), insulin-like growth factor (IGF), transforming Growth Factor (TGF), nerve Growth Factor (NGF), platelet-derived growth factor (PDGF), bone Morphogenic Protein (BMP), fibroblast growth factor, somatostatin, growth hormone, somatostatin, calcitonin, parathyroid hormone, colony Stimulating Factor (CSF), coagulation factors, tumor necrosis factor, interferon, interleukins, gastrointestinal peptides Vasoactive Intestinal Peptide (VIP), enteropancreatic peptide (CCK), gastrin, secretin, erythropoietin, hormone, antidiuretic hormone, octreotide, pancreatic enzyme, superoxide dismutase, thyroid stimulating hormone releasing hormone (TRH), thyroid stimulating hormone, luteinizing Hormone Releasing Hormone (LHRH), tissue type plasminogen activator, interleukin-1, interleukin-15, receptor antagonist (IL-1 RA), glucagon-like peptide-1 (GLP-1), leptin, auxin, granulocyte colony stimulating factor (GM-CSF), interleukin-2 (IL-2), adenosine deaminase, uricase, human growth hormone, macrophage activation, chorionic gonadotropin, heparin, atrial natriuretic peptide, hemoglobin, retroviral vectors, relaxin, cyclosporine, oxytocin, vaccines, monoclonal antibodies, single chain antibodies, ankyrin repeat proteins, affibodies, and the like.
Preferably, in the step (1), the protein and the initiator are connected through a covalent bond, and a linking site of the covalent bond is positioned at an N-end, a C-end or a site far away from an active site of the protein and not interfering with the activity of the protein, so that the biological property of the obtained protein macromolecular initiator is stable, and the multidimensional structure and the biological activity of the protein are completely reserved.
Preferably, in step (1), the protein is Bovine Serum Albumin (BSA), and the linkage site of the covalent bond is located at cysteine 34 on BSA, and the coupling ratio of the protein to the initiator is 1:1, realizing the modification of site selection, and ensuring that the protein macromolecular initiator has a definite chemical structure and uniform molecular weight of the product.
Preferably, in the step (2), the in-situ polymerization is an atom transfer radical polymerization, and the polymerization is carried out for 24-72 hours at 35 ℃ under the condition of inert gas atmosphere and magnetic stirring, so that the complete conversion rate of monomers in the polymerization is ensured, and the obtained polymer chain segment sequence is accurate and has high reactivity.
Preferably, in the step (2), the in-situ polymerization is performed under the catalysis of cuprous bromide (CuBr) and 1,1,4,7,10,10-Hexamethyltriethylenetetramine (HMTETA), wherein the molar ratio of the protein macromolecular initiator to the monomers of each section of sequence to the CuBr to the HMTETA is 1:20-120:40:80, the in-situ polymerization reaction efficiency is high, the monomer conversion rate is complete, and the sequence accuracy is ensured.
Preferably, the method further comprises the step of pre-reacting the initiator dissolved in the organic solvent a with the protein dissolved in the buffer solution at 7 ℃ for 24 hours.
The invention also provides a protein-macromolecule conjugate with a precise sequence structure, which is prepared by the preparation method of the protein-macromolecule conjugate.
Preferably, in the protein-macromolecule conjugate with the precise sequence structure, the chain segment sequence of the protein-macromolecule conjugate is regulated and controlled by regulating and controlling the feeding sequence of the monomers, so that the in-vivo and in-vitro biological performance of the protein-macromolecule conjugate is regulated and controlled. Further, the sequence structure is selected from at least one of BSA-MMMMMM, BSA-ooooooo, BSA-HHHHHH, BSA-MMOOHH, BSA-MOHMOH, and BSA-OMOHHM, wherein m=20m ', o=20o', h=20h ', wherein M', O ', H' correspond to the acrylic monomers shown in any one of formulas (2) to (4) of claim 3, respectively.
The invention also provides application of the protein-macromolecule conjugate with the precise sequence structure in preparation of protein medicines.
Compared with the prior art, the invention has the beneficial effects that:
1. The invention adopts a one-pot in-situ active polymerization method, which not only has simple operation, high polymerization reaction efficiency and strong universality, but also does not influence the biological activity of protein medicines.
2. In-vivo and in-vitro experiments, the protein-macromolecule conjugate of the invention has obvious sequence dependence on cell entry behavior, in-vivo organ distribution and pharmacokinetics, and has higher biological safety and biological activity.
3. According to the invention, through accurately regulating and controlling the segment sequences of the macromolecules, the biological stability, tissue specificity, organ targeting and in-vivo long circulation capacity of the protein medicine are further optimized, the clinical curative effect of the protein medicine can be improved to the maximum extent, and a new idea is provided for subsequent personalized treatment.
Compared with the existing preparation method of the protein-polymer conjugate, the preparation method provided by the invention is simple and efficient, the protein-polymer conjugate with an accurate sequence structure can be obtained without intermediate purification steps, and the in-situ preparation method is easy for mass production and site selection functionalization of the protein-polymer conjugate. The prepared protein-macromolecule conjugate with accurate sequence has good biocompatibility, biostability, controllable in-vivo organ distribution behavior and obviously improved in-vivo circulation capacity, and has good application prospects in the fields of personalized protein medicines, accurate medical treatment and the like.
Drawings
FIG. 1 shows the synthetic route of protein-polymer conjugates with precise sequence structure in examples.
FIG. 2 shows the results of a volume exclusion chromatography (SEC) of protein-polymer conjugates with precise sequence structure in the examples.
FIG. 3 shows the result of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of protein-polymer conjugates having a precise sequence structure in the examples.
FIG. 4 is a hydrated particle size characterization of a protein-polymer conjugate with a precise sequence structure in the examples.
FIG. 5 is a biosafety characterization of protein-macromolecule conjugates with precise sequence structure in examples.
FIG. 6 shows the effect of cell entry of protein-polymer conjugates with precise sequence structure in the examples.
FIG. 7 shows the biological distribution of protein-polymer conjugates with precise sequence structure in mice in the examples.
FIG. 8 shows the in vivo circulation half-life of the protein-polymer conjugate having a precise sequence structure in the examples.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the present invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications could be made by those skilled in the art without departing from the inventive concept. These are all within the scope of the present invention.
The experimental methods used in the following examples are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
L929 and NIH-3T3 in the examples below were purchased from tumor cell banks of the national academy of sciences.
The cell culture medium in the examples described below is a product of the company Sieimer.
SD rats and BALB/c mice in the following examples were purchased from Shanghai university animal experiment centers, SD rats are hereinafter abbreviated as rats, and BALB/c mice are hereinafter abbreviated as mice.
The molecular weight of protein-macromolecule conjugates with precise sequence structure was analyzed using SEC and SDS-PAGE in the following examples; measuring the hydrated particle size of protein-polymer conjugates of different sequences using a dynamic light scattering particle sizer (DLS); cell uptake experiments and cytotoxicity are utilized to test the cell entry performance and biosafety of protein-macromolecule conjugates with different precise sequence structures; verifying in vivo circulation half-lives of protein-macromolecule conjugates with different precise sequences by using SD rats; BALB/c mice were used to test the biodistribution of protein-polymer conjugates with different precise sequence structures in mice.
The quantitative experiments in the examples below were performed in triplicate, and the results averaged, unless otherwise indicated.
Example 1
The preparation method of the protein-macromolecule conjugate with six different precise sequence structures is as follows:
1. Preparation of protein macroinitiator BSA-Macro
(1) Synthesis of small molecule initiator BMP
Firstly, carrying out esterification reaction on maleimide alcohol and bromoisobutyryl bromide to obtain a small molecular initiator EBMP with the yield of 74％,1H NMR(400MHz,CDCl3,298K)δ＝6.72(s,1H),4.32(t,J＝5.2Hz,1H),3.85(t,J＝5.2Hz,1H),1.88(s,3H).
(2) Synthesis of BSA-Macro
BSA (0.35 mM,9.0 mL) was dissolved in PBS buffer, maleimide modification initiator EBMP (126 mM,0.8 mL) was added to the solution by using the free thiol group of cysteine at position 34 on bovine serum albumin, and the mixture was left to stand at 7℃for further reaction for 24 hours. After the reaction, the reaction mixture was dialyzed once with 5mM PBS buffer (pH=7.4, 10% DMSO was added in volume fraction) using a dialysis bag having a dialysis retention of 10kDa, then twice with 20mM PBS buffer (pH=7.4), and finally lyophilized to obtain a high-purity protein macroinitiator.
2. Preparation of protein-Polymer conjugates with precise sequence Structure
In the synthesis of BSA-precise sequence multiblock polymer conjugates, for example BSA-MOHMOH, BSA-Macro (0.1 mM,1 eq.) and the first sequence of acrylate monomer M' (16 mM) were added to a solution of 5% DMF in ultrapure water. Then, the mixed system was deoxygenated (bubbling N 2 for 30 min). Next, HMTETA and CuBr were added sequentially, wherein [ BSA-Macro ]: [ CuBr ]: the molar ratio of [ HMTETA ] was 1:40:80. After 72h, a second sequence of the corresponding acrylate monomers O' (16 mM) was added, each sequence of acrylate monomers being 20 molar equivalents. After six reactions, the reaction mixtures were exposed to air and then dialyzed twice against 5mM PBS buffer (ph=7.4) containing 10% DMSO, followed by 20mM PBS buffer (ph=7.4), the target BSA-MOHMOH product was obtained after lyophilization and then stored at-20 ℃.
Example 2
Physicochemical characterization of the BSA-precise sequence multiblock polymer conjugate is as follows:
Successful synthesis of six BSA-precise sequence multiblock polymer conjugates was indicated by SEC and SDS-PAGE. FIG. 2 shows SEC results, showing that the retention time of six BSA-precise sequence multiblock polymer conjugates is shortened to a different extent compared with BSA and BSA-Macro, demonstrating the increase in the molecular weight of the conjugates. FIG. 3 shows the result of SDS-PAGE, channel 1 is a protein standard, channel 2 is unmodified BSA, channel 3 is BSA-Macro, the latter channels correspond to six BSA-precise sequence multiblock polymer conjugates, and it can be seen that dispersion bands appear in the latter six channels, demonstrating successful polymerization.
FIG. 4 shows the results of DLS, the hydrated particle sizes of unmodified BSA and six BSA-precise sequence multiblock polymer conjugates were measured by dynamic light scattering on Malvern Zetasizer Nano-zs90, and samples were diluted in PBS buffer and filtered through a 0.22 μm pore size filter before testing.
Example 3
The biological safety and the cell entry effect of the BSA-precise sequence multi-block polymer conjugate are verified as follows:
The biosafety of six BSA-precise sequence multiblock polymeric conjugates was determined by MTT: l929 and NIH-3T3 cell lines were selected, cells were cultured in DMEM containing 10% FBS, 50U/mL penicillin and 50. Mu.g/mL streptomycin for a while, and then diluted into 96-well plates (20. Mu.L, 5000 per well), negative controls (without BSA-precise sequence multiblock polymer conjugate) and blank controls (with culture broth alone) were set, and incubated at 37℃for 24 hours with 5% CO 2, 20. Mu.L/well of MTT lysate (Promega) was added, and after 3 hours the absorbance at 490nm wavelength of each well was measured with an enzyme-labeled instrument, and the degree of proliferation of cells after treatment with different samples was compared. FIG. 5 shows that the BSA-precise sequence multiblock polymer conjugate has good biosafety below 500. Mu.g/mL.
After L929 cells were cultured in DMEM containing 10% FBS, 50U/mL penicillin and 50. Mu.g/mL streptomycin for a period of time, a cell suspension (50000 cells) was inoculated in a 35mm glass bottom dish, and cyanine dye Cy5.5-modified BSA-precise sequence multiblock polymer conjugate (2. Mu.M Cy5.5) was added to the cells for culturing for 0.5h, 1h, 2h and 4h. The culture broth was then discarded, washed 2 times with PBS, then fixed with 4% paraformaldehyde, then washed with PBS, nuclei were stained with 200. Mu.L of DAPI, then washed with PBS, observed with LEICA TCS SP laser scanning confocal microscope, and excitation emission wavelengths of DAPI and Cy5.5 were set at 360/460nm and 673/707nm. For further determination, L929 was seeded in a 6-well plate, and a cyanine dye Cy5.5 modified BSA-precise sequence multiblock polymer conjugate (2. Mu.M Cy5.5) was also added to the cells, which were cultured for 30min and 4h, collected, and then analyzed by BD FACS ARIA III flow cytometer. As shown in FIG. 6, both confocal microscopy and flow cytometry showed sequence-dependent cellular behavior of BSA-precise sequence multiblock polymer conjugates.
Example 4
Biodistribution and in vivo circulation time test of the BSA-precise sequence multiblock polymer conjugate are specifically as follows:
All animal experiments below were performed under the guidelines of the university of Shanghai transportation for animal experiments. Biodistribution tests were performed using BALB/c mice randomly grouped into eight groups, 200. Mu.L of 10. Mu.g/mL Cy5.5, cy5.5 modified BSA and Cy5.5 modified BSA-precise sequence multiblock polymer conjugates were injected, respectively, and fluorescent images at 0, 0.5h, 1h, 2h, 4h and 8h were acquired using IVIS LuminaII in vivo imaging system (Perkinelmer). As a result, as shown in FIG. 7, fluorescence after injection was distributed throughout the body and concentrated mainly in the liver, and the distribution of fluorescence intensity in each tissue organ was varied with the change in sequence as time passed. The SD rats were used for in vivo circulation time testing, the rats were randomly divided into seven groups, 30mg/kg BSA and BSA-precise sequence multiblock polymer conjugates were injected respectively, and orbital blood collection was performed at different times up to 6 days. Then, the content of BSA protein in serum is detected by adopting a rat BSA detection ELISA kit, and the BSA protein is analyzed by using a two-chamber model, and the result is shown in figure 8, wherein the in vivo circulation time of the BSA-precise sequence multiblock macromolecule conjugate is enhanced relative to that of the original BSA, and the in vivo circulation time variation degree is different according to different sequences.
The foregoing is a preferred embodiment of the present invention, but the present invention should not be limited to the disclosure of this embodiment. So that equivalents and modifications will fall within the scope of the invention, all within the spirit and scope of the invention as disclosed.
Claims (10)
1. The preparation method of the protein-macromolecule conjugate with the precise sequence structure is characterized by comprising the following steps:
(1) Covalently connecting an initiator dissolved in an organic solvent with protein dissolved in a buffer solution to obtain a protein macromolecular initiator;
(2) The protein macromolecular initiator is subjected to in-situ polymerization reaction with the monomer of the first sequence; after the monomer of the first sequence is completely reacted, adding the monomer of the second sequence to perform in-situ polymerization reaction; repeating the above operation until the protein-macromolecule conjugate with accurate sequence structure is obtained.
2. The method for producing a protein-polymer conjugate having a precise sequence structure according to claim 1, wherein in the step (1), the organic solvent is dimethyl sulfoxide;
And/or the volume ratio of the protein macroinitiator to the monomer of the first sequence in dimethylformamide and ultrapure water is 1:4, carrying out in-situ polymerization reaction in the mixed solution.
3. The method for producing a protein-polymer conjugate having a precise sequence structure according to claim 1, wherein in the step (1), the initiator is one or more selected from the group consisting of halides, halogenated esters, halogenated ketones, and halogenated nitriles;
and/or the monomer is an acrylic monomer selected from chemical structures shown in any one of the following formulas (2) - (4):
and/or the protein comprises a protein and/or an antibody selected from one or more of an interferon, insulin, monoclonal antibody, colony stimulating factor, growth hormone, therapeutic vaccine and enzyme;
And/or the protein is linked to the initiator by a covalent bond, the linking site of the covalent bond being located at the N-terminus, or C-terminus, or a site remote from the active site of the protein and not interfering with the activity of the protein.
4. The method for producing a protein-polymer conjugate having a precise sequence structure according to claim 3, wherein in the step (1), the initiator is a compound represented by the formula (1):
and/or the protein is bovine serum albumin;
And/or the linking site of the covalent bond is located at cysteine 34 on BSA;
and/or the coupling ratio of the protein to the initiator is 1:1.
5. The method for preparing a protein-polymer conjugate with a precise sequence structure according to claim 1, wherein in the step (2), the in-situ polymerization is an atom transfer radical polymerization, and the polymerization is carried out for 24-72 hours at 35 ℃ under the condition of inert gas atmosphere and magnetic stirring.
6. The method for preparing a protein-polymer conjugate having a precise sequence structure according to claim 1, wherein in step (2), the in-situ polymerization is performed under the catalysis of CuBr and HMTETA; wherein:
The molar ratio of the protein macromolecular initiator to the monomers of each section of sequence to the CuBr to the HMTETA is 1:20-120:40:80.
7. The method for preparing a protein-polymer conjugate having a precise sequence structure according to claim 1, further comprising the step of pre-reacting the initiator dissolved in the organic solvent a with the protein dissolved in the buffer solution at 7 ℃ for 24 hours.
8. A protein-polymer conjugate having a precise sequence structure, characterized by being prepared by the method for preparing a protein-polymer conjugate according to any one of claims 1 to 7.
9. The protein-polymer conjugate with a precise sequence structure according to claim 8, wherein the sequence structure is at least one selected from the group consisting of BSA-MMMMMM, BSA-ooooo, BSA-HHHHHH, BSA-MMOOHH, BSA-MOHMOH, and BSA-OMOHHM, wherein m=20m ', o=20o', and h=20h ', wherein M', O ', and H' correspond to the acrylic monomers shown in any one of formulas (2) to (4) of claim 3, respectively.
10. Use of a protein-macromolecule conjugate of any one of claims 1 to 7 having a precise sequence structure for the preparation of a protein drug.
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