CN115944714A - Nano material capable of simultaneously resisting interleukin 1 and tumor necrosis factor inflammation and preparation method and application thereof - Google Patents
Nano material capable of simultaneously resisting interleukin 1 and tumor necrosis factor inflammation and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a nano material capable of resisting interleukin 1 and tumor necrosis factor inflammation simultaneously and a preparation method and application thereof. The nano material adopted by the invention has large specific surface area, is coupled with two medicines of IL-1Ra and sTNFRI at the same time, has the advantage of high medicine carrying capacity, prolongs the retention time of a protein medicine in vivo, improves the targeting property of the protein medicine at inflammation, can enhance the medicine effect of the protein medicine, obtains better treatment effect in a CIA (collagen-induced rheumatoid arthritis) mouse body, and provides a potential method and strategy for the treatment of the rheumatoid arthritis.
Description
Technical Field
The invention relates to a nano material capable of resisting interleukin 1 and tumor necrosis factor inflammation simultaneously, and a preparation method and application thereof, and belongs to the technical field of medicines.
Background
Rheumatoid Arthritis (RA) is an autoimmune disease, a chronic inflammatory disease, which can cause joint inflammation, synovial hyperplasia, pannus formation, bone and cartilage destruction, and the like. Severe patients can cause cardiovascular, pulmonary, psychological and skeletal diseases. At present, the clinical medicines for treating rheumatoid arthritis mainly comprise non-steroidal anti-inflammatory drugs, glucocorticoids and disease-improving antirheumatic drugs. The disease-improving antirheumatic drugs comprise traditional disease-improving antirheumatic drugs, immunosuppressants and biological disease-improving antirheumatic drugs. The biological agent mainly inhibits cell factors related to the progress of the rheumatoid arthritis and can effectively inhibit inflammatory reaction in the pathogenesis process of the rheumatoid arthritis.
During the attack of rheumatoid arthritis, the content of interleukin 1 (IL-1) in joints is obviously increased, and further local inflammatory reaction is triggered. The interleukin 1 receptor antagonist (IL 1 Ra) is used for inhibiting the combination of the IL-1 receptor and the IL-1, so that the inflammatory reaction caused by the IL-1 can be inhibited, and the disease development can be effectively slowed down. Tumor necrosis factor-alpha (TNF- α) is a multifunctional cytokine that plays a key role in rheumatoid arthritis. In the process of rheumatoid arthritis development, TNF-alpha secreted by type 1 helper T cells and macrophages activates synovial fibroblasts, promotes epidermal hyperplasia and recruits inflammatory cells. TNF-alpha inhibitors are biological DMARDs which are well-documented and widely used for treating RA.
However, some patients may develop severe tolerance to a single biologic, particularly TNF, IL-1 antibody therapy. And the free IL-1Ra and sTNFRI have short half-life in vivo, short blood circulation time, low enrichment in inflammatory joints and poor curative effect.
Nano-drugs generally use nano-particles as carriers to load various drugs. The nano material is not only convenient to synthesize, but also has good degradability in vivo. Polyethylene glycol (PEG) has good water solubility and biocompatibility, and is a synthetic polymer material with the lowest level of protein and cell adsorption known at present. PEG can be directly discharged out of the body through metabolism, and when the PEG-modified polymer material is applied to drug-loaded microspheres, micelles and the like, the PEG-modified polymer material can be effectively prevented from being phagocytized by an RES system, so that long circulation in the body is realized. polylactic-co-Glycolic Acid (PLGA) is formed by random polymerization of two monomers, namely Lactic Acid (LA) and Glycolic Acid (GA), and is a degradable functional polymer organic compound. The products after PLGA hydrolysis are lactic acid and glycolic acid, which can participate in human metabolism, and finally form carbon dioxide and water to be discharged out of the body. PLGA has been recognized by the Food and Drug Administration (FDA) as having good biocompatibility and safety and is widely used in human clinical medical research.
There are researchers using pegylated soluble tumor necrosis factor receptor type I (PEG-sTNFRI) for the treatment of chronic inflammatory diseases. And their clinical phase I/II and early phase III data indicate that PEG-sTNFRI is non-immunogenic, and weekly use of the drug can relieve joint pain and joint swelling in patients with rheumatoid arthritis. Other clinical trials of PEG-sTNFRI are underway to determine optimal dose and time and to further evaluate the efficacy, safety and potential immunogenicity of PEG-sTNFRI.
Although many strategies for the transport of proteins have been successfully designed to date, they are often limited by reduced protein activity, and therefore the effect of biomaterial fusion on receptor binding affinity should be carefully considered in the design of new therapies. At present, no patent or literature reports exist at home and abroad about products and methods for integrating IL-1Ra and sTNFRI.
Disclosure of Invention
In order to solve the technical problems, the invention provides a nano material simultaneously connecting IL-1Ra and sTNFRI, which enhances the drug effect and reduces the tolerance of patients.
The first purpose of the invention is to provide a nano material capable of resisting interleukin 1 and tumor necrosis factor inflammation simultaneously, wherein the nano material is obtained by connecting human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI and human interleukin 1 receptor antagonist hs-IL1Ra to PLGA-PEG-NHS nano particles.
Further, the human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI and the human interleukin 1 receptor antagonist hs-IL1Ra are connected with the PLGA-PEG-NHS nano-particles through ester cross-linking reaction.
Furthermore, the amino acid sequence of the human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI is shown in SEQ ID NO. 1.
LVPHLGDREKRDSVCPQGKYIHPQNNSICCTKCHKGTYLYNDCPGPGQDTDCRECESGSFTASENHLRHCLSCSKCRKEMGQVEISSCTVDRDTVCGCRKNQYRHYWSENLFQCFNCSLCLNGTVHLSCQEKQNTVCTCHAGFFLRENECVSCSNCKKSLECTKLCLPQIENVKGTEDSGTT
Furthermore, the amino acid sequence of the human interleukin 1 receptor antagonist hs-IL1Ra is shown in SEQ ID NO. 2. RPSGRKSSKMQAFFRIWNQKTFYLRNQLVAGYLQGPNVNLEEKIDVPEPHALFLGIHGKMCLVKSDEFLQLEAVNITDLSENRKQDKRAFRFAFIRSDPTTSFEACPGWFLCTAMEADQPVSLTNMEGVMVTKFYFQEDEE
The second purpose of the invention is to provide a preparation method of the nanometer material, which comprises the following steps:
s1, dissolving PLGA-PEG-NHS powder in an organic solvent, dialyzing in a buffer solution, and removing the organic solvent after dialysis to obtain a PLGA-PEG-NHS nanoparticle solution;
and S2, adding human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI and human interleukin 1 receptor antagonist hs-IL1Ra into the PLGA-PEG-NHS nano-particle solution obtained in the step S1 to perform ester crosslinking reaction, and performing ultrafiltration and centrifugation to remove unbound protein after the reaction to obtain the nano-material.
Further, in the S2 step, the concentration of PLGA-PEG-NHS nanoparticles in the PLGA-PEG-NHS nanoparticle solution is 10-20 mg/mL.
Further, in the S2 step, the human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI is added according to the final concentration of 1-2 mg/mL, and the human interleukin 1 receptor antagonist hs-IL1Ra is added according to the final concentration of 1-2 mg/mL.
Further, the organic solvent is one or more of tetrahydrofuran, DMSO, dichloromethane and acetone.
Further, the buffer is HEPES buffer, phosphate, carbonate-bicarbonate or borate buffer.
Further, the ester crosslinking reaction is carried out in a buffer solution with the pH value of 7.2-8.5 for 0.5-4 hours at the temperature of 0-30 ℃.
The third purpose of the invention is to provide the application of the nano material in preparing the medicine for treating rheumatoid arthritis.
The invention has the beneficial effects that:
the nano material adopted by the invention has the advantages of large specific surface area, high drug loading capacity due to the coupling of two drugs of IL-1Ra and sTNFRI, prolonged retention time of protein drugs in vivo, improved targeting property of the protein drugs at inflammation, enhanced drug effect of the protein drugs and reduced tolerance of patients. Cell experiments prove that the cross-linking reaction involved in the invention does not destroy the activity of proteins sTNFRI and IL1Ra, so that the nanoparticle prepared by the method can maintain the biological activity of binding sTNFRI and TNF-alpha and the biological activity of binding IL1Ra and IL-1 beta.
Drawings
FIG. 1 is the construction of the expression vector of the human interleukin 1 receptor antagonist (hs-IL 1 Ra) gene; A. amplifying a target gene hs-IL1Ra; B. carrying out enzyme digestion identification on the recombinant plasmid; m is DNA marker;
FIG. 2 is the construction of the expression vector of human soluble tumor necrosis factor receptor I extracellular domain (hs-sTNFRI) gene; A. amplifying a target gene hs-sTNFRI; B. carrying out enzyme digestion identification on the recombinant plasmid; m is DNA marker;
FIG. 3 is an electrophoretogram after induction expression of recombinant protein; A. electrophoresis picture after induction expression of recombinant protein hs-IL1Ra; B. electrophoresis chart of recombinant protein hs-sTNFRI after induction expression; 1,3, crushing a supernatant without inducing recombinant bacteria; 2,4, crushing the supernatant of the induced recombinant bacteria; m is protein molecule standard marker;
FIG. 4 shows the protein electrophoresis results and immunoblot analysis of recombinant protein hs-IL1Ra gradient eluents; m is protein molecule standard marker; s, cell lysis supernatant; FF is a permeate;
FIG. 5 shows the results of protein electrophoresis and immunoblot analysis of the recombinant protein hs-sTNFRI gradient eluate; m is protein molecule standard marker; s, cell lysis supernatant; FF, permeate;
FIG. 6 is a standard curve of enterokinase cleavage fusion protein and endotoxin detection; a: cleaving 0.5mg of the hs-IL1Ra protein with 1.5U enterokinase at 4 ℃; b, cutting 0.5mg of protein hs-sTNFRI by 1.5U of enterokinase at 4 ℃;1,3: collecting samples for electrophoresis before protease digestion; 2,4: collecting samples for electrophoresis after the protease digestion for 48 hours; c: endotoxin standard curve;
FIG. 7 shows the measurement of the biological activity of the purified protein in vitro; cck-8 measures the proliferation level of thymocytes to reflect the biological activity of hs-IL1Ra; cck-8 measures the proliferation level of L929 to reflect the biological activity of hs-sTNFRI;
FIG. 8 is the characterization of the nanoparticles PLGA-PEG-NHS and the detection of biocompatibility; the particle size and the particle size distribution measured by DLS; b, TEM image of PLGA-PEG-NHS; c, cell activity experiments after incubation of the nanoparticles with different concentrations and L929; d, detecting the particle size of the nano particles at different time;
FIG. 9 is a biological activity study of hs-IL1Ra and hs-sTNFRI loaded nanoparticles; a, the connection efficiency of the nanoparticles and the proteins with different concentrations; b, detecting the biological activity of the hs-IL1Ra nano-particles; c, detecting the biological activity of the hs-sTNFRI nano-particles;
FIG. 10 is a study of the in vivo retention experiment of free protein or protein nanocomplexes; a, performing fluorescence imaging on legs of mice after injecting free protein or protein nano-composite labeled by Cy5.5 into joint cavities of the mice; b, fluorescence semi-quantitative analysis of A picture;
FIG. 11 is a study of the in vivo retention experiment of free protein or protein nanocomplexes; a, fluorescence imaging pictures of different time points after the tail of the mouse is injected with drugs in a vein; b, fluorescence semi-quantitative analysis of A picture;
FIG. 12 is a preliminary exploration of protein nanocomposite treatment for arthritic mice; a, an experimental flow chart; b, representative pictures of hind paws of different groups of mice after treatment; and C, mouse paw arthritis clinical score.
Detailed Description
The present invention is further described below in conjunction with specific examples to enable those skilled in the art to better understand the present invention and to practice it, but the examples are not intended to limit the present invention.
Example 1: constructing human soluble tumor necrosis factor receptor I extracellular segment (hs-sTNFRI) gene expression vector and human interleukin 1 receptor antagonist (hs-IL 1 Ra) gene expression vector
1.1 primer design and PCR
The extracellular domain of human tumor necrosis factor receptor (hs-sTNFRI) found by UNIPROT is (22 AA-211 AA), and the calculated base sequence corresponding to NCBI database is:
CTGGTCCCTCACCTAGGGGACAGGGAGAAGAGAGATAGTGTGTGTCCCCAAGGAAAATATATCCACCCTCAAAATAATTCGATTTGCTGTACCAAGTGCCACAAAGGAACCTACTTGTACAATGACTGTCCAGGCCCGGGGCAGGATACGGACTGCAGGGAGTGTGAGAGCGGCTCCTTCACCGCTTCAGAAAACCACCTCAGACACTGCCTCAGCTGCTCCAAATGCCGAAAGGAAATGGGTCAGGTGGAGATCTCTTCTTGCACAGTGGACCGGGACACCGTGTGTGGCTGCAGGAAGAACCAGTACCGGCATTATTGGAGTGAAAACCTTTTCCAGTGCTTCAATTGCAGCCTCTGCCTCAATGGGACCGTGCACCTCTCCTGCCAGGAGAAACAGAACACCGTGTGCACCTGCCATGCAGGTTTCTTTCTAAGAGAAAACGAGTGTGTCTCCTGTAGTAACTGTAAGAAAAGCCTGGAGTGCACGAAGTTGTGCCTACCCCAGATTGAGAATGTTAAGGGCACTGAGGACTCAGGCACCACA(SEQ ID NO.3)
the length is 546bp. Selecting EcoRI and HindIII on a plasmid vector pET-32a as restriction enzyme cutting sites of restriction enzyme, and designing a primer sequence as follows:
F:5’-CGGAATTCGACGACGACGACAAGCGCCCTTCTGGGAAAAGACC-3’；
R:5’-CCCAAGCTTCTATTGGTCTTCCTGGAAGTAG-3’。
the base sequence of human interleukin 1 receptor antagonist (hs-IL 1 Ra) found in NCBI database, the sequence of CDS region except signal peptide is the target sequence, the base sequence corresponding to NCBI database is:
CGACCCTCTGGGAGAAAATCCAGCAAGATGCAAGCCTTCAGAATCTGGGATGTTAACCAGAAGACCTTCTATCTGAGGAACAACCAACTAGTTGCTGGATACTTGCAAGGACCAAATGTCAATTTAGAAGAAAAGATAGATGTGGTACCCATTGAGCCTCATGCTCTGTTCTTGGGAATCCATGGAGGGAAGATGTGCCTGTCCTGTGTCAAGTCTGGTGATGAGACCAGACTCCAGCTGGAGGCAGTTAACATCACTGACCTGAGCGAGAACAGAAAGCAGGACAAGCGCTTCGCCTTCATCCGCTCAGACAGTGGCCCCACCACCAGTTTTGAGTCTGCCGCCTGCCCCGGTTGGTTCCTCTGCACAGCGATGGAAGCTGACCAGCCCGTCAGCCTCACCAATATGCCTGACGAAGGCGTCATGGTCACCAAATTCTACTTCCAGGAGGACGAGTAG(SEQ IDNO.4)
the total length is 459bp. Selecting EcoRI and HindIII on a plasmid vector pET-28a as restriction enzyme cutting sites of restriction enzyme, and designing a primer sequence as follows:
F:5’-CGGAATTCGACGACGACGACAAGCGACCCTCTGGGAGAAAATCCA-3’；
R:5’-CCCAAGCTTCTACTCGTCCTCCTGGAAGTAG-3’。
since the objective fragment is ligated to a plasmid vector and the promoter for gene transcription and the 6 XHis tag for protein purification are both on the plasmid upstream of the objective fragment, a plasmid portion is ligated in front of the objective fragment in addition to the objective gene, and therefore an enterokinase cleavage site (GACGACGACGACAAG) is preset in designing an upstream primer for the purpose of excising an excess portion at the time of later purification.
1.2 double digestion of target fragment and vector and ligation, transformation and verification of recombinant plasmid
1) And carrying out double enzyme digestion on the recombinant plasmid and the target fragment.
2) Connecting the plasmids of the target fragments which are cut by enzyme, transforming the plasmids into escherichia coli DH5 alpha, and extracting the plasmids to obtain the needed recombinant plasmids. After the recombinant plasmid is subjected to double digestion, the plasmid showing successful digestion is sent to a company for sequence sequencing identification. The sequencing results of the companies were aligned, showing that the sequencing was correct. The success of the recombinant plasmid construction is shown. The results are shown in FIGS. 1 and 2.
Example 2: induction expression and purification of recombinant proteins hs-IL1Ra and hs-sTNFRI
1) The recombinant expression vector with correct sequencing was transformed into e.coli BL21 (DE 3).
2) Single clones were picked for inducible expression with a final concentration of 0.1mM IPTG. The expression results are shown in FIG. 3. Prokaryotic expression offers the possibility of mass production. As can be seen from the figure, the recombinant proteins hs-IL1Ra and hs-sTNFRI were successfully expressed.
3) After the bacteria are broken by ultrasound, ni columns are used for protein affinity chromatography, and purer recombinant protein is obtained by a step-by-step elution mode. The electrophoresis pattern of the affinity purification SDS-PAGE and the protein Western are shown in FIGS. 4 and 5.
Example 3: enterokinase enzyme digestion of recombinant proteins hs-IL1Ra and hs-sTNFRI and detection of endotoxin content
1) The excess recombinant protein was excised using enterokinase, and 0.5mg of the desired protein was cleaved with 1.5U enterokinase at 4 ℃ for 48 hours to maintain the protein activity as much as possible. Then the Ni column is used again for protein affinity chromatography to obtain the target protein. The cleavage results are shown in FIGS. 6A and B.
2) The expressed and purified protein needs to be detected with endotoxin content before being subjected to subsequent cell experiments. Recombinant factor C endotoxin detection kits were used. A standard curve is drawn according to the endotoxin standard substance, the absorbance of the recombinant protein (1 mu g/ml) to be detected is substituted into the standard curve (C in figure 6), and the endotoxin content of the two purified proteins is respectively calculated to be 0.58 EU/mu g and 0.675 EU/mu g. The detection result meets the endotoxin standard of cell experiments.
Example 4: detection of biological activity of target proteins hs-IL1Ra and hs-sTNFRI in vitro
Method for determining the in vitro activity of hs-IL1 Ra: conA stimulated mouse thymocytes were assayed for hs-IL1Ra biological activity. Mouse thymocytes express IL-1R under stimulation, IL-1 beta synergizes with ConA to promote proliferation of T cells in the presence of IL-1 beta, and hs-IL1Ra competitively binds with IL-1R to antagonize proliferation of cells, and biological activity of hs-IL1Ra detected by IL-1 is reflected according to proliferation level of cells after administration. Method for determining the in vitro activity of hs-sTNFRI: and (3) detecting the inhibition effect of hs-sTNFRI on TNF-alpha killer cells by taking L929 cells as target cells. The experimental result is shown in A in figure 7, the purified protein hs-IL1Ra can obviously inhibit the proliferation of thymocytes, and B in figure 7 shows that the purified protein hs-sTNFRI can obviously save the killing of TNF-alpha to L929.
Example 5: preparation and characterization of nano-particle PLGA-PEG-NHS and detection of biocompatibility
Principle of nanoparticle PLGA-PEG-NHS connexin: NHS esters are reactive groups formed by carbodiimide activation of carboxylate molecules. NHS ester activated cross-linkers react with primary amines under physiological to slightly basic conditions (pH 7.2 to 9) to form stable amide bonds. The reaction releases N-hydroxysuccinimide (NHS). Because PLGA is insoluble in water and easily soluble in organic solvents, 20mg of PLGA-PEG-NHS freeze-dried powder purchased by a company is weighed and dissolved in 200 mu L of tetrahydrofuran, and is fully dissolved for about 1 hour by using a magnetic stirrer and then is dropwise added into 800 mu L of double distilled water for dilution. Since the NHS-ester cross-linking reaction is most commonly carried out at room temperature or 4 ℃ in phosphate, carbonate-bicarbonate, HEPES or borate buffer at pH 7.2-8.5, the nano solution is packed into dialysis bags and dialyzed in HEPES buffer overnight to obtain a nano particle concentration of 20mg/mL.
The particle size measured by dynamic laser light scattering (DLS) is shown in A of FIG. 8. B in FIG. 8 is TEM image of PLGA-PEG-NHS. Since the main obstacle of protein as a therapeutic drug is the toxicity of the transport material, after the nanoparticles are prepared, the toxicity of the nanoparticles with different concentrations to L929 is firstly detected, so as to select the appropriate material concentration for subsequent experiments. C in FIG. 8 is the toxicity analysis result of L929 with PLGA-PEG-NHS at different concentrations, and it can be seen from C in FIG. 8 that the cell activity was not significantly different from that of the control group without the material even when the nanoparticles were used at a concentration of 5.4 mg/mL. This result indicates that the nanoparticle has little cytotoxicity and good biocompatibility, which indicates that the particle is safe as a protein transport carrier. In fig. 8, D is the measurement of the particle size of the nanoparticles at different times, and the results show that there is no significant difference in the particle size of the nanoparticles over a certain period of time, which indicates that the particles are relatively stable as protein transport carriers.
Example 6: biological activity study of nanoparticles loaded with human soluble tumor necrosis factor receptor I extracellular domain (hs-sTNFRI) protein and human interleukin 1 protein receptor antagonist (hs-IL 1 Ra)
In order to ensure the efficiency of protein-nanoparticle connection under certain conditions, we first use nanoparticles and proteins with different concentrations to connect, and the result is shown in fig. 9A, the connection rate of nanoparticles and proteins with 15mg/mL is the highest, and the nanoparticles required in the subsequent experiments all use this concentration. Next, we prepared hs-IL1 Ra-loaded Nanoparticles (hs-IL 1Ra Nanoparticles), hs-sTNFRI-loaded Nanoparticles (hs-sTNFRI Nanoparticles), and Nanoparticles loaded with both hs-IL1Ra and hs-sTNFRI proteins (hs-IL 1Ra + hs-sTNFRI Nanoparticles). The loading method comprises the following steps: and (2) putting 200 mu L of PLGA-PEG-NHS nanoparticle solution (15 mg/mL) into an EP tube, and respectively adding 100 mu L of purified human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI (1.5 mg/mL) and 100 mu L of human interleukin 1 receptor antagonist hs-IL1Ra (1.5 mg/mL). Lightly blowing and uniformly mixing by using a liquid transfer gun, and then putting the mixture into a refrigerator and shaking table for reaction overnight. The next day the reaction product was added to an ultrafiltration centrifuge tube containing 4mL of HEPES buffer and centrifuged to remove unbound protein. Gently suck about 400. Mu.L of the solution above the ultrafiltration tube and store in a refrigerator at 4 ℃. And (3) detecting the residual protein concentration by using a kit for detecting the protein concentration to finally obtain a nanoparticle (7-7.5 mg/mL) solution which is simultaneously connected with two proteins (350-400 mu g/mL).
Then, cell experiments are carried out to detect the activity of the polypeptide.
1) Method for determining hs-IL1Ra in vitro Activity: conA stimulated thymocytes for hs-IL1Ra biological activity. 6-8 week C57 mice, thymus were prepared as cell suspension with cell density of 1.5 x 10 6 Into 96-well plates. ConA (3. Mu.g/mL), IL-1. Beta. (1 ng/mL) were added. Then, hs-IL1Ra (5. Mu.g/mL), hs-IL1Ra nanoparticules (5. Mu.g/mL), hs-IL1Ra + hs-sTNFRRI nanoparticules (5. Mu.g/mL), BSA nanoparticules (5. Mu.g/mL) were added, respectively. After 72 hours cck-8 was added and the absorbance at 450nm was measured.
2) Method for determining hs-sTNFRI in vitro activity: l929 by 1 x 10 4 The cell density of (2) was inoculated into a 96-well plate, and TNF-. Alpha. (150 ng/mL) was added at 24 hours later, followed by hs-sTNFRRI (16. Mu.g/mL), hs-sTNFRRI Nanoparticles (16. Mu.g/mL), hs-IL1Ra + hs-sTNFRRI Nanoparticles (16. Mu.g/mL), BSA Nanoparticles (16. Mu.g/mL), respectively. After 24 hours cck8 was added and the absorbance at 450nm was measured.
The results of the experiment are shown in FIG. 9. Panel B shows that the inhibitory effect of hs-IL1Ra loaded nanoparticles is not much different than hs-IL1Ra protein itself, indicating that modification etc. has no effect on the original biological activity of hs-IL1Ra during nanoparticle formation. Meanwhile, as a negative control, BSA-loaded nanoparticles did not inhibit IL-1 β proliferation on thymocytes. Panel C shows that the hs-sTNFRI loaded nanoparticles, although slightly lower than the hs-sTNFRI protein itself, can also significantly rescue the killing of L929 by TNF-alpha. Also as a negative control, BSA-loaded nanoparticles did not inhibit killing of L929 by TNF- α. In summary, the nanostructure can be used as a material for protein transport carriers. The biological activity of the two protein nanocomplexes has been clarified above, and then we have performed the same cell experiments after simultaneously attaching the two proteins to the nanoparticles. The results of the experiment are shown in FIG. 9, panel B, which shows that the inhibition of hs-IL1Ra and sTNFRI loaded nanoparticles is not much different than the hs-IL1Ra protein itself. Panel C shows that nanoparticles loaded with hs-IL1Ra and sTNFRI, although slightly lower than the hs-sTNFRI protein, can also significantly rescue the killing of L929 by TNF-alpha. In conclusion, the nanoparticles simultaneously compounding two proteins also have biological activity.
Example 7: in vivo retention experiments of protein nanocomposites
Experimental C57BL/6J (8-10 weeks old) mice were selected and 5 μ g Cy5.5 labeled free protein hs-IL1Ra + sTNFRI or nanoparticles loaded with hs-IL1Ra and hs-sTNFRI in a volume of 20 μ L were injected into the right knee after deep anesthesia with 4% chloral hydrate. Mice were imaged In Vivo at predetermined time points using an Imaging system (IVIS Spectrum In Vivo Imaging system, perkinElmer, USA).
Protein-loaded nanoparticles are used to maintain the activity of the protein and to increase the residence time of the protein at the desired site, if the therapeutic effect is desired. Therefore, we labeled the protein with Cy5.5 to monitor the retention of the protein-loaded nanoparticle and the protein, respectively, in vivo. The results are shown in fig. 10, where the entire imaging process lasted 8 days. Upon injection on the first Day, we can see, in conjunction with Day 0 in the a, B plots, that the initial fluorescence intensities of the loaded protein nanoparticle group and the free protein group are the same, which is the basic condition that needs to be met for the experiment to proceed. After one day, the fluorescence intensity of the free protein group decreased, and the fluorescence intensity of the protein-loaded nanoparticle group, although also decreased compared to the initial injection, was significantly higher than that of the free protein group (. About.. P < 0.001). Differences between the two groups were still evident at day 4 and day 8. These results show that after the protein enters into the body, the protein of the free proteome is cleared away quickly due to the metabolic clearance in the body, while the protein half-life period is prolonged obviously due to the protective effect of the nanoparticles on the protein, so that the protein stays in the body for a longer time.
Example 8: establishment of Zymosan Induced Arthritis (ZIA) mouse model and targeting research of protein nano-composite
In order to establish a ZIA mouse model, a certain amount of zymosan powder is weighed according to a plan, then 25mg/mL zymosan solution is prepared by PBS, the zymosan is boiled by heating and then continuously boiled for 5-10 minutes to form emulsion, and then the emulsion can be used after being subjected to ultrasonic treatment for 10 minutes. C57BL/6 mice left leg joint cavity injection 20 u L zymosan emulsion, right leg injection PBS control. After 24 hours, obvious swelling of knee joints of the mice can be seen, and the ZIA mouse model is established for experiments. To investigate the passive targeting ability of protein nanocomplexes at the site of inflammation in ZIA mice, 24 hours after the ZIA mouse model was established, the mice were divided into two groups (4 per group), and 200 μ L of cy 5.5-labeled protein solution or protein nanocomplexes of the same protein mass (5 μ g) were injected via tail vein and the mice were imaged in vivo at predetermined time points.
In order to study the targeting of the human protein nanocomposite to the inflammatory site, we established the above-mentioned ZIA model in the left leg joint of mice. And then, respectively injecting free protein and protein nano-complex marked by Cy5.5 into a mouse body through tail vein, and observing the change of the distribution of the medicament in main organs and inflammation parts of the mouse along with time through in vivo fluorescence imaging. The results show that protein drug can be rapidly enriched in the mouse left leg RA joint, with the average enrichment peaking at 8h and subsequently decreasing (fig. 11B). Within 32 hours of observation, the fluorescence value of the RA joint of the left leg of the free protein group is obviously lower, the enrichment of the protein nano-complex in the RA joint is about 2 times that of the RA joint of the free protein group, the retention time is longer, and the high fluorescence intensity is still maintained for 32 hours. This shows that the nanoparticles loaded with proteins hs-IL1Ra and hs-sTNFRI have stronger capability of passively targeting inflammatory sites in vivo, and lay the foundation for further exerting the anti-inflammatory effect of the proteins.
Example 9: establishment of mouse collagen-induced arthritis (CIA) model
Mouse collagen-induced arthritis (CIA) is one of the commonly used models during clinical trials, and a strategy for constructing collagen-induced arthritis: the bovine type II collagen solution was mixed with Freund's complete adjuvant 1 to prepare an emulsifier, and 100. Mu.g of collagen was injected subcutaneously into the tail of DBA/1 mouse. After 21 days, the tail of the mice was injected subcutaneously with 100. Mu.g of an emulsifier mixed with collagen and Freund's incomplete adjuvant 1, and then the mice were observed daily for the progress of arthritis.
After the CIA model is successfully constructed, the progress of arthritis is observed every day and scored according to the following table. In the mouse CIA model, four paws of the mouse can be affected, and the severity of arthritis of the whole mouse is obtained by adding scores of the four paws (total score of 16).
TABLE 1
| Severity scoring | |
| 0 | Evidence of no erythema and |
| 1 | Erythema and mild swelling localized to the midfoot or |
| 2 | Erythema and mild swelling spread from the ankle to the |
| 3 | Erythema and moderate swelling spread from the ankle to the |
| 4 | Erythema and severe swelling including ankle, foot and toe |
CIA mice received different treatments starting on day 35 after receiving the first immune stimulation, and each mouse was injected intravenously with 200 μ L of solution in the tail vein. The specific grouping is as follows: PBS, free protein (Soluble Hs-IL1Ra + sTNFRI), nanoparticles (BSA nanoparticules), protein-only nanocomplexes (Hs-IL 1Ra nanoparticules), protein-only nanocomplexes (Hs-sTNFRI nanoparticules), two protein-nanocomplexes (Hs-IL 1Ra + sTNFRI nanoparticules). The drug was administered every 5 days for a total of 5 times starting on day 35, and the flow chart is shown as a in fig. 12. After the treatment, the claws of the CIA mice of each treatment group were photographed and stored. The experimental results are shown in fig. 12B, and after the CIA model was established, the ankle, foot and toe swelling of the mice was significant. Joint scores were significantly reduced after protein drug treatment (. P < 0.05), while CIA mouse joint scores in nanoparticle-treated groups that simultaneously complex both proteins were significantly reduced compared to the protein drug group (. P < 0.05). And the therapeutic effect of the nanoparticle which simultaneously compounds two proteins is obviously better than that of a protein nano-complex (p < 0.05). No significant improvement was seen in CIA mice of the control PBS group and the nano group. The results preliminarily show that the nanoparticles simultaneously compounding the two proteins have a remarkable treatment effect on the CIA mice.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Claims (10)
1. The nanometer material capable of resisting interleukin 1 and tumor necrosis factor inflammation simultaneously is prepared through connecting human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI and human interleukin 1 receptor antagonist hs-IL1Ra to PLGA-PEG-NHS nanometer particle.
2. The nanomaterial of claim 1, wherein the human soluble tumor necrosis factor receptor-I extracellular domain protein hs-sTNFRI and the human interleukin 1 receptor antagonist hs-IL1Ra are linked to PLGA-PEG-NHS nanoparticles by ester cross-linking reaction.
3. The nanomaterial of claim 1, wherein the amino acid sequence of the human soluble tumor necrosis factor receptor-I extracellular domain protein hs-sTNFRI is shown in SEQ ID NO. 1.
4. The nanomaterial of claim 1, wherein the amino acid sequence of the human interleukin 1 receptor antagonist hs-IL1Ra is shown in SEQ ID No. 2.
5. A method for preparing a nanomaterial according to any one of claims 1 to 4, comprising the steps of:
s1, dissolving PLGA-PEG-NHS powder in an organic solvent, dialyzing in a buffer solution, and removing the organic solvent after dialysis to obtain a PLGA-PEG-NHS nanoparticle solution;
and S2, adding human soluble tumor necrosis factor receptor I extracellular domain protein hs-sTNFRI and human interleukin 1 receptor antagonist hs-IL1Ra into the PLGA-PEG-NHS nano-particle solution obtained in the step S1 to perform ester cross-linking reaction, and performing ultrafiltration and centrifugation to remove unbound protein after the reaction to obtain the nano-material.
6. The method of claim 5, wherein in the S2 step, the concentration of PLGA-PEG-NHS nanoparticles in the PLGA-PEG-NHS nanoparticle solution is 10 to 20mg/mL, the human soluble TNF receptor I extracellular domain protein hs-sTNFRI is added at a final concentration of 1 to 2mg/mL, and the human interleukin 1 receptor antagonist hs-IL1Ra is added at a final concentration of 1 to 2 mg/mL.
7. The method according to claim 5, wherein the organic solvent is one or more of tetrahydrofuran, DMSO, dichloromethane, and acetone.
8. The method of claim 5, wherein the buffer is HEPES buffer, phosphate, carbonate-bicarbonate or borate buffer.
9. The method according to claim 5, wherein the ester crosslinking reaction is carried out at 0 to 30 ℃ for 0.5 to 4 hours in a buffer solution having a pH of 7.2 to 8.5.
10. Use of a nanomaterial according to any one of claims 1 to 4 in the manufacture of a medicament for the treatment of rheumatoid arthritis.
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| US20060275868A1 (en) * | 1989-09-05 | 2006-12-07 | Immunex Corporation | Fusion proteins comprising tumor necrosis factor receptor |
| CN103386114A (en) * | 2013-07-05 | 2013-11-13 | 中国人民解放军军事医学科学院野战输血研究所 | Application of artificial platelet PLAG-PEG-RCD to preparing systemic nanometer styptic for veins |
| WO2021237891A1 (en) * | 2020-05-25 | 2021-12-02 | Beijing Vdjbio Co., Ltd. | An interleukin-1 receptor antagonist and a fusion protein containing the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060275868A1 (en) * | 1989-09-05 | 2006-12-07 | Immunex Corporation | Fusion proteins comprising tumor necrosis factor receptor |
| CN103386114A (en) * | 2013-07-05 | 2013-11-13 | 中国人民解放军军事医学科学院野战输血研究所 | Application of artificial platelet PLAG-PEG-RCD to preparing systemic nanometer styptic for veins |
| WO2021237891A1 (en) * | 2020-05-25 | 2021-12-02 | Beijing Vdjbio Co., Ltd. | An interleukin-1 receptor antagonist and a fusion protein containing the same |
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| CN115944714B (en) | 2023-12-15 |
| WO2024082347A1 (en) | 2024-04-25 |
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