CN109550056B - Matrix metalloproteinase inhibitory polypeptide gold nanoparticle composition and application and preparation method thereof - Google Patents
Matrix metalloproteinase inhibitory polypeptide gold nanoparticle composition and application and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a pharmaceutical preparation and a preparation method thereof, in particular to a matrix metalloproteinase inhibitory polypeptide gold nanoparticle composition, which is characterized in that the composition is formed by connecting matrix metalloproteinase-2/9 inhibitory polypeptide and gold nanoparticles; the composition is prepared by the following steps: mixing the chloroauric acid solution and the polypeptide solution, and uniformly stirring; adding a sodium borohydride solution into the mixed solution, and stirring in an ice water bath in a dark place; and dialyzing the reaction product solution to remove the polypeptide and other byproducts which do not participate in the reaction to obtain a polypeptide nanoparticle composition solution, and freeze-drying to obtain the composition. The nanoparticles prepared by the invention have high stability and good pharmaceutical activity.
Description
Technical Field
The invention belongs to the field of pharmaceutical preparations, and relates to a polypeptide nanoparticle composition capable of inhibiting matrix metalloproteinase-2/9 and a preparation method thereof.
Background
With the development of biomedical technology, more and more polypeptide protein drugs are researched, developed and applied to clinic, and meanwhile, new challenges are brought to pharmaceutical preparations. Because of the instability and deterioration of polypeptide and protein drugs, it is a complex and arduous task to make them stable, safe and efficient pharmaceutical preparations. At present, a great deal of research is being conducted on the delivery systems of polypeptide protein drugs, and a variety of novel delivery systems for different routes of administration have been reported.
First, tumor metastasis
According to the report of the World Health Organization (WHO), tumors are the main cause of morbidity and mortality, 880 ten thousands of people die in 2015, the number of new cases is expected to increase by about 70% in 20 years in the future, and the death of nearly 1/6 is caused by the tumors in global condition. Research finds that tumor metastasis is the cause of death of 90% of tumor patients, and is a worldwide problem in clinical tumor treatment. Most tumor patients do not die from primary tumors but from metastatic tumors. Metastasis of a tumor is a multistep, multistage process of a series of factors, gene interactions, whose basic process is: 1) vessel neogenesis: the tumor cells of the primary focus are proliferated in a large quantity, and new blood vessels grow; 2) degradation of extracellular matrix: tumor cells degrade the extracellular matrix by secreting various enzymes that eliminate the barrier around the tumor, the most important of which are matrix metalloproteinases; 3) shedding of primary tumor cells: shedding of tumor cells from primary foci; 4) tumor cells infiltrate blood or lymph vessels: the shed tumor cells invade the basement membrane and then infiltrate into blood vessels or lymphatic vessels; 5) circulation and survival: a small number of tumor cells are able to survive in the circulatory system, migrating with the blood stream, lymph stream, to another distant site or organ; 6) the tumor cells invaded and transferred are adhered to the endothelium and the basement membrane of a target organ to form a micro metastasis, the cells proliferate and generate new blood vessels, and further secondary metastasis tumors are formed, and the tumor cells can be invaded and transferred again.
The extracellular matrix plays a key role in tumor cell invasion and metastasis, and the balance of tumor cells and the environment outside the matrix of host tissues where the tumor cells live is a key factor in tumor metastasis. In the process of participating in the invasion and metastasis regulation of tumor cells, proteolytic enzymes (proteases) play an important role, especially extracellular proteolytic enzymes, because they are directly involved in the enzymolysis between tumor cells and surrounding stromal cells and stromal components, changing their inherent equilibrium state, resulting in the deterioration of tumor cells, such as metalloprotease, urokinase, Furin protease, etc. Among them, Matrix Metalloproteinases (MMPs) are important enzymes that degrade extracellular matrix, are highly expressed in various tumors, are closely related to invasion and metastasis of tumors, affect proliferation, migration and invasion of tumors through various mechanisms [5], and are a great research hotspot in tumor treatment. In view of this, the search and development of effective drugs with anti-tumor metastasis effects would be an important strategy to reduce the mortality of tumor diseases.
Tumor metastasis is a multi-step complex process, and matrix metalloproteases play a very important role in the process and are one of the most important regulatory molecules in the process of tumor invasion and metastasis. If the target is the matrix metalloproteinase inhibitor (MMPIs) capable of inhibiting tumor metastasis, the matrix metalloproteinase inhibitor has great social significance and economic value.
II, matrix metalloprotease
1 matrix Metalloproteinase
MMPs are a class of zinc ion and calcium ion dependent proteolytic enzymes that catalyze the hydrolysis of extracellular matrices. MMPs and tissue metalloproteinase inhibitors (TIMPs) together regulate the degradation of extracellular matrix, and imbalance of the regulation mechanism can excessively degrade the extracellular matrix, resulting in a series of diseases such as emphysema, multiple sclerosis, arthritis, periodontitis, invasion and metastasis of tumors, and the like. The research of traditional antitumor drugs emphasizes that the drugs are used for directly killing tumor cells, and the metastasis of tumors is the main cause of death of tumor patients. Degradation of extracellular matrix and basement membrane is a key link in tumor invasion and metastasis, and MMPs are important enzymes known to degrade extracellular matrix at present. Therefore, MMPs are a new target for tumor treatment, and the development of drugs capable of inhibiting the generation and activity of MMPs is beneficial to inhibiting the progress of tumors and improving the tumor treatment effect.
MMPs are a class of enzymes that degrade the extracellular matrix and require activation by zinc and calcium ions to exert their activity. In 1962, Gross discovered the enzyme for the first time in the tadpole tail degeneration process, and human beings discovered more than 20 MMPs. Based on the substrate specificity and structural features of MMPs, MMPs can be divided into 6 classes: collagenase, gelatinase, matrix degrading enzyme, stromelysin, membrane type matrix metalloproteinase (MT-MMP), and other MMPs. The signal peptide region, propeptide region, catalytically active region, hinge region and carboxy-terminal region together constitute the basic structure of MMPs. The signal peptide is positioned at the amino terminal of the protease, and the newly generated peptide enters cytoplasm through the guide of the signal peptide sequence; the propeptide domain contains a cysteine switch, which can bind to zinc, so that the zymogen of the MMPs cannot be activated; the catalytic domain has 3 histidines bound to the zinc ion at its active center; a proline-rich hinge region connects the catalytically active region to the carboxy-terminal region, which recognizes specific substrates or ligands, and binds to endogenous inhibitors, helping to localize subcellular localization and induce activation or inhibition of various MMPs. In addition to these 5 basic structures, the structures of mammalian MMPs continue to evolve, and some MMPs have other specific structures.
2 matrix Metalloproteinase 2/9
Matrix metalloprotease MMPs are proteases which degrade almost all extracellular matrix and are most closely related to tumor metastasis, wherein MMP-2 is also called gelatinase-A, MMP-9 is also called gelatinase-B, and the catalytic domains of MMP-2 and MMP-9 contain collagen binding regions and can specifically degrade collagen type IV which is the main component of Basement Membrane (BM) and extracellular matrix (ECM), so that the MMPs are considered as the most important matrix metalloproteases in tumor invasion and metastasis. MMP-2 and MMP-9 are synthesized, are secreted from the inside to the outside of the cell in an inactive form, and are cleaved by a specific protease to generate activity. Wherein, when MMP-2 is in an inactive form, the molecular weight is 72kD, and the molecular weight after activation is 66 kD; MMP-9 has molecular weight of 92kD and 83kD respectively when it is inactive and active. The research reports that the two compounds can specifically degrade the main component type IV collagen of extracellular matrix and also can up-regulate the expression of angiogenesis-related factors, thereby participating in the angiogenesis of tumor tissues and further promoting the metastasis of tumors.
3 matrix metalloproteinase inhibitors
The development of MMPIs has been particularly rapid since the 90s of the 20 th century. To date, scientists have designed and synthesized a large number of MMPIs, some of which have entered clinical trials. MMPIs can be classified into 3 classes according to source: 1) endogenous inhibitors, i.e., inhibitors of MMPs present in the body, such as TIMPs in humans; 2) artificially synthesized or expressed inhibitors including peptide-based inhibitors of MMPs, bisphosphates, monoclonal antibodies, and the like; 3) the inhibitor separated from natural products, such as tetracycline and its derivatives, bryostatin, neovastat, and some flavonoids.
The design and synthesis of MMPIs has been known for about 20 years. To date, a number of MMPIs have also been studied as clinical candidates for anti-tumor, but due to lack of selectivity, the clinical efficacy of some MMPIs is poor. Early MMPIs mostly exerted MMP inhibitory effect by chelating zinc ion chelating groups in their own structures with zinc ions of MMPs, and this structure of zinc ion chelating groups itself was the reason for poor MMPIs selectivity. These MMPIs inhibit MMPs at the tumor site, as well as other MMPs that exert physiological effects. In addition, different types of tumors, and the same tumor at different stages of development, have different MMPs, and therefore, identification of members of the MMP family in a tumor is critical to the implementation of precise therapy. In recent years, with the intensive research on MMPIs, people find more and more MMPIs which are non-zinc ion chelating agents, and also MMPIs which can specifically inhibit a certain MMP, and modification is carried out on the MMPIs, so that high-selectivity MMPIs are expected to be obtained. It is believed that with the continued understanding and research on MMPs, the success rate of the design and development of highly selective anti-tumor MMPIs will increase dramatically.
III, matrix metalloproteinase inhibitors
In view of the critical role of MMPs in physiological and pathological processes, research and development of matrix metalloproteinase inhibitors is essential. MMPIs have developed particularly rapidly since the last century of 1990 s. To date, scientists have designed and synthesized a large number of MMPIs, and some have entered clinical trials. Inhibitors are classified into three classes according to the source of MMPIs: a first endogenous inhibitor, MMP inhibitory factor present in the body, such as TIMP in the human body; artificially synthesized or expressed inhibitors including peptide-based MMP inhibitors, bisphosphates, monoclonal antibodies and the like; and the inhibitor is separated from natural products, such as tetracycline and derivatives thereof, bryostatin, Neovastat, flavonoid compounds and the like.
In recent years, the subject group carries out a series of researches on the relation between MMP and tumor invasion and metastasis, and designs and synthesizes a series of matrix metalloproteinase inhibitory polypeptides.
Because the polypeptide has the advantages of good biocompatibility, easy absorption by human bodies and the like, more and more scientists develop polypeptide medicaments, but because of the first pass effect of human body absorption, poor stability of the polypeptide under physiological conditions and the like, the polypeptide is not generally used for oral administration, usually adopts a multi-injection mode for administration, and brings inconvenience to patients in the actual clinical application process. In recent years, with the development of nanotechnology, the fusion of nanotechnology and biotechnology has led to significant progress in nanotechnology, and nanoparticles can be used in various ways such as oral administration, injection, pulmonary inhalation, etc. as carriers. For polypeptide drugs that are administered orally or by pulmonary inhalation, improving the mucoadhesive properties of the nanoparticles clearly contributes to improved efficacy and prolonged duration of action.
Among a plurality of nano materials, the gold nanoparticle AuNP has the advantages of biological safety, uniform physicochemical property, easy surface modification and the like, so that the gold nanoparticle AuNP is widely applied to the research of a nano drug system. The nano gold is used as a mature nano carrier in the nano particles, and can directionally deliver and release targeted drugs. The polypeptide is modified on the surface of the nanogold, so that the polypeptide nano-drug is constructed, the stability of the polypeptide can be enhanced, the absorption of the polypeptide drug is promoted, the nano-drug is further modified, and the targeting property of the nano-drug can be improved. Research shows that the nano particle with connected biological molecule and nano gold has high biocompatibility and is one excellent carrier for transmembrane transmission of extracellular matter. With the rapid development of nano biotechnology, nano drugs and nano delivery systems have been primarily applied in the fields of basic research and clinical medicine, wherein the anti-tumor drug Aurimune (CYT-6091) has already completed phase I clinical trials and is undergoing phase II clinical trials. The medicinal research of the nano-gold is developed rapidly, and the nano-gold shows good application prospect.
Based on the research, the invention designs and prepares the matrix metalloproteinase nanometer inhibitory polypeptide, and the polypeptide is modified on the surface of the nanometer gold through Au-S bonds, so as to obtain the novel polypeptide gold nanoparticle composition. The literature reports that the polypeptide is connected with the gold nanoparticles, so that the half-life period of the polypeptide in vivo is remarkably prolonged, the polypeptide is not easily hydrolyzed by in vivo protease, and the stability is improved, thereby exerting the activity to the maximum extent, and being expected to develop a medicament with stronger inhibition effect on tumor invasion and metastasis.
Disclosure of Invention
The invention aims to prepare a polypeptide nanoparticle composition capable of inhibiting matrix metalloproteinase-2/9. Polypeptide drugs capable of inhibiting matrix metalloproteinase-2/9 are connected to gold nanoparticles with good biocompatibility to prepare the nanoparticles. The nanoparticles prepared by the invention have high stability and good pharmaceutical activity.
A matrix metalloproteinase inhibitory polypeptide gold nanoparticle composition is characterized in that the composition is formed by connecting matrix metalloproteinase-2/9 inhibitory polypeptide and gold nanoparticles; the composition is prepared by the following steps:
the first step is as follows: mixing the chloroauric acid solution and the polypeptide solution, and uniformly stirring;
the second step is that: adding a sodium borohydride solution into the mixed solution, and stirring in an ice water bath in a dark place;
the third step: and (3) dialyzing the reaction product solution obtained in the second step to remove the polypeptide and other byproducts which do not participate in the reaction to obtain a polypeptide nanoparticle composition solution, and freeze-drying to obtain the composition.
The composition is characterized in that the polypeptide is a matrix metalloproteinase-2/9 inhibitory polypeptide and comprises any one or a mixture of more than two of the following polypeptides:
ProArgTrpPheXaa(D-Arg)GlyGluGlyGlyGlyGlyGluCysGly，
ProArgTyrHisGly(D-His)Xaa(D-Arg)GlyGluGlyGlyGlyGlyGluCysGly，
ProArgHisGly(D-His)Xaa(D-Arg)GlyGluGlyGlyGlyGlyGluCysGly，
wherein Xaa ═ D-Bip, D-Bip is diphenylalanine of form D.
The composition is characterized in that the particle size of the gold nanoparticles in the composition is 1-1000 nm.
The composition is applied to preparing antitumor drugs.
The polypeptide nanoparticle composition can inhibit migration and invasion of tumors and has anti-tumor proliferation activity.
The preparation method of the polypeptide nanoparticle composition is characterized by comprising the following steps:
the first step is as follows: and mixing the chloroauric acid solution and the polypeptide solution, and uniformly stirring.
The second step is that: and adding a sodium borohydride solution into the mixed solution, and stirring in an ice water bath in a dark place.
The third step: and (3) dialyzing the reaction product solution obtained in the second step to remove the polypeptide and other byproducts which do not participate in the reaction to obtain a polypeptide nanoparticle composition solution, and freeze-drying to obtain the polypeptide nanoparticle composition.
Has the advantages that:
the polypeptide nanoparticle composition is a brand new sequence, and the pharmacodynamic analysis is carried out in the form of the polypeptide nanoparticle composition for the first time, and the result shows that the polypeptide nanoparticle composition has higher inhibitory activity on the migration and invasion of tumor cells compared with polypeptide or nanogold.
Drawings
FIG. 1 is an electron microscope image of a polypeptide nanoparticle composition
Figure 2 polypeptide nanoparticle compositions inhibition of MMP2/9 enzyme activity (× P <0.05, × P <0.01, × P <0.001vs control)
FIG. 3 is a photograph of the migration of human gastric cancer cells MGC803 with AuNP-PG16 polypeptide nanoparticle composition of different concentrations
FIG. 4 shows that polypeptide nanoparticle composition AuNP-PG16 with different concentrations inhibits human gastric cancer cell MGC803 cell migration
FIG. 5 shows that the polypeptide nanoparticle composition AuNP-PG16 with different concentrations inhibits the human gastric cancer cell MGC803 cell invasion
Detailed Description
Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention.
Example 1
Preparation of polypeptide nanoparticle composition
Dissolving a polypeptide (ProArgTrpPheXaa (D-Arg) GlyGlyGlyGlyGlyGlyCysGly, wherein Xaa ═ D-Bip, and D-Bip is D-type diphenylalanine) in deionized water, mixing a polypeptide solution (1mg/mL, 4mL) with a chloroauric acid (HAuCl4, 0.2428M, L0 μ L) solution, and stirring in an ice-water bath in the dark for 10 min; adding sodium borohydride (NaBH4, 0.20M, 125 mu L), stirring for 4h to obtain polypeptide nanoparticle composition solution, dialyzing, freeze-drying, and refrigerating at 4 deg.C. The morphology of the polypeptide nanoparticle composition is shown in fig. 1.
Fluorescence detection method for detecting inhibitory activity of polypeptide nanoparticle composition on MMP enzyme
(1) Addition of MMP-2/9 enzyme: adding 10 μ L of diluted recombinant human matrix metalloproteinase MMP-2 (or MMP-9 enzyme) into a totally black 96-well plate;
(2) adding a sample to be tested: and adding different samples to be detected with different concentrations, which are prepared in advance, and setting a corresponding blank control group.
(3) Addition of fluorogenic substrate ES 001: add 10. mu.L of diluted fluorogenic substrate ES001 to each well, and after addition, repeatedly blow-suck with pipette, mix well quickly.
(4) Detection of fluorescent signal: and detecting the change condition of the fluorescence signal by using a multifunctional microplate reader under the excitation wavelength of 328nm and the emission wavelength of 392 nm.
(5) The specific result is shown in fig. 2, the polypeptide nanoparticle composition has stronger inhibition capability on the activity of recombinant human matrix metalloproteinase-9 (MMP-9).
Example 2
Preparation of polypeptide nanoparticle composition
Dissolving a polypeptide (ProArgTyrHisGly (D-His) Xaa (D-Arg) GlyGlyGlyGlyGlyGlyCysGly, wherein Xaa ═ D-Bip and D-Bip are D-type diphenylalanine) in deionized water, mixing a polypeptide solution (1mg/mL, 3mL) with a chloroauric acid (HAuCl4, 0.2428M, L0 μ L) solution, and stirring in ice-water bath for 10min in the absence of light; adding sodium borohydride (NaBH4, 0.20M, 125 mu L), stirring for 4h to obtain polypeptide nanoparticle composition solution, dialyzing, freeze-drying, and refrigerating at 4 deg.C.
Research on influence of polypeptide nanoparticle composition on tumor cell migration
(1) Preparing the medicine: the medicines are all prepared by serum-free DMEM medium, and gradient dilution is carried out by adopting an equal-time dilution method; blank control group: drug-free serum-free DMEM; positive control group: 0.20 μ M Avastin injection; two duplicate wells were set for each dose.
(2) Digestion of cells: digesting the tumor cells cultured to logarithmic growth phase with trypsin, centrifuging, resuspending in serum-free DMEM medium, counting with cell counter, and adjusting cell concentration to 1 × 105Per mL; cells were seeded in the chamber at 100. mu.L/chamber;
(3) adding medicine: adding the prepared medicines of different groups into corresponding chambers, wherein each chamber is 100 mu L;
(4) in 24-well plates, 600. mu.L of DMEM complete medium containing 10% FBS was added to each well, and the 24-well plates were placed in 5% CO2Culturing in an incubator at 37 ℃ for 24 hours;
(5) after 24h, the culture medium in the transwell chamber and the 24-well plate was discarded; transwell chamber was rinsed 2 times with PBS; placing the small chamber in a 24-pore plate containing 600 μ L of precooled absolute ethyl alcohol, and fixing at normal temperature for 30 min; dyeing the crystal violet with 0.2 percent of the total dye at normal temperature for 10min, and rinsing the crystal violet with clear water; the non-migrated cells on the upper layer of the cell membrane were gently wiped off with a cotton swab, observed under a microscope and counted by photographing in four fields randomly. Mobility Inhibition Rate (MIR) was calculated according to the formula:
where Ntest is the number of cells migrated in the administration group and Ncontrol is the number of cells migrated in the blank control group.
(6) Results of the experiment
Specific results are shown in fig. 3 and 4, and the polypeptide nanoparticle composition has stronger inhibitory activity on the migration capacity of tumor cells, and is similar to the activity of a positive control (Avastin).
Example 3
Preparation of polypeptide nanoparticle composition
Dissolving a polypeptide (ProArgHisGly (D-His) Xaa (D-Arg) GlyGlyGlyGlyGlyGlyCysGly, wherein Xaa ═ D-Bip, and D-Bip is D-type diphenylalanine) in deionized water, mixing a polypeptide solution (1mg/mL, 3mL) with a chloroauric acid (HAuCl4, 0.2428M, L0 μ L) solution, and stirring in ice water in the dark for 10 min; adding sodium borohydride (NaBH4, 0.20M, 125 mu L), stirring for 4h to obtain polypeptide nanoparticle composition solution, dialyzing, freeze-drying, and refrigerating at 4 deg.C.
Research on influence of polypeptide nanoparticle composition on tumor cell invasion
(1) Paving glue in the small chamber: one day before the experiment, 10mg/mL Matrigel was diluted 1:3 in an EP tube in serum-free DMEM, repeatedly blown and mixed by a gun head, and placed on an ice box to prevent solidification. Taking out the clean and sterile transwell chamber, sucking the diluted Matrigel, and uniformly coating the Matrigel on a transwell chamber membrane by 20 mu L/chamber; after the glue spreading is finished, placing the small chamber in a super clean bench, airing for 2 hours at room temperature by using small wind, covering a cover after the air-drying, placing the small chamber in an incubator at 37 ℃ overnight, and using the small chamber after ultraviolet irradiation for half an hour in the super clean bench before the use on the next day;
(2) the remaining steps are the same as cell migration.
(3) Results of the experiment
The specific result is shown in fig. 5, the polypeptide nanoparticle composition has stronger inhibition activity on the invasion capacity of tumor cells, and the activity is similar to that of a positive control (Avastin).
Sequence listing
<110> university of Chinese pharmacy
<120> matrix metalloproteinase inhibitory polypeptide gold nanoparticle composition, and application and preparation method thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 15
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 1
Pro Arg Trp Phe Xaa Arg Gly Glu Gly Gly Gly Gly Glu Cys Gly
1 5 10 15
<210> 2
<211> 17
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 2
Pro Arg Thr His Gly His Xaa Arg Gly Glu Gly Gly Gly Gly Glu Cys
1 5 10 15
Gly
<210> 3
<211> 16
<212> PRT
<213> Artificial sequence (2 Ambystoma latex x Ambystoma jeffersonia)
<400> 3
Pro Arg His Gly His Xaa Arg Gly Glu Gly Gly Gly Gly Glu Cys Gly
1 5 10 15
Claims (5)
1. A matrix metalloproteinase inhibitory polypeptide gold nanoparticle composition is characterized in that the composition is formed by connecting matrix metalloproteinase-2/9 inhibitory polypeptide and gold nanoparticles; the composition is prepared by the following steps:
the first step is as follows: mixing the chloroauric acid solution and the polypeptide solution, and uniformly stirring;
the second step is that: adding a sodium borohydride solution into the mixed solution, and stirring in an ice water bath in a dark place;
the third step: dialyzing the reaction product solution obtained in the second step to remove the polypeptide and other byproducts which do not participate in the reaction to obtain a polypeptide nanoparticle composition solution, and freeze-drying to obtain a composition;
the polypeptide is a matrix metalloproteinase-2/9 inhibitory polypeptide, and comprises any one or a mixture of the following polypeptides:
ProArgTrpPheXaa(D-Arg)GlyGluGlyGlyGlyGlyGluCysGly，
ProArgTyrHisGly(D-His) Xaa (D-Arg)GlyGluGlyGlyGlyGlyGluCysGly，
ProArgHisGly(D-His) Xaa(D-Arg)GlyGluGlyGlyGlyGlyGluCysGly，
wherein Xaa = D-Bip, and D-Bip is diphenylalanine in form D.
2. The composition of claim 1, wherein the gold nanoparticles in the composition have a particle size of 1 to 1000 nm.
3. Use of the composition according to claim 1 for the preparation of an antitumor medicament.
4. Use of a composition according to claim 1 in the manufacture of a medicament for inhibiting matrix metalloproteinase-9 to thereby treat inflammation.
5. The method for preparing the gold nanoparticle composition as claimed in claim 1, wherein the steps are as follows:
the first step is as follows: mixing the chloroauric acid solution and the polypeptide solution, and uniformly stirring;
the second step is that: adding a sodium borohydride solution into the mixed solution, and stirring in an ice water bath in a dark place;
the third step: and (3) dialyzing the reaction product solution obtained in the second step to remove the polypeptide and other byproducts which do not participate in the reaction to obtain a polypeptide nanoparticle composition solution, and freeze-drying to obtain the polypeptide nanoparticle composition.
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| CN102961756A (en) * | 2012-10-16 | 2013-03-13 | 常州大学 | Synthesis method of polypeptide-nanogold particle drug carrier |
| CN107175129A (en) * | 2017-06-01 | 2017-09-19 | 江苏大学 | A kind of lithotroph composite nano-catalyst Au@A β2535Preparation method |
| CN108484729A (en) * | 2018-03-23 | 2018-09-04 | 中国药科大学 | A kind of peptide inhibitor of matrix metalloproteinase 9 and its application |
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| CN102961756A (en) * | 2012-10-16 | 2013-03-13 | 常州大学 | Synthesis method of polypeptide-nanogold particle drug carrier |
| CN107175129A (en) * | 2017-06-01 | 2017-09-19 | 江苏大学 | A kind of lithotroph composite nano-catalyst Au@A β2535Preparation method |
| CN108484729A (en) * | 2018-03-23 | 2018-09-04 | 中国药科大学 | A kind of peptide inhibitor of matrix metalloproteinase 9 and its application |
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