CN117159745B - Clathrate compound of dendritic water-soluble tetrabiphenyl [4] arene and active polypeptide, and preparation method and application thereof - Google Patents
Clathrate compound of dendritic water-soluble tetrabiphenyl [4] arene and active polypeptide, and preparation method and application thereof Download PDFInfo
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a dendritic water-soluble tetrabiphenyl [4]]Clathrate of aromatic hydrocarbon and active polypeptide, and its preparation method and application are provided. The preparation method of the inclusion compound has simple operation, mild and efficient reaction conditions and is beneficial to industrial production. Dendritic tetrabiphenyl [4]]The arene has good water solubility and biocompatibility, can be well complexed with the active polypeptide Nap-FFGVRKKP, and has a complexing constant ofK a =(1.156±0.098)×10 7 M ‑1 . The inclusion compound can effectively promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), increase bone mass by increasing the volume fraction and thickness of trabecula, and improve osteoporosis caused by glucocorticoid.
Description
Technical Field
The invention belongs to the field of supermolecular chemistry and biological medicine, and relates to a dendritic water-soluble tetrabiphenyl [4] arene and active polypeptide inclusion compound, a preparation method thereof and application thereof as an osteoporosis treatment medicine.
Background
The bioactive polypeptide has great potential in aspects of drug delivery, disease treatment, regenerative medicine, immunoregulation and the like by virtue of the advantages of easy preparation and synthesis, strong specificity, good biocompatibility and the like. However, the problems associated with poor stability, short half-life, low retention rate, etc. remain significant challenges that prevent their further in vivo use or even clinical transformation. For this reason, scientists have been devoted to the development of various carriers for delivering bioactive polypeptides, and most of the carriers are physically encapsulated by using nanomaterials or chemically coupled by polymers, but the problems of low encapsulation efficiency, reduced bioactivity and the like are unavoidable.
In recent years, the manner in which polypeptide delivery is achieved based on host-guest interactions has become an important area of research for supramolecular chemists. Traditional macrocycles, represented by cyclodextrins, calixarenes, cucurbiturils, and pillar arenes, typically utilize hydrophobic cavities to anchor intrinsic amino acid residues to drive delivery of the entire polypeptide chain. However, this approach is very disadvantageous for the delivery of biologically active polypeptides, mainly because their activity is exerted mainly by the close binding of amino acid residues to the receptor. Encapsulation of amino acid residues by the macrocyclic cavity tends to affect the binding of the biologically active polypeptide to the corresponding receptor and even its biological activity. Thus, the delivery vehicle for the biologically active polypeptide is selected not only to have a strong complexation constant with the polypeptide (for improved stability) but also to not hinder exposure of its active site (to facilitate its binding to the receptor and thus the biological activity). The supermolecule strategy based on the supermacrocyclic host-guest complex which is recently proposed by the subject group has two points, and suggests that the biphenyl arene supermacrocyclic is a very promising candidate molecule for delivering bioactive polypeptide.
The incidence of brittle fracture caused by osteoporosis is high, especially for middle-aged and elderly women. 1 case of osteoporosis caused fracture occurs every 3 seconds worldwide, and disabling mortality is high. It is estimated that by 2050, the medical cost for osteoporosis fracture in China will reach 254.3 hundred million dollars, bringing great economic burden to society. The current clinical medicines for preventing and treating osteoporosis mainly comprise bone absorption inhibitors, bone formation promoters, other mechanism medicines, traditional Chinese medicines and the like. Growth factors are proteins which can be specifically combined with cell membrane receptors to regulate cell growth and function, and are potential medicines for treating osteoporosis. Platelet Derived Growth Factors (PDGFs) have been widely used for tissue repair and regeneration, and have a critical role in regulating proliferation, apoptosis, migration, etc. of fibroblasts and other mesenchymal-derived cells. Previous studies have shown that the active polypeptide Nap-ffgvrkp is a good performing PDGF mimetic that can be obtained by standard solid phase synthesis methods. The method selects the tetrabiphenyl [4] arene as a main body, increases the depth of a cavity of the tetrabiphenyl [4] arene by modifying a branched hydrophobic chain, and introduces a plurality of carboxyl groups at the tail end of the hydrophobic chain to increase the solubility of the tetrabiphenyl [4] arene in aqueous solution, thereby obtaining the dendritic water-soluble tetrabiphenyl [4] arene. We selected them as representatives of the active polypeptide and the macrocycle, respectively, to prepare inclusion compounds of the oversized-ring-encapsulated bioactive polypeptide and explored their therapeutic effect on glucocorticoid-induced osteoporosis through proliferation and osteogenic differentiation experiments of bone marrow mesenchymal stem cells (BMSCs).
Disclosure of Invention
In order to achieve the above object, the present invention discloses the following technical contents:
a clathrate compound formed by mixing dendritic water-soluble tetrabiphenyl [4] arene shown in a formula I and PDGF active polypeptide (Nap-FFGVRKKP) shown in a formula II, wherein the clathrate compound contains two components and is characterized by having the following structural characteristics:
the invention further discloses a preparation method of the inclusion compound, which is characterized by comprising the following steps: adding two kinds of dendritic water-soluble tetrabiphenyl [4] arene shown in a formula I and PDGF active polypeptide (Nap-FFGVRKKP) shown in a formula II into PBS buffer solution with pH value of=5.0-9.0 according to a molar ratio of 1:1, regulating the pH value to 6.0-7.0 by using sodium carbonate solution or hydrochloric acid solution, heating to 90 ℃ to completely dissolve the compounds, and cooling to room temperature to obtain the clathrate compound.
The invention further discloses application of the inclusion compound in preparation of a medicament for treating osteoporosis, and experimental results show that the inclusion compound can promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) through two signal paths of Wnt/beta-catenin and Hippo/YAP, and can keep bioactivity of a delivery peptide to the greatest extent, so that damage of glucocorticoid dexamethasone (Dex) to proliferation and differentiation of BMSCs is effectively reduced. The subsequent animal experiments are carried out by the inventor, the in vivo curative effect of the compound is evaluated by adopting a microcomputer tomography (Micro-CT) technology and histological staining, the bone mass of mice of the inclusion compound group is obviously increased, the trabecular bone volume fraction (BV/TV) is increased by 40.55%, the average trabecular thickness (Tb.Th) is increased by 17.03%, the in vivo effect of promoting bone formation is obvious, and the compound can effectively treat osteoporosis. The strategy of forming inclusion compound with the bioactive polypeptide by utilizing the ultra-large ring provides a general method for delivering the bioactive polypeptide and opens up a new idea for drug development based on the polypeptide.
The invention discloses a clathrate compound of dendritic water-soluble tetrabiphenyl [4] arene and active polypeptide, a preparation method and application thereof, and has the positive effects that:
(1) The inclusion compound has definite chemical structure, and the preparation method is simple to operate and is beneficial to industrial production.
Dendritic tetrabiphenyl [4]]The arene has good water solubility, can realize stronger complexation with bioactive polypeptide Nap-FFGVRKKP (bonding constant is (1.156 +/-0.098) multiplied by 10 7 M -1 ）。
(2) The strategy of forming inclusion compound with bioactive polypeptide provides new thought and method for delivering bioactive polypeptide and feasible scheme for constructing in vivo delivery system of bioactive polypeptide medicine molecule.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 dendritic water soluble tetrabiphenyl [4]]Competitive fluorescence titration and fitting of arene and bioactive polypeptide Nap-FFGVRKKP; wherein A is a continuous concentration of 1×10 -6 Rho123 and dendritic water soluble tetrabiphenyl [4] of M]Aromatic hydrocarbon 1:1 (ph=7.4) and Rho123 (excitation wavelength 500 nm), B is the binding constant of the corresponding fluorescence intensity value at 525 nm and active polypeptide concentration in a, fitted according to a 1:1 competitive binding model;
FIG. 2 is a graph showing the proliferation promoting effect of inclusion compound on bone marrow mesenchymal stem cells (BMSCs); wherein A is the optimal concentration of bioactive polypeptide Nap-FFGVRKKP detected by using a CCK-8 kit for promoting the proliferation of BMSCs, B is the cell proliferation condition of the BMSCs after the inclusion compound detected by using the CCK-8 kit is incubated and cultured for 1-3 days, and C is the cell proliferation rate of the BMSCs detected by using an EdU staining method;
FIG. 3 is a graph showing the effect of inclusion compounds on bone marrow mesenchymal stem cells (BMSCs) on bone differentiation; wherein A is a photograph of Alizarin Red (ARS) staining observed under an optical microscope 21 days after osteogenic differentiation induction, B is a histogram of statistics of alizarin red relative staining areas in the A graph by Image J software, C is a photograph of alkaline phosphatase (ALP) staining observed under an optical microscope 7 days after osteogenic differentiation induction, and D is a histogram of statistics of alkaline phosphatase relative staining areas in the C graph by Image J software;
FIG. 4 is a schematic diagram showing the mechanism of action of inclusion compound on bone marrow mesenchymal stem cells (BMSCs) to promote proliferation and osteogenic differentiation; wherein A is immunofluorescence staining photograph of beta-catenin in BMSCs, B is expression level of beta-catenin protein in BMSCs, C is immunofluorescence staining photograph of YAP in BMSCs, and D is relative expression level of p-YAP and YAP protein in BMSCs;
FIG. 5 is a graph showing the in vivo therapeutic effect of the inclusion compound on mouse osteoporosis; wherein A is a photograph of bone density of mice in different treatment groups analyzed and detected by a microcomputer tomography (Micro-CT), B is a photograph of average trabecular thickness (Tb.Th) of mice in different treatment groups measured by a mu CT evaluation program, C is a photograph of trabecular bone volume fraction (BV/TV) of mice in different treatment groups measured by a mu CT evaluation program, D is H & E and Masson staining of the trabecular structure of proximal tibia bone of mice in different treatment groups, and OPN, OCN, p-YAP expression level is detected by an immunohistochemical staining method.
Detailed Description
The invention is described below by means of specific embodiments. The technical means used in the present invention are methods well known to those skilled in the art unless specifically stated. Further, the embodiments should be construed as illustrative, and not limiting the scope of the invention, which is defined solely by the claims. Various changes or modifications to the materials ingredients and amounts used in these embodiments will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The raw materials and reagents used in the invention are as follows: the reagents such as rhodamine 123, phosphate buffered saline (PBS buffer), alpha-MEM basal medium, fetal bovine serum, bone marrow mesenchymal stem cells (BMSCs), various antibodies, and detection kits are all commercially available.
Dendritic water-soluble tetrabiphenyl [4]]Aromatic hydrocarbons (see journal literature: zhao, l.; chen, j.; tian, l.; zhang, y.; chen, l.; du, x.; ma, m.; li, j.; meng, q.; li, c. Supramolecular Detoxification of Macromolecular Biotoxin through theComplexation by a Large-Sized Macrocycle).Adv. Healthcare Mater.2022, 11, 2200270）
Active polypeptide Nap-FFGVRKKP (see invention: polypeptide derivative and nanofiber capable of simulating platelet derived factor biological activity and application thereof. ZL 201911146406. X)
In order to make the technical scheme and advantages of the present invention more apparent, embodiments of the present invention will be described in further detail with reference to the accompanying drawings.
Example 1
The experimental steps of competitive fluorescence titration of dendritic water-soluble tetrabiphenyl [4] arene and bioactive polypeptide Nap-FFGVRKP are carried out:
(1) Preparation of dendritic Water-soluble Tetrabiphenyls [4]]The mixed solution of aromatic hydrocarbon and rhodamine 123 is used for standby: accurately weighing 0.409 mg dendritic water-soluble tetrabiphenyl [4] by using balance]Aromatic hydrocarbon and 0.038 mg rhodamine 123 were dissolved in 100 mL phosphate buffer saline (PBS buffer) to give a concentration of 1X 10 -6 M and the mixture of M.
(2) Preparing mother liquor of bioactive polypeptide Nap-FFGVRKKP for later use: accurately weighing 0.115. 0.115 mg bioactive polypeptide Nap-FFGVRKKP in 10 mL PBS buffer solution with concentration of 1×10 -4 M mother liquor.
(3) The cuvette was first filled with 2 mL at a concentration of 1X 10 -6 M mixture obtained in step (1), then 1. Mu.L, 2. Mu.L, 4. Mu.L, 8. Mu.L, 16. Mu.L, 32. Mu.L, 64. Mu.L are sequentially added to the cuvette with a micropipette at a concentration of 1X 10 -4 The mother liquor of the bioactive polypeptide Nap-FFGVRKKP of M is subjected to fluorescence spectrum test under the conditions of excitation wavelength Ex=507 nm and emission wavelength Em=529 nm, and a series of fluorescence spectrograms are obtained.
As shown in FIG. 1, with the biologically active polypeptide Nap-FThe addition of FGVRKKP mother liquor, the fluorescence of rhodamine 123 is gradually recovered and the intensity is gradually increased, which proves that the bioactive polypeptide Nap-FFGVRKP can lead rhodamine 123 to be separated from dendritic water-soluble tetrabiphenyl [4]]The cavities of the main body of the arene macrocycle compete out. Dendritic water-soluble tetrabiphenyl [4] is calculated through nonlinear fitting]The bonding constant of aromatic hydrocarbon and bioactive polypeptide Nap-FFGVRKKP is (1.156 + -0.098) x 10 7 M -1 . The result shows that dendritic water-soluble tetrabiphenyl [4]]The arene is a good delivery carrier of the bioactive polypeptide Nap-FFGVRKKP, and the strong complexation can improve the stability of the bioactive polypeptide Nap-FFGVRKKP, thereby laying a foundation for improving the in-vitro and in-vivo curative effect of the bioactive polypeptide.
Example 2
Experimental procedure of proliferation promoting effect of inclusion compound on mesenchymal stem cells (BMSCs):
(1) Heating the water bath to 37 ℃ in advance, placing a culture medium, serum and the like into the water bath for preheating, and simultaneously turning on an ultra-clean bench ultraviolet lamp for irradiation for 30 min; (2) Frozen bone marrow mesenchymal stem cells (BMSCs) are taken out from a liquid nitrogen tank, quickly placed in a water bath at 37 ℃ to defrost the cells, and then quickly transferred into an ultra clean bench for the following operations: carefully transferring the cell-containing solution into a centrifuge tube containing a culture medium by using a pipette, centrifuging for 3 min, removing supernatant, re-suspending by using an alpha-MEM culture medium containing 10% of fetal bovine serum, transferring into a culture dish, and then placing into a culture box at 37 ℃ for culture; (3) The cell state is observed the next day, and after the cell state is good, the following experiment is carried out after the first generation; (4) Adding 2 mL pancreatin, gently shaking, uniformly covering, tapping the bottom of the culture dish, digesting at 37deg.C for 3 min, observing most of the cell suspension under microscope, blowing with 1 mL gun for 5-10 min, adding 2 mL culture medium, uniformly blowing, collecting cell culture solution, sucking into centrifuge tube, centrifuging at 1000 rpm for 3 min, removing supernatant solution, adding 10% fetal bovine serum-containing alpha-MEM culture medium, uniformly blowing with gun, and counting with cell counting plate to 5×10 per ml 7 Individual cells.
(5) Cells were resuspended in 96-well plates and 100. Mu.L of alpha-MEM medium containing 10% fetal bovine serum per well of 5000 cells were incubated overnight in an incubator at 37 ℃. The next day the alpha-MEM medium was aspirated and replaced with serum-free alpha-MEM medium, starvation treatment was performed for 4 h, after which 100 μl of serum-free alpha-MEM medium containing 50 nM, 100 nM, 200 nM, 400nM, 700 nM, 1 mM inclusion compound and 500uM Dex was added per well, 100 μl of any inclusion compound-free and serum-free alpha-MEM medium was added to the control group. After incubation in an incubator at 37℃for 48 h, 10. Mu.L of CCK-8 and 90. Mu.L of serum-free alpha-MEM medium were added to each well, and after 4. 4 h the OD at 450. 450 nm was measured by a microplate reader. As shown in fig. 2A, the OD value increased and then decreased with increasing clathrate concentration, which means that the cell number also increased and then decreased, and thus the optimum clathrate concentration for promoting cell proliferation was selected to be 400 nM.
(6) Cells were incubated 48 h with serum-free alpha-MEM medium containing 400nM clathrate and 500uM Dex, respectively, followed by 10 h with EdU working solution (1:1000), followed by fixation with 4% paraformaldehyde for 30 min at room temperature and permeation with 0.3% Triton X-100 for 10min in the same manner as step (5). EdU staining solution was added to the wells under shading for 30 min, and nuclei were stained with DAPI for 5 min. Finally, photographing by using a laser scanning confocal fluorescence microscope, and counting the number of the EdU marked cells.
As shown in FIG. 2A, the OD values of the clathrate compound concentration representing the cell number within the range of 50 nM-1 mu M are higher than those of the control group, and the clathrate compound is proved to be capable of promoting cell proliferation, the OD values are increased and then decreased along with the increase of the clathrate compound concentration, and the OD value is the maximum when the OD value is 400nM, so that the optimal concentration of the clathrate compound for promoting cell proliferation is 400nM; as shown in FIG. 2B, the number of cells in the 500uM Dex group alone was significantly less than that in the control group, while the number of cells in the 400nM clathrate and 500uM Dex group was significantly more than that in the 500uM Dex group alone, thus demonstrating that the clathrate was effective against apoptosis by Dex.
Example 3
Experimental procedure of the inclusion compound for bone marrow mesenchymal stem cells (BMSCs) contributing to bone differentiation effect:
(1) Cells were resuspended in 96-well plates and 100. Mu.L of alpha-MEM medium containing 10% fetal bovine serum per well of 5000 cells were incubated overnight in an incubator at 37 ℃. The next day the alpha-MEM medium was aspirated and replaced with serum-free alpha-MEM medium, starvation treatment was performed for 4 h, after which 100. Mu.L of serum-free alpha-MEM medium containing 500uM Dex, 400nM clathrate and 500uM Dex was added per well, and 100. Mu.L of serum-free alpha-MEM medium without any substance was added to the control group. Osteogenic differentiation and calcium deposition were assessed on days 7 and 21 after osteoinduction by alkaline phosphatase (ALP) staining and Alizarin Red (ARS) staining, respectively.
(2) For ALP staining, the various treated BMSCs were fixed with 4% formaldehyde at room temperature for 20 min and stained according to the alkaline phosphatase chromogenic kit procedure.
(3) For ARS staining, the differently treated BMSCs were fixed with 95% alcohol at room temperature for 10min and stained according to alizarin red staining kit procedure.
(4) Finally, photographing under an inverted optical microscope, and counting the area of the dyeing area.
As shown in fig. 3A-B, the significant decrease in alizarin red after the addition of Dex demonstrated a significant decrease in calcium deposition, while the significant improvement in this trend after the addition of Dex + inclusion compound resulted in a significant increase in the area of alizarin red from calcium deposition, thus demonstrating that inclusion compound can promote calcium deposition; as shown in FIGS. 3C-D, the apparent decrease in the area of alkaline phosphatase after Dex addition demonstrated that the tendency to bone differentiation was inhibited, while the apparent increase in the area of alkaline phosphatase after Dex+ inclusion complex addition demonstrated that the inclusion complex was able to promote osteogenic differentiation.
Example 4
Experimental procedure of action mechanism of clathrate compound on bone marrow mesenchymal stem cells (BMSCs) proliferation promotion and osteogenic differentiation:
cells were fixed with 4% paraformaldehyde for 30 min at room temperature, then permeabilized with 0.3% Triton X-100 in PBS buffer for 10min, followed by blocking 1 h with 5% goat serum. Then, the goat anti-rabbit secondary antibody labeled with anti- β -catenin, YAP was incubated overnight at 4 ℃ followed by 1 h with Alexa Fluor 488 at room temperature at 37 ℃. Finally, incubation with DAPI was performed for 5 min at room temperature. Images were observed and photographed under a laser scanning confocal fluorescence microscope, and the average value of three times was counted.
As shown in fig. 4A-B, the fluorescence micrograph shows that the fluorescence intensity of the beta-catenin is obviously reduced after the Dex is added, which indicates that the expression of the beta-catenin is inhibited, the expression of the p-beta-catenin corresponding to the beta-catenin is obviously enhanced, and the fluorescence intensity of the beta-catenin is obviously enhanced after the Dex+ inclusion compound is added, and the expression of the p-beta-catenin corresponding to the beta-catenin is obviously reduced, thereby indicating that the inclusion compound can promote the expression of the beta-catenin and inhibit the expression of the p-beta-catenin; similar to FIGS. 4A-B, the inclusion complex promotes YAP expression and inhibits p-YAP expression.
Example 5
The experimental steps of the in vivo treatment effect of the inclusion compound on the osteoporosis of the mice comprise:
30 male C57BL/6 mice, each 20-25g at 8 weeks of age, are selected for establishing an osteoporosis model, and the bone regeneration performance of the inclusion compound is evaluated in vivo. The C57BL/6 mice were randomly divided into 3 groups of 10 mice each. Wherein, dex (8 mg/kg/day) is injected into the lower limbs of 20 mice randomly, a glucocorticoid-induced osteoporosis model is established by continuous injection for 6 weeks, and the rest 10 mice are used as control groups and physiological saline is injected according to the method. Over the next 4 weeks, 20 osteoporosis mice were randomly divided into 2 groups, and each of them was intramuscular injected with either Dex (8 mg/kg/day, n=10) or Dex+ clathrate (8 mg/kg/day; 50 uM clathrate, n=10), and the control group continued to be given saline injection. All mice were sacrificed at week 10 and hind limbs were immobilized with 4% paraformaldehyde and bone mass was assessed by microcomputer tomography (CT). Proximal sections of tibia were taken after decalcification for hematoxylin and eosin (H & E) staining, masson (Masson) staining and Immunohistochemical (IHC) staining analysis.
The initial data were collected using a vivaCT80 scanner with an exposure time of 200 ms using the μct tomograph V6.4-2 software and the trabecular bone was reconstructed three-dimensionally using the μ CT Evaluation Program V6.6.6 software. To assess proximal tibial microstructure, trabecular bone was defined along the boundary between cortical and cancellous bone as a region of interest (ROI). The average trabecular thickness (tb.th) and trabecular bone volume fraction (BV/TV) within the defined ROI were quantified using ancillary software.
The femur and tibia of the mice were decalcified at 0.5M EDTA for 1 month, the decalcification solution was changed every 3 days, and paraffin was embedded after dehydration. A series of 3 μm thick slices were cut along the coronal plane. Histological observations were performed with hematoxylin and eosin (H & E) staining. During immunohistochemical staining, OPN, OCN and p-YAP antibodies detected the expression of the corresponding proteins in tissue sections. The collagen fibers were observed by Masson (Masson) staining to evaluate the content of the new bone tissue.
As shown in fig. 5A-B, the mean trabecular thickness (tb.th) and trabecular bone volume fraction (BV/TV) were significantly reduced in the Dex group, while the bone mass was significantly increased in the Dex + clathrate group, 40.55% increase in BV/TV, 17.03% increase in tb.th, and substantial recovery of tb.th values to the level of the control group compared to the Dex group; IHC staining (FIG. 5C) showed that the Dex treated mice had reduced OPN and OCN expression and increased p-YAP expression, which was effectively reversed after inclusion of the inclusion complex. HE and Masson staining further showed that the trabeculae of the Dex + clathrate mice were aligned and that the collagen fiber network representing the new bone tissue was more evident.
In conclusion, the clathrate compound of the dendritic water-soluble tetrabiphenyl [4] arene and the active polypeptide prepared by the invention has obvious effect on treating osteoporosis caused by glucocorticoid.
The foregoing description is only for the convenience of those skilled in the art to understand the technical solution of the present invention, and is not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (2)
1. Dendritic water-soluble tetrabiphenyl [4] shown in formula I]Application of clathrate compound formed by mixing aromatic hydrocarbon and PDGF active polypeptide (Nap-FFGVRKKP) shown in formula II in preparation of medicine for treating osteoporosis, wherein the clathrate compound contains two components and has the following structural characteristics: 。
2. the use according to claim 1, wherein the process for the preparation of the inclusion compound comprises the steps of: adding two kinds of dendritic water-soluble tetrabiphenyl [4] arene shown in a formula I and PDGF active polypeptide (Nap-FFGVRKKP) shown in a formula II into PBS buffer solution with pH value of=5.0-9.0 according to a molar ratio of 1:1, regulating the pH value to 6.0-7.0 by using sodium carbonate solution or hydrochloric acid solution, heating to 90 ℃ to completely dissolve the compounds, and cooling to room temperature to obtain the clathrate compound.
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| CN106109466A (en) * | 2016-07-26 | 2016-11-16 | 中国人民解放军第四军医大学 | The application of ligustrazine glucocorticoid-induced osteoporosis |
| CN110790822A (en) * | 2019-11-21 | 2020-02-14 | 南开大学 | Polypeptide derivative capable of simulating biological activity of platelet-derived factor, nanofiber and application of polypeptide derivative and nanofiber |
| CN115057797A (en) * | 2022-02-24 | 2022-09-16 | 上海大学 | Synthesis of water-soluble biphenyl aromatic hydrocarbon and application of biotoxin detoxification |
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| CN106109466A (en) * | 2016-07-26 | 2016-11-16 | 中国人民解放军第四军医大学 | The application of ligustrazine glucocorticoid-induced osteoporosis |
| CN110790822A (en) * | 2019-11-21 | 2020-02-14 | 南开大学 | Polypeptide derivative capable of simulating biological activity of platelet-derived factor, nanofiber and application of polypeptide derivative and nanofiber |
| CN115057797A (en) * | 2022-02-24 | 2022-09-16 | 上海大学 | Synthesis of water-soluble biphenyl aromatic hydrocarbon and application of biotoxin detoxification |
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