阅读说明：本技术 Cca-cd共组装体在制备大分子毒素解毒药物中的应用 (Application of CCA-CD co-assembly in preparation of macromolecular toxin detoxification drug ) 是由 郭东升 潘雨辰 岳宇昕 李华斌 于 2021-06-16 设计创作，主要内容包括：本发明涉及一种CCA-CD共组装体在制备大分子毒素解毒药物中的应用。该共组装体CCA-CD能够有效络合蜂毒肽、蜘蛛毒肽和蛇毒肽,缓解它们的细胞毒性,具有治疗这些毒性大分子中毒的潜力。CCA-CD各组成成分化学结构明确、批次重现性好,CCA-CD具有很高的化学稳定性和热稳定性,且不影响生物体内重要蛋白的活性,具有良好的生物相容性。(The invention relates to an application of a CCA-CD co-assembly in preparing a macromolecular toxin detoxification medicine. The CCA-CD can effectively complex melittin, spider venom peptide and snake venom peptide, relieve cytotoxicity of melittin, spider venom peptide and snake venom peptide, and has potential of treating toxic macromolecule poisoning. The CCA-CD has definite chemical structure of each component and good batch reproducibility, has high chemical stability and thermal stability, does not influence the activity of important protein in organisms, and has good biocompatibility.)

Application of CCA and CD nano supermolecule co-assembly in preparing detoxification drugs of melittin.

Application of CCA and CD nano supermolecule co-assembly in preparation of antidotes for spider venom peptides.

Application of CCA and CD nano supermolecule co-assembly in preparing antidote for snake venom peptide.

4. A CCA-CD according to any of claims 1 to 3, wherein: CCA of formula C₁₀₀H₁₅₀O₁₅CD is of the formula C₁₈₂H₃₅₀O₅₆S₇The molar ratio of the two substances is 1:1, and the supermolecular assembly is constructed by weak pi-pi interaction and hydrophobic interaction, and the supermolecular assembly is a vesicle with nano-scale and spherical appearance.

5. The method of claim 4, wherein the CCA-CD is prepared by: the preparation steps are as follows:

(1) dissolving CCA and CD in chloroform according to a metering ratio respectively to prepare CCA and CD chloroform solutions respectively;

(2) fully mixing the CCA chloroform solution and the CD chloroform solution, placing the mixture in a vacuum drier for vacuum drying, and completely evaporating the organic solvent after 4 to 5 hours to obtain a layer of uniform CCA and CD mixture film on the wall of the container;

(3) adding buffer aqueous solution into a container, placing the container in an ultrasonic instrument at 75-80 ℃ for ultrasonic treatment for 2-4 hours, wherein the solution becomes clear, and the laser pen is used for irradiating the solution to have obvious Tyndall effect, and then a CCA-CD supermolecule co-assembly is formed.

Technical Field

The invention belongs to the technical field of nano supermolecule materials, and relates to a supermolecule co-assembly, in particular to a nano supermolecule co-assembly which utilizes amphiphilic calixarene CCA and amphiphilic cyclodextrin CD to realize strong bonding with macromolecular toxins rich in basic amino acid and hydrophobic amino acid through the heteropolyvalence effect, and can be used for detoxifying the macromolecular toxins.

Background

Poisoning is one of the main causes of emergency and intensive care units, and for severe poisoning, the current treatment methods mainly include gastric lavage, enema, dialysis, and the like, see: 1) megarbane, b.; oberlin, m.; alvarez, j.c.; et al, ann. intensive Care 2020,10,157.2) Mokhlesi, b.; leiken, j.b.; murray, p.; et al Chest 2003,123,577 and 592 one of the treatments of recent interest has been the injection of antidotes to block the biological activity of toxic substances. This method has less requirements for toxins (e.g. distribution range of toxins, molecular weight, affinity for proteins in serum, etc.) and therefore has a wide application space, see: leroux, j.c. nat. nanotechnol.2007,2, 679-: 1) pan, Y. -C.; hu, x. -y.; guo, d. — s.angelw.chem.int.ed.2020, 60,2768-2794.2) Ma, x.; zhao, y.chem.rev.2015,115,7794-7839.3) Chen, y.; huang, z.; zhao, h.; et al, ACSAppl.Mater.interfaces 2017,9,8602-8608.4) ZHEN, Z.; geng, w.c.; xu, z.; et al, isr.j.chem.2019,59,913-927.5) Geng, w. -c.; huang, q.; xu, z.; et al, theranostics 2019,9, 3094-: 1) deng, c.l.; murkli, s.l.; isaacs, l.d.chem.soc.rev.2020,49,7516-7532.2) Yin, h.; zhang, x.; wei, J.; theranostics 2021,11, 1513-.

However, the substrates for current supramolecular detoxification are limited to small molecules. This is because only small molecules can be efficiently entrapped by supramolecular bodies and impede their interaction with biological targets. Besides small molecule toxins, many biological macromolecules are also highly toxic, such as amatoxins and melittin, see: 1) diaz, J.H.wild.environ.Med.2018,29,111-118.2) Habermann, E.science 1972,177,314-322 it is a great challenge to design supramolecular bodies to achieve selective strong bonding to these biomacromolecules because these macromolecules have large size, flexible conformation and complex and diverse sites, see: 1) hossain, m.a.; schneider, h. — j.j.am.chem.soc.1998,120,11208-11209.2) Wright, a.t.; anslyn, e.v.; McDevitt, J.T.J.Am.chem.Soc.2005,127,17405-17411.

Disclosure of Invention

The invention aims to solve the problem of strong specific bonding on macromolecular toxins, thereby effectively relieving the toxicity of the macromolecular toxins. The CCA-CD is a multivalent recognition co-assembly body constructed by mainly utilizing amphiphilic calixarene CCA and amphiphilic cyclodextrin CD, can selectively and strongly complex melittin, spider venom peptide (latarcin 1) and snake venom peptide (crotalicidin), and successfully relieves the cytotoxicity of the macromolecular toxins. Taking melittin as an example, CCA-CD is further verified to be capable of inhibiting the action of melittin and cell membranes, relieving hemolytic toxicity of melittin and improving survival rate of melittin poisoned mice.

The invention uses amphiphilic carboxyl calixarene CCA and amphiphilic beta cyclodextrin CD as construction units, and the construction units can form the co-assembly vesicle CCA-CD in a certain mode under the coexistence condition of a water solution. The surface of the vesicle is enriched with a cavity of calixarene and cyclodextrin. Calixarenes have strong bonds to basic amino acids, such as arginine and lysine, and cyclodextrins have strong bonds to hydrophobic amino acids, such as tyrosine, tryptophan, and phenylalanine. Toxic macromolecules such as melittin and the like which can destroy cell membranes are usually rich in amino acids and have a heteropoly effect with the co-assembly, so that the co-assembly realizes extremely strong bonding on the toxic macromolecules, the effect of the toxic macromolecules and the cell membranes is inhibited, and the potential for treating the toxic macromolecules is possessed.

The technical scheme for realizing the invention is as follows:

the carboxyl-modified amphiphilic calixarene CCA and the nano supermolecule co-assembly CCA-CD of the amphiphilic cyclodextrin CD are used for complexing melittin to relieve cytotoxicity and hemolytic toxicity of melittin, and can be used for preparing melittin detoxification drugs.

CCA-CD building unit CCA chemical formula is C₁₀₀H₁₅₀O₁₅CD is of the formula C₁₈₂H₃₅₀O₅₆S₇The molar ratio of the two substances is 1:1, and the supermolecular assembly is constructed by weak pi-pi interaction and hydrophobic interaction, and the supermolecular assembly is a vesicle with nano-scale and spherical appearance. CCA and CD are of the formula:

the preparation steps are as follows:

(1) the CCA and the CD are respectively dissolved in chloroform according to the metering ratio, and are respectively prepared into 1-5 millimole per liter of CCA and CD chloroform solution (mother liquor).

(2) After 500. mu.l of CCA chloroform solution and 500. mu.l of CD chloroform solution were mixed well, they were placed in a vacuum dryer and vacuum-dried. After 4-5 hours, the organic solvent had evaporated completely, resulting in a uniform film of the mixture of CCA and CD on the walls of the container.

(3) 5 ml of buffer aqueous solution is added into a container, the container is placed in an ultrasonic instrument at the temperature of 75-80 ℃ for ultrasonic treatment for 2-4 hours, the solution becomes clear, the laser pen is used for irradiating the solution to have obvious Tyndall effect, and then a CCA-CD supermolecule co-assembly body is formed.

The invention also protects the application of the amphiphilic calixarene GCA and amphiphilic cyclodextrin CD nano supermolecule co-assembly in the preparation of the spider venom peptide detoxification drug.

The invention also protects the application of the amphiphilic calixarene GCA and amphiphilic cyclodextrin CD nano supermolecule co-assembly in the preparation of the snake venom peptide detoxification drug.

The detoxification principle of the CCA-CD of the invention is as follows:

a plurality of calixarenes and cyclodextrins are enriched on the surface of the CCA-CD co-assembly, and provide a multivalent effect for the recognition of complex biological macromolecules, such as polypeptides and proteins. In addition, CCA and CD can recognize different amino acids, so that CCA-CD can further recognize biological macromolecules through the heteropoly effect and capture more sites on the biological macromolecules, thereby obtaining stronger acting force;

melittin, spider venom peptide and snake venom peptide are rich in positive charge and hydrophobic amino acid, CCA can effectively contain the positive charge amino acid, CD can effectively contain the hydrophobic amino acid, and the CCA-CD as a co-assembly body has strong interaction on the melittin through the heteropoly recognition of the melittin. The CCA-CD strongly complexes melittin, spider venom peptide and snake venom peptide, so that the melittin, the spider venom peptide and the snake venom peptide cannot act with cell membranes, the cell membranes are protected from being damaged, and the cytotoxicity and hemolytic toxicity of the melittin, the spider venom peptide and the snake venom peptide are relieved. In addition, CCA-CD has good biocompatibility, which makes CCA-CD a possibility for clinical application.

The invention has the advantages and beneficial effects that:

the CCA-CD can effectively complex melittin, spider venom peptide and snake venom peptide, relieve cytotoxicity of melittin, spider venom peptide and snake venom peptide, and has the potential of treating toxic macromolecule poisoning. The CCA-CD has definite chemical structure of each component and good batch reproducibility, has high chemical stability and thermal stability, does not influence the activity of important protein in organisms, and has good biocompatibility.

Drawings

FIG. 1 shows the amino acid sequence of melittin and the measurement of the complexation of melittin by CCA-CD as a co-assembly by fluorescence titration. Wherein (a) is melittin amino acid sequence, (b) is fluorescence titration graph, and (c) is fluorescence titration data fitting curve and result.

FIG. 2 shows the amino acid sequence of spider venom peptide and the measurement of the complexation of the co-assembly CCA-CD to spider venom peptide by fluorescence titration. Wherein (a) is spider venom peptide amino acid sequence, (b) is fluorescence titration graph, and (c) is fluorescence titration data fitting curve and result.

FIG. 3 shows the amino acid sequence of snake venom peptide and the measurement of the complexation of the snake venom peptide by the co-assembly CCA-CD by fluorescence titration. Wherein (a) is snake venom peptide amino acid sequence, (b) is fluorescence titration graph, and (c) is fluorescence titration data fitting curve and result.

FIG. 4 is a CCK-8 assay to determine the relief effect of CCA-CD on the cytotoxicity of melittin, arachnid and snake venom peptides, and the cytotoxicity of CCA-CD itself. Wherein (a) is the effect of CCA-CD in alleviating melittin cytotoxicity, (b) is the effect of CCA-CD in alleviating arachidide cytotoxicity, (c) is the effect of CCA-CD in alleviating venom peptide cytotoxicity, and (d) is the cytotoxicity of CCA-CD itself. The significant difference analysis results are expressed as: p <0.05, p <0.01, p < 0.001.

FIG. 5 is a lactate dehydrogenase leakage experiment to verify the inhibitory effect of the co-assembly CCA-CD on the action of melittin and cell membranes. The significant difference analysis results are expressed as: p < 0.001.

FIG. 6 is a hemolysis experiment demonstrating the mitigating effect of the co-assembly CCA-CD on melittin hemolytic toxicity. The significant difference analysis results are expressed as: p < 0.001.

FIG. 7 is the survival rate of melittin poisoned mice over 24 hours with CCA-CD as an antidote injected at different times. Wherein (a) is the survival rate of the mice in the case of CCA-CD detoxification by injection immediately after melittin injection, and (b) is the survival rate of the mice in the case of CCA-CD detoxification by re-injection 10, 20 or 40 minutes after melittin injection. The significant difference analysis results are expressed as: p < 0.01.

FIG. 8 is a graph of the results of a routine injection of CCA-CD detoxified or non-detoxified blood in melittin poisoned mice. Wherein (a) is the number of leukocytes, (b) is the number of lymphocytes, (c) is the number of neutrophils, (d) is the number of erythrocytes, (e) is the hemoglobin content, and (f) is the hematocrit. The significant difference analysis results are expressed as: p <0.01, p < 0.001.

FIG. 9 shows the biochemical results of blood detoxification or non-detoxification of melittin poisoned mice injected with CCA-CD. Wherein (a) is aspartate aminotransferase content, (b) is alanine aminotransferase content, (c) is alkaline phosphatase content, (d) is total protein content, (e) is creatinine content, (f) is alpha-hydroxybutyrate dehydrogenase content, (g) is lactate dehydrogenase content, and (h) is creatine kinase MB type isozyme content. The significant difference analysis results are expressed as: p <0.05, p <0.01, p < 0.001.

FIG. 10 is the result of injection of CCA-CD detoxified or detoxified histopathological sections of melittin poisoned mice.

Detailed Description

The present invention is further illustrated by the following specific examples, which are intended to be illustrative, not limiting and are not intended to limit the scope of the invention.

A co-assembly CCA-CD capable of realizing heteropoly value identification has the potential of treating poisoning of macromolecular toxins such as melittin and the like. An amphiphilic calixarene CCA modified by carboxyl and amphiphilic cyclodextrin CD are taken as a construction unit of a heteropoly identification platform,

wherein CCA has the chemical formula C₁₀₀H₁₅₀O₁₅CD is of the formula C₁₈₂H₃₅₀O₅₆S₇The molar ratio of the two substances is 1:1, and the supermolecular assembly is constructed by weak pi-pi interaction and hydrophobic interaction, and the supermolecular assembly is a vesicle with nano-scale and spherical appearance. CCA and CD are of the formula:

the preparation steps are as follows:

(1) the CCA and the CD are respectively dissolved in chloroform according to the metering ratio, and are respectively prepared into 1-5 millimole per liter of CCA and CD chloroform solution (mother liquor).

(2) After 500. mu.l of CCA chloroform solution and 500. mu.l of CD chloroform solution were mixed well, they were placed in a vacuum dryer and vacuum-dried. After 4-5 hours, the organic solvent had evaporated completely, resulting in a uniform film of the mixture of CCA and CD on the walls of the container.

(3) 5 ml of buffer aqueous solution is added into a container, the container is placed in an ultrasonic instrument at the temperature of 75-80 ℃ for ultrasonic treatment for 2-4 hours, the solution becomes clear, the laser pen is used for irradiating the solution to have obvious Tyndall effect, and then a CCA-CD supermolecule co-assembly body is formed.

CCA-CD can realize strong bonding on three toxic polypeptides from bees, spiders and snakes, and has the function of relieving the cytotoxicity of the three toxic polypeptides.

Measurement of binding constant of CCA-CD to toxic polypeptide: the lucigenin is selected as a fluorescent probe, the lucigenin can be wrapped by CCA, the fluorescence of the lucigenin is quenched, and after the polypeptide is complexed with CCA, the lucigenin is replaced out of a CCA cavity, and the fluorescence is recovered. By the change in the fluorescence of lucigenin, we were able to quantitatively know the amount of polypeptide bound to the surface of CCA-CD. In addition, when hydrophobic amino acids on the polypeptide are bonded with CD, the distance between the positively charged amino acids and CCA is shortened, the amino acids are promoted to replace the lucigenin in CCA cavities, and therefore the interaction between the hydrophobic amino acids and CD can be represented by the change of the fluorescence of the lucigenin. And adding a mixed solution of lucigenin and CCA-CD into the quartz cell. Preparing a mixed solution containing lucigenin and CCA-CD with the same concentration as that in the quartz pool and high-concentration polypeptide. The solution was added dropwise to a quartz cell and the change in fluorescence (excitation wavelength 368 nm) before and after each addition was recorded. The fluorescence change at 507 nm was fitted using the competition fit formula, see: 1) d, S.Guo, V.D.Uzunova, X.Su, Y.Liu, W.M.Nau, chem.Sci.2011,2,1722-1734.2) G.Ghale, A.G.Lancet, H.T.Kreissl, M.H.Jacob, H.Weingart, M.Winterhalter, W.M.Nau, Angew.chem.int.Ed.2014,53,2762-2765. the complexation of GCA-CD to some interfering proteins, such as serum albumin, was determined using the same method. The CCA-CD co-assembly was extremely strong binding to the three polypeptides (fig. 1-3) with a binding constant at least 2 orders of magnitude greater than the other proteins assayed.

The mitigating effects of CCA-CD on cytotoxicity of three polypeptides: 293FT cells (human renal epithelial cell line) were cultured in 96-well plates. After attachment, a mixture of polypeptides (5 micromoles per liter melittin and 40 micromoles per liter for the other two polypeptides) or polypeptides (5 micromoles per liter for melittin and 40 micromoles per liter for the other two polypeptides) with different concentrations of CCA-CD was added to the cell culture medium. After culturing at 37 ℃ for 24 hours, the medium in the wells was discarded, and CCK-8 (cell proliferation assay kit) solution was added to continue culturing at 37 ℃ for 2 hours. The absorbance at 450 nm was measured by a microplate reader, and the survival rate of the cells in each well was calculated from the absorbance. Cell viability was scored as 100% using cells not incubated with the polypeptide as control groups. The presence of CCA-CD significantly ameliorated the cytotoxicity of these three toxic polypeptides, while CCA-CD itself was almost non-toxic (fig. 4).

Taking melittin as an example, the CCA-CD has further studied the effects of inhibiting melittin and cell membrane, relieving hemolytic toxicity, and treating melittin poisoning mice.

Inhibition of melittin disruption by CCA-CD: when melittin interacts with the cell membrane, it disrupts the cell membrane, resulting in the efflux of intracellular material. We examined the disruption of the cell membrane by melittin by detecting leakage of lactate dehydrogenase in the cells. 293FT cells were cultured in 96-well plates and after adherence, polypeptides (5. mu. mol/l) or a mixture of polypeptides (5. mu. mol/l) with CCA-CD at different concentrations was added to the cell culture medium. After incubation at 37 ℃ for 24 hours, the various reagents provided in the kit were added in sequence according to the instructions of the lactate dehydrogenase detection kit. The absorbance at 440 nm was measured by a microplate reader, and the leakage of lactate dehydrogenase was calculated. The presence of CCA-CD significantly eases the leakage of melittin treated cellular lactate dehydrogenase, suggesting that CCA-CD effectively inhibits melittin damage to cell membranes, while CCA-CD itself does not cause leakage of lactate dehydrogenase (FIG. 5).

Remission of CCA-CD on melittin hemolytic toxicity: blood from healthy BALB/c mice was placed in an anticoagulation tube and diluted with physiological saline. The mixture was centrifuged at 1500rpm for 10 minutes, and the red blood cells in the lower layer of the centrifuge tube were collected and washed four times with physiological saline. Subsequently, the red blood cells were diluted 50-fold with physiological saline for use. 29.4. mu.l of melittin or a mixed solution of melittin and CCA-CD was added to 700. mu.l of the above red blood cells, and incubated at 37 ℃ for 1.5 hours. Centrifuge at 2000rpm for 10 minutes. The supernatant was transferred to a quartz dish, and the absorbance at 545 nm was measured with an ultraviolet spectrophotometer to calculate the degree of hemolysis of the red blood cells. The presence of CCA-CD significantly ameliorated the hemolytic toxicity of melittin, while CCA-CD itself did not cause hemolysis of red blood cells (fig. 6).

Therapeutic effect of CCA-CD on melittin poisoned mice: 60 female BALB/c mice at 5-6 weeks were divided into 5 groups of 12 mice each. The first group of mice were injected intravenously with 50 μ l of melittin (600 μmol per liter, 4.30 mg per kg) at the tail. A second group of mice was injected with 50 microliters of melittin (600 micromoles per liter, 4.30 milligrams per kilogram) immediately followed by 150 microliters of CCA-CD (200 micromoles per liter, 7.25 milligrams per kilogram). The tail vein of mice in the third to fifth groups was injected with 50 μ l melittin (600 μmol per l, 4.30 mg per kg) and 150 μ l CCA-CD (200 μmol per l, 7.25 mg per kg) at 10 min, 20 min and 40 min intervals, respectively. After injection, mice were returned to cages and tested for 24 hours. After 24 hours, all the living mice of the first and second groups were sacrificed, their blood was collected for blood routine and blood biochemical tests, and their major organs (heart, liver, spleen, lung, kidney) were collected for histopathological analysis. The mortality rate of the first group of mice exceeded 50% and the mortality rate of the remaining four groups of mice was below 30% (fig. 7), indicating that CCA-CD has a very good in vivo detoxification effect on melittin. The blood routine results showed that the first group of mice all had a significant increase in the number of leukocytes, lymphocytes, neutrophils, erythrocytes, hemoglobin and erythrocyte volume, while the second group of mice had a significant decrease in these indices (fig. 8), indicating that CCA-CD successfully alleviated melittin-induced inflammation. The blood biochemical results showed that injection of CCA-CD significantly ameliorated the abnormal levels of aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total protein, creatinine, alpha-hydroxybutyrate dehydrogenase, lactate dehydrogenase, and creatine kinase MB-type isozymes in the blood of the poisoned mice (fig. 9), indicating that CCA-CD ameliorated melittin-induced liver, kidney, and heart damage. The results of the histopathological analysis also support this conclusion (fig. 10).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the inventive concept, and these changes and modifications are all within the scope of the present invention.