CN113278050B - Controllable disassembled and assembled supermolecule and preparation method and application thereof - Google Patents
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
- C07K5/1002—Tetrapeptides with the first amino acid being neutral
- C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y40/00—Manufacture or treatment of nanostructures
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
Abstract
The invention provides a controllable disassembled and assembled supermolecule and a preparation method and application thereof. The controlled disassembled supermolecule is nanofiber prepared by dephosphorylation and self-assembly of polypeptide supermolecule precursor, and amino acid motif in the polypeptide supermolecule precursor selects amino acid with D configuration; the polypeptide supermolecule precursor has a structure shown in a formula II. The controllable disassembled supermolecule provided by the invention can be used for delivering living drugs, can be stored for a long time at the temperature of minus 20 ℃, and solves the biological safety problem of polypeptide supermolecule assemblies in the current drug delivery system.
Description
Technical Field
The invention relates to the field of nano supermolecule materials, in particular to a controllable disassembled and assembled supermolecule and a preparation method and application thereof.
Background
A drug delivery system is a device for releasing a drug in a targeted and quantitative manner using a carrier or an active substance, which can improve the delivery efficiency, targeting property, absorption rate and reduce the use of the drug, and has been rapidly developed since it was proposed. Nanomaterials are considered as a main material used as drug delivery carriers, and the design and modification of nanomaterials make them suitable for different delivery environments, so that they have been widely researched. As more and more nanomaterials are used as delivery vehicles, the attendant problems of nanomaterial safety have also raised intense concerns. A large number of researches show that the nano material has biotoxicity and can exist in organisms for a long time to generate potential safety hazards. The mechanism of toxicity of nanomaterials is not completely understood, and most studies agree that the generation of Reactive Oxygen Species (ROS) is the most important toxic effect. Nanomaterials can stimulate the production of ROS, an imbalance between ROS production and clearance can lead to a variety of diseases, and nanomaterials can accumulate in various organs for a long period of time through blood circulation and can interact with cellular contents to cause cell death.
The delivery of most cancer therapeutic drugs is unsatisfactory, mainly due to insufficient accumulation of the drug at the tumor site, metabolism of most of the drug into the body through the metabolic system, and off-target toxicity occurring at sites other than the tumor site. Enzyme-catalyzed supramolecular self-assembly (EISA) has become a novel method of tumor diagnosis and treatment. Tumor cells generally have higher metabolic levels than normal cells and therefore overexpress certain enzymes. Enzymatic supramolecular self-assembly is based on the deregulation of enzymes in tumors, achieving the selective construction inside tumor cells. Bioorthogonal reactions are a class of rapid specific covalent reactions that can be performed in the physiological environment of living cells with little impact on physiological activity. There is a need to provide a novel controllably assembled supramolecular assembly to solve the problems of biological safety of polypeptide supramolecular assemblies in current drug delivery systems.
Disclosure of Invention
The invention provides a controlled disassembled supermolecule and a preparation method and application thereof. The controllable disassembled supermolecule provided by the invention can be used for delivering living drugs, can be stored for a long time at the temperature of minus 20 ℃, and solves the biological safety problem of polypeptide supermolecule assemblies in the current drug delivery system.
The invention provides a controllable disassembled supermolecule which has a structure shown as a formula I:
in the invention, the supermolecule can be controllably disassembled and assembled, so that the potential safety hazard caused by long-term existence of large-size assemblies in vivo is solved. The supermolecular assembly has good biocompatibility and certain selectivity, and has more accumulation and larger cytotoxicity in cancer cells. The assembly can still exist in the organism for a long time after cancer cells die, and the cytotoxicity generated by the assembly can be eliminated by disassembling the assembly, can be used for delivering medicaments to the living body, can be stored for a long time at the temperature of minus 20 ℃, and solves the biological safety problem of the polypeptide supramolecular assembly in the current medicament delivery system.
According to the controllable disassembled supermolecule provided by the invention, the controllable disassembled supermolecule is a nanofiber with the molecular weight of 1000-1300 g/mol, preferably 1028.1 g/mol; preferably, the diameter of the nanofibers is between 8nm and 15nm, preferably 10 nm.
The invention provides a preparation method of the controllable disassembled supermolecule, wherein the controllable disassembled supermolecule is prepared by dephosphorylation and self-assembly of a polypeptide supermolecule precursor; the polypeptide supramolecular precursor is a phosphorylated polypeptide supramolecular precursor modified by tetrazine.
In the invention, the detachable supramolecular precursor is designed and constructed, and can perform self-assembly through enzyme catalytic reaction to perform targeted delivery and enrichment of cargoes. Followed by the release of the cargo and the disassembly of the assembly by bio-orthogonal reactions. In particular, enrichment of targeted cancer cells using Tz-modified prodrug supramolecules with weak assembly forces, and subsequent addition of TCO can bio-orthogonally react with Tz-coupled prodrugs to release cargo and create environmental perturbations. This perturbation does not affect normal physiological activities but is sufficient to disrupt the pi-pi stacking of the major intermolecular forces of the assembly, breaking the assembly down into small molecules that can be metabolized from the body. The release and spontaneous disintegration of the cargo from such an assembly is controllable.
According to the method for preparing the controlled disassembled supramolecules provided by the invention, the polypeptide supramolecular precursor comprises Tz- D F D F D Yp D K (tz) -OH; preferably, the polypeptide supramolecular precursor has a structure shown in formula II:
according to the invention, the tetrazine (Tz) modified supermolecular precursor can be subjected to phosphorylation under the catalysis of alkaline phosphatase, self-assembly is carried out, hydrogel with a nanofiber structure is formed, and enrichment is carried out in cells. Then trans-cyclooctene (TCO) is added, cargo can be released through the bioorthogonal reaction between Tz and TCO, environmental disturbance is generated to destroy the pi-pi accumulation of the main intermolecular force of the assembly, and the large-size assembly can be disassembled into small molecules to be metabolized. Among them, phenylalanine (F) is an aromatic amino acid capable of forming intermolecular π - π stacking. The greater the number of phenylalanines in the polypeptide sequence, the stronger the assembly ability and the more stable the aggregates formed. In addition, the FF sequence is also the core sequence of Alzheimer disease-causing amyloid. Phosphorylated tyrosines are capable of specifically responding to highly expressed phosphatases of tumor cells. Lysine can provide an additional amino group on a polypeptide branch for coupling to another Tz molecule. Thus, two Tz groups will be present on one prodrug molecule, and twice the amount of reaction will result in more drastic environmental perturbation and better disassembly than if only one Tz were orthogonally reacted on the molecule. The amino acid motif of the polypeptide is selected from D configuration amino acid, and researches show that D configuration based polypeptide supramolecular assembly can exist more stably in organisms because enzymes for degrading D configuration amino acid are lacked in organisms. Cancer cell-targeted drug delivery or treatment using polypeptides based on D-configuration amino acids can cause higher damage to cancer cells. But the same fact is that the assemblies remaining in the body can be more toxic. The detachable supramolecules designed by the invention can be used as a delivery carrier more stably, and meanwhile, the biotoxicity remained in the body can be eliminated controllably.
According to the method for preparing the controlled disassembled supramolecules, the preparation of the polypeptide supramolecular precursor comprises the following steps: synthesizing a target polypeptide sequence by adopting a solid phase synthesis method, and then coupling a tetrazine functional group on the target polypeptide sequence.
In the invention, the enzyme-catalyzed supramolecular self-assembly is adopted as a carrier for in-vivo delivery, so that the delivery efficiency and targeting property of goods can be improved, and the drug use or off-target toxicity is reduced; particularly, by adopting the supermolecule controllable solution and assembly, the technical problems of long-term retention and metabolism of the nano material in a living body can be better solved, so that the biological toxicity hidden danger of the nano material is eliminated, and the release efficiency and accuracy of goods are further improved by adopting biological orthogonal reaction.
The invention provides a controlled disassembly and assembly method of supermolecules, which comprises the following steps:
1) performing self-assembly on the polypeptide supramolecular precursor under the catalytic action of alkaline phosphatase to obtain hydrogel;
2) TCO-Gly is added into the hydrogel, and controllable disassembly and assembly are carried out through bioorthogonal reaction.
In the invention, the polypeptide supermolecule precursor is dephosphorylated in the presence of alkaline phosphatase, so that a nanofiber structure is formed by self-assembly, and pi-pi accumulation among molecules can be destroyed through bio-orthogonal reaction so as to disassemble an assembly. The disassembly method of the invention has wide applicability, and other polypeptide sequences with weak assembly force are also suitable. Other polypeptide molecule sequences tested by the inventors such as FYpK, FFYpK, D F D Yp D K also successfully achieves enzyme-catalyzed self-assembly and bio-orthogonal reaction-induced disassembly.
According to the method for disassembling the controllable disassembled supermolecule, the hydrogel is the controllable disassembled supermolecule, and the molecular structure obtained after the bioorthogonal reaction is as shown in a formula III:
according to the invention, the bioorthogonal reaction between tetrazine (Tz) and trans-cyclooctene (TCO) is a rapid specific covalent reaction, which proceeds in the physiological environment of living cells and has little effect on physiological activities. The molecule based on the bioorthogonal reaction is specifically marked, so that the precise release of goods, the real-time tracking of physiological activities and the super-resolution observation of living cells can be realized. In the invention, supermolecules can generate dephosphorylation under the existence of alkaline phosphatase so as to self-assemble to form a nanofiber structure, and pi-pi accumulation among molecules can be destroyed through bio-orthogonal reaction so as to disassemble an assembly.
According to the controlled disassembly and assembly supermolecule disassembly and assembly method provided by the invention, the critical gelling concentration of the hydrogel is 0.8-1.5 mg/ml, preferably 1 mg/ml.
According to the method for disassembling the controllable disassembled supermolecule, step 1), the polypeptide supermolecule precursor is dissolved in PBS (phosphate buffer solution) at the concentration of 2mg/ml, and the pH value is adjusted to 7.4 by using 1M NaOH; adding 20U/ml alkaline phosphatase, and standing at room temperature for at least 2h to obtain hydrogel; and/or, in the step 2), TCO-Gly is added into the hydrogel, and the hydrogel is placed at room temperature overnight; the molar ratio of the hydrogel to the TCO-Gly is at least 1: 2.
The invention provides the use of said controllably disassembled supramolecules or the method of disassembly of said controllably disassembled supramolecules in the delivery of drugs for the treatment of cancer.
In the invention, the supermolecule nano material is used for delivering the material targeting cancer, and simultaneously, the problem of toxic and side effects of the nano assembly body on organisms is solved. The invention solves the potential toxicity problem of the assembly body through the controllable disassembly of the assembly body. The invention provides a method for destroying an assembly body by generating environmental disturbance through bioorthogonal reaction, and provides a new idea for the controllable disassembly of materials with similar nanofiber structures.
The invention has the beneficial effects that: most cancer-targeting polypeptide supramolecular self-assembly has certain off-target toxicity, and the assembly can exist in vivo for a long time and is degraded or metabolized slowly. The controllable disassembled supermolecule provided by the invention can be used for delivering living drugs, can be stored for a long time at the temperature of minus 20 ℃, and solves the biological safety problem of polypeptide supermolecule assemblies in the current drug delivery system. The invention solves the potential toxicity problem of the assembly body through the controllable disassembly of the assembly body.
Drawings
In order to more clearly illustrate the technical solutions of the present invention or the prior art, the drawings needed for the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and those skilled in the art can also obtain other drawings according to the drawings without creative efforts.
FIG. 1 is a NMR spectrum of a supramolecular precursor of a polypeptide in an embodiment of the invention;
FIG. 2 is a mass spectrum (orthophotom) of a supramolecular precursor of a polypeptide in an embodiment of the invention;
FIG. 3 is a diagram showing the change of molecular structure during the dynamic assembly of polypeptide supramolecules in the embodiment of the invention;
FIG. 4 is a schematic diagram of the dynamic assembly process of polypeptide supramolecules in an embodiment of the invention;
fig. 5 is a schematic diagram showing the experimental results of supramolecular hydrogel in the experimental example of the present invention (a. phosphorylated supramolecular precursor, b. enzyme catalyzed self-assembly to form stable hydrogel, c. bio-orthogonal reaction induced disassembly);
FIG. 6 is a schematic diagram showing the experimental results of a transmission electron microscope (B compound on the left and C compound on the right) in the experimental example of the present invention;
FIG. 7 is a diagram showing the results of fluorescence measurement of THT dye in the experimental example of the present invention;
FIG. 8 is a schematic diagram of a rotational rheological test result in an experimental example of the present invention;
FIG. 9 is a graph showing the results of cell accumulation experiments in the experimental examples of the present invention;
FIG. 10 is a diagram showing the results of cytotoxicity experiments in the experimental examples of the present invention;
FIG. 11 is a diagram showing the results of cytotoxicity experiments in the experimental examples of the present invention;
FIG. 12 is a diagram showing the results of cytotoxicity experiments in the experimental examples of the present invention;
FIG. 13 is a graph showing the results of detecting cell viability in the experimental examples of the present invention;
FIG. 14 is a diagram illustrating the results of detecting cell viability in the experimental examples of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is obvious that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be obtained by a person skilled in the art without inventive step based on the embodiments of the present invention, are within the scope of protection of the present invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The instruments and the like are conventional products which are purchased by normal distributors and are not indicated by manufacturers. The process is conventional unless otherwise specified, and the starting materials are commercially available from the open literature. The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications.
Example 1
This example provides a controlled disassembled supramolecule with molecular weight of 1028.1 g/mol; the supermolecule assembled in a controllable way is nanofiber with the diameter of 10 +/-1 nm, and has the structure shown in the formula I:
the present invention also provides a polypeptide supramolecular precursor for preparing the above-mentioned controllably disassembled supramolecules, having a structure as shown in formula ii:
the present invention also provides a method for preparing the supramolecular precursor of the polypeptide, which comprises the following steps:
1) synthesizing a target polypeptide sequence by a solid phase synthesis method;
2) Tz-NHS was mixed with polypeptide and N, N-Diisopropylethylamine (DIPEA) at a ratio of 1.6: 2: 1 is dissolved in trichloromethane, and stirred for 24 hours at the temperature of 30 ℃;
3) purifying by semi-preparative HPLC (phase A water, phase B acetonitrile, both mobile phases containing one thousandth of TFA), lyophilizing by a lyophilizer to obtain light purple powdery supramolecular precursor, and storing at-20 deg.C for a long time;
4) determining whether the obtained molecule is a target molecule or not by nuclear magnetic resonance and mass spectrometry;
5) (E) -Cyclooct-2-enol (TCO), pyridine and 4-nitrophenylchloroformate in a molar ratio of 4:14:7 in 25mL of CH in an ice-water bath 2 Cl 2 The preparation method comprises the following steps of (1) performing; the reaction was stirred at room temperature overnight and the reaction was purified by column chromatography (PE: EA ═ 10: 1) to give (E) -cyclooct-2-en-1-yl (4-nitrophenyl) carbonate as a pale yellow solid. (E) -cyclooct-2-en-1-yl (4-nitrophenyl) carbonate and glycine were dissolved in 2mL of TFA/H in a molar ratio of 1:2 2 And (4) in O. The mixture was stirred at 30 ℃ overnight, the solvent of the product was removed by rotary evaporator and the residual liquid was purified by column chromatography (PE: EA ═ 5: 1) to give TCO-Gly.
The nuclear magnetic and mass spectrum results of the supramolecular precursor of the polypeptide in the example are shown in FIGS. 1-2;
fig. 2-3 are schematic diagrams illustrating the dynamic assembly process of polypeptide supramolecules in the examples.
The embodiment of the invention provides a method for disassembling supermolecule in a controllable way, which comprises the following specific steps: the (Tz-conjugated) polypeptide supramolecular precursors were dissolved in PBS at a concentration of 2mg/ml, adjusted to pH 7.4 with 1M NaOH. Adding alkaline phosphatase 20U/ml into the solution, standing at room temperature for more than 2h, and sufficiently dephosphorylating to obtain hydrogel. A slight excess (molar ratio of hydrogel to TCO-Gly of at least 1:2) of TCO-Gly was added to the hydrogel to allow for sufficient bioorthogonal reaction and left overnight at room temperature. The purple stabilized hydrogel faded to a colorless transparent solution with good fluidity, and fig. 4 shows the hydrogel experimental results (a. phosphorylated supramolecular precursor, b. enzyme-catalyzed self-assembly to form stabilized hydrogel, c. bio-orthogonal reaction induced disassembly).
Experimental example 1
The supramolecules provided in example 1 were subjected to the following experiments:
1. transmission electron microscope experiment: a large number of nanofiber structures exist in the stable hydrogel through the preparation of a transmission electron microscope sample for the compound B and the compound C in the figure 4 and the observation of the transmission electron microscope, and the nanofiber structures cannot be seen in the sample prepared by the compound C under the electron microscope. (in FIG. 6, TEM images of Compound B (left) and Compound C (right) of sample B).
2. And (3) testing by a fluorescence spectrometer: thioflavin t (tht) is a commonly used fluorescent dye for labeling the beta-sheet structure of amyloid fibrils. THT produces strong fluorescence after binding to amyloid fibers and little fluorescence in aqueous solution. The dye can be excited at 440nm to produce a maximum emission wavelength around 480 nm. 10 μ M THT was added to the sample of FIG. 5.B, the excitation wavelength was set at 440nm, and the emission wavelength detection range was 460-490 nm. As shown by the solid line in fig. 7 (fluorescence test of THT dye), THT slightly blue-shifted after mixing with the assembly, and a significant increase in fluorescence intensity around 475nm evidences the presence of the nanofiber structure. Whereas in the sample of fig. 5. C10 μ M THT was added and the peak at 475nm was drawn and disappeared, demonstrating the disappearance of the nanofibrous structure. As a control, an aqueous solution of THT showed no significant fluorescence upon excitation at a wavelength of 440nm as shown by the following dotted line.
3. Rotational rheology experiments: the rotational rheological experiment can detect the mechanical strength of a substance, and the change of the microstructure can be reflected through the mechanical strength. As shown in fig. 8, the left side shows the test results of the sample of fig. 5.B, which has a dynamic storage modulus (G') one order of magnitude higher than the dynamic loss modulus (G "), indicating that the supramolecular precursor forms a stable hydrogel with nanofiber structure through the enzyme-catalyzed self-assembly and the hydrogel has a certain mechanical strength. The right panel is the test results for the sample of fig. 5.C, which shows a sharp drop in mechanical strength and comparable dynamic storage modulus (G') as dynamic loss modulus (G "), demonstrating destruction of the nanofiber structure.
4. Cell accumulation experiments: next, the invention selects the cervical cancer HeLa cell and the uterine epithelial cell HcerEpic cell to detect the accumulation of supramolecular compounds in different cells, the left graph in figure 9 shows the accumulation relationship of the compounds with incubation concentration, and the right graph shows the accumulation relationship of different compounds with time. The compound can be found to have certain selectivity and higher accumulation in cancer cells with high expression of phosphatase. The amount accumulated is directly proportional to the incubation time and incubation concentration. After 6h incubation of the cells with the compound, the maximum accumulation was essentially reached.
5. Cytotoxicity test: the toxicity of the four compounds on different cells was examined using the MTT method, and cervical cancer cells (HeLa) and uterine epithelial cells (HcerEpic) were also selected for testing. The resulting absorbance data were processed and analyzed by Origin software. The calculation formula used was (experimental-zeroed wells)/(control-zeroed wells). The results are shown in FIGS. 10-12, where cytotoxicity increased with increasing compound incubation concentration and incubation time. Polypeptide supramolecular peptides based on amino acids in D configuration have significant cytotoxicity to both cells after 24h incubation, since intracellular assemblies destroy cellular structures. The more assemblies formed, the longer the assemblies are present and the lower the survival rate of the cells.
6. The disassembly solves the cytotoxicity problem: the invention proves that the designed supermolecule has certain cell selectivity through previous experiments and can be self-assembled in cells, and the formed assembly can destroy organelles in the cells to cause cell death. Cell viability was also measured by the MTT method under different incubation conditions, as shown in fig. 13-14. A represents cells incubated in the medium as a blank control, B represents cells incubated with 200. mu.M TCO-Gly for 24 h; c is that the uterine epithelial cells and HeLa cells are incubated for 24h by using a compound culture medium containing 100 mu M, both cells obviously die, and the cell survival rate is obviously higher than that of a group without TCO-Gly (column D) when TCO-Gly is added for incubation for 24h after the cells are incubated for 6h by using the compound, which indicates that the cell toxicity generated by the assembly body can be solved by the disassembly.
Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
Claims (7)
1. A controlled disassembled supramolecule, which is a nanofiber prepared by dephosphorylation and self-assembly of a polypeptide supramolecular precursor, wherein an amino acid motif in the polypeptide supramolecular precursor selects an amino acid with a D configuration; the polypeptide supramolecular precursor has a structure shown in a formula II:
2. the controllably disassembled supramolecule as claimed in claim 1, wherein said nanofibers have a diameter of 8-15 nm.
3. The controllably disassembled supramolecules as claimed in claim 2, characterized in that said nanofibers have a diameter of 10 nm.
4. A method for the preparation of controllably disassembled supramolecules as claimed in claims 1 to 3, characterized in that said controllably disassembled supramolecules are prepared by autophosphorylation of precursor supramolecules of said polypeptides.
5. Method for the preparation of controllably disassembled supramolecules as claimed in claim 4, characterized in that the preparation of polypeptide supramolecular precursors comprises: synthesizing a target polypeptide sequence by adopting a solid phase synthesis method, and then coupling a tetrazine functional group on the target polypeptide sequence.
6. A method for controlled disassembly of supramolecules as claimed in any of claims 1 to 5, comprising:
1) the polypeptide supermolecule precursor is subjected to self-assembly under the catalysis of alkaline phosphatase to obtain hydrogel;
2) TCO-Gly is added into the hydrogel, and controllable disassembly and assembly are carried out through a bioorthogonal reaction;
in the step 1), the polypeptide supramolecular precursor is dissolved in PBS (phosphate buffer solution) at the concentration of 2mg/ml, and the pH is adjusted to 7.4 by using 1M NaOH; adding 20U/ml alkaline phosphatase, and standing at room temperature for at least 2h to obtain hydrogel; in the step 2), TCO-Gly is added into the hydrogel, and the hydrogel is placed at room temperature overnight; the molar ratio of the hydrogel to the TCO-Gly is 1: 2.
7. A method of controlled disassembly of supramolecules as claimed in claim 6, characterized in that said hydrogel has a critical gel forming concentration of 1.0 mg/ml.
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