CN112876673B - PH-responsive nano copolymer carrier and preparation method and application thereof - Google Patents
PH-responsive nano copolymer carrier and preparation method and application thereof Download PDFInfo
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- CN112876673B CN112876673B CN202110101524.XA CN202110101524A CN112876673B CN 112876673 B CN112876673 B CN 112876673B CN 202110101524 A CN202110101524 A CN 202110101524A CN 112876673 B CN112876673 B CN 112876673B
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
- C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
- C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
- C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
- C08G69/10—Alpha-amino-carboxylic acids
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/445—Non condensed piperidines, e.g. piperocaine
- A61K31/4515—Non condensed piperidines, e.g. piperocaine having a butyrophenone group in position 1, e.g. haloperidol
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- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
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- A61K41/0052—Thermotherapy; Hyperthermia; Magnetic induction; Induction heating therapy
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- 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
- A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
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- A—HUMAN NECESSITIES
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- 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
- A61K47/56—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
- A61K47/58—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 macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
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- A61K49/0017—Fluorescence in vivo
- A61K49/0019—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules
- A61K49/0021—Fluorescence in vivo characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules the fluorescent group being a small organic molecule
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Abstract
The invention relates to the technical field of medicines, in particular to a pH-responsive nano-copolymer carrier and a preparation method and application thereof. The HP @ PHIs/LC NPs provided by the invention are used for preparing a tumor microenvironment targeted preparation, and expression of cancer stem cells is detected, so that the nano-carrier can be dissociated in response to tumor tissues to release HP, and accurate position release is realized to inhibit the cancer stem cells; the results of anti-tumor experiments show that the HP @ PHIS/LC NPs nano preparation greatly improves the anti-tumor effect, effectively blocks cells from epithelial-mesenchymal transition, reduces the invasion and metastasis of tumors, and relieves the tolerance of photodynamic therapy.
Description
Technical Field
The invention relates to the technical field of medicines, in particular to a pH-responsive nano-copolymer carrier and a preparation method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Tumor stem cells are a subset of very small stem cell-like cancer cells with unlimited proliferation potential in tumor tissue, and are a group of tumor cells with the ability to self-renew, initiate and reconstitute the tumor tissue phenotype. The normal tumor cells are in a balanced state of self-renewal and differentiation, but the tumor stem cells are in an unbalanced state and have strong self-renewal capacity, thereby remarkably promoting the growth of tumors. Tumor stem cells also play an important role in tumor invasion, metastasis and drug resistance. Tumor invasion and metastasis refer to the process in which tumor cells are shed from primary sites and tumors of the same nature grow out at different target sites through blood circulation. However, in addition to the tumor stem cells inherent in the tumor tissue, epithelial-mesenchymal transition during tumor progression also induces the generation of tumor stem cells, thereby promoting tumor growth, invasion and metastasis.
Epithelial-mesenchymal transition refers to the transition from epithelial cells to mesenchymal cells, plays a key role in the development process of tumors, and not only endows the tumor cells with the capabilities of metastasis and invasion, but also obtains the characteristics of stem cells. On one hand, the characteristics of the tumor cells can be changed, so that the expression of the connecting molecules among the tumor cells is lost, the polarity is lost, the adhesion is weakened, the motility is improved, and the invasion capacity of the tumor cells is enhanced; on the other hand, epithelial-mesenchymal transition can also endow the characteristics of tumor cell stem cells and promote the generation of the tumor stem cells. The inventor researches and finds that the cancer stem cells can be completely eliminated only by inhibiting the two processes of the intrinsic cancer stem cells and the epithelial-mesenchymal transition, and the invasion and the metastasis of tumors are avoided.
Disclosure of Invention
In order to solve the problems of single method for eliminating or inhibiting cancer stem cells and unobvious effect of reducing tumor growth and metastasis in the prior art, the invention provides a pH responsive nano copolymer carrier, a preparation method and application thereof, and a multifunctional responsive nano drug delivery system with high drug loading capacity is prepared, so that clinical application of eliminating tumor stem cells and reducing tumor growth and metastasis is realized.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the present invention, a pH-responsive nano-copolymer carrier is provided, which has a chemical structural formula as follows:
the value of n ranges from 1 to 16.
In a second aspect of the present invention, a method for preparing a pH-responsive nano-copolymer carrier is provided, wherein an amino group of polyethyleneimine is used as an initiator to polymerize N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine to form the pH-responsive nano-copolymer carrier, namely polyhistidine PHis.
In a third aspect of the present invention, a pH-responsive nanomicelle comprising a pH-responsive nanocopolymer carrier and a cancer stem cell inhibitor is provided.
In the fourth aspect of the present invention, a method for preparing a pH-responsive nanomicelle is provided, wherein the pH-responsive nanomicelle is obtained by self-assembling a pH-responsive nano copolymer carrier and a cancer stem cell inhibitor in water by a dialysis method.
In the fifth aspect of the invention, a hierarchical responsive nano-composite is provided, wherein the pH-responsive nano-micelle is used as the core of the composite, and the copolymer of low-molecular heparin and chlorin e6 is used as the shell of the composite.
In a sixth aspect of the present invention, a preparation method of a hierarchical responsive nanocomposite is provided, wherein a copolymer of low molecular heparin and chlorin e6 is adsorbed on the surface of a pH responsive nanomicelle through an electrostatic effect, i.e., the hierarchical responsive nanocomposite is provided.
In a seventh aspect of the invention, there is provided a nano-formulation comprising a graded-response nano-composite.
In the eighth aspect, the invention provides a pH-responsive nano copolymer carrier, a pH-responsive nano micelle and an application of a graded-responsive nano composite in preparation of an anti-tumor medicament.
In an eighth aspect, the present invention provides an application of haloperidol in promoting differentiation of cancer stem cells into tumor cells.
One or more embodiments of the present invention have the following advantageous effects:
(1) the invention firstly uses polyethyleneimine to polymerize N- (tert-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine to synthesize the pH-responsive nano copolymer carrier PHIs. The carrier overcomes the defects that N- (tert-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine is strong in hydrophobicity and cannot carry medicine, can be protonated in an acid environment to complete the conversion from hydrophobicity to hydrophilicity, realizes accurate target point release of loaded medicine, realizes long-circulation and tumor microenvironment responsive release, and plays an anti-tumor role in a long time.
(2) The invention firstly uses the copolymer (LC) of the nano-carrier low-molecular heparin with negative electricity and chlorin e6 to be adsorbed on the surface of the pH responsive nano-micelle through static electricity to synthesize the hierarchical responsive nano-composite. The preparation not only realizes the internal inhibition of cancer stem cells and the external induction of the cancer stem cells, but also effectively realizes the epithelial-mesenchymal transition blocking and photodynamic killing effects, inhibits the growth, invasion and metastasis of tumors, and realizes the in vivo tracking of the medicine through the fluorescence signal of Ce 6.
(3) The pH-responsive nano HP @ PHIs micelle prepared by the method has high drug loading, the drug loading of HP to PHIs is up to 33.0% when the mass ratio of HP to PHIs is 5:1, and the pH-responsive nano HP @ PHIs micelle is easy to transport and store and provides favorable conditions for industrial storage.
(4) The nano preparation prepared by the invention has uniform shape, the grain diameter is less than 200nm, and the nano preparation is suitable for intravenous injection and can be accumulated in tumor parts through passive targeting effect.
(5) The hierarchical responsive nano-composite prepared by the invention has pH responsiveness and can realize multi-target administration.
(6) Experiments show that when the cancer stem cell inhibitor is Haloperidol (HP), the prepared HP @ PHIs/LC has a strong cancer stem cell inhibiting effect, and the preparation has good biocompatibility and a strong epithelial-mesenchymal transition blocking effect.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention. Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:
FIG. 1 is a nuclear magnetic spectrum of polyhistidine of example 1 of the present invention;
FIG. 2 is a graph showing the experimental characterization of HP @ PHIs/LC for inhibiting cancer stem cells in example 3 of the present invention;
FIG. 3 is a graph showing the change in tumor volume in the in vivo antitumor effect of HP @ PHIs/LC in example 4 of the present invention;
FIG. 4 is a graphical representation of tumor weight in the in vivo anti-tumor effect of HP @ PHIs/LC of example 4 of the present invention;
FIG. 5 is a graph showing the experimental characterization of HP @ PHIs/LC as inhibiting epithelial-mesenchymal transition in vivo in example 5 of the present invention.
Detailed Description
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not noted in the following examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of exemplary embodiments according to the invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
In order to solve the problems of single method for eliminating or inhibiting cancer stem cells and unobvious effect of reducing tumor growth and metastasis in the prior art, the invention provides a pH responsive nano copolymer carrier, a preparation method and application thereof, and a multifunctional responsive nano drug delivery system with high drug loading capacity is prepared, so that clinical application of eliminating tumor stem cells and reducing tumor growth and metastasis is realized.
Specifically, the invention is realized by the following technical scheme:
in a first aspect of the present invention, a pH-responsive nano-copolymer carrier is provided, which has a chemical structural formula as follows:
the value of n is in the range of 1 to 16, preferably 4 to 16.
The method synthesizes the pH responsive nano copolymer carrier PHIS by polymerizing N- (tert-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine with polyethyleneimine for the first time. The carrier not only overcomes the defects that N- (tert-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine is strong in hydrophobicity and can not carry medicine, but also can complete the conversion from hydrophobicity to hydrophilicity by protonation in an acid environment, and realizes accurate target point release of the loaded medicine.
In a second aspect of the present invention, a method for preparing a pH-responsive nano-copolymer carrier is provided, wherein an amino group of polyethyleneimine is used as an initiator to polymerize N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine to form the pH-responsive nano-copolymer carrier, namely polyhistidine PHis.
The experimental process is as follows:
preferably, the polyethyleneimine is branched polyethyleneimine, derived from michelin, model 25987-06-8, and has the structural formula:
In one or more embodiments of the invention, N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine is dissolved in a first solvent, thionyl chloride is added, precipitation and centrifugation are performed to obtain N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride, N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride is dissolved in a second solvent, stirring is performed under a protective atmosphere, polyethyleneimine is added for reaction, and impurity removal, dialysis and freeze-drying are performed to obtain the compound;
preferably, the first solvent is selected from the group consisting of tetrahydrofuran, dimethyl sulfoxide, N, N dimethylformamide, N, N dimethylacetamide,
preferably, the molar ratio of N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine to thionyl chloride is 0.5-2.5:8-15, preferably 2-3:11, more preferably 2.37: 11;
preferably, the precipitation process uses diethyl ether for precipitation;
preferably, the centrifugation step further comprises a purification step of dissolving the crude N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride salt with acetone and precipitating with diethyl ether to obtain purified N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride salt;
preferably, the molar ratio of N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic acid anhydride hydrochloride to polyethyleneimine is from 4:1 to 6:1, preferably 5: 1;
preferably, the second solvent is selected from tetrahydrofuran, dimethyl sulfoxide, N dimethylformamide, N dimethylacetamide;
preferably, the stirring is room temperature stirring, and the stirring time is 2-4 days, preferably 3 days;
preferably, the step of removing impurities is removing Boc group, i.e., t-butyloxycarbonyl group, using 2-mercaptoethanol.
In a third aspect of the present invention, a pH-responsive nanomicelle comprising a pH-responsive nanocopolymer carrier and a cancer stem cell inhibitor is provided.
The mass ratio of the cancer stem cell inhibitor to the pH-responsive nano-copolymer carrier is 1:1-6:1, preferably 5: 1.
Preferably, the cancer stem cell inhibitor is selected from haloperidol, chlorpromazine, trifluoperazine and perphenazine, preferably haloperidol.
Preferably, the pH-responsive nano-copolymer carrier entraps the cancer stem cell inhibitor by hydrophobic interaction to synthesize the pH-responsive nano-micelle.
When the cancer stem cell inhibitor is haloperidol, the prepared pH-responsive nano-micelle is HP @ PHIs.
In the fourth aspect of the present invention, a method for preparing a pH-responsive nanomicelle is provided, wherein the pH-responsive nanomicelle is obtained by self-assembling a pH-responsive nano copolymer carrier and a cancer stem cell inhibitor in water by a dialysis method.
In one or more embodiments of the invention, the cancer stem cell inhibitor solution and the pH-responsive nano copolymer carrier solution are mixed and stirred, water is added for reaction, and pH-responsive nano micelles are obtained by water dialysis;
preferably, the solvent of the cancer stem cell inhibitor solution is dimethyl sulfoxide;
preferably, the solvent of the pH-responsive nano-copolymer carrier solution is formamide;
preferably, the mass ratio of the cancer stem cell inhibitor to the pH-responsive nano-copolymer carrier is 1:1-6:1, preferably 5: 1.
Preferably, the cancer stem cell inhibitor is selected from haloperidol, chlorpromazine, trifluoperazine and perphenazine, preferably haloperidol;
preferably, the water adding mode is dropwise water adding;
preferably, the reaction time is from 1 to 3 hours, preferably 2 hours,
preferably, the water dialysis is performed with a diameter of 0.45mm3The aqueous membrane is dialyzed to obtain pH-responsive nano-micelles with uniform sizes.
The cancer stem cell inhibitor is encapsulated by the pH responsive nano copolymer carrier, so that the problems that N- (tert-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine is high in hydrophobicity and cannot carry medicine can be solved, the conversion from hydrophobicity to hydrophilicity can be completed by protonation in an acidic environment, and the cancer stem cell inhibitor can be released at an accurate target point.
In the fifth aspect of the invention, a hierarchical responsive nano-composite is provided, wherein the pH-responsive nano-micelle is used as the core of the composite, and the copolymer of low-molecular heparin and chlorin e6 is used as the shell of the composite.
Preferably, the core pH-responsive nanomicelle of the hierarchical responsive nanocomposite is positively charged and the copolymer of the shell low molecular heparin and chlorin e6 is negatively charged;
preferably, the mass ratio of the low-molecular heparin and chlorin e6 copolymer to the pH-responsive nano-micelle is 4:1-1:1, and preferably 2: 1.
In a sixth aspect of the present invention, a preparation method of a hierarchical responsive nanocomposite is provided, wherein a copolymer of low molecular heparin and chlorin e6 is adsorbed on the surface of a pH responsive nanomicelle through an electrostatic effect, i.e., the hierarchical responsive nanocomposite is provided.
In one or more embodiments of the invention, in the copolymer (LC) of low molecular heparin and chlorin e6, the mass ratio of the low molecular heparin to chlorin e6 is 1:1 to 3:1, preferably 2: 1;
the preparation method of the copolymer of low molecular weight heparin and chlorin e6 comprises the following steps: chlorin e6, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved in an organic solvent and stirred. Adding 4-Dimethylaminopyridine (DMAP) and Low Molecular Weight Heparin (LMWH), reacting for a period of time, dialyzing, and lyophilizing to obtain LMWH-Ce6 (LC).
Preferably, the molar ratio of chlorin e6, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) is 5-10:13-16:15-20, preferably 8:15.6: 17.4;
preferably, the organic solvent is selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, dimethylsulfoxide.
Preferably, the agitation is at N2Stirring for 3-6 hours, preferably 4 hours;
preferably, the mass ratio of the 4-dimethylaminopyridine DMAP to the low molecular heparin LMWH is 12.217: 100;
preferably, the dialysis is a dialysis reaction by using N, N-dimethylformamide and water;
preferably, the mass ratio of the low-molecular heparin and chlorin e6 copolymer to the pH-responsive nano micelle is 4:1-1:1, preferably 2: 1;
preferably, the pH-responsive nanomicelle is haloperidol @ pH-responsive nano copolymer carrier, i.e., HP @ PHIs;
preferably, the incubation temperature is 37 ℃ and the time is 10-50min, preferably 20 min;
preferably, the centrifugation parameters are 8000-.
The invention synthesizes the hierarchical responsive nano-composite HP @ PHIs/LC by using the negatively charged molecular heparin and chlorin e6 copolymer (LC) to be adsorbed on the surface of amphiphilic nano-micelle HP @ PHIs through static electricity. The preparation not only realizes the internal inhibition of cancer stem cells and the external induction of the cancer stem cells, but also effectively realizes the epithelial-mesenchymal transition blocking and photodynamic killing effects, and inhibits the growth, invasion and metastasis of tumors.
In a seventh aspect of the invention, there is provided a nano-formulation comprising a graded-response nano-composite.
In one or more embodiments of the invention, the nano-formulation is an intravenous formulation.
In the eighth aspect, the invention provides an application of the pH-responsive nano copolymer carrier and/or the pH-responsive nano micelle and/or the graded-responsive nano composite in preparing an anti-tumor medicament.
Preferably, the antineoplastic drug is selected from alkylating agents, alkaloids, antibacterial and antineoplastic sulfonamides, platinum drugs and antimetabolites;
preferably, the antineoplastic agent further comprises a phototherapy agent selected from indocyanine green, neoindocyanine green, chlorin e6, IR780 iodide.
In an eighth aspect, the present invention provides an application of haloperidol in promoting differentiation of cancer stem cells into tumor cells.
Tumors cannot be completely eradicated under various therapeutic strategies due to the presence of cancer stem cells. In addition, the tumor stem cells are in a non-equilibrium state, have strong self-renewal capacity and weak differentiation capacity, and play an important role in driving tumor invasion, metastasis and drug resistance. Cancer stem cells are far more dangerous than tumor cells, so that the cancer stem cells are eradicated by a promising strategy of differentiating the cancer stem cells into the tumor cells, and then the tumor cells are completely eliminated under the action of phototherapy. The invention discovers that the haloperidol can promote cancer stem cells to be differentiated into tumor cells, so that the harm of the cancer stem cells is reduced, and the drug resistance is avoided.
The present invention is described in further detail below with reference to specific examples, which are intended to be illustrative of the invention and not limiting.
Example 1
Synthesis of pH-responsive nano micelle HP @ PHIs
2g of N- (tert-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine (Boc-His (DNP) -OH) was dissolved in 15mL of tetrahydrofuran, followed by thionyl chloride (1.6mL) added dropwise. Finally, the product is obtained by ether precipitation and centrifugation. To further purify the product, the purified product N- (tert-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride (Boc-His (DNP) -NCA) was obtained by dissolving the product with acetone and precipitating with diethyl ether. Boc-His (DNP) -NCA (1g, 2.6mmol) was dissolved in 20mL dry dimethylformamide and washed with N2Stir for 1h at ambient. Polyethyleneimine PEI (0.52mmol) was added to the mixture and reacted for 3 days. Thereafter, Boc was removed using 2-mercaptoethanol (3 mL). By means of H2O dialyzed and then lyophilized to collect the Product (PHIs). 4mg of PHIs was then dissolved in formamide (1mL) and a 20mg HP solution in DMSO was added to the PHIs mixture with stirring. Then, H is reacted with2O was added dropwise to the reaction. After 2 hours, by H2O dialysis to obtain the product HP @ PHIs.
FIG. 1 is a nuclear magnetic spectrum of polyhistidine in example 1 of the present invention, and the successful preparation of the nanocarrier PHIs is demonstrated by nuclear magnetic hydrogen spectrum data analysis.
Example 2
Preparation of hierarchical responsive nanocomposite HP @ PHIs/LC
The preparation method of the low molecular weight heparin and chlorin e6 copolymer (LC) comprises the following steps:
ce6(0.08mmol, 50mg), EDC (0.156mmol) and NHS (0.174mmol) were dissolved in dry DMF (6mL) in N2Stirred for 4 hours. DMAP (0.1mmol) and LMWH (100mg) were then added to the above mixture. After 48 hours, the dialysis reaction was carried out using DMF and water, respectively. Finally, LMWH-Ce6(LC) was obtained by lyophilization.
The HP @ PHIs/LC NPs are obtained by the electrostatic interaction of pH-responsive nanomicelle HP @ PHIs and a copolymer (LC) of low-molecular heparin and chlorin e 6. The LC was mixed with HP @ phil at a mass ratio of 2:1 and incubated for 20 minutes. Finally, centrifugation (10000rpm, 5 minutes) was carried out to obtain the graded responsive nanocomposite HP @ PHIs/LC.
Example 3
Graded response nano-composite HP @ PHIs/LC inhibition cancer stem cell proliferation experiment
Cancer stem cells (2X 10)3) Incubate in ultra low attachment six well plates for 3 days. Thereafter, the cells were treated with PBS buffer, haloperidol HP, HP @ PHIs/LC (pH 6.5) and HP @ PHIs/LC (pH 7.4). After 3 days, the number of cancer stem cells was observed by a fluorescence inverted microscope.
FIG. 2 is a representation of HP @ PHIs/LC experiments for inhibiting cancer stem cells, comparing the PBS buffer group with the HP group, the number of cancer stem cells can be significantly reduced after HP is used, which indicates that cancer stem cells have differentiated into tumor cells. Because the tumor stem cells are in an unbalanced state, the self-renewal capacity is strong, the growth of tumors is promoted, and the tumor stem cells play an important role in tumor invasion, metastasis and drug resistance, namely the cancer stem cells have stronger harmfulness than the tumor cells. The characteristic that HP promotes cancer stem cells to be differentiated into tumor cells is utilized, so that the harm of the cancer stem cells can be reduced, and meanwhile, the drug resistance is reduced.
The HP @ PHIs/LC NPs can responsively release HP in a tumor microenvironment to further act on tumor stem cells, and compared with cancer stem cells only using PBS buffer solution, the HP @ PHIs/LC NPs can promote the differentiation of the cancer stem cells into the tumor cells and reduce the number of the cancer stem cells.
When the cancer stem cells and PBS buffer solution are incubated together, the cancer stem cells still maintain strong self-renewal potential. The differences between the HP @ PHIs/LC (pH 6.5) and HP @ PHIs/LC (pH 7.4) groups are mainly attributed to the fact that the dissociation of micelles is realized at pH 6.5, and then the cancer stem cells are released to be accurately targeted to dopamine receptors on the surfaces of the cancer stem cells, and the cancer stem cells are promoted to be differentiated into tumor cells.
Compared with HP, HP @ PHIS/LC has the advantages that the nano preparation is synthesized through a self-assembly strategy, enrichment of the nano preparation in tumor tissues is improved, and meanwhile, under the strategy of cancer stem cell elimination, photodynamic therapy is adopted, so that growth and metastasis of tumors can be weakened, and in-vivo tracking of drugs can be achieved through fluorescence signals of Ce 6.
Example 4
In vivo anti-tumor effect of hierarchical responsive nano-composite HP @ PHIs/LC
In-vivo anti-tumor experiments of the example show that HP @ PHIs/LC has a strong anti-tumor effect.
4T1 cells (1X 10)6) Injected into the right axilla of the mice. When the tumor reaches about 100mm3On every other day, mice were given drug-treated normal saline NS (0.2mL), HP (7.5mg/kg), chlorin e6(Ce6) (2.5mg/kg), low molecular heparin LMWH and HP @ PHIs/LC NPs, respectively. After 6 hours, at a rate of 0.1W/cm2The HP @ PHIs/LC NPs and free Ce6 groups of mice were treated with near infrared light (660nm, 10 min). Changes in tumor volume were recorded during treatment. After 7 treatments, mice were sacrificed and tumors were removed and weighed.
As can be seen from the figures 3 and 4, the accumulation of the photosensitizer Ce6 at the tumor site is improved due to the targeted therapy of the HP @ PHIs/LC NPs, the cancer cells are killed by active oxygen, the cancer stem cell inhibitor HP is accurately released in the tumor tissues, the cancer stem cells are promoted to be differentiated into the common tumor cells, the tolerance to photodynamic therapy is relieved, and the volume and the weight of the tumor are remarkably reduced, so that the tumor inhibition effect of the HP @ PHIs/LC NPs preparation is obviously superior to that of Ce 6.
Example 5
In-vivo epithelial-mesenchymal transition inhibition effect of hierarchical responsive nano-composite HP @ PHIs/LC
4T1 cells (1X 10)6) Injected into the right axilla of the mice. When the tumor reaches about 100mm3On every other day, mice were given drug-treated normal saline NS (0.2mL), HP (7.5mg/kg), chlorin e6(Ce6) (2.5mg/kg), low molecular heparin LMWH and HP @ PHIs/LC NPs, respectively. After 6 hours, at a rate of 0.1W/cm2The HP @ PHIs/LC NPs and free Ce6 groups of mice were treated with near infrared light (660nm, 10 min).
15 days after treatment of mice, all groups of tumors were collected and digested to detect expression of E-cadherin and N-cadherin.
As can be seen from FIG. 5, for HP @ PHIs/LC NPs, under the action of LMWH, the epithelial-mesenchymal transition process is effectively inhibited, and compared with the control group (NS), the expression of E-cadherin protein of epithelial cells is obviously increased, and the expression of N-cadherin protein of mesenchymal cells is simultaneously reduced.
The construction and application of the hierarchical responsive nano-composite HP @ PHIs/LC disclosed by the embodiment solve the problem of in-vivo delivery of hydrophobic Ce6 and HP, and realize accurate release of a heterotarget drug, inhibition of tumor stem cells and reduction of invasion and metastasis of tumors.
Although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (56)
1. A hierarchical responsive nano-composite is characterized in that pH responsive nano-micelle is used as the core of the composite, and low molecular heparin and chlorin e6 copolymer are used as the shell of the composite;
the pH-responsive nano-micelle comprises a pH-responsive nano-copolymer carrier and a cancer stem cell inhibitor;
the chemical structural formula of the pH-responsive nano copolymer carrier is as follows:
the value of n ranges from 1 to 16.
2. The graded responsive nanocomposite of claim 1, wherein the value of n ranges from 4 to 16.
3. The hierarchical responsive nanocomposite according to claim 1, wherein the method for preparing the pH responsive nano-copolymer carrier comprises polymerizing N- (t-butyloxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine with an amino group of polyethyleneimine as an initiator to produce the pH responsive nano-copolymer carrier, namely polyhistidine Phis.
4. The graded-response nanocomposite according to claim 3, wherein the polyethyleneimine is a branched polyethyleneimine.
5. The graded responsive nanocomposite as claimed in claim 3, wherein N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine is dissolved in a first solvent, thionyl chloride is added, precipitation and centrifugation are performed to obtain N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride, N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride is dissolved in a second solvent, stirring is performed under a protective atmosphere, polyethyleneimine is added for reaction, and impurity removal, dialysis and freeze-drying are performed to obtain the graded responsive nanocomposite.
6. The fractionated responsive nanocomposite according to claim 5, wherein the first solvent is selected from tetrahydrofuran, dimethylsulfoxide, N, N dimethylformamide, N, N dimethylacetamide.
7. The graded responsive nanocomposite according to claim 5, wherein the molar ratio of N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine to thionyl chloride is 0.5-2.5: 8-15.
8. The graded responsive nanocomposite according to claim 5, wherein the molar ratio of N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine to thionyl chloride is 2-2.5: 11.
9. The graded responsive nanocomposite of claim 5, wherein the molar ratio of N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine to thionyl chloride is 2.37: 11.
10. The fractionated responsive nanocomposite of claim 5, wherein the precipitation process uses ether precipitation.
11. The graded responsive nanocomposite according to claim 5, further comprising a purification step after the centrifugation, wherein the crude product N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride is dissolved with acetone and subjected to ether precipitation to obtain purified N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic anhydride hydrochloride.
12. The graded responsive nanocomposite of claim 5, wherein the molar ratio of N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic acid anhydride hydrochloride to polyethyleneimine is 4:1-6: 1.
13. The graded responsive nanocomposite of claim 5, wherein the molar ratio of N- (tert-butoxycarbonyl) -1- (2, 4-dinitrophenyl) -L-histidine carboxylic acid anhydride hydrochloride to polyethyleneimine is 5: 1.
14. The fractionated responsive nanocomposite according to claim 5, wherein the second solvent is selected from tetrahydrofuran, dimethylsulfoxide, N, N dimethylformamide, N, N dimethylacetamide.
15. The graded responsive nanocomposite according to claim 5, wherein the stirring is room temperature stirring for 2-4 days.
16. The graded responsive nanocomposite according to claim 5, wherein the stirring is room temperature stirring for 3 days.
17. The graded responsive nanocomposite according to claim 5, wherein the step of removing impurities is removing 2, 4-dinitrobenzene using 2-mercaptoethanol.
18. The hierarchical responsive nanocomposite of claim 1, wherein the cancer stem cell inhibitor is selected from haloperidol, chlorpromazine, trifluoperazine, and perphenazine.
19. The hierarchical responsive nanocomposite according to claim 1, wherein the pH-responsive nanocopolymer carrier entraps cancer stem cell inhibitor by hydrophobic interaction to synthesize pH-responsive nanomicelles.
20. The hierarchical responsive nano-composite according to claim 1, wherein the preparation method of the pH responsive nano-micelle comprises self-assembling the pH responsive nano-copolymer carrier and the cancer stem cell inhibitor in water by a dialysis method to obtain the pH responsive nano-micelle.
21. The hierarchical responsive nanocomposite according to claim 20, wherein the cancer stem cell inhibitor solution and the pH responsive nano-copolymer carrier solution are mixed and stirred, water is added for reaction, and pH responsive nano-micelles are obtained by water dialysis.
22. The fractionated responsive nanocomposite according to claim 20, wherein the solvent of the cancer stem cell inhibitor solution is dimethyl sulfoxide.
23. The graded-response nanocomposite according to claim 20, wherein the solvent of the pH-responsive nanocomposite carrier solution is formamide.
24. The fractionated responsive nanocomposite according to claim 20, wherein the mass ratio of the cancer stem cell inhibitor to the pH responsive nano copolymer carrier is 1:1-6: 1.
25. The fractionated responsive nanocomposite according to claim 20, wherein the mass ratio of the cancer stem cell inhibitor to the pH responsive nano copolymer carrier is 5: 1.
26. The hierarchical responsive nanocomposite according to claim 21, wherein the water addition is dropwise water addition.
27. The graded-response nanocomposite according to claim 21, wherein the reaction time is 1-3 h.
28. The graded-response nanocomposite according to claim 1, wherein the reaction time is 2 h.
29. The graded-response nanocomposite according to claim 21, wherein the water dialysis employs 0.45mm3The aqueous membrane is dialyzed to obtain pH-responsive nano-micelles with uniform sizes.
30. The hierarchical responsive nanocomposite according to claim 1, wherein the core pH-responsive nanomicelle of the hierarchical responsive nanocomposite is positively charged and the copolymer of the shell low molecular heparin and chlorin e6 is negatively charged.
31. The hierarchical responsive nanocomposite according to claim 1, wherein the mass ratio of the low molecular heparin and chlorin e6 copolymer to the pH responsive nanomicelle is 4:1 to 1: 1.
32. The hierarchical responsive nanocomposite according to claim 1, wherein the low molecular heparin and chlorin e6 copolymer to pH responsive nanomicelle mass ratio is 2: 1.
33. The method of claim 1, wherein the copolymer of low molecular heparin and chlorin e6 is electrostatically adsorbed on the surface of the pH-responsive nanomicelle, thereby obtaining the hierarchical responsive nanocomposite.
34. The method for preparing the hierarchical responsive nanocomposite according to claim 33, wherein the hierarchical responsive nanocomposite is obtained by mixing a low molecular heparin and chlorin e6 copolymer with the pH-responsive nanomicelle, incubating, and centrifuging.
35. The method for preparing the hierarchical responsive nanocomposite according to claim 34, wherein the low molecular heparin and chlorin e6 copolymer has a mass ratio of the low molecular heparin to the chlorin e6 of 1:1 to 3: 1.
36. The method for preparing a hierarchical responsive nanocomposite according to claim 34, wherein the low molecular heparin/chlorin e6 copolymer has a mass ratio of 2:1 between low molecular heparin/chlorin e 6.
37. The method for preparing the hierarchical responsive nanocomposite according to claim 34, wherein the method for preparing the copolymer of low molecular heparin and chlorin e6 comprises: dissolving chlorin e6, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS in an organic solvent, and stirring; adding 4-dimethylaminopyridine DMAP and low molecular weight heparin LMWH, reacting for a period of time, dialyzing, and freeze-drying to obtain the copolymer of low molecular weight heparin and chlorin e 6.
38. The method for preparing a hierarchical responsive nanocomposite according to claim 37, wherein the molar ratio of chlorin e6, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS is 5-10:13-16: 15-20.
39. The method for preparing a hierarchical responsive nanocomposite according to claim 37, wherein the molar ratio of chlorin e6, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride EDC and N-hydroxysuccinimide NHS is 8:15.6: 17.4.
40. The method of claim 37, wherein the organic solvent is selected from the group consisting of N, N-dimethylformamide, N-dimethylacetamide, and dimethylsulfoxide.
41. The method of claim 37, wherein the stirring is at N2Stirring for 3-6 hr.
42. The method of claim 37, wherein the stirring is at N2Stirring was carried out for 4 hours.
43. The method of claim 37, wherein the mass ratio of 4-Dimethylaminopyridine (DMAP) to low molecular heparin (LMWH) is 12.217: 100.
44. The method of claim 37, wherein the dialysis is a dialysis reaction using N, N-dimethylformamide and water.
45. The method for preparing the hierarchical responsive nano-composite according to claim 37, wherein the mass ratio of the copolymer of the low molecular heparin and the chlorin e6 to the pH responsive nano-micelle is 4:1-1: 1.
46. The method for preparing the hierarchical responsive nanocomposite according to claim 37, wherein the mass ratio of the copolymer of the low molecular heparin and the chlorin e6 to the pH-responsive nanomicelle is 2: 1.
47. The method of claim 37, wherein the pH-responsive nanomicelle is haloperidol @ pH-responsive nano-copolymer carrier, HP @ phil.
48. The method of claim 34, wherein the incubation temperature is 37 ℃ and the incubation time is 10-50 min.
49. The method of claim 34, wherein the incubation temperature is 37 ℃ and the incubation time is 20 min.
50. The method for preparing a hierarchical responsive nanocomposite as claimed in claim 34, wherein the centrifugation parameter is 8000-15000rpm, 3-10 min.
51. The method of claim 34, wherein the centrifugation parameter is 10000rpm, 5 min.
52. A nano-formulation comprising the graded-response nano-composite of claim 1.
53. The NanoPrepration of claim 52, wherein the NanoPrepration is an intravenous formulation.
54. Use of the hierarchical responsive nanocomposite according to claim 1 for preparing an antitumor drug.
55. The use according to claim 54, wherein said antineoplastic agent is selected from the group consisting of alkylating agents, alkaloids, antibacterial antineoplastic sulfonamides, platinum-based agents, antimetabolites.
56. The use of claim 54, wherein said antineoplastic agent further comprises a phototherapeutic agent selected from the group consisting of indocyanine green, neoindocyanine green, chlorin e6, IR780 iodide.
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