CN109771662B - Curcumin spice system, preparation method and application - Google Patents
Curcumin spice system, preparation method and application Download PDFInfo
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Abstract
The curcumin spice system comprises gold nanoparticles with molecular imaging advantages positioned in a core, a polymer material coated on the surfaces of the gold nanoparticles, a double-stage targeting molecule connected with the polymer material, and curcumin spice chemically bonded with the polymer material through response release. The curcumin perfume system of the invention passes through a blood brain barrier, targets neurons, and controllably releases curcumin perfume at affected parts, thereby solving the problems of low bioavailability and short retention time in vivo of the curcumin perfume, reducing the dosage of the curcumin perfume, and greatly improving the efficiency of the curcumin perfume in treating Parkinson's disease.
Description
Technical Field
The invention belongs to the field of medicines, and particularly relates to a curcumin spice system, a preparation method and application.
Background
Parkinson's disease is a common neurodegenerative disease characterized clinically by resting tremor, muscular rigidity, bradykinesia, and postural reflex disorder. The disease is caused by dopaminergic neurotransmitter deficiency due to degeneration and death of dopaminergic neurons in substantia nigra in brain, thereby causing motor and non-motor symptoms such as resting tremor, myotonia, neuropsychiatric symptoms, autonomic nerve dysfunction and the like. The related data show that the number of the worldwide sick people is close to more than 400 million people, wherein the number of the Chinese sick people is as high as more than 200 million. Parkinson's disease seriously affects the life of patients, with disability rates as high as 60% in 5-9 years, and even death of patients. The neurodegenerative diseases are widely concerned, and the reasonable medicine system method is urgently proposed.
Curcumin (curmin, CUR for short) is a yellow spice extracted from rhizomes of plants in Zingiberaceae, has wide source, low price, high efficiency and low toxicity, and has great research value. Curcumin flavors have a wide range of applications: such as antioxidation, anti-inflammation, anti-tumor, anti-mutation, neuronal protection after brain damage, etc. In 2006, researchers found that curcumin, a combination of diacetylcurcumin and manganese can inhibit epileptic seizure and reduce generation of inducible Nitric Oxide Synthase (NOS), and the neuroprotective effect of curcumin is exerted through the antioxidation and NOS scavenging activities of curcumin, so that the Parkinson's disease is treated.
However, curcumin has a plurality of active groups (such as beta-diketone structure) in the structure, so that the curcumin has poor in-vivo and in-vitro stability. In practical application, curcumin is found to have low solubility, poor stability, low absorption rate, easy conversion to glucuronic acid, sulfonic acid and other complexes in intestinal tracts, fast metabolism, short half-life, and the existence of the problems causes the low bioavailability of the curcumin and limits the application of the curcumin in the fields of food and medicines. For example, the oral dosage of the composition for rats is 0.1-1 g/kg-1In this case,. rho.max is less than 1. mu.g/mL-1In the case of experiments on human bodies, the ρ max was only 50ng · mL despite oral administration of up to 12g-1Left and right. Therefore, how to improve the stability and bioavailability of the flavin perfume is urgent, which is one of the key problems to be solved urgently in the process of developing the curcumin perfume into a preparation for treating the Parkinson disease.
Disclosure of Invention
In view of the above, the main object of the present invention is to provide a curcumin flavor system, a preparation method and an application, which aim to at least partially solve at least one of the above mentioned technical problems.
In order to achieve the purpose, the technical scheme of the invention is as follows:
as an aspect of the present invention, there is provided a curcumin flavor system, comprising a substance comprising at least curcumin flavor, a first-stage targeting molecule, and a second-stage targeting molecule, covalently bonded to a polymeric material, wherein the polymeric material comprises a poly-t-butyl betaine methacrylate polymer with a thiol group, and the curcumin flavor is bonded with a hydrogen peroxide-responsive chemical bond as a linking group, the linking group that achieves controlled release of curcumin in response to hydrogen peroxide is a mixture of any one or at least two of a propylene sulfide group, a thioether ketone group, a selenium-containing block copolymer group, an arylborate group, or a derivative thereof.
Preferably, the primary targeting molecule comprises one or more of a cell-penetrating peptide, a cell-penetrating peptide; the secondary targeting molecule comprises one or more of short peptide, horse indole or derivatives thereof.
As a further aspect of the present invention, there is provided a process for the preparation of a curcumin flavor system as described above, comprising the steps of:
(1) dissolving curcumin spice, polymer material and chemical molecules capable of realizing hydrogen peroxide response in an organic solvent, and reacting to obtain hydrophobic H2O2Responsive curcumin polymer a;
(2) the hydrophobicity H prepared in the step (1)2O2Mixing the responding curcumin polymer a and a first-stage targeting molecule dissolved in an organic solvent, and dialyzing to obtain a solution b;
(3) the hydrophobicity H prepared in the step (1)2O2Mixing the responding curcumin polymer a and a second-stage targeting molecule dissolved in an organic solvent, and dialyzing to obtain a solution c;
(4) will be hydrophobic H2O2Responsive curcumin polymer a, solution b and solution c mixingAnd obtaining the curcumin spice system after ultrasonic treatment.
Preferably, in the step (2), the molar ratio of the first-stage targeting molecule to the curcumin pro-flavorant is 1: 1-1: 10;
in the step (3), the molar ratio of the second-stage targeting molecule to the curcumin polymer responded by the hydrophobic H2O2 is 1: 1-1: 10;
in step (4), hydrophobicity H2O2The responding curcumin polymer a, the solution b and the solution c are added according to the molar ratio of curcumin of 1: 1. Preferably, in step (4), the hydrophobicity is towards H2O2And adding gold nanoparticles into the mixed solution of the responsive curcumin polymer a, the solution b and the solution c, carrying out ultrasonic treatment, standing overnight to obtain a solution e, acidifying the solution e, and centrifuging to obtain the polymer gold nanoparticles.
Preferably, the method further comprises the step (5) of adding siRNA into the polymer gold nanoparticles to obtain the curcumin perfume system through compounding.
Preferably, in step (4), the hydrophobicity H2O2The mass ratio of the mixed solution of the responsive curcumin polymer a, the solution b and the solution c to the gold nanoparticles is 1: 1-1: 5; the particle size of the gold nanoparticles is 10-50 nm; the gold nanoparticle solution may be prepared by a preparation method conventional in the art, and more preferably, by a trisodium citrate reduction method.
Preferably, in the step (5), the ratio of N to P of the polymer gold nanoparticles to siRNA is 1: 100-1: 500.
As a further aspect of the invention, there is also provided the use of a curcumin perfume system as described above in the manufacture of a medicament for the treatment of parkinson's disease.
Compared with the prior art, the invention has the beneficial effects that:
the curcumin perfume system can be condensed with carboxyl of a polymer material to form an ester bond, and forms a thioether bond through the addition reaction of a double bond and a sulfydryl, so that the response release of curcumin can be realized under hydrogen peroxide, the slow release effect is achieved, the medicine dosage is reduced, and the efficiency of treating the Parkinson disease by the curcumin perfume is greatly improved;
the invention uses two-stage targeting molecules, the first stage targeting molecule enables the curcumin perfume system to penetrate through a blood brain barrier, and the second stage targeting molecule enables the curcumin system to be further concentrated at a cerebral lesion part, so that the curcumin concentration of the perfume is increased, the problems of low bioavailability and short in-vivo retention time of the curcumin are solved, and the curcumin perfume has an obvious treatment effect on the Parkinson's disease, so that the curcumin perfume has an ideal application prospect.
Drawings
FIG. 1 is a transmission electron micrograph of a curcumin flavor system prepared in example 1 of the present invention;
FIG. 2A is a graph of the cell level first-order targeting effect of curcumin flavor system prepared in example 1 of the present invention;
FIG. 2B is a graph of the second level targeting effect on the cell level of the curcumin flavor system prepared in example 1 of the present invention;
fig. 3 shows the response release effect of the curcumin flavor system prepared in example 1 of the present invention;
fig. 4 is a graph of the cellular level therapeutic effect of the curcumin flavor system prepared in example 1 of the present invention.
Detailed Description
In order that the objects, technical solutions and advantages of the present invention will become more apparent, the present invention will be further described in detail with reference to the accompanying drawings in conjunction with the following specific embodiments.
The preparation method of the curcumin perfume system for treating the Parkinson disease comprises the following steps:
(1) dissolving curcumin spice, polymer material and chemical molecules capable of realizing hydrogen peroxide response in an organic solvent, and reacting to obtain hydrophobic H2O2Responsive curcumin polymer a;
(2) the hydrophobicity H prepared in the step (1)2O2Mixing the responding curcumin polymer a and a first-stage targeting molecule dissolved in an organic solvent, and dialyzing to obtain a solution b;
(3) the hydrophobicity H prepared in the step (1)2O2Responsive curcumin polymers a andmixing and dialyzing the second-stage targeting molecules dissolved in the organic solvent to obtain a solution c;
(4) will be hydrophobic H2O2And mixing the responding curcumin polymer a, the solution b and the solution c, and performing ultrasonic treatment to obtain the curcumin perfume system.
The first-stage targeting molecule comprises one or more of a cell-penetrating peptide, a cell penetrating peptide or a derivative thereof; the secondary targeting molecule comprises one or more of short peptide, horse indole or derivatives thereof.
The cell-penetrating peptide is selected from (SynB); the cell penetrating peptide is selected from (cell penetrating peptide B6); the short peptide has its conventional meaning in the art, and a short peptide (RVG) having 29 amino acid residues is used in the present invention.
Preferably, in step (2), the first targeting molecule is conjugated with hydrophobic H2O2The molar ratio of the responding curcumin polymer is 1: 1-1: 10;
in step (3), the second targeting molecule is conjugated to the hydrophobic moiety H2O2The molar ratio of the responding curcumin polymer is 1: 1-1: 10;
in step (4), hydrophobicity H2O2The responding curcumin polymer a, the solution b and the solution c are added according to the molar ratio of curcumin of 1: 1. Preferably, in step (4), the hydrophobicity is towards H2O2And adding gold nanoparticles into the mixed solution of the responsive curcumin polymer a, the solution b and the solution c, carrying out ultrasonic treatment, standing overnight to obtain a solution e, acidifying the solution e, and centrifuging to obtain the polymer gold nanoparticles. Hydrophobic H2O2The mass ratio of the mixed solution of the responsive curcumin polymer a, the solution b and the solution c to the gold nanoparticles is 1: 1-1: 5; the particle size of the gold nanoparticles is 10-50 nm. The gold nanoparticles may be prepared by a conventional preparation method in the art, and more preferably, by a sodium citrate reduction method.
Preferably, the method further comprises the step (5) of adding siRNA into the polymer gold nanoparticles to obtain the curcumin perfume system through compounding. The ratio of N to P of the polymer gold nanoparticles to siRNA is 1: 100-1: 500.
The curcumin spice system prepared by the method has double-stage targeting molecules connected with the polymer material, and spice curcumin chemically bonded with the polymer material through response release.
The inventor of the application discovers in research that a Blood Brain Barrier (BBB) is a Barrier between cerebral vessels and Brain parenchyma and can prevent most substances from entering the Brain, a first-level targeting molecule such as cell-penetrating peptide can be selectively compatible with the Blood Brain Barrier, a spice system is promoted to penetrate through the Blood Brain Barrier, the bioavailability of curcumin is improved, the retention time in a spice body is prolonged, and the dosage required by the beneficial effect of the spice is reduced.
After entering the brain, the second-level targeting molecules such as mazindol can be compatible with lesion tissues, so that the system has the capability of continuously targeting lesion tissues, and more spice curcumin is further concentrated at the lesion part of the brain, thereby increasing the concentration of the spice curcumin, continuously improving the bioavailability of the spice curcumin, increasing the retention time in the body and reducing the dosage required by the beneficial effect.
The perfume curcumin has a polyol structure, can be condensed with carboxyl of a polymer material to form an ester bond, and forms a thioether bond through the addition reaction of a double bond and a sulfydryl, so that the response of hydrogen peroxide is realized. The hydrogen peroxide concentration of the lesion part of the Parkinson disease is increased relative to the hydrogen peroxide concentration of the normal tissue part, and the curcumin perfume system can realize the responsive release of the turmeric under the hydrogen peroxide, thereby achieving the slow release effect, reducing the drug dosage and greatly improving the efficiency of the perfume curcumin for treating the Parkinson disease.
Further, the curcumin spice system prepared by the method also comprises gold nanoparticles with molecular imaging advantages, which are positioned in the core, and the surfaces of the gold nanoparticles are coated with the polymer material. The gold nanoparticles have the advantages of molecular imaging, can track a curcumin perfume system, and investigate the imaging effect of the curcumin perfume on the lesion part of the Parkinson disease.
Furthermore, siRNA is loaded on the surface of the polymer gold nanoparticle to form a biomolecule-chemical molecule composite preparation. RNA interference (RNAi) refers to specific degradation of intracellular mRNA mediated by endogenous or exogenous double-stranded RNA (dsrna), thereby resulting in silencing of expression of a target gene and causing a phenomenon of loss of a corresponding functional phenotype, thereby inhibiting expression of a related gene and achieving a gene therapy effect. Gene therapy suppresses "poison" sources, reducing neuronal death; the curcumin nerve protection spice reduces alpha-Syn aggregates and generated toxicants, and gene therapy and chemical molecule therapy are complementary to obtain the best treatment effect.
The following examples are provided to further illustrate the practice and technical effects of the present invention.
In the following examples, the experimental materials used were obtained from conventional commercial sources or prepared by manufacturers conventional in the art.
In the following examples, the operations of stirring, dialysis, freezing and vacuum drying are all routine experimental methods in the art.
Example 1
In this example, a curcumin flavor system was prepared by the following method, specifically including the steps of:
(1) adding curcumin 500mg into round bottom flask, dissolving with 25ml THF (tetrahydrofuran), adding 330ul triethylamine, and stirring for 30 min. 20ul of methacrylic anhydride was taken and dissolved in 1.25ml of tetrahydrofuran. And (4) dropwise adding the reaction system, stirring vigorously, and slowly dropwise adding until the dropwise adding is finished. The temperature was increased to 71 ℃ overnight under nitrogen. The product is obtained by post-treatment and then separated by a chromatographic column. Taking 41.9mg of the product, adding DMF (dimethyl formamide) for dissolving, adding 5ul of triethylamine, dropwise adding 5.8mg of poly-tert-butyl betaine methacrylate polymer with sulfydryl and a structure shown as the following formula, and stirring for 24 hours to obtain hydrophobic H2O2Responsive curcumin polymer a.
Structural formula of poly-tert-butyl betaine methacrylate polymer with sulfydryl
(2) GetThe hydrophobicity H prepared in the step (1)2O2The responding curcumin polymer (20 mg) was dissolved in DMF (anhydrous dimethyl sulfoxide), 4.2mg of PYBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) and 2.2ml of DIEA (diisopropylethylamine) were added, 15mg of a DMF solution of cell-penetrating peptide (CGHKAKGPRK, 99%) was added dropwise, and the mixture was dialyzed against ethanol and water, respectively, to obtain a solution b.
(3) Taking the hydrophobic H prepared in the step (1)2O2The corresponding curcumin polymer (20 mg) was dissolved in DMF, DMAP (1.16 mg), EDCCL (1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride) (2.2 mg) was added, a DMF solution of 4.5mg of mazindol was added dropwise, stirred for 24h, and dialyzed against ethanol and water, respectively, to give solution c.
(4) By adopting a sodium citrate reduction method, 10mg of tetrachloroauric acid is dissolved in 100mL of deionized water, and the solution is heated to boiling while being vigorously stirred. Then adding 3mL of trisodium citrate solution with the mass fraction of 1%, and continuing to react for 30min to obtain a gold nanoparticle solution d with the particle size of 14 nm.
(5) The hydrophobicity H prepared in the step (1) is2O2And (3) mixing the responding curcumin polymer, the solution b prepared in the step (2) and the solution c prepared in the step (3) with the gold nanoparticles according to the mass ratio of 1: 4, carrying out ultrasonic treatment for 1 hour, and standing overnight to obtain the polymer gold nanoparticles with uniform particle size.
(6) Firstly, acidifying polymer gold nanoparticles, centrifuging the obtained product at 1.5ml14000rpm, adding 750ul of buffer solution with the pH value of 2.5, and compounding with 0.02OD siRNA, wherein the ratio of N to P is 1:100, and obtaining the curcumin perfume system after 30 min.
The curcumin flavor system prepared in example 1 was characterized by transmission electron microscopy, and the results are shown in fig. 1, where the particle size of the obtained product is 20-50nm, which is the particle size of the synthesized curcumin flavor system.
The two-stage targeting effect was verified at the cell level for the curcumin flavor system prepared in example 1. In vitro Blood Brain Barrier (BBB) model experiment: to construct an in vitro BBB model, a non-contact co-culture model was designed. Mouse brain microvascular endothelial cells mimicking the blood brain barrier(bEND.3) at 2X 105Cell/cell, cultured on the upper layer of the cell chamber (pore size: 0.4 μm), and cultured in a 2X 10 manner using a human bone marrow neuroblastoma cell line (SHSY5Y cells)5Cell/chamber, cultured in the lower layer of the cell chamber. Adding curcumin perfume system and PBS into upper layer of cell chamber, culturing, and detecting with flow cytometer after 0.5, 1, 2, 4, 6, 8 and 12 h. The results, as shown in fig. 2A and 2B, demonstrate the high efficiency of dual-stage targeting at the cellular level: quantitative analysis is carried out on the endocytosis level of the SHSY5Y cell mediated by the double-stage targeting, and the result shows that the bEND.32h, 4h and 6h mediated by the first-stage targeting cell-penetrating peptide are obviously absorbed, and the absorption rate is improved by more than 70% in 6 h; the second-stage targeting short peptide mediated SHSY5Y cells take the protein significantly for 2h, 4h and 6h, and the protein is improved by more than 80% at 6 h. The curcumin spice system prepared in the example 1 obviously improves the accumulation of curcumin spices in affected parts, and lays a foundation for the curcumin spices to exert the drug effect to the maximum extent to achieve the treatment effect.
The curcumin release effect of the curcumin flavor system prepared in example 1 was determined using High Performance Liquid Chromatography (HPLC). The reaction was carried out under 4 conditions, PBS solution, pH7.4, PBS solution, pH5.0, hydrogen peroxide 1mM, pH7.4, and hydrogen peroxide 1mM, pH5.0, respectively. 1.5ml of curcumin perfume system solution was placed in a dialysis bag with molecular weight of 3500. They were placed in 1000ml bottles, to which 800ml of PBS and hydrogen peroxide were added, respectively. The vial was placed in a constant temperature shaker, the temperature was adjusted to 37 ℃, the rotation speed was adjusted to 150rpm, and 1ml of sample was taken from the vial at a preset time (0h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h, 20h, 25h, 30h, 35h, 40 h). After the sample was taken, 1ml of the corresponding buffer was added. Curcumin concentrations were calculated from the standard curve and release curves were plotted. The results are shown in fig. 3, the curcumin drug is not released basically in the solution without hydrogen peroxide, but the curcumin is released rapidly and continuously under the environment with the hydrogen peroxide concentration of 1mM and the pH value of 5.0, and the release amounts reach 30% and 60% after 10h and 30h respectively. The release result shows that the curcumin spice system prepared in the example 1 has hydrogen peroxide responsiveness and spice controlled-release capacity, improves the effect of the curcumin spice on the affected part, and lays a foundation for the curcumin spice to play a role to the maximum to achieve a treatment effect.
Example 2
The preparation method is the same as that in example 1, except that: in step (6), no siRNA was added, but the acidified polymeric gold nanoparticles were used directly as a sole curcumin flavor system.
The cell level treatment effect of the curcumin spice system prepared in the example 1 and the example 2 is verified by the cell model of the Parkinson disease transformed over-expression EGFP-a-syn in a laboratory. Respectively carrying out comparison tests with a blank control, curcumin, siRNA and an siRNA system, wherein the siRNA system test method comprises the following steps: firstly, nano-scale gold nanoparticles are synthesized, and gold nanoparticles with uniform particle size are prepared by a trisodium citrate reduction method. The cell-penetrating peptide B6/maindole modified prodrug polymer molecule is formed by connecting a first-level targeting molecule cell-penetrating peptide (CGHKAKGPRK, 99%) and a second-level targeting molecule maindole to the surface of a polymer carrier through an amide bonding ester bond connection mode by adopting a 1-ethyl- (3-dimethylaminopropyl) carbodiimide N-hydroxysuccinimide activation method. Then preparing the polymer modified gold nanoparticles prepared on the surface by a ligand displacement method. Finally, siRNA is loaded on the surface of the polymer carrier by utilizing the electrostatic action.
And (3) paving the Parkinson disease change overexpression EGFP-a-syn cell model in a laser confocal cell culture dish, adding 1mL of culture medium into each hole, and culturing at 37 ℃ for 24h to make the cells adhere to the wall. Then, a curcumin spice system and PBS are respectively added into the hole of the cell model of the Parkinson disease overexpressed EGFP-a-syn cell to be continuously cultured for 24 hours. Washing with water for three times, fixing with 4% paraformaldehyde in dark at room temperature for 15min, washing with PBS for three times, adding DAPI solution, dyeing in dark at room temperature for 20min, washing with PBS for three times, and performing laser confocal detection. The results are shown in fig. 4, compared with the parkinsonism change model PBS group, the a-syn expression of the curcumin perfume system prepared in the example 1 and the example 2 is reduced after the curcumin perfume system is added, which shows that the curcumin perfume system inhibits the a-syn aggregation and has obvious treatment effect on parkinsonism. Compared with the single curcumin spice system group and the single siRNA system group prepared in the embodiment 2, the curcumin spice/siRNA system a-syn prepared in the embodiment 1 has the advantages of obviously reduced expression and most obvious treatment effect, and the gene therapy and the chemical molecule therapy are complementary to obtain the optimal treatment effect.
Example 3
In this example, a curcumin flavor system was prepared by the following method, specifically including the steps of:
1) adding curcumin 500mg into round bottom flask, dissolving with 25ml THF (tetrahydrofuran), adding 330ul triethylamine, and stirring for 30 min. 20ul of methacrylic anhydride was taken and dissolved in 1.25ml of tetrahydrofuran. And (4) dropwise adding the reaction system, stirring vigorously, and slowly dropwise adding until the dropwise adding is finished. The temperature was increased to 71 ℃ overnight under nitrogen. The product is obtained by post-treatment and then separated by a chromatographic column. Taking 41.9mg of the product, adding DMF (dimethyl formamide) for dissolving, adding 5ul of triethylamine, dropwise adding 5.8mg of poly-tert-butyl betaine methacrylate polymer with sulfydryl, stirring for 24H to obtain hydrophobic H2O2Responsive curcumin polymer a.
(2) Taking the hydrophobic H prepared in the step (1)2O220mg of the responsive curcumin polymer was dissolved in DMF (anhydrous dimethyl sulfoxide), 4.2mg of PYBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) and 2.2ml of DIEA (diisopropylethylamine) were added, 15mg of a solution of SynB1 in DMF was added dropwise, and the mixture was dialyzed against ethanol and water, respectively, to obtain a solution b.
(3) Taking the hydrophobic H prepared in the step (1)2O2The corresponding curcumin polymer (20 mg) was dissolved in DMF, DMAP (1.16 mg), EDCCL (1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride) (2.2 mg) was added, a DMF solution of 4.5mg of mazindol was added dropwise, stirred for 24h, and dialyzed against ethanol and water, respectively, to give solution c.
(4) By adopting a sodium citrate reduction method, 10mg of tetrachloroauric acid is dissolved in 100mL of deionized water, and the solution is heated to boiling while being vigorously stirred. Then adding 3mL of trisodium citrate solution with the mass fraction of 1%, and continuing to react for 30min to obtain a gold nanoparticle solution d with the particle size of 14 nm.
(5) The hydrophobicity H prepared in the step (1) is2O2Responsive curcumin polymer prepared in step (2)And (3) mixing the obtained solution b, the mixed solution of the solution c prepared in the step (3) and the gold nanoparticles according to the mass ratio of 1: 4, carrying out ultrasonic treatment for 1 hour, and standing overnight to obtain the polymer gold nanoparticles with uniform particle size.
(6) Firstly, acidifying polymer gold nanoparticles, centrifuging the obtained product at 1.5ml14000rpm, adding 750ul of buffer solution with the pH value of 2.5, and compounding with 0.02OD siRNA, wherein the ratio of N to P is 1:100, and obtaining the curcumin perfume system after 30 min. The particle size is 20-30nm as characterized by an electron microscope, and the curcumin spice system prepared in the example 3 is verified to inhibit a-syn aggregation through the existing Parkinson disease over-expression EGFP-a-syn cell model in a laboratory, so that the curcumin spice system has an obvious treatment effect on Parkinson disease.
Example 4
In this example, a curcumin flavor system was prepared by the following method, specifically including the steps of:
(1) adding curcumin 500mg into round bottom flask, dissolving with 25ml THF (tetrahydrofuran), adding 330ul triethylamine, and stirring for 30 min. 20ul of methacrylic anhydride was taken and dissolved in 1.25ml of tetrahydrofuran. And (4) dropwise adding the reaction system, stirring vigorously, and slowly dropwise adding until the dropwise adding is finished. The temperature was increased to 71 ℃ overnight under nitrogen. The product is obtained by post-treatment and then separated by a chromatographic column. And (2) taking 41.9mg of the product, adding DMF (dimethylformamide) to dissolve the product, adding 5ul of triethylamine, dropwise adding 5.8mg of poly-tert-butyl betaine methacrylate polymer with sulfydryl, and stirring for 24 hours to obtain the curcumin polymer a responding to hydrophobic H2O 2.
(2) Taking the hydrophobic H prepared in the step (1)2O2The responding curcumin polymer (20 mg) was dissolved in DMF (anhydrous dimethyl sulfoxide), 4.2mg of PYBOP (benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate) and 2.2ml of DIEA (diisopropylethylamine) were added, 15mg of a DMF solution of cell-penetrating peptide (CGHKAKGPRK, 99%) was added dropwise, and the mixture was dialyzed against ethanol and water, respectively, to obtain a solution b.
(3) Taking the hydrophobic H prepared in the step (1)2O2The corresponding curcumin polymer, 20mg, was dissolved in DMF and DMAP, 1.16mg, EDCCL (1-ethyl- (3-dimethylaminopropyl) carbonyldiimine hydrochloride), 2.2mg, 4.5mg dropwise with 29 amino groupsA DMF solution of the short peptide of acid Residue (RVG) was stirred for 24 hours and dialyzed against ethanol and water, respectively, to obtain a solution c.
(4) By adopting a sodium citrate reduction method, 10mg of tetrachloroauric acid is dissolved in 100mL of deionized water, and the solution is heated to boiling while being vigorously stirred. Then adding 3mL of trisodium citrate solution with the mass fraction of 1%, and continuing to react for 30min to obtain a gold nanoparticle solution d with the particle size of 14 nm.
(5) The hydrophobicity H prepared in the step (1) is2O2And (3) mixing the responding curcumin polymer, the solution b prepared in the step (2) and the solution c prepared in the step (3) with the gold nanoparticles according to the mass ratio of 1: 4, carrying out ultrasonic treatment for 1 hour, and standing overnight to obtain the polymer gold nanoparticles with uniform particle size.
(6) Firstly, acidifying polymer gold nanoparticles, centrifuging the obtained product at 1.5ml14000rpm, adding 750ul of buffer solution with the pH value of 2.5, and compounding with 0.02OD siRNA, wherein the ratio of N to P is 1:100, and obtaining the curcumin perfume system after 30 min.
The particle size is 20-40nm as characterized by an electron microscope, and the curcumin spice system prepared in the example 3 is verified to inhibit a-syn aggregation through the existing Parkinson disease over-expression EGFP-a-syn cell model in a laboratory, so that the curcumin spice system has an obvious treatment effect on Parkinson disease.
The results of the above embodiments show that the curcumin perfume system prepared by the invention has an obvious treatment effect on Parkinson's disease, and compared with the prior art, the curcumin perfume system greatly improves the bioavailability and the in-vivo retention time, and has an excellent application prospect.
In addition, in the above embodiments of the present invention, the organic solvent may also be other organic solvents with similar properties in the art, which can also achieve substantially equivalent effects, and is not limited thereto.
The hydrophobicity H in the step (1)2O2The responsive curcumin polymer can also be prepared by replacing methacrylic anhydride with an arylboronic acid ester, and the molecule for realizing the controllable release of curcumin through hydrogen peroxide response is phenylboronic acid.
In step (2), the second-stage targeting molecule may also be any one or a combination of two or more of a derivative of a short peptide, mazindol or a derivative thereof, and can also achieve substantially equivalent effects, but not limited thereto.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A curcumin flavor system is formed by bonding substances at least comprising curcumin flavors, a first-stage targeting molecule and a second-stage targeting molecule with a polymer material through covalent bonds, wherein the first-stage targeting molecule comprises cell-penetrating peptide or cell penetrating peptide, the second-stage targeting molecule comprises mazindol or short peptide RVG29, the polymer material comprises poly-tert-butyl betaine methacrylate polymer with sulfydryl shown in formula (1), and a connecting group which takes a hydrogen peroxide response chemical bond as a connecting group to bond curcumin flavors, and the connecting group which realizes the controllable release of curcumin through hydrogen peroxide response is an acryl sulfide group;
2. a curcumin flavor system as claimed in claim 1 wherein said primary targeting molecule is SynB1 or CPP B6.
3. A curcumin flavor system as claimed in claim 1 further comprising gold nanoparticle particles having molecular imaging advantage in the core of said polymeric material.
4. A process for the preparation of a curcumin flavor system as claimed in any one of claims 1 to 3 comprising the steps of:
(1) dissolving curcumin spice, polymer material and chemical molecules capable of realizing hydrogen peroxide response in an organic solvent, and reacting to obtain hydrophobic H2O2Responsive curcumin polymer a;
(2) the hydrophobicity H prepared in the step (1)2O2Mixing the responding curcumin polymer a and a first-stage targeting molecule dissolved in an organic solvent, and dialyzing to obtain a solution b;
(3) the hydrophobicity H prepared in the step (1)2O2Mixing the responding curcumin polymer a and a second-stage targeting molecule dissolved in an organic solvent, and dialyzing to obtain a solution c;
(4) will be hydrophobic H2O2And mixing the responding curcumin polymer a, the solution b and the solution c, and performing ultrasonic treatment to obtain the curcumin perfume system.
5. The method of claim 4, wherein in step (2), the primary targeting molecule is conjugated to a hydrophobic moiety H2O2The molar ratio of the responsive curcumin polymer is 1: 1-1: 10;
in step (3), the second targeting molecule is conjugated to the hydrophobic moiety H2O2The molar ratio of the responsive curcumin polymer is 1: 1-1: 10;
in step (4), hydrophobicity H2O2The responding curcumin polymer a, the solution b and the solution c are added according to the molar ratio of curcumin of 1:1: 1.
6. The method according to claim 4, wherein in the step (4), the hydrophobicity H is changed to2O2And adding gold nanoparticles into the mixed solution of the responsive curcumin polymer a, the solution b and the solution c, carrying out ultrasonic treatment, standing overnight to obtain a solution e, acidifying the solution e, and centrifuging to obtain the polymer gold nanoparticles.
7. The preparation method according to claim 6, further comprising a step (5) of adding siRNA to the polymeric gold nanoparticles and complexing to obtain a curcumin flavor system.
8. The method according to claim 6, wherein in the step (4), the hydrophobicity H is2O2The mass ratio of the mixed solution of the responsive curcumin polymer a, the solution b and the solution c to the gold nanoparticles is 1: 1-1: 5; the particle size of the gold nanoparticles is 10-50 nm; the gold nanoparticle solution is prepared by a trisodium citrate reduction method.
9. The method according to claim 7, wherein in the step (5), the ratio of the polymer gold nanoparticles to the N: p is 1:100 to 1: 500.
10. Use of a curcumin flavor system as claimed in any one of claims 1 to 3 in the manufacture of a medicament for the treatment of parkinson's disease.
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| CN103720675A (en) * | 2014-01-06 | 2014-04-16 | 天津大学 | Curcumin prodrug micelle with oxidation and reduction sensitivity, micellar monomer and preparation method of micellar monomer |
| CN107050465A (en) * | 2017-01-17 | 2017-08-18 | 中国药科大学 | A kind of feature Nano medication of curcumin and its application for brain diseases diagnosis and treatment |
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| CN103720675A (en) * | 2014-01-06 | 2014-04-16 | 天津大学 | Curcumin prodrug micelle with oxidation and reduction sensitivity, micellar monomer and preparation method of micellar monomer |
| CN107050465A (en) * | 2017-01-17 | 2017-08-18 | 中国药科大学 | A kind of feature Nano medication of curcumin and its application for brain diseases diagnosis and treatment |
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