CN112656960B - Mitochondria-controlled iron-based magnetic coordination polymer nanoparticle and preparation method and application thereof - Google Patents
Mitochondria-controlled iron-based magnetic coordination polymer nanoparticle and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a mitochondria-controlled iron-based magnetic coordination polymer nanoparticle and a preparation method and application thereof, belonging to the technical field of medicines. The polymeric nanoparticle comprises: dopamine-modified hyaluronic acid, iron oxide nanoparticles and dichloroacetic acid-modified cisplatin; wherein: the hydroxyl of the catechol on the dopamine is in coordination complexing with the ferric oxide, and the polycarboxyl on the hyaluronic acid is in coordination complexing with the platinum atom. The polymer nanoparticles combine dichloroacetic acid and cisplatin, so that intracellular H can be greatly increased through mitochondria and NOX double-acting sites in tumor cells 2 O 2 Of (1) containsThe high-toxicity active oxygen hydroxyl free radicals are generated by catalyzing the high-toxicity active oxygen hydroxyl free radicals with magnetic iron oxide, so that tumor cell apoptosis is initiated; in addition, magnetic iron oxide can produce T 2 The magnetic resonance imaging effect can accurately obtain the spatial position and size information of the tumor.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a mitochondria-regulated iron-based magnetic coordination polymer nanoparticle and a preparation method and application thereof.
Background
Cisplatin, as a broad-spectrum anticancer drug, is one of the most widely used chemotherapy drugs in clinical practice, and is commonly used for treating lung cancer, testicular cancer, ovarian cancer, sarcoma and other diseases. However, the low therapeutic effect and high toxic and side effect of cisplatin, especially the drug resistance of cisplatin, often limit the application of cisplatin in clinical treatment. At present, other chemotherapy drugs or biological similar drugs are often combined with cisplatin in clinic to improve the treatment effect. Therefore, there is a need to develop a therapeutic system that can improve the sensitivity of tumors to cisplatin.
After cisplatin enters tumor cells, it usually targets the nucleus to react with the nucleophilic site of DNA to form an adduct, resulting in DNA cross-linking to disrupt its ability to transcribe and replicate, thereby exerting its anti-tumor effect. In addition, cisplatin can also activate Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase (NOX) in tumor cells to convert O into oxygen 2 Conversion to O 2 • - And generating H under the action of superoxide dismutase (SOD) 2 O 2 Increasing the oxidation level in the cells. The introduction of the iron-based material can utilize Fenton's reaction to convert H 2 O 2 Converted into high-toxicity hydroxyl free radicals, and further enhances the anti-tumor curative effect of the cisplatin. However, cisplatin alone produces insufficient hydrogen peroxide to induce a strong fenton reaction, and thus it is difficult to significantly improve its antitumor efficacy.
Dichloroacetic acid (DCA) is a small molecule compound that is often used clinically to treat lactic acidosis because it can target mitochondria, inhibit Pyruvate Dehydrogenase Kinase (PDK), activate Pyruvate Dehydrogenase (PDH) activity, and transfer glucose oxidation from glycolysis to the aerobic oxidation pathway. The mitochondria is an important place for intracellular oxidation, and the dichloroacetic acid is introduced into tumor cells, so that the oxidation function of the mitochondria can be activated, and the intracellular oxidation level is greatly enhanced. Dichloroacetic acid has been widely used in the treatment of certain cancers, such as lung cancer, breast cancer, glioblastoma, and the like. In addition, dichloroacetic acid can activate the oxidation activity of mitochondria and promote the release of factors related to mitochondrial apoptosis, and the substances can further increase the sensitivity of tumor cells to apoptosis.
Therefore, the present invention contemplates the design of therapeutic systems that can increase the sensitivity of tumors to cisplatin.
Disclosure of Invention
The invention aims to provide a mitochondria-controlled iron-based magnetic coordination polymer nanoparticle for enhancing cis-platinum anti-tumor sensitivity. The polymer nanoparticles form a coordination polymer by using hyaluronic acid modified by dopamine and magnetic iron oxide, and load cisplatin modified by dichloroacetic acid, so that the mitochondrial regulation type iron-based magnetic coordination nano polymer is prepared.
In order to achieve the above purpose, the technical scheme of the invention is as follows:
a mitochondria-regulated iron-based magnetic coordination polymer nanoparticle, comprising: dopamine-modified hyaluronic acid, iron oxide nanoparticles and dichloroacetic acid-modified cisplatin;
wherein: the hydroxyl of the catechol on the dopamine is in coordination complexing with the ferric oxide, and the polycarboxyl on the hyaluronic acid is in coordination complexing with the platinum atom.
Furthermore, the particle size of the coordination polymer nanoparticles is 140-180 nm.
The preparation method of the coordination polymer nanoparticle comprises the following steps:
step 3, administering cisplatinIs added to H 2 O 2 Reacting in the solution to obtain hydroxylated cisplatin, adding the hydroxylated cisplatin into acetone, dropwise adding dichloroacetyl chloride under stirring to react, and dropwise adding the reaction liquid into diethyl ether after the reaction is finished to obtain dichloroacetic acid modified cisplatin;
and 4, adding dichloroacetic acid modified cis-platinum into the iron oxide-dopamine modified hyaluronic acid coordination polymer obtained in the step 2, and stirring for reaction to obtain coordination polymer nanoparticles.
Further, the feeding molar ratio of the hyaluronic acid, the EDC and the NHS in the step 1 is 1: 5-10: 5-10, wherein the feeding molar ratio of dopamine hydrochloride to hyaluronic acid is 5-10: 1, the reaction conditions are 20-25 ℃ and 12-24 h.
Further, in the step 2, the feeding mass ratio of the iron oxide nanoparticles to the dopamine-modified hyaluronic acid is 1: 2-4, and the reaction conditions are 20-25 ℃ and 2-6 h.
Further, in step 3, cisplatin and H 2 O 2 The feeding molar ratio of (1): 2-4, the reaction conditions are 50-70 ℃ and 3-6 h; the feeding molar ratio of the hydroxylated cisplatin to the dichloroacetyl chloride is 1: 2-4, the reaction conditions are 20-25 ℃ and 12-24 h.
Further, in the step 4, the feeding mass ratio of the dichloroacetic acid modified cisplatin to the iron oxide-dopamine modified hyaluronic acid coordination polymer is 1: 5-10, and reacting for 48-72 h.
The coordination polymer nanoparticle takes dopamine hyaluronic acid as a core, and is loaded with magnetic iron oxide and cisplatin for modifying dichloroacetic acid, and the loading is realized by coordination complexation of o-diphenol hydroxyl on dopamine and iron oxide and coordination of polycarboxyl on hyaluronic acid and platinum atoms. Intracellular H enhancement by acidolysis and reduction, cisplatin and dichloroacetic acid release, respectively, by NOX and PDH enzymatic reactions in tumor cells 2 O 2 Content (c); the iron oxide can release free iron ions in the acidic environment of tumor cells, and can effectively catalyze H 2 O 2 Generating highly toxic hydroxyl radicals. In addition, cisplatin can interfere with nuclear DNA (deoxyribonucleic acid) which is a key target of cisplatin, and dichloroacetic acid can activate mitochondrial apoptosis pathway and is used for controlling mitochondrial membrane potentialThe outflow of the altered and pro-apoptotic factors further promotes the killing effect of tumors. Meanwhile, the nanoparticle also has T 2 The magnetic resonance imaging function can be used for diagnosing and treating tumors.
Drawings
FIG. 1 is a schematic diagram of synthesis of dopamine-modified hyaluronic acid (A) and dichloroacetic acid-modified cisplatin (B).
FIG. 2 is a diagram showing hydrogen nuclear magnetic resonance spectroscopy (A) for dopamine-modified hyaluronic acid, hydrogen nuclear magnetic resonance spectroscopy (B) for dichloroacetic acid-modified cisplatin, and mass spectrometry (C) for dichloroacetic acid-modified cisplatin
FIG. 3 is a diagram of the related in vitro characterization of the mitochondria-regulated iron-based magnetically coordination polymer nanoparticles: particle size distribution graph (A), particle size stability graph (B).
FIG. 4 is a zeta potential diagram of the iron-based magnetic coordination polymer nanoparticle of the mitochondria regulation type.
FIG. 5 is a diagram of the efficiency of the iron-based magnetic coordination polymer nanoparticle in vitro catalysis of hydrogen peroxide in the mitochondria-controlled manner.
FIG. 6 is a graph showing the in vitro release of iron-based magnetic coordination polymer nanoparticles of the mitochondria-controlled type.
FIG. 7 is an imaging diagram of the mitochondrial modulation type iron-based magnetic coordination polymer nanoparticle external magnetic resonance.
FIG. 8 is a cytotoxicity diagram of mitochondria-regulated iron-based magnetic coordination polymer nanoparticles.
FIG. 9 is a representation of the cell correlation after the action of the mitochondria-regulated iron-based magnetic coordination polymer nanoparticles: mitochondrial membrane potential map (A) and intracellular active oxygen content map (B).
FIG. 10 is the image of magnetic resonance imaging in mitochondria of iron-based magnetic coordination polymer nanoparticles of mitochondrial regulation type.
Fig. 11 is a graph of in vivo relevant pharmacodynamic data for mitochondria-regulated iron-based magnetic coordination polymer nanoparticles.
Detailed Description
In the invention, the mitochondrial regulation type iron-based magnetic coordination polymer nanoparticle for enhancing cis-platinum anti-tumor sensitivity is provided, dopamine hyaluronic acid is taken as a core,and meanwhile, the cisplatin loaded with magnetic iron oxide and modified dichloroacetic acid realizes the loading of the medicament by coordination complexation of o-diphenol hydroxyl on dopamine and iron oxide and coordination of polycarboxyl on hyaluronic acid and platinum atoms. Intracellular H enhancement by acidolysis and reduction, cisplatin and dichloroacetic acid release, respectively, by NOX and PDH enzymatic reactions in tumor cells 2 O 2 The content; the iron oxide can release free iron ions in the acidic environment of tumor cells, and can effectively catalyze H 2 O 2 Generating highly toxic hydroxyl radicals. In addition, cisplatin can further promote the killing effect of tumors by interfering the key target of the cisplatin, namely nuclear DNA, and dichloroacetic acid can activate mitochondrial apoptosis pathways through the change of mitochondrial membrane potential and the outflow of pro-apoptotic factors. Meanwhile, the nanoparticle also has T 2 The magnetic resonance imaging function can be used for diagnosing and treating tumors.
The coordination polymer nanoparticles are prepared by firstly forming a coordination polymer by dopamine-modified hyaluronic acid and magnetic iron oxide nanoparticles through coordination, and then loading dichloroacetic acid-modified cisplatin in the polymer through coordination of platinum atoms and carboxyl groups. The particle size of the coordination polymer nanoparticles is 140-180 nm, and preferably 160-170 nm; the drug loading rate is 6-10%, and 8% is preferred.
The coordination polymer nanoparticles are prepared by the following method:
(1) dissolving hyaluronic acid, EDC and NHS in PBS buffer solution with pH6.8 according to a molar ratio of 1: 5-10, and reacting for 15-30 min.
(2) Dissolving dopamine hydrochloride in ultrapure water, adding the solution into the mixed solution obtained in the step (1), and reacting for 12-24 hours under the protection of nitrogen, wherein the feeding molar ratio of dopamine hydrochloride to hyaluronic acid is 5-10: 1.
(3) and (3) adding the reaction solution obtained in the step (2) into a dialysis bag of 8000-12000 Da, dialyzing in ultrapure water for 24h, and freeze-drying the dialyzed solution to obtain the off-white dopamine modified hyaluronic acid.
(4) Dissolving the iron oxide nanoparticles in ultrapure water to obtain an iron oxide nanoparticle aqueous solution.
(5) Dissolving the dopamine-modified hyaluronic acid obtained in the step (3) in ultrapure water, and adding the ultrapure water into the iron oxide nanoparticle aqueous solution obtained in the step (4), wherein the feeding mass ratio of the iron oxide nanoparticles to the dopamine-modified hyaluronic acid is 1: 2-4, adjusting the pH value to 6.0 by using NaOH solution, and stirring and reacting for 2-6 h at room temperature to form iron oxide-dopamine modified hyaluronic acid coordination polymer solution.
(6) Addition of cisplatin to H 2 O 2 In solution, cisplatin and H 2 O 2 The feeding molar ratio of (1): and 2-4, reacting for 3-6 hours at 50-70 ℃, and removing the solution through rotary evaporation to obtain the hydroxylated cis-platinum.
(7) Adding the hydroxylated cisplatin in the step (6) into an acetone solvent, dropwise adding a dichloroacetyl chloride solution under the stirring condition, and reacting for 12-24h, wherein the feeding molar ratio of the hydroxylated cisplatin to the dichloroacetyl chloride is 1: 2 to 4.
(8) Dropwise adding the reaction solution obtained in the step (7) into an ether solution, wherein the volume ratio of the reaction solution to the ether solution is 1: 10, obtaining yellow precipitate, and drying to obtain dichloroacetic acid modified cisplatin.
(9) Dissolving the dichloroacetic acid modified cisplatin in the step (8) in ultrapure water, and adding the solution into the coordination polymer nanoparticle solution in the step (5) under continuous stirring, wherein the feeding mass ratio of the dichloroacetic acid modified cisplatin to the coordination polymer nanoparticles is 1: and 5-10, reacting for 48-72 hours to prepare a crude coordination polymer nanoparticle solution.
(10) And (4) dialyzing the crude product solution in the step (9) by using a dialysis bag with the molecular weight of 3500Da to remove the unloaded free drug, so as to obtain the final coordination polymer nanoparticle solution.
The technical solutions of the present invention are further described in detail below with reference to the drawings and the specific embodiments, but should not be construed as limiting the present invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
Example 1
The nanoparticles are prepared by the following method:
(1) 100mg of Hyaluronic Acid (HA), 120mg of EDC and 70mg of NHS were dissolved in 25mL of PBS buffer (pH6.8), and reacted in a 50mL round-bottomed flask for 15 min.
(2) 120mg of dopamine hydrochloride (Dopa) is dissolved in ultrapure water and added into the mixed solution in the step (1) to react for 12 hours under the protection of nitrogen.
(3) And (3) adding the reaction solution obtained in the step (2) into a dialysis bag of 8000-12000 Da, dialyzing in ultrapure water for 24h, changing water every 2h, and freeze-drying the dialyzed solution to obtain the off-white dopamine modified hyaluronic acid (DPA).
(4) 1mg of iron oxide nanoparticles was dissolved in 5mL of ultrapure water to obtain an iron oxide nanoparticle aqueous solution.
(5) And (3) dissolving 10mg of dopamine-modified hyaluronic acid in the step (3) in 5mL of ultrapure water, adding the solution into the iron oxide nanoparticle aqueous solution in the step (4), adjusting the pH to 6.0 by using a NaOH solution, and stirring and reacting at room temperature for 2 hours to form a coordination polymer nanoparticle solution (FeO. DPA).
(6) Mu. mol cisplatin (Pt) was added to 10mL H 2 O 2 In solution, reacted at 60 ℃ for 4h, and the solution was removed by rotary evaporation to obtain hydroxylated cisplatin (Pt-OH).
(7) The hydroxylated cisplatin in step (6) was added to 7mL of acetone solvent, and 300. mu. mol of dichloroacetyl chloride solution was added dropwise with stirring and reacted for 12 hours.
(8) And (3) dropwise adding the reaction liquid in the step (7) into 25mL of diethyl ether solution to obtain yellow precipitate, and drying to obtain dichloroacetic acid modified cisplatin (DPt).
(9) And (3) dissolving 1mg of dichloroacetic acid modified cisplatin in the step (8) in 1mL of ultrapure water, adding the solution into the coordination polymer nanoparticle solution in the step (5) under continuous stirring, and reacting for 48 hours to prepare a crude coordination polymer nanoparticle solution.
(10) And (3) dialyzing the crude product solution in the step (9) by using a dialysis bag with the molecular weight of 3500Da, dialyzing for 6h, and replacing water every 2h to remove the free drug which is not loaded, so as to obtain the final coordination polymer nanoparticle solution (DPt @ FeO. DPA).
FIG. 1 is a schematic diagram of a synthesis scheme of dopamine-modified hyaluronic acid (A) and a schematic diagram of a synthesis scheme of dichloroacetic acid-modified cisplatin (B).
FIG. 2 is a nuclear magnetic resonance hydrogen spectrum (A) of dopamine-modified hyaluronic acid, wherein characteristic hydrogen peaks of dopamine and hyaluronic acid are shown, and the successful modification of dopamine on hyaluronic acid is proved; and a dichloroacetic acid modified cis-platinum nuclear magnetic resonance hydrogen spectrogram (B) and a mass spectrogram (C) show an amino hydrogen spectrogram peak of cis-platinum, a hydrogen spectrogram peak of dichloromethyl and dichloroacetic acid modified cis-platinum, and prove that the dichloroacetic acid modified cis-platinum is successfully synthesized.
FIG. 3 is a diagram of the related in vitro characterization of the mitochondria-regulated iron-based magnetically coordination polymer nanoparticles: particle size distribution graph (A), particle size stability graph (B). The average particle size of the prepared drug-loaded coordination polymerization nanoparticles is 167.6nm, and the stability in pH7.4PBS buffer solution and 10% FBS culture medium is good for 48 h.
FIG. 4 is a zeta potential diagram of the iron-based magnetic coordination polymer nanoparticle of the mitochondria regulation type. It can be seen from the figure that the average zeta potential of the finally prepared drug-loaded coordination polymer nanoparticles is-41.9 mV.
FIG. 5 is a diagram of the efficiency of the iron-based magnetic coordination polymer nanoparticle in vitro catalysis of hydrogen peroxide in the mitochondria-controlled manner. Adding methylene blue, methylene blue and H into 4 ep tubes respectively 2 O 2 Methylene blue + DPt @ FeO DPA, and methylene blue + H 2 O 2 + DPt @ FeO & DPA, and after reacting for 2-6 h, placing each group of solution in an ultraviolet absorption spectrophotometer to measure an absorption spectrum of 400-800 nm. As can be seen in the figure, H alone 2 O 2 And the coordination polymer nanoparticles can not reduce the characteristic ultraviolet absorption peak of Methylene Blue (MB) when H is reduced 2 O 2 After the coordination polymer nanoparticles are incubated together, the characteristic ultraviolet absorption peak of Methyl Blue (MB) can be greatly reduced, which indicates that H is catalyzed by the coordination polymer nanoparticles 2 O 2 The decomposition produces hydroxyl radical OH with high oxidizing power.
FIG. 6 is a graph showing the in vitro release of iron-based magnetic coordination polymer nanoparticles of the mitochondria-controlled type. DPt @ FeO.DPA is respectively put into 30-50 ml release medium (pH7.4, 5.0PBS buffer solution), and the temperature is controlled to be 37 +/-1 ℃. 2ml of release medium were removed at 1, 2, 4, 6, 8, 10, 12, 24, 36h, respectively, and 2ml of fresh medium was replenished, and the release profile was plotted over time by measuring the cisplatin content by atomic absorption spectrophotometer. It can be seen from the figure that the release of cisplatin is pH sensitive, and can release 60.5% under the condition of pH5.0, and can only release 38.5% of the drug under the condition of pH 7.4.
FIG. 7 is an imaging diagram of the mitochondrial regulated iron-based magnetic coordination polymer nanoparticle external magnetic resonance. DPt @ FeO.DPA containing 0.5mM iron ions is prepared, diluted into a series of gradient concentrations respectively, and placed on a 7.0T magnetic resonance imager for scanning to obtain T 2 Magnetic resonance image, each set of time is read separately, and the reciprocal (1/T) is used 2 ) And (4) making a scatter diagram of the concentration of the Fe ions and performing linear fitting. It can be seen from the figure that the relaxation coefficient r2 of the coordination polymer nanoparticles prepared is 217.42Mm -1 s -1 Having excellent T 2 Imaging effect as efficient T 2 A contrast agent.
FIG. 8 is a cytotoxicity diagram of mitochondria-regulated iron-based magnetic coordination polymer nanoparticles. 4T1 and MB-231 tumor cells were cultured in 96-well plates, respectively, and Pt, DPt, Pt @ FeO.DPA, and DPt @ FeO.DPA were prepared at different platinum contents. And when the cells grow to 80-90%, adding the solution, and evaluating the cell killing effect by adopting a standard MTT method. As can be seen from the figure, the final drug-loaded coordination polymer nanoparticle group remarkably enhances the killing effect of 4T1 and MB-231 tumor cells, reduces the cis-platinum IC50 value by 48.39 percent and 82.18 percent respectively, and remarkably enhances the anti-tumor sensitivity of the cis-platinum.
FIG. 9 is a representation of cells after the action of the mitochondria-regulated iron-based magnetic coordination polymer nanoparticles. Adding 4T1 tumor cells into a 6-well plate, and adding PBS, FeO & DPA, Pt, DPt, Pt @ FeO & DPA and DPt @ FeO & DPA when the cells grow to 80% -90%. After culturing for 18-30 h, washing each culture hole by PBS, respectively adding a mitochondrial membrane potential probe RDM-123 and an active oxygen probe DCFH-DA, and observing the fluorescence intensity of each group under a fluorescence microscope. After the mitochondrial membrane potential diagram (A) is indicated by using RDM-123, the fluorescence intensity of the drug-loaded coordination polymer nanoparticle group can be obviously reduced, which shows that the drug-loaded coordination polymer nanoparticles can obviously reduce the mitochondrial membrane potential; and (B) indicating the active oxygen content by using a DCFH-DA probe, wherein the fluorescence intensity of the drug-loaded coordination polymer nanoparticle group is obviously enhanced, which shows that the drug-loaded coordination polymer nanoparticles can obviously enhance the active oxygen content in the cells.
FIG. 10 is the image of magnetic resonance imaging in mitochondria of iron-based magnetic coordination polymer nanoparticles of mitochondrial regulation type. Randomly selecting 3 tumor-bearing mice, injecting the coordination polymer nanoparticle solution prepared by tail vein injection, and then placing the mice on a magnetic resonance imaging scanner for MRI imaging at 0h, 6h and 12 h. It can be seen from the figure that after the prepared coordination polymer nanoparticles are injected intravenously, the tumor part of a mouse is obviously darker than the tumor part before the nanoparticles are injected, the contrast between the tumor tissue and the surrounding normal tissue is also more obvious, and the tumor boundary is clearer along with the time extension, which indicates that the nanoparticles have good in vivo magnetic resonance imaging effect.
FIG. 11 is a graph of the in vivo related pharmacodynamic data of the iron-based magnetic coordination polymer nanoparticle of mitochondria-controlled type. The evaluation was carried out using a 4T1 tumor-bearing mouse model, which was divided into six groups, PBS group, FeO & DPA group, Pt group, DPt group, Pt @ FeO & DPA group, and DPt @ FeO & DPA group. Randomly selecting 30 tumor-bearing mice, 5 mice in each group, injecting the solution on 0, 2, 4, 6 and 8 days, and measuring the tumor size with a vernier caliper every other day for 21 days. After the experiment was completed, the mice were sacrificed by spinal cord dissection, and tumors of each group of mice were dissected and collected, weighed and photographed. The tumors of each group were sectioned and stained with H & E, and finally the tumor tissue morphology of each group was observed with a biological inverted microscope and photographed. Obtaining a tumor inhibition effect graph (A), a tumor inhibition curve graph (B) and a tumor H & E staining graph (C). It can be seen from fig. a and B that the drug-loaded coordination polymer nanoparticles prepared by intravenous injection can significantly inhibit tumor growth compared to other control groups. As can be seen from the graph C, the tumor of the final drug-loaded coordination polymer nanoparticle group is obviously damaged, the intercellular space of the tumor tissue area is larger, and the cell nucleus is more compact.
Example 2
The nanoparticle is prepared by the following method:
(1) 100mg of Hyaluronic Acid (HA), 150mgEDC and 111mgNHS were dissolved in 25mL of PBS buffer (pH6.8), and reacted in a 50mL round-bottomed flask for 20 min.
(2) 180mg of dopamine hydrochloride (Dopa) is dissolved in ultrapure water and added into the mixed solution in the step (1) to react for 18h under the protection of nitrogen.
(3) And (3) adding the reaction solution obtained in the step (2) into a dialysis bag of 8000-12000 Da, dialyzing in ultrapure water for 24h, changing water every 2h, and freeze-drying the dialyzed solution to obtain the off-white dopamine modified hyaluronic acid (DPA).
(4) 1mg of iron oxide nanoparticles was dissolved in 5mL of ultrapure water to obtain an iron oxide nanoparticle aqueous solution.
(5) And (3) dissolving 20mg of dopamine-modified hyaluronic acid in the step (3) in 5mL of ultrapure water, adding the solution into the iron oxide nanoparticle aqueous solution in the step (4), adjusting the pH to 6.0 by using a NaOH solution, and stirring and reacting at room temperature for 3 hours to form a coordination polymer nanoparticle solution (FeO. DPA).
(6) Mu. mol cisplatin (Pt) was added to 15mL H 2 O 2 In the solution, the reaction was carried out at 60 ℃ for 5 hours, and the solution was removed by rotary evaporation to obtain hydroxylated cisplatin (Pt-OH).
(7) The hydroxylated cisplatin in step (6) was added to 7mL of an acetone solvent, and a 400. mu. mol dichloroacetyl chloride solution was added dropwise with stirring and reacted for 18 hours.
(8) And (3) dropwise adding the reaction liquid in the step (7) into 25mL of diethyl ether solution to obtain yellow precipitate, and drying to obtain dichloroacetic acid modified cisplatin (DPt).
(9) And (3) dissolving 1.5mg of dichloroacetic acid modified cisplatin in the step (8) in 1mL of ultrapure water, adding the solution into the coordination polymer nanoparticle solution in the step (5) under continuous stirring, and reacting for 60 hours to prepare a crude coordination polymer nanoparticle solution.
(10) And (4) dialyzing the crude product solution in the step (9) by using a dialysis bag with the molecular weight of 3500Da, dialyzing for 10h, and replacing water every 2h to remove the unsupported free drug to obtain the final coordination polymer nanoparticle solution (DPt @ FeO. DPA).
Example 3
The nanoparticle is prepared by the following method:
(1) 100mg of Hyaluronic Acid (HA), 180mgEDC and 133mgNHS were dissolved in 25mL of PBS buffer (pH6.8), and reacted in a 50mL round-bottomed flask for 30 min.
(2) 220mg of dopamine hydrochloride (Dopa) is dissolved in ultrapure water and added into the mixed solution in the step (1) to react for 24 hours under the protection of nitrogen.
(3) And (3) adding the reaction solution obtained in the step (2) into a dialysis bag of 8000-12000 Da, dialyzing the solution in ultrapure water for 24 hours, changing the water every 2 hours, and freeze-drying the dialyzed solution to obtain the off-white dopamine modified hyaluronic acid (DPA).
(4) 1mg of iron oxide nanoparticles was dissolved in 5mL of ultrapure water to obtain an iron oxide nanoparticle aqueous solution.
(5) And (3) dissolving 20mg of dopamine-modified hyaluronic acid in the step (3) in 5mL of ultrapure water, adding the solution into the iron oxide nanoparticle aqueous solution in the step (4), adjusting the pH to 6.0 by using a NaOH solution, and stirring and reacting at room temperature for 6 hours to form a coordination polymer nanoparticle solution (FeO. DPA).
(6) Mu. mol cisplatin (Pt) was added to 20mL H 2 O 2 In the solution, the reaction was carried out at 60 ℃ for 6 hours, and the solution was removed by rotary evaporation to obtain hydroxylated cisplatin (Pt-OH).
(7) The hydroxylated cisplatin in step (6) was added to 7mL of an acetone solvent, and a 500. mu. mol dichloroacetyl chloride solution was added dropwise with stirring and reacted for 24 hours.
(8) And (3) dropwise adding the reaction liquid in the step (7) into 25mL of diethyl ether solution to obtain yellow precipitate, and drying to obtain dichloroacetic acid modified cisplatin (DPt).
(9) And (3) dissolving 2.0mg of dichloroacetic acid modified cisplatin in the step (8) in 1mL of ultrapure water, adding the solution into the coordination polymer nanoparticle solution in the step (5) under continuous stirring, and reacting for 48-36 h to prepare a crude coordination polymer nanoparticle solution.
(10) And (3) dialyzing the crude product solution in the step (9) by using a dialysis bag with the molecular weight of 3500Da, dialyzing for 12h, and replacing water every 2h to remove the free drug which is not loaded, so as to obtain the final coordination polymer nanoparticle solution (DPt @ FeO. DPA).
Claims (3)
1. A mitochondria-controlled iron-based magnetic coordination polymer nanoparticle is characterized in that:
the method comprises the following steps: the composition comprises hyaluronic acid modified by dopamine, iron oxide nanoparticles and cisplatin modified by dichloroacetic acid; wherein: the o-diphenol hydroxyl on the dopamine is in coordination complexing with the iron oxide, and the polycarboxyl on the hyaluronic acid is in coordination complexing with the platinum atom;
the preparation method of the coordination polymer nanoparticles comprises the following steps:
step 1, dissolving hyaluronic acid, EDC and NHS in PBS buffer solution with pH6.8, adding dopamine hydrochloride aqueous solution, and reacting under the protection of nitrogen, wherein the feeding molar ratio of hyaluronic acid, EDC and NHS is 1: 5-10: 5-10, wherein the feeding molar ratio of dopamine hydrochloride to hyaluronic acid is 5-10: 1, obtaining dopamine modified hyaluronic acid under the reaction conditions of 20-25 ℃ and 12-24 hours;
step 2, adding the aqueous solution of the iron oxide nanoparticles into the aqueous solution of dopamine-modified hyaluronic acid, adjusting the pH value to 6.0, and stirring for reaction, wherein the feeding mass ratio of the iron oxide nanoparticles to the dopamine-modified hyaluronic acid is 1: 2-4, reacting at 20-25 ℃ for 2-6 h to obtain the iron oxide-dopamine modified hyaluronic acid coordination polymer;
step 3, adding cisplatin to H 2 O 2 In solution, cisplatin and H 2 O 2 The feeding molar ratio of (1): 2-4, reacting at 50-70 ℃ for 3-6 h to obtain hydroxylated cis-platinum, and reacting hydroxylAdding cisplatin into acetone, dropwise adding dichloroacetyl chloride to react under the stirring condition, wherein the feeding molar ratio of the hydroxylated cisplatin to the dichloroacetyl chloride is 1: 2-4, the reaction conditions are 20-25 ℃ and 12-24 hours, and after the reaction is finished, the reaction liquid is dripped into ether to obtain dichloroacetic acid modified cis-platinum;
and 4, adding dichloroacetic acid modified cis-platinum into the iron oxide-dopamine modified hyaluronic acid coordination polymer obtained in the step 2, wherein the feeding mass ratio of the dichloroacetic acid modified cis-platinum to the iron oxide-dopamine modified hyaluronic acid coordination polymer is 1: and 5-10, stirring for reaction, and reacting for 48-72 h to obtain the coordination polymer nanoparticles.
2. The polymeric nanoparticle of claim 1, wherein: the particle size of the coordination polymer nanoparticles is 140-180 nm.
3. The use of the polymeric nanoparticles of claim 1 for the preparation of a medicament for the treatment and/or diagnosis of tumors.
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