CN113616806A - Platinum-icodextrin-polycaprolactone macromolecular compound, nano drug delivery system and application thereof - Google Patents

Platinum-icodextrin-polycaprolactone macromolecular compound, nano drug delivery system and application thereof Download PDF

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CN113616806A
CN113616806A CN202110990139.5A CN202110990139A CN113616806A CN 113616806 A CN113616806 A CN 113616806A CN 202110990139 A CN202110990139 A CN 202110990139A CN 113616806 A CN113616806 A CN 113616806A
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icodextrin
polycaprolactone
platinum
compound
drug
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CN113616806B (en
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李子福
杨祥良
官建坤
万江陵
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Huazhong University of Science and Technology
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Huazhong University of Science and Technology
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Abstract

The invention belongs to the field of nano drug-carrying systems, and discloses a platinum-icodextrin-polycaprolactone macromolecular compound, a nano drug-carrying system and application thereof, wherein the compound is a polycaprolactone-modified icodextrin-platinum conjugate, the icodextrin-platinum conjugate is formed by connecting a carboxylated tetravalent platinum compound to icodextrin through an ester bond, and the polycaprolactone is coupled with the icodextrin through the ester bond; wherein the molecular weight of the polycaprolactone is 1kDa-10kDa, and the molecular weight of the icodextrin is 10kDa-20 kDa; the grafting rate of polycaprolactone on icodextrin is 0.5-1, and the mass percentage of platinum element in the macromolecular compound is 1-5%. The compound of the invention contains hydrophilic segment icodextrin, hydrophobic segment polycaprolactone and tetravalent platinum, is used as a tumor treatment drug, reduces the toxic and side effect of cisplatin, and improves the stability of circulation in blood, thereby improving the bioavailability of platinum and greatly enhancing the anti-tumor effect.

Description

Platinum-icodextrin-polycaprolactone macromolecular compound, nano drug delivery system and application thereof
Technical Field
The invention belongs to the technical field of nano drug delivery systems, and particularly relates to a platinum-icodextrin-polycaprolactone macromolecular compound, a nano drug delivery system and application thereof.
Background
Tumors are the most major health-threatening factor in humans. Chemotherapy is one of the most effective means for treating tumors at present. Although many common chemotherapeutic drugs have certain therapeutic effects, a great number of chemotherapeutic drugs on the market at present have great defects. The selectivity of bivalent platinum drugs on tumors mainly comes from high nutrient requirement of the tumors, but the selectivity is very weak, and other rapidly growing tissues (such as bone marrow, hair follicles and the like) are toxic due to the increased uptake; meanwhile, Pt (II) drugs are usually cleared through the kidney, so certain hepatotoxicity and nephrotoxicity are generated. In addition, Pt (ii) class drugs are unstable and they readily react with nucleophiles (especially human serum albumin) in the blood circulation, resulting in reduced bioavailability and increased toxic side effects. The tetravalent platinum prodrug is a low-spin regular octahedral complex obtained by oxidizing and adding divalent platinum into two axial ligands, and the regular octahedral structure of the tetravalent platinum prodrug enables the tetravalent platinum prodrug not to easily react with other ligands, but can be reduced by a reducing agent to release the two axial ligands, so that the original divalent platinum drug is released. This allows the tetravalent platinum prodrug to remain stable in the blood circulation, but to be rapidly converted to divalent platinum in the cells, functioning. The tetravalent platinum complex can reduce the toxic and side effects of divalent platinum, but is also small molecule, and has problems in drug delivery, such as poor water solubility, too fast blood clearance, insufficient accumulation at tumor sites, low bioavailability and the like.
Through the technological development of decades, the nano drug delivery system becomes one of effective means capable of effectively solving the problems. At present, a plurality of drug-loaded nano preparations enter the market, and in addition, a large number of nano preparations are in clinical research and preclinical research stages. The amphiphilic polymer drug-loaded nano system is one of nano drug-loaded systems which are researched more at present. It can provide a hydrophobic core solubilization hydrophobic drug molecule, and its hydrophilic shell can reduce protein adsorption, reduce phagocytic clearance of reticuloendothelial system and prolong the half-life period of drug in vivo. However, at present, few studies on platinum nano drug delivery systems exist, and an effective scheme for solving the problems of low bioavailability and the like caused by poor water solubility and high blood clearance rate of platinum prodrugs is lacking.
On the other hand, the drug-loaded nano-particle size of the nano-drug-loaded system has a great influence on the anti-tumor activity, generally speaking, when the particle size is small, the nano-drug-loaded system is not easily phagocytized by a reticuloendothelial system, and is more favorable for the drug to penetrate through tumor cells to play an anti-tumor role, however, the nano-drug size of the amphiphilic nano-drug-loaded system in the prior art is generally large, for example, the particle size of the hydroxyethyl starch amphiphilic nano-drug-loaded system which is researched by the inventor in the previous period of the subject group is generally the lowest particle size of only about 200nm, so that the drug is rapidly cleared by blood when the hydroxyethyl starch amphiphilic nano-drug-loaded system is used for treating tumors, and the drug utilization rate is poor.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a platinum-icodextrin-polycaprolactone macromolecular compound, a nano drug delivery system and application thereof.
In order to achieve the above object, the present invention provides a platinum-icodextrin-polycaprolactone macromolecular compound, which is a polycaprolactone-modified icodextrin-platinum conjugate, wherein the icodextrin-platinum conjugate is formed by connecting a carboxylated tetravalent platinum compound to icodextrin through an ester bond, and the polycaprolactone is coupled with the icodextrin through an ester bond;
wherein the molecular weight of the polycaprolactone is 1kDa-10kDa, and the molecular weight of the icodextrin is 10kDa-20 kDa; the grafting rate of the polycaprolactone on the icodextrin is 0.5-1, and the mass percentage of the platinum element in the macromolecular compound is 1-5%.
According to another aspect of the present invention, there is provided a method for preparing a platinum-icodextrin-polycaprolactone macromolecular compound, comprising the steps of:
s1, oxidizing cisplatin into a tetravalent platinum compound by using hydrogen peroxide, mixing the tetravalent platinum compound with acid anhydride for reaction, and separating and purifying to obtain a compound Pt-COOH;
s2, carrying out esterification reaction on carboxyl on polycaprolactone and hydroxyl on icodextrin, and separating and purifying to obtain a compound ICO-PCL;
s3, carrying out esterification reaction on the carboxyl on the Pt-COOH compound obtained in the step S1 and the hydroxyl on the icodextrin in the ICO-PCL compound obtained in the step S2, and separating and purifying to obtain the platinum-icodextrin-polycaprolactone macromolecular compound.
According to another aspect of the present invention, there is provided a use of the platinum-icodextrin-polycaprolactone macromolecular compound of the present invention for the preparation of a medicament for the treatment and/or prevention of cancer.
According to another aspect of the invention, an icodextrin platinum prodrug assembled nano-medicament is provided, which comprises nanoparticles, wherein the nanoparticles contain the platinum-icodextrin-polycaprolactone macromolecular compound.
Preferably, the nanoparticles are loaded with a photosensitizer and/or a fluorescent molecule.
Preferably, the photosensitizer is 1,1' -octacosyl-3, 3,3',3' -tetramethylindolylcarbonyl iodide.
Preferably, the nanoparticle further comprises a folic acid-icodextrin-polycaprolactone macromolecular compound;
the folic acid-icodextrin-polycaprolactone macromolecular compound is a folic acid modified icodextrin-polycaprolactone conjugate which is formed by connecting polycaprolactone to icodextrin through an ester bond, and the folic acid is coupled with the icodextrin through the ester bond; wherein the molecular weight of the polycaprolactone is 1kDa-10kDa, and the molecular weight of the icodextrin is 10kDa-20 kDa; the grafting rate of the polycaprolactone on the icodextrin is 0.5-1, and the grafting rate of the folic acid on the icodextrin is 5-10.
Preferably, the mass ratio of the platinum-icodextrin-polycaprolactone macromolecular compound to the folic acid-icodextrin-polycaprolactone macromolecular compound is (2-9): 1.
Preferably, the mass ratio of the platinum-icodextrin-polycaprolactone macromolecular compound to the folic acid-icodextrin-polycaprolactone macromolecular compound is (2.5-5): 1.
Preferably, the mass ratio of the platinum-icodextrin-polycaprolactone macromolecular compound to the folic acid-icodextrin-polycaprolactone macromolecular compound is 4: 1.
Preferably, the preparation method of the folic acid-icodextrin-polycaprolactone macromolecular compound comprises the following steps:
sa, performing esterification reaction on carboxyl on polycaprolactone and hydroxyl on icodextrin, and separating and purifying to obtain a compound ICO-PCL;
and Sb, carrying out esterification reaction on carboxyl on folic acid and hydroxyl on the icodextrin in the compound ICO-PCL in the step Sa, and separating and purifying to obtain the folic acid-icodextrin-polycaprolactone macromolecular compound.
According to another aspect of the present invention, there is provided a method for preparing an icodextrin platinum prodrug assembled nano-drug, comprising the steps of: dissolving 1,1' -octacosyl-3, 3,3',3' -tetramethylindole carbonyl iodide, platinum-icodextrin-polycaprolactone macromolecular compound and folic acid-icodextrin-polycaprolactone macromolecular compound in an organic solvent at the same time, dialyzing and drying to obtain the icodextrin platinum prodrug assembled nano-drug.
According to another aspect of the present invention, there is provided a use of the icodextrin platinum prodrug assembly nano-drug of the present invention in the preparation of a medicament for treating and/or preventing cancer.
According to another aspect of the invention, the invention also provides the application of the icodextrin platinum prodrug assembled nano-drug in tumor imaging.
Generally, compared with the prior art, the above technical solution conceived by the present invention has the following beneficial effects:
(1) the icodextrin is peritoneal dialysis solution which is used in large quantities clinically, the icodextrin which is naturally sourced and has excellent biocompatibility is used as a hydrophilic segment, the polycaprolactone is used as a lipophilic segment, and the tetravalent platinum prodrug is combined to prepare the amphiphilic nano drug-carrying system, so that the toxic and side effects of divalent platinum are effectively reduced, and meanwhile, as a macromolecular compound, the platinum has long circulation stability in a blood environment, can be well enriched in tumor cells, improves the bioavailability of the platinum, and achieves an excellent anti-tumor effect.
(2) According to the invention, polycaprolactone and carboxylated tetravalent platinum are sequentially connected to icodextrin through ester bonds to prepare the platinum-icodextrin-polycaprolactone macromolecular compound, so that the structural components of the macromolecular compound can be well controlled, the platinum drug loading rate can be controlled, and meanwhile, the macromolecular compound has good solubility.
(3) The invention utilizes the platinum-icodextrin-polycaprolactone macromolecular compound to prepare the nano drug-carrying system with the particle size of about 70nm, uniform distribution and stable structure, reduces the toxicity of platinum drugs and improves the stability of the drug circulation in blood, thereby leading the platinum drugs to be absorbed by tumor cells in large quantity; compared with the existing amphiphilic drugs, the drug has small particle size, is easier to penetrate tumor cells, can be quickly released after entering the tumor cells so as to achieve excellent tumor treatment effect, and has remarkably enhanced activity of killing the tumor cells.
(4) According to the invention, the photosensitizer 1,1' -octacosyl-3, 3,3',3' -tetramethyl indole carbonyl iodide is loaded in the nano-drug, so that the in-vivo metabolism of the nano-drug can be monitored in real time, and meanwhile, compared with single chemotherapy, the photothermal effect brought by the photosensitizer can obviously enhance the tumor treatment effect.
(5) According to the invention, the high expression characteristic of tumor cell folate receptors is utilized, and the specific ligand-folic acid of the amphiphilic polymer nano drug-loading system is modified on the surface of the amphiphilic polymer nano drug-loading system, so that the tumor targeting property of the drug can be remarkably improved, the drug can be rapidly and massively delivered to tumor tissues, the uptake of the drug-loading nano particles by tumor cells is promoted, the drug is rapidly released under the high reducing condition in the tumor cells, the systemic toxic and side effects are reduced, and the anti-tumor curative effect is enhanced.
(6) According to the invention, the ratio of the platinum-icodextrin-polycaprolactone macromolecular compound and the folic acid-icodextrin-polycaprolactone macromolecular compound in the nano-medicament assembled by the icodextrin platinum prodrug is adjusted so as to assemble the nano-medicament which has a proper particle size, is uniformly distributed and is easier to be absorbed by tumor cells.
(7) The method for preparing the icodextrin platinum prodrug assembled nano-drug is simple and easy to implement, mild in preparation conditions and low in cost, and a nano-drug delivery system with a stable structure and an excellent tumor treatment effect can be prepared.
(8) According to the prepared icodextrin platinum prodrug assembled nano-drug, the evaluation of in vivo and in vitro tumor targeting and antitumor drug effect shows that the icodextrin platinum prodrug assembled nano-drug has good tumor targeting performance on breast cancer, can rapidly deliver more drugs to tumor tissues, promotes the uptake of drug-loaded nanoparticles by breast tumor cells, rapidly releases the drugs in the tumor cells, and plays a better tumor killing activity; meanwhile, the toxicity of platinum drugs is remarkably reduced, compared with cisplatin, the weight of a mouse is not remarkably reduced, the survival state of the mouse is good, and the tumor volume and the tumor weight of breast cancer tumors are remarkably inhibited; under the same administration dosage, the nano-drug shows better anti-tumor activity than free drugs and non-folic acid targeted drug-loaded systems, obviously inhibits the tumor volume and tumor weight of breast cancer, obviously inhibits the proliferation of tumor cells in the breast cancer, and promotes the apoptosis of the tumor cells; in addition, the nano-drug can be rapidly heated up under the irradiation of near-infrared laser, can be heated up to 43 ℃ in vivo and in vitro, and can significantly improve the anti-tumor effect of pure platinum chemotherapy through mild photo-heat.
(9) The icodextrin platinum prodrug assembled nano-drug provided by the invention is coated with small-molecule fluorescent particles, so that when the drug is transferred in vivo, the in-vivo imaging of the tumor can be realized by the fluorescent staining of the tumor, and a doctor can directly observe the tumor by naked eyes to determine the position, the size and the boundary of the tumor, thereby facilitating the targeted anti-tumor treatment.
(10) The amphiphilic macromolecule provided by the invention has good biocompatibility. A fluorescent small-molecular compound 1,1' -octacosyl-3, 3,3',3' -tetramethyl indole carbonyl iodide is entrapped to the hydrophobic core of the polymer nano-particle by a dialysis method to form the drug-loaded nano-particle with the particle size of about 70nm and uniform distribution. The drug-loaded nanoparticles have good stability, can circulate in a blood system for a long time, and can be quickly and specifically enriched on tumor parts through the targeting effect of folic acid. Under the mediation of a folate receptor, the tumor cells take the drug-loaded nanoparticles into the cells. Under the action of reducing substances in tumor cells, tetravalent platinum is reduced into divalent platinum, and divalent platinum raw drug with stronger cell killing capacity is rapidly released, so that tumors are killed. Meanwhile, the fluorescent characteristic of the encapsulated micromolecules is utilized to carry out in-vivo imaging on the tumor, and the temperature of the tumor part can be quickly raised by utilizing the near infrared light irradiation, so that the precise tumor thermotherapy and chemotherapy combined treatment guided by the fluorescent imaging is realized. In conclusion, the nano drug delivery system which is coated with the 1,1' -octacosyl-3, 3,3',3' -tetramethylindolylcarbonyl iodide and is assembled based on the platinum-icodextrin-polycaprolactone macromolecular compound and the folic acid-icodextrin-polycaprolactone macromolecular compound has good application prospect.
Drawings
FIG. 1 is a scheme showing the preparation of compound PtIP and compound FIP according to example 1 of the present invention;
FIG. 2 is a diagram of nuclear magnetic resonance (H) spectrum (600M) of icodextrin, polycaprolactone, ICO-PCL, PtIP and FIP in example 1 of the present invention;
FIG. 3 is an X-ray diffraction photoelectron spectrum of cisplatin, Pt-COOH, and PtIP in example 1 of the present invention;
FIG. 4 is an infrared spectrum of cisplatin, Pt-COOH, and PtIP in example 1 of the present invention;
FIG. 5 is a graph showing cell viability in culture of compound ICO-PCL and compound FA-ICO-PCL measured by MTT method in example 1 of the present invention;
FIG. 6 is a graph showing the distribution of the hydration particle size of DPtFIP in different proportions measured by a dynamic light scattering instrument in example 2 of the present invention;
FIG. 7 is a graph showing the detection of the cell uptake of DPtFIP with different ratios by tumor cells (content A) and the relative quantitative detection (content B) by a flow cytometer in example 2 of the present invention;
FIG. 8 is a graph of DPtFIP morphology using electron microscopy as described in example 3 of the present invention;
FIG. 9 is a diagram showing the distribution of the hydration particle size of PtFIP and DPtFIP nanoparticles detected by a dynamic light scattering instrument in example 3 of the present invention;
FIG. 10 shows the stability of the nanoparticle of DIP, DFIP, PtIP, PtFIP, DPtIP and DPtFIP within two weeks measured by a dynamic light scattering instrument in example 3 of the present invention;
FIG. 11 is a chart showing the UV-VIS absorption spectra of DiR, DPtIP and DPtFIP in example 4 of the present invention;
FIG. 12 shows fluorescence emission spectra of DiR, DPtIP and DPtFIP in different solvents in example 5;
FIG. 13 is a graph showing the release profiles of PtIP and PtFIP under normal ambient and high reducing conditions in example 6 of the present invention;
FIG. 14 is a graph (content A) of temperature rise and a thermal imaging graph (content B) of DPtFIP with different concentrations under laser irradiation in example 7 of the present invention;
FIG. 15 is a graph of temperature rise of DPtFIP under different laser power irradiation in example 7 of the present invention;
FIG. 16 is a temperature rise curve chart of different nanoparticles irradiated by the same laser power at the same concentration in example 7 of the present invention;
FIG. 17 is a graph showing the cytocidal activity of Pt-COOH, PtIP and PtFIP on tumor cells in example 8 of the present invention;
FIG. 18 is a graph showing the cytocidal effect of DPtIP and DPtFIP on tumor cells in the presence or absence of laser light in example 9 of the present invention;
FIG. 19 is a diagram illustrating the measurement of the cellular uptake of different nanoparticles by tumor cells by confocal laser microscopy in example 10 of the present invention;
FIG. 20 is a graph showing the detection of the cell uptake of different nanoparticles by tumor cells using flow cytometry (content A) and the relative quantitative detection (content B) in example 11 of the present invention;
FIG. 21 is an in-vivo imaging graph (content A) and a relative quantitative detection graph (content B) of different nanoparticles enriched in mouse tumor sites in example 12 of the present invention;
FIG. 22 is a fluorescence imaging diagram (content A) and a relative quantitative detection diagram (content B) of different nanoparticles enriched in organs of a mouse in example 12 of the present invention;
FIG. 23 is a graph showing the temperature rise of a tumor site of a mouse irradiated with laser light (content A) and a graph showing the thermal imaging (content B) of the tumor site of the mouse treated with different drugs in example 13 of the present invention;
FIG. 24 is a graph showing the change in body weight of mice treated with different drugs in example 14 of the present invention;
FIG. 25 is a graph showing the change in tumor volume of mice treated with different doses in example 14 of the present invention;
FIG. 26 is a graph showing the ex vivo tumor weights of mice treated with different doses in example 14 of the present invention;
FIG. 27 is a photograph showing the sizes of the released tumors of the mice treated with different drugs in example 14 of the present invention;
FIG. 28 is a photograph showing the staining of the dissected tumor sections of mice treated with different doses in example 14 of the present invention;
FIG. 29 is a graph showing the relative quantitative determination of the fluorescence intensity of Ki67 staining (Contents A) and TUNEL staining (Contents B) of exfoliated tumors following various dosing treatments in mice according to example 14 of the present invention;
FIG. 30 is a sectional view showing HE staining of organs of a mouse treated with different drugs in example 14 of the present invention;
FIG. 31 is a graph showing the measurement of the amount of leukocytes, the amount of erythrocytes, the amount of platelets and the amount of creatinine, the amount of glutamic-pyruvic transaminase, the amount of glutamic-oxalacetic transaminase, the amount of creatinine kinase and the amount of urea nitrogen in the blood of mice treated with different administration in example 14 of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a platinum-icodextrin-polycaprolactone macromolecular compound (in the invention, Pt-ICO-PCL is used for representation and is abbreviated as PtIP), the macromolecular compound is a polycaprolactone-modified icodextrin-platinum conjugate, the icodextrin-platinum conjugate is formed by connecting a carboxylated tetravalent platinum compound to icodextrin through an ester bond, and the polycaprolactone is coupled with the icodextrin through the ester bond;
wherein the molecular weight of the polycaprolactone is 1kDa-10kDa, and the molecular weight of the icodextrin is 10kDa-20 kDa; the grafting rate of polycaprolactone on icodextrin is 0.5-1; the mass percentage content of the platinum element in the macromolecular compound (namely the drug loading of platinum) is 1-5%.
In a preferred embodiment, the molecular weight of polycaprolactone is 7.5kDa, the molecular weight of icodextrin is 16kDa, the grafting ratio of polycaprolactone on icodextrin is 0.98, and the mass percentage content of platinum element in the macromolecular compound is 2.5%.
The invention also provides a preparation method of the platinum-icodextrin-polycaprolactone macromolecular compound, which comprises the following steps:
s1, oxidizing cisplatin into a tetravalent platinum compound by using hydrogen peroxide, mixing the tetravalent platinum compound with acid anhydride for reaction, and separating and purifying to obtain a compound Pt-COOH;
s2, carrying out esterification reaction on carboxyl on polycaprolactone and hydroxyl on icodextrin, and separating and purifying to obtain a compound ICO-PCL;
s3, carrying out esterification reaction on the carboxyl on the Pt-COOH compound obtained in the step S1 and the hydroxyl on the icodextrin in the ICO-PCL compound obtained in the step S2, and separating and purifying to obtain the platinum-icodextrin-polycaprolactone macromolecular compound.
In some embodiments, step S1 specifically includes: mixing cisplatin with hydrogen peroxide for reaction to oxidize bivalent platinum in the cisplatin into tetravalent platinum, and separating and purifying to obtain a tetravalent platinum compound; then the tetravalent platinum compound and succinic anhydride are subjected to substitution reaction to obtain an intermediate product of carboxylated tetravalent platinum Pt-COOH.
In some embodiments, step S2 specifically includes: mixing polycaprolactone, a carboxyl activating reagent and an acid-binding agent, dissolving the mixture in an organic solvent, and performing substitution reaction on carboxyl contained in a polycaprolactone structure to convert the carboxyl to generate activated ester; then the activated ester and the icodextrin are subjected to substitution reaction and are connected through ester bond to obtain the icodextrin-polycaprolactone (ICO-PCL).
In some embodiments, step S3 specifically includes: mixing the carboxylated tetravalent platinum in the step S1, a carboxyl activating reagent and an acid binding agent, dissolving the mixture in an organic solvent, and performing substitution reaction on carboxyl in the carboxylated tetravalent platinum to generate activated ester; and carrying out substitution reaction on the activated ester and an intermediate product ICO-PCL in the step S2, and connecting the activated ester and the intermediate product ICO-PCL through an ester bond to obtain the platinum-icodextrin-polycaprolactone macromolecular compound (Pt-ICO-PCL).
The inventive steps S1, S2 and S3 can react in a wide temperature range, such as 20-60 ℃. In steps S2 and S3, the carboxyl activating reagent is dicyclohexylcarbodiimide or 1-ethyl-3 (3-dimethylpropylamine) carbodiimide; the acid-binding agent is one or more of 4-dimethylamino pyridine, pyridine and triethylamine; the organic solvent is dimethyl sulfoxide and/or tetrahydrofuran. The feeding molar ratio of the polycaprolactone, the icodextrin and the carboxylated tetravalent platinum is (2-6):1 (20-40), preferably (3-5):1 (25-35), and more preferably 4:1: 30. The products of step S2 and step S3 were isolated and purified by the following steps: and precipitating the obtained product by using dichloromethane or methanol, dissolving the precipitate, dialyzing by using ultrapure water, and finally freeze-drying to obtain the corresponding product.
It is found that excessive polycaprolactone grafting rate can cause the solubility of the prepared macromolecular compound to be reduced. The acid-binding agent is also used as a catalyst, the dosage of the acid-binding agent is equivalent to that of carboxylated tetravalent platinum or polycaprolactone, and the organic solvent is used for dissolving the raw materials. Meanwhile, experiments show that if the carboxylated tetravalent platinum and the polycaprolactone are mixed with the icodextrin at the same time for reaction, rather than sequentially connecting the polycaprolactone and the carboxylated tetravalent platinum to the icodextrin, the solubility of the prepared macromolecular compound is influenced, and the drug loading capacity of the cisplatin on the macromolecular compound is difficult to control.
The invention also provides the application of the platinum-icodextrin-polycaprolactone macromolecular compound in preparing a medicament for treating and/or preventing cancers, and the prepared nano medicament-carrying system can obviously reduce the toxic and side effects of platinum medicaments, has good water solubility, can maintain long-term stability in a blood environment and improves the bioavailability of the platinum medicaments.
The invention provides an icodextrin platinum prodrug assembled nano-drug, which comprises nanoparticles, wherein the nanoparticles contain the platinum-icodextrin-polycaprolactone macromolecular compound.
Preferably, the nanoparticles are loaded with a photosensitizer and/or a fluorescent molecule. The photosensitizer can be any conventional photosensitizer with photothermal effect, and is not limited to porphyrin compounds, phthalocyanine and its derivatives, hypericin, and the like. The nano-drug coated with the photosensitizer can be used for photo-thermal-chemotherapy combination treatment, has better anti-tumor effect and has no obvious toxic or side effect. The fluorescent molecule preferably has an emission wavelength in a near infrared region (650nm-900nm), and has higher signal-to-noise ratio, stronger penetrability and higher detection sensitivity compared with the fluorescent molecule detected by conventional visible light, so that the introduction of the fluorescent molecule can carry out living body imaging on tumors, and the resolution is good, and the fluorescent molecule is not limited to chlorins, Cy5 fluorescent dyes and the like. And may have, but is not limited to, the same molecule with both photothermal and fluorescent properties, such as 1,1' -octacosyl-3, 3,3',3' -tetramethylindolylcarbonyl iodide (DiR), indocyanine green, and IR-780 iodide.
In some embodiments, the nanoparticle further comprises a folic acid-icodextrin-polycaprolactone macromolecular compound; the folic acid-icodextrin-polycaprolactone macromolecular compound is a folic acid modified icodextrin-polycaprolactone conjugate which is formed by connecting polycaprolactone to icodextrin through an ester bond, and the folic acid is coupled with the icodextrin through the ester bond; wherein the molecular weight of the polycaprolactone is 1kDa-10kDa, and the molecular weight of the icodextrin is 10kDa-20 kDa; the grafting rate of polycaprolactone on icodextrin is 0.5-1, and the grafting rate of folic acid on icodextrin is 5-10.
Preferably, the molecular weight of polycaprolactone is 7.5kDa and the molecular weight of icodextrin is 16 kDa. The grafting rate of polycaprolactone on icodextrin is 0.98; the grafting ratio of folic acid on icodextrin was 8.
In some embodiments, the mass ratio of the platinum-icodextrin-polycaprolactone macromolecular compound to the folic acid-icodextrin-polycaprolactone macromolecular compound is (2-9):1, and the particle size of the assembled nano-drug is 60nm-100 nm; the mass ratio of the two is preferably (2.5-5):1, and more preferably 4:1, the nano-drug assembled according to the mass ratio has a particle size of about 70nm, the nano-drug is uniformly distributed, and the smaller particle size has stronger penetrability in tumor cells and is easier to be taken up by the tumor cells.
The invention also provides a preparation method of the icodextrin platinum prodrug assembled nano-drug, which comprises the following steps: dissolving 1,1' -octacosyl-3, 3,3',3' -tetramethyl indole carbonyl iodide and platinum-icodextrin-polycaprolactone macromolecular compound in an organic solvent at the same time, dialyzing and drying to obtain the icodextrin platinum prodrug assembled nano-drug.
In some embodiments, the 1,1' -octacosyl-3, 3,3',3' -tetramethylindolcarbonyl iodide, the platinum-icodextrin-polycaprolactone macromolecular compound and the folic acid-icodextrin-polycaprolactone macromolecular compound are simultaneously dissolved in an organic solvent, and the three are dialyzed and dried to obtain an icodextrin platinum prodrug assembled nano-drug; the preparation method of the folic acid-icodextrin-polycaprolactone macromolecular compound comprises the following steps:
sa, performing esterification reaction on carboxyl on polycaprolactone and hydroxyl on icodextrin, and separating and purifying to obtain a compound ICO-PCL;
and Sb, carrying out esterification reaction on carboxyl on folic acid and hydroxyl on the icodextrin in the compound ICO-PCL in the step Sa, and separating and purifying to obtain the folic acid-icodextrin-polycaprolactone macromolecular compound.
In some embodiments, step Sa specifically includes: mixing polycaprolactone, a carboxyl activating reagent and an acid-binding agent, dissolving the mixture in an organic solvent, and performing substitution reaction on carboxyl contained in a polycaprolactone structure to convert the carboxyl to generate activated ester; then the activated ester and the icodextrin are subjected to substitution reaction and are connected through ester bond to obtain the icodextrin-polycaprolactone (ICO-PCL).
In some embodiments, step Sb is specifically: mixing folic acid, a carboxyl activating reagent and an acid-binding agent, dissolving the mixture in an organic solvent, and performing substitution reaction on carboxyl contained in the folic acid to convert the carboxyl into activated ester; and then carrying out substitution reaction on the activated ester and an intermediate product ICO-PCL in the step Sa, and connecting the activated ester and the intermediate product ICO-PCL through ester bonds to obtain the folic acid-icodextrin-polycaprolactone macromolecular compound (FA-ICO-PCL).
Preferably, the carboxyl activating reagent is dicyclohexylcarbodiimide or 1-ethyl-3 (3-dimethylpropylamine) carbodiimide; the acid-binding agent is one or more of 4-dimethylamino pyridine, pyridine and triethylamine; the organic solvent is dimethyl sulfoxide and/or tetrahydrofuran. The feeding molar ratio of the polycaprolactone to the icodextrin to the folic acid is (2-6) to 1 (20-60), and the feeding molar ratio is preferably 4 to 1 to 40.
The invention also provides application of the icodextrin platinum prodrug assembled nano-drug in preparation of a drug for treating and/or preventing cancer. The type of cancer treated or prevented may be breast cancer, liver cancer, colon cancer, ovarian cancer or melanoma. The medicine has wide application dosage form, and can be made into injection, powder for injection, oral preparation, spray, capsule or suppository. The prepared anti-cancer medicine can be added with pharmaceutically acceptable additives.
The invention also provides application of the icodextrin platinum prodrug assembled nano-drug in tumor imaging. Because the nano-drug is loaded with small fluorescent particles, drug delivery and tumor imaging can be simultaneously realized, and a doctor can directly observe the tumor by naked eyes by dyeing the tumor.
In the preferred embodiment of the invention, the drug-loaded nanoparticles which contain the drug DiR and have the particle size of about 70nm, uniform distribution and stable structure are prepared by selecting icodextrin as a hydrophilic segment, polycaprolactone as a hydrophobic segment, folic acid as a tumor specific targeting molecule, platinum as a tumor treatment drug and DiR as a fluorescence imaging and photothermal treatment drug. Compared with the nano-particle without folate targeting, the nano-particle remarkably increases the enrichment speed and the enrichment amount of the nano-particle at the tumor part, promotes the uptake of the nano-particle by tumor cells, shows better anti-tumor effect in various breast cancer 4T1 mouse models, and simultaneously reduces toxic and side effects. The nano medicine-carrying system provided by the invention can image tumors in real time through a living body imaging system, and can quickly raise the temperature of the tumor part by irradiating the tumor part with laser, so that the combined treatment of photothermal therapy and chemotherapy is realized, and the anti-tumor curative effect is enhanced.
The above technical solution is described in detail below with reference to specific examples.
EXAMPLE 1 preparation and validation of Compounds PtIP and FIP
In this example, compound PtIP was prepared according to the scheme shown in FIG. 1, with the following specific steps:
(1) 1g of Cisplatin (Cisplatin, 3.33mmol) was weighed into a 100mL round-bottom flask, and then 35mL of 30% hydrogen peroxide (10-fold excess) and 25mL of ultrapure water were added to the flask in this order. The reaction was refluxed at 50 ℃ for 1 h. And after the reaction is finished, carrying out ice bath, recrystallizing to separate out a product, centrifuging to collect supernatant, washing with ethanol and diethyl ether sequentially for three times, and carrying out vacuum drying to obtain white solid powder, namely the cisplatin oxidation product.
(2) 334mg (1mmol) of the cisplatin oxidation product and 100mg (1mmol) of succinic anhydride in the previous step were weighed accurately and added to a 50mL round-bottomed flask, and then 30mL of molecular sieve-dried anhydrous Dimethylsulfoxide (DMSO) was added to the round-bottomed flask. The reaction was stirred at 45 ℃ for 12 h. After completion of the reaction, the reaction product was precipitated with a large amount of ether (10 times the amount of DMSO) and a small amount of ethanol, and then centrifuged at 9000rpm for 7min to collect the precipitate. Washing the precipitate with acetone and ethyl ether for three times, and vacuum drying to obtain the cisplatin prodrug (Pt (IV) -COOH).
(3) 1.6g (0.1mmol) of Icodextrin (Icodextrin, Mw 16000Da) was weighed into a 25mL round-bottomed flask, and 4mL of Dimethylsulfoxide (DMSO) which had been dried beforehand with a molecular sieve was added to dissolve. Another 300mg (0.4mmol) of Polycaprolactone (PCL), 768mg (4mmol) of 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI) and 488mg (4mmol) of 4-Dimethylaminopyridine (DMAP) were weighed out and dissolved in 2mL of dichloromethane (double distilled water). Then the latter solution is added into the former solution dropwise while stirring, and the mixture is stirred and reacted for 48 hours at the temperature of 35 ℃. After completion of the reaction, the reaction system was dropped into 40mL of methylene chloride to precipitate the product, and the precipitate was separated by centrifugation (8000rpm, 5min) and washed three times with methylene chloride. Redissolving the obtained precipitate with DMSO, dialyzing with ultrapure water (MWCO cut-off in dialysis bag: 3500) for three days, and freeze-drying to obtain icodextrin-polycaprolactone (ICO-PCL).
(4) 554mg (about 30. mu. mol) of the reaction product ICO-PCL from step (3) was weighed into a 50mL round-bottomed flask, dissolved by adding 20mL of dried DMSO, and then 434mg of cisplatin prodrug (1mmol), 768mg of EDCI (4mmol) and 488mg of DMAP (4mmol) were added in this order, and the reaction was stirred at 45 ℃ for 48 hours. After the reaction is finished, the reaction system is dripped into 200mL of methanol to precipitate a product, the product is separated by centrifugation (8000rpm, 5min), the obtained precipitate is washed by methanol for 2-3 times and then redissolved by DMSO, and then dialyzed in ultrapure water (MWCO: 3500Da) for three days and freeze-dried to obtain the Pt-icodextrin-polycaprolactone macromolecular compound Pt-ICO-PCL (PtIP).
This example also prepares compound FIP according to the scheme shown in figure 1, with the following specific operating steps:
(1) 1.6g,0.1mmol of icodextrin (Mw 16000Da) were weighed into a 25mL round-bottomed flask, and 4mL of DMSO, which had been previously dried with molecular sieves, were added to dissolve. The corresponding masses of PCL (300mg, 0.4mmol), EDCI (768mg, 4mmol) and DMAP (488mg, 4mmol) were weighed out separately and dissolved in 2mL of dichloromethane (water was distilled off). Then the latter solution is added into the former solution dropwise while stirring, and the mixture is stirred and reacted for 48 hours at the temperature of 35 ℃. After completion of the reaction, the reaction system was dropped into 40mL of methylene chloride to precipitate the product, and the precipitate was separated by centrifugation (8000rpm, 5min) and washed three times with methylene chloride. Redissolving the obtained precipitate with DMSO, dialyzing with ultrapure water (MWCO cut-off in dialysis bag: 3500) for three days, and freeze-drying to obtain icodextrin-polycaprolactone (ICO-PCL).
(2) 529mg of folic acid (FA, 1.2mmol), 230mg of EDCI (1.2mmol) and 144mg of DMAP (1.2mmol) were put into a 50mL round-bottomed flask, 20mL of DMSO was added, the mixture was dissolved by stirring with heating at 45 ℃ and 554mg of ICO-PCL (about 30. mu. mol) was added to the reaction system after the folic acid was dissolved. Stirring and reacting for 48h at 45 ℃, after the reaction is finished, dropwise adding the reaction system into 200mL of methanol to precipitate a product, centrifuging (8000rpm, 5min) to separate the precipitate, washing the obtained precipitate for 2-3 times by using methanol, redissolving the precipitate by using DMSO, dialyzing the precipitate in ultrapure water (MWCO: 3500Da) for three days, and freeze-drying to obtain the folic acid-icodextrin-polycaprolactone macromolecular compound FA-ICO-PCL (FIP).
Performing nuclear magnetic resonance test on the compound obtained by separation and purification,1h NMR (600MHz) data are shown in figure 2, a nuclear magnetic resonance hydrogen spectrum of PtIP not only has a hydroxyl proton peak on an icodextrin glucose ring, namely a multiple peak with chemical shift between 4.5ppm and 6ppm, but also can find a methylene proton peak of polycaprolactone with chemical shift between 1ppm and 2ppm, and can also find a proton peak of an amino group on platinum with chemical shift between 6ppm and 7ppm, and the nuclear magnetic resonance spectrum confirms that the PtIP has been successfully prepared. As shown in figure 2, the nuclear magnetic resonance hydrogen spectrum of FIP not only has a hydroxyl proton peak on an icodextrin glucose ring, namely a multiple peak with chemical shift between 4.5ppm and 6ppm, but also can find a methylene proton peak of polycaprolactone with chemical shift between 1ppm and 2ppm, and can also find a proton peak on a folic acid benzene ring with chemical shift between 6ppm and 8ppm, and the nuclear magnetic resonance spectrogram proves that FIP is successfully prepared.
And detecting the change of the Pt valence in the cisplatin, the Pt-COOH and the Pt-ICO-PCL by an X-ray diffraction photoelectron spectrometer. From the XPS spectrum shown in FIG. 3, it can be seen that cisplatin is divalent platinum, the binding energies of the outer electrons are 73eV and 76.5eV, respectively, the platinum in the cisplatin prodrug is oxidized to be tetravalent, and the binding energies of the outer electrons are increased to be 75.6eV and 79.1eV, respectively, which is consistent with the report in the prior art. After the cisplatin prodrug is grafted on the ICO-PCL, the binding energy has absorption peaks at 73eV, 76eV and 79eV, which indicates that the Pt-ICO-PCL sample contains bivalent platinum and tetravalent platinum at the same time, and the reason for this is that each icodextrin molecule has an aldehyde group, and a part of the cisplatin prodrug is reduced by the aldehyde group to be bivalent platinum.
The absorption conditions of cisplatin, Pt-COOH and Pt-ICO-PCL to infrared light with different wavelengths are measured by an infrared spectrometer, so that the structure of the compound is determined. As can be seen from FIG. 4, the infrared spectrum of Pt-COOH showed several new absorption peaks, wherein 3450cm was observed, compared to cisplatin-1The weak absorption peak is specially belonged to the expansion vibration peak of C ═ O bond in ester bond, 1350cm-1The middle absorption peak is the stretching vibration peak of C-O in carboxylic acid, 900cm-1The medium absorption peak is ascribed to the in-plane bending vibration peak of-OH in carboxylic acid. This indicates that cisplatin prodrug has more ester bonds and carboxyl groups than cisplatin, and thus succinic acid can be judged to be successfully coupled to cisplatin. PtIP at 300cm compared to Pt-COOH-1-3600cm-1The strong absorption peak shows that ICO-PCL is successfully coupled with Pt-COOH.
The biocompatibility of ICO-PCL and FA-ICO-PCL was examined by MTT assay. As can be seen from FIG. 5, both the compound ICO-PCL and the compound FA-ICO-PCL have good biocompatibility.
Example 2 preparation and characterization of encapsulated DiR based on PtIP and FIP co-assembled nanoparticles DPtFIP
In this embodiment, several nanoparticles with different Pt-ICO-PCL and FA-ICO-PCL ratios were prepared by dialysis, and the specific operation method is as follows:
(1) weighing two samples according to the mass ratio of Pt-ICO-PCL to FA-ICO-PCL of 9:1, 8:2 and 7:3 respectively to make the total mass of the two samples be 5 mg.
(2) 3mL of dimethyl sulfoxide dissolved with 3mgDiR is added into each mixture ratio in the step (1) for dissolution, and then samples are respectively added into dialysis bags with the molecular weight cutoff of 3500 Da. And (3) stirring and dialyzing in ultrapure water for 24 hours at room temperature, replacing fresh ultrapure water every 2 hours, and collecting the solution in the dialysis bag after dialysis. Small amount of concentrate by ultrafiltration (cut off molecular weight 10kDa) to a final volume of 5 mL; most were freeze dried for use.
The particle size distributions of several samples were measured by a dynamic light scattering particle sizer, and the results are shown in fig. 6. The results show that the particle sizes of the three nanoparticles with different proportions are all between 60nm and 100nm, and the distribution is uniform.
The nanoparticle ratio is optimized by in vitro cellular uptake. Triple negative breast cancer 4T1 cells were ranked as 1X 104The density of each cell per well was seeded in 4 6-well plates overnight; respectively incubating the cells for 2h by using DPtFIP cell culture solutions containing the same DiR amount and containing PBS, 9:1, 8:2 and 7:3 in different proportions; the cells were then digested with pancreatin and detected by flow cytometry. The results are shown in fig. 7, and it is seen from the results that nanoparticles with a ratio of 8:2 are easier to be taken up by tumor cells than nanoparticles with a ratio of 9:1 and 7:3, and therefore the mass ratio of PtIP to FIP in the drug-loaded nanoparticles is preferably 8: 2.
Example 3 preparation of non-encapsulated DiR and encapsulated DiR based on PtIP, FIP co-assembled nanoparticles PtFIP and DPtFIP this example prepared DPtFIP with a mass ratio of PtIP to FIP of 8:2 by dialysis method, the specific operating method was as follows:
(1) the following samples were weighed separately in different proportions:
ratio a (for preparing PtIP): weighing 5mg of Pt-ICO-PCL;
proportioning b (for preparing PtFIP): weighing two compounds according to the mass ratio of Pt-ICO-PCL to FA-ICO-PCL of 8:2 to ensure that the total mass of the two compounds is 5 mg;
ratio c (for preparing DPtIP): weighing 5mg of Pt-ICO-PCL, and then weighing 300 mu g of DiR;
ratio d (for preparing DPtFIP): weighing two compounds according to the mass ratio of Pt-ICO-PCL to FA-ICO-PCL of 8:2 to ensure that the total mass of the two compounds is 5mg, and then weighing DiR300 mu g;
ratio e (for preparation of DIP): weighing 5mg ICO-PCL, and then weighing 300 mu g of DiR;
proportioning f (for preparing DFIP): 5mg of FA-ICO-PCL was weighed, and 300. mu.g of DiR was weighed.
(2) 3mL of dimethyl sulfoxide is added into each mixture ratio in the step (1) to be dissolved, and then samples are respectively added into dialysis bags with the molecular weight cutoff of 3500 Da. And (3) stirring and dialyzing in ultrapure water for 24 hours at room temperature, replacing fresh ultrapure water every 2 hours, and collecting the solution in the dialysis bag after dialysis. The small amount was concentrated by ultrafiltration (molecular weight cut-off 10kDa) to a final volume of 5 mL. Most were freeze dried for use.
20 mu L of the DPtFIP dispersion liquid is dropped on a net, dyed by 0.1 percent phosphotungstic acid and naturally dried at room temperature, and then the appearance of the DPtFIP dispersion liquid is observed by a transmission electron microscope (TEM, H-700FA, HITACHI), and the acceleration voltage is 20KV-125 KV. As can be seen approximately from fig. 8, the particle size of DPtFIP is not greater than 100 nm.
The particle size distribution of PtFIP and DPtFIP prepared was measured using a dynamic light scattering particle sizer, and the results are shown in fig. 9. The particle diameters of DIP, PtIP, DPtIP, DFIP, PtFIP and DPtFIP were measured with a dynamic light scattering particle sizer every day for two consecutive weeks, and the results are shown in FIG. 10. The experimental result shows that the particle sizes of PtIP, DPtIP, DFIP, PtFIP and DPtFIP are all between 60nm and 100nm, the particle size of DIP is about 150nm, the samples can be kept stable within two weeks, and no obvious agglomeration or depolymerization phenomenon occurs.
Example 4 UV-VIS absorption Spectroscopy detection of DPtIP and DPtFIP nanoparticles
0.5mg/mL of an aqueous solution of DPtIP and DPtFIP and a solution of dimethyl sulfoxide were prepared using ultrapure water and dimethyl sulfoxide, respectively. The absorption spectra of the two samples were measured using an ultraviolet-visible spectrophotometer with dimethylsulfoxide as a reference, and the scanning wavelength range was 300nm to 850nm, and the scanning step was 1nm, and the results are shown in fig. 11.
The UV-VIS spectrum results showed that free DiR (10. mu.g/mL in dimethylsulfoxide) had an absorption maximum at 756nm, that DPtIP had an absorption maximum at 756nm as did DPtFIP (in dimethylsulfoxide), and that the three almost coincided in the 600nm-800nm range, indicating that DiR has indeed been successfully entrapped in DPtIP and DPtFIP nanoparticles.
Example 5 fluorescence emission spectroscopy detection of DPtIP and DPtFIP nanoparticles
0.5mg/mL of an aqueous solution of DPtIP and DPtFIP and a solution of dimethyl sulfoxide were prepared using ultrapure water and dimethyl sulfoxide, respectively. Fluorescence spectra of the two samples in DMSO and water were measured by fluorescence spectrometer, with an excitation wavelength of 750nm and an emission spectrum scan range of 760nm-850nm, and the results are shown in FIG. 12.
From the fluorescence spectra, DPtIP and DPtFIP showed almost no fluorescence in water, whereas in DMSO the fluorescence intensity was similar to the same concentration of DiR. This indicates that DiR is in an aggregated state and quenched in fluorescence after being entrapped in the nanoparticles, whereas in dimethylsulfoxide, both dpti p and DPtFIP nanoparticles are in a completely dissolved state and DiR fluorescence is not quenched.
Example 6 detection of PtIP and PtFIP nanoparticle Release in PBS and DTT
In this example, release conditions of PtIP and PtFIP nanoparticles in PBS solution and 10mM Dithiothreitol (DTT) solution are detected, and the specific operation method is as follows:
(1) two release mediums were configured. 5g of Tween-80 was dissolved in 1000mL of PBS to obtain a PBS solution containing 0.5% Tween-80, 500mL of the PBS solution was taken out, 0.77g of DTT was added thereto, and ultrasonic dissolution was carried out to obtain a 10mM DTT solution.
(2) A10 mg/mL solution of PtIP and PtFIP was prepared in 7mL each using a PBS solution containing 0.5% Tween-80. The concentration of platinum in both sample solutions was around 200. mu.g/mL.
(3) The two samples were added to pre-cooked dialysis bags (MWCO: 3500Da) 1mL each, and 6 bags each. The dialysis bags containing the two samples were sealed and placed into 50mL centrifuge tubes containing 29mL of different release media.
(4) The centrifuge tubes were incubated at 37 ℃ with a shaker at 200 rpm. 2mL of release solution was taken out of each centrifuge tube at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h, 48h, 72h, respectively, while 2mL of the corresponding release medium was supplemented.
(5) The withdrawn 2mL was added to a different glass sample bottle, another 2mL of ultrapure water was added to another sample bottle as a blank, and then 2mL of nitric acid and 0.1mL of perchloric acid were added to each sample bottle. After digesting for 2 hours at about 300 ℃ on an electric heating plate, adding a certain amount of 2% dilute nitric acid solution for dissolving, and then using 2% dilute nitric acid to fix the volume of each sample and blank control to 10 mL. The concentration of platinum in the release solution was measured by inductively coupled plasma emission spectrometer, and the results are shown in fig. 13.
The results show that the two nanoparticles of PtIP and PtFIP are slowly released in PBS, and the release efficiency of the two nanoparticles of Pt is only 39.5% and 37.4% within 72 h; the release rates of the PtIP and PtFIP nanoparticles in 10mM DTT are obviously accelerated, the release efficiency of 12 hours reaches 61.1 percent and 51.1 percent respectively, and the release efficiency of 72 hours reaches 89.7 percent and 81.0 percent respectively. The slow release of the nanoparticles in PBS is attributed to hydrolysis of the ester bond in water, whereas the fast, large release of the nanoparticles in 10mM DTT is attributed to reduction of tetravalent platinum to divalent by DTT. The reduction responsive drug release of the nanoparticles in a reduction medium is beneficial to ensuring the relative stability of the drug in storage and blood circulation, and the drug is released rapidly and in large quantity after entering cells, thereby achieving good treatment effect.
Example 7 in vitro temperature increase Effect detection of different samples under different conditions
Dissolving the freeze-dried DPtFIP nanoparticles in ultrapure water, and respectively diluting the solution into samples with a series of concentrations according to the concentration of DiR: 40. mu.g/mL, 20. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL, 0. mu.g/mL. Taking 1mL into a 1.5mL EP tube, 808 using a laser at 1.5W/cm2The power was applied and the temperature change of the liquid in the EP tube was detected by a thermal imager, and the temperature was recorded every 30 seconds and the temperature change was continuously recorded for 360 seconds at room temperature of 25 ℃ as shown in FIG. 14.
The freeze-dried DPtFIP nanoparticles were dissolved in ultrapure water and diluted to 20. mu.g/mL according to the concentration of DiR. Prepare multitubular 1mL samples in 1.5mL EP tubes, 808 laser 1.5W/cm respectively2、1W/cm2、0.75W/cm2And 0.5W/cm2Irradiating with power, detecting the temperature change of the liquid in the EP tube with a thermal imager,the temperature was recorded every 30s and a temperature change of 360s was continuously recorded at room temperature of 25 ℃ as shown in FIG. 15.
DiR, DIP, DFIP, DPtIP and DPtFIP nanoparticles were dissolved in ultrapure water, respectively, and diluted to 20. mu.g/mL in accordance with the concentration of DiR. Taking 1mL of sample in a 1.5mL EP tube, 808 laser using 1.5W/cm2The temperature change of the liquid in the EP tube was detected by a thermal imager, and the temperature was recorded every 30 seconds and the temperature change was continuously recorded for 360 seconds at room temperature of 25 ℃ as shown in FIG. 16.
The results showed that the concentration was 20. mu.g/mL, and the laser power was 1.5W/cm2In time, DPtFIP exhibits better in vitro warming capability; DIP, DFIP, DPtIP and DPtFIP have better in-vitro heating effect than free DiR.
Example 8 in vitro antitumor Activity assay of Pt-COOH, PtIP and PtFIP
In this example, the MTT method is used to detect the in vitro anti-tumor activities of Pt-COOH, PtIP and PtFIP, and the specific operation method is as follows:
cell pretreatment is performed first. Digesting and centrifugally collecting 4T1 cells with good growth state, and diluting to 5 × 104Cells in suspension per mL were then plated in 96-well plates at 100 μ L per well and incubated overnight in an incubator to adhere. All media containing Pt-COOH, PtIP and PtFIP were prepared at Pt concentrations of 20. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL and 0. mu.g/mL, respectively. Old media in 96-well plates was aspirated and 200. mu.L of prepared new media containing Pt was added, one column per group (6 wells). The blank control and the no cell control were added with new whole medium. Then put into an incubator to be incubated for 24 h. After incubation was complete 20. mu.L of 5mg/mL MTT solution (in sterile PBS) was added to each well. The 96-well plate was then placed in an incubator for incubation. After 4h of MTT incubation, medium was aspirated from 96-well plates and 150. mu.L of dimethyl sulfoxide was added and the plates were incubated at 37 ℃ for 30min to completely dissolve formazan formed. Finally, absorbance was measured by a microplate reader at a wavelength of 492nm, and the results are shown in FIG. 17.
The result shows that compared with Pt-COOH, PtIP and PtFIP have obviously enhanced tumor cell killing activity; the killing activity of the tumor cells of the PtFIP nanoparticles with folic acid targets is obviously stronger than that of the PtIP nanoparticles without folic acid targets, and the killing activity of the cells is enhanced by 1 time.
Example 9 in vitro anti-tumor Activity assays for DPtIP and DPtFIP
In this example, the MTT method is used to detect the in vitro anti-tumor activities of dpti p and DPtFIP, and the influence of laser irradiation on the anti-tumor activities is detected, and the specific operation method is as follows:
cell pretreatment is performed first. Digesting and centrifugally collecting 4T1 cells with good growth state, and diluting to 5 × 104Cells in suspension per mL were then plated in 96-well plates at 100 μ L per well and incubated overnight in an incubator to adhere. The culture medium solutions containing DPtIP and DPtFIP were prepared at Pt concentrations of 20. mu.g/mL, 10. mu.g/mL, 5. mu.g/mL, 2. mu.g/mL, 1. mu.g/mL, and 0. mu.g/mL, respectively, corresponding to DiR concentrations of 10. mu.g/mL, 5. mu.g/mL, 2.5. mu.g/mL, 1. mu.g/mL, 0.5. mu.g/mL, and 0. mu.g/mL. Old medium was aspirated from the 96-well plate and 200. mu.L of prepared new medium containing DPtIP and DPtFIP was added, one column for each group, for 6 wells. The blank control and the no cell control were added with new whole medium. Adding two 96-well plates for DPtIP and DPtFIP respectively, leaving one plate untreated, and irradiating the other plate with 808nm laser at power of 1.5W/cm after administration for 2h2Irradiation was performed for 3min per well. After incubation was complete 20. mu.L of 5mg/mL MTT solution (in sterile PBS) was added to each well. The 96-well plate was then placed in an incubator for incubation. After 4h of MTT incubation, medium was aspirated from 96-well plates and 150. mu.L of dimethyl sulfoxide was added and the plates were incubated at 37 ℃ for 30min to completely dissolve formazan formed. Finally, absorbance was measured by a microplate reader at a wavelength of 492nm, and the results are shown in FIG. 18.
The result shows that the killing activity of the tumor cells with the folate targeting nanoparticles DPtFIP and DPtFIP + Laser is obviously stronger than that of the tumor cells without the folate targeting nanoparticles DPtIP and DPtIP + Laser; the cell killing of the DPtFIP + Laser and the DPtIP + Laser which are irradiated by the Laser is stronger than that of the DPtFIP and the DPtIP which are not irradiated by the Laser respectively.
Example 10 in vitro uptake of different nanoparticles by tumor cells Using confocal laser microscopy
In this embodiment, a confocal laser microscope is used to study the uptake of 4T1 cells to different drug-loaded nanoparticles, and the specific operation method is as follows:
4T1 cells with good growth status were digested and collected and plated on confocal dishes at 5X 10/dish4Individual cells were allowed to adhere by overnight incubation in an incubator. The solutions of DPtIP and DPtFIP were then prepared in serum-free medium to give a final DiR concentration of 5. mu.g/mL. The DPtFIP solution was prepared in serum-free medium containing 100. mu.g/mL of folic acid, and the concentration of DiR was similarly adjusted to 5. mu.g/mL. 1mL of the nanoparticle-containing medium was added to each of the pretreated 4T1 cells, and then incubated in an incubator for 2 hours. After incubation, 1mL of 4% paraformaldehyde was added to each dish and fixed for 15 min. Then paraformaldehyde was aspirated and 1mL of 10. mu.g/mL DAPI was added for staining. After 15min DAPI was aspirated and washed three times with PBS. The DiR fluorescence intensity in 4T1 cells was measured using confocal laser microscopy, and the results are shown in fig. 19.
The uptake of folate-targeted DPtFIP nanoparticles by 4T1 cells is stronger than that of folate-free targeted DPtIP nanoparticles, and free folic acid and DPtFIP compete for targeting 4T1 cells, so that the uptake of folate-co-incubated DPtFIP + FA by 4T1 cells is lower than that of folate-free co-incubated DPtFIP, which indicates that the folate-targeted molecules connected to the nanoparticles enhance the uptake of the nanoparticles by tumor cells.
Example 11 detection of DPtFIP nanoparticle uptake in vitro by tumor cells Using flow cytometry
In this embodiment, a flow cytometer is used to study the uptake of DPtFIP nanoparticles by 4T1 cells, and the specific operation method is as follows:
a blank medium containing DiR, DPtIP, DPtFIP and DPtFIP + 100. mu.g/mL folic acid was prepared in advance so that the final DiR concentration was 1. mu.g/mL. Well-grown 4T1 cells were digested, collected and counted, and then plated in 6-well plates, each plated at 5X 105One cell, seeded into 15 wells. The cells were incubated overnight at 37 ℃ in a 5% carbon dioxide incubator. Blank 1640 medium, DiR and three nanoparticle solutions were added to 6-well plates, 1mL per well, and three replicate wells per sample, respectively. After incubation at 37 ℃ for 2h, the samples were aspirated, washed three times with PBS and digested for 3min with 0.5mL pancreatin per well. After digestion, the serum-containing medium was added to stop digestion, and cells were collected by centrifugation at 1200rpm for 3 min. The collected cells were washed with PBS 23 times. Finally, the collected cells were blown up with 200. mu.L of PBS. The fluorescence intensity of DiR was measured by flow cytometry, and the results are shown in fig. 20.
The results show that 4T1 cells uptake folate-targeted DPtFIP nanoparticles more strongly than folate-free targeted DPtFIP nanoparticles, and 4T1 cells uptake folate-co-incubated DPtFIP + FA less strongly than folate-co-incubated DPtFIP. Consistent with the results of example 10, it is demonstrated that folate targeting molecules enhance nanoparticle uptake by tumor cells.
Example 12 in vivo fluorescence imaging and drug tissue distribution study of nanoparticles
BALB/c females were purchased and six weeks old, weighing between 15g-17 g. After one week of acclimatization in the laboratory animal house, the mice were shaved clean of hair around the right hind limb. The 4T1 cells were cultured, and when the number of cells was sufficient and in log phase, the cells were collected by digestion and centrifugation. Washed once with PBS and resuspended in PBS to 107And (4) placing the cell suspension in an ice box for standby. 100 μ L of cell suspension was injected subcutaneously over the right hind limb of each mouse using a syringe. And (5) continuing feeding after the injection is finished. The formula for calculating the tumor volume is as follows: v ═ 2 (L × W ^2)/2, where V denotes the tumor volume, L denotes the long diameter of the tumor, and W denotes the short diameter. Until the tumor volume reaches 300mm3Thereafter, tumor-bearing mice were randomly divided into 3 groups of 5 mice each. Free DiR, dptiip and DPtFIP nanoparticle solutions (in PBS or saline) were administered separately into the tail vein at a dose of 1mg/kg, calculated as DiR. The mice were anesthetized at 0.5h, 1h, 2h, 4h, 8h, 12h, 24h and 48h before and after administration, fluorescence was imaged and the fluorescence intensity was measured by a small animal living body imager, the ICG channel was selected for the fluorescence channel, excitation was 745nm, emission was 830nm, and the results are shown in fig. 21. To further study the distribution behavior of the nano-drug in vivo, at 48h after the administration, the mice were sacrificed and the heart, liver, spleen, lung, kidney and tumor were removed, fluorescence imaging was performed using a small animal in vivo imager and the fluorescence intensity was measured, and the results are shown in fig. 22.
The result shows that the DiR endows the nanoparticle with the capability of in-vivo imaging, and can image the tumor in real time. As shown in FIG. 21, the fluorescence intensity at the tumor sites of the three groups of mice increased with the time of administration, and there was no significant change in DiR after 1h, while the fluorescence maxima appeared 24h after the administration in the DPtIP and DPtFIP groups. Therefore, the irradiation time points selected when performing photothermal experiments were 24h after administration, except that DiR was 1h after administration. At each time point when the fluorescence photograph was taken, the fluorescence of the DiR group mice was weak, and there was hardly any expression on the fluorescence photograph. This is mainly because DiR is a small molecule, which can be easily removed directly by direct injection into the body via the liver or kidney, and because the half-life of blood circulation is very short, only a small amount of DiR can reach the tumor site to produce fluorescence. Compared with the DiR group, the dptid and DPtFIP groups were more enriched at the tumor site, and the average fluorescence intensity at 48h was 13.3-fold and 25.3-fold higher than that of the DiR group. This is mainly because the surface hydrophilicity increases the blood circulation time of nanoparticles, and the nanoparticles are enriched in tumor sites under the influence of enhanced permeability and retention Effect (EPR) of solid tumors. Compared with dpti, the DPtFIP group was enriched more at the tumor site, and the mean fluorescence intensity at the tumor site was 1.9 times higher than that of the dpti group at 48h after the administration. This is mainly caused by two aspects: firstly, the targeting effect of folic acid enables the DPtFIP nanoparticles to be more enriched at the tumor part; second, dptiip is positively charged and is easily cleared by opsonization, while DPtFIP is more nearly neutral and therefore has a longer blood half-life. Fig. 22, a and B, respectively, show fluorescence photographs of each organ of each group of mice taken 48h after administration and results of semiquantification of fluorescence of each organ, and it can be seen from the results that nanoparticles of DPtFIP group have higher enrichment at tumor sites than DiR and dpti p groups, 17 times and 2 times of DiR and dpti p groups, respectively. The above results show that compared with DiR and dptiip, DPtFIP nanoparticles have higher enrichment at tumor sites, which means that DPtFIP nanoparticles have higher imaging sensitivity and better imaging effect.
Example 13 in vivo photothermal Effect detection of nanoparticles
BALB/c females were purchased and six weeks old, weighing between 15g-17 g. One week after adaptive breeding in laboratory animal house, mice were placed around the right hind limbThe surrounding hair is shaved clean. The 4T1 cells were cultured, and when the number of cells was sufficient and in log phase, the cells were collected by digestion and centrifugation. Washed once with PBS and resuspended in PBS to 107And (4) placing the cell suspension in an ice box for standby. 100 μ L of cell suspension was injected subcutaneously over the right hind limb of each mouse using a syringe. And (5) continuing feeding after the injection is finished. The formula for calculating the tumor volume is as follows: v ═ 2 (L × W ^2)/2, where V denotes the tumor volume, L denotes the long diameter of the tumor, and W denotes the short diameter. When the tumor grows to 200mm3Left and right, mice were randomly divided into 6 groups of 3 mice each. Physiological Saline (Saline), DiR, DPtIP and DPtFIP were administered separately into the tail vein at a dose of 1.25mg/kg calculated as DiR. The laser irradiation was carried out 24h after administration of the drug to the groups other than the group 1h after administration of DiR, and the laser power was 1.5W/cm2Irradiation was carried out for 5min, during which time the temperature change of the tumor site was recorded with a hand-held thermal imaging camera, and the results are shown in FIG. 23.
The result shows that the DiR endows the nanoparticles with the capability of generating photo-heat, and can carry out photo-heat treatment on the tumor in real time. Using a 808 laser at 1.5W/cm2The tumor site was irradiated at power for 5min, and the temperature rise curve in DiR and nanoparticle tumor was shown as a in fig. 23. The temperature of the tumor part of the normal saline group is increased by about 3 ℃, which is mainly due to the absorption of near infrared light by animal tissues; the temperature of the tumor part of the free DiR group is increased from about 31.5 ℃ to about 38.4 ℃ which is far higher than that of the normal saline group, which shows that a small part of the free DiR group is enriched in the tumor part and generates photothermal effect; because the tumor part enrichment of DPtIP is less than that of DPtFIP nanoparticles, the temperature rise effect of the DPtIP nanoparticles is poorer than that of the DPtFIP nanoparticles, the tumor part of a DPtIP group can be heated to about 41.8 ℃ within 5min, and the temperature of DPtFIP can be raised to about 43.5 ℃. The temperature raising capability of the nanoparticles at the tumor part is higher than that of free DiR, and the temperature raising capability of the folic acid targeted DPtFIP at the tumor part is higher than that of folic acid-free DPtIP.
Example 14 evaluation of in vivo antitumor Effect of different nanoparticles
BALB/c females were purchased and six weeks old, weighing between 15g-17 g. One week after acclimatization in laboratory animal house, the right hind limb of the mouse wasThe surrounding hair is shaved clean. The 4T1 cells were cultured, and when the number of cells was sufficient and in log phase, the cells were collected by digestion and centrifugation. Washed once with PBS and resuspended in PBS to 107And (4) placing the cell suspension in an ice box for standby. 100 μ L of cell suspension was injected subcutaneously over the right hind limb of each mouse using a syringe. And (5) continuing feeding after the injection is finished. The formula for calculating the tumor volume is as follows: v ═ 2 (L × W ^2)/2, where V denotes the tumor volume, L denotes the long diameter of the tumor, and W denotes the short diameter. When the tumor grows to 100mm3On the left and right, the mice were randomly divided into 9 groups of 8 mice each and given (1) physiological saline, (2) DiR, (3) cisplatin, (4) DIP (DiR @ ICO-PCL), (5) DFIP (DiR @ FA-ICO-PCL), (6) PtIP, (7) PtFIP, (8) DPtIP and (9) DPtFIP, respectively. The administration was via the tail vein at a dose of 2.5mg/kg platinum and 1.25mg/kg DiR. The drugs were administered once every three days for a total of four times and were irradiated after the first dose, the DiR group was irradiated 1 hour after the dose, and DIP, DFIP, DPtIP and DPtFIP were irradiated 24 hours after the dose, with an irradiation power of 1.5W/cm2The illumination time is 10 min. During the experiment, the body weight and tumor volume of the mice were measured every two days, and the results are shown in fig. 24 and fig. 25, respectively. Mice were sacrificed on day 14 post-dose, tumors were dissected, and ex vivo tumors were weighed and photographed, and the results are shown in fig. 26 and fig. 27, respectively. Tumors were fixed with 4% paraformaldehyde, sections embedded with paraffin, and HE stained, Ki67 and TUNEL immunofluorescence stained as shown in figure 28; and the mean fluorescence intensity in Ki67 and TUNEL sections was quantified, and the results are shown in fig. 29. The heart, liver, spleen, lung and kidney were fixed with 4% paraformaldehyde, embedded in paraffin, and HE-stained as shown in fig. 30. After mice were sacrificed, two whole blood samples were collected from each mouse, one for routine (anticoagulation) and the other for biochemical (anticoagulation) measurement. The whole blood for measuring the blood biochemical index can be directly detected by a blood cell analyzer, the whole blood for measuring the blood biochemical index is placed at 4 ℃ overnight, centrifuged at 3000rpm for 5min, and detected by using a corresponding kit after serum is collected, and the result is shown in figure 31.
As can be seen in FIG. 24, cisplatin was very toxic and the body weight of cisplatin mice dropped dramatically after administration; the body weight of the mice in the cisplatin group is reduced from about 16g to about 12g by the eighth day, the average reduction exceeds 25 percent, and the hairs are upright and dull and are not eaten. From an animal experimental ethical point of view, we sacrificed cisplatin groups of mice on day eight. The cisplatin group mice lost too much weight, the body was unable to provide nutrition, and tumor growth was arrested, but this did not reflect the efficacy of cisplatin, so the efficacy of cisplatin was not compared in the present invention. As can be seen from fig. 25 to 27, even if the temperature of the tumor site in the free DiR group can only be raised to about 38 ℃, photothermal therapy can still exert a certain curative effect, and the tumor inhibition rate is 18.4%; compared with the normal saline group, the single chemotherapy (PtIP and PtFIP) has limited drug effect, and the tumor inhibition rates are respectively 33.3 percent and 33.6 percent; because the photothermal temperature rise is controlled not to exceed 45 ℃, the single photothermal (DIP and DFIP) treatment effect is not obvious, and the tumor inhibition rates are only 30.3 percent and 38.6 percent respectively; compared with the monotherapy, the photothermal-chemotherapy combination treatment can obviously enhance the anti-tumor curative effect, and the tumor inhibition rates of the DPtIP group and the DPtFIP group respectively reach 55.3 percent and 80.7 percent.
The staining results in fig. 28 show that the dpti p and DPtFIP groups showed a larger tumor necrosis area. Cell proliferation at the tumor site was relatively quantitatively examined by Ki67 staining and TUNEL staining, and as seen in fig. 29, the dptid and DPtFIP groups had the lowest Ki67 fluorescence, indicating that cell proliferation within the tumor was the weakest; DPtFIP has the highest TUNEL fluorescence, indicating that apoptosis is strongest inside the tumor. The above results are consistent with the evidence that the DPtFIP group has the best antitumor effect.
The damage condition of different drugs to each organ of the mouse is evaluated through an HE stained section, and the result of figure 30 shows that cisplatin causes severe damage to the heart, liver, spleen, lung and kidney of the mouse; the other medicines do not obviously damage organs of the mice and have no obvious toxicity.
The results in figure 31 also show that the groups other than the cisplatin group did not cause significant toxicity to the mice.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims (10)

1. A platinum-icodextrin-polycaprolactone macromolecular compound is characterized in that: the macromolecular compound is a polycaprolactone-modified icodextrin-platinum conjugate, the icodextrin-platinum conjugate is formed by connecting a carboxylated tetravalent platinum compound to icodextrin through an ester bond, and the polycaprolactone is coupled with the icodextrin through the ester bond;
wherein the molecular weight of the polycaprolactone is 1kDa-10kDa, and the molecular weight of the icodextrin is 10kDa-20 kDa; the grafting rate of the polycaprolactone on the icodextrin is 0.5-1, and the mass percentage of the platinum element in the macromolecular compound is 1-5%.
2. A method for preparing the platinum-icodextrin-polycaprolactone macromolecular compound of claim 1, characterized by comprising the following steps:
s1, oxidizing cisplatin into a tetravalent platinum compound by using hydrogen peroxide, mixing the tetravalent platinum compound with acid anhydride for reaction, and separating and purifying to obtain a compound Pt-COOH;
s2, carrying out esterification reaction on carboxyl on polycaprolactone and hydroxyl on icodextrin, and separating and purifying to obtain a compound ICO-PCL;
s3, carrying out esterification reaction on the carboxyl on the Pt-COOH compound obtained in the step S1 and the hydroxyl on the icodextrin in the ICO-PCL compound obtained in the step S2, and separating and purifying to obtain the platinum-icodextrin-polycaprolactone macromolecular compound.
3. Use of the platinum-icodextrin-polycaprolactone macromolecular compound according to claim 1 for the preparation of a medicament for the treatment and/or prevention of cancer.
4. An icodextrin platinum prodrug assembled nano-drug is characterized in that: comprising nanoparticles comprising the platinum-icodextrin-polycaprolactone macromolecular compound according to claim 1.
5. The icodextrin platinum prodrug assembling nano-drug of claim 4, wherein: the nanoparticles are loaded with a photosensitizer and/or a fluorescent molecule.
6. The icodextrin platinum prodrug assembling nano-drug of claim 4, wherein: the nanoparticle also comprises a folic acid-icodextrin-polycaprolactone macromolecular compound;
the folic acid-icodextrin-polycaprolactone macromolecular compound is a folic acid modified icodextrin-polycaprolactone conjugate which is formed by connecting polycaprolactone to icodextrin through an ester bond, and the folic acid is coupled with the icodextrin through the ester bond; wherein the molecular weight of the polycaprolactone is 1kDa-10kDa, and the molecular weight of the icodextrin is 10kDa-20 kDa; the grafting rate of the polycaprolactone on the icodextrin is 0.5-1, and the grafting rate of the folic acid on the icodextrin is 5-10.
7. The icodextrin platinum prodrug assembling nano-drug of claim 6, wherein: the mass ratio of the platinum-icodextrin-polycaprolactone macromolecular compound to the folic acid-icodextrin-polycaprolactone macromolecular compound is (2-9):1, and preferably (2.5-5): 1.
8. The icodextrin platinum prodrug assembled nano-drug according to claim 6, wherein the preparation method of the folic acid-icodextrin-polycaprolactone macromolecular compound comprises the following steps:
sa, performing esterification reaction on carboxyl on polycaprolactone and hydroxyl on icodextrin, and separating and purifying to obtain a compound ICO-PCL;
and Sb, carrying out esterification reaction on carboxyl on folic acid and hydroxyl on the icodextrin in the compound ICO-PCL in the step Sa, and separating and purifying to obtain the folic acid-icodextrin-polycaprolactone macromolecular compound.
9. Use of icodextrin platinum prodrug assembly nano-drug as claimed in any one of claims 4-8 for the preparation of a medicament for the treatment and/or prevention of cancer.
10. The use of icodextrin platinum prodrug assembly nano-drug of claim 5 in tumor imaging.
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