CN112386695A - Chitosan-based nano prodrug carrying indocyanine green and platinum drugs and preparation method thereof - Google Patents
Chitosan-based nano prodrug carrying indocyanine green and platinum drugs and preparation method thereof Download PDFInfo
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Abstract
A chitosan-based nano prodrug carrying indocyanine green and platinum drugs and a preparation method thereof are disclosed, wherein 3,3' -dithiodipropylhydrazide is modified on a chitosan derivative by utilizing a Diels-Alder reaction to obtain a hydrazide chitosan derivative; and then mixing the hydrazide chitosan derivative solution with indocyanine green and platinum drugs, and obtaining the nano prodrug through a self-assembly process. The particle size of the nano prodrug prepared by the invention can be regulated and controlled within the range of 100-200nm, has the dual stimulation responsiveness of glutathione and pH, has the advantages of no toxicity of chitosan, good biocompatibility and the like, can be used for combined therapy of phototherapy and chemotherapy of tumors, and has the characteristics of overcoming the multidrug resistance of tumor cells, having small toxic and side effects, having good effect of inhibiting the proliferation of the tumor cells and the like. The preparation method has the advantages of easily available raw materials, simple drug loading process, easy purification and the like, and the prepared nano prodrug can be used for phototherapy/chemotherapy combination treatment of various solid tumors.
Description
Technical Field
The invention belongs to the field of biological materials, and particularly relates to a chitosan-based nano prodrug carrying indocyanine green and platinum drugs and a preparation method thereof.
Background
Cancer has now become the second leading cause of death worldwide after cardiovascular disease. Chemotherapy remains one of the most prominent cancer treatments among the numerous cancer treatments. Platinum antineoplastic drugs are one of the most important first-line antineoplastic drugs, and are widely applied to the treatment of various tumors. However, the platinum antineoplastic drugs have great toxic and side effects on normal tissues and are easy to generate drug resistance on tumor cells due to the lack of tumor targeting function and systemic medication, so that the treatment effect is severely restricted. In order to overcome the above disadvantages, nanotechnology based combination therapy has received great attention in recent years.
Combination therapy, which generally reduces the amount of individual drugs used to reduce toxic side effects and enhances therapeutic efficacy through synergistic or additive effects between the different drugs, is one of the most promising strategies. Particularly, the combination of the medicines with different action mechanisms or the combination of different treatment modes can effectively overcome the problems of multi-medicine tolerance of the tumor and the like. In recent years, the phototherapy technique for tumor has received much attention because of its advantages such as non-invasive, good biocompatibility, and remote space-time control. Phototherapy is divided into photothermal therapy (PTT) and photodynamic therapy (PDT). PTT is that a photo-thermal agent absorbs light energy and converts the light energy into heat energy, and apoptosis is promoted by raising local temperature, so that tumor cells are killed; PDT involves three key factors, namely light, photosensitizer and oxygen, and the photosensitizer, under the excitation of light with a specific wavelength, generates oxygen-dependent photochemical reaction to generate highly cytotoxic Reactive Oxygen Species (ROS), thereby triggering oxidative stress of tumor cells and leading to death of the tumor cells. Indocyanine green (ICG) is currently the only near-infrared imaging agent approved for clinical use by the U.S. food and drug administration, while being capable of generating heat for PTT upon photo-excitation and ROS for PDT. Therefore, ICG has the dual functions of both a photo-thermal agent and a photosensitizer, and can realize PTT/PDT synergistic treatment on tumors. However, ICG has problems of poor stability in aqueous media, concentration-dependent aggregation, short half-life in vivo, and the like, so that its application effect is greatly limited. Therefore, it would be of great interest to develop a carrier material that overcomes the drawbacks of free ICG and that can be combined with platinum drugs.
Nano drug delivery systems are a very promising approach to achieve the above goals. The nano-drug delivery system can improve the solubility of the drug, enhance the accumulation effect of the tumor, prolong the blood circulation time and the like, and can effectively improve the tumor treatment effect and reduce the toxic and side effects on normal tissues. Especially, the nano-carrier capable of simultaneously loading two or more drugs has development value. In previous reports, chitosan has attracted extensive attention in the development of nano-drug delivery systems due to its advantages of cationic property, abundant sources, safety, non-toxicity, good biocompatibility and biodegradability. However, chitosan itself is poorly soluble in water, greatly restricting the drug loading effect and the application range.
Disclosure of Invention
The invention aims to provide a chitosan-based nano prodrug carrying indocyanine green and platinum drugs and a preparation method thereof, the particle size of the nano prodrug prepared by the preparation method is within the range of 100-200nm, and the chitosan-based nano prodrug has drug release behavior with dual stimulation responsiveness of glutathione and pH and can be used for phototherapy/chemotherapy combination therapy of tumors.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a preparation method of a chitosan-based nano prodrug carrying indocyanine green and platinum drugs comprises the steps of bonding 3,3' -dithiodipropyl hydrazide to a chitosan derivative by utilizing a Diels-Alder reaction to obtain a hydrazide chitosan derivative, mixing a hydrazide chitosan derivative solution with the indocyanine green and the platinum drugs, and carrying out a self-assembly process to obtain the chitosan-based nano prodrug carrying the indocyanine green and the platinum drugs.
The invention has the further improvement that the method specifically comprises the following steps:
(1) mixing the aldehyde group furan solution and the water-soluble chitosan derivative solution, carrying out Schiff base reaction to form an imine bond, and then carrying out reduction reaction by adopting a cyano sodium borohydride solution to obtain a furan group chitosan derivative;
activating carboxyl in maleimide fatty acid by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and bonding 3,3' -dithiodipropylhydrazide to obtain maleimide hydrazide; carrying out Diels-Alder reaction on the furan chitosan derivative and maleimide hydrazide to obtain a hydrazide chitosan derivative;
(2) dissolving the hydrazide chitosan derivative in water with the pH value of 6.4-6.8 to obtain an aqueous solution of the hydrazide chitosan derivative, sequentially adding an aqueous solution of indocyanine green and an aqueous solution of platinum drugs under the ice bath ultrasonic condition, reacting for 0.5-72 hours in a shaking table at the temperature of 25-50 ℃, and then dialyzing and freeze-drying to obtain the chitosan-based nano prodrug carrying the indocyanine green and the platinum drugs.
In a further improvement of the invention, the aldehyde furan in the step (1) is 3- (2-furyl) acrolein, 2-furfural, 2-furaldehyde, 5-methyl-2-furaldehyde, 3- (5-methyl-2-furyl) butyraldehyde or 5-methylfuran-2-propionaldehyde;
the water-soluble chitosan derivative in the step (1) is hydroxyethyl chitosan, hydroxypropyl chitosan, carboxymethyl chitosan or carboxypropyl chitosan;
the molecular weight of the water-soluble chitosan derivative in the step (1) is 2-200 kDa, and the deacetylation degree is 60-90%.
In a further improvement of the invention, the maleimide-based fatty acid in the step (1) is 2-maleimide-based acetic acid, 3-maleimide-based propionic acid, 4-maleimide-based butyric acid, 5-maleimide-based valeric acid or 6-maleimide-based caproic acid;
the conditions for performing the Schiff base reaction in the step (1) are as follows: in N2Reacting for 1-12 h at the temperature of 20-40 ℃ under the atmosphere.
In a further development of the invention, the ratio of the amount of imine bonds formed in step (1) to the amount of substance of sodium cyanoborohydride is 1: (1.1-1.5);
the ratio of the amount of the aldehyde furan in the step (1) to the amount of the amino substance in the water-soluble chitosan derivative is 10: (1-10).
In a further improvement of the present invention, the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in step (1) is 1: (1-1.5) the amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride added is 5-60% of the amount of the carboxyl substance in the maleimide-based fatty acid.
In a further improvement of the invention, the molar ratio of the maleimide-based fatty acid to the 3,3' -dithiodipropylhydrazide in the step (1) is 1: (1-10);
the Diels-Alder reaction in the step (1) is carried out at the temperature of 35-55 ℃ for 24-72 h.
The further improvement of the invention is that the ratio of the amount of the platinum drug to the amount of the hydrazide group substance in the hydrazide chitosan derivative in the step (2) is 1 (1-10);
the ratio of the amount of the indocyanine green to the amount of the hydrazide group substance in the hydrazide chitosan derivative in the step (2) is 1 (1-10).
In a further improvement of the present invention, the platinum-based drug in step (2) is cisplatin, carboplatin, nedaplatin or lobaplatin.
The chitosan-based nano prodrug carrying indocyanine green and platinum drugs, which is obtained by the method, is characterized in that the particle size of the nano prodrug is adjustable within the range of 100-200nm, and the chitosan-based nano prodrug presents a glutathione and pH dual stimulus-responsive drug release behavior.
Compared with the prior art, the invention has the beneficial effects that: (1) the chitosan-based nano prodrug carrying indocyanine green and platinum drugs has the particle size range of 100-200nm, uniform particle size distribution and nearly spherical appearance; (2) the chitosan-based nano prodrug carrying indocyanine green and platinum drugs is formed by the electrostatic interaction between anions and cations, the metal-hydrazide coordination action and the self-assembly process, so that the drug solubility, the loading efficiency and the drug utilization rate are effectively improved, and the chitosan-based nano prodrug has good biological safety; (3) the electrostatic compound, the coordination bond and the disulfide bond in the chitosan-based nano prodrug carrying indocyanine green and platinum drugs have responsiveness to acidic pH and reducibility, and the stimulus-responsive release behavior of the two drugs can be realized; (4) the nano prodrug prepared by the invention can realize the purpose of treating tumors by combining phototherapy and chemotherapy; (5) the nano prodrug prepared by the invention has outstanding functionality, easily obtained raw materials, simple drug loading process and simple purification process, and is suitable for nano combined treatment of various solid tumors.
Drawings
FIG. 1 shows a method for synthesizing a chitosan-based carrier material carrying indocyanine green and a platinum-based drug in example 11H-NMR spectrum. Wherein A is furan hydroxyethyl chitosan; b is hydrazide hydroxyethyl chitosan.
Fig. 2 is a particle size distribution diagram of the chitosan-based nano prodrug carrying indocyanine green and platinum-based drugs synthesized in example 1.
Fig. 3 is a transmission electron microscope image of the chitosan-based nano prodrug carrying indocyanine green and platinum-based drugs synthesized in example 1.
Fig. 4 shows the release behavior of indocyanine green and cisplatin under different pH and glutathione conditions of the chitosan-based nano-prodrug carrying indocyanine green and platinum drugs synthesized in example 1. Wherein A is indocyanine green, and B is cisplatin.
Fig. 5 shows photothermal properties of the chitosan-based nano prodrug supporting indocyanine green and a platinum-based drug synthesized in example 1.
Fig. 6 shows the effect of intracellular active oxygen generation after the chitosan-based nano prodrug carrying indocyanine green and platinum drugs, synthesized in example 1, is treated with HepG2 cells.
Fig. 7 shows the cell killing effect of the chitosan-based prodrug carrying indocyanine green and platinum-based drugs synthesized in example 1.
Detailed Description
The present invention is further described below by way of examples, but the present invention is not limited thereto.
The preparation method of the chitosan-based nano prodrug carrying indocyanine green and platinum drugs comprises the following steps: bonding 3,3' -dithiodipropylhydrazide on a chitosan derivative by using a Diels-Alder reaction to obtain a hydrazide chitosan derivative, then mixing a hydrazide chitosan derivative solution with indocyanine green and a platinum drug, and obtaining the nano prodrug by a self-assembly process, wherein the method specifically comprises the following steps:
(1) preparation of hydrazide chitosan derivative: mixing the aldehyde furan solution with the water-soluble chitosan derivative solution, carrying out Schiff base reaction on aldehyde groups and amino groups, and reducing Schiff base bonds formed by the reaction into stable C-N bonds by using a sodium cyanoborohydride solution to obtain furan chitosan derivatives;
then, activating carboxyl in the maleimide-based fatty acid by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, wherein the adding molar weight ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1: (1-1.5), adding 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in an amount of 5-60% of the molar amount of carboxyl in the activated maleimide-based fatty acid, and bonding 3,3' -dithiodipropylhydrazide by using the hydrochloride to obtain maleimide hydrazide;
and finally, carrying out Diels-Alder reaction on the furan chitosan derivative and maleimide hydrazide to obtain the hydrazide chitosan derivative.
(2) Preparation of chitosan-based nano prodrug carrying indocyanine green and platinum drugs: preparing a hydrazide chitosan derivative into an aqueous solution with the pH value of 6.4-6.8, sequentially adding an indocyanine green aqueous solution and a platinum drug aqueous solution under the ice bath ultrasonic condition, reacting for 0.5-72 h in a shaking table at the temperature of 25-50 ℃, and then performing dialysis and freeze drying processes to obtain the chitosan-based nano prodrug.
The aldehyde furan in the step (1) is 3- (2-furyl) acrolein, 2-furfural, 2-furaldehyde, 5-methyl-2-furaldehyde, 3- (5-methyl-2-furyl) butyraldehyde or 5-methyl furan-2-propionaldehyde and the like.
The water-soluble chitosan derivative in the step (1) is hydroxyalkyl chitosan, such as hydroxyethyl chitosan and hydroxypropyl chitosan, and carboxylated chitosan, such as carboxymethyl chitosan and carboxypropyl chitosan.
The molecular weight of the water-soluble chitosan derivative in the step (1) is 2-200 kDa, and the deacetylation degree is 60-90%.
The maleimide fatty acid in the step (1) is 2-maleimide acetic acid, 3-maleimide propionic acid, 4-maleimide butyric acid, 5-maleimide valeric acid or 6-maleimide caproic acid, etc.
The formation conditions of the imine bond in the step (1) are 20-40 ℃ and N2Reacting for 1-12 h under the atmosphere.
The mass ratio of imine bonds to sodium cyanoborohydride formed in step (1) is 1: (1.1-1.5).
The ratio of the amount of the aldehyde furan to the amount of the amino substance on the water-soluble chitosan derivative in the step (1) is 10: (1-10).
The molar ratio of the maleimide-based fatty acid to the 3,3' -dithiodipropylhydrazide in the step (1) is 1: (1-10).
The Diels-Alder reaction in the step (1) is carried out at 35-55 ℃ for 24-72 h.
The ratio of the amount of the platinum drug to the amount of the hydrazide group substance on the hydrazide chitosan derivative in the step (2) is 1 (1-10).
The ratio of the amounts of the indocyanine green and the hydrazide group substances on the hydrazide chitosan derivative in the step (2) is 1 (1-10).
The platinum drugs in the step (2) are platinum anti-tumor drugs such as cisplatin, carboplatin, nedaplatin, lobaplatin and the like.
The operation of the indocyanine green or the platinum-based drug in the step (2) is carried out under the condition of keeping out light.
The chitosan-based nano prodrug carrying indocyanine green and platinum drugs has adjustable particle size within the range of 100-200nm, presents a glutathione and pH dual stimulation response drug release behavior, and can be used for phototherapy/chemotherapy combination treatment of various solid tumors.
The following are specific examples.
Example 1
(1) Preparation of hydrazide hydroxyethyl chitosan: dripping 0.33% 3- (2-furyl) acrolein solution into 1.0% hydroxyethyl chitosan (MW 170kDa) solution, mixing, stirring, and heating at 30 deg.C and N2Reacting for 12h under the atmosphere, adding a sodium cyanoborohydride solution with the mass concentration of 0.6%, continuing the reduction reaction for 12h to form a crude product of the furan-modified hydroxyethyl chitosan, dialyzing for 72h in deionized water by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying to obtain the furan-modified hydroxyethyl chitosan; activating 1.0 mass percent maleimide caproic acid by 1.09 mass percent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.65 mass percent N-hydroxysuccinimide, adding the activated maleimide caproic acid into 1.0 mass percent 3,3' -dithiodipropyl hydrazide, and reacting at room temperature for 12 hours to obtain maleimide hydrazide; the method comprises the steps of uniformly mixing 0.4% of furan hydroxyethyl chitosan and 4.0% of maleimide hydrazide, carrying out Diels-Alder reaction at 40 ℃ under the condition of magnetic stirring, dialyzing for 48 hours by using a dialysis bag with the molecular weight cutoff of 3500Da in deionized water, and freeze-drying to obtain hydrazide hydroxyethyl chitosan.
(2) Preparation of a chitosan-based nano prodrug carrying indocyanine green and cisplatin: firstly, preparing hydrazide hydroxyethyl chitosan with the mass concentration of 0.2% into an aqueous solution with the pH value of 6.5, then adding indocyanine green with the mass concentration of 0.1% under the ice bath and ultrasonic conditions, then adding cisplatin aqueous solution with the mass concentration of 0.1% and uniformly mixing, reacting in a shaker at 37 ℃ for 24 hours, dialyzing in a dark place in deionized water for 36 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying and collecting the nano prodrug. Wherein the ratio of the amount of the hydrazide group substances in the indocyanine green and the hydrazide-based hydroxyethyl chitosan is 1:4, and the ratio of the amount of the hydrazide group substances in the cisplatin and the hydrazide-based hydroxyethyl chitosan is 1: 4.
The structural formula of the chitosan-based nano prodrug carrying indocyanine green and cisplatin synthesized in the embodiment is as follows:
the hydrazide hydroxyethyl chitosan synthesized in this example, which1The H-NMR spectrum is shown in FIG. 1. As can be seen from A and B in FIG. 1, the chemical shifts of the protons of the components are well identified, which indicates that the synthesis of the synthesized core-shell structure type chitosan-based nano prodrug carrier material hydrazide hydroxyethyl chitosan which carries doxorubicin and platinum drugs together is successful.
The chitosan-based nano prodrug carrying indocyanine green and cisplatin synthesized by the embodiment can be self-assembled in water to form nanoparticles, and a particle size distribution graph measured by a dynamic light scattering method is shown in fig. 2. The particle size of the synthesized chitosan-based nano prodrug carrying indocyanine green and platinum drugs is concentrated to 120nm, the particle size distribution range is narrow, the chitosan-based nano prodrug is ideal, and the requirement of nano drugs on the particle size is met.
The indocyanine green and cisplatin-loaded chitosan-based nano prodrug synthesized by the embodiment can be self-assembled in water to form nanoparticles, and as can be seen from fig. 3, when the morphology of the synthesized indocyanine green and platinum-based drug-loaded chitosan-based nano prodrug is observed under a transmission electron microscope, the nanoparticles are uniformly distributed in a spherical shape, the particle size is distributed near 120nm, and the requirements of the nano drugs on the particle size are met.
Study of in vitro drug release behavior. The in vitro drug release capacity of the chitosan-based nano prodrug carrying indocyanine green and cisplatin synthesized in the example was evaluated by a dialysis method. The specific operation is as follows: preparing a drug-loaded nanoparticle dispersion solution with the concentration of 1mg/mL by ultrasonic dispersion, respectively filling 5mL of the dispersion solution into four dialysis bags with the molecular weight cutoff of 1000Da, respectively putting the dialysis bags into 100mL of PBS buffer solution containing glutathione and having the pH value of 5.5 and 7.4 and the simple pH value of 5.5 and 7.4, and placing the dialysis bags on a shaking table for shaking. And at a set time node, taking out 2mL of dialysate, replacing with 2mL of fresh PBS solution, taking out the absorbance of the liquid at 780nm, namely the absorbance of indocyanine green by using an ultraviolet and visible light spectrophotometer, and measuring the content of platinum element by using inductively coupled plasma mass spectrometry. FIG. 4 shows the in vitro drug release profiles of the synthesized indocyanine green and cisplatin loaded chitosan-based nano-prodrug under different pH and glutathione environments; as can be seen from a and B in fig. 4, indocyanine green supported on the nanoparticles by electrostatic interaction is rapidly released under acidic conditions. Under the action of glutathione, the disulfide bond is broken, so that cisplatin bonded by the disulfide bond is rapidly released, and the pH and the reduction responsiveness of the synthesized chitosan-based nano prodrug carrying indocyanine green and platinum drugs are shown.
And (3) in-vitro photothermal property research. The photothermal properties of the nanoparticles were evaluated using the change in temperature of the nanoparticles during near infrared irradiation. Firstly, PBS, free indocyanine green and chitosan-based nano-prodrug carrying indocyanine green and cisplatin are applied to near infrared (1.0W/cm) with the wavelength of 808nm2) The aqueous solution 480s was irradiated and the temperature change was monitored. As can be seen from fig. 5, within the first 3min, the temperature of the nanoparticle solution formed by the free indocyanine green and the chitosan-based nano-prodrug carrying indocyanine green and platinum-based drugs is rapidly raised, and then the temperature of the free indocyanine green group is slowly lowered due to quenching between the free indocyanine green molecules; the temperature of the solution of the nanoparticles formed by the chitosan-based nano prodrug carrying the indocyanine green and the platinum drugs continues to rise until 5min later, and the highest temperatures of the nanoparticles reach 58.5 ℃, which shows that the nanoparticles improve the stability of the free indocyanine green. And instead the nanoparticle embedded indocyanine green can more effectively produce a near-infrared dependent temperature increase than a free indocyanine green solution. In addition, as a control, PBS had only a slight temperature increase upon near infrared irradiation. These show that the synthesized chitosan-based nano prodrug carrying indocyanine green and platinum drugs has good photothermal effect.
The production of ROS. Human liver tumor cells (HepG2) under 808nm NIR light irradiation were stained for ROS production by the DCFH-DA method. HepG2 cells were culturedDensity of 5X 104One/well was inoculated in 6-well plates containing 5% CO at 37 deg.C2Is cultured in an incubator for 24 hours, then is incubated with free indocyanine green and chitosan-based nano prodrug loaded with indocyanine green and cisplatin for 24 hours, is incubated with 20 mu M DCFH-DA for 20 minutes, and is incubated with 1.0W/cm2Irradiating 6-well plate with near infrared radiation for 5 min. The fluorescence intensity of DCF was then recorded by flow cytometry to quantify ROS production. As can be seen from fig. 6, compared with the free indocyanine green group, the synthesized indocyanine green and platinum-based drug-loaded chitosan-based nanoparticule-induced HepG2 cell group shows stronger fluorescence intensity, indicating that it can induce more active oxygen. The synthesized chitosan-based nano prodrug carrying indocyanine green and platinum drugs has the potential of photodynamic cancer treatment.
Cytotoxicity test of chitosan-based nano-prodrug loaded with indocyanine green and platinum-based drugs. The conventional tetramethyl azodicarbonamide salt trace enzyme reaction colorimetric method (MTT method) is adopted to detect the capability of the chitosan-based nano prodrug carrying indocyanine green and platinum drugs for inhibiting the proliferation of human liver tumor cells (HepG 2). HepG2 cells were first seeded onto 96-well plates. It was placed at 37 ℃ in 5% CO2After culturing for 12h under the condition, adding nanoparticle solutions with different concentrations to continue culturing for 48 h. After culturing for 4 hours, 20. mu.L of MTT solution (5mg/mL) was added to each well of the culture dish, and the supernatant was aspirated and 200. mu.L of dimethyl sulfoxide was added to dissolve formazan crystals formed. The absorbance (OD) at 492nm was measured for each well using a microplate reader. The test results are shown in fig. 7, and it can be seen that the chitosan-based nano prodrug carrying indocyanine green and platinum-based drugs treated HepG2 cell group showed better tumor cell inhibition effect compared to the free drug group.
Example 2
(1) Preparation of hydrazide hydroxyethyl chitosan: dripping 0.33% 3- (2-furyl) acrolein solution into 1.0% hydroxyethyl chitosan (MW 200kDa), mixing, stirring, and heating at 30 deg.C and N2Reacting for 12h under the atmosphere, adding a cyano sodium borohydride solution with the mass concentration of 0.6 percent, and reacting at the temperature of 20 ℃ and N2The reduction reaction is continued for 1h under the atmosphere to form furanDialyzing the crude product of the ethoxyl chitosan by a dialysis bag with the molecular weight cutoff of 3500Da for 72h in deionized water, and freeze-drying to obtain furan hydroxyethyl chitosan; activating 1.0 mass percent maleimide caproic acid by 1.09 mass percent 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.65 mass percent N-hydroxysuccinimide, adding the activated maleimide caproic acid into 1.0 mass percent 3,3' -dithiodipropyl hydrazide, and reacting at room temperature for 12 hours to obtain maleimide hydrazide; the method comprises the steps of uniformly mixing 0.4% of furan hydroxyethyl chitosan and 4.0% of maleimide hydrazide, carrying out Diels-Alder reaction at 35 ℃ under the condition of magnetic stirring, dialyzing for 48 hours by using a dialysis bag with the molecular weight cutoff of 3500Da through deionized water after 24 hours of reaction, and freeze-drying to obtain hydrazide hydroxyethyl chitosan.
(2) Preparation of a chitosan-based nano prodrug carrying indocyanine green and cisplatin: firstly, preparing hydrazide hydroxyethyl chitosan with the mass concentration of 0.2% into an aqueous solution with the pH value of 6.5, then adding indocyanine green with the mass concentration of 0.2% under the ice bath and ultrasonic conditions, then adding cisplatin aqueous solution with the mass concentration of 0.2% and uniformly mixing, reacting in a shaker at 37 ℃ for 24 hours, dialyzing in a dark place in deionized water for 36 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying and collecting the nano prodrug. Wherein the ratio of the amount of the hydrazide group substances in the indocyanine green and the hydrazide-type hydroxyethyl chitosan is 1:7, and the ratio of the amount of the hydrazide group substances in the cisplatin and the hydrazide-type hydroxyethyl chitosan is 1: 4.
Example 3
(1) Preparation of hydrazide hydroxyethyl chitosan: dripping 0.33% 3- (2-furyl) acrolein solution into 1.0% hydroxyethyl chitosan (MW 20kDa) solution, mixing, stirring, and heating at 30 deg.C and N2Reacting for 12h under the atmosphere, adding a cyano sodium borohydride solution with the mass concentration of 0.6 percent, and reacting at the temperature of 30 ℃ and N2Continuing reduction reaction for 6h under the atmosphere to form a crude product of the furan hydroxyethyl chitosan, dialyzing with deionized water for 72h by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying to obtain furan hydroxyethyl chitosan; maleimide caproic acid with the mass concentration of 1.0 percent is added into the mixtureActivating 1.09% of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.65% of N-hydroxysuccinimide, adding the activated 1.09% of 1.0% of 3,3' -dithiodipropylhydrazide, and reacting at room temperature for 12 hours to obtain maleimidohydrazide; the method comprises the steps of uniformly mixing 0.4% of furan hydroxyethyl chitosan and 4.0% of maleimide hydrazide, carrying out Diels-Alder reaction at 55 ℃ under the condition of magnetic stirring, dialyzing for 48 hours by using a dialysis bag with the molecular weight cutoff of 3500Da through deionized water after 72 hours of reaction, and freeze-drying to obtain hydrazide hydroxyethyl chitosan.
(2) Preparation of a chitosan-based nano prodrug carrying indocyanine green and cisplatin: firstly, preparing hydrazide hydroxyethyl chitosan with the mass concentration of 0.05% into an aqueous solution with the pH value of 6.5, then adding indocyanine green with the mass concentration of 0.05% under the ice bath and ultrasonic conditions, then adding cisplatin aqueous solution with the mass concentration of 0.2% and uniformly mixing, reacting in a shaker at 37 ℃ for 24 hours, dialyzing in a dark place in deionized water for 36 hours by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying and collecting the nano prodrug. Wherein the ratio of the amount of the hydrazide group substances in the indocyanine green and the hydrazide-type hydroxyethyl chitosan is 1:3, and the ratio of the amount of the hydrazide group substances in the cisplatin and the hydrazide-type hydroxyethyl chitosan is 1: 2.
Example 4
(1) Preparation of hydrazide hydroxypropyl chitosan: dripping 0.33% 2-furaldehyde solution into 1.0% hydroxypropyl chitosan (MW 20kDa, deacetylation degree 60%) solution, mixing, stirring, and heating at 30 deg.C and N2And reacting for 12 hours under an atmosphere to form imine bonds, adding a sodium cyanoborohydride solution with the mass concentration of 0.6%, wherein the mass ratio of the imine bonds to the sodium cyanoborohydride is 1: 1.1, at 30 ℃ and N2Continuing reduction reaction for 6h under the atmosphere to form a furylated hydroxypropyl chitosan crude product, dialyzing the product for 72h by using a dialysis bag with the molecular weight cutoff of 3500Da in deionized water, and freeze-drying the product to obtain the furylated hydroxypropyl chitosan;
activating a 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride solution with the mass concentration of 1.09% and an N-hydroxysuccinimide solution with the mass concentration of 0.65% in a 2-maleimidoacetic acid solution with the mass concentration of 1.0%, adding the activated solution into a 3,3' -dithiodipropyl hydrazide solution with the mass concentration of 1.0%, and reacting at room temperature for 12 hours to obtain maleimidohydrazide; uniformly mixing 0.4% of furylated hydroxypropyl chitosan solution and 4.0% of maleimide hydrazide solution, carrying out Diels-Alder reaction at 55 ℃ under the condition of magnetic stirring, dialyzing for 48h by using a dialysis bag with the molecular weight cutoff of 3500Da in deionized water, and freeze-drying to obtain the hydrazide hydroxypropyl chitosan. Wherein the molar ratio of the 2-maleimide acetic acid solution to the 3,3' -dithiodipropylhydrazide is 1: 1; the adding molar weight ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1: the 1, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was used in an amount of 30% by mole of the carboxyl groups in the activated maleimide-based fatty acid.
(2) Preparation of a chitosan-based nano prodrug carrying indocyanine green and cisplatin: under the dark condition, firstly, preparing hydrazide hydroxypropyl chitosan into an aqueous solution with the pH value of 6.5 and the mass concentration of 0.05%, then adding a indocyanine green solution with the mass concentration of 0.05% under the ice bath and ultrasonic conditions, then adding a cisplatin aqueous solution with the mass concentration of 0.2% and uniformly mixing, reacting in a shaking table at 25 ℃ for 50h, dialyzing in deionized water for 36h in the dark condition by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying and collecting the nano prodrug. Wherein the amount ratio of the indocyanine green to the hydrazide group substances in the hydrazide hydroxypropyl chitosan is 1:6, and the amount ratio of the cisplatin to the hydrazide group substances in the hydrazide hydroxypropyl chitosan is 1: 1.
Example 5
(1) Preparation of hydrazide carboxymethyl chitosan: dripping 5-methyl-2-furan acrolein with mass concentration of 0.33% into carboxymethyl chitosan (MW 200kDa, deacetylation degree of 80%) solution with mass concentration of 1.0%, mixing and stirring uniformly, and adding N2Reacting for 12 hours at 20 ℃ under an atmosphere to form imine bonds, adding a sodium cyanoborohydride solution with the mass concentration of 0.6%, wherein the mass ratio of the imine bonds to the sodium cyanoborohydride is 1: 1.5, N2The reduction reaction is continued for 6h at 30 ℃ under the atmosphere to formDialyzing the crude product of the furylated carboxymethyl chitosan for 72 hours by using a dialysis bag with the molecular weight cutoff of 3500Da in deionized water, and freeze-drying to obtain the furylated carboxymethyl chitosan;
activating a 1.09 mass percent solution of 3-maleimidopropionic acid by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and 0.65 mass percent N-hydroxysuccinimide, adding the activated solution into 1.0 mass percent solution of 3,3' -dithiodipropylhydrazide, and reacting at room temperature for 12 hours to obtain maleimidohydrazide; the preparation method comprises the steps of uniformly mixing 0.4% of furylated carboxymethyl chitosan and 4.0% of maleimide hydrazide, carrying out Diels-Alder reaction at 35 ℃ under the condition of magnetic stirring, dialyzing for 48 hours by using a dialysis bag with the molecular weight cutoff of 3500Da through deionized water after 72 hours of reaction, and freeze-drying to obtain the hydrazide carboxymethyl chitosan. Wherein the molar ratio of the 3-maleimidopropionic acid to the 3,3' -dithiodipropylhydrazide is 1: 5; the adding molar weight ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1: 1.5, the amount added is 5% of the molar amount of carboxyl groups in the activated maleimide-based fatty acid.
(2) Preparation of a chitosan-based nano prodrug carrying indocyanine green and carboplatin: under the condition of keeping out of the sun, firstly, preparing hydrazide carboxymethyl chitosan into an aqueous solution with the pH value of 6.4 and the mass concentration of 0.05%, then adding an indocyanine green solution with the mass concentration of 0.05% under the conditions of ice bath and ultrasound, then adding a carboplatin aqueous solution with the mass concentration of 0.2% and uniformly mixing, reacting for 0.5h in a shaking table at 50 ℃, dialyzing for 36h in the deionized water in the absence of the sun by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying and collecting the nano prodrug. Wherein the ratio of the amount of the indocyanine green to the amount of the hydrazide group substances in the hydrazide carboxymethyl chitosan is 1:10, and the ratio of the amount of the carboplatin to the amount of the hydrazide group substances in the hydrazide carboxymethyl chitosan is 1: 5.
Example 6
(1) Preparation of hydrazide carboxypropyl chitosan: 3- (5-methyl-2-furyl) butyraldehyde with the mass concentration of 0.33 percent is dripped into a solution of 1.0 percent of carboxypropyl chitosan (MW 100kDa, the deacetylation degree is 90 percent) to be mixed and stirredHomogeneous, N2Reacting for 1h at 40 ℃ under an atmosphere to form imine bonds, adding a sodium cyanoborohydride solution with the mass concentration of 0.6%, wherein the mass ratio of the imine bonds to the sodium cyanoborohydride is 1: 1.3, N2Continuously carrying out reduction reaction for 6h at 30 ℃ under the atmosphere to form a crude product of the furylated carboxypropyl chitosan, dialyzing the crude product for 72h by using a dialysis bag with the molecular weight cutoff of 3500Da in deionized water, and freeze-drying the dialyzed product to obtain the furylated carboxypropyl chitosan;
activating a 5-maleimidovaleric acid solution with the mass concentration of 1.0% by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride with the mass concentration of 1.09% and N-hydroxysuccinimide with the mass concentration of 0.65%, adding the activated solution into 3,3' -dithiodipropionylhydrazide with the mass concentration of 1.0%, and reacting at room temperature for 12 hours to obtain maleimidohydrazide; the preparation method comprises the steps of uniformly mixing 0.4% of furylated carboxypropyl chitosan and 4.0% of maleimide hydrazide, carrying out Diels-Alder reaction at 40 ℃ under the condition of magnetic stirring, dialyzing for 45 hours by using a dialysis bag with the molecular weight cutoff of 3500Da in deionized water for 48 hours, and freeze-drying to obtain the hydrazide carboxypropyl chitosan. Wherein the molar ratio of the 5-maleimide valeric acid to the 3,3' -dithiodipropylhydrazide is 1: 10; the adding molar weight ratio of the 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to the N-hydroxysuccinimide is 1: 1.2, the amount added is 60% of the molar amount of carboxyl groups in the activated maleimide-based fatty acid.
(2) Preparation of a chitosan-based nano prodrug carrying indocyanine green and nedaplatin: under the condition of keeping out of the sun, firstly, preparing hydrazide carboxypropyl chitosan into an aqueous solution with the pH value of 6.8 and the mass concentration of 0.05%, then adding a indocyanine green solution with the mass concentration of 0.05% under the conditions of ice bath and ultrasound, then adding a nedaplatin aqueous solution with the mass concentration of 0.2% and uniformly mixing, reacting in a shaking table at 42 ℃ for 40 hours, dialyzing in the deionized water for 36 hours in the absence of the sun by using a dialysis bag with the molecular weight cutoff of 3500Da, and freeze-drying and collecting the nano prodrug. Wherein the ratio of the amount of the hydrazide group substances in the indocyanine green and the hydrazide-based chitosan is 1:1, and the ratio of the amount of the nedaplatin to the amount of the hydrazide group substances in the hydrazide-based chitosan is 1: 10.
Claims (10)
1. A preparation method of a chitosan-based nano prodrug carrying indocyanine green and platinum drugs is characterized in that 3,3' -dithiodipropylhydrazide is bonded to a chitosan derivative through a Diels-Alder reaction to obtain a hydrazide chitosan derivative, then a hydrazide chitosan derivative solution is mixed with the indocyanine green and the platinum drugs, and the chitosan-based nano prodrug carrying the indocyanine green and the platinum drugs is obtained through a self-assembly process.
2. The preparation method of the indocyanine green and platinum drug-loaded chitosan-based nano prodrug as claimed in claim 1, which is characterized by comprising the following steps:
(1) mixing the aldehyde group furan solution and the water-soluble chitosan derivative solution, carrying out Schiff base reaction to form an imine bond, and then carrying out reduction reaction by adopting a cyano sodium borohydride solution to obtain a furan group chitosan derivative;
activating carboxyl in maleimide fatty acid by using 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, and bonding 3,3' -dithiodipropylhydrazide to obtain maleimide hydrazide; carrying out Diels-Alder reaction on the furan chitosan derivative and maleimide hydrazide to obtain a hydrazide chitosan derivative;
(2) dissolving the hydrazide chitosan derivative in water with the pH value of 6.4-6.8 to obtain an aqueous solution of the hydrazide chitosan derivative, sequentially adding an aqueous solution of indocyanine green and an aqueous solution of platinum drugs under the ice bath ultrasonic condition, reacting for 0.5-72 hours in a shaking table at the temperature of 25-50 ℃, and then dialyzing and freeze-drying to obtain the chitosan-based nano prodrug carrying the indocyanine green and the platinum drugs.
3. The method for preparing a chitosan-based nano-prodrug supporting indocyanine green and a platinum-based drug according to claim 2, wherein the aldehyde-based furan in the step (1) is 3- (2-furyl) acrolein, 2-furfural, 2-furaldehyde, 5-methyl-2-furaldehyde acrolein, 3- (5-methyl-2-furyl) butyraldehyde or 5-methylfuran-2-propionaldehyde;
the water-soluble chitosan derivative in the step (1) is hydroxyethyl chitosan, hydroxypropyl chitosan, carboxymethyl chitosan or carboxypropyl chitosan;
the molecular weight of the water-soluble chitosan derivative in the step (1) is 2-200 kDa, and the deacetylation degree is 60-90%.
4. The method for preparing a chitosan-based nano-prodrug carrying indocyanine green and a platinum-based drug, according to claim 2, wherein the maleimide-based fatty acid in the step (1) is 2-maleimidoacetic acid, 3-maleimidopropionic acid, 4-maleimidobutyric acid, 5-maleimidovaleric acid, or 6-maleimidocaproic acid;
the conditions for performing the Schiff base reaction in the step (1) are as follows: in N2Reacting for 1-12 h at the temperature of 20-40 ℃ under the atmosphere.
5. The method for preparing a chitosan-based nano-prodrug carrying indocyanine green and a platinum-based drug, according to claim 2, wherein the amount of the imine bond to sodium cyanoborohydride substance formed in the step (1) is in a ratio of 1: (1.1-1.5);
the ratio of the amount of the aldehyde furan in the step (1) to the amount of the amino substance in the water-soluble chitosan derivative is 10: (1-10).
6. The preparation method of a chitosan-based nano-prodrug carrying indocyanine green and a platinum-based drug, according to claim 2, wherein the molar ratio of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide in the step (1) is 1: (1-1.5) the amount of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride added is 5-60% of the amount of the carboxyl substance in the maleimide-based fatty acid.
7. The preparation method of a chitosan-based nano-prodrug carrying indocyanine green and a platinum-based drug, according to claim 2, wherein the molar ratio of the maleimide-based fatty acid to the 3,3' -dithiodipropylhydrazide in the step (1) is 1: (1-10);
the Diels-Alder reaction in the step (1) is carried out at the temperature of 35-55 ℃ for 24-72 h.
8. The preparation method of the indocyanine green and platinum drug-loaded chitosan-based nano prodrug as claimed in claim 2, wherein the ratio of the amount of the platinum drug to the amount of hydrazide group species in the hydrazide chitosan derivative in step (2) is 1 (1-10);
the ratio of the amount of the indocyanine green to the amount of the hydrazide group substance in the hydrazide chitosan derivative in the step (2) is 1 (1-10).
9. The preparation method of the indocyanine green and platinum drug-loaded chitosan-based nano prodrug as claimed in claim 2, wherein the platinum drug in the step (2) is cisplatin, carboplatin, nedaplatin or lobaplatin.
10. The chitosan-based nano prodrug carrying indocyanine green and platinum drugs, which is obtained by the method of any one of claims 1 to 9, is characterized in that the particle size of the nano prodrug is adjustable within the range of 100-200nm, and the chitosan-based nano prodrug exhibits the drug release behavior of dual stimulus responses of glutathione and pH.
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