CN110746598A

CN110746598A - Completely degradable GSH/ROS double-sensitive polymer and preparation method and application thereof

Info

Publication number: CN110746598A
Application number: CN201911088965.XA
Authority: CN
Inventors: 康洋; 吴钧; 李丹丹; 张铷和
Original assignee: Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
Current assignee: Seventh Affiliated Hospital Of Sun Yat Sen University Shenzhen
Priority date: 2019-11-08
Filing date: 2019-11-08
Publication date: 2020-02-04
Anticipated expiration: 2039-11-08
Also published as: CN110746598B

Abstract

The invention discloses a GSH/ROS double-sensitive polymer capable of being completely degraded, and a preparation method and application thereof. The invention successfully synthesizes a novel carrier material polymer Cys-0E with an oxalate-like ester bond and a disulfide bond by taking natural cystine ester containing a disulfide bond and oxalyl chloride as raw materials; the polymer material can be successfully assembled into a nano-targeting delivery system for efficiently loading various hydrophilic and hydrophobic drugs. The drug-loaded nanoparticles constructed by the nano precipitation method have obvious GSH/ROS dual responsiveness, small particle size, high drug loading capacity, good in-vivo stability and specific targeting on tumors; under the action of tumor redox environment, the carrier is completely degraded, and the entrapped drug is quickly and massively released, so that the effect of efficiently inhibiting tumors in vivo and in vitro is achieved. Meanwhile, the invention has the advantages of simple synthesis, convenient application, environmental protection and no way in the field of biological medicine.

Description

Completely degradable GSH/ROS double-sensitive polymer and preparation method and application thereof

Technical Field

The invention belongs to the technical field of functional polymer materials and biological medicines. More particularly, relates to a GSH/ROS double-sensitive polymer capable of being completely degraded, a preparation method and an application thereof.

Background

Cancer is also called malignant tumor, which is a disease seriously harming human life and health. Cancer is increasingly valued in view of its high morbidity and mortality. At present, the common treatment methods for cancer mainly comprise surgery, radiotherapy, chemotherapy and biological treatment, and the methods are usually combined clinically for tumor treatment. Statistically, the cancer patients who undergo surgery and radiotherapy account for 1/3, and chemotherapy accounts for 2/3. Therefore, chemotherapy plays an important role in combination therapy as an adjuvant therapy to surgery or radiotherapy. However, the systemic administration mode of chemotherapy and the hydrophobicity of the common anticancer drugs greatly limit the clinical application of chemotherapy, often resulting in many problems, such as lack of selectivity to tumor cells, high toxicity and side effects, low bioavailability, drug resistance, etc., and therefore, in order to overcome many of the disadvantages of the conventional chemotherapy, a new administration mode of anticancer drugs needs to be researched.

With the advent and advancement of nanotechnology, a number of platforms for the delivery of antitumor drugs (DDSS) have been developed to address the many obstacles encountered in the clinical application of antitumor drugs. The drug delivery systems can collect the antitumor drugs at the tumor part as much as possible, prolong the blood circulation time of the drugs, improve the curative effect and avoid the toxic and side effects of the drugs. Through the efforts of a second generation of researchers, nanocarriers have evolved from liposomes to smart nanoparticles. In recent years, in order to ensure the stability of a controlled drug release system in the circulation process and the targeted release of anticancer drugs, a stimulation response group related to tumor treatment is introduced to achieve the treatment target. These unique tumor environments and some exogenous stimuli include temperature, redox substances, light, enzymes, etc.

It is well known that the tumor microenvironment has abnormal redox levels, including Glutathione (GSH) or Glutathione (GSH) in tumor tissuesReactive Oxygen Species (ROS) expression levels were higher than normal tissues. According to recent decades of research, ROS have been found to be overexpressed in cancer. In particular, H in normal tissue₂O₂The concentration is typically 20nM, whereas in tumor tissue the concentration is due to H₂O₂The concentration of the (D) is up to 50-100 mu M. Based on this abnormal level of reactive oxygen species, researchers have explored many reactive oxygen species-responsive drug nanocarriers. In addition, glutathione, a thiol-containing tripeptide, is the major reducing substance in cells. The reported concentration of the compound in cancer cells (10-40 mM) is more than 1000 times higher than that in normal blood environment (2-20 mu M) and more than 4 times higher than that in normal cells, and the huge concentration difference provides a new idea for targeted release of intracellular drugs. At present, a plurality of redox response type drug delivery platforms have been developed based on tumor specific microenvironment, but problems such as carrier materials can not be completely degraded into harmless substances, and rapid response of single redox sensitivity to complex tumor microenvironment can not be realized still exist, and further improvement is needed.

Disclosure of Invention

The technical problem to be solved by the invention is to overcome the defects and shortcomings of the prior art and provide synthesis and application of a poly (dithioamide) polymer (Cys-0E) which is formed by amide reaction of cystine ester and is double-sensitive to Glutathione (GSH) and Reactive Oxygen Species (ROS). The material is simple to synthesize, and has good biological safety and complete biodegradability; the nano drug delivery system taking Cys-0E as a carrier material to load chemotherapeutic drugs has small particle size, high drug loading, good in-vivo stability, capability of specifically targeting tumors and obvious tumor inhibition effect.

The first purpose of the invention is to provide a GSH/ROS double sensitive polymer Cys-0E which can be completely degraded.

The second purpose of the invention is to provide a preparation method of the GSH/ROS double-sensitive polydithioamide polymer Cys-0E which can be completely degraded.

The third purpose of the invention is to provide the application of the polymer Cys-0E or the polymer Cys-0E prepared by the method in the aspect of being used as or preparing a drug delivery carrier.

The fourth purpose of the invention is to provide a GSH/ROS double-sensitive drug delivery carrier which can be completely degraded and a preparation method thereof.

The fifth purpose of the invention is to provide a GSH/ROS double-sensitive nano drug delivery system which can be completely degraded.

The sixth purpose of the invention is to provide a preparation method of the GSH/ROS double-sensitive nano drug delivery system which can be completely degraded.

The above purpose of the invention is realized by the following technical scheme:

a fully degradable GSH/ROS dual sensitive polydithioamide polymer Cys-0E, the polymer Cys-0E having the formula (I):

wherein n is 5 to 1000. Products within this range of degrees of polymerization can assemble to form stable nanoparticles.

The polymer Cys-0E can be assembled in water to form nanoparticles to form a stable colloid system, and experiments show that the polymer Cys-0E nanoparticles have no obvious biotoxicity, can be completely degraded, have GSH/ROS dual responsiveness, have the advantages of simple operation, few reaction steps, no pollution of products and the like, and the preparation method is simple, efficient and economical.

The invention also relates to a preparation method of the completely degradable GSH/ROS double-sensitive poly-dithioamide polymer Cys-0E, which comprises the following steps: dissolving cystine ester with an organic solvent, and reacting with an oxalyl chloride solution at-20-100 ℃ for 0.5-48 h under the protection of inert gas to obtain the GSH/ROS double-sensitive polymer Cys-0E.

The invention successfully synthesizes a novel carrier material polymer Cys-0E with an oxalate-like ester bond and a disulfide bond by taking natural cystine ester containing a disulfide bond and oxalyl chloride as raw materials; the polymer material can be successfully assembled into a nano-targeting delivery system for efficiently loading various hydrophilic and hydrophobic anticancer drugs. The drug-loaded nanoparticles constructed by a nano precipitation method have small particle size, high drug loading, good in-vivo stability and specific targeting on tumors; under the action of tumor redox environment, the carrier is completely degraded, and the entrapped drug is quickly and massively released, so that the effect of efficiently inhibiting tumors in vivo and in vitro is achieved. Meanwhile, the invention has the advantages of simple synthesis, convenient application, environmental protection and wide prospect in the field of biological medicine.

In a preferred embodiment of the present invention, the molar ratio of the cystine ester to oxalyl chloride is 1-10: 1-10; preferably 1: 1-5; more preferably 1: 1.2. in this ratio, the product produced is guaranteed to be in the appropriate molecular mass range. If the cystine ester content is too low, it will be more difficult to form a polymer; if the cystine ester content is too high, the resulting polymer cannot be assembled into stable nanoparticles.

In a preferred embodiment of the present invention, the reaction temperature is 0-20 ℃ and the reaction time is 4-6 h. The reaction time is too long under the condition of being lower than 0 ℃, the polymerization degree does not change obviously after the time is prolonged, the energy is wasted, and the production cost is increased; if the reaction time is too short, the reaction cannot be sufficiently carried out, and the polymer component is reduced, so that the response property of the obtained product is lowered, and the yield of the polymer is low. When the reaction time is 4-6 h at the temperature of 0-20 ℃, the reaction can be carried out fully and rapidly.

In a preferred embodiment of the invention, the cystine ester is cystine ester hydrochloride; before dissolving cystine ester hydrochloride, the cystine ester hydrochloride is subjected to hydrochloric acid removal, extraction and drying treatment.

In the preferred embodiment of the invention, the cystine ester hydrochloride is selected from one or more of L-cystine dimethyl ester dihydrochloride, L-cystine ethyl ester dihydrochloride, L-cystine benzyl ester dihydrochloride or L-cystine bis (tert-butyl ester) dihydrochloride; preferably, L-cystine dimethyl ester dihydrochloride is used as a synthesis raw material, a stable disulfide bond source can be provided, the yield of synthesis with oxalyl chloride is high, nanoparticles can be formed by being assembled in water to form a polymer Cys-0E of a stable colloid system, the source is wide, and the cost is low.

In the present invention, the desalting acid agent may be selected from a large number of agents, and may be one or more selected from sodium carbonate, sodium bicarbonate, sodium hydroxide, triethylamine and the like. In one embodiment of the invention, sodium carbonate is preferred, which is cheap and easy to obtain, and the desalting conditions are mild, and the pollution of the desalting by-products is small.

In the invention, the organic solvent is selected from one or more of dichloromethane, ethyl acetate, chloroform or tetrahydrofuran. In one embodiment of the invention, the organic solvent is preferably chloroform, so that high extraction yield can be achieved, and the synthesized polymer is easy to separate from the polymer and remove.

In the present invention, the inert gas is nitrogen or argon or other inert gas. In one embodiment of the invention, the inert gas is preferably nitrogen, and nitrogen is the gas with the largest content in the atmosphere, so the extraction is convenient, the cost is low, and the nitrogen is not easy to react with the cystine ester and oxalyl chloride which are reaction raw materials and the synthesized polymer Cys-0E, and the purpose of isolating oxygen to protect the reaction raw materials and the reaction products can be achieved.

The invention also relates to the application of the polymer Cys-0E in the aspect of being used as or preparing a drug delivery carrier.

The polymer Cys-0E can be prepared into nanoparticles with uniform and stable particle size and loaded with different hydrophilic and hydrophobic drugs by a nano precipitation method or a single emulsion/double emulsion method.

In the invention, the polymer Cys-0E can be independently used as a drug carrier, and pharmaceutically acceptable auxiliary materials can be added into the polymer Cys-0E to prepare the drug carrier. Such as distearoylphosphatidylethanolamine-polyethylene glycol stabilizers (DSPE-PEG), stabilizers such as polyvinyl alcohol (PVA) or phospholipid molecules, vitamin C derivatives, and the like, further provide active oxygen content.

In one embodiment of the invention, the preparation method of the completely degradable GSH/ROS double-sensitive nano drug delivery carrier comprises the following steps: dissolving the polymer Cys-0E in an organic solvent to obtain a Cys-0E solution; dropwise adding the Cys-0E solution into an aqueous solution containing a stabilizer under the stirring condition, and assembling the aqueous solution into nanoparticles; or dissolving the polymer Cys-0E and the stabilizer in an organic solvent, and dropwise adding the obtained mixed solution into water to assemble the nanoparticles.

As an improvement, in one embodiment of the invention, the preparation method of the completely degradable GSH/ROS double-sensitive nano drug delivery carrier comprises the following steps: dissolving polymer Cys-0E, stabilizer and vitamin C derivative in organic solvent to obtain oil phase; and (3) under the condition that the stirring speed is 800-1200 rpm, dropwise adding the mixed oil phase solution into the water phase at the speed of 5-10 mu L/s, performing ultrafiltration, and freeze-drying to obtain the GSH/ROS double-sensitive nano-drug carrier.

The invention also relates to a GSH/ROS double-sensitive nano drug delivery system capable of being completely degraded, which comprises the polymer Cys-0E and a carried drug. In one embodiment, the loaded drug comprises a hydrophobic drug or a hydrophilic drug. The invention has no special limitation on the type of the loaded medicine, and can be hydrophobic or hydrophilic antitumor medicines, anti-inflammatory medicines, cardiovascular disease medicines or immunologic adjuvants and the like. Wherein, the anti-tumor drug includes but is not limited to chemotherapeutic drugs, nucleic acid drugs, protein polypeptide drugs and the like. Such anti-inflammatory drugs include, but are not limited to, dexamethasone and the like. The cardiovascular disease drugs include but are not limited to aspirin, dipyridamole, and the like.

The drug delivery carrier/drug delivery system can obviously improve the solubility of the hydrophobic drug, greatly improve the availability of the hydrophobic drug and also greatly improve the circulation time of the nanoparticles in blood, thereby improving the drug accumulation of tumor parts and improving the treatment effect. In a preferred embodiment of the invention, the loaded drug is preferably an anti-tumor drug. Wherein, the hydrophilic drugs include, but are not limited to, doxorubicin hydrochloride, gemcitabine hydrochloride, irinotecan hydrochloride, fluorouracil or lentinan and other drugs. The hydrophobic drugs include, but are not limited to, Docetaxel (DTX), Paclitaxel (PTX), methotrexate, camptothecin, adriamycin and the like.

In one embodiment, the nano drug delivery system is prepared from the polymer Cys-0E, a stabilizer, a vitamin C derivative and a loaded drug, and the stability of the drug carrier and the application effect of the drug carrier can be further improved by adding the stabilizer and the vitamin C derivative.

In a preferred embodiment of the present invention, the nano drug delivery system is prepared by the following method: and (3) co-dissolving the polymer Cys-0E, the stabilizer, the vitamin C derivative and the carried medicine in an oil phase solvent according to a proportion, dispersing the mixed oil phase solution into a water phase under a stirring state, and performing ultrafiltration to obtain the compound oil phase. The nano drug delivery system has the characteristics that under the redox action of abnormally high glutathione and active oxygen in human tumor cells, disulfide bonds and oxalate-like bonds are broken, the hydrophilic/hydrophobic anticancer drugs are released, and the nano drug delivery system has redox response, controlled release and slow release and toxic and side effects.

In a preferred embodiment of the invention, the mass ratio of the polymer Cys-0E to the loaded drug is 5: 1-3; preferably 5: 2. if the proportion of the polymer Cys-0E is too large, the drug loading rate is low, and the number of the formed nanoparticles is small; if the proportion of the polymer Cys-0E is too small, the encapsulation efficiency is low, the waste of the medicine is caused, and the obtained nanoparticles are unstable and are easy to precipitate. The invention controls the mass ratio of polymer Cys-0E to the loaded medicine to be 5: 1-3, the small encapsulation rate can be avoided, the stability and the drug-loading rate of a drug delivery system are ensured, and a product with uniform particle size, moderate encapsulation rate and drug-loading rate and good stability is obtained. Wherein when the mass ratio of the polymer Cys-0E to the carried medicine is 5: 2, the prepared nano drug delivery system has small particle size, high drug loading rate of 10 percent, good in vivo stability, capability of specifically targeting tumors (good GSH and ROS responsiveness) and obvious tumor inhibition effect.

In a preferred embodiment of the invention, the vitamin C derivative comprises one or more of vitamin C, sodium ascorbate, magnesium ascorbate, ascorbyl phosphate, ascorbyl acetate, ascorbyl propionate, ascorbyl stearate, ascorbyl palmitate or ascorbyl dipalmitate. In the invention, the vitamin C derivative is added to generate more active oxygen, promote the disintegration of a drug carrier in cells, particularly tumor cells, and quickly release a large amount of drugs, thereby achieving the purpose of improving the treatment effect.

In some embodiments of the invention, the stabilizer is selected from one or more of distearoylphosphatidylethanolamine-polyethylene glycol-based stabilizer (DSPE-PEG), polyvinyl alcohol (PVA), or phospholipid molecules. In one embodiment of the invention, the stabilizer is preferably DSPE-PEG2000, so that the optimal long-circulating effect of the nano-drug can be realized, and the therapeutic index can be improved.

In a preferred embodiment of the present invention, the mass ratio of the stabilizer to the vitamin C derivative is 1-5: 1. in the proportion, the generation of enough hydrogen peroxide in tumor cells can be ensured, and the effective release of the medicine is realized. If the content of the vitamin C derivative is too low, the medicine is slowly released; if the content of the vitamin C derivative is too high, the drug loading of the nano-drug can be influenced.

In a preferred embodiment of the invention, the volume ratio of the oil phase solution to the water phase is 1: 25 to 100 parts; preferably 1: 100. Under the proportion, a large amount of nano particles with smaller particle size and uniform particle size can be ensured to be formed, the specific surface area is improved, and the drug loading capacity and the drug release effect are improved. If the content of the oil phase solution is too low, fewer nanoparticles are formed; if the content of the oil phase solution is too high, nanoparticles with too large particle size can be formed.

In the present invention, the oil phase solvent is an organic reagent that is miscible with water, and is preferably one or more of dimethylsulfoxide, N-dimethylformamide, tetrahydrofuran, and dioxane. In one embodiment of the invention, the oil phase solvent is preferably dimethyl sulfoxide, so that a large amount of hydrophobic drugs can be dissolved, and the drug loading rate of the carrier can be effectively improved.

In one embodiment, the mixed oil phase solution is dispersed into the water phase in a dropping mode, and the dropping speed is 5-10 mu L/s. This speed allows complete dispersion of the oil phase droplets in the aqueous phase.

In one embodiment, the ultrafiltration speed is 1000 to 4000rpm, preferably 3000 rpm. The nanoparticles are easy to agglomerate due to the overlarge ultrafiltration speed, the specific surface area of the drug-loaded nanoparticles is reduced, and the action effect of the drug-loaded nanoparticles is influenced; if the ultrafiltration speed is too low, organic solvent is not easy to remove, and the responsiveness of the drug-loaded nanoparticles is affected.

The nano drug delivery system can be quickly disintegrated, and the encapsulated drug can be quickly released, so that the nano drug delivery system has a good application prospect in the aspect of inhibiting the proliferation of related tumor cells.

Compared with the prior art, the invention has the following beneficial effects:

(1) the GSH/ROS double-sensitive polymer Cys-0E has good biological safety and complete biodegradability, and the synthesis process is simple. According to the invention, by optimizing an improved nano precipitation method, the double-sensitive polymer can be assembled into nano particles with smaller particle size and uniform size in a water phase under the participation of a proper amount of a stabilizer.

(2) The invention also develops the double-sensitive polymer into a novel intelligent nano-drug carrier, the drug carrier takes hydrophobic anticancer drugs as model drugs, and the nano-particles with hydrophobic inner cores and hydrophilic shells are constructed by a nano-precipitation method, and the obtained drug-loaded nano-particles have small particle size, high drug loading capacity, good in-vivo stability and capability of specifically responding to tumors; the high permeability and high retention (EPR effect) of tumor tissues are adopted to realize the large interception of the nanoparticles on the tumor, so that the nanoparticles have a passive targeting function; the effects of tumor redox microenvironment are utilized to realize the complete degradation of the carrier in the tumor tissue and the rapid and large-scale release of the entrapped drug (as shown in figure 1), realize the specific release of the drug-loaded nanoparticles in tumor cells, achieve the result of high-efficiency inhibition of tumors in vivo and in vitro, and have good application prospect and wide development space in the field of medicine.

Drawings

FIG. 1 is a schematic of the synthesis scheme of the present invention; wherein A) is a synthesis scheme of a redox double sensitive copolymer (Cys-0E); B) the polymer is assembled into DTX-loaded nanoparticles and a schematic diagram of the tumor inhibition effect in the nanoparticles.

FIG. 2 is a nuclear magnetic hydrogen spectrum of Cys-0E, a polymer synthesized according to the present invention.

FIG. 3 shows the synthesized polymer Cys-0E and raw material L-cystine dimethyl ester dihydrochloride ((H-Cys-OMe)₂2HCl) in the sample.

FIG. 4 is a graph of Dynamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) test data for Docetaxel (DTX) loaded Cys-0E @ DTX nanoparticles of the present invention.

FIG. 5 shows Docetaxel (DTX) -loaded Cys-0E @ DTX nanoparticles of the invention at different concentrations of Glutathione (GSH) and hydrogen peroxide (H) at 37 deg.C₂O₂) Graph of DTX release results under action.

FIG. 6 is a graph of cytotoxicity results of Cys-0E @ DTX nanoparticles prepared in accordance with the present invention, Cys-0E carrier material and free DTX at various concentrations on mouse colon cancer cells (CT-26cells) after 48 h.

FIG. 7 is a graph of the results of antitumor experiments with different concentrations of Cys-0E @ DTX nanoparticles, Cys-0E carrier material, and free DTX prepared according to the present invention in tumor-bearing mice (xenografted mouse colon carcinoma).

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

EXAMPLE 1 Synthesis of fully degradable GSH/ROS bis-sensitive Polysulfamide Polymer Cys-0E

The synthesis of the GSH/ROS double sensitive polymer Cys-0E comprises the following steps:

(1) l-cystine dimethyl ester dihydrochloride ((L-Cys-OMe)₂2HCl) desalting acid treatment: placing L-cystine dimethyl ester dihydrochloride (5g, 14.66mmol) into a conical flask, adding 20mL of saturated sodium carbonate solution to dissolve, and stirring at room temperature for 20 min; after the reaction was completed, the above solution was extracted with dichloromethane and repeated 3 times; drying the extract by using a certain amount of anhydrous sodium sulfate for 3 hours, and finally performing rotary evaporation to obtain a yellow oily dehydrohydrochloric acid product with the yield of 54.96%;

(2) synthesis of Cys-0E: putting the desalted acid product (5.33g, 19.88mmol) into a 100mL round-bottom flask, adding a certain amount of chloroform to dissolve, and slowly dropping a solution of oxalyl chloride (2mL, 23.86mmol) dissolved in chloroform in an ice bath under the protection of nitrogen, wherein the molar ratio of the cystine ester dehydrohydrochloric acid product to the oxalyl chloride is controlled to be 1: 1.2; after the solution is dropwise added, stirring the reaction solution at room temperature for 4 hours; after the reaction is finished, carrying out rotary evaporation on the solution to remove chloroform; the resulting product was recrystallized 3 times and finally lyophilized to give Cys-0E 3.13g (58.66% yield) as a white powder polymer.

EXAMPLE 2 Synthesis of fully degradable GSH/ROS bis-sensitive Polysulfamide Polymer Cys-0E

1. The synthesis of the GSH/ROS double sensitive polymer Cys-0E comprises the following steps:

(1) l-cystine dimethyl ester dihydrochloride ((L-Cys-OMe)₂2HCl) desalting acid treatment: placing L-cystine dimethyl ester dihydrochloride (5g, 14.66mmol) into a conical flask, adding 30mL of saturated sodium carbonate solution to dissolve, and stirring at room temperature for 30 min; after the reaction was completed, the above solution was extracted with dichloromethane and repeated 3 times; drying the extract by using a certain amount of anhydrous sodium sulfate for 5 hours, and finally performing rotary evaporation to obtain a yellow oily dehydrohydrochloric acid product with the yield of 56.86%;

(2) synthesis of Cys-0E: putting the dehydrochlorination product (5.33g, 19.88mmol) into a 100mL round-bottom flask, adding a certain amount of chloroform to dissolve, and slowly dropping the solution into a solution of oxalyl chloride dissolved in chloroform in an ice bath under the protection of nitrogen, wherein the molar ratio of the cystine ester dehydrochlorination product to the oxalyl chloride is controlled to be 1: 10; after the solution is dropwise added, stirring the reaction solution at room temperature for 6 hours; after the reaction is finished, carrying out rotary evaporation on the solution to remove chloroform; the resulting product was recrystallized 3 times and finally freeze-dried to give polymer Cys-0E as a white powder (57.84% yield).

EXAMPLE 3 Synthesis of fully degradable GSH/ROS bis-sensitive Polysulfamide Polymer Cys-0E

(1) and (3) hydrochloric acid removal treatment of L-cystine ethyl ester dihydrochloride: l-cystine ethyl ester dihydrochloride (14.66mmol) was placed in a conical flask, dissolved by adding 30mL of saturated sodium bicarbonate solution and stirred at room temperature for 30 min; after the reaction was completed, the above solution was extracted with dichloromethane and repeated 3 times; drying the extract by using a certain amount of anhydrous sodium sulfate for 4 hours, and finally performing rotary evaporation to obtain a yellow oily dehydrohydrochloric acid product with the yield of 53.69 percent;

(2) synthesis of Cys-0E: putting the dehydrochlorination product (5.33g, 19.88mmol) into a 100mL round-bottom flask, adding a certain amount of dichloromethane for dissolution, and slowly dropping the solution into a dichloromethane-dissolved oxalyl chloride solution in an ice bath under the protection of nitrogen, wherein the molar ratio of the cystine ester dehydrochlorination product to the oxalyl chloride is controlled to be 10: 1; after the solution is dropwise added, stirring the reaction solution at room temperature for 8 hours; after the reaction is finished, carrying out rotary evaporation on the solution to remove dichloromethane; the resulting product was recrystallized 3 times and finally freeze-dried to give polymer Cys-0E as a white powder (57.63% yield).

2. Results

The chemical structure of the polymer Cys-0E prepared in example 1-3 was determined by nuclear magnetic hydrogen spectroscopy. As shown in FIG. 2, the signals at 9.22 to 9.34ppm are different from those of the raw material (L-Cys-OMe)₂A proton absorption peak of newly formed amide group of 2HCl, 4.58 to 4.77ppm represent a proton absorption peak on a methylene group, 3.66 to 3.69ppm represent a proton absorption peak of a methyl group, and 2.83 to 3.24ppm represent a proton absorption peak of a methylene group. The above analysis results show that Cys-0E was successfully synthesized.

In addition, the success of the polymer Cys-0E prepared in examples 1-3 was further confirmed by Fourier infrared spectroscopy. As shown in FIG. 3, the newly synthesized polymers Cys-0E of examples 1 to 3 appeared to be different from the starting material (L-Cys-OMe)₂Characteristic peaks of 2HCl, which are characteristic peaks of an amine group in a newly formed amide group (3287 cm)^-1) And a characteristic peak belonging to carbonyl group (1664 cm)^-1)。

EXAMPLE 4 preparation of a Redox GSH/ROS Dual responsive drug Carrier (i.e., blank nanoparticles (Cys-0E-NPs))

The preparation method of the drug carrier Cys-0E-NPs by the following nano precipitation method comprises the following steps:

(1) 5mg of carrier material Cys-0E, 5mg of stabilizer distearoylphosphatidylethanolamine-polyethylene glycol (DSPE-PEG2000), 1mg of L-ascorbyl Palmitate (PA) were dissolved in 1.1mL of DMSO to serve as an oil phase; under the condition that the stirring speed is 1000rpm, 100 mu L of the mixed solution is dropwise added into 9mL of deionized water at the speed of 10 mu L/s, and 1mL of 10 xPBS is added to obtain a stable blank nanoparticle solution;

(2) and (3) carrying out ultrafiltration concentration on the nanoparticle solution by using a Millipore ultrafiltration centrifugal tube (15mL/10000D) and fixing the volume to 1mL to obtain a blank nanoparticle concentrated solution, and freeze-drying to obtain the redox GSH/ROS dual-response drug carrier Cys-0E-NPs.

Example 5 preparation and characterization of drug-loaded nanoparticles (Cys-0E @ DTX NPs) with double-response of redox GSH/ROS loaded with DTX

1. The drug-loaded nanoparticles Cys-0E @ DTX NPs are prepared by a nano precipitation method, and the method comprises the following specific steps:

(1) 5mg of the polymer Cys-0E from example 1, 5mg of the stabilizer DSPE-PEG2000, 1mg of L-ascorbyl Palmitate (PA) and 2mg of Docetaxel (DTX) were dissolved in 1.2mL of DMSO as an oil phase; under the condition that the stirring speed is 3000rpm, dropwise adding 100 mu L of the mixed solution into 9mL of deionized water at the speed of 10 mu L/s, and adding 1mL of 10 xPBS to obtain a stable drug-loaded nanoparticle solution;

(2) and (3) carrying out ultrafiltration concentration on the drug-loaded nanoparticle solution by using a Millipore ultrafiltration centrifugal tube (15mL/10000D) and fixing the volume to 1mL to obtain a drug-loaded nanoparticle concentrated solution, and freeze-drying to obtain the drug-loaded nanoparticle Cys-0E @ DTX NPs.

2. The performance of the prepared drug-loaded nanoparticles Cys-0E @ DTX NPs is characterized, and the test result is as follows:

the performance characterization of the drug-loaded nanoparticles Cys-0E @ DTX NPs comprises that the particle size and the particle size distribution are measured by a dynamic light scattering particle size analyzer, and the DLS result is shown as a picture in figure 4, so that the drug-loaded nanoparticles with the diameter of 140nm and uniform particle size distribution are obtained; the apparent morphology of Cys-0E @ DTX NPs is observed by a transmission electron microscope, a graph b in FIG. 4 shows a TEM result of the drug-loaded nanoparticles Cys-0E @ DTX NPs, and the drug-loaded nanoparticles Cys-0E @ DTX NPs are observed to be spherical and have the granularity consistent with a DLS result.

3. Evaluation of Redox responsiveness of Redox Biresponse Cys-0E @ DTX NPs

(1) Method of producing a composite material

In the invention, the in-vitro release of the anticancer drug in the drug-loaded nanoparticles is analyzed by using a dialysis method. The drug-loaded nanoparticles prepared in the invention are respectively placed in dialyzates of glutathione (0, 1 mu M, 10 mu M, 1mM and 10mM) with different concentrations and hydrogen peroxide (0, 1 mu M, 10 mu M, 1mM and 10mM) with different concentrations, and are placed on a shaking table to be incubated for a period of time at 37 ℃, and samples are taken at preset time points, and HPLC (high performance liquid chromatography) detects the amount of DTX (discontinuous transmission) encapsulated by the drug-loaded nanoparticles in the corresponding dialyzates to calculate the cumulative release amount.

(2) Results

The graphs a and b in FIG. 5 are the above experimental results, and the experimental data was analyzed to find that GSH or H was present at low concentration₂O₂At the horizontal level, the drug-loaded nanoparticles show good stability along with GSH or H₂O₂The concentration is improved, and the drug release rate and the cumulative release amount of the drug-loaded nanoparticles are obviously changed, so that the system is proved to have obvious oxidation reduction GSH/ROS dual sensitivity and excellent sustained-release and controlled-release effects.

4. In vitro cytotoxicity test of redox dual-response Cys-0E @ DTX NPs

(1) Method of producing a composite material

In-vitro cytotoxicity experiments of the carrier material polymer Cys-0E and the drug-loaded nanoparticles Cys-0E @ DTX NPs prepared by the method are researched on mouse colon cancer (CT26) cells by a tetrazolium salt (MTT) colorimetric method.

The method comprises the following steps: CT26 cells suspended in the medium were seeded at a density of 5000 cells/well in a 96-well plate and incubated in a carbon dioxide incubator for 12h to attach. Subsequently, the original medium was replaced with 200. mu.L of fresh medium containing different concentrations of Cys-0E, free DTX and Cys-0E @ DTX NPs, 5 parallel wells were set at each concentration, and co-incubation was continued for 48 h. After incubation, 20. mu.L of MTT solution (5mg/mL in PBS) was added to each well and the cells were incubated at 37 ℃ for an additional 4 h. Finally, 200. mu.L DMSO was used instead of the medium to dissolve the bluish purple crystals. The 96-well plate was shaken for 15min and the absorbance value of each well was measured with a microplate reader at 490 nm.

(2) Results

FIG. 6 is a graph showing the results of the experiment after the preparations of each group act on CT26 tumor cells for 48 hours. FIG. 6 shows that the carrier material polymer Cys-0E has no obvious toxic effect on CT26 tumor cells, and the survival rate of cells in each concentration group reaches > 90%, which indicates that the polymer Cys-0E has good biological safety and biocompatibility; meanwhile, the free DTX group and the drug-loaded nanoparticle Cys-0E @ DTX NPs group in FIG. 6 show concentration-dependent cytotoxicity; compared with a free DTX group, the drug-loaded nanoparticles Cys-0E @ DTXNPs have stronger tumor cell inhibition effect.

5. In vivo antitumor activity experiment of redox dual-response Cys-0E @ DTX NPs

(1) Method of producing a composite material

The in-vivo tumor inhibition effect of the drug-loaded nanoparticles Cys-0E @ DTX NPs is verified by using xenograft tumor mice, specifically, CT26 cells are selected to be inoculated on the right lower limb thigh of a male BALB/c mouse, and each mouse is inoculated with 10 percent of the drug-loaded nanoparticles⁵～10⁶Each cell, until the tumor volume reaches 50-100 cm³The administration is carried out at the same time. Tumor-bearing mice were randomly assigned, 5 mice per group, and the administration and grouping were as follows:

① PBS, &lTtTtransformation = &gTt & &lTt/T &gTt carrier material Cys-0E (5mg/kg), ③ free DTX (5mg/kg), ④ Cys-0E @ DTX NPs (5mg/kg), ⑤ Cys-0E @ DTX NPs (10 mg/kg).

Administering 1 time every 1 day for 5 times, observing mouse state every day, measuring tumor volume until the tumor volume reaches 2500cm³The experiment was terminated.

(2) Results

The change in tumor volume of each group of tumor-bearing mice in the tumor inhibition experiment is shown in FIG. 7. The carrier material polymer Cys-0E does not show any tumor inhibition effect, and compared with a control group, each group of DTX preparation group shows obvious tumor inhibition effect, wherein the tumor inhibition effect of the drug-loaded nanoparticles Cys-0E @ DTX NPs is optimal and the effect is more obvious along with the increase of the dosage.

Example 6 preparation of drug-loaded nanoparticles with dual responses of oxidation-reduction GSH/ROS

The other conditions of the preparation process were the same as in example 5, except that: the load medicine is adriamycin hydrochloride (hydrophilic medicine); the stabilizer is phospholipid molecules, and the vitamin C derivative is ascorbic acid phosphate; the molar ratio of the polymer Cys-0E to the antitumor drug is 5: 1. the method comprises the following specific steps:

(1) 5mg of the polymer Cys-0E from example 1, 5mg of the stabilizer phospholipid molecule, 1mg of ascorbic acid phosphate ester and 1mg of doxorubicin hydrochloride were dissolved in 1.2mL of DMSO as an oil phase; under the condition that the stirring speed is 4000rpm, dropwise adding 100 mu L of the mixed solution into 9mL of deionized water at the speed of 10 mu L/s, and adding 1mL of 10 xPBS to obtain a stable drug-loaded nanoparticle solution;

(2) and (3) carrying out ultrafiltration concentration on the drug-loaded nanoparticle solution by using a Millipore ultrafiltration centrifugal tube (15mL/10000D) and fixing the volume to 1mL to obtain a drug-loaded nanoparticle concentrated solution, and freeze-drying to obtain the drug-loaded nanoparticles capable of loading hydrophilic drugs.

Example 7 preparation of drug-loaded nanoparticles with dual responses of redox GSH/ROS

The method comprises the following specific steps:

(1) 5mg of the polymer Cys-0E from example 1, 5mg of the stabilizer polyvinyl alcohol, 1mg of L-ascorbyl palmitate and 3mg of camptothecin were dissolved in 1.2mL of N, N-dimethylformamide as an oily phase; under the condition that the stirring speed is 1000rpm, 100 mu L of the mixed solution is dropwise added into 2mL of deionized water at the speed of 5 mu L/s, and 0.5mL of 10 xPBS is added to obtain a stable drug-loaded nanoparticle solution;

(2) and (3) carrying out ultrafiltration concentration on the drug-loaded nanoparticle solution by using a Millipore ultrafiltration centrifugal tube (15mL/10000D), and fixing the volume to 1mL to obtain a drug-loaded nanoparticle concentrated solution, and freeze-drying to obtain the drug-loaded nanoparticles.

Example 8 other preparation methods of drug-loaded nanoparticles with dual responses of oxidation-reduction GSH/ROS

1. The preparation method of the drug-loaded nanoparticles by a single emulsion method, which are used for loading hydrophobic drugs, comprises the following steps:

(1) dissolving polymer Cys-0E and hydrophobic drug in dichloromethane together, and emulsifying in ice-water mixture according to certain instrument parameters to form an oil phase;

(2) adding a certain amount of polyvinyl alcohol (PVA) aqueous solution (with the concentration of 0.5%) into the oil phase, and emulsifying in an ice-water mixture according to certain instrument parameters;

(3) adding a small amount of isopropanol solution, stirring at room temperature overnight to volatilize dichloromethane and solidify the surface of the nanoparticles;

(4) adding deionized water, washing for many times, centrifuging, collecting, and freeze-drying to obtain stable and uniform redox double-responsive drug-loaded nanoparticles.

2. Redox double-responsive drug-loaded nanoparticles can also be prepared by a double emulsion method and used for loading hydrophilic/hydrophobic drugs, and the preparation steps of the hydrophilic/hydrophobic drug-loaded nanoparticles are consistent; the hydrophobic drug-loaded nanoparticles are prepared by dissolving a hydrophobic drug in an oil phase, and the hydrophilic drug-loaded nanoparticles are prepared by dissolving a hydrophilic drug in a water phase. The preparation method of the hydrophilic drug-loaded nanoparticle comprises the following specific steps:

(1) dissolving a polymer Cys-0E in dichloromethane, and emulsifying in an ice-water mixture according to certain instrument parameters to form an oil phase;

(2) adding water phase dissolved with water soluble medicine into the oil phase, emulsifying in ice water mixture according to certain instrument parameters to form water-in-oil type emulsion

(3) Adding a certain amount of polyvinyl alcohol (PVA) aqueous solution (with the concentration of 0.5%) into the oil phase, and emulsifying at low temperature according to certain instrument parameters to form water-in-oil-in-water emulsion;

(4) adding a small amount of isopropanol solution, stirring at room temperature overnight to volatilize dichloromethane and solidify the surface of the nanoparticles;

(5) adding deionized water, washing for many times, centrifuging, collecting, and lyophilizing to obtain stable and uniform drug-loaded nanoparticles with redox dual responses.

The type of the drug loaded in the above embodiments of the present invention is not particularly limited, and the drug may be a hydrophobic or hydrophilic anti-tumor drug, anti-inflammatory drug, cardiovascular disease drug, or immune adjuvant. The loaded antitumor drug can be chemotherapeutic drugs, nucleic acid drugs, protein polypeptide drugs and other drugs. The anti-inflammatory drug can be dexamethasone, rofecoxib, celecoxib and other steroidal or non-steroidal anti-inflammatory drugs. The loaded cardiovascular disease medicines can be aspirin, dipyridamole and the like. The loaded hydrophilic antitumor drug can be doxorubicin hydrochloride, gemcitabine hydrochloride, irinotecan hydrochloride, fluorouracil or lentinan and the like. The loaded hydrophobic antitumor drug can be Docetaxel (DTX), Paclitaxel (PTX), methotrexate, camptothecin, adriamycin, etc.

The above detailed description is of the preferred embodiment for the convenience of understanding the present invention, but the present invention is not limited to the above embodiment, that is, it is not intended that the present invention necessarily depends on the above embodiment for implementation. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.

Claims

1. A fully degradable GSH/ROS dual sensitive polydithioamide polymer Cys-0E, wherein said polymer Cys-0E has the formula (I):

wherein n is 5 to 1000.

2. A preparation method of a GSH/ROS double-sensitive poly (bisamide) polymer Cys-0E capable of being completely degraded is characterized in that cystine ester is dissolved by an organic solvent and then reacts with an oxalyl chloride solution at a temperature of-20-100 ℃ for 0.5-48 h under the protection of inert gas, so that the double-sensitive poly (bisamide) polymer Cys-0E can be obtained.

3. The preparation method according to claim 2, wherein the molar ratio of the cystine ester to the oxalyl chloride is 1-10: 1 to 10.

4. The process according to claim 2, wherein the cystine ester is cystine ester hydrochloride; before dissolving cystine ester hydrochloride, the cystine ester hydrochloride is subjected to hydrochloric acid removal, extraction and drying treatment.

5. The preparation method according to claim 4, wherein the cystine ester hydrochloride is selected from one or more of dimethyl L-cystine dihydrochloride, ethyl L-cystine dihydrochloride, benzyl L-cystine dihydrochloride or bis (tert-butyl) L-cystine dihydrochloride.

6. Use of the polymer Cys-0E according to claim 1 or the polymer Cys-0E obtained by the method according to any one of claims 2 to 5 as or in the preparation of a drug delivery vehicle.

7. A fully degradable GSH/ROS double sensitive nano drug delivery system, which comprises the polymer of claim 1 or the polymer prepared by the method of any one of claims 2 to 4, and a drug carried by the polymer.

8. The nano-delivery system of claim 7, which is prepared by the following method: and (3) co-dissolving the polymer Cys-0E, the stabilizer, the vitamin C derivative and the carried medicine in an oil phase solvent according to a ratio, dispersing the mixed oil phase solution into a water phase under a stirring state, and performing ultrafiltration to obtain the compound vitamin C-containing water-soluble chitosan/chitosan composite material.

9. The nano drug delivery system according to claim 7 or 8, wherein the mass ratio of the polymer Cys-0E to the loaded drug is 5: 1-3; the loaded drug comprises a hydrophobic drug or a hydrophilic drug; the loaded medicine is preferably an anti-tumor medicine.

10. The nano drug delivery system according to claim 8, wherein the mass ratio of the stabilizer to the vitamin C derivative is 1-5: 1.