CN113521030B - ROS (reactive oxygen species) -sensitive polyethylene glycol-polyester copolymer nano drug delivery system and application thereof - Google Patents

Abstract

The invention provides a ROS-sensitive polyethylene glycol-polyester nano drug delivery system, characterized in that it is sensitive to ROS, and the nano drug delivery system contains polyethylene glycol hydrophilic segment A and polyester hydrophobic segment B , and A-B, B-A-B copolymers are composed of ROS-sensitive bonds linking hydrophilic and hydrophobic segments. The ROS-sensitive material provided by the present invention has at least one of the following advantages: the polymer material provided by the present invention can be used to prepare nano drug-loaded materials, carry out drug encapsulation through polymer self-assembly, can stabilize drug molecules, and release sensitively in ROS response, and Realize oral administration, etc.

Description

ROS sensitive polyethylene glycol-polyester copolymer nano drug delivery system and its application

technical field

The invention relates to the field of biomedicine, in particular to the protection of ionizing radiation damage, in particular to an amphiphilic polymer material, a nanometer drug-carrying system and applications thereof.

Background technique

Radiation damage is tissue damage caused by ionizing radiation (IR), which usually occurs in nuclear leak accidents, radioactive source loss accidents, and radiotherapy for tumor patients. Instantaneous exposure to high-dose rays or prolonged exposure to low-dose rays can cause tissue damage. Ionizing radiation can generate reactive oxygen species (ROS) free radicals in tissues and cells, interfere with DNA, protein and other macromolecules, induce cell damage and abnormal cell function, and eventually lead to functional disorders and pathological changes in various organs of the body Even cause the body to mutate or die.

So far, most medical efforts aimed at mitigating radiation damage have remained experimental. The types of radioprotectants mainly include aminothiols, phenols, polysaccharides, hormones, cytokines, vitamins, and natural products. Amifostine is the only chemical drug with radiation protection effect approved by the FDA, but its effective time is short, and it cannot be taken orally and can only be used by injection. When taken orally, the effect is poor (N.P. Praetorius, T.K. Mandal, J. Pharm. Pharmacol. 60 (2008) 809-815). Oral administration can improve patient compliance. Therefore, finding a radioprotectant that can be taken orally, has an ideal effect and has no obvious side effects on the human body has always been an important issue in the fields of radiation biology and medicine.

Contents of the invention

In view of this, the present invention provides a ROS-sensitive polymer material, the polymer material forms nanoparticles through self-assembly, the preparation process is simple, and the drug can be effectively stabilized and released in time when it encounters radiation damage.

The present invention is realized through the following technical scheme, the ROS sensitive polyethylene glycol-polyester copolymer nano drug delivery system is characterized in that the polymer is composed of a hydrophilic polyethylene glycol segment with a number average molecular weight of 600 to 10000 A, the polyester with a hydrophobic segment with a number average molecular weight of 250 to 20000 is connected to block A and The end groups of B are connected to form an A-B or B-A-B copolymer. The hydrophilic-hydrophobic ratio is (0.5-5):1.

The above-mentioned polyethylene glycol is selected from dihydroxy polyethylene glycol, monohydroxy polyethylene glycol, polyoxyethylene and polypropylene block copolymers and polymer diols and unit alcohols containing polyethylene glycol blocks. A sort of.

The polyesters mentioned above are polycaprolactone, polylactic acid or polylactide, polyglycolic acid or glycolide, polyhydroxybutyric acid or copolymers of mixtures of the abovementioned polymer units.

The present invention provides a nano drug-carrying system residing in the above-mentioned ROS-sensitive material. The preparation method includes: preparing the nano-particle drug-carrying system by double emulsification (W/O/W) and solution volatilization method with the above-mentioned polymer material and a small molecule radiation protection drug.

In a specific embodiment of the present invention, the small molecule radioprotectant is selected from amifostine, WR-1065 or other aminomercapto compounds.

In a specific embodiment of the present invention, the preparation method of the pharmaceutical composition includes adding the small molecule radioprotectant and the nano drug-loading material into an organic solvent for encapsulation.

In one embodiment of the present invention, the organic solvent is a suitable solvent commonly used in organic reactions, for example, including but not limited to aliphatic and aromatic, optionally hydrocarbon or halogenated hydrocarbon (such as pentane, hexane , heptane, cyclohexane, petroleum ether, gasoline, volatile oil, benzene, toluene, xylene, methylene chloride, dichloroethane, chloroform, carbon tetrachloride, chlorobenzene and o-dichlorobenzene), aliphatic and Aromatic, optional alcohols (e.g. methanol, ethanol, propanol, isopropanol, tert-butanol, ethylene glycol, etc.), ethers (e.g. diethyl ether and dibutyl ether, ethylene glycol dimethyl ether and diethylene glycol dimethyl ether alcohol dimethyl ether, tetrahydrofuran and dioxane, etc.), esters (such as methyl acetate or ethyl acetate, etc.), nitriles (such as acetonitrile or propionitrile, etc.), ketones (such as acetone, butanone, etc.), amides (such as di methylformamide, dimethylacetamide and N-methylpyrrolidone, etc.), and dimethyl sulfoxide, tetramethylene sulfone and hexamethylphosphoric triamide and N,N-dimethylpropylene urea (DMPU) etc.

In a specific embodiment of the present invention, the preparation method of the nanoparticle drug-carrying system specifically includes adding ROS-sensitive materials into dichloromethane, adding a small molecule radioprotectant into water, ultrasonic emulsification, and spin-drying the solution, dry.

In a specific embodiment of the present invention, the nano-drug delivery system further includes one or more pharmaceutically acceptable vehicles, adjuvants, auxiliary agents or diluents.

In a specific embodiment of the present invention, the dosage forms of the nanoparticle drug-carrying system include but are not limited to injections, emulsions, microemulsions, submicroemulsions, nanoparticles, tablets, capsules, pills, inhalants, troches, Gel, powder, suppository, suspoemulsion, cream, jelly, or spray.

In a specific embodiment of the present invention, the administration methods that the nano drug delivery system can take include but are not limited to: subcutaneous injection, intramuscular injection, intravenous injection, oral administration, rectal administration, vaginal administration, nasal cavity administration, Transdermal, subconjunctival, intraocular, orbital, retrobulbar, retinal, choroidal, or intrathecal.

Another aspect of the present invention provides the use of the above-mentioned ROS-sensitive polymer material, or the above-mentioned nano drug-carrying system in the preparation of drugs for treating and/or preventing radiation damage and/or chemotherapy damage.

In a specific embodiment of the present invention, the radiation damage includes damage caused by ionizing radiation, non-ionizing radiation or multiple types of radiation; wherein ionizing radiation includes but not limited to alpha rays, beta rays, gamma rays, X rays, Proton or neutron radiation.

In a specific embodiment of the present invention, the radiation damage includes, but is not limited to, the reduction of leukocytes and/or platelets and/or red blood cells in the peripheral blood of mammals caused by radiation.

In a specific embodiment of the invention, said drug is administered alone or in combination with known radioprotectants.

The ROS-sensitive polymer material provided by the present invention has at least one of the following advantages: the polymer material provided by the present invention can be used to prepare a nano drug-carrying system, which can stabilize drug molecules and release them at a time when they encounter radiation damage through self-assembly and encapsulation of drugs , and can be administered orally.

Description of drawings

Fig. 1 shows the nuclear magnetic spectrum of the ROS-sensitive polymer provided by the embodiment of the present invention. It proved that the polymer was prepared successfully.

Fig. 2 shows the particle size distribution diagram (A) and TEM diagram (B) of the nanoparticles of the nano-drug loading system provided by the embodiment of the present invention.

Figure 3 shows the NMR image of the polymer under the action of H ₂ O ₂ in the embodiment of the present invention, which proves that the polymer is sensitive to ROS degradation.

Figure 4 shows the stability results of WR-1065-loaded nano-drug delivery system and free WR-1065 in simulated gastric juice (pH1.2) provided by the embodiment of the present invention. It can be seen that the nano-system can protect the drug from being destroyed by gastric juice.

Fig. 5 is a diagram showing the experimental results of the survival rate of mice within 30 days after whole body irradiation with ¹³⁷ Cs provided by the embodiment of the present invention.

Fig. 6 is a graph showing the results of radiation protection of major organs provided by the embodiment of the present invention loaded with WR-1065 nanoparticles.

Detailed ways

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Specifically, as used herein, "comprising" is synonymous with "including", "containing" or "characterized by", and is inclusive or open-ended and does not exclude additional unstated elements or means step. The term "comprising" in any expression herein, especially when describing the method, use or product of the present invention, should be understood as including consisting essentially of said components or elements or steps and consisting of said components or elements or Those products, methods and uses that consist of steps. The invention exemplarily described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

The terms and expressions which have been employed herein are used as terms of description rather than limitation, and in the use of such terms and expressions it is not intended to exclude any equivalents of the features shown and described, or parts thereof, and various modifications are to be recognized. It is possible within the scope of the claimed invention. Accordingly, it should be understood that although the invention has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be employed by those skilled in the art and that such modifications and variations are considered to be defined within the scope of the invention.

"Radiation damage" as used herein refers to damage caused by various rays in the electromagnetic spectrum, such as damage caused by microwave, infrared, visible light, ultraviolet, X-ray, beta-ray, gamma-ray, neutron or proton beam irradiation.

In order to illustrate the present invention more clearly, the following examples are now described in detail, but these examples are only exemplary descriptions of the present invention and should not be construed as limitations on the present application.

Example 1 ROS sensitive polymer preparation

Add thioketal TK (1.0 g) into the reaction flask, and add 3 mL of acetic anhydride to dissolve it, and react at room temperature for 2 hours under nitrogen protection. Then 20 mL of toluene was added to the system, and dried under reduced pressure for 3 times. Add 10 mL of dichloromethane into the reaction bottle, and add polyethylene glycol (3.0 g) with a relative molecular weight of 2000, and react overnight. The product solution was washed 3 times with water, the oil phase was concentrated, and the product was dried. TK-PEG-TK is obtained. In a dry reaction flask, add TK-PEG-TK (1.4g), EDCI (0.55g) and HoBt (0.38g) dissolved in 20mL of dichloromethane, and react for half an hour. Then 1.4 g of polycaprolactone (PCL2000) were added. React at room temperature for 24 hours. The reaction solution was concentrated, dialyzed in distilled water for 24 hours, and the product was freeze-dried for 24 hours to obtain the target product PCL-TK-PEG-TK-PCL. (referred to as I-1). The NMR spectra of TK, TK-PEG-TK and I-1 are shown in Fig. 1 .

According to the method of Example 1, by changing the type and composition of polyethylene glycol and polyester, a variety of B-A-B and A-B TK-linked ROS-sensitive polymers can be obtained. The specific parameters are shown in Table 1.

Table 1B-A-B type and A-B type (TK bond) ROS sensitive polyethylene glycol-polyester copolymer

M _PEG : number average relative molecular weight of PEG/mPEG; M _PE : number average relative molecular weight of polyester; W _AB : mass ratio of hydrophobic segment/hydrophilic segment. PCLA: Copolymer of caprolactone and lactide; PLGA: Copolymer of lactide and glycolide.

Example 2 (II-1) Preparation of disulfide bond-linked ROS sensitive compounds

In a dry reaction flask, add cystine (1.0 g), EDCI (0.55 g) and HoBt (0.38 g) dissolved in 20 mL of dichloromethane, and react for half an hour. Add polyethylene glycol (1.0 g) with a relative molecular weight of 2000 into the reaction flask, and add 3 mL of acetic anhydride to dissolve it, and react at room temperature for 2 hours under nitrogen protection. Then 20 mL of toluene was added to the system, and dried under reduced pressure for 3 times. Add 10 mL of dichloromethane into the reaction flask, and add methyl-terminated polyethylene glycol (3.0 g) with a relative molecular weight of 2000, and react overnight. The product solution was washed 3 times with water, the oil phase was concentrated, and the product was dried. Obtain mPEG-TK. In a dry reaction vial, add mPEG-TK (1.4g),

EDCI (0.55g) and HoBt (0.38g) were dissolved in 20mL of dichloromethane and reacted for half an hour. Then 1.4 g of polycaprolactone (PCL2000) were added. React at room temperature for 24 hours. The reaction solution was concentrated, dialyzed in distilled water for 24 hours, and the product was freeze-dried for 24 hours to obtain the target product mPEG-TK-PCL. (referred to as II-1).

According to the method in Example 2, by changing the type and composition of polyethylene glycol and polyester, a variety of B-A-B and A-B disulfide-linked ROS-sensitive polymers can be obtained. The specific parameters are shown in Table 2.

The ROS sensitive polyethylene glycol-polyester copolymer of table 2B-A-B type and A-B type (disulfide bond)

Copolymer A/M_PEG Polyester B/M_PE W_AB II-1 2000 PCL/2000 1.00 II-2 2000 PCL/1000 0.50 II-3 600 PCL/2500 4.16 II-4 1500 PCL/1000 0.67 II-5 4000 PCL/4000 1.00 II-6 1500 PCLA/1500 1.00 II-7 1000 PCLA/2500 2.50 II-8 5000 PCLA/20000 4.00 II-9 2000 PLA/3000 1.50 II-10 1000 PLA/1000 1.00 II-11 2000 PLAG/2000 1.00 II-12 4000 PLAG/5000 1.25 II-13 250 PLAG/400 1.60

M _mPEG : number average relative molecular weight of PEG; M _P E : number average relative molecular weight of polyester; W _AB : mass ratio of hydrophobic segment/hydrophilic segment. PCLA: Copolymer of caprolactone and lactide; PLGA: Copolymer of lactide and glycolide.

The preparation of embodiment 3 drug-loaded nanoparticles

The polymers prepared in Examples 1-2 above can be combined with various bioactive molecules to form drug-loaded nanoparticles for radiation protection. In the following, only amifostine (Am) and its metabolite WR-1065 are used as examples for illustration.

The ROS-sensitive polymer self-assembly was prepared by double emulsion (W/O/W) method.

Drug-loaded nanoparticles 1: Preparation of I-1/WR-1065 nanoparticles

Dissolve 50mg of WR-1065 in 200mL of distilled water to form an inner water phase (W1), and dissolve 100mg of I-1 polymer in 2mL of dichloromethane to form an oil phase (O). Add W1 to O and ultrasonicate for 3 minutes in an ice-water bath , forming the first emulsion (W1/O). Add colostrum into 10 mL of distilled water (W2), and sonicate for 3 minutes in an ice-water bath to form a W1/O/W2 double emulsion. The dichloromethane was removed by rotary evaporation, and the drug-loaded nanoparticle solution was freeze-dried for 24 hours. The particle size distribution and morphology of the prepared nanoparticles were detected by dynamic light scattering and TEM, respectively, and the results are shown in Figure 2. The drug loading is shown in Table 3.

Table 3 Drug-loaded nanoparticles and drug loading

According to the method in Example 3, various drug-loaded nanoparticles can be prepared by changing the types and ratios of polymers and drugs.

The degradation research of embodiment 4ROS sensitive polymer

The blank ROS-sensitive polymer freeze-dried powder was placed in different concentrations of hydrogen peroxide, and the degradation behavior of the polymer was investigated by NMR. The test results are shown in Figure 3. It shows that the synthesized polymer has ROS sensitive degradation behavior.

Example 5 Research on the protective effect of drug-loaded nanoparticles on drugs

Using WR-1065 as a control, compare the stability of loaded WR-1065 nanoparticles in the simulated gastric juice environment. The specific process is as follows: 10 mg of WR-1065 and nanoparticles containing 10 mg of WR-1065 were weighed and dissolved in 10 mL of simulated gastric juice (pH 1.2), and placed at 37°C. The solutions were taken out at different time intervals, and the changes in drug content were detected by HPLC. The results are shown in Figure 4. It shows that WR-1065 alone will be degraded in the environment of gastric juice, and the polymer carrier can protect the drug from being degraded by gastric juice.

In vivo radiation protection of embodiment 6 drug-loaded nanoparticles

An in vivo mouse radiation model was established to study the in vivo radiation protection effect of I-1/WR-1065.

The mice were irradiated by whole body irradiation with ¹³⁷ Cs, and the effects of ROS-sensitive radioprotective drugs on the survival rate and body weight of mice were observed. C57BL/6 mice were grouped according to body weight randomized block method, and the non-irradiated group consisted of two groups: the blank group (normal saline gavage) and the I-1/WR-1065 oral gavage group. The irradiation group was divided into 4 groups, namely the irradiation group (8.0Gy) (denoted as a): receiving 8.0Gy whole-body irradiation, intragastric administration of normal saline 1 hour before irradiation; intragastric administration of I-1/WR-1065; irradiation + ammonia Fostin group (amifostine irradiated intraperitoneal injection group, denoted as b): received 8.0Gy whole-body irradiation, and administered amifostine intraperitoneally 1 h before irradiation; irradiation+WR-1065 group (WR-1065 irradiated oral group, Denoted as c): Received 8.0Gy whole-body irradiation, and administered WR-1065 orally to mice 1h before irradiation; irradiation+I-1/WR-1065 group (WR-1065 nanoparticle irradiated oral group, denoted as d): accepted 8.0Gy whole body irradiation, mice were given I-1/WR-1065 orally 1h before irradiation. During the irradiation, the mice received a total body irradiation (TBI) with ¹³⁷ Cs source, the irradiation dose was 8.0Gy, and the dose rate was 0.99Gy/min. The death situation and body weight of the mice were recorded every day, and the 30-day survival rate of the mice was counted. The results are shown in FIG. 5 . As can be seen from Figure 5, the non-irradiated group, the I-1/WR-065 nanoparticle administration group mouse survival rate was 100%, indicating that the drug-loaded nanoparticle has good biocompatibility, and in the irradiation group, I -1/WR-1065 has a higher 30-day survival rate than other administration groups, indicating that it has a good radiation protection effect.

Example 7 The protective effect of drug-loaded nanoparticles on major organs

Establish an in vivo mouse radiation model to study the protective effect of I-1/WR-1065 on major organs.

Whole-body irradiation with ¹³⁷ Cs was used to establish a mouse radiation model. C57BL/6 mice were divided into random groups according to body weight. b): Received 7.2Gy whole body irradiation, gavage with normal saline 1h before irradiation; irradiation+WR1065 group (WR-1065 irradiated oral group, denoted as c): received 7.2Gy whole body irradiation, and administered WR1065 by gavage 1h before irradiation ; Irradiation+I-1/WR-1065 group (WR-1065 nanoparticles were irradiated orally, denoted as d): received 7.2Gy whole body irradiation, and the mice were administered I-1/WR-1065 orally 1h before irradiation. During irradiation, the mice received a total body irradiation (TBI) with ¹³⁷ Cs source, the irradiation dose was 7.2Gy, and the dose rate was 0.99Gy/min. After 7 days of irradiation, the main organs of the mice were removed, and the tissue sections were analyzed pathologically. The results are shown in Figure 6. It can be seen from Figure 6 that lung fibrosis and intestinal damage caused by radiation can be significantly improved.

The ROS-sensitive polymer nanocarrier provided in the present invention stabilizes the small molecule radioprotectant through self-assembly, and realizes oral administration. The ROS-sensitive nanosystem prepared by it can be used as a radioprotectant, which can prolong the survival period of animals after lethal dose irradiation and reduce the mortality rate, and can improve lung fibrosis and intestinal damage caused by radiation. The drug compound can be used alone as a radiation damage protection and rescue drug, and can also be used in combination with radiotherapy, which can alleviate and prevent adverse reactions caused by radiotherapy, and can also be used in combination with known radioprotectants, such as radiotherapy agents, to prevent and treat Both effects, thereby enhancing the prevention and/or treatment of radiation-induced damage.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention within.

Claims (4)

1. The nano drug-loaded system is characterized in that the preparation method of the nano drug-loaded system comprises the following steps: self-assembling the ROS sensitive polyethylene glycol-polyester copolymer material with a small molecular radiation protective agent by a double-emulsion method to form nanoparticles, wherein the small molecular radiation protective agent is loaded in holes of the nano drug-loaded material, and is selected from amifostine, WR-1065 and an amino sulfhydryl compound;

the ROS sensitive polyethylene glycol-polyester copolymer material is an A-B, B-A-B copolymer formed by connecting ase:Sub>A hydrophilic polyethylene glycol block A with the number average molecular weight of 600-10000 and ase:Sub>A polyester segment B with the number average molecular weight of 250-20000 through ROS sensitive bonds; wherein the mass ratio of the mass of the hydrophobic section B to the mass of the hydrophilic section A is (0.5-5): 1;

the administration mode of the nano medicine carrying system is oral administration;

the polyethylene glycol is selected from one of dihydroxy polyethylene glycol, monohydroxy polyethylene glycol, polyoxyethylene and polypropylene block copolymer, polymer dihydric alcohol containing polyethylene glycol block and monohydric alcohol;

the polyester is polycaprolactone, polylactic acid or polylactide, polyglycolic acid or glycolide, polyhydroxy butyric acid or a copolymer of the polymer unit mixture;

the ROS-sensitive bond refers to: thioketal, disulfide bond, phenylboronic acid chemical bond.

2. Use of the nanopharmaceutical system of claim 1 in the manufacture of a medicament for the treatment and/or prevention of radiation and/or chemotherapy injury.

3. The use of claim 2, wherein the radiation damage comprises damage caused by ionizing radiation, non-ionizing radiation, or multiple types of radiation together; wherein the ionizing radiation comprises alpha rays, beta rays, gamma rays, X rays, protons, or neutron radiation.

4. The use of claim 2, wherein said radiation damage comprises a decrease in peripheral blood leukocytes and/or platelets and/or erythrocytes in a mammal due to radiation; the drugs for chemotherapy-induced injury include antineoplastic drugs acting on DNA, RNA and tubulin, and the drugs are administered alone or in combination with known radioprotectants.

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