CN113521030A

CN113521030A - ROS (reactive oxygen species) -sensitive polyethylene glycol-polyester copolymer nano drug delivery system and application thereof

Info

Publication number: CN113521030A
Application number: CN202010287206.2A
Authority: CN
Inventors: 蔺晓娜; 田红旗
Original assignee: Institute of Radiation Medicine of CAMMS
Current assignee: Institute of Radiation Medicine of CAMMS
Priority date: 2020-04-13
Filing date: 2020-04-13
Publication date: 2021-10-22
Anticipated expiration: 2040-04-13
Also published as: CN113521030B

Abstract

The invention provides an ROS-sensitive polyethylene glycol-polyester nano drug delivery system which is characterized by comprising ROS sensitivity, wherein the nano drug delivery system contains a polyethylene glycol hydrophilic segment A and a polyester hydrophobic segment B, and an ROS sensitive bond is linked with the hydrophilic and hydrophobic segments to form an A-B, B-A-B copolymer. The ROS sensitive material provided by the invention has at least one of the following advantages: the high molecular material provided by the invention can be used for preparing a nano drug-loaded material, drug encapsulation is carried out through polymer self-assembly, drug molecules can be stabilized, ROS response sensitive release is realized, and oral administration is realized.

Description

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

Technical Field

The invention relates to the field of biomedicine, in particular to the aspect of ionizing radiation injury protection, and specifically relates to an amphiphilic polymer material, a nano drug-loading system and application thereof.

Background

Radiation damage is damage to body tissue caused by Ionizing Radiation (IR), which is commonly encountered in nuclear leakage accidents, radioactive source loss accidents, and in radiotherapy for tumor patients. Tissue damage can be caused by either a large dose of radiation being delivered instantaneously or a low dose of radiation being delivered for a long period of time. Ionizing radiation can generate Reactive Oxygen Species (ROS) free radicals in tissues and cells, interfere macromolecules such as DNA (deoxyribonucleic acid), protein and the like, induce cell damage and cell function abnormity, and finally cause functional disorder, pathological changes and even body mutation or death of various organs of a body.

To date, most medical actions aimed at reducing radiation damage remain in the experimental phase. The types of radioprotectants mainly include amino sulfhydryls, phenols, polysaccharides, hormones, cytokines, vitamins, natural products, and the like. Amifostine is the only chemical drug which has radioprotective effect and is passed by FDA, but the effective time is short, and the amifostine can not be taken orally and only can be used by injection. When orally taken, the effect is poor (N.P.Praetorius, T.K.Mandal, J.Pharm.Pharmacol.60(2008) 809-815). Oral administration can improve compliance of patients. Therefore, the search for radioprotectors that can be orally administered, that are efficacious and that have no significant side effects on the human body has been a major concern in the fields of radiobiology and medicine.

Disclosure of Invention

In view of the above, the invention provides a ROS-sensitive polymer material, which forms nanoparticles through self-assembly, has a simple preparation process, can effectively stabilize a drug, and can be released at a fixed time when suffering from radiation damage.

The ROS sensitive polyethylene glycol-polyester copolymer nano drug delivery system is realized by the following technical scheme and is characterized in that the polymer is formed by connecting a hydrophilic polyethylene glycol section A with the number average molecular weight of 600-10000 and a hydrophobic polyester section with the number average molecular weight of 250-20000 with the end groups of a block A and a block B through ROS sensitive bonds (thioketal (TK), disulfide bond (S-S), azobenzene, phenylboronic acid and the like) to form an A-B or B-A-B copolymer. Wherein the hydrophilic-hydrophobic ratio is (0.5-5): 1.

The polyethylene glycol is selected from one of dihydroxy polyethylene glycol, monohydroxy polyethylene glycol, polyoxyethylene and polypropylene block copolymer, polymer diol containing polyethylene glycol block, and monohydric alcohol.

The polyester is polycaprolactone, polylactic acid or polylactide, polyglycolic acid or glycolide, polyhydroxybutyric acid or a copolymer of a mixture of the above polymer units.

The invention provides a nano drug delivery system residing in the ROS sensitive material, and the preparation method comprises the following steps: the polymer material and micromolecular radiation protection medicines are prepared into a nanoparticle medicine carrying system through a double emulsification (W/O/W) method and a solution volatilization method.

In one embodiment of the invention, the small molecule radioprotectant is selected from the group consisting of amifostine, WR-1065 or other amine sulfhydryl compounds.

In one embodiment of the invention, the preparation method of the pharmaceutical composition comprises the step of adding the small-molecule radioprotectant and the nano drug-loaded 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 (e.g., pentane, hexane, heptane, cyclohexane, petroleum ether, gasoline, volatile oil, benzene, toluene, xylene, dichloromethane, dichloroethane, chloroform, carbon tetrachloride, chlorobenzene, and o-dichlorobenzene), aliphatic and aromatic, optionally alcohol (e.g., methanol, ethanol, propanol, isopropanol, t-butanol, ethylene glycol, etc.), ether (e.g., diethyl ether and dibutyl ether, ethylene glycol dimethyl ether and diethylene glycol dimethyl ether, tetrahydrofuran and dioxane, etc.), ester (e.g., methyl acetate or ethyl acetate, etc.), nitrile (e.g., acetonitrile or propionitrile, etc.), ketone (e.g., acetone, butanone, etc.), amide (e.g., dimethylformamide, dimethylacetamide, N-methylpyrrolidone, etc.), etc.), And dimethyl sulfoxide, tetramethylene sulfone and hexamethylphosphoric triamide and N, N-dimethylpropylene urea (DMPU) and the like.

In a specific embodiment of the invention, the preparation method of the nanoparticle drug delivery system specifically comprises the steps of adding the ROS sensitive material into dichloromethane, adding the small-molecule radiation protective agent into water, carrying out ultrasonic emulsification, spin-drying the solution, and drying.

In one embodiment of the present invention, the drug delivery system further comprises one or more pharmaceutically acceptable vehicles, adjuvants or diluents.

In one embodiment of the present invention, the nanoparticle drug delivery system includes, but is not limited to, injection, emulsion, microemulsion, submicron emulsion, nanoparticle, tablet, capsule, pill, inhalant, buccal tablet, gel, powder, suppository, suspension emulsion, cream, jelly or spray.

In one embodiment of the present invention, the drug delivery system can be administered by the following methods including but not limited to: subcutaneous injection, intramuscular injection, intravenous injection, oral administration, rectal administration, vaginal administration, nasal administration, transdermal administration, subconjunctival administration, intra-ocular administration, orbital administration, retrobulbar administration, retinal administration, choroidal administration or intrathecal injection.

In another aspect, the invention provides the ROS-sensitive polymer material or the drug delivery nanosystem as described above for use in the preparation of a medicament for the treatment and/or prevention of radiation damage and/or chemotherapy damage.

In one embodiment of the invention, the radiation damage comprises damage caused by ionizing radiation, non-ionizing radiation, or multiple types of radiation together; wherein ionizing radiation includes, but is not limited to, alpha rays, beta rays, gamma rays, X rays, protons, or neutron radiation.

In one embodiment of the invention, the radiation damage includes, but is not limited to, a decrease in peripheral blood leukocytes and/or platelets and/or erythrocytes in mammals due to radiation.

In one embodiment of the invention, the medicament is administered alone or in combination with known radioprotectants.

The ROS sensitive polymer material provided by the invention has at least one of the following advantages: the polymer material provided by the invention can be used for preparing a nano drug delivery system, can stabilize drug molecules and release the drug molecules at regular time when suffering from radiation damage by self-assembly coating of drugs, and can realize oral administration and the like.

Drawings

FIG. 1 shows nuclear magnetic spectra of ROS sensitive polymers provided in the examples of the present invention. The success of polymer preparation was demonstrated.

Fig. 2 shows a nanoparticle size distribution diagram (a) and a TEM diagram (B) of a nano drug delivery system provided by an embodiment of the present invention.

FIG. 3 shows an embodiment of the present invention at H₂O₂And (3) under the action of the polymer, performing a nuclear magnetic diagram to prove that the polymer ROS is sensitively degraded.

Fig. 4 shows the stability results of the WR-1065 loaded nano drug delivery system and the free WR-1065 in simulated gastric fluid (ph1.2) provided by the embodiment of the invention, and it can be seen that the nano system can protect the drug from being damaged by the gastric fluid.

FIG. 5 illustrates the use of whole body irradiation according to an embodiment of the present invention¹³⁷Experimental results plot of survival of mice within 30 days after Cs method.

Fig. 6 is a graph showing the results of the WR-1065 loaded nanoparticle radiation protection on the major organs according to the embodiment of the present invention.

Detailed Description

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.

In particular, as used herein, "comprising" is synonymous with "including," "containing," or "characterized by," and is inclusive or open-ended and does not exclude additional unrecited elements or method steps. The term "comprising" in any of the expressions herein, particularly in describing the method, use or product of the invention, is to be understood as including those products, methods and uses which consist essentially of and consist of the recited components or elements or steps. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein.

The terms and expressions which have been employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

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

For a more clear illustration of the invention, reference is now made in detail to the following examples, which are intended to be purely exemplary of the invention and are not to be interpreted as limiting the application.

Example 1 ROS-sensitive Polymer preparation

The thioketal TK (1.0g) was charged into a reaction flask, and 3mL of acetic anhydride was added to dissolve it, followed by reaction at room temperature for 2 hours under nitrogen. Then, 20mL of toluene was added to the system, and the mixture was dried under reduced pressure 3 times. 10mL of methylene chloride was added to the reaction flask, and polyethylene glycol (3.0g) having a relative molecular weight of 2000 was added thereto, and the reaction was allowed to proceed overnight. The product solution was washed 3 times with water, the oil phase was concentrated and the product was dried. Thus obtaining the TK-PEG-TK. In a dry reaction flask TK-PEG-TK (1.4g), EDCI (0.55g) and HoBt (0.38g) were added and dissolved in 20mL dichloromethane for half an hour. Then 1.4g of polycaprolactone (PCL2000) was added. The reaction was carried out at room temperature for 24 hours. And concentrating the reaction solution, dialyzing in distilled water for 24 hours, and freeze-drying the product for 24 hours to obtain a target product PCL-TK-PEG-TK-PCL. (abbreviated as I-1). The nuclear magnetic maps of TK, TK-PEG-TK and I-1 are shown in figure 1.

By changing the types and compositions of polyethylene glycol and polyester according to the method of example 1, a plurality of ROS sensitive polymers linked by TK bonds of B-A-B type and A-B type can be obtained, and the specific parameters are shown in Table 1.

TABLE 1 ROS-sensitive polyethylene glycol-polyester copolymers of type B-A-B and type A-B (TK linkage)

Copolymer	A/M_PEG	Polyester B/M_PE	W_AB
				I-1	PEG/2000	PCL/2000	2.00
I-2	mPEG/2000	PCL/2000	1.00
				I-3	PEG/600	PCL/250	0.83
I-4	mPEG/1500	PCL/2000	1.33
				I-5	PEG/4000	PCL/1000	0.50
I-6	PEG/1500	PCLA/3000	2.00
				I-7	PEG/1000	PCLA/2500	5.00
I-8	PEG/5000	PCLA/10000	4.00
				I-9	mPEG/2000	PLA/3000	1.50
I-10	mPEG/1000	PLA/2000	2.00
				I-11	PEG/2000	PLAG/2000	2.00
I-12	mPEG/4000	PLAG/5000	1.25
				I-13	PEG/1000	PLAG/400	0.80

M_PEG: number average relative molecular weight of PEG/mPEG; m_PE: number average relative molecular weight of the polyester; w_AB: mass ratio of hydrophobic segment/hydrophilic segment. PCLA: copolymers of caprolactone and lactide; PLGA: copolymers of lactide and glycolide.

Example 2(II-1) preparation of disulfide-linked ROS-sensitive Compounds

In a dry reaction flask, cystine (1.0g), EDCI (0.55g) and HoBt (0.38g) were added dissolved in 20mL of dichloromethane and reacted for half an hour. Polyethylene glycol (1.0g) having a relative molecular weight of 2000 was added to the reaction flask, and 3mL of acetic anhydride was added to dissolve it, followed by reaction at room temperature for 2 hours under nitrogen. Then, 20mL of toluene was added to the system, and the mixture was dried under reduced pressure 3 times. 10mL of methylene chloride was added to the reaction flask, and terminal methyl polyethylene glycol (3.0g) having a relative molecular weight of 2000 was added thereto, and the reaction was allowed to proceed overnight. The product solution was washed 3 times with water, the oil phase was concentrated and the product was dried. Thus obtaining mPEG-TK. In a dry reaction flask, mPEG-TK (1.4g) was added,

EDCI (0.55g) and HoBt (0.38g) were dissolved in 20mL of dichloromethane and reacted for half an hour. Then 1.4g of polycaprolactone (PCL2000) was added. The reaction was carried out at room temperature for 24 hours. And concentrating the reaction solution, dialyzing the reaction solution in distilled water for 24 hours, and freeze-drying the product for 24 hours to obtain a target product mPEG-TK-PCL. (abbreviated as II-1).

According to the method of example 2, various ROS sensitive polymers linked by disulfide bonds of B-A-B type and A-B type can be obtained by changing the types and compositions of polyethylene glycol and polyester, and the specific parameters are shown in Table 2.

TABLE 2 ROS sensitive polyethylene glycol-polyester copolymers of type B-A-B and type A-B (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_PE: number average relative molecular weight of the polyester; w_AB: mass ratio of hydrophobic segment/hydrophilic segment. PCLA: copolymers of caprolactone and lactide; PLGA: copolymers of lactide and glycolide.

Example 3 preparation of drug-loaded nanoparticles

The polymer prepared in the embodiment 1-2 can be combined with various bioactive molecules to form a drug-loaded nanoparticle for radiation protection. The following is only an exemplary description of amifostine (Am) and its metabolite WR-1065.

The ROS sensitive polymer self-assembly is prepared by a double emulsification (W/O/W) method.

Drug-loaded nanoparticles 1: preparation of I-1/WR-1065 nano-particle

50mg WR-1065 was dissolved in 200mL distilled water to form an internal aqueous phase (W1), 100mg I-1 polymer was dissolved in 2mL methylene chloride to form an oil phase (O), W1 was added to O, and sonication was performed in an ice water bath for 3 minutes to form a primary emulsion (W1/O). The colostrum was added to 10mL of distilled water (W2) and sonicated in an ice water bath for 3 minutes to form a W1/O/W2 double emulsion. And (4) removing dichloromethane by rotary evaporation to obtain a drug-loaded nanoparticle solution, and freeze-drying for 24 hours. The particle size distribution and morphology of the prepared nanoparticles are detected by dynamic light scattering and TEM respectively, and the results are shown in FIG. 2. Drug loading was as shown in table 3.

TABLE 3 drug-loaded nanoparticles and drug-loading rates

According to the method of the embodiment 3, various drug-loaded nanoparticles can be prepared by changing the types and the proportions of the polymer and the drug.

Example 4 degradation study of ROS-sensitive polymers

Blank ROS sensitive polymer freeze-dried powder is taken and respectively placed in hydrogen peroxide with different concentrations, and nuclear magnetism is utilized to investigate the degradation behavior of the polymer. The test results are shown in fig. 3. Indicating that the synthesized polymer has ROS sensitive degradation behavior.

Example 5 study of protective action of drug-loaded nanoparticles on drugs

WR-1065 was used as a control to compare the stability of the nanoparticles loaded with WR-1065 in a simulated gastric environment. The specific process is as follows: 10mg WR-1065 and nanoparticles containing 10mg WR-1065 were dissolved in 10mL simulated gastric fluid (pH1.2) and left at 37 ℃. Solutions were removed at different time intervals and changes in drug content were detected using HPLC, the results are shown in fig. 4. The WR-1065 alone is shown to degrade in the gastric environment, and the polymeric carrier protects the drug from degradation by gastric fluid.

Example 6 in vivo radioprotection of drug-loaded nanoparticles

An in-vivo mouse radiation model is established, and the in-vivo radiation protection effect of I-1/WR-1065 is researched.

By whole body irradiation¹³⁷And the Cs method is used for establishing a mouse radiation model and observing the influence of the ROS sensitive radiation protection medicament on the survival rate and the weight of the mouse. The C57BL/6 mice were grouped according to the weight randomized block method, and the non-irradiated group had 2 groups: the group was a blank group (gavage with physiological saline) and an oral gavage group I-1/WR-1065. The irradiation groups were divided into 4 groups, each irradiation group (8.0Gy) (denoted as a): receiving 8.0Gy whole body irradiation, and irrigating the stomach with physiological saline 1h before irradiation; I-1/WR-1065 administration by intragastric administration; irradiation + amifostine group (amifostine irradiated intraperitoneal injection group, noted as b): 8.0Gy systemic irradiation is carried out, and 1h before the irradiation of the mice is carried out, the administration of the amifostine is carried out by intraperitoneal injection; irradiation + WR-1065 group (WR-1065 irradiation oral group, noted as c): receiving 8.0Gy systemic irradiation, and carrying out intragastric administration for 1h WR-1065 before the irradiation of the mice; irradiation + I-1/WR-1065 group (WR-1065 nanoparticle irradiation oral group, noted as d): the mice were subjected to systemic irradiation with 8.0Gy and gavage administration of 1h I-1/WR-1065 prior to irradiation. Mice received one time of irradiation¹³⁷And (3) Cs source whole body irradiation (TBI), wherein the irradiation dose is 8.0Gy, and the dose rate is 0.99 Gy/min. Mice were daily recorded for mortality and body weight and the survival rate of the mice was counted for 30 days, which was The results are shown in FIG. 5. As can be seen from FIG. 5, the survival rate of mice in the non-irradiated group and the I-1/WR-065 nanoparticle administration group is 100%, which indicates that the drug-loaded nanoparticles have good biocompatibility, and in the irradiated group, the I-1/WR-1065 has a higher survival rate for 30 days than other administration groups, which indicates that the radiation protection effect is good.

Example 7 protective action of drug-loaded nanoparticles on major visceral organs

An in-vivo mouse radiation model is established, and the protection effect of I-1/WR-1065 on main organs is researched.

By whole body irradiation¹³⁷Cs method, establishing mouse radiation model, grouping C57BL/6 mice according to weight random block method, blank group (marked as a): normal saline is infused into the stomach without irradiation; irradiation group (7.2Gy) (noted b): receiving 7.2Gy general irradiation, and irrigating the stomach with physiological saline 1h before irradiation; irradiation + WR1065 group (WR-1065 irradiation oral group, noted c): receiving 7.2Gy systemic irradiation, and carrying out intragastric administration on the mice 1h WR1065 before irradiation; irradiation + I-1/WR-1065 group (WR-1065 nanoparticles irradiation oral, noted as d): the mice were subjected to 7.2Gy systemic irradiation and gavage before mouse irradiation at 1h I-1/WR-1065. Mice received one time of irradiation¹³⁷And (3) Cs source whole body irradiation (TBI), wherein the irradiation dose is 7.2Gy, and the dose rate is 0.99 Gy/min. After 7 days of irradiation, the major organs of the mice were harvested, and the tissue sections were analyzed for pathology, the results of which are shown in fig. 6. As can be seen from fig. 6, the radiation-induced pulmonary fibrosis and intestinal injury can be significantly improved.

The ROS sensitive polymer nano-carrier provided by the invention stabilizes the small molecular radiation protective agent through self-assembly, and realizes oral administration. The ROS sensitive nano system prepared by the method can be used as a radiation protective agent, has the effects of prolonging the life cycle of animals irradiated by lethal dose and reducing the death rate, and can improve pulmonary fibrosis and intestinal injury caused by radiation. The pharmaceutical compound can be independently used as a radiation injury protection and treatment medicine, can also be used in combination with radiotherapy, has the functions of relieving and preventing adverse reactions caused by radiotherapy, and can also be used in combination with known radiation protective agents, such as radiation therapeutic agents, so as to achieve the effect of preventing and treating radiation injury, thereby enhancing the prevention and/or treatment of radiation injury.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and the like that are within the spirit and principle of the present invention are included in the present invention.

Claims

1. The ROS sensitive polyethylene glycol-polyester copolymer material is characterized in that the copolymer is an A-B copolymer and a B-A-B copolymer which are formed by connecting a hydrophilic polyethylene glycol block A with the number average molecular weight of 600-10000 and 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.

2. The ROS-sensitive polyethylene glycol-polyester copolymer material of claim 1, wherein the polyethylene glycol is selected from the group consisting of bishydroxypolyethylene glycol, monohydroxypolyethylene glycol, block copolymers of polyoxyethylene and polypropylene, and polymeric glycols and monoalcohols containing polyethylene glycol blocks.

3. The ROS-sensitive polyethylene glycol-polyester copolymer material of claim 1, wherein said polyester is a polycaprolactone, a copolymer of polylactic acid or polylactide, polyglycolic acid or glycolide, polyhydroxybutyric acid, or a mixture of the foregoing polymer units.

4. The ROS-sensitive polyethylene glycol-polyester copolymer material of claim 1, wherein the ROS-sensitive bond refers to: ketone dithiol (TK), disulfide bond (S-S), azobenzene, phenylboronic acid, etc.

5. The ROS-sensitive polyethylene glycol-polyester copolymer material of claim 1, wherein the polyethylene glycol-polyester copolymer is an a-B, B-a-B copolymer.

6. A nano drug delivery system is characterized in that the preparation method of the nano drug delivery system comprises the following steps: mixing the polymer as described in claims 1-5 with small molecule radioprotectant by double emulsion method (O/W/O) or solution evaporation method, etc., wherein the small molecule radioprotectant is loaded in the nanometer drug loading material hole, preferably the small molecule radioprotectant is selected from the group consisting of amifostine, WR-1065, amine sulfhydryl compound.

7. Use of the polymeric material of any one of claims 1-5, or the nanopharmaceutical system of claim 6, in a medicament for the treatment and/or prevention of radiation damage and/or chemotherapy damage.

8. The use of claim 7, wherein the radiation damage comprises damage caused by ionizing radiation, non-ionizing radiation, or multiple types of radiation together; wherein ionizing radiation includes, but is not limited to, alpha rays, beta rays, gamma rays, X rays, protons, or neutron radiation.

9. The use of claim 7, wherein the radiation damage includes, but is not limited to, mammal peripheral blood leukocyte and/or platelet and/or erythrocyte depletion due to radiation; such agents for chemotherapeutic damage include, but are not limited to, antineoplastic agents acting on DNA, RNA and tubulin, and optionally, the agents are administered alone or in combination with known radioprotectants.