Active oxygen response type polymer micelle as well as preparation method and application thereof

Abstract

The invention discloses an active oxygen response type polymer prodrug micelle, and further discloses a polymer prodrug micelle loaded with oridonin or dihydroartemisinin and a polymer prodrug micelle loaded with the oridonin and the dihydroartemisinin at the same time. The invention further provides a preparation method of the polymer prodrug micelle. The ORI and DHA co-loaded active oxygen responsive polymer prodrug micelle provided by the invention can significantly improve the water solubility and biocompatibility of ORI and DHA, can effectively eliminate reduced glutathione in gastric cancer cells AGS cells and amplify active oxygen, and finally causes the generation of a large amount of lipid peroxides, thereby killing tumor cells and inducing cell ferroptosis, and has a good application prospect. And finally, tumor proliferation is inhibited.

Description

Technical Field

[0001] The present invention relates to the technical field of stimulus-responsive polymer materials, and in particular to an active oxygen-responsive polymer prodrug micelle and a preparation method and application thereof. Background Art

[0002] Gastric cancer is the fifth most commonly diagnosed cancer in the world and the third leading cause of cancer-related death. Late diagnosis is one of the main reasons for the high mortality rate of gastric cancer, resulting in limited treatment options and poor treatment effects. Currently, chemotherapy is an indispensable treatment for patients with clinical gastric cancer. However, traditional chemotherapy drugs have poor clinical treatment effects due to poor water solubility, low tumor tissue accumulation, toxic side effects and drug resistance.

[0003] Traditional Chinese medicine and Chinese medicine extracts have been widely used in anti-tumor treatment in recent years due to their low toxicity and good efficacy. For example, Oridonin (ORI) is the main component of Oridonium, a plant of the genus Oridonium of the Lamiaceae family, and is a diterpenoid of the kaurene class. Studies have shown that Oridonin has anti-tumor, antibacterial, anti-inflammatory and analgesic effects. In particular, Oridonin can inhibit the proliferation of tumor cells by consuming reduced glutathione in tumor cells, destroying the intracellular oxygen reduction balance, and inducing cell ferroptosis. In addition, dihydroartemisinin (DHA) is an active metabolite of artemisinin derivatives and is an antimalarial drug well tolerated by millions of patients. In recent years, studies have shown that it can inhibit tumor cell proliferation by inducing the expansion of reactive oxygen species in cancer cells. DHA contains an endoperoxide bridge, which reacts with ferrous catalysts to produce free radicals and cause oxidative stress. Tumor cell iron metabolism changes, increasing uptake and reducing efflux, leading to an increase in the iron pool, thereby increasing the tumor-specific activity of DHA. However, the efficacy of both drugs is limited by poor water solubility, low bioavailability, and rapid metabolism. In addition, the high metabolic environment of tumor cells allows them to quickly increase and decrease endogenous substances, adjust the redox balance homeostasis, and significantly reduce the efficacy of ORI or DHA. Therefore, developing new methods to improve the bioavailability of ORI and DHA, increase their water solubility, prolong blood circulation, and enhance their ability to break the intracellular redox homeostasis may be an effective way to improve gastric cancer treatment.

[0004] Stimuli-responsive polymer prodrug micelles have the advantages of designable structure and function, high drug loading, and significantly improved drug bioavailability. They can effectively prevent drug leakage in the systemic circulation and improve drug efficacy. They have been a hot topic of research at home and abroad in recent years. Therefore, the development of stimulus-responsive polymer prodrug micelles to co-deliver ORI and DHA may be a traditional Chinese medicine method for the treatment of gastric cancer. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide an active oxygen responsive polymer prodrug micelle and its preparation method and application in view of the deficiencies in the prior art. The polymer prodrug micelle provided by the present invention can co-deliver ORI and DHA, improve the biocompatibility of the two drugs, and effectively treat gastric cancer through synergistic effects.

[0006] In order to solve the above technical problems, the present invention discloses an active oxygen responsive polymer micelle, characterized in that its structural formula is as shown in formula (I):

[0007]

[0008] Wherein TK represents thioketal, x represents the number of structural units, and the value of x ranges from 4 to 20.

[0009] The present application further provides a method for preparing the active oxygen responsive polymer micelles, comprising the following steps: reacting thioketal with acetic anhydride to obtain thioketal anhydride; reacting polyethylene glycol-polylysine with thioketal anhydride to obtain polyethylene glycol-polylysine-thioketal, wherein the mass ratio of thioketal: acetic anhydride: polyethylene glycol-polylysine is (3-5): (20-40): (6-10).

[0010] The present application further provides an active oxygen responsive polymer prodrug micelle, which comprises the above active oxygen responsive polymer micelle and a drug.

[0011] In some specific embodiments, the reactive oxygen species responsive polymer prodrug micelle is Rubescensine A or dihydroartemisinin, and ORI and DHA can synergistically treat ferroptosis.

[0012] The present application further proposes a method for preparing the above-mentioned active oxygen responsive polymer prodrug micelles, wherein the active oxygen responsive polymer micelles are reacted with a drug under the action of a catalyst, and after the reaction is completed, the reaction solution is precipitated in cold ether, the obtained precipitate is dissolved with dimethyl sulfoxide and transferred to a dialysis bag, and after dialysis with dimethyl sulfoxide for 40 to 60 hours, it is further dialyzed with water for 20 to 30 hours, and the polymer prodrug micelles are obtained after freeze-drying; wherein the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dimethylaminopyridine.

[0013] The mass ratio of the drug, active oxygen responsive polymer micelles, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dimethylaminopyridine is (10-30):(20-40):(5-15):(1-10); the reaction temperature is 20-40°C; and the reaction time is 40-60h.

[0014] Furthermore, the present application proposes an active oxygen responsive polymer prodrug micelle co-loaded with ORI and DHA, which comprises polymer prodrug micelles represented by formula (II) and formula (III):

[0015] Formula (II):

[0016]

[0017] Formula (III):

[0018]

[0019] Wherein, TK represents thioketal, and n, m, x and y are the numbers of structural units, which are all in the range of 4 to 20.

[0020] The present invention also provides a method for preparing the above-mentioned active oxygen responsive polymer prodrug micelles co-loaded with ORI and DHA. First, the polymer prodrug micelles described in formula (II) and formula (III) are prepared, and the two are mixed in a mass ratio of (1 to 10): (1 to 10) and dissolved in dimethyl sulfoxide, and then loaded into a dialysis bag. After dialyzing in deionized water for a period of time, the active oxygen responsive polymer prodrug micelles co-loaded with ORI and DHA are obtained by filtering with a filter membrane.

[0021] Preferably, the molecular weight cutoff of the dialysis bag is 3.5 kDa; the dialysis time is 4 to 10 hours; the dialysis temperature is 20 to 40° C.; and the pore size of the filter membrane is 0.2 to 0.5 μm.

[0022] The present invention further proposes the use of the above-mentioned active oxygen responsive polymer prodrug micelles in the preparation of drugs for treating gastric cancer, specifically, inducing the generation of active oxygen and elimination of reduced glutathione in gastric cancer cells, as well as the use of anti-gastric cancer in vivo and in vitro.

[0023] Beneficial effects: The reactive oxygen responsive polymer prodrug micelles co-loaded with ORI and DHA provided by the present invention can significantly improve the water solubility and biocompatibility of ORI and DHA, can effectively eliminate reduced glutathione in gastric cancer cell AGS cells and amplify reactive oxygen, ultimately leading to the production of a large amount of lipid peroxides, thereby killing tumor cells, inducing cell ferroptosis, and ultimately inhibiting tumor proliferation. At the same time, the inhibition of gastric cancer cells and tumor-bearing mouse tumors by single ORI and DHA polymer prodrug mice is weaker than that by the two-drug co-loaded polymer prodrug micelles, indicating that ORI and DHA co-loaded polymer prodrug micelles can effectively deliver the two drugs to tumor tissues, exert a synergistic anti-tumor effect, and ultimately inhibit tumor proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 To screen the optimal combined dosage of ORI and DHA;

[0025] Figure 2 A preparation route for ORI and DHA polymer prodrugs PEG-b-PLL-TK-ORI and PEG-b-PLL-TK-DHA;

[0026] Figure 3 It is the nuclear magnetic resonance hydrogen spectrum of PEG-b-PLL-TK-ORI and PEG-b-PLL-TK-DHA;

[0027] Figure 4 The hydrodynamic particle sizes of ORI polymer prodrug micelles ORI-M, DHA polymer prodrug micelles DHA-M and two-drug co-loaded polymer prodrug micelles OD-M;

[0028] Figure 5 is the reduced glutathione content in AGS cells after being treated with ORI, DHA and OD-M;

[0029] Figure 6 is the content of reactive oxygen species in AGS cells after treatment with ORI, DHA and OD-M;

[0030] Figure 7 is the content of lipid peroxide DMA in AGS cells after being treated with ORI, DHA and OD-M;

[0031] Figure 8 is the survival rate of AGS cells after treatment with ORI, DHA and OD-M;

[0032] Fig. 9 Figure 2 shows the changes in tumor volume in AGS tumor-bearing mice after treatment with ORI, DHA and OD-M. DETAILED DESCRIPTION

[0033] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments, and the above and / or other advantages of the present invention will become more clear.

[0034] Example 1 Preparation of PEG-b-PLL-TK.

[0035] PEG-b-PLL was prepared according to the method disclosed by XU et al. (J. Mater. Chem. B, 2021, 9, 3068–3078). Specifically, 5.0 g of PEG-NH2 was dissolved in 80.0 mL of dry N, N-dimethylformamide (DMF) to obtain solution A; 9.2 g of Lys-NCA was dissolved in 100.0 mL of dry DMF to obtain solution B, and solution A was mixed with solution B and vigorously stirred at 40 ° C and N2 for 72 hours. After the reaction, the solution was concentrated under vacuum and then precipitated into an excess of ice-cold ether to obtain PEG-b-PLLZ. Subsequently, 15.0 mL of HBr / acetic acid (33 wt%) was added to a mixture of 5.0 g of PEG-b-PLLZ and 50.0 mL of trifluoroacetic acid. The mixture was incubated at 25 ° C for 1 hour with slight stirring to remove the protecting group. Then, the mixture was precipitated in an excess of ice-cold ether to obtain a crude product. The crude product was dried under vacuum, dissolved in DMSO, dialyzed against ultrapure water, and then freeze-dried to obtain pure PEG-b-PLL.

[0036] Weigh 2.9g of TK and dissolve it in 25mL of acetic anhydride, and stir and react at 30℃ for 2.5h. Then, add 15mL of toluene and remove the solvent by rotary evaporation. Repeat this step five times to obtain TK anhydride. The obtained product is immediately dissolved in 25mL of anhydrous N,N-dimethylformamide and mixed with 20mL of N,N-dimethylformamide solution containing 5.8g of PEG-b-PLL. The mixed solution is stirred and reacted at 30℃ for 12h. After the reaction is completed, the reaction solution is precipitated with 150mL of pre-cooled ether. The precipitate is dissolved in 5mL of dimethyl sulfoxide and loaded into a dialysis bag with a molecular weight cutoff of 3.5kDa. It is dialyzed with dimethyl sulfoxide for 48h, then dialyzed with deionized water for 24h, and lyophilized to obtain PEG-b-PLL-TK.

[0037] Example 2 Screening of the optimal combined dosage of ORI and DHA.

[0038] The common MTT method was used to screen the optimal combined dosage of ORI and DHA. AGS cells were seeded into 96-well plates at 5,000 cells per well and cultured overnight. The culture medium was then removed, 200 μL of culture medium dissolved with ORI and DHA was added, and the culture was continued for 48 hours. The mass ratios of ORI and DHA were 4:1, 2:1, 1:1, 1:2, and 1:4, respectively. The drug concentrations were 0.01, 0.1, 1, 10, 25, 50, and 100 μg / mL. After 48 hours, 20 μL of commercially available MTT reagent (5 mg / mL) was added to each well, and the culture was continued for 4 hours. The culture medium was discarded, and 200 μL of dimethyl sulfoxide was added and shaken on a shaker for 15 minutes. The absorbance value was then read at 450 nm on an enzyme reader, and the Graph IC was used. 50Analysis method to calculate IC at each ratio 50 The result is Figure 1 shown.

[0039] Example 3 Preparation of PEG-b-PLL-TK-ORI and PEG-b-PLL-TK-DHA.

[0040] like Figure 2 As shown, PEG-b-PLL-TK 2.8g, EDCI 1.0g, DMAP 0.7g and ORI 2.0g were weighed and dissolved in 40mL of anhydrous N,N-dimethylformamide, and then nitrogen was introduced and stirred at 30°C for 48 hours. After the reaction, the reaction solution was added dropwise to 150mL of cold ether for precipitation, and the resulting precipitate was dissolved in dimethyl sulfoxide and transferred to a dialysis bag. After dialyzing with dimethyl sulfoxide for 48 hours, it was further dialyzed with water for 24 hours, and then freeze-dried to obtain the ORI polymer prodrug. PEG-b-PLL-TK-DHA was prepared in the same manner, wherein the amounts of PEG-b-PLL-TK, EDCI, DMAP and DHA or ORI were 2.8g, 1.0g, 0.7g and 1.6g, respectively. The obtained prodrug was verified by hydrogen nuclear magnetic resonance spectroscopy, and the results are shown as follows. Figure 3 shown.

[0041] Example 4 Preparation of polymer prodrug micelles co-loaded with ORI and DHA.

[0042] The present invention prepares polymer drug precursor micelles through the dialysis method, which is a conventional method for preparing nano drug delivery micelles. The specific process is as follows: weigh 18.1 mg of PEG-b-PLL-TK-ORI and 16.2 mg of PEG-b-PLL-TK-DHA prepared in Example 3, dissolve them with 1 mL of dimethyl sulfoxide, and then transfer the solution to a dialysis bag. Under nitrogen protection, dialyze directly with water for 4 hours. After the liquid is filtered with a 0.4 μm filter membrane, ORI and DHA co-loaded polymer prodrug micelles are obtained. The same method is used to prepare oxidation-responsive polymer prodrug micelles ORI-M and DHA-M loaded with ORI and DHA alone. The average particle sizes of the three micelles OD-M, ORI-M and DHA-M are 62 nm, 54 nm, and 55 nm, respectively (e.g. Figure 4 shown).

[0043] The in vitro anti-tumor experiment investigated the effect of ORI and DHA co-loaded reactive oxygen species-responsive polymer prodrug micelles on the removal of reduced glutathione ( Figure 5 ), amplification of reactive oxygen species ( Figure 6 ) and induce cell ferroptosis ( Figure 7 ) ability, the inhibitory effect on the proliferation of gastric cancer AGS cells (such as Figure 8 shown).

[0044] Specifically, after treating gastric cancer cell AGS with the corresponding drugs, the amount of active oxygen amplification was detected using a commercial active oxygen detection kit, the elimination rate of reduced glutathione was detected using a commercial total glutathione detection kit; the cell lipid peroxidation was detected using a commercial malondialdehyde (MDA) kit; the cell survival rate was detected using a commercial MTT reagent; and 4-6 week nude mice were used as experimental subjects to record the changes in mouse tumor volume and mouse weight. Among them, the amount of drugs used and the treatment conditions refer to the use of commercial kits.

[0045] from Figures 5 to 7 It can be seen that compared with free ORI and DHA, or micelles loaded with ORI and DHA alone, polymer prodrug micelles OD-M co-loaded with ORI and DHA can significantly eliminate intracellular reduced glutathione, amplify reactive oxygen species, and induce cell ferroptosis.

[0046] The inhibitory effect of ORI- and DHA-co-loaded reactive oxygen species-responsive polymer prodrug micelles on the proliferation of gastric cancer AGS cells was investigated by in vitro antitumor experiments (e.g. Figure 8 As shown in the figure, it can be seen that the anti-tumor effect of the active oxygen responsive polymer prodrug micelles co-loaded with ORI and DHA is better than that of free ORI and DHA, or micelles loaded with only ORI and DHA.

[0047] In vivo antitumor experiments showed that the reactive oxygen species-responsive polymer prodrug micelles co-loaded with ORI and DHA could significantly enhance the therapeutic efficiency (e.g. Fig. 9 shown).

[0048] In summary, the reactive oxygen species-responsive polymer prodrug micelles co-loaded with ORI and DHA provided by the present invention can significantly improve the water solubility and biocompatibility of ORI and DHA, and effectively remove reduced glutathione in gastric cancer cells, amplify reactive oxygen species, induce cell ferroptosis, and ultimately inhibit tumor proliferation.

[0049] The present invention provides a concept and method for an active oxygen responsive polymer prodrug micelle co-loaded with ORI and DHA. There are many methods and approaches to implement the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention. All components not specified in this embodiment can be implemented using existing technologies.

Claims

1. An active oxygen responsive polymer micelle, characterized in that: Its structural formula is shown in formula (I): Wherein TK represents thioketal, x represents the number of structural units, and the value of x ranges from 4 to 20.

2. The method for preparing the active oxygen responsive polymer micelles according to claim 1, characterized in that: The method comprises the following steps: reacting thioketal with acetic anhydride to obtain thioketal anhydride; reacting polyethylene glycol-polylysine with thioketal anhydride to obtain polyethylene glycol-polylysine-thioketal, wherein the mass ratio of thioketal: acetic anhydride: polyethylene glycol-polylysine is (3-5): (20-40): (6-10).

3. An active oxygen responsive polymer prodrug micelle, characterized in that: The invention comprises the active oxygen responsive polymer micelle according to claim 1 or 2 and a drug.

4. The active oxygen responsive polymer prodrug micelle according to claim 3, characterized in that: The drug is oridonin or dihydroartemisinin.

5. The method for preparing the active oxygen responsive polymer prodrug micelles according to claim 3 or 4, characterized in that The active oxygen responsive polymer micelles according to claim 1 are reacted with a drug under the action of a catalyst. After the reaction is completed, the reaction solution is precipitated in cold ether, the obtained precipitate is dissolved with dimethyl sulfoxide and transferred to a dialysis bag, dialyzed with dimethyl sulfoxide for 40 to 60 hours, and then dialyzed with water for 20 to 30 hours, and freeze-dried to obtain polymer prodrug micelles; wherein the catalyst is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dimethylaminopyridine.

6. The preparation method according to claim 6, characterized in that: The mass ratio of the drug, active oxygen responsive polymer micelles, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and dimethylaminopyridine is (10-30):(20-40):(5-15):(1-10); the reaction temperature is 20-40°C; and the reaction time is 40-60h.

7. An active oxygen responsive polymer prodrug micelle co-loaded with ORI and DHA, characterized in that: It comprises polymer prodrug micelles represented by formula (II) and formula (III): Formula (II): Formula (III): Wherein, TK represents thioketal, and n, m, x and y are the numbers of structural units, which are all in the range of 4 to 20.

8. The method for preparing the active oxygen responsive polymer prodrug micelles according to claim 7, characterized in that: The polymer prodrug micelles of formula (II) and formula (III) are prepared respectively by the method described in claim 5 or 6, and the two are mixed in a mass ratio of (1-10): (1-10) and dissolved in dimethyl sulfoxide, and then loaded into a dialysis bag. After dialyzing in deionized water for a period of time, the active oxygen responsive polymer prodrug micelles co-loaded with ORI and DHA are obtained by filtering with a filter membrane.

9. The preparation method according to claim 8, characterized in that: The molecular weight cutoff of the dialysis bag is 3.5 kDa; the dialysis time is 4 to 10 hours; the dialysis temperature is 20 to 40° C.; and the pore size of the filter membrane is 0.2 to 0.5 μm.

10. Use of the active oxygen responsive polymer prodrug micelle according to claim 3 or claim 7 in the preparation of a drug for treating gastric cancer.

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