X-ray response controlled-release diselenide prodrug and preparation method thereof

Abstract

The invention provides an X-ray response controlled-release diselenide prodrug and a preparation method thereof.The diselenide prodrug is a copolymer P (Se-DOX), and the structural formula of the diselenide prodrug is as follows: # imgabs0 #. The synthesis method comprises the steps that DOX is dissolved in anhydrous dichloromethane CH2Cl2, then 2, 2-dienyldiphenylacetic acid, EDCI and DMAP are sequentially added, and stirring is conducted for a period of time at the room temperature; removing the solvent to obtain a CH2Cl2 solution containing a product, dropwise adding the CH2Cl2 solution containing the product into a CH2Cl2 solution containing ethylenediamine, and stirring for reaction to synthesize NH2-Se2-DOX; the preparation method comprises the following steps: dissolving polyacrylic acid PAA, NHS and EDCI in DMF, and reacting in N2 atmosphere; then, adding NH2-Se2-DOX into the DMF (Dimethyl Formamide); dialyzing the final solution in distilled water to remove unreacted reactants and DMF (Dimethyl Formamide); and carrying out freeze drying to obtain the copolymer P (Se-DOX). According to the diselenide prodrug, the release amount of DOX can be adjusted through X-ray dose stimulation conditions, more sensitive and accurate drug-loaded release response is achieved, the treatment effect is improved, and meanwhile damage of the drug to normal tissue is reduced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a diselenide prodrug with X-ray-responsive controlled drug release and a preparation method thereof.

Background Art

[0002] Stimulus-responsive probes are a series of intelligent systems that can respond to external stimuli and produce corresponding changes in structure or physical and chemical properties. Currently, they have become a research hotspot in the field of tumor diagnosis and treatment. Stimulus-responsive probes change their physical properties or chemical structures according to subtle external changes, so as to achieve targeted, quantitative, and timed drug release at the tumor site, achieving the purpose of efficient treatment; or they can enhance the imaging contrast, bringing a leap-forward progress to tumor diagnosis and treatment. However, the research and development of stimulus-responsive probes are still in its infancy, and the ever-changing development of tumor clinical diagnosis and treatment technologies has put forward higher requirements for relevant researchers. Many stimulus-responsive probes have problems such as low sensitivity, slow response speed, low drug loading capacity, and the biological safety needs to be further investigated. In order to achieve the purpose of clinical application, the constructed intelligent-responsive probes need to have the following characteristics: (1) good biocompatibility, (2) degradable or metabolizable, (3) high drug loading capacity; and maintain low drug leakage during the in vivo circulation process, and at the same time can preferentially accumulate in the tumor site and rapidly change their physical and chemical properties under various environmental stimuli to achieve the purpose of precise imaging and treatment. In addition, stimulus-responsive nanoprobes integrating multiple functions can more sensitively make "responses" suitable for different environments, precisely control the physical and chemical properties of the nanoprobes as needed, which is the future research direction of stimulus-responsive probes and has important significance. At the same time, selenium is an essential trace element for the human body, which can regulate the redox balance in the body. Some selenium-containing small molecules are used clinically as anti-cancer and adjuvant drugs. Previous studies have shown that selenious acid can be used as a chemotherapeutic drug to induce cancer cells to produce reactive oxygen species (ROS) and induce apoptosis. The diselenide bond is sensitive to ionizing radiation and is easily broken and oxidized to generate selenious acid or selenic acid. Therefore, compounds containing diselenide bonds are potential candidates for a radiosensitizer.

Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a diselenide prodrug with X-ray-responsive controlled drug release and a preparation method thereof. The diselenide prodrug can regulate the release amount of DOX under X-ray dose stimulation conditions, realize a more sensitive and precise drug release response, improve the treatment effect, and at the same time reduce the damage of the drug to normal tissues.

[0004] The present invention is realized as follows:

[0005] A diselenide prodrug with X-ray-responsive controlled drug release, the diselenide prodrug is the copolymer P(Se-DOX), and its structural formula is as follows:

[0006]

[0007] Furthermore, a preparation method of a diselenide prodrug with controllable X-ray-responsive drug release is as follows:

[0008] Step 1: Synthesize NH 2 -Se 2 -DOX: Dissolve DOX in CH 2 Cl 2 . Then, successively add 2,2-diallyldiphenylacetic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI, and 4-dimethylaminopyridine DMAP, and stir at room temperature for a period of time; then remove the solvent under reduced pressure, and drop the obtained CH 2 Cl 2 solution containing the product into the CH 2 Cl 2 solution containing ethylenediamine, and stir to react; after the reaction is completed, wash with saturated NaCl, and finally dry with Na 2 SO 4 .

[0009] Step 2: Preparation of copolymer P(Se-DOX): Dissolve polyacrylic acid PAA, N-hydroxysuccinimide NHS, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI in N,N-dimethylformamide DMF, and react under N 2 atmosphere; then add NH 2 -Se 2 -DOX to DMF; finally, dialyze the solution in distilled water to remove unreacted reactants and DMF; then freeze-dry the obtained solution to obtain copolymer P(Se-DOX).

[0010] Furthermore, in Step 1, the molar ratio of the reactants is DOX: 2,2-diallyldiphenylacetic acid: EDCI: DMAP = 0.1 - 0.4: 0.5 - 2: 0.6 - 2.4: 0.03 - 0.12.

[0011] Furthermore, in Step 1, the molar ratio of the solute in the CH 2 Cl 2 solution containing the product and the CH 2 Cl 2 solution containing ethylenediamine is 0.05 - 0.2: 1 - 4.

[0012] Furthermore, in Step 2, the molar ratio of the reactants is PAA: NHS: EDCI: NH 2 -Se 2-DOX = 0.5 to 2: 1 to 4: 1 to 4: 1 to 4.

[0013] Further, the DOX can be replaced with other chemotherapeutic drugs containing amino or carboxyl groups.

[0014] Further, the synthesis method of 2,2'-diallyldiphenylacetic acid is as follows:

[0015] Add ethanol to selenium powder, and then drop the sodium borohydride (NaBH 4 ) ethanol solution into the suspension containing selenium powder under an argon atmosphere, and stir the mixture in an ice bath; after the solution turns colorless, continue to add selenium powder, quickly heat the mixture to 80 °C and keep it for 30 min to obtain reddish-brown Na 2 Se 2 ; then add bromoacetic acid ethanol solution to the Na 2 Se 2 solution, stir and react overnight; finally, filter the mixture and dilute it in water, extract with ethyl acetate, and then dry the ethyl acetate layer over Na 2 SO 4 and rotary evaporate to obtain the product.

[0016] Further, in the synthesis step of 2,2'-diallyldiphenylacetic acid, the molar ratio of the reactants is the first addition of selenium powder: NaBH 4 : the second addition of selenium powder: bromoacetic acid ethanol = 5 to 20: 10 to 40: 5 to 20: 5 to 20.

[0017] The present invention has the following advantages:

[0018] The diselenide prodrug copolymer P(Se-DOX) of the present invention is X-ray-responsive and controllable drug release, that is, the release amount of DOX can be regulated by specific X-ray dose stimulation conditions to achieve more sensitive and precise response; the present invention has high biosafety. In addition to DOX, it can also combine with other chemotherapeutic drugs containing amino or carboxyl groups to deliver the active molecules of chemotherapeutic drugs to the tumor site, and quickly achieve sensitive and precise drug release under X-ray stimulation, improving the therapeutic effect while reducing the damage of drugs to normal tissues, and has broad application prospects in in vivo imaging and treatment.

Description of the Drawings

[0019] The following further describes the present invention with reference to the drawings in conjunction with the embodiments.

[0020] Figure 1 is the 1 H NMR spectrum of P(Se-DOX) in the embodiment of the present invention.

[0021] Figure 2TEM images of the preparation in the embodiments of the present invention, where (a) is the transmission electron microscope (TEM) image of DCNPs; (b) is the TEM image of DCNP modified with P(Se-DOX) (DCNP@P(Se-DOX)), and (c) is the high-resolution TEM image of DCNP@P(Se-DOX).

[0022] Figure 3 Energy-dispersive X-ray spectroscopy (EDS) elemental distribution map of DCNP@P(Se-DOX) in the embodiments of the present invention.

[0023] Figure 4 Hydrated particle sizes of DCNPs and DCNP@P(Se-DOX) in the embodiments of the present invention (a) and UV-vis-NIR spectrum of DCNP@P(Se-DOX) (b).

[0024] Figure 5 Near-infrared II (NIR-II) fluorescence (FL) spectrum of DCNP@P(Se-DOX) in the embodiments of the present invention.

[0025] Figure 6 FL spectra of DOX released after irradiating DCNP@P(Se-DOX) with different doses of X-rays in the embodiments of the present invention, where the corresponding FL imaging diagrams are shown in the insets.

Detailed implementation manners

[0026] The technical solutions of the present invention will be clearly and completely described below in conjunction with the attached Figure 1-6 drawings and specific implementation manners. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the scope of protection of the present invention. For those not specified in the embodiments, the conventional conditions or the conditions recommended by the manufacturer are followed. For the reagents or instruments not specified by the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0027] Embodiment

[0028] Step A: Synthesize 2,2'-diene diphenylacetic acid. Add selenium powder (0.79 g, 10 mmol) to a three-necked round-bottom flask, and then add ethanol. Then, under an argon atmosphere, drop the ethanol solution of NaBH 4 (0.76 g, 20 mmol) into the suspension containing selenium powder, and stir the mixture in an ice bath. After the solution turns colorless, continue to add selenium powder (0.79 g, 10 mmol), and quickly heat the mixture to 80 °C and keep it for 30 min to obtain reddish-brown Na 2 Se 2 . Then add Na 2 Se 2Bromoacetic acid ethyl ester (2.78 g, 20 mmol) was added to the solution, and the mixture was stirred overnight. Then the mixture was filtered and diluted with water, and extracted three times with ethyl acetate. The ethyl acetate layer was dried over Na 2 SO 4 , and the solvent was removed by rotary evaporation to obtain 1.2 g of the product with a yield of about 30.7%.

[0029] Step B: Synthesis of NH 2 -Se 2 -DOX. DOX (0.2 mmol) was dissolved in anhydrous dichloromethane (CH 2 Cl 2 ), and then 2,2-diallyldiphenylacetic acid (1 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI (1.2 mmol) and 4-dimethylaminopyridine DMAP (0.06 mmol) were added successively. After stirring at room temperature for 12 h, the mixture was washed twice with saturated NaCl solution, and then dried over Na 2 SO 4 . The solvent was removed under reduced pressure, and the resulting CH 2 Cl 2 solution containing the product (0.1 mmol) was added dropwise to the CH 2 Cl 2 solution containing ethylenediamine (2 mmol). The mixture was stirred for 24 h, then washed twice with saturated NaCl solution, and finally dried over Na 2 SO 4 .

[0030] Step C: Polyacrylic acid PAA (0.01 mmol), N-hydroxysuccinimide NHS (0.02 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI (0.02 mmol) were dissolved in 5 mL of N,N-dimethylformamide DMF, and the reaction was carried out under N 2 atmosphere for 6 h (room temperature); then NH 2 -Se 2 -DOX (0.02 mmol) was added to 1 mL of DMF. The final solution was dialyzed against distilled water (MWCO: 10000) for 48 h to remove unreacted reactants and DMF; then the resulting solution was freeze-dried to obtain the copolymer P(Se-DOX). The structural formula is as follows:

[0031]

[0032] The 1 H NMR spectrum of the product is as shown in Figure 1 , and the characterization of the product is as follows:

[0033] 11H NMR (400 MHz, CDCl 3 ): δ 8.231 (s, 3H, -CO-NH-), 7.011 - 7.101 (m, 3H, Ar-H, J = 7 Hz), 4.185 (s, 4H, -CO-CH 2 -Se-), 4.183 (m, 1H, O-CH-), 4.180 (s, 3H, CH 3 -O-), 4.178 (d, 2H, O-CH 2 -, J = 7.5 Hz), 3.714 (m, 2H, Ar-CH 2 -C), 3.742 (s, 1H, -CH-O-), 3.667 (s, 2H, -CH-N-), 2.532 (m, 1H, -CH-), 2.491 (m, 1H, -CH 2 -), 1.96 (m, 1H, -CH-), 1.831 (m, 2H, -CH 2 -), 1.821 (m, 2H, -CH 2 -), 1.821 (m, 23H, -CH-), 1.818 (m, 1H, -CH-), 1.354 (m, 50H, -CH 2 -), 1.121 (m, 3H, -CH 3 -).

[0034] Performance Test:

[0035] DCNPs were synthesized by high-temperature pyrolysis method, and P(Se-DOX) was coupled with DCNPs through non-covalent bonds to obtain DCNP@P(Se-DOX). As shown in Figure 2 a, the prepared DCNPs had a particle size of 6.8 ± 1.2 nm and good dispersibility. It was observed by TEM that the particle size of DCNP@P(Se-DOX) increased slightly by about 1 nm ( Figure 2 b), and at the same time, the high-magnification TEM image could clearly observe the P(Se-DOX) layer modified on the surface of DCNP ( Figure 2 c). The results of STEM-mapping elemental distribution ( Figure 3 ) showed that Na, Yb, F, and Er were evenly distributed in the nanoparticles, corresponding to the elements of DCNP; at the same time, Se, C, N, and O corresponded to the elements of P(Se-DOX); these results indicated that DCNP@P(Se-DOX) nanoparticles were successfully prepared. The results of dynamic light scattering test showed that the particle size of DCNP@P(Se-DOX) increased slightly compared with DCNPs ( Figure 4 a). The absorption spectrum of DCNP@P(Se-DOX) showed a strong characteristic absorption peak of DOX ( Figure 4b), indicating its high efficiency in loading the chemotherapeutic drug DOX. After calculation, the drug loading rate of DOX reached 18.40%. The NIR-II FL spectrum of DCNP@P(Se-DOX) showed almost no change compared with that of DCNPs ( Figure 5 ), indicating that the surface modification of DCNP with P(Se-DOX) hardly changed its NIR-II FL emission performance.

[0036] Verify the release of DOX from DCNP@P(Se-DOX) under X-ray triggering: Irradiate the DCNP@P(Se-DOX) solution (dimethyl sulfoxide solution) with different doses of X-rays, and record the FL spectrum and FL imaging map of the solution under 480 nm excitation ( Figure 6 ). The results showed that with the increase of the X-ray irradiation dose, DCNP@P(Se-DOX) continuously released DOX. However, under the irradiation of X-ray doses of 2 Gy or 4 Gy, the difference in the DOX release amount of DCNP@P(Se-DOX) was not significant, which could be attributed to the limited amount of DOX modified on DCNP@P(Se-DOX). Therefore, the released amount of DOX could not increase infinitely with the increase of the irradiation dose. It can be seen that the release amount of DOX in DCNP@P(Se-DOX) is related to the X-ray dose, and the controllable drug release effect can be achieved by using X-ray.

[0037] In summary, the diselenide prodrug copolymer P(Se-DOX) of the present invention enables X-ray-responsive controlled release, that is, the release amount of DOX can be regulated by specific X-ray dose stimulation conditions to achieve more sensitive and precise response; the present invention has high biosafety. In addition to DOX, it can also combine with other chemotherapeutic drugs containing amino or carboxyl groups, deliver the active molecules of chemotherapeutic drugs to the tumor site, and quickly achieve sensitive and precise drug loading and release under X-ray stimulation, improving the therapeutic effect while reducing the damage of the drug to normal tissues, and having broad application prospects in in vivo imaging and treatment.

[0038] Although the specific embodiments of the present invention have been described above, those skilled in the art should understand that the specific embodiments we described are illustrative rather than used to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be covered by the scope protected by the claims of the present invention.

Claims

1. A diselenide prodrug of an X-ray responsive controlled release drug, characterized in that: The diselenide prodrug is the copolymer P(Se-DOX), and its structural formula is as follows:

2. A method for preparing a diselenide prodrug of an X-ray responsive controlled release drug, characterized in that: The method steps are as follows: Step 1, synthesis of NH2-Se2-DOX: DOX was dissolved in CH2Cl2, followed by sequential addition of 2,2-diphenylacetic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI and 4-dimethylaminopyridine DMAP, and stirred at room temperature for a period of time; then the solvent was removed under reduced pressure, and the obtained CH2Cl2 solution containing the product was added dropwise to the CH2Cl2 solution containing ethylenediamine, and stirred for reaction; after the reaction was completed, it was washed with saturated NaCl and finally dried with Na2SO4; Step 2, preparation of copolymer P (Se-DOX): polyacrylic acid PAA, N-hydroxysuccinimide NHS and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide EDCI are dissolved in N,N-dimethylformamide DMF and reacted under N2 atmosphere; then NH2-Se2-DOX is added to DMF; the final solution is dialyzed in distilled water to remove unreacted reactants and DMF; then the obtained solution is freeze-dried to obtain copolymer P (Se-DOX).

3. The method for preparing a diselenide prodrug of an X-ray responsive controlled release drug according to claim 1, characterized in that: The molar ratio of the reactants in step 1 is DOX: 2,2-diphenylacetic acid: EDCI: DMAP = 0.1-0.4: 0.5-2: 0.6-2.4: 0.03-0.

12.

4. The method for preparing a diselenide prodrug of an X-ray responsive controlled release drug according to claim 1, characterized in that: The molar ratio of the solute substance in the CH2Cl2 solution containing the product and the CH2Cl2 solution containing ethylenediamine in step 1 is 0.05-0.2:1-4.

5. The method for preparing a diselenide prodrug of an X-ray responsive controlled release drug according to claim 1, characterized in that: The molar ratio of the reactants in step 2 is PAA:NHS:EDCI:NH2-Se2-DOX=0.5~2:1~4:1~4:1~4.

6. The method for preparing a diselenide prodrug of an X-ray responsive controlled release drug according to claim 1, characterized in that: The DOX can be replaced by other chemotherapeutic drugs containing amino or carboxyl groups.

7. The method for preparing a diselenide prodrug of an X-ray responsive controlled release drug according to claim 1, characterized in that: The synthesis of 2,2'-diphenylacetic acid is as follows: Add ethanol to the selenium powder, then add the sodium borohydride ethanol solution dropwise to the selenium powder suspension under argon atmosphere, and stir the mixture in an ice bath; after the solution becomes colorless, continue to add selenium powder, quickly heat the mixture to 80°C and maintain for 30 minutes to obtain reddish-brown Na2Se2; then add bromoacetic acid ethanol solution to the Na2Se2 solution, stir and react overnight; finally, filter the mixture and dilute it in water, extract it with ethyl acetate, and then dry the ethyl acetate layer over Na2SO4, and rotary evaporate to obtain the product.

8. The method for preparing a diselenide prodrug of an X-ray responsive controlled release drug according to claim 6, characterized in that: The molar ratio of the reactants in the synthesis step of 2,2'-diphenylacetic acid is as follows: first added selenium powder: NaBH4: second added selenium powder: bromoacetic acid ethanol = 5-20: 10-40: 5-20: 5-20.

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