Organic polymer composite nano-material for ultrasonically exciting copper death as well as preparation method and application of organic polymer composite nano-material
By developing organic polymer composite nanomaterials, using ultrasound to stimulate reactive oxygen production and release copper death drugs, the problems of poor biosafety and low yield of traditional inorganic sound-sensitizers are solved, and efficient tumor cell killing and new tumor treatment methods are achieved.
Patent Information
- Application Number
- CN202510171889.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-17
- Publication Date
- 2025-05-23
AI Technical Summary
Traditional inorganic sound sensitizers have poor biosafety, low yield of ultrasound production of reactive oxygen species, and poor ultrasound stability, making it difficult to effectively treat tumor cells.
Develop an organic polymer composite nanomaterial, which encapsulates the sound-sensitizer PBD NPs and the copper death drug ESCu through nanosphere shells, uses ultrasound excitation to generate singlet oxygen and release copper death drug to achieve precise killing of tumor cells.
This nanomaterial can quickly and efficiently produce reactive oxygen species in an ultrasonic environment, with high biosafety and stability, significantly improves the killing effect on tumor cells, and provides a new tumor treatment method.
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Figure CN120022253A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of nano materials and biomedicine, and in particular to an organic polymer composite nano material for ultrasonically stimulating copper death, and a preparation method and application thereof. Background Art
[0002] Copper death refers to the fact that copper ions can bind to lipoyl proteins in the tricarboxylic acid cycle (TCA), causing abnormal oligomerization of lipoyl proteins. In addition, copper ions can also reduce the level of Fe-S cluster proteins. The two together induce protein toxicity stress response and ultimately lead to cell death.
[0003] Sonodynamic therapy (SDT) is an emerging non-invasive treatment method that achieves therapeutic purposes by integrating ultrasound technology with sonosensitizers. Ultrasound is a mechanical wave with good tissue penetration. When ultrasound propagates in biological tissues, it can interact with tissues to produce cavitation effects, thermal effects, etc. The cavitation effect refers to the formation of tiny bubbles in the liquid under the action of ultrasound. These bubbles will expand and contract rapidly under the action of ultrasound and eventually burst, producing local extreme physical conditions such as high temperature, high pressure and shock waves. Sonosensitizers are a class of substances that can absorb ultrasound energy and produce chemical reactions. Under the action of ultrasound, sonosensitizers are activated and change from the ground state to the excited state. The excited sonosensitizer has high energy and can react with the surrounding oxygen molecules to produce reactive oxygen species (ROS) such as singlet oxygen. Reactive oxygen species such as singlet oxygen have strong oxidizing ability and can oxidize various biological macromolecules in cells, such as lipids, proteins and nucleic acids, resulting in damage to the structure and function of cells. At the same time, reactive oxygen can also activate the apoptosis signaling pathway in cells and induce cell apoptosis, thereby achieving the purpose of treating diseases. The advantage of sonodynamic therapy is that ultrasound can penetrate the skin and tissues, without the need for surgical incisions, reducing the patient's pain and risk of infection. Sonosensitizers can selectively accumulate in diseased tissues, achieving precise killing of diseased cells with less damage to surrounding normal tissues.
[0004] However, traditional inorganic sonosensitizers have poor biosafety, low yield of reactive oxygen species generated by ultrasound, and poor ultrasound stability. Therefore, it is very important to develop a new sonosensitizer and a new tumor treatment method with good biosafety, high ultrasound reactive oxygen species yield and good ultrasound stability. Summary of the invention
[0005] Purpose of the invention: In view of the problems of poor biological safety, low yield of active oxygen generated by ultrasound and poor ultrasonic stability of traditional inorganic sonosensitizers, the present invention provides an organic polymer composite nanomaterial for ultrasonically induced copper death and its preparation method and application.
[0006] To achieve the purpose, the present invention adopts the following technical scheme: a method for preparing an organic polymer composite nanomaterial for ultrasound-induced copper death treatment of tumor cells, wherein the organic polymer composite nanomaterial is made of an outer shell wrapped around an inner core, the inner core is a sonosensitizer and a copper death drug, and the outer shell is a nano spherical shell;
[0007] The nanosphere shell is formed by the reaction of compound 1 and N-isopropylacrylamide (NIPAM); the compound 1 is formed by the reaction of 2-methylallylamine (2-MAA) and thioketal (TK), and the reaction structure is as follows:
[0008]
[0009] Furthermore, the preparation method of compound 1 is specifically as follows: 2-methylallylamine (2-MAA), thioketal (TK), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS), and then an appropriate amount of ultrapure water is added to fully dissolve them, react at room temperature for about 3 hours, and spin dry and purify through a column to obtain compound 1. Among them, the molar ratio of 2-MAA, TK, EDC, and NHS is 2:1:2:2.
[0010] Furthermore, the sonosensitizer is a sonosensitizer PBDNPs aqueous solution, and the preparation steps are as follows:
[0011] 1) 4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]-2,6-bis(trimethylstannyl)benzo[1,2-b:4,5-b']dithiophene is reacted with 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-hexyldecyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione to form an organic conjugated polymer PBD.
[0012] The preparation method of the organic conjugated polymer PBD is as follows: 4,8-bis[5-(2-ethylhexyl)thiophene-2-yl]-2,6-bis(trimethylstannyl)benzo[1,2-b:4,5-b']dithiophene, 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-hexyldecyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione and a catalyst are added into a Schlenk tube, anhydrous toluene is then added to dissolve the mixture, the mixture is subjected to multiple freezing cycles for deoxygenation, the mixture is reacted at 110°C in the dark for 4 hours, and after the reaction is completed, the mixture is added dropwise into ice methanol for precipitation, the mixture is centrifuged for washing and purification, and the precipitate is dried in a vacuum oven for standby use to obtain the organic conjugated polymer PBD; the catalyst is 2,6-di-tert-butylphenol and tetrakis(triphenylphosphine)palladium.
[0013] The weight average molecular weight of the organic conjugated polymer PBD is 16735, and the ultraviolet absorption peaks are at 680 and 760 nm.
[0014] 2) The obtained organic conjugated polymer PBD is dissolved in tetrahydrofuran, then injected into ultrapure water under ultrasound, and nitrogen is introduced to remove the tetrahydrofuran to obtain a PBD NPs aqueous solution.
[0015] Furthermore, the specific preparation steps of the organic polymer composite nanomaterial are as follows:
[0016] An aqueous solution of N-isopropylacrylamide, an aqueous solution of compound 1, an aqueous solution of bis(2-methylpropylene)ethoxydisulfide, and an aqueous solution of sodium dodecyl sulfate were added to the reaction flask respectively; an aqueous solution of sonosensitizer PBD NPs and an aqueous solution of copper-killing drug ESCu were then added to the reaction flask, and after nitrogen was passed to remove oxygen, ammonium persulfate was added to react at 72°C for 4 hours, followed by centrifugation, washing, and purification to obtain an organic polymer composite nanomaterial.
[0017] In the specific preparation steps of the organic polymer composite nanomaterial, the volume ratio of the aqueous solution of N-isopropylacrylamide, the aqueous solution of compound 1, the aqueous solution of bis(2-methylpropylene)ethoxy disulfide, the aqueous solution of sodium dodecyl sulfate, the aqueous solution of sonosensitizer PBD NPs, the aqueous solution of copper-death drug ESCu, and ammonium persulfate is 6:0.67:1.2:0.1; 3:1:1; the concentrations of the aqueous solution of N-isopropylacrylamide, the aqueous solution of compound 1, the aqueous solution of bis(2-methylpropylene)ethoxy disulfide, and the aqueous solution of sodium dodecyl sulfate are all 100mM. In the aqueous solution of sonosensitizer PBD NPs, the concentration of PBD NPs is 60μg / mL; in the aqueous solution of copper-death drug ESCu, the concentration of ESCu is 100μg / mL.
[0018] Furthermore, the sonosensitizer generates singlet oxygen under the action of ultrasound.
[0019] The organic polymer composite nanomaterial prepared by the present invention can be used as a medicine for sonodynamic therapy of tumors. In the organic polymer composite nanomaterial, the nanosphere shell wraps the sonosensitizer PBD NPs and the copper-death drug ESCu. During sonodynamic therapy of tumors, ultrasound is used by an ultrasonic therapeutic device, and the sonosensitizer PBD NPs generates singlet oxygen. The nanosphere shell can react with the singlet oxygen and then disintegrate, and the copper-death drug ESCu is released. Both the copper-death drug ESCu and the singlet oxygen can kill tumor cells.
[0020] Compared with the prior art, the present invention has the following advantages:
[0021] The organic polymer composite nanomaterial prepared by the present invention can generate active oxygen at a very fast speed in an ultrasonic environment and has a high active oxygen yield. More importantly, it has extremely high biological safety, which makes it have broad application prospects in the fields of tumor cell therapy and in vivo therapy.
[0022] When comparing common photosensitizers, the sonosensitizer used in the present invention has significant advantages. Its penetration depth far exceeds that of ordinary photosensitizers, which means that it can act more deeply on lesions in the body without being restricted too much by tissue depth. At the same time, the sonosensitizer has good controllability and can accurately control its action time and range according to actual treatment needs, thereby effectively improving the treatment effect. Moreover, the sonosensitizer has fewer side effects and can greatly reduce adverse effects. It is worth mentioning that it can effectively avoid the problem of light damage, which is a common problem in the treatment of photosensitizers, which undoubtedly provides patients with a safer treatment option.
[0023] Under the action of ultrasound, the singlet oxygen produced by the sonosensitizer PBD NPs plays a key role. On the one hand, it can induce apoptosis of tumor cells, directly causing a fatal blow to tumor cells, and inhibiting the growth and spread of tumors. On the other hand, singlet oxygen can also cause the sulfide bonds in the nanoshell to respond by breaking and disintegrating, thereby promoting the release of the copper-death drug ESCu. This unique mechanism realizes the synergistic effect of sonodynamics and copper-death. The two modes of action complement each other and work together to attack tumor cells, thereby killing tumor cells more efficiently, providing a new and powerful means to overcome the problem of tumors. BRIEF DESCRIPTION OF THE DRAWINGS
[0024] Figure 1 Schematic diagram of the synthesis of organic conjugated polymer PBD;
[0025] Figure 2 GPC test characterization of organic conjugated polymer PBD;
[0026] Figure 3 It is the ultraviolet absorption spectrum of organic conjugated polymer PBD;
[0027] Figure 4 is the ultraviolet absorption spectrum of sonosensitizer PBD NPs;
[0028] Figure 5 This is the transmission electron microscope TEM image of organic polymer composite nanomaterials;
[0029] Figure 6 This is the particle size distribution diagram of organic polymer composite nanomaterials;
[0030] Figure 7 Schematic diagram for characterizing the in vitro reactive oxygen generation ability of organic polymer composite nanomaterials;
[0031] Figure 8 Schematic diagram for characterizing the intracellular reactive oxygen generation ability of organic polymer composite nanomaterials;
[0032] Fig. 9 Schematic diagram of the characterization of cytotoxicity of organic polymer composite nanomaterials. DETAILED DESCRIPTION
[0033] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.
[0034] Example 1: Preparation of organic conjugated polymer PBD
[0035] Weigh 90.5 mg of 4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]-2,6-bis(trimethylstannyl)benzo[1,2-b:4,5-b']dithiophene (BDTT), 90.7 mg of 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-hexyldecyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione (DPP), 10 mg of 2,6-di-tert-butylphenol and 10 mg of tetrakis(triphenylphosphine)palladium, add them into a Schlenk tube, then add 5 mL of anhydrous toluene to dissolve it, deoxygenate it after 3 freezing cycles, react it at 110°C in the dark for 4 h, add it dropwise into ice methanol to precipitate after the reaction, wash it by centrifugation and purify it 3 times, then dry the precipitate in a vacuum oven for standby use to obtain a semiconductor organic conjugated polymer PBD. Figure 1 The schematic diagram of the synthesis of organic conjugated polymer PBD is shown in FIG. Figure 2 As shown, the weight average molecular weight of the prepared organic conjugated polymer PBD was measured by gel chromatography GPC to be 16735. Figure 3 As shown, the ultraviolet absorption peaks of the organic conjugated polymer PBD are measured at 680 and 760 nm by ultraviolet absorption spectrometer.
[0036] Example 2: Preparation of PBD NPs
[0037] The semiconductor organic conjugated polymer PBD was added to tetrahydrofuran and fully dissolved to prepare a 100 μg / mL PBD tetrahydrofuran solution. A 20 mL glass bottle was added with 5 mL of ultrapure water and placed in an ultrasonicator for ultrasonication. 3 mL of 100 μg / mL PBD tetrahydrofuran solution was quickly injected into the ultrapure water and ultrasonication was continued for 10 min. Nitrogen was then blown until the tetrahydrofuran was completely removed to obtain an aqueous solution of sonosensitizer PBD NPs. The concentration of PBD NPs was 60 μg / mL. The ultraviolet absorption spectrum of sonosensitizer PBD NPs is shown in the figure below. Figure 4 shown.
[0038] Example 3: Preparation of Compound 1
[0039] 2-Methylallylamine (2-MAA) and thioketal (TK) were weighed in a molar ratio of 2:1, added to a flask, and twice the molar amount of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and twice the molar amount of N-hydroxysuccinimide (NHS) of TK were added, and then an appropriate amount of ultrapure water was added to fully dissolve it, reacted at room temperature for about 3 hours, and then spin-dried and separated and purified by column for later use.
[0040] Example 4: Preparation of organic polymer composite nanomaterials
[0041] N-isopropylacrylamide (NIPAM), compound 1, bis(2-methylpropylene)ethoxy disulfide (DSDMA), sodium dodecyl sulfate (SDS), and ammonium persulfate (APS) were respectively prepared into 100mM aqueous solutions and ultrasonically dissolved; 6mL N-isopropylacrylamide (NIPAM), 0.67mL compound 1, 1.2mL bis(2-methylpropylene)ethoxy disulfide (DSDMA), and 100μL sodium dodecyl sulfate (SDS) were added to a 100mL reaction bottle, respectively; then 3mL of an aqueous solution of sonosensitizer PBDNPs (the concentration of PBD NPs was 60μg / mL) was added, and 1mL of an aqueous solution of copper-killing drug ESCu (100μg / mL) was added. After nitrogen was used to fully remove oxygen for 30min, 1mL of ammonium persulfate (APS) was added and reacted at 72℃ for 4h. Subsequently, after centrifugation and purification for 3 times, an organic polymer composite nanomaterial was obtained.
[0042] The method for preparing the aqueous solution of the copper-killing drug ESCu is as follows: ESCu is prepared by coordinating the purchased Elesclomol (ES) and copper chloride in a 1:1 ratio in dimethyl sulfoxide (DMSO) (this preparation method is the prior art), and then adding ultrapure water to prepare a 100 μg / mL aqueous solution.
[0043] Figure 5 This is the transmission electron microscope TEM image of organic polymer composite nanomaterials. Figure 6 This is the particle size distribution diagram of organic polymer composite nanomaterials. Figure 5 and Figure 6 It can be seen that the organic polymer composite nanomaterial prepared by the method of the present invention has good dispersibility, stable morphology and uniform size.
[0044] Test Example 1: In vitro reactive oxygen generation level test
[0045] Taking 1,3-diphenylisobenzofuran (DPBF) as the active oxygen fluorescence probe, 1 mL of PBD NPs (PBD NPs concentration of 60 μg / mL) prepared in Example 2 and 2 mL of ultrapure water were added into a 3 mL quartz cuvette, and the concentration of PBD NPs in the quartz cuvette was maintained at 20 μg / mL.
[0046] Add 10 μL of 500 μM 1,3-diphenylisobenzofuran DPBF solution (solvent is dimethyl sulfoxide) into the quartz cuvette, and start the test after it is fully dissolved. The ultrasonic therapy instrument parameters are set to an ultrasonic power of 1 W / cm 3 , the frequency is 50kHz, the duty cycle is 50%, and the test records are performed every 2 minutes. The data obtained are as follows Figure 7 As shown, it can be clearly observed that the ultraviolet peak of DPBF is decreasing, indicating that the organic polymer composite nanomaterial has good acoustic dynamic properties.
[0047] Test Example 2: Test of intracellular reactive oxygen generation level
[0048] Breast cancer cells were prepared into cell suspensions and plated in 4 confocal dishes (each confocal dish contained 2×10 5 cells), set up a control group, a control + ultrasound group, an organic polymer composite nanomaterial, and an organic polymer composite nanomaterial + ultrasound group. After incubation overnight, remove the old culture medium in the four confocal dishes, wash them with PBS solution, add new culture medium, and add 100uL of 100μg / mL organic polymer composite nanomaterial aqueous solution to the organic polymer composite nanomaterial group and the organic polymer composite nanomaterial + ultrasound group. Continue to incubate overnight, remove the old culture medium, add new culture medium, and add intracellular active oxygen fluorescent probe dichlorodihydrofluorescein (DCFH-DA) to the four confocal dishes (the amount of DCFH-DA added was 5μL, and the concentration was 1mg / mL) and incubate with the cells for 30min. The size of the confocal dish was 15mm.
[0049] The control group did not add organic polymer composite nanomaterials and did not undergo ultrasonic treatment. The two ultrasound groups were then treated with an ultrasonic therapeutic device, and the parameters of the ultrasonic therapeutic device were set to a power of 1 W / cm 3 , the frequency was 50kHz, the air ratio was 50%, each ultrasound was 2min, and then the laser confocal microscope was used for observation, such as Figure 8 As shown, the organic-polymer composite nanomaterial + ultrasound group has obvious green fluorescence generation, indicating that the organic-polymer composite nanomaterial can produce singlet oxygen after ultrasound.
[0050] Test Example 3: Cytotoxicity Test
[0051] Breast cancer cells were seeded in 96-well plates (cell suspension concentration 1×10 4 / well), incubate overnight, set up organic polymer composite nanomaterials, organic polymer composite nanomaterials + ultrasound groups, remove the old culture medium. Add 150 μL of organic polymer composite nanomaterial culture medium mixed solution of different concentrations, i.e. 0, 5, 10, 20, 30, 40 μg / mL, to the wells, and set 6 replicate wells for each concentration.
[0052] After incubation overnight, the organic polymer composite nanomaterial + ultrasound group was subjected to ultrasound, and the ultrasound therapy instrument parameters were set to a power of 1 W / cm 3 , frequency is 50kHz, empty ratio is 50%, each ultrasound is 2min, then culture for 12h, then remove the culture medium, add MTT cytotoxicity test reagent (MTT reagent and diluent are configured in a ratio of 1:5), add 50μL to each well, incubate for 4h, remove the cytotoxicity test reagent, add 150μL dimethyl sulfoxide (DMSO) to each well, place on a shaker to fully shake for 30min, and test with an enzyme marker. The test results are as follows Fig. 9 As shown, the organic polymer composite nanomaterials + ultrasound group has the best killing effect on tumor cells.
[0053] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention.
Claims
1. A method for preparing an organic polymer composite nanomaterial for ultrasonically induced copper death, characterized in that: The organic polymer composite nanomaterial is made of a core wrapped by a shell, the core is a sonosensitizer and a copper-death drug, and the shell is a nanosphere shell; The nanosphere shell is formed by the reaction of compound 1 and N-isopropylacrylamide; the compound 1 is generated by the reaction of 2-methylallylamine and thioketal, and the reaction structure is as follows:
2. The preparation method according to claim 1, characterized in that: The sonosensitizer is a sonosensitizer PBD NPs aqueous solution, and the preparation steps are as follows: Step (1), reacting 4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]-2,6-bis(trimethylstannyl)benzo[1,2-b:4,5-b']dithiophene with 3,6-bis(5-bromo-2-thienyl)-2,5-bis(2-hexyldecyl)pyrrolo[3,4-c]pyrrole-1,4(2H,5H)-dione to generate an organic conjugated polymer PBD; Step (2), dissolving the obtained organic conjugated polymer PBD with tetrahydrofuran, then injecting it into ultrapure water under ultrasound, and then introducing nitrogen to remove the tetrahydrofuran to obtain a PBD NPs aqueous solution.
3. The preparation method according to claim 2, characterized in that: The specific preparation steps of organic polymer composite nanomaterials are as follows: An aqueous solution of N-isopropylacrylamide, an aqueous solution of compound 1, an aqueous solution of bis(2-methylpropylene)ethoxydisulfide, and an aqueous solution of sodium dodecyl sulfate were added to the reaction flask respectively; an aqueous solution of sonosensitizer PBD NPs and an aqueous solution of copper-killing drug ESCu were then added to the reaction flask, and after nitrogen was passed to remove oxygen, ammonium persulfate was added to react at 72°C for 4 hours, followed by centrifugation, washing, and purification to obtain an organic polymer composite nanomaterial.
4. The preparation method according to claim 3, characterized in that: The specific preparation steps of organic polymer composite nanomaterials are: The volume ratios of aqueous solution of N-isopropylacrylamide, aqueous solution of compound 1, aqueous solution of bis(2-methylpropylene)ethoxydisulfide, aqueous solution of sodium dodecyl sulfate, aqueous solution of sonosensitizer PBD NPs, aqueous solution of copper-killing drug ESCu, and ammonium persulfate are 6:0.67:1.2:0.1; 3:1:1; The concentrations of the aqueous solution of N-isopropylacrylamide, the aqueous solution of compound 1, the aqueous solution of bis(2-methylpropylene)ethoxydisulfide, and the aqueous solution of sodium dodecyl sulfate were all 100 mM; The concentration of PBD NPs in the sonosensitizer PBD NPs aqueous solution is 60 μg / mL; the concentration of ESCu in the copper-killing drug ESCu aqueous solution is 100 μg / mL.
5. The preparation method according to claim 2, characterized in that: The weight average molecular weight of the organic conjugated polymer PBD is 16735, and the ultraviolet absorption peaks are at 680 and 760 nm.
6. The preparation method according to claim 2, characterized in that: The sonosensitizer generates singlet oxygen under the action of ultrasound.
7. An organic polymer composite nanomaterial for ultrasonically induced copper death obtained by the preparation method according to any one of claims 1 to 6.
8. Use of the organic polymer composite nanomaterial according to claim 7 in sonodynamic tumor therapy agents.