ROS (reactive oxygen species) responsive nanoparticles as well as preparation method and application thereof
Through nanoparticles composed of oxaliplatin polyprodrug and semiconductor polymer, the efficient ROS response release and photothermal photodynamic synergistic treatment of oxaliplatin are achieved, solving the problem of poor chemotherapy for ovarian cancer in the prior art, and enhancing the targeting and immune response of tumor treatment.
Patent Information
- Application Number
- CN202510602372.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-12
- Publication Date
- 2025-07-22
AI Technical Summary
In the prior art, the oxaliplatin nanodelivery system lacks an efficient ROS response and release mechanism, and the synergistic effect with semiconductor polymers is insufficient, resulting in limited chemotherapy effect of ovarian cancer, weak targeting and significant chemotherapy resistance.
Nanoparticles composed of oxaliplatin polyprodrug OTP and semiconductor polymer SP are used to responsively release oxaliplatin through ROS-sensitive bonds, combining photothermal and photodynamic effects to achieve synergistic treatment.
It enhances the anti-tumor effect of ovarian cancer treatment, improves the targeted and immune response of chemotherapy through drug enrichment and responsive release at the tumor site, and achieves tumor cell killing and immunogenic cell death.
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Figure CN120346318A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and more specifically, to a kind of nanoparticle with ROS-responsive function, its preparation method and application. Background Art
[0002] Ovarian cancer is one of the most lethal malignant tumors in the female reproductive system. Although the traditional chemotherapy drug oxaliplatin can induce the death of tumor cells, its poor water solubility, weak targeting and chemotherapy drug resistance significantly limit the curative effect. The nano-drug delivery system has become a key strategy to improve the chemotherapy effect by enhancing the drug enrichment at the tumor site (EPR effect) and responsive release. As a new type of organic photonic material, semiconductor polymer (SP) has both photothermal effect (PTT) and photodynamic effect (PDT), and can generate reactive oxygen species (ROS) or heat through laser irradiation to induce the death of tumor cells and activate the immune response. However, the anti-tumor effect of single therapy is limited, and there is an urgent need to develop a synergistic treatment system combining chemotherapy, photothermal and photodynamic effects. In the prior art, the nano-delivery system of oxaliplatin lacks an efficient ROS-responsive release mechanism, and the synergistic effect with semiconductor polymer has not been studied enough. Therefore, there is an urgent need to develop a new treatment strategy that can improve the efficacy of oxaliplatin immunotherapy to achieve the purpose of effectively eliminating tumors and enhancing the immune response. Summary of the Invention
[0003] The purpose of the present invention is to provide a kind of ROS-responsive nanoparticle to solve the deficiencies of the prior art. Through the synergistic effect of the responsive release of oxaliplatin prodrug and the photothermal / photodynamic effect of semiconductor polymer, the anti-tumor efficacy is enhanced, especially suitable for the treatment of ovarian cancer.
[0004] Another purpose of the present invention is to provide a preparation method of the ROS-responsive nanoparticle.
[0005] Another purpose of the present invention is to provide an application of the ROS-responsive nanoparticle.
[0006] The present invention adopts the following technical solution to achieve the above purpose: A kind of ROS-responsive nanoparticle, characterized in that it mainly consists of oxaliplatin prodrug OTP and semiconductor polymer SP; the molecular structure of the oxaliplatin prodrug includes oxaliplatin, a molecule containing a ROS-sensitive responsive bond, a linking molecule and a polyether derivative; the semiconductor polymer has the following molecular structure of formula I: , formula I; in formula I, R is an organic group for increasing solubility and compatibility.
[0007] As a further description of the above solution, the molecular weight of the oxaliplatin prodrug OTP is 15,000-35,000, and the platinum element content is 4.0-5.0%.
[0008] Furthermore, the oxaliplatin is oxaliplatin oxide; the ROS-sensitive responsive bond is one or more of a thioacetal bond, a thioether bond, and a borate ester bond; the linking molecule is L-lysine diisocyanate; the molecular weight of the polyether derivative is 200-10,000; the structure of the polyether derivative is at least one of a linear or branched polyether derivative; the linear polyether derivative is one or more of methoxypolyethylene glycol, aminomethoxypolyethylene glycol, and mercaptomethoxypolyethylene glycol.
[0009] Furthermore, in the formula I structure, R is one or more of an alkyl group, a polyethylene glycol derivative, and a polypropylene glycol derivative.
[0010] A method for preparing ROS-responsive nanoparticles, characterized in that it comprises the following specific steps:
[0011] S1. Prepare oxaliplatin prodrug OTP:
[0012] S11. Suspend oxaliplatin in 30% hydrogen peroxide solution, stir at room temperature for 16-30 hours, after drying, dissolve the product in methanol, precipitate with cold ether, filter and collect the precipitate, and dry in vacuum to obtain oxaliplatin oxide;
[0013] S12. Quickly add the linking molecule to the anhydrous DMF solution of the oxaliplatin oxide obtained in step S1 and the molecule containing the Ros-sensitive responsive bond, stir at room temperature for 16-30 hours, then add the polyether derivative to the reaction mixture, and magnetically stir at 45-55 °C for 40-55 hours. After that, dialyze the reaction solution in a dialysis bag (cut-off molecular weight: 8000-14000 Da) for 60-72 hours; freeze-dry the solution to obtain oxaliplatin prodrug OTP;
[0014] S2. Prepare semiconductor polymer SP:
[0015] Disperse 4,7-dibromo-5,6-difluorobenzo[c][1,2,5]thiadiazole, 4-octyl-2,6-bis(trimethylstannyl)-4H-dithieno[3,2-b:2',3'-d]pyrrole, and tetrakis(triphenylphosphine)palladium in toluene solution, add potassium carbonate and then degas by freezing-pumping, and carry out Stille polycondensation reaction under an argon atmosphere. The reaction temperature is 90-110 °C and the reaction time is 40-55 hours. Drop the resulting mixture into methanol, centrifuge to obtain a dark precipitate, wash with methanol at least three times, and dry in vacuum to obtain semiconductor polymer SP;
[0016] S3. Dissolve the oxaliplatin prodrug OTP and the semiconductor polymer SP into dimethyl sulfoxide respectively, mix and stir, slowly drop the mixture into deionized water under ultrasonic oscillation, continue ultrasonic treatment for 15 - 30 min, place the mixed solution in a dialysis bag with a molecular weight cut-off of 3000 - 5000 and dialyze for 5 - 10 h to obtain particles with a particle size of 100 - 200 nm.
[0017] Furthermore, the mass ratio of the oxaliplatin prodrug OTP to the semiconductor polymer SP is 20:1, and they self-assemble to form nanoparticles OSN with a particle size of 100 - 200 nm.
[0018] An application of ROS-responsive nanoparticles, characterized in that it is applied in drug delivery and the preparation of anti-tumor drugs.
[0019] Furthermore, the tumor is ovarian cancer.
[0020] The beneficial effects that can be achieved by the present invention adopting the above technical solutions are:
[0021] The present invention uses the oxaliplatin prodrug OTP and the semiconductor polymer SP to self-assemble into nanoparticles with a particle size of 100 - 200 nm in water, and passively accumulate in tumor sites through the enhanced permeability and retention effect of solid tumors, increasing the drug concentration in tumor tissues; the oxaliplatin prodrug OTP can respond to high levels of ROS and GSH in tumor cells, release oxaliplatin to kill tumor cells and induce immunogenic cell death; the semiconductor polymer SP can generate reactive oxygen species and photothermal effects under 660 nm laser irradiation, perform photodynamic therapy and photothermal therapy to further kill tumor cells, and enhance the immunogenic cell death induced by oxaliplatin. Description of the Drawings
[0022] Figure 1 1H NMR characterization of the oxaliplatin prodrug OTP in Example 1 of the present invention;
[0023] Figure 2 1H NMR characterization of the semiconductor polymer SP in Example 1 of the present invention;
[0024] Figure 3 Particle size, potential characterization and electron microscopy images of the nanoparticles OSN in Example 2 of the present invention;
[0025] Figure 4 Stability of the nanoparticles OSN in PBS and FBS solutions in Example 2 of the present invention;
[0026] Figure 5 Inhibitory effect on the viability of ID8 ovarian cancer cells after the nanoparticles OSN act for 24 h in Example 3 of the present invention;
[0027] Figure 6 In Example 3 of the present invention, nanoparticles OSN increased the apoptosis of ID8 ovarian cancer cells after acting for 24 h;
[0028] Figure 7 In Example 4 of the present invention, in vivo imaging of nanoparticles OSN in a subcutaneous tumor model of ID8 ovarian cancer in mice;
[0029] Figure 8 In Example 4 of the present invention, in vivo photothermal effect of nanoparticles OSN in a subcutaneous tumor of ID8 ovarian cancer in mice;
[0030] Figure 9 In Example 5 of the present invention, in vivo therapeutic effect of nanoparticles OSN in a subcutaneous tumor of ID8 ovarian cancer in mice. Detailed implementation manners
[0031] In the description of the present invention, it should be noted that for orientation terms, such as the terms "center", "horizontal", "longitudinal", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", etc., the orientation and position relationships indicated are based on the orientation or position relationships shown in the drawings. It is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and should not be construed as limiting the specific protection scope of the present invention.
[0032] In addition, such terms as "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance or implicitly specifying the quantity of technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include one or more of such features. In the description of the present invention, the meaning of "at least" is one or more than one, unless otherwise specifically defined.
[0033] In the present invention, unless otherwise clearly specified and limited, such terms as "assembled", "connected" and "joined" should be understood in a broad sense. For example, it may be a fixed connection, a detachable connection or an integral connection; it may also be a mechanical connection; it may be directly connected or connected through an intermediate medium, and it may be internally connected and communicated between two elements. For those of ordinary skill in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.
[0034] In the invention, unless otherwise specified and defined, the first feature being "above" or "below" the second feature may include direct contact between the first and second features, or may include the first and second features not being in direct contact but in contact through additional features therebetween. Moreover, the first feature being "above", "below", and "on top of" the second feature includes the first feature being directly above and diagonally above the second feature, or merely indicating that the horizontal height of the first feature is higher than that of the second feature. The first feature being "above", "below", and "beneath" the second feature includes the first feature being directly below or diagonally below the second feature, or merely indicating that the horizontal height of the first feature is lower than that of the second feature.
[0035] The following further describes the specific embodiments of the present invention in conjunction with the accompanying drawings of the specification, making the technical solutions and their beneficial effects of the present invention clearer and more definite. The embodiments described below by referring to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as limiting the present invention.
[0036] As Figures 1-9 shown, the present invention is a ROS-responsive nanoparticle, which mainly consists of an oxaliplatin-containing prodrug OTP and a semiconductor polymer SP; the molecular structure of the oxaliplatin prodrug includes oxaliplatin, a molecule containing a ROS-sensitive responsive bond, a linking molecule, and a polyether derivative; the semiconductor polymer has the molecular structure of Formula I as follows: , Formula I; in Formula I, R is an organic group for increasing solubility and compatibility. The molecular weight of the oxaliplatin prodrug OTP is 15,000 - 35,000, and the platinum element content therein is 4.0 - 5.0%. The oxaliplatin is oxaliplatin oxide; the ROS-sensitive responsive bond is one or more of a thioketal bond, a thioether bond, and a borate ester bond; the linking molecule is L-lysine diisocyanate; the molecular weight of the polyether derivative is 200 - 10,000; the structure of the polyether derivative is at least one of a linear or branched polyether derivative; the linear polyether derivative is one or more of methoxypolyethylene glycol, aminomethoxypolyethylene glycol, and mercaptomethoxypolyethylene glycol. In the structure of Formula I, R is one or more of an alkyl group, a polyethylene glycol derivative, and a polypropylene glycol derivative. Example
[0037] I. Synthesis of oxaliplatin prodrug OTP and novel semiconductor polymer SP.
[0038] First, oxaliplatin (0.5 g, 1.26 mmol) was suspended in 10 mL of 30% hydrogen peroxide solution and stirred at room temperature for 24 hours. Then it was dried with a freeze dryer. The product was dissolved in methanol, precipitated with cold diethyl ether, the precipitate was collected by filtration, and dried in vacuo to obtain white powdery oxidized oxaliplatin (505 mg, yield 93%). Then, L-lysine diisocyanate (250 mg, 1.1 mmol) was quickly added to an anhydrous DMF solution of oxidized oxaliplatin (216 mg, 0.5 mmol) and the ROS-sensitive linker 2,2'-(propane-2,2-diylbis(sulfinyl))diethanol (98 mg, 0.5 mmol). After magnetic stirring at room temperature for 24 hours, mPEG5000-OH (550 mg, 0.11 mmol) was added to the reaction mixture. After magnetic stirring at 50 °C for 48 hours, the reaction solution was dialyzed in a dialysis bag (cut-off molecular weight: 8000 - 14000 Da) for 72 hours. Subsequently, the solution was freeze-dried under reduced pressure to obtain light yellow powdery OTP. The NMR spectrum of the obtained OTP product is as shown in Figure 1 shown.
[0039] 4,7-Dibromo-5,6-difluorobenzo[c][1,2,5]thiadiazole (133 mg, 0.405 mmol), 4-octyl-2,6-bis(trimethylstannyl)-4H-dithieno[3,2-b:2',3'-d]pyrrole (250 mg, 0.405 mmol) and tetrakis(triphenylphosphine)palladium (20 mg) were added, and then toluene (20 mL). Potassium carbonate (20 mg) was dissolved in a small amount of water and added to the reaction tube. Then the mixture was degassed by three freeze-pump-thaw cycles. Subsequently, a Stille polycondensation reaction was carried out at 100 °C under an argon atmosphere for 48 hours. The resulting mixture was dropped into methanol, and a dark precipitate was obtained after centrifugation. The obtained solid was washed three times with methanol and then purified, and then dried in vacuo to obtain the final product SP. The NMR spectrum of the obtained SP product is as shown in Figure 2 shown.
[0040] II. Preparation and characterization of nanoparticles OSN.
[0041] Oxaliplatin prodrug OTP and semiconductor polymer SP were accurately weighed and dissolved in 1 mL of dimethyl sulfoxide respectively. Nanoparticles OSN were prepared by the nanoprecipitation method according to different ratios. The drug ratios of OTP and SP are shown in Table 1. The oxaliplatin prodrug OTP and semiconductor polymer SP were self-assembled at an appropriate feeding ratio of 20:1 by mass to form the most ideal nanoparticle size and similar drug concentrations. As shown in Figure 3As shown, inspection of the particle size and potential by a particle size analyzer revealed that the particle size of OSN was 157.47 ± 4.5 nm and the potential was -13.48 ± 0.79 mV. Transmission electron microscopy images showed that the nanoparticles OSN were dispersed and had a uniform shape. Figure 4 The stability test indicated that the nanoparticles OSN could stably exist in phosphate buffered saline solution (PBS) and 10% fetal bovine serum solution (FBS) for 7 days, suggesting that OSN may have good stability in vivo.
[0042] Table 1 Characteristics of nanoparticles OSN prepared with different drug ratios .
[0043] III. In vitro therapeutic effect of nanoparticles OSN.
[0044] The antitumor effect of nanoparticles OSN in ovarian cancer ID8 cells was further investigated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. As Figure 5 shown, the half inhibitory concentration (IC50) of oxaliplatin was 9.68 μg / ml, and that of OSN was 8.71 μg / ml. However, after laser irradiation, the IC50 value of the SP + light irradiation group was 4.92 μg / ml, and that of the OSN + light irradiation group was 0.47 μg / ml. The cytotoxicity of both groups was higher than the IC50 value of 1.172 μg / ml of the oxaliplatin + SP + light irradiation group. Subsequently, Annexin V-FITC / PI staining was used to further detect apoptosis. As Figure 6 shown, consistent with the MTT results, after treatment with 1.25 μg / ml of platinum or SP, the apoptosis rate of the OSN + light irradiation group was the highest (53.1 ± 4.3%). In contrast, the apoptosis rate of the OSN group without laser irradiation was only 11.5 ± 1.3%, indicating that laser irradiation could significantly activate apoptosis induced by nanoparticles OSN.
[0045] IV. In vivo distribution of nanoparticles OSN.
[0046] The biodistribution of nanodrugs is a prerequisite for their efficacy and biosafety. We established an ID8 tumor mouse model to evaluate the biodistribution of nanoparticles OSN, studied the targeting ability of OSN by real-time fluorescence imaging, and used ID8 tumor mice injected with free drug SP via the tail vein as a control. As Figure 7 shown in A, after intravenous injection of nanoparticles OSN, the effective accumulation and persistent retention ability in tumor tissues were superior to those of free drug SP. The animals were sacrificed after 48 hours, the main organs were collected, and the biodistribution was determined by fluorescence imaging. As Figure 7As shown in Figure B, some OSNs are present in the liver, but a large amount of OSNs are also detected in tumor tissues. Intravenous injection of SP mainly accumulates in the liver, and only a small amount of the drug is observed in tumors. In vitro and in vivo fluorescence imaging and semi-quantification of fluorescence intensity confirmed the tumor targeting ability of the nanoparticle OSN, indicating that the nanoparticle OSN can effectively accumulate at the tumor site, thereby exerting an anti-tumor effect.
[0047] V. In vivo therapeutic effect of nanoparticle OSN.
[0048] The ability of nanoparticle OSN to generate a photothermal effect under laser irradiation was studied in an ID8 tumor mouse model. Mice were intravenously injected with PBS, free drug SP, and nanoparticle OSN, and then the tumor site was irradiated with a laser (660 nm, 0.6 W / cm 2 ) to monitor the temperature change at the tumor site. As Figure 8 shown, laser irradiation itself increased the tumor temperature by about 3 °C, but the nanoparticle OSN group could increase the tumor temperature by 20 °C within 4 minutes after irradiation, significantly higher than the 9 °C increase in the free drug SP group. These results indicate that the accumulation effect of nanoparticle OSN at the tumor site is better than that of SP, and it can produce a significant photothermal effect at the tumor site under laser irradiation. Further study on the inhibitory effect of nanoparticle OSN + light irradiation on tumor growth. On the 1st, 3rd, and 5th days, animals were intravenously injected with different drugs. 12 hours after injection, the tumor site was irradiated with a laser (660 nm, 0.6 W / cm 2 , 2 minutes). As Figure 9 shown, the oxaliplatin group and the nanoparticle OSN group without laser irradiation caused a slight reduction in tumor volume, while laser irradiation led to a significant remission of the tumor. Among them, nanoparticle OSN + laser irradiation was the most effective in inhibiting tumors.
[0049] Compared with the prior art, the present invention adopts ROS-responsive nanoparticles self-assembled from oxaliplatin prodrug and semiconductor polymer; the oxaliplatin prodrug releases drugs in response to the tumor microenvironment through ROS-sensitive bonds, and the semiconductor polymer has both photothermal and photodynamic effects, synergistically inducing tumor cell death; the preparation method includes prodrug synthesis, semiconductor polymer preparation, and nanoparticle self-assembly, with a particle size of 100 - 200 nm, suitable for the treatment of ovarian cancer, and having the advantages of targeted enrichment, responsive release, and synergistic treatment.
[0050] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the inventive concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention.
Claims
1. A ROS-responsive nanoparticle, characterized in that, It mainly consists of an oxaliplatin-containing prodrug OTP and a semiconductor polymer SP; in the molecular structure of the oxaliplatin prodrug, it includes oxaliplatin, a molecule containing a ROS-sensitive response bond, a linking molecule, and a polyether derivative; the semiconductor polymer has the molecular structure of formula I as follows: , Formula I; in Formula I, R is an organic group that increases solubility and compatibility.
2. The ROS-responsive nanoparticles according to claim 1, characterized in that, The molecular weight of the oxaliplatin prodrug OTP is 15,000 - 35,000, and the platinum element content is 4.0 - 5.0%.
3. The ROS-responsive nanoparticles according to claim 1, characterized in that, The oxaliplatin is oxaliplatin oxide; the ROS-sensitive response bond is one or more of thioacetal bond, thioether bond, and borate ester bond; the linking molecule is L-lysine diisocyanate; the molecular weight of the polyether derivative is 200 - 10,000; the structure of the polyether derivative is at least one of linear or branched polyether derivatives; the linear polyether derivative is one or more of methoxypolyethylene glycol, aminomethoxypolyethylene glycol, and mercaptomethoxypolyethylene glycol.
4. A ROS-responsive nanoparticle according to claim 1, characterized in that, In the structure of formula I, R is one or more of an alkyl group, a polyethylene glycol derivative, and a polypropylene glycol derivative.
5. A preparation method corresponding to the ROS-responsive nanoparticles described in any one of claims 1-4, characterized in that, It includes the following specific steps: S1. Prepare the oxaliplatin prodrug OTP: S11. Suspend oxaliplatin in 30% hydrogen peroxide solution, stir at room temperature for 16 - 30 hours, after drying, dissolve the product in methanol, precipitate with cold diethyl ether, filter and collect the precipitate, and dry it under vacuum to obtain oxaliplatin oxide; S12. Quickly add the linking molecule to the anhydrous DMF solution of the oxaliplatin oxide obtained in step S1 and the molecule containing a Ros-sensitive response bond, stir at room temperature for 16 - 30 hours, then add the polyether derivative to the reaction mixture, magnetically stir at 45 - 55 °C for 40 - 55 hours, and dialyze the reaction solution in a dialysis bag (cut-off molecular weight: 8000 - 14000 Da) for 60 - 72 hours; freeze-dry the solution to obtain the oxaliplatin prodrug OTP; S2. Prepare the semiconductor polymer SP: Disperse 4,7-dibromo-5,6-difluorobenzo[c][1,2,5]thiadiazole, 4-octyl-2,6-bis(trimethylstannyl)-4H-dithieno[3,2-b:2',3'-d]pyrrole, and tetrakis(triphenylphosphine)palladium in toluene solution, add potassium carbonate and then degas by freezing-pumping, carry out Stille polycondensation reaction under an argon atmosphere, the reaction temperature is 90 - 110 °C, the reaction time is 40 - 55 hours, drop the resulting mixture into methanol, centrifuge to obtain a dark precipitate, wash it with methanol at least three times, and dry it in vacuo to obtain the semiconductor polymer SP; S3. Dissolve the oxaliplatin prodrug OTP and the semiconductor polymer SP into dimethyl sulfoxide respectively, mix and stir, slowly drop it into deionized water under ultrasonic oscillation, continue ultrasonic for 15 - 30 min, dialyze the mixed solution in a dialysis bag with a cut-off molecular weight of 3000 - 5000 for 5 - 10 h to obtain particles with a particle size of 100 - 200 nm.
6. The preparation method of a ROS-responsive nanoparticle according to claim 5, characterized in that, The mass ratio of the oxaliplatin prodrug OTP to the semiconductor polymer SP is 20:1, and they self-assemble to form nanoparticles OSN with a particle size of 100 - 200 nm.
7. Use of the ROS-responsive nanoparticles as described in claims 1-5, characterized in that, It is applied in drug delivery and the preparation of anti-tumor drugs.
8. Use of the ROS-responsive nanoparticles according to claim 7, characterized in that, The tumor is ovarian cancer.