A degradable polymer, nanoparticles and a preparation method and application thereof

Abstract

This invention belongs to the field of biochemical technology, specifically relating to a biodegradable polymer, nanoparticles, their preparation method, and applications. The biodegradable polymer is copolymerized from a conjugated dibromomonone, a bistin monomer, and a responsive dibromomonone; wherein the responsive dibromomonone contains at least two bromine groups and includes chemical bonds or functional groups that can break in response to oxidative, reducing, hypoxic, or acid-base conditions; the bistin monomer contains two trimethyltin groups; and the conjugated dibromomonone has a conjugated structure and at least two bromine groups. The biodegradable polymer provided by this invention possesses excellent degradation performance, fluorescence imaging capabilities, and sonodynamic effects, and can be used to construct a safe integrated diagnostic and therapeutic platform.

Description

Technical Field

[0001] This invention belongs to the field of biochemical technology, and specifically relates to a biodegradable polymer, nanoparticles, their preparation methods and applications. Background Technology

[0003] Over the past few decades, nanomaterials have been extensively studied in the biomedical field, particularly in drug delivery and bioimaging. Due to their ease of modification, good solubility, and multifunctionality, nanomaterials are considered a promising platform for integrating diagnosis and treatment.

[0004] Currently, some organic conjugated polymers possess excellent optical properties or sonodynamic effects and can be prepared into nanoparticles through encapsulation with amphiphilic polymers, showing great potential in the field of tumor diagnosis and treatment. However, because these conjugated polymers typically have stable chemical structures, they are difficult to degrade in vivo, posing a risk of long-term retention, which severely limits their application in vivo. Existing organic conjugated polymers cannot simultaneously achieve good biodegradability and efficient fluorescence imaging and sonodynamic therapy performance, thus hindering the construction of a safe integrated diagnostic and therapeutic platform. Summary of the Invention

[0005] To address the challenge of existing organic conjugated polymers simultaneously achieving biodegradability and therapeutic performance (fluorescence imaging / sonodynamic therapy), this invention provides a biodegradable polymer, nanoparticles, their preparation method, and applications. The biodegradable polymer provided by this invention exhibits excellent biodegradability, further enhancing its biosafety, and also possesses good fluorescence imaging and sonodynamic effects, making it suitable for constructing safe therapeutic platforms.

[0006] The first aspect of the present invention provides a biodegradable polymer obtained by polymerization of a conjugated dibromo monomer, a bistin monomer, and a responsive dibromo monomer; The responsive dibromo monomer has chemical bonds or groups that can be broken in response to oxidative, reducing, hypoxic, or acid-base conditions; the responsive dibromo monomer also has at least two bromine groups; The bistin monomer contains two trimethyltin groups; The conjugated dibromo monomer has a conjugated structure and at least two bromine groups.

[0007] The present invention involves the polymerization of a responsive dibromo monomer and a ditin-containing monomer via a Stille coupling reaction in the presence of a catalyst to obtain the biodegradable polymer of the present invention. This biodegradable polymer can degrade in response to the cellular microenvironment.

[0008] The biodegradable polymer provided by this invention can generate ROS under ultrasound, emit fluorescence to guide treatment, and damage tumor cells.

[0009] In some embodiments, the chemical bonds that can break in response to oxidizing conditions, reducing conditions, hypoxic conditions, or acid-base conditions include at least one of disulfide bonds, diselenide bonds, and thioacetate bonds. In some embodiments, the groups that can be broken in response to oxidizing conditions, reducing conditions, hypoxic conditions or acid-base conditions include at least one of thioacetal and acetal. In some embodiments, the degradable polymer is a block copolymer.

[0010] In some embodiments, the groups that can be cleaved in response to oxidizing conditions, reducing conditions, hypoxic conditions, or acid-base conditions include , , , , At least one of them.

[0011] In some embodiments, the ratio of the total molar number of the responsive dibromomon and the conjugated dibromomon to the molar number of the bistin monomer is (1-1.2):(1-1.2). In some embodiments, the ratio of the total molar number of the responsive dibromomon and the conjugated dibromomon to the molar number of the bistin monomer is 1:1; In some embodiments, the molar ratio of the responsive dibromomon to the conjugated dibromomon is (0.1-0.5):(0.5-0.9).

[0012] In some embodiments, the responsive dibromo monomer is selected from at least one of the following groups of compounds: , , , , , , ; In some embodiments, the conjugated dibromo monomer is selected from at least one of the following groups of compounds: , , , , , , , , , , , .

[0013] In some embodiments, the bistin monomer is selected from at least one of the following groups of compounds: , , , , , , .

[0014] In some embodiments, the biodegradable polymer is obtained by polymerization of a conjugated dibromo monomer, a bistin monomer, and a responsive dibromo monomer; In some embodiments, the biodegradable polymer is obtained by polymerization of a conjugated dibromo monomer, a bistin monomer, and two responsive dibromo monomers; In some embodiments, the biodegradable polymer includes a first monomer unit, a second monomer unit, and a third monomer unit; In some embodiments, the first monomer unit is selected from at least one of the following groups of compounds: , , , , , , ; In some embodiments, the second monomer unit is selected from at least one of the following groups of compounds: , , , , , , , , , , , .

[0015] In some embodiments, the third monomer unit is selected from at least one of the following groups of compounds: , , , , , , .

[0016] In some embodiments, the biodegradable polymer comprises at least one selected from the group consisting of: Formula I Formula II Formula III Formula IV Formula V Formula VI Formula VII The molar ratio of m to n is (0.1-0.5):(0.5-0.9). In the biodegradable polymer structural formula of this invention, r (random) indicates that the two monomer units connected to it are randomly arranged on the polymer backbone.

[0017] A second aspect of the present invention provides a method for preparing the above-mentioned biodegradable polymer, comprising the following steps: The biodegradable polymer is obtained by polymerizing conjugated dibromo monomers, ditin monomers, and responsive dibromo monomers in an organic solvent under catalytic conditions.

[0018] In some embodiments, the organic solvent includes toluene; In this invention, the completion of the reaction is mainly determined by whether the reaction system undergoes a significant color change. In some embodiments, the polymerization reaction temperature is 105-115℃ and the polymerization reaction time is 5-20 min; in some embodiments, the polymerization reaction temperature is 110℃ and the polymerization reaction time is 10 min.

[0019] In some embodiments, the ratio of the bistin monomer to the organic solvent is (0.1-0.2):5, in mmol / mL.

[0020] In some embodiments, the catalyst comprises at least one of tri-tert-butylphosphine (P(t-Bu)3) and tris(dibenzylideneacetone)palladium (Pd2(dba)3); the ratio of the total molar amount of the catalyst to the total molar amount of the conjugated dibromo monomer, the bistin monomer and the responsive dibromo monomer is (0.01-0.03):(0.2-0.35).

[0021] A third aspect of the present invention provides the application of the above-described biodegradable polymer or the biodegradable polymer prepared by the above-described preparation method in the preparation of nanoparticles with fluorescence imaging and sonodynamic therapy functions.

[0022] A fourth aspect of the present invention provides a nanoparticle comprising: a) an amphiphilic polymer; b) the above-described biodegradable polymer or a biodegradable polymer prepared by the above-described preparation method.

[0023] The nanoparticles provided by this invention are prepared by coating the aforementioned biodegradable polymer with an amphiphilic polymer. After intravenous injection and blood circulation, they accumulate at the tumor site and are then treated with ultrasound to kill tumor cells. In the tumor microenvironment, the amphiphilic polymer-coated polymer is biodegradable under oxidative conditions, achieving better biocompatibility.

[0024] In some embodiments, the amphiphilic polymer is an amphiphilic polymer capable of loading radionuclides.

[0025] In some embodiments, the mass ratio of the amphiphilic polymer to the degradable polymer is (5-15):1; In some embodiments, the amphiphilic polymer includes distearate-phosphatidylethanolamine-polyethylene glycol (DSPE-PEG 2000), methoxy-polyethylene glycol-polylactic acid block copolymer (mPEG-PLA), methoxy-polyethylene glycol-polylactic-glycolic acid copolymer (mPEG-PLGA), and polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer (PEG-b-PPG-b-PEG). At least one of them; In some embodiments, the amphiphilic polymer is In addition to being amphiphilic, it also has the functions of being degradable and capable of loading nuclides.

[0026] The fifth aspect of this invention provides a novel amphiphilic polymer capable of loading nuclides, having the following structure:

[0027] This amphiphilic biodegradable polymer with DOTA-branched side chains is biodegradable under ultrasound conditions. The radionuclide chelating unit (DOTA) is chemically bonded to the side chains of the biodegradable polymer, achieving stable loading of radionuclides. Unlike existing technologies that commonly use physically mixed radionuclide ligands or non-degradable carrier materials, this amphiphilic biodegradable polymer combines biodegradability with robust radionuclide integration capabilities in a single molecular structure. This aims to reduce the potential risks of long-term retention of amphiphilic carriers and minimize non-specific release of radionuclides in vivo. This polymer can self-assemble with the aforementioned biodegradable polymer into nanoparticles, enabling multimodal imaging and integrated diagnosis and treatment in conjunction with radionuclide imaging. Compared with the prior art, the technical solution of the present invention has the following advantages: 1. The biodegradable polymer provided by this invention possesses unique chemical bonds that are sensitive to oxidative environments and are easily broken in such environments. This allows for degradation at the tumor site, promoting its metabolic clearance in vivo, significantly improving biocompatibility, reducing long-term toxicity, and thus enhancing biosafety. Furthermore, it exhibits sonodynamic therapy effects, efficiently generating reactive oxygen species (ROS) under ultrasound excitation, enabling deep tissue penetration and damage to tumor tissue. Simultaneously, this biodegradable polymer exhibits strong absorption in the 250-600 nm range, allowing it to be excited by lasers and emit fluorescence for fluorescence imaging. The biodegradable polymer provided by this invention simultaneously achieves controlled degradation, fluorescence imaging, and sonodynamic therapy, enabling the construction of a safe, integrated diagnostic and therapeutic platform.

[0028] 2. The biodegradable polymer provided by this invention can be coated and self-assembled into nanoparticles by a novel amphiphilic biodegradable polymer capable of loading radionuclides (the amphiphilic biodegradable polymer has DOTA chelating agents branched on its side chains, and the radionuclide chelating units (DOTA) are stably fixed on the side chains of the biodegradable polymer through chemical bonding, thereby achieving stable loading of radionuclides) to improve the stability of drugs and can load radionuclides, thereby achieving multimodal imaging and integrated diagnosis and treatment in conjunction with radionuclide imaging. 3. The preparation method of the biodegradable polymer provided by the present invention has a simple process and the product has high stability.

[0029] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0030] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0031] Figure 1 The preparation of monomers for Example 1 of the invention 1 H-NMR spectrum (DMSO-d6, 400MHz); Figure 2 The copolymer prepared for Example 2 of the invention 1 H-NMR spectrum (CDCl3-d6, 400MHz); Figure 3 The copolymer prepared for Example 3 of the present invention 1H-NMR spectrum (DMSO-d6, 400MHz); Figure 4 The ultraviolet absorption spectrum of the copolymer prepared in Example 2 of this invention. Figure 5 The fluorescence spectrum of the copolymer prepared in Example 2 of this invention; Figure 6 These are before and after GPC degradation of the copolymer prepared in Example 2 of this invention; Figure 7 The image shows the ultraviolet absorption spectrum of the nanoparticles prepared in Example 4 of this invention. Figure 8 The fluorescence spectrum of the nanoparticles in Example 4 of this invention is shown below. Figure 9 This is a DPBF test image of the nanoparticles in Example 4 of the present invention; Figure 10 The EPR spectrum of the nanoparticles prepared in Example 4 of this invention, which capture hydroxyl radicals using DMPO; Figure 11 The EPR spectrum of the nanoparticles prepared in Example 4 of this invention, which capture singlet oxygen using TEMP; Figure 12 The figure shows the particle size and potential characterization results of the nanoparticles before and after degradation in Example 4 of the present invention; Figure (A) shows the comparison of the particle size of the nanoparticles before and after degradation, and Figure (B) shows the potential characterization results of the nanoparticles before degradation. Figure 13 The image shows the morphology characterization results of the nanoparticles in Example 4 of this invention. Figure (A) shows the morphology characterization results of the nanoparticles before degradation; Figure (B) shows the morphology characterization results of the nanoparticles after degradation. Figure 14 The results of the cytotoxicity evaluation of the nanoparticles in Example 4 of this invention against human nasopharyngeal carcinoma cells C666 are shown. Figure 15 The cellular ROS flow cytometry results of the nanoparticles in Example 4 of this invention; Figure 16 The images show the in vivo fluorescence imaging and tumor fluorescence intensity quantification curve of the nanoparticles in Example 4 of this invention. Figure (A) shows the in vivo fluorescence imaging of tumor-bearing mice at different time points after injection of nanoparticles; (B) shows the change curve of fluorescence intensity in the tumor area at each time point. Figure 17 This is a SPECT / CT image of the nanoparticles of Example 4 of the present invention in a mouse. Detailed Implementation

[0032] Exemplary embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0033] It should be understood that the terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein may also include the plural forms. The terms “comprising,” “including,” “containing,” and “having” are inclusive and therefore indicate the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein are not construed as requiring them to be performed in a particular order described or illustrated unless the order of performance is explicitly indicated. It should also be understood that additional or alternative steps may be used.

[0034] In the description of the embodiments of this invention, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this invention, "multiple" means two or more, unless otherwise explicitly defined.

[0035] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0036] In the description of the embodiments of this invention, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0037] In the description of the embodiments of the present invention, the term "multiple" refers to two or more (including two), similarly, "multiple groups" refers to two or more (including two groups), and "multiple pieces" refers to two or more (including two pieces).

[0038] In the description of the embodiments of the present invention, unless otherwise explicitly specified and limited, the technical terms such as "installation," "connection," "joining," and "fixing" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in the embodiments of the present invention according to the specific circumstances.

[0039] The following explanations of some terms used in this invention are provided to enable those skilled in the art to understand them.

[0040] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained commercially or through existing methods; unless otherwise specified, the amounts of experimental reagents used are the amounts used in conventional experimental operations; unless otherwise specified, the experimental methods are conventional methods. It should be further noted that the following descriptions are merely exemplary and not intended to limit the specific scope of the invention. Moreover, the comparative examples below are selected to compare with the technical solutions of the present invention to demonstrate the advancement of the present invention, and do not necessarily represent prior art in this technical field.

[0041] Example 1 This embodiment provides a dibromothioacetate-responsive monomer, the structure of which is shown below:

[0042] This embodiment provides a method for synthesizing the above-mentioned dibromothioacetate responsive monomer; Its synthetic route:

[0043]

[0044] .

[0045] The specific synthesis steps include the following: Thioglycolic acid (19.82 g, 200 mmol) and acetone (5.0 g, 86 mmol) were added to a round-bottom flask, followed by 50 μL of trifluoroacetic acid. The mixture was stirred at room temperature until it became a white, viscous liquid. The system was then heated to 85 °C, and acetonitrile was added in portions until the white, viscous liquid was completely dissolved, becoming transparent and reaching saturation. The heating device was removed, and the system was allowed to cool to room temperature. Recrystallization yielded the dicarboxylated thioacetate response monomer 2,2'-[propane-2,2-dimethylbis(thio)]diacetic acid.

[0046] Lithium aluminum hydride (0.38 g, 22.4 mmol) was placed in a two-necked flask, both necks were sealed, and a vacuum was applied, purging with nitrogen to create a protective atmosphere. An appropriate amount of anhydrous tetrahydrofuran was added to dissolve the lithium aluminum hydride. Separately, 2,2'-[propane-2,2-diylbis(thio)]diacetic acid (2.24 g, 10 mmol) was dissolved in 200 mL of anhydrous tetrahydrofuran, and this solution was slowly added dropwise to the reaction system under ice bath conditions. After the addition was complete, the ice bath was removed, and the reaction was stirred at room temperature for 12 h. After the reaction was complete, 100 mL of a tetrahydrofuran / water mixture (v:v = 1:1) was slowly added to the system until no more bubbles were generated. Then, 40 mL of an acetonitrile / isopropanol mixture (v:v = 1:1) was added, and stirring was continued for 30 min to quench any remaining lithium aluminum hydride. Insoluble matter was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 1:1, v / v) to obtain the target product 2,2'-(propane-2,2-diylbis(thionyl))diethanol.

[0047] 2,2'-(propane-2,2-diylbis(thionidyl))diethanol (196 mg, 1 mmol), 5-bromothiophene-2-carboxylic acid (455 mg, 2.2 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (422 mg, 2.2 mmol), and 4-dimethylaminopyridine (DMAP) (366 mg, 3 mmol) were dissolved in an appropriate amount of N,N-dimethylformamide (DMF), and the mixture was stirred at room temperature for 24 h in a round-bottom flask. After the reaction was completed, the reaction solution was concentrated under reduced pressure to remove most of the solvent. The crude product was purified by silica gel column chromatography (eluent: petroleum ether / ethyl acetate = 5:1, v / v) to finally obtain the target product, the dibromothioacetate response monomer. ,That 1 H-NMR spectrum as shown Figure 1 As shown.

[0048] Example 2 This embodiment provides a biodegradable polymer having the structure shown in Formula I:

[0049] This embodiment provides a method for a biodegradable polymer as shown in Formula I; Synthesis route: ; The specific steps include: Weigh out the tin monomer (100.0 mg, 0.16 mmol), dibromo monomer (78.8 mg, 0.14 mmol, CAS: 198964-46-4), the dibromo monomer prepared in Example 1 (9.2 mg, 0.02 mmol), P(t-Bu)3 (3.9 mg, 0.013 mmol), and Pd2(dba)3 (5.8 mg, 0.006 mmol) were dissolved in 5 mL of degassed toluene. Under nitrogen protection, the reaction was carried out at 110 °C for 10 min. The reaction system was observed to change from orange-yellow to purple, indicating the reaction was complete. While still hot, the reaction system was added dropwise to 500 mL of anhydrous methanol. After standing for 30 min, the mixture was filtered to obtain a dark purple precipitate. After drying, the product, namely the biodegradable polymer shown in Formula I, was obtained (where r represents the random arrangement of the two monomer units on the polymer backbone, and m is...). The total molar percentage of the blocks, n is The total molar percentage of the blocks; m = 0.1, n = 0.9), its 1 H-NMR spectrum (CDCl3-d6, 400MHz) as shown Figure 2 As shown.

[0050] The biodegradable polymer provided in this embodiment is a terpolymer synthesized via Stille coupling reaction. Its molecular chain is composed of 9,9-di-n-octylfluorene (DOF) units, 4,7-bis(thiophene-2-yl)-2,1,3-benzothiadiazole (DTBT) units, and oxidation-responsive thioketene linker units. The thioketene linker units embedded in the backbone exhibit oxidation-sensitive responsiveness, specifically responding to and breaking down in the high levels of reactive oxygen species (ROS) in the tumor microenvironment. This facilitates degradation at the tumor site, promotes metabolic clearance in vivo, significantly improves biocompatibility, reduces long-term toxicity, and thus enhances biosafety. The alternating arrangement of DOF and DTBT units forms a narrow-bandgap conjugated backbone with a strong push-pull electron effect. This strong push-pull electron structure not only efficiently generates ROS under ultrasound excitation, enabling deep tissue penetration for sonodynamic therapy to damage tumor tissue, but also provides strong absorption in the 250-600 nm range, allowing it to be excited by lasers and emit fluorescence for fluorescence imaging.

[0051] The UV-Vis absorption spectra of the biodegradable polymer shown in Formula I in tetrahydrofuran (THF) were determined using a UV-Vis-NIR spectrophotometer. The results are as follows: Figure 4 As shown, the copolymer exhibits strong absorption in the wavelength range of 250-600 nm.

[0052] The fluorescence emission spectrum of this copolymer in tetrahydrofuran (THF) was determined using transient fluorescence spectroscopy, and the results are as follows: Figure 5 As shown.

[0053] To investigate its degradation performance, 10 mM hydrogen peroxide solution was added to the THF solution of the copolymer, and the mixture was incubated at 37°C for 0, 24, and 48 hours, respectively. The molecular weight change was monitored using gel permeation chromatography (GPC), and the results are as follows: Figure 6 As shown in the figure, after incubation under oxidative conditions for 48 hours, the molecular weight of the copolymer decreased significantly from 19.0 kDa to 7.0 kDa, indicating that it has good oxidative degradation characteristics.

[0054] Example 3 This embodiment provides a copolymer TK-PEG-DOTA, having the following structure:

[0055] This embodiment provides a method for synthesizing the copolymer TK-PEG-DOTA: The synthetic route includes: .

[0056] Specifically, the steps include the following: Weigh the dihydroxy monomers as shown above. 392 mg, 2 mmol; 1,2,4,5-cyclohexanetetracarboxylic dianhydride (493 mg, 2.2 mmol) was dissolved in 5 ml DMF and stirred at room temperature for 24 h. mPEG5000 (2 g, 0.4 mmol) was added, and the reaction continued for another 24 h. The mixture was dialyzed and lyophilized to obtain a white powder. The above white powder (500 mg, 0.036 mmol), DOTA-tri-tert-butyl-C2-amino (219.3 mg, 0.36 mmol, CAS: 173308-19-5), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) (68.4 mg, 0.36 mmol), and N-hydroxysuccinimide (NHS) (41 mg, 0.36 mmol) were dissolved in N,N-dimethylformamide (DMF) and stirred at 50 °C for 24 h. Then 1 ml of trifluoroacetic acid was added, and stirring was continued at room temperature for 24 hours. The mixture was then dialyzed and lyophilized to obtain the above-mentioned TK-PEG-DOTA polymer as a white powder. 1H-NMR spectrum (DMSO-d6, 400MHz) as shown Figure 3 As shown.

[0057] Example 4 This embodiment provides a method for preparing nanoparticles, including the following steps: (1) Weigh 100 mg of the TK-PEG-DOTA polymer prepared in Example 3 and 10 mg of the biodegradable polymer of Formula I prepared in Example 2, and dissolve them in 1 ml of tetrahydrofuran (THF). (2) Quickly add the above solution to 10 ml of distilled water under ultrasonic conditions.

[0058] (3) Continue sonication for 5 minutes and then dialyze to remove organic solvents to obtain nanoparticle dispersion.

[0059] The nanoparticles were measured using a UV-Vis-NIR spectrophotometer, and their absorption spectra are as follows: Figure 7 As shown. By Figure 7 It can be seen that the nanoparticles exhibit strong absorption in the wavelength range of 250-600 nm. Further analysis using fluorescence spectroscopy to determine their excitation and emission spectra yielded the following results: Figure 8 As shown.

[0060] Under ultrasonic irradiation, the nanoparticles prepared in Example 4 can effectively generate reactive oxygen species (ROS). First, singlet oxygen was detected using a consumption method based on 1,3-diphenylisobenzofuran (DPBF). 1 O2), the results are as follows Figure 9 As shown, the characteristic absorption intensity of DPBF decreased rapidly with increasing sonication time, preliminarily confirming that nanoparticles can effectively generate ROS under ultrasonic conditions. Further EPR spectroscopy analysis revealed that the generated ROS included not only hydroxyl radicals (·OH), but also those captured by 5,5-dimethyl-1-pyrrolidone N-oxide (DMPO), the characteristic signals of which are shown below. Figure 10 It also includes singlet oxygen ( 1 O2), captured by 2,2,6,6-tetramethyl-4-piperidinone (TEMP), characteristic signal see Figure 11 .

[0061] The obtained nanoparticles (denoted as NP) were characterized for size and potential using dynamic light scattering (DLS), and the results are as follows: Figure 12 As shown, its average particle size (z-average) is 103 nm, its polymer dispersion index (PDI) is 0.159, and its potential is -25.0 mV.

[0062] Subsequently, to investigate the dissociation behavior of the nanoparticles under oxidizing conditions, 10 mM hydrogen peroxide solution was added to the NP dispersion and incubated at 37°C for 24 h. The results were then measured again by DLS (labeled as NP+H2O2), and are as follows: Figure 12 As shown in the particle size distribution diagram, new, smaller particle size peaks appear, while the main peak broadens significantly, indicating that the nanoparticles undergo dissociation under oxidative conditions. The morphology of the nanoparticles and their changes before and after degradation were directly observed using transmission electron microscopy (TEM), and the results are as follows. Figure 13 As shown, where Figure 13 Figure (A) shows the morphology characterization results of the nanoparticles before degradation; Figure 13 Figure (B) shows the morphology characterization results of the degraded nanoparticles. The characterization results show that... Figure 13 In Figure (A), the nanoparticles before degradation exhibit a complete and regular spherical morphology with a relatively uniform size distribution. However, after degradation... Figure 13 As shown in Figure (B), the morphology of the nanoparticles has changed significantly, and their structural integrity has decreased. Irregular and broken shapes can be observed, which directly confirms the damage to their microstructure caused by the degradation process.

[0063] Example 5 In this embodiment, the MTT assay was used to detect the effect of nanoparticles (denoted as NP) combined with ultrasound on the viability of C666 cells. Human nasopharyngeal carcinoma cells C666 were cultured in RPMI-1640 medium containing 10% fetal bovine serum in a conventional incubator at 37°C and 5% CO2. Four experimental groups were set up: (1) PBS group (negative control group); (2) PBS+US group (ultrasound treatment control); (3) NP group (nanoparticle toxicity group); (4) NP+US group (ultrasound-enhanced treatment group). Cells in the logarithmic growth phase were seeded at a density of 5 × 10³ cells per well in 96-well plates and cultured for 24 h to allow the cells to adhere completely. The old culture medium was discarded. For the NP and NP+US groups, 100 μL / well of fresh culture medium containing different concentrations of nanoparticles (nanoparticle concentrations were calculated based on the biodegradable polymer shown in Formula I, at 0 μg / mL, 0.25 μg / mL, 2.5 μg / mL, 50 μg / mL, 100 μg / mL, and 200 μg / mL, respectively) was added. For the PBS and PBS+US groups, 100 μL / well of fresh culture medium containing an equal volume of phosphate-buffered saline (PBS) was added. Four replicates were set for each concentration. After 6 h of incubation, the PBS+US and NP+US groups were sonicated (parameters: 1.0 MHz, 1.5 W / cm²). 2The first group was incubated at 50% duty cycle for 2 min, while the remaining groups were placed in the same environment without sonication. All cells were cultured for 48 h after treatment, then 10 μL of thiazolyl blue (MTT) solution (5 mg / mL) was added to each well, and incubation continued for 4 h. The culture medium in the wells was carefully aspirated, and 200 μL of SDS-HCl solution (10% sodium dodecyl sulfate (SDS), 0.01 M HCl) was added directly to each well. The cells were incubated overnight (12 h) at 37°C in the dark to allow the formazan crystals to dissolve completely. The absorbance of each well was measured at 570 nm using a microplate reader, and calibrated using 650 nm as the reference wavelength.

[0064] The average absorbance of the PBS group was set at 100% cell viability, and the relative cell viability (%) of each experimental group was calculated. The experiment was independently repeated three times, and data are expressed as mean ± standard deviation. Results are as follows: Figure 14 As shown.

[0065] Example 5 This embodiment uses the DCFH-DA fluorescent probe method to detect the ability of nanoparticles (denoted as NP) to generate reactive oxygen species (ROS) in tumor cells under ultrasound triggering. The experiment was set up in four groups: (1) PBS (negative control) group: containing only cells and probes, used to determine the baseline fluorescence level; (2) PBS+US group: to explore the effect of ultrasound itself on the intracellular ROS level; (3) NP group: to explore the inherent oxidative stress of nanoparticles in cells without ultrasound; (4) NP+US group: the core experimental group, to explore the effect of ultrasound triggering nanoparticles to generate ROS.

[0066] The specific experimental steps include: Human nasopharyngeal carcinoma cells C666 were injected at a concentration of 1×10⁶ cells / well. 4 Cells were seeded at a density of [number] cells / well in 96-well plates using RPMI-1640 medium containing 10% fetal bovine serum and incubated at 37°C with 5% CO2 for 24 hours to allow complete cell adhesion. The old medium was discarded, and four groups were established. The NP and NP+US groups were replaced with 100 μL / well of fresh serum-free medium containing nanoparticles (nanoparticle concentration, calculated as 100 μg / mL of the biodegradable polymer shown in Formula I). ​​The PBS and PBS+US groups were replaced with 100 μL / well of fresh serum-free medium containing an equal volume of PBS. All groups were in triplicate. Cells were returned to the incubator and incubated for another 8 hours to ensure adequate nanoparticle uptake.

[0067] After incubation, discard the culture medium. Gently wash the cells once with pre-warmed PBS. Add 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) working solution diluted with serum-free medium (final DCFH-DA concentration 10 μM) to each well and incubate at 37°C in the dark for 30 minutes. After incubation, gently wash the cells twice with pre-warmed PBS to thoroughly remove any extracellular probes that have not yet entered the cells. Immediately sonicate the wells of the PBS+US group and the NP+US group (parameters: 1.0 MHz, 1.5 W / cm²). 2 (50% duty cycle, 2 min). The remaining groups were placed in the same environment but without sonication. After treatment, the cells were digested with trypsin without EDTA and the digestion was terminated with serum-containing medium. The cell suspension was collected in a flow cytometry tube, centrifuged at 500×g for 5 min, the supernatant was discarded, the cells were resuspended in pre-cooled PBS and stored on ice in the dark.

[0068] Flow cytometry was used for detection. Fluorescence was excited using a 488 nm laser, and the green fluorescence signal of DCF was collected through the FITC channel. The results are as follows: Figure 15 As shown in the figure, the fluorescence signal intensity of the NP+US treatment group was significantly higher than that of the PBS, PBS+US, and NP control groups. This demonstrates that the prepared nanoparticles can efficiently generate ROS in tumor cells under ultrasound triggering, thereby exerting a significant killing effect; while under conditions without ultrasound, the nanoparticles themselves exhibit good biocompatibility.

[0069] Example 6 In vivo accumulation fluorescence imaging experiment: Female C3H mice were selected, and murine squamous cell carcinoma cell line SCC7 (approximately 5 × 10⁻⁶) was subcutaneously inoculated into their right back. 5 (each cell). Wait until the tumor volume grows to approximately 100-150 mm. 3 For imaging experiments, tumor-bearing mice were injected via tail vein with a nanoparticle solution at a dose of 1 mg / mL (based on the biodegradable polymer shown in Formula I), with an injection volume of 100 µL. Time-series fluorescence images were acquired using a small animal in vivo optical imaging system (IVIS Spectrum CT) at 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, and 48 h post-administration. Mice were anesthetized with isoflurane gas before each imaging session. Imaging parameters were set as follows: excitation wavelength 640 nm, emission filter 700 nm, and exposure time 60 s.

[0070] Image processing and quantitative analysis were performed using the instrument's accompanying Living Image® software. On each image, the tumor region was manually delineated as the region of interest, and its average fluorescence intensity was recorded. The fluorescence intensity values ​​of the tumor region at different time points were obtained through analysis, and time-intensity change curves were plotted. The in vivo fluorescence imaging images of tumor-bearing mice at different time points after nanoparticle injection are shown below. Figure 16 Figure (A) shows the fluorescence intensity variation curves of the tumor region at each time point (n=3). Figure 16 As shown in Figure (B).

[0071] Example 7 In vivo radionuclide imaging experiment: Female C3H mice were selected, and murine squamous cell carcinoma cell line SCC7 (approximately 5 × 10⁻⁶) was subcutaneously inoculated into the right back of the mice. 5 (each cell). Wait until the tumor volume grows to approximately 100-150 mm. 3 At that time, it was used for imaging experiments. The nanoparticles prepared according to the method in Example 4 above were used with Na... 99m Radiolabeled TcO4 was purified to obtain 99m Tc-labeled nanoparticle imaging agents (denoted as Tc-labeled nanoparticle imaging agents) 99m Tc NP). Simultaneously, set Na. 99m TcO4 solution was used as a control group. Tumor-bearing mice were injected with the above solution via the tail vein. 99m Tc NP or Na 99m The TcO4 solution was injected at a dose of 1.5-2.0 mCi (radioactivity) per mouse. Mice were anesthetized with isoflurane gas at 0.5h, 4h, 12h, and 24h after administration, and images were acquired using the Mediso nanoScan SPECT / CT system. The imaging results are as follows: Figure 17 As shown. By Figure 17 It can be seen that, with free Na 99m Compared to the TcO4 group 99m The Tc NP group showed significant radioactive signal enrichment at the tumor site (circled in red), demonstrating that the nanoparticles have good passive targeting and accumulation capabilities for tumor imaging.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A biodegradable polymer, characterized in that, The biodegradable polymer is obtained by polymerization of conjugated dibromo monomers, ditin monomers, and responsive dibromo monomers; The responsive dibromo monomer has chemical bonds or groups that can be broken in response to oxidative, reducing, hypoxic, or acid-base conditions; the responsive dibromo monomer also has at least two bromine groups; The bistin monomer contains two trimethyltin groups; The conjugated dibromo monomer has a conjugated structure and at least two bromine groups.

2. The biodegradable polymer according to claim 1, characterized in that, The chemical bonds that can break in response to oxidative, reducing, hypoxic, or acid-base conditions include at least one of disulfide bonds, diselenide bonds, and thioacetate bonds. Optionally, the groups that can be broken in response to oxidizing conditions, reducing conditions, hypoxic conditions or acid-base conditions include at least one of thioacetal and acetal. Optional groups that can be cleaved in response to oxidizing, reducing, hypoxic, or acid-base conditions include: , , , , At least one of them.

3. The biodegradable polymer according to claim 1 or 2, characterized in that, The ratio of the total molar number of the responsive dibromomon and the conjugated dibromomon to the molar number of the bistin monomer is (1-1.2):(1-1.2). Preferably, the molar ratio of the responsive dibromomon to the conjugated dibromomon is (0.1-0.5):(0.5-0.9).

4. The biodegradable polymer according to any one of claims 1-3, characterized in that, The responsive dibromo monomer is selected from at least one of the following groups of compounds: ， ， ， ， ， ， ； Preferably, the conjugated dibromo monomer is selected from at least one of the following groups of compounds: ， ， ， ， ， ， ， ， ， ， ， ； Preferably, the bistin monomer is selected from at least one of the following groups of compounds: ， ， ， ， ， ， 。 5. The biodegradable polymer according to any one of claims 1-4, characterized in that, The biodegradable polymer is obtained by polymerization of a conjugated dibromo monomer, a ditin monomer, and a responsive dibromo monomer; Alternatively, the biodegradable polymer is obtained by polymerization of a conjugated dibromo monomer, a ditin monomer, and two responsive dibromo monomers; Preferably, the degradable polymer comprises at least one selected from the group consisting of: Formula I Formula II Formula III Formula IV Formula V Formula VI Formula VII The molar ratio of m to n is (0.1-0.5):(0.5-0.9).

6. A method for preparing the biodegradable polymer according to any one of claims 1-5, characterized in that, Includes the following steps: The biodegradable polymer is obtained by polymerizing conjugated dibromo monomers, ditin monomers, and responsive dibromo monomers in an organic solvent under catalytic conditions.

7. The method for preparing the biodegradable polymer according to claim 6, characterized in that, The organic solvent includes toluene; Preferably, the polymerization reaction temperature is 105-115℃ and the polymerization reaction time is 5-20 min; Preferably, the ratio of the bistin monomer to the organic solvent is (0.1-0.2):5, in mmol / mL; Preferably, the catalyst comprises at least one of tri-tert-butylphosphine and tris(dibenzylideneacetone)palladium; the ratio of the total molar amount of the catalyst to the total molar amount of the conjugated dibromo monomer, the bistin monomer and the responsive dibromo monomer is (0.01-0.03):(0.2-0.35).

8. The application of the biodegradable polymer according to any one of claims 1-5 or the biodegradable polymer prepared by the preparation method according to claim 6 or 7 in the preparation of nanoparticles with fluorescence imaging and sonodynamic therapy functions.

9. A nanoparticle, characterized in that, The nanoparticles include: a) an amphiphilic polymer; b) a degradable polymer according to any one of claims 1-5 or a degradable polymer prepared by the preparation method according to claim 6 or 7.

10. The nanoparticles according to claim 9, characterized in that, The mass ratio of the amphiphilic polymer to the biodegradable polymer is (5-15):1; Preferably, the amphiphilic polymer includes distearylphosphatidylethanolamine-polyethylene glycol, methoxy polyethylene glycol-polylactic acid block copolymer, methoxy polyethylene glycol-polylactic acid-glycolic acid copolymer, polyethylene glycol-polypropylene glycol-polyethylene glycol block copolymer, etc. At least one of them; Preferably, the amphiphilic polymer is 。

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