Potassium ion response type aggregated gold nanoparticles and preparation method thereof

Abstract

The invention discloses a potassium ion response type aggregable gold nanoparticle and a preparation method thereof, the aggregable gold nanoparticle is grafted on the surface of the gold nanoparticle by a potassium ion recognition type copolymer, the structural formula of the copolymer is as follows: x / (x + y + z) = 0.1-0.3, y / (x + y + z) = 0.1-0.3, z / (x + y + z) = 0.4-0.8, and x + y + z = 10-300. The aggregable gold nanoparticles are prepared by the following steps: dissolving a potassium ion recognition type copolymer in deionized water, and co-incubating the dissolved potassium ion recognition type copolymer and gold nanoparticles which are prepared by a sodium citrate reduction method and have good monodispersity at normal temperature under the protection of nitrogen. After the polymer recognizes abnormal potassium ion concentration in a tumor microenvironment, the polymer is converted from hydrophilicity to hydrophobicity, the gold nanoparticles are agglomerated, and enhanced photoacoustic signals and photothermal effects are presented at the same time. And the preparation method is simple, has good biocompatibility and can be used for treating different types of tumors.

Description

Technical Field

[0001] This invention relates to a potassium ion-responsive aggregateable gold nanoparticle and its preparation method, specifically a novel liposome that can specifically respond to abnormal concentrations (40-50 mM) of potassium ions in the tumor microenvironment and undergo self-assembly. Background Technology

[0002] Cancer treatment remains a formidable task and challenge for humanity. With the rapid development of nanoscience, many new technologies for tumor diagnosis and treatment have emerged. Photoacoustic imaging (PAI) is a non-invasive biomedical imaging modality with high imaging contrast and excellent tissue penetration, showing promising application prospects in cancer tissue imaging. Photothermal therapy (PAA) is a non-invasive local treatment method that uses photothermal converters to convert light energy into heat energy to kill cancer cells, effectively reducing damage to normal tissues during treatment. PAI and PAA, as novel imaging and treatment methods, have been extensively studied in the field of tumor diagnosis and treatment. Both require photothermal converters to convert light energy into acoustic and thermal energy, respectively, for cancer imaging and treatment. Gold nanoparticles (AuNPs), as commonly used photothermal converters, possess excellent biocompatibility, strong light absorption, good photothermal conversion ability, and easily modifiable surface properties, making them highly promising for applications in tumor diagnosis and treatment.

[0003] To maximize the imaging capabilities and therapeutic efficacy of AuNPs, researchers have recently developed a series of tumor microenvironment-responsive aggregateable AuNPs by modifying them with specific ligands such as polymers, small molecules, peptides, and proteins, targeting specific pathological features of the tumor microenvironment that differ from normal tissues, such as low pH, high enzyme expression, high glutathione content, and high H2O2 concentration. These AuNPs exhibit a monodisperse state in normal tissues, but upon reaching the tumor microenvironment, their physicochemical properties change in response to pathological features, causing the nanoparticles to aggregate. This allows for targeted enrichment at the tumor site, enhancing therapeutic efficacy. Furthermore, AuNP aggregates exhibit optical properties significantly different from dispersed individual particles. The shortened interparticle distance within the aggregates generates a strong surface plasmon resonance (LSPR) effect, resulting in superior PAI and PTT effects compared to monodisperse nanoparticles, demonstrating superior performance in tumor imaging and treatment. However, current common pH, enzyme and other stimulus-responsive AuNPs still have problems such as insufficient stimulus response precision and limited substrate contact efficiency. Therefore, it is necessary to design and develop novel pathological feature stimulus-responsive AuNPs.

[0004] Potassium ions (K) +Potassium ions (K+) are an important ionic component for maintaining the body's osmotic pressure. In a normal body, the vast majority of potassium ions are located inside cells, specifically in the intracellular fluid. + The concentration is approximately 150 mM, normal serum / extracellular fluid K + The concentration is 3.5–5.5 mM. Tumor development involves extensive cell necrosis, leading to the release of high concentrations of potassium ions from within the cells into the extracellular fluid, resulting in an excess of potassium in the tumor microenvironment. + The concentration abnormally increased to approximately 40–50 mM. Therefore, this invention targets the abnormally elevated concentration of K in the tumor microenvironment. + Construct a K-specific response + Aggregated AuNPs exhibit superior PAI and PTT effects compared to monodisperse nanoparticles, providing a new approach and method for tumor diagnosis and treatment. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, this invention proposes a gold nanoparticle that can specifically recognize and aggregate abnormal concentrations (40-50 mM) of potassium ions in the tumor microenvironment, and its preparation method. The gold nanoparticle aggregates exhibit superior PAI and PTT effects compared to monodisperse nanoparticles, improving imaging and therapeutic effects, and providing a new technology for tumor imaging and treatment.

[0006] To achieve the above objectives, the present invention provides a potassium ion-responsive aggregateable gold nanoparticle, wherein the aggregateable gold nanoparticle is composed of a potassium ion-recognizing copolymer grafted onto the surface of the gold nanoparticle, and the structural formula of the copolymer is as follows:

[0007]

[0008] Among them, x / (x+y+z)=0.1~0.3, y / (x+y+z)=0.1~0.3, z / (x+y+z)=0.4~0.8, x+y+z=10~300.

[0009] Furthermore, the grafted gold nanoparticles have a particle size of 1–100 nm.

[0010] This invention also discloses a series of methods for preparing the aggregateable gold nanoparticles. Specifically, the aggregateable gold nanoparticles are prepared by the following steps:

[0011] S1. Weigh the aforementioned potassium ion recognition copolymer, add deionized water to dissolve it, and obtain solution A for later use;

[0012] S2. Gold nanoparticles B with good monodispersity were prepared by sodium citrate reduction method and are ready for use;

[0013] S3. Add solution A to particles B from step S2 and incubate them together at room temperature under nitrogen protection to obtain potassium ion-responsive aggregateable gold nanoparticles.

[0014] Furthermore, the gold nanoparticles are prepared by the following steps:

[0015] ST1. Dissolve tetrachloroauric acid trihydrate in ultrapure water to prepare an aqueous solution of tetrachloroauric acid for later use;

[0016] ST2. Dissolve trisodium citrate dihydrate in ultrapure water to prepare an aqueous solution of sodium citrate for later use;

[0017] ST3. Add pure water to a three-necked flask, heat to boiling, add the prepared tetrachloroauric acid aqueous solution while stirring vigorously, and then quickly add an equal volume of the prepared sodium citrate aqueous solution.

[0018] ST4. After vigorous stirring under reflux for 10 min, stirring was continued at room temperature for 15 min, and the mixture was allowed to stand at room temperature to obtain gold nanoparticles (AuNPs).

[0019] Furthermore, the synthesis steps of the potassium ion-recognizing copolymer include:

[0020] STEP 1. Weigh N-isopropylacrylamide, benzo15-crown5acrylamide, azobisisobutyronitrile, N,N-dimethylacrylamide, and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid, and add 1,4-dioxane to dissolve them completely.

[0021] STEP2. Introduce nitrogen gas into the solution from step STEP1 to remove oxygen from the solution, and seal the reaction at 60℃~80℃ for 4h~24h.

[0022] STEP3. After the reaction in step STEP2 is completed, a polymer solution is obtained. It is cooled to room temperature and diluted with tetrahydrofuran. The polymer solution is then added dropwise to anhydrous diethyl ether or methyl tert-butyl ether while stirring until all the white flocculent precipitate is precipitated. Then, stirring is stopped, and the mixture is allowed to stand and filtered.

[0023] STEP 4. Dissolve the filtered white flocculent precipitate completely in tetrahydrofuran, then add it dropwise into anhydrous diethyl ether or methyl tert-butyl ether to precipitate again. Filter and repeat this process three times to obtain a white polymer precipitate.

[0024] STEP5. Place the white polymer precipitate obtained in step STEP4 into a vacuum drying oven and dry it to obtain product C;

[0025] STEP 6. Dissolve product C in methanol, purge with nitrogen to remove oxygen from the solution, then add sodium borohydride methanol solution and stir to react for 6 to 48 hours.

[0026] STEP 7. After the reaction in step STEP 6 is completed, the copolymer is purified by dialysis with deionized water and then freeze-dried under vacuum to obtain the copolymer.

[0027] Furthermore, in step STEP4, the amount of tetrahydrofuran added is just enough to completely dissolve the white polymer precipitate.

[0028] Furthermore, in step STEP1, the molar ratio of N-isopropylacrylamide, azobisisobutyronitrile, and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid is (20-200):(0.1-1):1. The molar amount of benzo

[15] crown-5-acrylamide accounts for 10%-30% of the sum of the molar amounts of benzo

[15] crown-5-acrylamide, N-isopropylacrylamide, and N,N-dimethylacrylamide. The molar amount of N,N-dimethylacrylamide accounts for 40%-80% of the sum of the molar amounts of benzo

[15] crown-5-acrylamide, N-isopropylacrylamide, and N,N-dimethylacrylamide. The amount of 1,4-dioxane used makes the molar concentration of benzo

[15] crown-5-acrylamide, N-isopropylacrylamide, and N,N-dimethylacrylamide in the mixture 0.1-0.3 mol / L.

[0029] Furthermore, the synthesis steps of the benzo

[15] crown-5 acrylamide include:

[0030] Step 1. Weigh 4'-nitrobenzene 15 crown 5 and dissolve it in slightly heated ethanol. Add catalyst Pd / C and purge the solution with nitrogen gas for reduction protection. Stir the solution and slowly add hydrazine hydrate dropwise. After the addition is complete, raise the reaction temperature to 70°C and reflux for 1.5 hours.

[0031] Step 2. After the reflux reaction is complete, filter while hot to remove Pd / C so that the solution is colorless and transparent. Remove the solvent under reduced pressure to obtain a yellow oily substance. Add dichloromethane to dissolve it, separate the organic layer, wash the organic layer with deionized water, separate the organic layer again, add anhydrous magnesium sulfate to remove water, and filter to obtain a dichloromethane solution containing benzo

[15] crown[5]acrylamide.

[0032] Step 3. Add triethylamine to the dichloromethane solution, stir and mix well, then purge with nitrogen gas for amino protection. After that, cool the reaction solution with an ice bath, and slowly add the acylation reagent dropwise with stirring. After the addition is complete, transfer to a 25°C constant temperature water bath and stir to react for 16 hours.

[0033] Step 4. After the stirring reaction is complete, filter to remove salt; wash the filtrate twice with deionized water to separate the organic layer, add an appropriate amount of anhydrous magnesium sulfate to the organic solution to remove water, filter, remove the solvent by rotary evaporation, and dry under vacuum at room temperature to obtain benzo

[15] crown-5 acrylamide.

[0034] The responsive aggregation mechanism of potassium ion-responsive gold nanoparticles is as follows:

[0035] The potassium ion-recognizing copolymer was synthesized via reversible addition-fragmentation chain transfer radical polymerization (RAFT). The polymer consisted of three monomers: N-isopropylacrylamide, benzo

[15] crown[5]acrylamide, and N,N-dimethylacrylamide. The benzo

[15] crown[5]acrylamide in this copolymer could selectively complex potassium ions (K+). + In complex K + Subsequently, the lower critical solution temperature (LSCT) of the copolymer shifts to lower temperatures, and the hydrophobicity of the copolymer increases. The potassium ion-recognizing copolymer gold nanoparticles of this invention, after entering the body circulation, are attracted by potassium ions in the blood / normal tissue / extracellular environment. + At lower concentrations (3.5–5.5 mmol / L), the copolymer responds better to low concentrations of K. + With low responsiveness and exhibiting hydrophilicity, the nanoparticles are in a monodisperse state; when the nanoparticles reach the tumor microenvironment, due to the K+ of the tumor microenvironment... + At higher concentrations (40–50 mmol / L), the copolymer on the surface of the nanoparticles specifically complexes K. + As LCST migrates to lower temperatures, the copolymer changes from hydrophilic to hydrophobic, and nanoparticles aggregate due to hydrophobic association.

[0036] The potassium ion-recognizing polymer described in this invention changes from hydrophilic to hydrophobic upon recognizing abnormal potassium ion concentrations in the tumor microenvironment, causing gold nanoparticles to aggregate and exhibiting enhanced photoacoustic signals and photothermal effects. This invention features a simple preparation method, good biocompatibility, and nanoparticles that self-assemble in response to potassium ions. It can be used for the treatment of various types of tumors, representing a convenient and versatile novel nanomedicine. Attached Figure Description

[0037] Figure 1 In the image, (A), (B), and (C) are the infrared spectra of B15C5Am, PNDB, and PNDB-SH, respectively.

[0038] Figure 2 In the image, (A) and (B) are the NMR spectra of PNDB and PNDB-SH, respectively.

[0039] Figure 3 The transmittance of PNDB dispersed in simulated extracellular fluid and simulated tumor microenvironment solutions as a function of temperature is shown.

[0040] Figure 4 Figure 1 shows the particle size distribution of AuNPs (A), transmission electron microscopy (B), and ultraviolet absorption spectrum (C). The scale bar in Figure 2 is 200 nm.

[0041] Figure 5The UV absorption spectra of PNDB-AuNPs and PEG-AuNPs dispersed in simulated extracellular fluid and simulated tumor microenvironment solutions, respectively.

[0042] Figure 6 Transmission electron microscopy images of PNDB-AuNPs and PEG-AuNPs dispersed in simulated extracellular fluid and simulated tumor microenvironment solutions, respectively. The scale bar is 200 nm.

[0043] Figure 7 Photoacoustic signal diagrams of PNDB-AuNPs and PEG-AuNPs dispersed in PBS and simulated tumor microenvironment solutions, respectively.

[0044] Figure 8 The image shows the photothermal effect of PNDB-AuNPs and PEG-AuNPs dispersed in PBS and simulated tumor microenvironment solutions, respectively. Detailed Implementation

[0045] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0046] Example 1

[0047] In this experimental example, a copolymer-poly(N-isopropylacrylamide-copolymer-benzo15crown5acrylamide-copolymer-N,N-dimethylacrylamide)-mercapto(PNDB-SH) was prepared, with the following structural formula:

[0048]

[0049] The specific preparation method is as follows:

[0050] 1) Weigh N-isopropylacrylamide (NIPAM), benzo

[15] crown[5]acrylamide (BCAm), azobisisobutyronitrile (AIBN), N,N-dimethylacrylamide (DMAm), and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid (DDMAT), and dissolve them in 1,4-dioxane.

[0051] 2) Nitrogen gas is introduced into the above solution to completely remove oxygen from the solution, and the reaction is carried out at 70°C in a sealed environment for 6 hours;

[0052] 3) After the reaction is complete, wait for the reaction solution to cool to room temperature, dilute the polymer solution with tetrahydrofuran, and add the cooled polymer solution to a large amount of methyl tert-butyl ether dropwise while stirring. A white flocculent precipitate will form. After the addition is complete, stop stirring and let stand for filtration.

[0053] 4) Dissolve the obtained white precipitate again with an appropriate amount of tetrahydrofuran, where the amount of tetrahydrofuran is just enough to completely dissolve the precipitate. Then, precipitate the solution again with a large amount of methyl tert-butyl ether, filter, and dissolve the precipitate three times in the above steps.

[0054] 5) The final white polymer precipitate was dried in a vacuum drying oven at 40°C to obtain copolymer poly(N-isopropylacrylamide-copolymer-benzo15crown5acrylamide-copolymer-N,N-dimethylacrylamide) (PNDB);

[0055] 6) Weigh an appropriate amount of the copolymer prepared in 5) and dissolve it in methanol, then remove oxygen with nitrogen.

[0056] 7) Add sodium borohydride methanol solution as a reducing agent and stir for 48 hours;

[0057] 8) After the reaction was completed, the copolymer PNDB-SH was obtained by dialysis purification and vacuum freeze drying.

[0058] In this embodiment, the molar ratio of NIPAM, AIBN, and DDMAT in step 1) is 20:0.5:1, the molar amount of B15C5Am accounts for 20% of the sum of the molar amounts of B15C5Am, NIPAM, and DMAm, and the molar amount of DMAm accounts for 60% of the sum of the molar amounts of B15C5Am, NIPAM, and DMAm.

[0059] The infrared spectra of B15C5Am, PNDB prepared in this embodiment, and PNDB-SH prepared in this embodiment are as follows: Figure 1 As shown in (A), (B), and (C), 1367cm -1 and 1386cm -1 (-CH(CH3)2) is the characteristic absorption peak of NIPAM, at 1513 cm⁻¹. -1 (arom.) and 1130cm -1 (CO) is the characteristic absorption peak of B15C5Am. The characteristic absorption peaks of NIPAM and B15C5Am both appear in the infrared spectra of PNDB and PNDB-SH, indicating that PNDB and the thiol-terminated polymer PNDB-SH have been successfully synthesized.

[0060] The NMR spectra of PNDB and PNDB-SH prepared in this embodiment are as follows: Figure 2 As shown in (A) and (B), a comparison is made between PNDB and PNDB-SH. 1The HNMR spectra all showed the following identical chemical shifts: the chemical shift of the hydrogen atom on the benzene ring in B15C5Am appeared around 7 ppm; the chemical shift of the hydrogen atom on the two methyl groups on the isopropyl group in NIPAM was around 1 ppm; the chemical shift of the hydrogen atom on the two methyl groups in DMAm was around 2.8 ppm; while the chemical shifts of the hydrogen atom in DDMAT in PNDB around 3.1 ppm and 1.1 ppm were not shown in the PNDB-SH spectrum, indicating that the DDMAT end chain had been successfully reduced to thiol groups. The NMR results further confirm that the PNDB and PNDB-SH copolymers were successfully prepared in this embodiment.

[0061] Example 2

[0062] In this embodiment, the potassium ion responsiveness of the PNDB copolymer prepared in Example 1 was measured.

[0063] The PNDB copolymer was characterized in simulated normal tissue fluid (5 mM K) using a temperature-controlled UV-Vis spectrophotometer. + +150mMNa + ) and tumor microenvironment tissue fluid (45mM K) + +110mM Na + The low critical solution temperature (LCST) of the copolymer was defined as the temperature at which the transmittance was initially 50%. The potassium ion responsiveness of the copolymer was investigated. The concentration of the copolymer in the samples in which the potassium ion responsiveness was measured was 0.5 wt%.

[0064] like Figure 3 As shown, the transmittance of PNDB gradually decreased in both simulated normal tissue fluid and tumor microenvironment tissue fluid with increasing temperature. The LCST of PNDB in simulated normal tissue fluid was approximately 47.8℃, while the LCST of PNDB in simulated tumor microenvironment tissue fluid showed a significant shift towards lower temperatures, with an LCST of approximately 30.9℃. PNDB exhibited good Kelvin transmittance. + Response performance. At 37°C, PNDB is hydrophilic in normal tissue simulation solution and hydrophobic in tumor microenvironment simulation solution, and can be used to construct a potassium ion responsive nanodelivery system under physiological conditions.

[0065] Example 3

[0066] In this embodiment, inorganic gold nanoparticles (AuNPs) are prepared using the following steps:

[0067] 1) Weigh an appropriate amount of tetrachloroauric acid trihydrate, dissolve it in ultrapure water, and prepare a 10 mM tetrachloroauric acid aqueous solution:

[0068] 2) Take an appropriate amount of trisodium citrate dihydrate, dissolve it in ultrapure water, and prepare a 38.8 mM sodium citrate aqueous solution;

[0069] 3) Add a large amount of pure water to a three-necked flask, heat to boiling, add 10 mM tetrachloroauric acid aqueous solution while stirring vigorously, and then quickly add an equal volume of 38.8 mM sodium citrate aqueous solution.

[0070] 4) Stir vigorously under reflux for 10 min, continue stirring at room temperature for 15 min, and let stand at room temperature to obtain AuNPs.

[0071] like Figure 4 As shown, the hydrated particle size distribution diagram (A) and transmission electron microscopy (B) show that the particle size of AuNPs is 10–20 nm. In addition, the ultraviolet curve (C) shows that there is maximum absorption at about 520 nm. These results indicate that AuNPs were successfully prepared and have good monodispersity.

[0072] Example 4

[0073] In this embodiment, potassium ion responsive gold nanoparticles were constructed using PNDB-SH and AuNPs prepared in Examples 1 and 3.

[0074] Take an appropriate volume of gold nanoparticle aqueous solution, add an equal volume of 2 mg / mL PNDB-SH aqueous solution under nitrogen protection, stir and react for 24 h at room temperature in the dark, filter after reaction, and centrifuge to purify and obtain polymer grafted nanoparticles PNDB-AuNPs.

[0075] In this embodiment, the molar ratio of PNDB-SH to AuNPs is 5:4.

[0076] Comparative Example 5

[0077] A control group of hydrophilic polymer-grafted nanoparticles without potassium ion responsiveness was prepared according to Example 4.

[0078] Take an appropriate volume of gold nanoparticle aqueous solution, add an equal volume of 2 mg / mL mPEG-SH aqueous solution under nitrogen protection, stir and react at room temperature in the dark for 24 h, filter after reaction, and centrifuge to purify and obtain polymer grafted nanoparticles PEG-AuNPs.

[0079] Example 6

[0080] In this embodiment, the potassium ion response properties of the two polymer grafted nanoparticles prepared in Examples 4 and 5 were measured.

[0081] The absorption spectra of PNDB-AuNPs and PEG-AuNPs (wavelength range: 400–900 nm) were measured using a UV-Vis spectrophotometer. Both PNDB-AuNPs and PEG-AuNPs were dispersed in simulated normal tissue fluid (5 mM K). + +150mM Na + ) and tumor microenvironment tissue fluid (45mM K) + +110mM Na + In this study, the dispersion of nanoparticles was determined by ultraviolet absorption. The morphology and dry particle size of PNDB-AuNPs and PEG-AuNPs were observed using transmission electron microscopy. Both PNDB-AuNPs and PEG-AuNPs were dispersed in simulated normal tissue fluid and tumor microenvironment tissue fluid, respectively, and samples were prepared for observation.

[0082] like Figure 5 As shown, both PEG-AuNPs samples and the PNDB-AuNPs sample dispersed in simulated normal tissue fluid exhibited absorption peaks at approximately 520 nm, consistent with the absorption peaks of AuNPs. However, when PNDB-AuNPs were dispersed in simulated tumor microenvironment tissue fluid, the characteristic absorption peak of PNDB-AuNPs red-shifted, appearing at approximately 565 nm, and exhibiting enhanced absorption in the near-infrared region. This is because the PNDB copolymer contains 15crown5 groups, which can recognize and capture K+. + This forms a stable 2:1 "sandwich" structure, which causes PNDB-AuNPs to aggregate in a high-concentration potassium ion solution. The localized electric field on the surface of a single particle couples with the electric field of the neighboring particles, thereby causing a change in the resonance frequency. This indicates that Example 4 successfully constructed PNDB-AuNPs with potassium ion responsiveness.

[0083] like Figure 6 The transmission electron microscopy results show that Figures (A) and (B) show PEG-AuNPs dispersed in simulated normal tissue fluid and tumor microenvironment tissue fluid, respectively. The nanoparticles are well dispersed and no nanoparticle aggregation occurs. Figures (C) and (D) show PNDB-AuNPs dispersed in simulated normal tissue fluid and tumor microenvironment tissue fluid, respectively. PNDB-AuNPs are well dispersed in simulated normal tissue fluid, but PNDB-AuNPs are significantly aggregated in simulated tumor microenvironment tissue fluid. This further demonstrates that Example 4 successfully constructed PNDB-AuNPs with potassium ion responsiveness.

[0084] Example 7

[0085] In this embodiment, the photothermal effect of the two polymer grafted nanoparticles prepared in Examples 4 and 5 was measured.

[0086] Based on the plasmon resonance that occurs when gold nanoparticles aggregate, resulting in enhanced light absorption in the near-infrared region, and considering that the contrast of photoacoustic imaging primarily depends on light absorption capacity and thermoacoustic conversion efficiency, aggregated nanoparticles with enhanced near-infrared absorption can improve photothermal conversion efficiency and have the potential to serve as photoacoustic contrast agents. Preparations were made by dispersing gold nanoparticles separately in PBS and tumor microenvironment tissue fluid (45 mM K). + +110mM Na + The PNDB-AuNPs and PEG-AuNPs samples in the sample were kept at a consistent gold nanoparticle concentration. The samples were irradiated with an 808nm laser to observe the photoacoustic signal and examine their imaging effect.

[0087] like Figure 7 As shown in the figure, (A) and (B) represent the photoacoustic images and average photoacoustic signal values ​​of PNDB-AuNPs and PEG-AuNPs under 808nm laser irradiation in PBS and simulated tumor microenvironment tissue fluid, respectively. The results show that as the concentration of gold nanoparticles gradually increases, the signal of PNDB-AuNPs under simulated tumor microenvironment tissue fluid gradually increases, while the signals of other groups remain basically unchanged. This indicates that Example 4 successfully constructed potassium ion responsive PNDB-AuNPs, and the aggregated PNDB-AuNPs exhibit a stronger photoacoustic signal than monodisperse nanoparticles.

[0088] Example 8

[0089] In this embodiment, the photoacoustic intensity of the two polymer nanoparticle grafts prepared in Examples 4 and 5 was measured.

[0090] Based on the principle that gold nanoparticles undergo plasmon resonance during aggregation, resulting in increased photothermal conversion efficiency, this not only enhances their photoacoustic signal but also their photothermal effect. Preparations were made by dispersing gold nanoparticles in PBS and tumor microenvironment tissue fluid (45 mM K) respectively. + +110mMNa + The PNDB-AuNPs and PEG-AuNPs samples in the study were kept at a consistent gold nanoparticle concentration. They were irradiated with an 808nm laser, and their temperature rise was observed by thermal imaging to investigate the photothermal effect.

[0091] like Figure 8 As shown in the figure, (A) and (B) represent PNDB-AuNPs and PEG-AuNPs in PBS and simulated tumor microenvironment tissue fluid, respectively, using an 808nm laser with 0.74W cm⁻¹. -2Infrared thermography and temperature curves obtained after 5 minutes of irradiation showed that the temperature of all samples increased to varying degrees under the same laser irradiation. PNDB-AuNPs exhibited a significantly greater temperature increase under simulated tumor microenvironment tissue fluid, with an increase approximately twice that of the control group. This indicates that aggregated nanoparticles do indeed improve photothermal conversion performance. This demonstrates that Example 4 successfully constructed potassium-ion-responsive PNDB-AuNPs, and that aggregated PNDB-AuNPs exhibit a stronger photothermal effect than monodisperse nanoparticles.

[0092] The technical solutions provided by the embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the embodiments of the present invention. The descriptions of the embodiments above are only for helping to understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the embodiments of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A potassium ion-responsive aggregateable gold nanoparticle, characterized in that: The aggregateable gold nanoparticles are composed of a potassium ion-recognizing copolymer grafted onto the surface of the gold nanoparticles. The structural formula of the copolymer is as follows: Among them, x / (x+y+z)=0.1~0.3, y / (x+y+z)=0.1~0.3, z / (x+y+z)=0.4~0.8, x+y+z=10~300.

2. The potassium ion-responsive aggregateable gold nanoparticles according to claim 2, characterized in that: The grafted gold nanoparticles have a particle size of 1–100 nm.

3. A method for preparing potassium ion-responsive aggregateable gold nanoparticles according to claim 1 or 2, characterized in that: The aggregateable gold nanoparticles were prepared by the following steps: S1. Weigh the aforementioned potassium ion recognition copolymer, add deionized water to dissolve it, and obtain solution A for later use; S2. Gold nanoparticles B with good monodispersity were prepared by sodium citrate reduction method and are ready for use; S3. Add solution A to particles B from step S2 and incubate them together at room temperature under nitrogen protection to obtain potassium ion-responsive aggregateable gold nanoparticles.

4. The method for preparing potassium ion-responsive aggregateable gold nanoparticles according to claim 3, characterized in that: The gold nanoparticles are prepared by the following steps: ST1. Dissolve tetrachloroauric acid trihydrate in ultrapure water to prepare an aqueous solution of tetrachloroauric acid for later use; ST2. Dissolve trisodium citrate dihydrate in ultrapure water to prepare an aqueous solution of sodium citrate for later use; ST3. Add pure water to a three-necked flask, heat to boiling, add the prepared tetrachloroauric acid aqueous solution while stirring vigorously, and then quickly add an equal volume of the prepared sodium citrate aqueous solution. ST4. After vigorous stirring under reflux for 10 min, stirring was continued at room temperature for 15 min, and the mixture was allowed to stand at room temperature to obtain gold nanoparticles (AuNPs).

5. The method for preparing potassium ion-responsive aggregateable gold nanoparticles according to claim 3, characterized in that: The synthesis steps of the potassium ion-recognizing copolymer include: STEP 1. Weigh N-isopropylacrylamide, benzo15-crown5acrylamide, azobisisobutyronitrile, N,N-dimethylacrylamide, and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid, and add 1,4-dioxane to dissolve them completely. STEP2. Introduce nitrogen gas into the solution from step STEP1 to remove oxygen from the solution, and seal the reaction at 60℃~80℃ for 4h~24h. STEP3. After the reaction in step STEP2 is completed, a polymer solution is obtained. It is cooled to room temperature and diluted with tetrahydrofuran. The polymer solution is then added dropwise to anhydrous diethyl ether or methyl tert-butyl ether while stirring until all the white flocculent precipitate is precipitated. Then, stirring is stopped, and the mixture is allowed to stand and filtered. STEP 4. Dissolve the filtered white flocculent precipitate completely in tetrahydrofuran, then add it dropwise into anhydrous diethyl ether or methyl tert-butyl ether to precipitate again. Filter and repeat this process three times to obtain a white polymer precipitate. STEP5. Place the white polymer precipitate obtained in step STEP4 into a vacuum drying oven and dry it to obtain product C; STEP 6. Dissolve product C in methanol, purge with nitrogen to remove oxygen from the solution, then add sodium borohydride methanol solution and stir to react for 6 to 48 hours. STEP 7. After the reaction in step STEP 6 is completed, the copolymer is purified by dialysis with deionized water and then freeze-dried under vacuum to obtain the copolymer.

6. The method for preparing potassium ion-responsive aggregateable gold nanoparticles according to claim 5, characterized in that: In step STEP4, the amount of tetrahydrofuran added should be just enough to completely dissolve the white polymer precipitate.

7. The method for preparing potassium ion-responsive aggregateable gold nanoparticles according to claim 5, characterized in that: In step STEP1, the molar ratio of N-isopropylacrylamide, azobisisobutyronitrile, and 2-(dodecyltrithiocarbonate)-2-methylpropionic acid is (20-200):(0.1-1):

1. The molar amount of benzo[15]crown-5-acrylamide accounts for 10%-30% of the sum of the molar amounts of benzo[15]crown-5-acrylamide, N-isopropylacrylamide, and N,N-dimethylacrylamide. The molar amount of N,N-dimethylacrylamide accounts for 40%-80% of the sum of the molar amounts of benzo[15]crown-5-acrylamide, N-isopropylacrylamide, and N,N-dimethylacrylamide. The amount of 1,4-dioxane used makes the molar concentration of benzo[15]crown-5-acrylamide, N-isopropylacrylamide, and N,N-dimethylacrylamide in the mixture 0.1-0.3 mol / L.

8. The method for preparing potassium ion-responsive aggregateable gold nanoparticles according to claim 5, characterized in that: The synthesis steps of the benzo[15]crown-5 acrylamide include: Step 1. Weigh 4'-nitrobenzene 15 crown 5 and dissolve it in slightly heated ethanol. Add catalyst Pd / C and purge the solution with nitrogen gas for reduction protection. Stir the solution and slowly add hydrazine hydrate dropwise. After the addition is complete, raise the reaction temperature to 70°C and reflux for 1.5 hours. Step 2. After the reflux reaction is complete, filter while hot to remove Pd / C so that the solution is colorless and transparent. Remove the solvent under reduced pressure to obtain a yellow oily substance. Add dichloromethane to dissolve it, separate the organic layer, wash the organic layer with deionized water, separate the organic layer again, add anhydrous magnesium sulfate to remove water, and filter to obtain a dichloromethane solution containing benzo[15]crown[5]acrylamide. Step 3. Add triethylamine to the dichloromethane solution, stir and mix well, then purge with nitrogen gas for amino protection. After that, cool the reaction solution with an ice bath, and slowly add the acylation reagent dropwise with stirring. After the addition is complete, transfer to a 25°C constant temperature water bath and stir to react for 16 hours. Step 4. After the stirring reaction is complete, filter to remove salt; wash the filtrate twice with deionized water to separate the organic layer, add an appropriate amount of anhydrous magnesium sulfate to the organic solution to remove water, filter, remove the solvent by rotary evaporation, and dry under vacuum at room temperature to obtain benzo[15]crown-5 acrylamide.

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