Tumor microenvironment response type drug-loaded nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses tumor microenvironment response type drug-loaded nanoparticles as well as a preparation method and application thereof, belongs to the technical field of medical application, and develops new application of the tumor microenvironment response type drug-loaded nanoparticles. Tumor microenvironment response type drug-loaded nanoparticles are prepared and are polypyrrole modified PNIPAM-BACy nanoparticles, the application of the tumor microenvironment response type drug-loaded nanoparticles in preparation of photothermal chemotherapy combined treatment drugs is explained by means of pharmacological experiments and results of the tumor microenvironment response type drug-loaded nanoparticles, and the tumor microenvironment response type drug-loaded nanoparticles can be applied to preparation of the photothermal chemotherapy combined treatment drugs through in-vitro biological experiment and in-vivo biological experiment results. It is confirmed that the tumor microenvironment response type drug-loaded nano particles have a good anti-tumor effect in photothermal therapy, and meanwhile, the tumor microenvironment response type drug-loaded nano particles can load chemotherapy drugs and play a combined role of photothermal therapy and chemotherapy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology, specifically relating to a tumor microenvironment-responsive drug-loaded nanoparticle, its preparation method, and its application. Background Technology

[0002] Cancer is the second leading cause of death worldwide. Current treatments such as surgery, radiotherapy, chemotherapy, and immunotherapy are generally characterized by high invasiveness, poor targeting, and significant side effects, limiting efficacy and impacting patients' quality of life. Therefore, developing highly efficient and low-toxicity multimodal therapeutic strategies has become a key research focus. Drug delivery systems (DDS) leverage the tumor microenvironment (such as weak acidity, high glutathione levels, and specific receptor overexpression) to achieve targeted drug accumulation and controlled drug release, offering possibilities for precision medicine.

[0003] Photothermal therapy (PTT), as an emerging physical therapy method, has received widespread attention in recent years due to its advantages such as ease of operation, non-invasiveness, and precise treatment. In the PTT process, photothermal agents (PTAs) play a crucial role. PTT utilizes photothermal conversion agents (PTAs) to convert the light energy of an external laser into heat energy, rapidly generating localized high temperatures at the tumor site. This heat sensitivity of cancer cells induces thermal ablation and apoptosis. Currently developed PTAs include small organic molecules, metal nanomaterials, and carbon-based nanomaterials. However, the therapeutic effect of PTT is highly dependent on temperature control, and some limitations still exist. On the one hand, mild hyperthermia induced by low-power laser irradiation at 39–43°C can improve treatment efficacy by enhancing drug delivery and tumor uptake while avoiding thermal damage to non-tumor tissues. However, its ability to kill tumor cells and tumor stem cells is insufficient, easily leading to tumor recurrence and metastasis. On the other hand, high-power laser irradiation induced by high-temperature hyperthermia at 48–60°C, while effectively killing tumors through thermal ablation, may lead to damage to normal tissues and overexpression of heat shock proteins, resulting in treatment resistance. These challenges limit the clinical translation of PTT.

[0004] While chemotherapy drugs such as doxorubicin (DOX) are widely used, they face limitations such as cardiotoxicity and drug resistance. Nanodelivery systems can improve their targeting and safety. Multimodal synergistic therapy strategies combining thermotherapy transluminal transluminal (PTT) with chemotherapy have shown super-additive (1+1>2) therapeutic effects. On the one hand, the thermal effect of PTT can enhance cellular uptake of chemotherapy drugs, promote drug penetration deep within tumors, and potentially reverse tumor drug resistance. On the other hand, chemotherapy drugs can complement the therapeutic effect of PTT, eliminating escaped tumor cells and preventing tumor recurrence. Furthermore, PTT can also be used to trigger the release of chemotherapy drugs from thermosensitive nanocarriers, achieving spatiotemporally controllable drug delivery. However, most current combination therapies simply combine different treatment modalities, failing to fully leverage the synergistic effects between them. Therefore, designing a nanodelivery system that intelligently responds to the tumor microenvironment and effectively integrates PTT and chemotherapy is a key challenge in promoting the clinical translation of multimodal therapy. Summary of the Invention

[0005] This invention provides tumor microenvironment-responsive drug-loaded nanoparticles, their preparation method, and applications. It develops new uses for tumor microenvironment-responsive drug-loaded nanoparticles and provides new drugs for combined photothermal chemotherapy.

[0006] To better understand the essence of this invention, the following pharmacological experiments and results of tumor microenvironment-responsive drug-loaded nanoparticles will be used to illustrate their application in the preparation of photothermal chemotherapy combination therapy drugs.

[0007] A tumor microenvironment-responsive drug-loaded nanoparticle is a PNIPAM-BACy nanogel modified with polypyrrole. The PNIPAM-BACy nanogel is synthesized by free radical copolymerization, which is formed by the polymerization of N-isopropylacrylamide (NIPAM) monomer and N,N'-bis(acryloyl)cysteine ​​(BACy) crosslinking agent containing disulfide bonds. This process forms a crosslinked network in which BACy structural units are integrated into the polymer backbone. The photothermal component polypyrrole (PPy) is loaded onto the PNIPAM-BACy nanogel by in-situ chemical oxidative polymerization. This process is completed in an acidic medium by electrostatic interaction-initiated polymerization of pyrrole monomers.

[0008] A method for preparing tumor microenvironment-responsive drug-loaded nanoparticles includes the following steps: Synthesis of PNIPAM-BACy: N-isopropylacrylamide, N,N'-methylenebisacrylamide, N,N'-bis(acryloyl)cysteine, and sodium dodecyl sulfonate were dissolved in a solvent, with the molar ratio of N-isopropylacrylamide, N,N'-methylenebisacrylamide, N,N'-bis(acryloyl)cysteine, and sodium dodecyl sulfonate being (30-34.8):(15-15.8):(2-2.6):1; after removing dissolved oxygen, potassium persulfate was added, the reaction system was quickly sealed, and the reaction was continuously stirred to ensure that the PNIPAM chain growth and BACy crosslinking were completed simultaneously. The sodium dodecyl sulfonate, as a surfactant, could effectively control the particle size and prevent agglomeration; after the reaction was completed, unreacted monomers and small molecule impurities were removed, and the mixture was freeze-dried to obtain a white powder. Synthesis of PNIPAM-BACy-PPY: The synthesized PNIPAM-BACy nanogel was dispersed in HCl solution, pyrrole monomer was added, and after sonication in an ice bath, APS solution was added dropwise. The reaction was carried out in the dark. After the reaction was completed, the unreacted monomer was removed, and the product was freeze-dried to obtain a black powder.

[0009] The tumor microenvironment-responsive drug-loaded nanoparticles prepared above can be used to prepare photothermal chemotherapy combined therapy drugs; for the synthesis of PNIPAM-BACy-PPY loaded with chemotherapy drugs; under irradiation with 808nm near-infrared light, the PPY component can efficiently convert light energy into heat energy, generating local high temperature to kill cancer cells; the photothermal effect can trigger the release of chemotherapy drugs and enhance the chemotherapy effect; the disulfide bond structure in the system can respond to the high concentration of glutathione in the tumor microenvironment, further promoting the release of chemotherapy drugs, and realizing drug delivery with dual stimulation response.

[0010] Beneficial effects: This invention provides tumor microenvironment-responsive drug-loaded nanoparticles, their preparation method, and applications, developing new uses for drug-loaded nanoparticles. Compared with existing technologies, it has the following advantages: 1. Under 808nm near-infrared light irradiation, the PPY component of the drug-loaded nanoparticles prepared in this invention can efficiently convert light energy into heat energy, generating local high temperature to kill cancer cells. 2. The drug-loaded nanoparticles prepared by this invention have sensitive stimulus response. Under the action of near-infrared light, the heat generated by polypyrrole can kill cancer cells and cause PNIPAM to undergo a phase transition, resulting in a burst release of the drug, thus enabling the nanoparticles to achieve a more effective active controlled release effect. 3. The drug-loaded nanoparticles prepared by this invention have photothermal therapeutic effects and can also load chemotherapy drugs, thereby further enhancing the combined treatment capability. 4. The drug-loaded nanoparticles prepared by this invention have high biosafety and strong practical value. Attached Figure Description

[0011] Figure 1 This is a flowchart illustrating the preparation process of tumor microenvironment-responsive drug-loaded nanoparticles in this embodiment of the invention.

[0012] Figure 2 This is an example of a multiple stimulus response test of PNIPAM-BACy in this invention, where a is a transmission electron microscope image of PNIPAM-BACy without 10 mM GSH treatment; b is a DLS measurement of the particle size of PNIPAM-BACy as a function of temperature; c is a transmission electron microscope image of PNIPAM-BACy treated with 10 mM GSH; and d is the particle size distribution of 100 randomly selected PNIPAM-BACy particles in GSH-treated and untreated samples.

[0013] Figure 3 The above are temperature rise tests of the photothermal performance of PNIPAM-BACy-PPY in this embodiment of the invention; where a is the temperature change of PNIPAM-BACy-PPY (200 μg / mL) under different laser power densities; b is the temperature change curve of PNIPAM-BACy-PPY at different concentrations under 808 nm laser irradiation; and c is the infrared thermal imaging image of PNIPAM-BACy-PPY at different irradiation times and concentrations.

[0014] Figure 4 This is a test of the photothermal conversion efficiency and photostability of PNIPAM-BACy-PPY in an embodiment of the present invention; where a is a graph showing the relationship between the temperature and time of PNIPAM-BACy-PPY during laser irradiation (808 nm, 1 W / cm). 2 a) and cooling (laser off) stage; b) linear time data obtained from the cooling period after laser off and ln(θ); c) temperature map of PNIPAM-BACy-PPY irradiated with 808 nm laser for 5 switching cycles; d) UV absorption curves of PNIPAM-BACy-PPY after different irradiation times.

[0015] Figure 5 In this embodiment of the invention, PNIPAM-BACy-PPY@DOX is tested under normal physiological conditions (37°C). c =0 mM), GSH-free (42℃); c =0 mM) and containing GSH (37℃; c =10 mM) and containing GSH (42℃; c =10 mM) and contains GSH and is exposed to near-infrared light midway ( c =10 mM; with NIR) drug release curve.

[0016] Figure 6This invention relates to the testing of the viability of 4T1 cells with and without 808nm near-infrared light irradiation using different concentrations of PNIPAM-BACy-PPY and PNIPAM-BACy-PPY@DOX in this embodiment of the invention.

[0017] Figure 7 This is an in vivo thermal imaging image of PNIPAM-BACy-PPY@DOX tumor-bearing mice in an embodiment of the present invention.

[0018] Figure 8 This refers to the in vivo antitumor effects of PNIPAM-BACy and PNIPAM-BACy-PPY@DOX in examples of this invention; where a represents the relative tumor volume of different treatment groups ( n =5); b is the organ coefficient of mice in different treatment groups ( n = 5, organ coefficient is the ratio of different organ weights to mouse body weight); c is a representative image of tumors in different treatment groups after 14 days; (I: Control-Dark, II: Control-Light, III: PNIPAM-BACy-PPY-Dark, IV: PNIPAM-BACy-PPY-Light, V: PNIPAM-BACy-PPY@DOX-Dark, VI: PNIPAM-BACy-PPY@DOX-Light); d is a representative image of tumors in different treatment groups ( n = 5) Tumor growth inhibition value (TGI) in mice; e is the tumor coefficient of mice in different treatment groups ( n = 5, the tumor coefficient is the ratio of tumor weight to mouse body weight; d represents different treatment groups ( n = 5) Relative tumor proliferation rate (T / C) in mice. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments: like Figure 1 As shown, a tumor microenvironment-responsive drug-loaded nanoparticle is a PNIPAM-BACy nanogel modified with polypyrrole. The thermosensitive and reduction-responsive nanogel PNIPAM-BACy is synthesized through a free radical copolymerization reaction, which is formed by the polymerization of N-isopropylacrylamide (NIPAM) monomer and N,N'-bis(acryloyl)cysteine ​​(BACy) crosslinking agent containing disulfide bonds. This process forms a crosslinked network in which BACy structural units are integrated into the polymer backbone, thereby providing both structural integrity and glutathione-triggered degradation capability. The photothermal component polypyrrole (PPy) is loaded onto the PNIPAM-BACy nanogel through in-situ chemical oxidative polymerization. This process is completed in an acidic medium by electrostatic interaction-initiated polymerization of pyrrole monomers.

[0020] Chemotherapy drugs (such as DOX, 5-fluorouracil, oxaliplatin, etc.) are mainly loaded into cross-linked networks through temperature-triggered physical encapsulation. This core mechanism is synergistically enhanced by the hydrophobic interaction between the drug and the polymer chain, and is completed in the initial stage with the assistance of electrostatic adsorption.

[0021] The preparation method of the above-mentioned tumor microenvironment-responsive drug-loaded nanoparticles includes the following steps: Synthesis of PNIPAM-BACy:

[0022] N-Isopropylacrylamide (NIPAM, 0.1485 g, 1.31 mmol), N,N'-methylenebisacrylamide (MBA, 0.07475 g), N,N'-bis(acryloyl)cysteine ​​(BACy, 0.017 g, 0.065 mmol), and sodium dodecyl sulfonate (SDS, 0.00865 g) were dissolved in water and magnetically stirred for 1 h until completely dissolved. N2 was then continuously bubbled through the solution for 1 h to completely remove dissolved oxygen. Potassium persulfate (KPS, 0.02973 g) was added, and the reaction system was quickly sealed and placed in a 70 °C oil bath with continuous stirring (400 rpm) for 4 h to ensure that PNIPAM chain growth and BACy crosslinking were completed simultaneously. The reaction solution was transferred to a dialysis bag (MWCO 3500 Da) and dialyzed in deionized water for 24 h to completely remove unreacted monomers and small molecule impurities. The solution was freeze-dried for 48 h to obtain a white powder.

[0023] Synthesis of PNIPAM-BACy-PPY:

[0024] PNIPAM-BACy nanogel (100 mg) was dispersed in 10 mL of 1 mmol HCl solution, and pyrrole monomer (10 μL, 0.1 mmol) (represented as a black circle in the preparation flow chart) was added. The mixture was sonicated in an ice bath for 10 min. APS solution (20 mg) was added dropwise at a rate of 0.5 mL / min, and the reaction was carried out at 4 °C in the dark for 6 h. The reaction solution was transferred to a dialysis bag (MWCO3500 Da) and dialyzed in deionized water at 4 °C for 48 h (to remove unreacted monomers). The solution was then freeze-dried for 48 h to obtain a black powder.

[0025] Synthesis of PNIPAM-BACy-PPY@DOX:

[0026] 6 mg of doxorubicin (represented by a red circle in the preparation flowchart) was prepared into a 1 mL solution using phosphate-buffered saline (PBS, pH 7.4) under light-protected conditions. This solution was then vortexed with PNIPAM-BACy-PPY nanogel for 5 min and incubated at 150 rpm for 6 h in a shaker at 4 °C. The sample was then transferred to a 37 °C water bath and allowed to stand for 1 h. Unloaded DOX was removed by centrifugation (14000 rpm, 10 min), and the sample was purified with deionized water. DOX-loaded PNIPAM-BACy-PPY (PNIPAM-BACy-PPY@DOX) was obtained by freeze-drying for 48 h.

[0027] The tumor microenvironment-responsive drug-loaded nanoparticles prepared above achieve photothermal chemotherapy by modifying the surface of PNIPAM-BACy with polypyrrole (PPY) and loading the chemotherapeutic drug doxorubicin (DOX). Under 808nm near-infrared light irradiation, the PPY component of the drug-loaded nanoparticles can efficiently convert light energy into heat energy, generating localized high temperatures to kill cancer cells. The heat release generated by the photothermal effect can cause PNIPAM to contract, triggering the release of DOX and enhancing the chemotherapy effect. The disulfide bond structure in the drug-loaded nanoparticles can respond to the high concentration of glutathione in the tumor microenvironment, further promoting DOX release and achieving dual-stimulation response drug delivery.

[0028] The application of the tumor microenvironment-responsive drug-loaded nanoparticles in photothermal chemotherapy combination therapy was carried out by subcutaneous injection, based on the results of in vitro and in vivo biological experiments. The drug concentration was 7 mg / mL, 100 μL was injected (tumor-bearing mouse experiment), and light irradiation was performed 2 hours after injection. The ultrasound power was 1 W / cm for 3 minutes.

[0029] The following tests were performed on the aforementioned tumor microenvironment-responsive drug-loaded nanoparticles: Test Example 1: Temperature Sensitivity and Glutathione Response Test of PNIPAM-BACy

[0030] Take appropriate amounts of PNIPAM-BACy treated with 10 mM glutathione and PNIPAM-BACy not treated with glutathione, disperse them separately in water, drop them onto a copper mesh, and let them air dry naturally. Figure 2As shown, after GSH treatment, the average particle size of PNIPAM-BACy decreased from 193 nm to 173 nm, indicating that GSH accelerated the degradation of the nanoparticles, ultimately leading to drug release. An appropriate amount of PNIPAM-BACy was dispersed in water, and the size distribution of the PNIPAM-BACy nanoparticles was evaluated using DLS. The change in hydrodynamic diameter was measured within a temperature range of 25-60 °C to determine the LCST. With increasing temperature, the PNIPAM-BACy particle size decreased from 330 nm to 230 nm, indicating particle size shrinkage and volume reduction, suggesting potential for drug release.

[0031] Test Example 2: In vitro temperature rise performance test of PNIPAM-BACy-PPY

[0032] The photothermal properties of PNIPAM-BACy-PPY solution were studied by irradiating it with an 808 nm laser (d=1 cm) and recording its temperature changes using an infrared thermal imager. First, 3 mL of PNIPAN-BACy-PPY (50, 100, and 200 μg / mL) was placed in a cuvette and irradiated with an 808 nm laser (1.0 W / cm²). 2 Irradiate the solution for 8 min, and record the temperature change using an infrared thermal imager; separately, take 3 mL of PNIPAM-BACy-PPY (1 mg / mL) in a cuvette and irradiate it with an 808 nm laser (0, 0.5, 1.0, 1.5 and 2 W / cm²). 2 The solution was irradiated for 8 minutes, and the temperature change was recorded using an infrared thermal imager. Figure 3 As can be seen, under near-infrared 808 nm laser irradiation, the temperature changes at concentrations of 0, 100, and 200 μg / mL were 9.8, 11.9, and 14.1 °C, respectively, indicating a positive correlation between the photothermal heating effect and the concentration of the nanomaterial. This is because the increase in photothermal conversion agent per unit volume leads to improved light energy absorption and heat energy conversion efficiency. Under near-infrared 808 nm laser irradiation at different powers, while keeping the PNIPAM-BACy-PPY concentration constant, the laser power was 0.5, 1, 1.5, and 2 W / cm². 2 The temperature changes were 16.9, 30.1, 39.2 and 47.1 °C, respectively, indicating that the temperature rise of PNIPAM-BACy-PPY is laser power dependent.

[0033] Test Example 3: In vitro photothermal conversion performance and stability test of PNIPAM-BACy-PPY

[0034] Using an 808nm laser (1.0W·cm) -2The photothermal conversion efficiency of PNIPAM-BACy-PPY solution was studied by irradiating it with an 808 nm laser (1.0 W / cm²) and recording the temperature changes simultaneously. First, 3 mL of PNIPAM-BACy-PPY (1 mg / mL) was placed in a cuvette and irradiated with an 808 nm laser (1.0 W / cm²). 2 Irradiate for 8 min, then turn off the 808 nm laser and allow to cool naturally. Record the temperature change of the solution using an infrared thermal imager. Then, perform UV-Vis absorption spectroscopy on the 1 mg / mL PNIPAM-BACy-PPY solution and calculate the photothermal conversion efficiency. Separately, take 3 mL of PNIPAM-BACy-PPY (1 mg / mL) in a cuvette and irradiate with an 808 nm laser (1.0 W / cm²). 2 Irradiate for 8 min, then turn off the 808 nm laser and allow to cool naturally. Repeat this process 5 times, recording the temperature change of the solution using an infrared thermal imager. Five identical PNIPAM-BACy-PPY solutions (1 mg / mL) were then treated with 1.0 W / cm² solution. 2 The photostability of PNIPAM-BACy-PPY was evaluated by irradiating it with an 808 nm laser for 0, 30, 60, 90, and 180 s, respectively. The photothermal conversion efficiency was measured using the following formula: (1) (2) (3) (4) T represents the temperature (°C) at different time points during cooling. max T represents the highest temperature (°C). min Let Q represent the ambient temperature (°C), τ be the system time constant (s), t be the cooling time (s), m be the solution mass, c be the specific heat capacity of the solution, and Q be the total heat dissipation power of the system. drug Q is the heat dissipation power of the photothermal reagent system. water I is the heat dissipation power of the pure water system, and A is the power density of the incident laser. 808 The absorbance of the sample at a wavelength of 808 nm is given.

[0035] The stability of photothermal conversion materials is a key indicator for evaluating their practical application value. Figure 4 As can be seen, after multiple cycles, the photothermal heating curve of the nanomaterial remained almost unchanged, and the highest temperature remained at a similar level, indicating that PNIPAM-BACy-PPY has excellent photothermal stability and repeatability, with an η value of 36.05%.

[0036] Test Example 4: Drug Release Experiment of PNIPAM-BACy-PPY@DOX

[0037] Typically, PNIPAM-BACy-PPY@DOX (4.0 mg) is dispersed in 4.0 mL of PBS buffer (pH = 7.4). The dispersion is divided into five equal portions, and each PNIPAM-BACy-PPY@DOX aliquot is transferred to a dialysis bag (molecular weight 3500 Da). Each aliquot is dialyzed against the appropriate buffer for 10 mL (with or without 10 mM GSH) and gently mixed at 37°C or 42°C. Furthermore, the DOX release behavior of the NIR laser-responsive PNIPAM-BACy-PPY@DOX solution (1 mg / mL) was measured by vigorous stirring at 37°C and irradiation for 10 minutes at each time point. As a control, a PNIPAM-BACy-PPY@DOX solution without NIR laser irradiation was used. Drug release is considered to have begun once the dialysis bag is immersed in the reservoir. At predetermined time intervals, 1 mL of solution was periodically collected from the reservoir, and the release of DOX from PNIPAM-BACy-PPY@DOX was analyzed by measuring the absorbance at 481 nm. To maintain a constant volume, 1 mL of fresh buffered medium was added to the reservoir after each sampling.

[0038] (7) In the formula, V is the sampling volume (mL), V0 is the total volume of the drug release medium (mL), and C i Let C be the concentration of DOX (mol / L) at sampling time i. n Let m be the concentration of DOX (mol / L) at sampling n, m be the total amount of DOX in PNIPAM-BACy-PPY@DOX, and n be the number of samplings.

[0039] The drug release behavior of PNIPAM-BACy-PPY@DOX under different conditions was studied. Dox release behavior was observed for 72 h at 37 ℃ and 42 ℃. Figure 5 As can be seen, the cumulative release of DOX in PNIPAM-BACy-PPY@DOX was 23% and 54%, respectively. Under the action of GSH, both the release amount and release rate of DOX were further increased. Therefore, this nanocomposite material can degrade under different environments and effectively achieve responsive release of the loaded drug.

[0040] Test Example 5: Cell viability assay of PNIPAM-BACy-PPY and PNIPAM-BACy-PPY@DOX

[0041] Using 4T1 cells as the research object, the effects of PNIPAM-BACy-PPY and PNIPAM-BACy-PPY@DOX on the presence and absence of near-infrared light (808 nm, 33.3 mW / cm²) were detected by CCK-8 assay. 2 Cytotoxicity under irradiation (3 min). In short, cells in the exponential growth phase were irradiated at 1 × 10⁻⁶ cells per well. 4 Cells were seeded in 96-well plates. When the cell density reached 70%, different concentrations of PNIPAM-BACy-PPY and PNIPAM-BACy-PPY@DOX were applied to the cells. For phototoxicity, the cells were irradiated with a laser after 16 hours of incubation. The untreated group was kept in darkness throughout the incubation period, and after another 24 hours, CCK-8 was added and incubated for 1 hour. The absorbance at 450 nm was then measured. Figure 6 As shown, regardless of sample concentration, 4T1 cell viability remained high after 24 hours of incubation with PNIPAM-BACy-PPY. Therefore, the PNIPAM-BACy-PPY nanocomposite material did not exhibit cytotoxicity as a nanocarrier for drug delivery. After NIR laser irradiation of 4T1 cells containing the PNIPAM-BACy-PPY@DOX nanocomposite, cell survival dropped sharply to 16%, lower than other groups. This result indicates that NIR laser-mediated chemophotothermal combined therapy has the best therapeutic effect.

[0042] Test Example 6: In vivo thermal imaging of PNIPAM-BACy-PPY@DOX

[0043] The in vivo photothermal imaging performance of PNIPAM-BACy-PPY@DOX was validated using an infrared thermal imaging system with a subcutaneous tumor model. PNIPAM-BACy-PPY@DOX was subcutaneously injected into each mouse as a control. PBS (100 μL) was also subcutaneously injected into the mice, and the tumor area was irradiated with an 808 nm laser for 3 minutes (1 W / cm²). 2 Afterwards, thermal images and corresponding temperature changes were recorded for each mouse. For example... Figure 7 As shown, mice treated with PBS exhibited the smallest temperature increase (40.6℃) after 5 minutes of laser irradiation, indicating that the photothermal effect of the laser alone is negligible. During the same irradiation cycle, the tumor temperature reached 50.8℃ after injection of PNIPAM-BACy-PPY@DOX.

[0044] Test Example 7: In vivo antitumor effect of PNIPAM-BACy-PPY@DOX

[0045] The antitumor effect of PNIPAM-BACy-PPY@DOX was evaluated using a mouse subcutaneous breast cancer tumor model. The specific experimental steps are as follows: 4T1 cells (2 × 10⁻⁶) were injected subcutaneously into the right rib area of ​​mice. 6 A tumor model was established using 100 µL of cells, with the tumor volume reaching 100 mm². 3 Treatment was initiated by dividing the tumor-bearing mice into 6 groups (5 mice in each group): Group I: Control-Dark: Subcutaneous injection of PBS (100 μL); Group II: Control-Light: Two hours after subcutaneous injection of PBS (100 μL), laser (808 nm, 1 W / cm²) was used. 2 (3 minutes) Group III: PNIPAM-BACy-PPY-Dark: Injection of PNIPAM-BACy-PPY (7 mg / mL, 100 μL); Group IV: PNIPAM-BACy-PPY-Light: Two hours after subcutaneous injection of PNIPAM-BACy-PPY (7 mg / mL, 100 μL), the patient was treated with a laser (808 nm, 1 W / cm²). 2 (3 minutes) Group V: PNIPAM-BACy-PPY@DOX-Dark: PNIPAM-BACy-PPY@DOX injection (7 mg / mL, 100 μL); Group VI: PNIPAM-BACy-PPY@DOX-Light: Two hours after subcutaneous injection of PNIPAM-BACy-PPY@DOX (7 mg / mL, 100 μL), the patient was treated with a laser (808 nm, 1 W cm⁻¹). −2 (3 minutes).

[0046] Fourteen days after treatment, tumors and major organ tissues (heart, liver, spleen, lung, and kidney) from all mice were collected, photographed, weighed, and recorded. The therapeutic effect of PNIPMA-BACy-PPY@DOX on subcutaneous breast cancer tumors in a mouse model is as follows: Figure 8 As shown, tumors grew rapidly in the Control group and the PNIPAM-BACy-PPY-Dark group, while tumors in the PNIPAM-BACy-PPY@DOX-Light group were effectively inhibited and regressed, exhibiting the strongest anti-tumor effect. The organ coefficients of PNIPAM-BACy-PPY and PNIPAM-BACy-PPY@DOX after treatment of a mouse subcutaneous breast cancer tumor model are shown in the figure. Figure 8As shown, the organ coefficients of heart, liver, lung, and kidney remained normal in all groups. Mice in the control group and PNIPAM-BACy-PPY-Dark group showed splenomegaly, which is one of the pathological features of the 4T1 tumor model. The splenomegaly in mice in the drug-treated and light-exposed group was effectively relieved, which indirectly indicates that it has excellent anti-tumor therapeutic effect.

[0047] In summary, the drug-loaded nanoparticles of the present invention have been applied in vitro and in vivo. In vitro tests predict their photothermal therapeutic effects, and in vitro cell experiments and in vivo experiments show that the nanoparticles have good photothermal therapy and chemotherapy combined therapy effects as well as strong biosafety.

[0048] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A tumor microenvironment-responsive drug-loaded nanoparticle, characterized in that, To modify the PNIPAM-BACy of polypyrrole, the PNIPAM-BACy is formed into a crosslinked network by polymerization of N-isopropyl acrylamide monomers and disulfide bond-containing crosslinking agent N,N'-bis(acryloyl)cystamine, wherein the BACy structural unit is integrated into the polymer main chain; and the polypyrrole is loaded on the PNIPAM-BACy by in-situ chemical oxidative polymerization.

2. A method for preparing a tumor microenvironment-responsive drug-loaded nanoparticle, characterized in that, The method comprises the following steps: Synthesis of PNIPAM-BACy: N-isopropyl acrylamide, N,N'-methylene bisacrylamide, N,N'-bis(acryloyl)cystamine, and sodium dodecyl sulfate are dissolved in a solvent, and after removal of dissolved oxygen, potassium persulfate is added, the reaction system is sealed, the reaction is continuously stirred, the growth of the PNIPAM chain is ensured to be synchronized with the crosslinking of BACy, after the reaction is completed, impurities are removed, and white powder is obtained by freeze-drying; Synthesis of PNIPAM-BACy-PPY: The synthesized PNIPAM-BACy is modified with polypyrrole to obtain PNIPAM-BACy-PPY drug-loaded nanoparticles.

3. The method of claim 2, wherein the tumor microenvironment-responsive drug-loaded nanoparticles are prepared by the steps of: (a) preparing a mixture of a tumor microenvironment-responsive polymer, a drug, and a solvent; (b) adding a non-solvent to the mixture; and (c) isolating the tumor microenvironment-responsive drug-loaded nanoparticles. The molar ratio of the N-isopropyl acrylamide, N,N'-methylene bisacrylamide, N,N'-bis(acryloyl)cystamine, and sodium dodecyl sulfate is (30-34.8):(15-15.8):(2-2.6):

1.

4. The method of claim 2, wherein the tumor microenvironment-responsive drug-loaded nanoparticles are prepared by the steps of: (a) preparing a mixture of a tumor microenvironment-responsive polymer, a drug, and a solvent; (b) adding a non-solvent to the mixture; and (c) isolating the tumor microenvironment-responsive drug-loaded nanoparticles. After the potassium persulfate is added, the reaction is continuously stirred at 70°C for 4h.

5. The method for preparing tumor microenvironment-responsive drug-loaded nanoparticles according to claim 2, characterized in that, In the synthesis of PNIPAM-BACy, the impurities are removed by dialysis in a dialysis bag in deionized water for 24h, so as to completely remove unreacted monomers and small molecular impurities.

6. The method for preparing tumor microenvironment-responsive drug-loaded nanoparticles according to claim 2, characterized in that, The specific process for synthesizing PNIPAM-BACy-PPY is as follows: the PNIPAM-BACy nanogel is dispersed in a 1 mmol HCl solution, pyrrole monomers are added, ice-bath ultrasonic treatment is performed for 10 min, an APS solution of 20 mg is added dropwise at a dropwise adding rate of 0.5 mL / min, reaction is performed at 4°C in the dark for 6h, the reaction liquid is transferred to a dialysis bag for dialysis in deionized water at 4°C for 48h to remove unreacted monomers, and black powder is obtained by freeze-drying for 48h. 7.The use of the tumor microenvironment-responsive drug-loaded nanoparticles of claim 1, wherein, The drug-loaded nanoparticles are used for preparing a photothermal-chemotherapy combined treatment drug, and the drug-loaded nanoparticles load a chemotherapy drug. 8.The use of the tumor microenvironment-responsive drug-loaded nanoparticles according to claim 7, wherein, The specific process for loading the chemotherapy drug is as follows: the chemotherapy drug is placed into a solution in a dark environment, is uniformly mixed with the PNIPAM-BACy-PPY nanogel by vortex mixing, is oscillated at 150 rpm in a constant-temperature shaking table at 4°C for 6h, is transferred to a constant-temperature water bath at 37°C for standing for 1h to remove un-loaded drugs, and after purification and freeze-drying, the PNIPAM-BACy-PPY loaded with the chemotherapy drug is obtained.

9. The use of the tumor microenvironment-responsive drug-loaded nanoparticles according to claim 7 or 8, characterized in that, Under irradiation of near-infrared light, the PPY component can efficiently convert light energy into heat energy to generate local high temperature to kill cancer cells, the photothermal effect triggers the release of the chemotherapy drug to enhance the chemotherapy effect, the disulfide bond structure in the drug-loaded nanoparticles responds to high-concentration glutathione in a tumor microenvironment to further promote the release of the chemotherapy drug, and double-stimulus-responsive drug delivery is achieved.

Images