Preparation and application of radio frequency thermally responsive platinum ion crosslinked black phosphorus nanogels

Abstract

This invention discloses the preparation and application of radiofrequency thermoresponsive platinum ion crosslinked black phosphorus nanogel. The nanogel has a core-shell structure, comprising a core composed of a radiofrequency thermosensitive agent and chemotherapeutic drugs, and a shell constructed from a thermosensitive nanopolymer. The thermosensitive nanogel BP-Pt@PNA prepared using the above components exhibits radiofrequency thermosensitive properties. Simultaneously, the introduction of the thermosensitive nanopolymer improves the stability of the radiofrequency thermosensitive agent and chemotherapeutic drugs, enabling long-term drug retention and fully leveraging the synergistic effect of non-invasive radiofrequency thermotherapy and chemotherapy. This drug-loaded nanogel can achieve multimodal treatment applications, including non-invasive radiofrequency thermotherapy-chemotherapy synergistic antitumor therapy and invasive radiofrequency ablation-chemotherapy-vascular embolization synergistic antitumor therapy, demonstrating significant clinical translational potential.

Description

Technical Field

[0001] This invention relates to the field of drug-loaded nanogel technology, and in particular to the preparation and application of radio frequency thermally responsive platinum ion crosslinked black phosphorus nanogels. Background Technology

[0002] Radiofrequency ablation (RFA) is a minimally invasive local treatment for solid tumors and is widely used clinically as a first-line treatment for early-stage liver cancer (BCLC 0). However, traditional invasive RFA has many limitations. It suffers from incomplete ablation of larger tumors, making single-treatment with RFA less than ideal. Invasive RFA requires the insertion of electrode needles, which is somewhat invasive and causes significant pain to patients. Furthermore, it cannot accurately distinguish between tumor tissue and surrounding healthy tissue, potentially causing irreversible damage to adjacent normal tissue.

[0003] The combination of RFA and chemotherapy has been widely practiced in clinical practice. For example, the classic combination of RFA and transarterial chemoembolization (TACE) for the treatment of liver cancer can significantly reduce the sublethal area formed by RFA alone, while achieving chemotherapy sensitization through TACE.

[0004] Non-invasive radiofrequency therapy (RF radiation field) is a promising anti-tumor treatment method that utilizes radiofrequency-responsive nanomaterials to convert radiofrequency field energy and exert radiofrequency-responsive thermal effects (RTT). Furthermore, compared to invasive radiofrequency ablation (RFA), it eliminates the need for needle insertion, allowing for flexible and precise ablation of tumor cells and sustained activation of anti-tumor immune responses. This effectively avoids non-selective damage to adjacent normal tissue cells, significantly alleviating patient suffering.

[0005] Nanomaterial-mediated non-invasive radiofrequency therapy, or RTT, is currently a hot research topic. It uses a radiofrequency field to heat tumor tissue treated with nanomaterials to selectively kill tumor cells while reducing side effects. However, the effect of RTT alone is not ideal, and there are problems such as incomplete ablation and recurrence and regrowth for larger tumors.

[0006] In summary, based on current clinical application needs, it is an urgent scientific problem to be solved to construct and design a treatment platform that can achieve synergistic enhancement of RTT and chemotherapy, effectively avoid non-selective damage to adjacent normal tissue cells, and allow free switching between invasive RFA and non-invasive RF therapy according to clinical application needs. Summary of the Invention

[0007] The purpose of this invention is to provide the preparation and application of radiofrequency thermally responsive platinum ion crosslinked black phosphorus nanogels. The prepared nanogel BP-Pt@PNA can realize multimodal treatment applications such as non-invasive radiofrequency thermotherapy-chemotherapy synergistic anti-tumor therapy and invasive radiofrequency thermal ablation-chemotherapy-vascular embolization synergistic anti-tumor therapy, which has great clinical translational prospects.

[0008] To achieve the above objectives, the present invention provides a platinum ion crosslinked black phosphorus nanogel with radio frequency thermal response, comprising a radio frequency responsive thermal sensitizer, a chemotherapeutic drug, and a thermosensitive nanopolymer.

[0009] The nanogel BP-Pt@PNA consists of a core composed of a radiofrequency-responsive thermal sensitizer and chemotherapeutic drugs, and a shell constructed from thermosensitive nanopolymers.

[0010] Preferably, the mass ratio of the radiofrequency responsive thermal sensitizer, the chemotherapy drug, and the thermosensitive nanopolymer is 1:1:100 to 1:6:100.

[0011] Preferably, the radio frequency responsive thermal effect sensitizer is black phosphorus nanoparticles (BPNP) with a particle size of 24.2-38.6 nm.

[0012] Preferably, the chemotherapy drug is nitrocisplatin (Nitro-Pt).

[0013] Preferably, the thermosensitive nanopolymer is a linear block polymer (PNA) synthesized from N-isopropylacrylamide monomer and acrylic acid monomer via RAFT polymerization, with a degree of polymerization of 100-100.

[0014] Preferably, the BP-Pt@PNA exhibits radio frequency thermal effects, which are positively correlated with radio frequency power and negatively correlated with radio frequency distance, respectively.

[0015] Preferably, BP-Pt@PNA exhibits controlled drug release behavior regulated by radiofrequency thermal effect.

[0016] Preparation of the above-mentioned radio frequency thermally responsive platinum ion crosslinked black phosphorus nanogel:

[0017] S1. Preparation of radio frequency responsive thermal effect sensitizer: Black phosphorus nanoparticles were subjected to liquid phase exfoliation by low-temperature probe ultrasound and non-contact water bath ultrasound. Unexfoliated black phosphorus nanoparticles were removed by multiple water washing and centrifugation. Finally, radio frequency responsive thermal effect sensitizer BPNP was obtained by freeze drying.

[0018] S2. Preparation of chemotherapy drugs: Cisplatin and silver nitrate were mixed at a molar ratio of 1:2, stirred in the dark, and centrifuged to remove silver chloride precipitate to obtain the chemotherapy drug Nitro-Pt.

[0019] S3. Preparation of thermosensitive nanopolymer: N-isopropylacrylamide monomer was added to DMSO solvent, stirred and dissolved, and then repeatedly frozen and thawed with liquid nitrogen. After freezing again, chain transfer agent was added, and after thawing, the mixture was stirred in a warm water bath. After freezing again, initiator was added and reacted. After evacuation and argon purging, the mixture was thawed, reacted in an oil bath at 70°C for 24 hours, and then dialyzed and freeze-dried. Acrylic acid monomer was added, and the reaction was continued for 24 hours. After dialyzing, the thermosensitive nanopolymer PNA was obtained by freeze-drying.

[0020] S4. Preparation of nanogel: The radio frequency responsive thermal sensitizer in S1 was added to the nitrocisplatin solution in S2. After stirring, mixing and centrifuging in the dark, the thermosensitive nanopolymer in S3 was added. The self-assembly reaction formed a co-coordinated nanogel. Subsequently, the concentrate was collected by ultrafiltration and centrifugation, and then freeze-dried to obtain the nanogel BP-Pt@PNA.

[0021] Preferably, in S1, the liquid phase stripping solvent during stripping is N-methylpyrrolidone.

[0022] Preferably, in S3, the initiator is azobisisobutyronitrile and the chain transfer agent is 4-cyano-4-(thiobenzoyl)valerate.

[0023] Preferably, in S4, the mass ratio of the radio frequency responsive thermal sensitizer, the chemotherapy drug, and the thermosensitive nanopolymer is 1:1:100 to 1:6:100.

[0024] Applications of the aforementioned radiofrequency thermally responsive platinum ion crosslinked black phosphorus nanogel BP-Pt@PNA: BP-Pt@PNA is applied to non-invasive radiofrequency thermotherapy-chemotherapy synergistic anti-tumor therapy and invasive radiofrequency ablation-chemotherapy-vascular embolization synergistic anti-tumor therapy.

[0025] Preferably, the non-invasive RF hyperthermia time is 15 min and the power is 300 W, and the invasive RFA ablation time is 2 min and the power is 100 W.

[0026] This invention utilizes Nitro-Pt as a grafting bridge to achieve co-coordination between the radiofrequency-responsive thermal sensitizer BPNP and the thermosensitive polymer PNA, constructing a non-invasive RF-responsive drug-loaded thermosensitive nanogel. Its thermal response and thermosensitive behavior under non-invasive RF are investigated. The in vitro and in vivo antitumor therapeutic effects of BP-Pt@PNA under non-invasive RF are evaluated by detecting the ICD effect of 4T1 breast cancer cells and the expression levels of Ki67 and TUNEL-positive cells in corresponding 4T1 tumor-bearing mice. The local, systemic, and immune memory effects mediated by BP-Pt@PNA under non-invasive RF radiation are confirmed in 4T1 tumor-bearing mice and lung metastasis models. The effective long-term retention of BP-Pt@PNA in 4T1 tumor-bearing mice is demonstrated, significantly improving mouse survival and biosafety compared to free chemotherapy drugs. In a VX2 tumor-bearing rabbit orthotopic liver cancer model, BP-Pt@PNA + RFA effectively inhibits the proliferation of orthotopic liver cancer. The prepared BP-Pt@PNA can be used for multimodal tumor therapy and has great potential for clinical translation.

[0027] The beneficial effects achieved by this invention are as follows:

[0028] 1. This invention utilizes the chemotherapy drug Nitro-Pt as a grafting bridge to achieve co-coordination of the radiofrequency responsive sensitizer black phosphorus nanoparticles BPNP and the thermosensitive polymer PNA, successfully preparing a thermosensitive nanogel BP-Pt@PNA loaded with radiofrequency responsive sensitizer and chemotherapy drug;

[0029] 2. The BP-Pt@PNA prepared by this invention has good thermosensitivity and sol-gel phase transition behavior. It can be gelled at 37°C and can be delivered by injection through a needle or microcatheter. At the same time, the gel complex formed can also achieve long-term drug retention and has good application in clinical TACE treatment.

[0030] 3. The BP-Pt@PNA prepared by this invention has good radio frequency response performance. Under non-contact radio frequency field irradiation, it can generate excellent radio frequency response thermal effect, and the radio frequency response thermal effect can be controlled by changing the concentration of BPNP, radio frequency power and radio frequency distance.

[0031] 4. The BP-Pt@PNA prepared in this invention can achieve controlled drug release under a non-contact radio frequency field;

[0032] 5. The BP-Pt@PNA prepared in this invention can promote tumor cell apoptosis and activate the ICD effect under a non-contact radiofrequency field. It can further promote lymphocyte infiltration by stimulating the maturation of dendritic cells, improve the immune microenvironment at the tumor site, activate the immune response, and thus inhibit tumor metastasis.

[0033] 6. The BP-Pt@PNA prepared in this invention can remain in mouse tumor tissue for a long time, and compared with free chemotherapy drugs, it significantly improves the survival time and biosafety of mice.

[0034] 7. The BP-Pt@PNA prepared in this invention can stimulate anti-tumor immune memory effects under a non-contact radio frequency field;

[0035] 8. The BP-Pt@PNA prepared in this invention can realize multimodal treatment of invasive radiofrequency ablation-chemotherapy-vascular embolization combined with antitumor therapy, and has great clinical translation prospects.

[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the process for preparing BP-Pt@PNA in Example 1 of the present invention;

[0038] Figure 2 This is a transmission electron microscope image of the BPNP prepared in Example 1 of the present invention;

[0039] Figure 3 This is a transmission electron microscope image of BP-Pt@PNA prepared in Example 1 of the present invention;

[0040] Figure 4 This is a composite diagram of experimental data for the products of each step in Example 1 of the present invention;

[0041] Figure 5 The graphs show the changes in particle size, transmittance, and potential of PNA and BP-Pt@PNA prepared in Example 1 of this invention as a function of temperature.

[0042] Figure 6 The graphs show the changes in elastic modulus, viscous modulus, and loss angle of BP-Pt@PNA and PNA prepared in Example 1 of this invention as a function of temperature.

[0043] Figure 7 The graph shows the shear viscosity variation curves of PNA and BP-Pt@PNA prepared in Example 1 of this invention at different shear rates.

[0044] Figure 8 This is a quantitative graph showing the uptake of Nitro-Pt and BP-Pt@PNA by 4T1 tumor cells in Example 6 of the present invention.

[0045] Figure 9 This is a cell survival rate graph of 4T1 tumor cells in Example 6 of the present invention;

[0046] Figure 10 This is a quantitative diagram showing the effect of cell apoptosis on 4T1 tumor cells in Example 6 of the present invention;

[0047] Figure 11 This is a quantitative diagram showing the effect of different materials on the CRT eversion of 4T1 tumor cells under the presence and absence of an RF field in Example 7 of the present invention;

[0048] Figure 12 This is a quantitative graph showing the different effects of different materials on 4T1 tumor cells in the presence and absence of an RF field in Example 7 of the present invention;

[0049] Figure 13 This illustrates the effect of different materials on DC cell maturation with and without an RF field in Example 7 of the present invention.

[0050] Figure 14 The images show the relative growth rate of 4T1 subcutaneous tumors in mice in Example 8 of this invention, as well as the actual tumor images on day 14 after treatment.

[0051] Figure 15 This refers to the change in mouse body weight in Example 8 of the present invention;

[0052] Figure 16 This is a quantitative immunofluorescence image of tumor tissue from mouse 4T1 subcutaneous tumors in Example 8 of this invention;

[0053] Figure 17 This is a data combination diagram of various experiments in Embodiments 9-11 of the present invention;

[0054] Figure 18 This is a quantitative graph showing the in vivo drug retention effect of BP-Pt@PNA in the 4T1 subcutaneous tumor model in Example 12 of this invention.

[0055] Figure 19 This is a graph showing the in vivo antitumor effect and immunofluorescence and histochemical quantitative analysis of the VX2 liver cancer tumor model in Example 13 of this invention.

[0056] Figure 20 Gross images of tumors in the isolated rabbit liver at different time points in Example 13 of this invention. Detailed Implementation

[0057] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0058] The present invention will be explained in more detail through the following embodiments. The purpose of disclosing the present invention is to protect all changes and modifications within the scope of the present invention. The present invention is not limited to the following embodiments.

[0059] Example 1

[0060] S1. Preparation of radio frequency responsive thermal sensitizer BPNP

[0061] Accurately weigh 30 mg of black phosphorus nanoparticles and place them in a sealed centrifuge tube containing 30 mL of N-methylpyrrolidone. Sonicate the mixture using a probe (800 W) for 8 h at a frequency of 19-25 kHz, with a 2s on / 4s off cycle. Continue sonicating the mixture in an ice-water bath at 1600 W for 20 h. Centrifuge the dispersion at 7000 rpm for 20 min to remove any unexfoliated black phosphorus nanoparticles. Collect the supernatant and wash it three times with water (12000 rpm, 20 min) to remove N-methylpyrrolidone solvent and impurities. Collect the precipitate and freeze-dry it under vacuum to obtain purified BP powder.

[0062] S2, Preparation of the chemotherapy drug Nitro-Pt

[0063] Traditional chemotherapy drug cisplatin has weak electropositivity, which may result in insufficient coordination with BPNP. To further expose the cation core of cisplatin, this invention utilizes a nitration reaction to replace the two chloride ions on cisplatin, thereby better exposing the Pt(II) cation and improving the electropositivity of cisplatin. The specific implementation steps are as follows: Accurately weigh cisplatin (300 mg, 1 mmol) and dissolve it in ultrapure water, then mix it with silver nitrate (340 mg, 2 mmol) at a molar ratio of 1:2. Stir overnight at room temperature in the dark, then centrifuge at 3000 rpm for 5 min to remove the AgCl precipitate. Collect the supernatant and purify it through a 0.22 μm filter. The concentration of the Nitro-Pt solution is then determined by ICP-OES and stored at 4°C in the dark for later use.

[0064] Preparation of S3 and temperature-sensitive nanopolymer PNA

[0065] Thermosensitive nanopolymer PNA was prepared using reversible addition-fragmentation chain transfer radical polymerization (RAFT), specifically including the following steps:

[0066] Accurately weigh 11.3 g (100 mmol) of N-isopropylacrylamide monomer NIPAM into a Stokes reaction tube equipped with a magnetic stir bar. Separately, accurately measure 35 mL of DMSO and add it to the reaction tube. Stir on a thermostatic magnetic stirrer until NIPAM is completely dissolved to obtain the sample. Transfer the sample to a liquid nitrogen bath and freeze for 5 min, then evacuate the vacuum pump for 5 min. Finally, place the sample in a warm water bath, stir, and purge with argon gas for 5 min to rapidly thaw the sample.

[0067] After argon gas introduction, the sample was frozen again with liquid nitrogen. When the liquid sample solidified, the chain transfer agent 4-cyano-4-(thiobenzoyl)valerate (CPT) (279 mg, 1 mmol) was added, and the liquid nitrogen freezing-vacuuming cycle was repeated. While argon gas was introduced, the sample was stirred in a warm water bath to thaw it and rapidly dissolve the chain transfer agent. After argon gas introduction, the sample was frozen again with liquid nitrogen. When the liquid sample solidified, an accurately weighed initiator azobisisobutyronitrile (AIBN) (2.6 mg, 0.01 mmol) was added, and the remaining vacuuming-argon gas introduction cycle was completed. After the reaction solution was completely thawed, the reaction tube was placed in a 70°C oil bath for 24 h.

[0068] After NIPAM monomer polymerization was completed, the product was dialyzed and lyophilized, and then acrylic acid monomer AA (100 mM, 7.206 mL) was added for a second RAFT polymerization reaction. The reaction was then continued in a 70℃ oil bath for 24 h. After the reaction was complete, the product was transferred to a 3500 kDa dialysis bag and dialyzed in pure water for 3-4 days. Finally, the dialysate was lyophilized to obtain purified PNA powder. PNA has a lower critical solubility temperature of 30.75℃, which is lower than the temperature inside the human body, allowing it to change from a hydrophilic to a hydrophobic state within the body.

[0069] S4. Synthesis of radio frequency thermally responsive platinum ion crosslinked black phosphorus nanogel (BP-Pt@PNA)

[0070] This nanogel utilizes Nitro-Pt as a grafting bridge, enabling stable co-loading with the lone pair electrons on the BPNP surface and the carboxyl groups in the thermosensitive polymer through coordination bonds. The specific steps include: adding the prepared BP and PNA powders stepwise to a Nitro-Pt solution, with a mass ratio of BPNP, Nitro-Pt, and PNA of 1:5:100. The mixture is stirred for 24 h under nitrogen protection in the dark. After the reaction is complete, the mixture is purified by ultrafiltration (4500 rpm, 10 min) using an ultrafiltration tube (MWCO: 3 kDa). The filtrate is collected and freeze-dried to obtain purified BP-Pt@PNA powder. Figure 1 ).

[0071] Example 2

[0072] Characterization of radio frequency thermally responsive platinum ion crosslinked black phosphorus nanogel (BP-Pt@PNA)

[0073] The prepared BP-Pt@PNA was prepared into a dispersion at a polymer concentration of 1 mg / mL for electron microscopy analysis. Figure 2 As shown, the prepared BPNP exhibits good dispersibility with an average diameter of 24.2-38.6 nm. The prepared BP-Pt@PNA material has a core-shell structure, as shown in the figure. Figure 3 As shown, it consists of BPNP, Nitro-Pt (core), and PNA (shell), with an average diameter of 300 nm.

[0074] Example 3

[0075] Characterization of RF response thermal effects of BP-Pt@PNA

[0076] 1.5 mL of BPNP at different concentrations was placed in a radio frequency field with the same power and radiation distance for 5 min. The highest real-time temperature of the sample was recorded using an infrared thermal imager every 1 min to characterize the strength of the radio frequency thermal effect of the sample.

[0077] 1.5 mL of each of BP-Pt@PNA, BPNP, Nito-Pt, PNA, physiological saline, and ultrapure water were placed in a non-invasive RF field with the same power and radiation distance for 5 min. The highest real-time temperature of the samples was recorded using an infrared thermal imager every 1 min to characterize the strength of the radio frequency thermal effect of the samples. Figure 4 ).

[0078] The radiation power of the radio frequency field ranges from 100W to 300W, and the irradiation distance ranges from 1.5 cm to 3 cm.

[0079] like Figure 4 As shown, Figure 4 b is a real-time infrared thermal image of BP-Pt@PNA, BPNP, Nito-Pt, PNA, and physiological saline prepared in Example 1 of this invention by radio frequency heating. Figure 4 c is a comparison chart of the radio frequency heating thermal efficiency of BP-Pt@PNA, BPNP, Nito-Pt, PNA, and physiological saline prepared in Example 1 of this invention. Figure 4 d is the radio frequency heating curve of BP-Pt@PNA, BPNP, Nito-Pt, PNA, and physiological saline prepared in Example 1 of the present invention; Figure 4 e represents the radio frequency (RF) heating curves of BPNP at different concentrations prepared in Example 1 of this invention. The RF thermal effect generated by BPNP increases with increasing BPNP concentration. Under the same BPNP concentration and the same RF radiation conditions, BP-Pt@PNA produces the most significant RF thermal effect compared to the control group sample.

[0080] The RF temperature rise curves of BP-Pt@PNA at different RF distances and powers are as follows: Figure 4As shown, the radio frequency (RF) thermal effect generated by BP-Pt@PNA gradually increases with increasing RF power; however, the RF thermal effect generated by BP-Pt@PNA gradually decreases with increasing RF irradiation distance. Therefore, the RF response thermal effect of BP-Pt@PNA exhibits BPNP concentration, RF power, and RF distance dependence characteristics.

[0081] Example 4

[0082] Investigation of controlled drug release behavior of BP-Pt@PNA in a non-invasive RF field

[0083] The BP-Pt@PNA prepared according to the method of this invention was irradiated in a non-invasive RF field with the same power and radiation distance for 0-15 min, and the sample supernatant was collected every 2 min. For the pulsed drug release experiment, BP-Pt@PNA was irradiated in a non-invasive RF field for 0.5, 1, 1.5, 2, 2.5, and 3 min, respectively, and then heated in a 37℃ water bath for 30 s, and the sample supernatant was collected at each time point. After high-temperature digestion, the content of Pt(NO3)2(NH3)2 in each supernatant was determined using inductively coupled plasma atomic emission spectrometry. The results show that the drug release behavior of BP-Pt@PNA in a non-invasive RF field is radio frequency power dependent. Furthermore, BP-Pt@PNA also exhibits pulsed drug release behavior triggered by a non-invasive RF field. Figure 4 ).

[0084] Example 5

[0085] Characterization of the thermosensitivity of BP-Pt@PNA

[0086] The PNA and BP-Pt@PNA prepared according to the method of this invention were prepared into dispersions at a polymer concentration of 1 mg / mL. The temperature sensitivity of the samples was characterized by measuring the particle size, potential, and transmittance of the samples using a particle size analyzer and a UV-Vis spectrophotometer within the temperature range of 25℃-45℃. The incubation time at each temperature point was 5 min. Figure 5 As shown, both PNA and BP-Pt@PNA exhibit good temperature sensitivity. As the temperature increases, the polymer gradually transforms from a sol state to a gel state, and the hydrophobicity of the polymer gradually increases. The water absorbed in the polymer is expelled, resulting in a slow decrease in particle size. Furthermore, the shrinkage of the polymer increases its specific surface area, leading to an increase in the absolute value of the Zeta potential. Simultaneously, with the change in the hydrophilicity of the polymer, the transmittance of both PNA and BP-Pt@PNA gradually decreases.

[0087] The PNA and BP-Pt@PNA prepared according to the method of this invention were formulated into dispersions at a polymer concentration of 100 mg / mL, and their thermosensitive sol-gel phase transition behavior was characterized using an advanced rotational rheometer. Rheological results ( Figure 6 This indicates that the storage modulus (G) of BP-Pt@PNA is... ， ) and loss modulus (G ，， The Pa values ​​increased from 1.2 and 0.31 at 25°C to 3700 and 65.8 at 50°C, respectively, and the G of PNA... ， and G ，， The presence of the same trend at 25℃ and 50℃ indicates that BP-Pt@PNA exhibits excellent gelation behavior, maintaining long-term drug retention while effectively embolizing blood vessels at the tumor site. On the other hand, as... Figure 7 As shown, both PNA and BP-Pt@PNA exhibit good shear-thinning properties. With increasing shear rate, the shear viscosity decreases significantly, indicating that the polymers have excellent injection flowability before gelation and are easy to inject.

[0088] Example 6

[0089] The effects of BP-Pt@PNA + RF on tumor cell apoptosis were investigated using cell uptake assays, CCK8 cell viability assays, and apoptosis assays.

[0090] 6.1 Cell uptake experiment

[0091] 4T1 cells in logarithmic growth phase were seeded into 6-well plates, 600,000 cells per well, and incubated in an incubator for adhesion. Nito-Pt and BP-Pt@PNA were prepared according to a Pt concentration gradient. After adhesion, the supernatant in the wells was replaced with medium containing Nito-Pt or BP-Pt@PNA, and the plates were incubated for another 6 hours. The cells were then digested, collected, and counted.

[0092] After high-temperature digestion of the cell digestion solution, the concentration of platinum ions in the cell digestion solution was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES), and the uptake of the chemotherapeutic drug Nito-Pt by the cells was calculated.

[0093] like Figure 8 As shown, due to the hydrophilic-hydrophobic transformation of BP-Pt@PNA in vivo, this transformation results in a higher cellular uptake of BP-Pt@PNA compared to free nitrocisplatin.

[0094] 6.2 CCK8 Cell Viability Assay

[0095] 4T1 cells in logarithmic growth phase were seeded into 24-well plates, 50,000 cells per well, and incubated in an incubator for adhesion. BPNP, Nitro-Pt, PNA, and BP-Pt@PNA were prepared at a Nito-Pt concentration of 12 ppm, along with corresponding amounts of culture medium as controls. After adhesion, the supernatant in the wells was replaced with culture medium containing the aforementioned materials, and the plates were incubated for another 6 h. Different experimental groups underwent non-invasive RF field irradiation treatment, and after 18 h of incubation, 100 μL of CCK8-containing culture medium was added to each well. After incubation in the dark for another 2 h, the absorbance of the sample wells at 450 nm was measured using a microplate reader.

[0096] like Figure 9 As shown, Figure 9 A is a graph showing the cell survival rate of 4T1 tumor cells using different materials in Example 6 of the present invention. Figure 9 B shows the cell survival rate of 4T1 tumor cells under the presence and absence of an RF field in Example 6 of this invention. Under a non-invasive RF field, BP-Pt@PNA can induce more radiofrequency thermal effects and effective Nitro-Pt release, thereby leading to more 4T1 cell death.

[0097] 6.3 Apoptosis Experiment

[0098] 4T1 cells in the logarithmic growth phase were seeded into 6-well plates, 100,000 cells per well, and incubated in an incubator for adhesion. BPNP, Nitro-Pt, PNA, and BP-Pt@PNA were prepared at a Nitro-Pt concentration of 12 ppm, along with corresponding amounts of culture medium as controls. After adhesion, the supernatant in the wells was replaced with culture medium containing the above materials, and the plates were incubated for another 6 h.

[0099] Different experimental groups were treated with and without non-invasive RF field irradiation. After incubation for 18 h, the cells in the well plates were digested and collected (the cells in the supernatant needed to be collected before digestion). The cells were then stained according to the apoptosis kit and the proportion of cells at different apoptotic stages was analyzed by flow cytometry.

[0100] like Figure 10 As shown, Figure 10 A is a flow cytometry diagram showing the effect of different materials on apoptosis of 4T1 tumor cells under the presence and absence of an RF field in Example 6 of the present invention. Figure 10 B is a quantitative graph showing the effect of different materials on apoptosis of 4T1 tumor cells under and without an RF field in Example 6 of this invention. Under an RF field, BP-Pt@PNA can induce more radiofrequency thermal effects and effective Nito-Pt release, effectively promoting early and late apoptosis of 4T1 cells.

[0101] Example 7

[0102] The efficacy of BP-Pt@PNA + RF synergistic activation of in vitro antitumor immune response was determined by detecting ICD biomarkers and dendritic cell (DC) maturation.

[0103] 7.1 ICD Biomarker Detection Experiment

[0104] 4T1 cells in logarithmic growth phase were seeded in 24-well plates at 120,000 cells per well and incubated for adhesion. BPNP, Nitro-Pt, PNA, and BP-Pt@PNA were prepared at a Nito-Pt concentration of 12 ppm, along with corresponding amounts of culture medium as controls. After adhesion, the supernatant in the wells was replaced with the aforementioned medium, and the plates were incubated for another 6 h. Different experimental groups underwent non-invasive RF field treatment with and without the treatment. After 18 h of incubation, the cells were digested and collected, and CRT expression in 4T1 cells was measured using flow cytometry. After another 18 h of incubation, the cell supernatant was collected, and the amounts of ATP and HMGB-1 released by 4T1 cells were measured using an ATP and HMGB-1 assay kit.

[0105] 7.2 Dendritic cell (DC) maturation detection experiment

[0106] 4T1 cells in logarithmic growth phase were seeded into 24-well plates, 100,000 cells per well, and incubated in an incubator for adhesion. BPNP, Nitro-Pt, PNA, and BP-Pt@PNA were prepared at a Nito-Pt concentration of 12 ppm, with corresponding amounts of culture medium as controls. After adhesion, the supernatant in the wells was replaced with culture medium containing the materials, and the plates were incubated for another 6 h. Different experimental groups underwent invasive RF field irradiation treatment, and after 18 h of incubation, the cell supernatant was collected and added to pre-seeded and adhered DC2.4 cells (600,000 cells per well in 12-well plates). After a total of 24 hours of incubation, the cells were digested and collected, and the maturation status of the DC2.4 cells was analyzed using flow cytometry. Figure 11 , 12 and Figure 13 As shown, the results indicate that the experimental groups containing the chemotherapy drug Nitro-Pt can significantly increase the eversion of CRT, enhance ATP secretion, and induce the release of HMGB1. Furthermore, BP-Pt@PNA + RF can induce the release of more DAMPs, enhance the ICD effect, promote DC cell maturation, and thus activate the in vitro anti-tumor immune response.

[0107] Example 8

[0108] After establishing a 4T1 subcutaneous tumor model in BALB / c mice (female, 6 weeks, 16-18 g), the synergistic in vivo antitumor effect of BP-Pt@PNA + RF was evaluated.

[0109] One day prior to non-invasive radiofrequency (RF) treatment, mice were injected intratumorally with drugs prepared at a Nitro-Pt concentration of 2 mg / mL in physiological saline, BPNP, Nitro-Pt, PNA, and BP-Pt@PNA, respectively, in 50 μL injection volumes. The tumors were then exposed to a 300 W non-invasive radiofrequency field for 15 min. Tumor volume and body weight were monitored every two days after treatment. On day 14 post-treatment, the remaining mice were sacrificed, tumors were dissected, and the excised tumors were weighed and photographed. The tumor tissues were then subjected to H&E staining, Ki67 and TUNEL immunofluorescence staining to investigate tumor cell proliferation and apoptosis in the mice.

[0110] like Figure 14 As shown, the results indicate that the volume increased from 161.98 ± 18.05 mm on day 0. 3 The value decreased to 25.56 ± 12.85 mm on day 14. 3 The average tumor weight was only 13.16 ± 7.72 mg, significantly lower than the 310.84 ± 55.86 mg in the control group mice. Due to the synergistic effect of radiofrequency thermal effect and chemotherapy, tumors were significantly inhibited in BP-Pt@PNA + RF.

[0111] Tumors were also inhibited to some extent in the Nitro-Pt + RF group, but due to the strong side effects of free Nitro-Pt, it had a significant impact on the body weight and health status of mice. Figure 15 ), while BP-Pt@PNA nanogel significantly inhibits the side effects of Nito-Pt. Furthermore, such as Figure 16 As shown, the immunofluorescence staining analysis of tumor tissue also indicates that BP-Pt@PNA +RF can significantly kill tumor cells, promote tumor cell apoptosis, and inhibit tumor cell proliferation.

[0112] Example 9

[0113] After establishing a 4T1 lung metastasis model in BALB / c mice (female, 6 weeks, 16-18 g), the in vivo antitumor metastasis effect of BP-Pt@PNA + RF was evaluated.

[0114] 4T1 cells (100 μL, 1 million) were injected into tumor-bearing mice via the tail vein. Twenty-four hours later, the tumors were injected intratumorally with drugs prepared with physiological saline, BPNP, Nitro-Pt, PNA, and BP-Pt@PNA at a concentration of 2 mg / mL nitrocisplatin, in 50 μL injection volumes. The tumors were then exposed to a 300 W non-invasive radiofrequency field for 15 min. On day 14 post-treatment, the remaining mice were sacrificed, lung tissue was dissected, weighed, stained with picric acid, and the number of lung nodules was counted. Images were taken and then subjected to H&E staining. Gross images, count and quantitative images, H&E staining images, and lung wet weight quantitative images of the mouse lung metastasis model are shown below. Figure 17 As shown in the il, the results indicate that after treatment with BP-Pt@PNA + RF, the number of lung nodules in mice was significantly reduced, and tumor metastasis was inhibited.

[0115] Example 10

[0116] After establishing a 4T1 subcutaneous tumor model in BALB / c mice (female, 6 weeks, 16-18 g), the synergistic in vivo antitumor immune effect of BP-Pt@PNA + RF was evaluated.

[0117] 4T1 tumor-bearing mice were administered the drug according to the protocol in Example 10, and on day 5 post-treatment (with or without invasive radiofrequency therapy), flow cytometry was used to analyze the maturity of dendritic cells (DCs) in the primary tumor and major organs, as well as CD8+. + Expression of cytotoxic T lymphocytes (TILs). Dendritic cells (DCs) are antigen-presenting cells that play an important role in ICD effects and immune responses. An analysis of the in vivo antitumor immune response in a 4T1 subcutaneous tumor model is shown in the figure. Figure 17 As shown in the figure, the number of mature dendritic cells (DCs) in the tumor tissue and lymph nodes of the BP-Pt@PNA + RF group was significantly higher than that in the control group, fully revealing the necessity of the synergistic activation of ICD effects by thermotherapy-chemotherapy. More importantly, the number of CD8+ cells in the tumor tissue, lymph nodes, and spleen of the BP-Pt@PNA + RF group was significantly higher. + The expression of cytotoxic T lymphocytes also reached the highest level among all groups. Therefore, the above results indicate that BP-Pt@PNA + RF-induced thermotherapy-chemotherapy can induce a significant tumor-specific immune response.

[0118] Example 11

[0119] After establishing a 4T1 subcutaneous tumor model in BALB / c mice (female, 6 weeks, 16-18 g), we evaluated the synergistic in vivo antitumor immune memory effect of BP-Pt@PNA + RF.

[0120] 4T1 tumor-bearing mice were administered the drug according to the protocol in Example 10, with or without non-invasive radiofrequency (RF) treatment. On day 60 post-treatment, flow cytometry was used to analyze the expression of effector memory T cells in the spleen. A second tumor graft was performed, and tumor growth was assessed within 14 days after the second graft. The in vivo anti-tumor immune memory effect analysis of the 4T1 subcutaneous tumor model is shown in the following figure. Figure 17 As shown in the mp, the expression of effector memory T cells in the spleen of the BP-Pt@PNA + RF group was significantly higher than that of the control group, and no significant tumor growth was observed in the BP-Pt@PNA + RF group within 14 days after the second tumor inoculation compared with the control group.

[0121] Example 12

[0122] After establishing a 4T1 subcutaneous tumor model in BALB / c mice (female, 6 weeks, 16-18 g), we evaluated the in vivo drug retention effect and biosafety of BP-Pt@PNA.

[0123] After administration of the drug according to the protocol in Example 10, tumor tissues and major organs of 4T1 tumor-bearing mice were collected on day 2 and day 14 of administration. The tissues were weighed, digested at high temperature, and the Nitro-Pt content in each tissue was determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). A quantitative graph evaluating the in vivo drug retention effect of BP-Pt@PNA in the 4T1 subcutaneous tumor model is shown below. Figure 18 As shown, after intratumoral drug administration, Nitro-Pt was mainly distributed in the tumor tissue compared to major organs; on day 2 of administration, the drug content in the tumor of the BP-Pt@PNA group was nearly twice that of the free Nitro-Pt group; even on day 14 of treatment, the drug content in the tumor of the BP-Pt@PNA group was significantly higher than that of the control group. This demonstrates the excellent intratumoral drug retention effect of BP-Pt@PNA.

[0124] Example 13

[0125] To establish a VX2 tumor-bearing rabbit orthotopic hepatocellular carcinoma model and evaluate the in vivo antitumor effect of BP-Pt@PNA + RFA.

[0126] The pre-prepared VX2 tumor fragment was implanted into the left lateral lobe of the liver (near the falciform ligament) of the experimental rabbits, and the rabbits were fed for 18 days. CT scan confirmed successful tumor implantation. BP-Pt@PNA, prepared at a Nitro-Pt concentration of 400 ppm with a drug volume of 0.3 mL, was used to treat the experimental rabbits via transarterial chemoembolization. Three days later, invasive radiofrequency ablation was performed at a power of 100 W for 2 minutes.

[0127] like Figure 19 and 20As shown, experimental rabbits were sacrificed on days 0, 4, 7, and 10, and tumors and surrounding normal liver tissue were collected. After photographing, the tumor tissue was subjected to H&E staining, Ki67 and TUNEL immunofluorescence staining, and HIF-1α, VEGF, CD31, HSP70, and CD8 staining. + Immunohistochemical staining. Based on the tumor volume change curves obtained from CT images, there was no statistically significant difference in tumor volume among the experimental groups on day 0. However, on day 10, the tumor volume in the BP-Pt@PNA + RFA group was significantly lower than that in the Saline group, Saline + RFA group, and BP-Pt@PNA group. Tumor H&E staining results also showed that, except for the BP-Pt@PNA + RFA group, viable tumor cells could be found in all other experimental groups at all time points. However, in the BP-Pt@PNA + RFA group, viable tumor cells were difficult to find after day 4. Furthermore, compared to other groups, the BP-Pt@PNA + RFA group showed the highest tumor necrosis rate, reaching 90%. In addition, tumor images and quantitative volume analysis at different time points also indicated that the BP-Pt@PNA + RFA group had the best tumor growth inhibition effect and the lowest number of intrahepatic metastatic lesions.

[0128] Immunofluorescence staining and immunohistochemical results of tumor tissue further demonstrated that TACE based on BP-Pt@PNA combined with invasive RFA achieved synergistic treatment of radiofrequency ablation-chemotherapy-vascular embolization. It has a good regulatory effect on the tumor immune microenvironment (HSP70 and CD8+ T cells), effectively inhibits tumor angiogenesis (HIF-1α, VEGF, CD31), suppresses tumor cell proliferation, and promotes tumor cell apoptosis.

[0129] 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 preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A radio frequency heat responsive platinum ion cross-linked black phosphorus nanogel characterized by: Including radiofrequency-responsive thermal sensitizers, chemotherapy drugs, and thermosensitive nanopolymers; The chemotherapy drug was nitrocisplatin; Thermosensitive nanopolymers are linear block polymers synthesized from N-isopropylacrylamide monomers and acrylic acid monomers via RAFT polymerization, with a degree of polymerization of 100-100.

2. The radio frequency heat responsive Pt4+ cross-linked black phosphorous nanohydrogel according to claim 1, wherein: The mass ratio of the radiofrequency responsive thermal sensitizer, the chemotherapy drug, and the thermosensitive nanopolymer is 1:1:100 to 1:6:

100.

3. The radio frequency heat responsive Pt4 ion cross-linked black phosphorous nanohydrogel according to claim 1, wherein: The radio frequency responsive thermal effect sensitizer is black phosphorus nanoparticles with a particle size of 24.2-38.6 nm.

4. The preparation of radiofrequency heat-responsive Pt4+-crosslinked black phosphorus nanohydrogel according to any one of claims 1-3, characterized in that: S1. Preparation of radio frequency responsive thermal effect sensitizer: Black phosphorus nanoparticles were exfoliated by low-temperature probe ultrasound and non-contact water bath ultrasound. Unexfoliated black phosphorus nanoparticles were removed by multiple water washing and centrifugation. Finally, radio frequency responsive thermal effect sensitizer was obtained by freeze drying. S2. Preparation of chemotherapy drugs: Cisplatin and silver nitrate were mixed at a molar ratio of 1:2, stirred in the dark, and centrifuged to remove silver chloride precipitate to obtain nitrocisplatin; S3. Preparation of thermosensitive nanopolymer: N-isopropylacrylamide monomer was added to DMSO solvent, stirred and dissolved, and then repeatedly frozen and thawed with liquid nitrogen. After freezing again, chain transfer agent was added, and after thawing, the mixture was stirred in a warm water bath. After freezing again, initiator was added and reacted. After vacuuming and purging with argon, the mixture was thawed, reacted in an oil bath at 70°C for 24 hours, and then dialyzed and freeze-dried. Acrylic acid monomer was added, and the reaction was continued for 24 hours. After dialyzing, the thermosensitive nanopolymer was obtained by freeze-drying. Preparation of S4 nanogel: The radio frequency responsive thermal sensitizer in S1 was added to the nitrocisplatin solution in S2. After stirring, mixing and centrifuging in the dark, the thermosensitive nanopolymer in S3 was added. The self-assembly reaction formed a co-coordinated nanogel. Subsequently, the concentrate was collected by ultrafiltration and centrifugation, and then freeze-dried to obtain the nanogel.

5. The preparation of radio frequency heat responsive Pt4+-crosslinked black phosphorus nanohydrogel according to claim 4, characterized by: In S3, the initiator is azobisisobutyronitrile, and the chain transfer agent is 4-cyano-4-(thiobenzoyl)valerate.

6. Use of a nanogel prepared according to the process of any one of claims 4-5, characterized in that: Nanogels are used in the preparation of drugs for non-invasive radiofrequency thermotherapy-chemotherapy synergistic anti-tumor therapy and drugs for invasive radiofrequency thermoablation-chemotherapy-vascular embolization synergistic anti-tumor therapy.

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