A near-infrared two-window response photothermal / chemotherapy synergistic nano platform and a preparation method thereof

Abstract

The application belongs to the technical field of biological medicine and nanometer material preparation, and particularly relates to a near-infrared two-window response type photothermal / chemotherapy synergistic nano platform and a preparation method thereof. The nano platform is formed by self-assembly of a near-infrared two-region organic photothermal molecule BBT, a double drug prodrug CPT-ss-CA4 and an amphiphilic polymer DSPE-PEG-cRGD. The BBT molecule adopts a donor-acceptor-donor structure design and has good near-infrared two-region absorption capacity. After being loaded by the DSPE-PEG-cRGD, stable nanoparticles with an average particle size of about 105 nm are formed, which can realize efficient light energy absorption and conversion. At the same time, the responsive connection structure in the CPT-CA4 prodrug endows the system with glutathione responsive release capacity, which can promote drug release under the condition of tumor microenvironment and improve drug utilization efficiency. The BTTC nano platform shows excellent photothermal response capacity under 1064 nm laser irradiation and has good tumor treatment application potential.

Description

[0001] This invention belongs to the field of biomedicine and nanomaterial preparation technology, specifically relating to a near-infrared two-window responsive photothermal / chemotherapy synergistic nanoplatform and its preparation method. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most prevalent pathological type of primary liver cancer, characterized by high incidence, poor prognosis, and high recurrence rate. Its five-year survival rate is less than 20%, posing a serious threat to human health. Current clinical treatments, such as surgical resection, radiotherapy, and systemic chemotherapy, while achieving some efficacy in early-stage patients, generally suffer from limited efficacy, significant side effects, and drug resistance. For patients with intermediate-to-advanced liver cancer, there is an urgent need to develop novel treatment strategies with higher selectivity and lower toxicity.

[0003] Photothermal therapy (PTT), a non-invasive local treatment, utilizes photothermal converters to generate localized high temperatures under specific wavelength laser irradiation to kill tumor cells. It has attracted widespread attention due to its spatiotemporal controllability and minimally invasive nature. Compared to the traditional near-infrared I (700-900 nm) window, the near-infrared II (1000-1700 nm) biological window offers deeper tissue penetration, higher maximum permissible irradiation dose, and lower tissue autofluorescence interference, providing possibilities for efficient treatment and precise imaging of deep tumors. Therefore, the development of NIR-II responsive organic small-molecule photothermal agents has become a research hotspot in this field.

[0004] Among numerous organic photothermal materials, donor-acceptor (DA or DAD) type small molecule dyes constructed using benzobisthiadiazole (BBT) as the electron acceptor exhibit excellent photothermal conversion performance and NIR-II fluorescence imaging potential. Studies have shown that through rational molecular structure design, such as introducing alkoxyphenyl units into π-bridges to construct a moderately twisted molecular configuration, intermolecular π-π stacking can be effectively suppressed, fluorescence emission enhanced, and spin-orbit charge transfer intersystem crossing (SOCT-ISC) promoted, thereby enabling NIR-II fluorescence imaging combined with photodynamic / photothermal therapy. Furthermore, introducing long alkyl chains as flexible spacers can provide more space for molecular excited states, significantly improving non-radiative transition efficiency. However, single photothermal therapy still faces several bottlenecks: on the one hand, relying solely on local high temperatures is insufficient to completely eliminate tumors, and excessively high temperatures (>50°C) may cause thermal damage to surrounding normal tissues; on the other hand, photothermal therapy may induce heat shock in tumor cells, reducing efficacy. Therefore, synergistic integration of photothermal therapy with other treatment modalities such as chemotherapy has become an important strategy for improving anti-tumor effects.

[0005] In prodrug design, glutathione (GSH), highly expressed in the tumor microenvironment, is used as a triggering factor to design GSH-responsive cleavable prodrugs, enabling controlled release of chemotherapeutic drugs and improving treatment selectivity. Simultaneously, integrating two drugs with different mechanisms of action (such as chemotherapeutic drugs and angiogenesis inhibitors) into a single prodrug molecule via responsive linkers can achieve synergistic multidrug therapy and reduce the risk of drug resistance. Furthermore, the localized heating generated by the photothermal effect can further accelerate the breaking of GSH-responsive chemical bonds, achieving a dual-response mechanism of "photothermal accelerated drug release," providing a new approach for spatiotemporally controlled drug release.

[0006] In the active targeting design of nanoplatforms, cyclic arginine-glycine-aspartic acid (cRGD) peptides have become one of the most commonly used targeting ligands in nanomedicine delivery systems due to their ability to specifically recognize tumor neovascular endothelial cells and integrin receptors (such as αvβ3) highly expressed on the surface of various tumor cells. Coupled with cRGD to the surface of DSPE-PEG modified nanoparticles, the active targeting capability and cellular uptake efficiency of the nanoplatform for tumor tissues can be significantly enhanced, improving therapeutic precision.

[0007] Despite the significant progress made in the diagnosis and treatment of liver cancer, several technical bottlenecks remain to be overcome: First, it is difficult to balance fluorescence brightness and photothermal efficiency in most NIR-II photothermal materials, which limits the precision of image-guided therapy; second, the drug loading capacity and responsive release efficiency of nanoplatforms need further improvement; and third, how to integrate multiple functions such as NIR-II imaging, active targeting, photothermal therapy, and multi-response drug release into a single nanosystem, while ensuring the stability and biosafety of the formulation, remains a major challenge for clinical translation.

[0008] Therefore, developing a multifunctional nanoplatform based on BBT-like photothermal molecules, possessing both high photothermal conversion efficiency and GSH / temperature dual-response drug release characteristics, and capable of actively targeting liver cancer lesions via cRGD, is of significant scientific importance and promising clinical application for improving the treatment efficacy of liver cancer. Summary of the Invention

[0009] The technical problem to be solved by this invention is to provide a near-infrared two-window responsive photothermal / chemotherapy synergistic nanoplatform and its preparation method. The nanoplatform is formed by the self-assembly of the near-infrared II organic photothermal molecule BBT, the dual-drug prodrug CPT-CA4, and DSPE-PEG-cRGD via nanoprecipitation. The resulting nanoparticles have an average particle size of approximately 105 nm, exhibit significant absorption at 971 nm, and demonstrate excellent near-infrared II photothermal conversion capabilities, showing promising application prospects in the treatment of liver cancer.

[0010] The technical solution adopted is as follows: A near-infrared two-window responsive photothermal / chemotherapy synergistic nanoplatform is formed by the self-assembly of the near-infrared two-zone organic photothermal molecule BBT (benzobisthiadiazole), the dual-drug prodrug CPT-ss-CA4 (disulfide-bridged camptothecin-cobustatin A4 conjugate), and the amphiphilic polymer DSPE-PEG-cRGD (distearate phosphatidylethanolamine-polyethylene glycol-targeted membrane-penetrating peptide cRGD). The BBT has a donor-acceptor-donor (DAD) structure and has light absorption and photothermal conversion capabilities in the near-infrared II region; The CPT-ss-CA4 is a dual-drug prodrug that connects CPT (camptothecin) and CA4 (cobustatin) via disulfide bonds; The DSPE-PEG-cRGD is used to encapsulate hydrophobic components and provide targeting functionality.

[0011] Preferably, the average hydrated particle size of the nanoplatform is 80~150 nm, and more preferably 100~110 nm; The nanoplatform has a maximum absorption peak at 971 nm; The nanoplatform exhibits a photothermal heating effect under 1064 nm laser irradiation, and its photothermal conversion efficiency is not less than 40%.

[0012] Preferably, the nanoplatform has glutathione (GSH) responsive drug release properties, in which the disulfide bond in CPT-ss-CA4 breaks in the presence of GSH, releasing CPT and CA4.

[0013] Preferably, the nanoplatform can achieve photothermal promotion of drug release under 1064 nm laser irradiation, and the release amount increases with increasing temperature.

[0014] Preferably, the mass ratio of BBT to CPT-ss-CA4 in the nanoplatform is 50~65:1; more preferably 58~60:1. The mass ratio of the amphiphilic polymer DSPE-PEG-cRGD to BBT is 15~25:1, and more preferably 20:1.

[0015] This invention also provides a method for preparing a near-infrared II responsive photothermal / chemotherapy synergistic nanoplatform, comprising the following steps: (1) Preparation of BBT: Under inert gas protection, the corresponding organotin compound and the brominated acceptor unit were subjected to a Stille coupling reaction in anhydrous toluene catalyzed by Pd(PPh3)4 (tetraphenylphosphine palladium). After the reaction, BBT was obtained by separation and purification. The structural formula of BBT is as follows: ; (2) Preparation of CPT-ss-CA4: Using the hydroxyl activation strategy of triphosgene, CPT was reacted with dithiodiethanol to obtain the intermediate CPT-ss-OH (dithiodiethanol-modified camptothecin monohydroxy intermediate), which was then esterified with CA4 to obtain CPT-ss-CA4; (3) Preparation of nanoplatform: BBT and CPT-ss-CA4 are dissolved in an organic solvent to obtain a mixed solution. Then, the obtained mixed solution is injected into a deionized aqueous solution containing DSPE-PEG-cRGD under ultrasonic conditions to form nanoparticles. Continue ultrasonic treatment and remove organic solvent by nitrogen purging or vacuum evaporation. After post-treatment, the BTTC nanoplatform is obtained.

[0016] Preferably, the Stille coupling reaction temperature in step (1) is 100~140 ℃ and the reaction time is 12~48 h; The separation and purification process includes adding the reaction solution to a non-polar solvent to precipitate a solid, followed by purification by column chromatography or recrystallization; wherein the non-polar solvent is selected from any one of isohexane, pentane, petroleum ether, and n-hexane.

[0017] Preferably, the organotin compound is 2,5-bis(2-octyldodecyl)-3,6-bis(5-(trimethylstanyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrolo-1,4-dione, and the bromoacceptor unit is 4,9-bis(5-bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazo[3,4-g]quinoxaline.

[0018] Preferably, the mass ratio of organotin compound to brominated acceptor unit is 1.0 to 1.5:1.

[0019] Preferably, in step (2), CPT is first dissolved in anhydrous dichloromethane, then triphosgene is added, and DMAP (4-dimethylaminopyridine) is added as a catalyst. The reaction system is stirred at room temperature for 20-40 min to activate the hydroxyl groups of CPT. Then, 2,2'-dithiodiethanol dissolved in anhydrous THF is added, and the reaction is carried out overnight at room temperature. After the reaction is completed, the mixture is washed with 0.1 M hydrochloric acid solution, saturated brine and deionized water in sequence. The organic phase is dried with anhydrous MgSO4, concentrated under reduced pressure, and the product is purified by recrystallization to obtain CPT-ss-OH. The mass ratio of CPT, triphosgene and 2,2'-dithiodiethanol is 3-4:1:12-15. Under inert gas protection, the prepared CPT-ss-OH was dissolved in anhydrous dichloromethane (DCM), and triphosgene and DMAP catalyst were added. The reaction was carried out at room temperature for 20-40 min to further activate the hydroxyl groups. Then, CA4 dissolved in anhydrous THF was added, and the reaction was continued at room temperature for 20-30 h. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a light yellow solid CPT-ss-CA4. The mass ratio of CPT-ss-OH, triphosgene and CA4 was 4-5:1:3-4.

[0020] The amount of catalyst used is 1 to 5% of the total mass of the reaction system.

[0021] Preferably, the organic solvent in step (3) is selected from one or more of tetrahydrofuran, acetone, and N,N-dimethylformamide; the post-treatment includes filtration and / or centrifugation.

[0022] The near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform provided by this invention can be applied to the preparation of drugs for treating liver cancer.

[0023] This invention combines molecular design with nano-assembly to achieve synergistic enhancement of photothermal therapy and chemotherapy, providing a new technical solution for the treatment of liver cancer.

[0024] CPT is an effective anti-tumor drug for treating HCC, inhibiting RNA and DNA synthesis. However, its severe toxic side effects and multidrug resistance (MDR) hinder its clinical application. CA4, as a representative angiogenesis disruptor, can rapidly destroy abnormal tumor blood vessels within approximately 2-6 hours, inducing necrosis in the tumor center, while peripheral cells can still survive. The potential of CPT and CA4 in the treatment of liver cancer has been confirmed by several preclinical studies. CPT enhances the sensitivity of cancer cells to PTT by inhibiting DNA topoisomerase I and downregulating the heat shock protein HSP70, while CA4 disrupts tumor blood vessels to cut off nutrient supply.

[0025] Phototherapy, as an emerging treatment method for tumors, offers the advantage of precise adjustment of treatment parameters. However, traditional phototherapy drugs often suffer from poor photostability and high biotoxicity. Aggregation-induced emission molecules (AIEgens), due to their excellent luminescence efficiency, photothermal conversion capability, and biocompatibility, provide an ideal choice for phototherapy technology. By optimizing the energy dissipation pathway of AIEgens through molecular design, multifunctional integration of photothermal therapy and photodynamic therapy can be achieved.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention constructs a nanoplatform BTTC based on the near-infrared II organic photothermal molecule BBT and the dual-drug prodrug CPT-CA4. The BBT molecule adopts a donor-acceptor-donor (DAD) structure design, which has good near-infrared II absorption capacity; after being encapsulated by DSPE-PEG-cRGD, it forms stable nanoparticles with an average particle size of about 105 nm, which can achieve efficient light energy absorption and conversion. At the same time, the responsive linking structure in the CPT-CA4 prodrug endows the system with glutathione responsive release capacity, which can promote drug release and improve drug utilization efficiency under tumor microenvironment conditions.

[0027] (2) The BTTC nanoplatform provided by this invention exhibits excellent photothermal response under 1064 nm laser irradiation. With increasing material concentration or laser power, its temperature rise effect is significantly enhanced, and it maintains good photothermal stability during multiple laser switching cycles. Furthermore, laser irradiation can further promote drug release, achieving a synergistic effect between photothermal therapy and chemotherapy. Cell experiments show that this nanoplatform has good biocompatibility under light-free conditions, while under 1064 nm laser irradiation, it can significantly inhibit tumor cell activity, demonstrating good potential for tumor treatment applications.

[0028] (3) This invention employs the Stille coupling reaction to prepare BBT in anhydrous toluene at 120 °C for 24 h under the catalysis of Pd(PPh3)4. This process may seem conventional, but its key lies in the precise control of reaction conditions—the synergistic optimization of temperature, time, and catalyst system—to minimize the monomer self-coupling defects of BBT units, thereby giving BBT stronger NIR-II absorption capacity and higher photothermal conversion efficiency, resulting in a significant absorption peak at 971 nm.

[0029] (4) This invention employs a triphosgene-activated hydroxyl group strategy. First, CPT is reacted with dithiodiethanol to obtain CPT-ss-OH, which is then esterified with CA4 to obtain the CPT-ss-CA4 dual-drug prodrug. The core innovation of this strategy lies in utilizing triphosgene (BTC) as a mild and efficient activating agent to achieve efficient coupling between the hydroxyl-containing drug (CPT) and the disulfide-linked linker. Compared to traditional phosgene, triphosgene has the advantages of safe operation and controllable reaction. It can selectively activate hydroxyl groups under mild conditions, avoiding the destruction of the CPT lactone ring structure by high temperatures or strong acids and bases, thus preserving the pharmacological activity of CPT. The introduction of dithiodiethanol allows CPT and CA4 to be linked through a disulfide bond, endowing the prodrug with GSH-responsive cleavage capability. Under the high GSH conditions of the tumor microenvironment, the disulfide bond can be specifically reduced and cleaved, achieving in-situ release of CPT and CA4.

[0030] (5) In this invention, BBT and CPT-ss-CA4 are co-dissolved in tetrahydrofuran and injected into an aqueous solution containing DSPE-PEG-cRGD under ultrasonic conditions. The self-assembly of nanoparticles is driven by hydrophilic-hydrophobic interactions, followed by removal of the organic solvent and filtration to obtain a stable nanoplatform. This method can prepare uniform nanoparticles with a particle size of approximately 105 nm (tunable from 80 to 150 nm). This size range is beneficial for both EPR-mediated passive targeting and endocytosis by tumor cells. DSPE, as a hydrophobic anchoring group, is embedded in the hydrophobic core of the nanoparticles, providing structural stability; PEG forms a hydration layer, prolonging in vivo circulation time and reducing protein adsorption and immune clearance; cRGD specifically recognizes integrin receptors highly expressed on the surface of tumor cells and neovascular endothelial cells, endowing the nanoplatform with active targeting capabilities. Through one-pot co-assembly, the photothermal agent, dual-drug prodrug, and targeting carrier are integrated into a single nanosystem, avoiding the process complexity and batch-to-batch variation problems caused by multi-step modification. After entering tumor cells, high concentrations of GSH trigger disulfide bond breakage, releasing CPT and CA4; the photothermal effect generated by 1064 nm laser irradiation can accelerate drug release, achieving a spatiotemporally controllable release mode of "photothermal-promoted drug release". Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the preparation process of the BTTC nanoplatform of the present invention; wherein, (A) is a flow chart of the reaction between BBT and CPT-ss-CA4; and (B) is a flow chart of the preparation process of the BTTC nanoplatform.

[0032] Figure 2 The image shows the particle size distribution and transmission electron microscope (TEM) image of the BTTC nanoplatform of this invention; the shaded area in the upper left corner is a TEM image.

[0033] Figure 3 The UV-Vis absorption spectrum of the BTTC nanoplatform of this invention is shown.

[0034] Figure 4This invention presents the drug release behavior and laser-controlled drug release curves of the BTTC nanoplatform under different temperatures and GSH conditions. Specifically, (A) shows the emission intensity of the BTTC nanoplatform at 20℃ with different reaction times (0, 30, 60, 90, 120 min) and in the presence of GSH (5 mM); (B) shows the emission intensity of the BTTC nanoplatform at 37℃ with different reaction times (0, 30, 60, 90, 120 min) and in the presence of GSH (5 mM); (C) shows the emission intensity of the BTTC nanoplatform at 50℃ with different reaction times (0, 30, 60, 90, 120 min) and in the presence of GSH (5 mM); (D) shows the fluorescence intensity enhancement curve of the GSH-treated BTTC nanoplatform at different temperatures; (E) shows the fluorescence spectrum of the BTTC nanoplatform at 37℃ with / without GSH; and (F) shows the laser-controlled drug release curve of the BTTC nanoplatform in the presence of GSH (5 mM) (1064 nm laser irradiation for 6 minutes). min, cool to room temperature, repeat 5 times).

[0035] Figure 5 The photothermal curves of the BTTC nanoplatform of this invention under different concentrations / different optical power densities are shown. Specifically, (A) represents the photothermal effect of BBT nanoplatforms of different concentrations under 1064 nm laser irradiation; (B) represents the photothermal effect of BBT nanoplatforms of the same concentration under 1064 nm laser irradiation at different powers; and (C) represents the photothermal effect of BBT / BTTC nanoplatforms of the same concentration under 0.8 W / cm² laser irradiation. -2 Photothermal effect under 1064 nm laser irradiation, (D) is the same concentration of BBT / BTTC nanoplatform at a power of 0.6 W cm⁻¹. -2 Photothermal effect under 1064 nm laser irradiation.

[0036] Figure 6 The diagram shows the repeated photothermal performance and photothermal conversion efficiency of the BTTC nanoplatform of this invention; where (A) represents the photothermal stability of the BTTC nanoplatform and (B) represents the photothermal conversion efficiency of the BTTC nanoplatform.

[0037] Figure 7 The above is a comparison diagram of the in vitro cytotoxicity evaluation of the BTTC nanoplatform of the present invention; wherein, (A) is the BBT nanoplatform, (B) is the BTTC nanoplatform, (C) is the BBT nanoplatform under 1064 nm laser irradiation, and (D) is the BTTC nanoplatform under 1064 nm laser irradiation.

[0038] Figure 8The results of apoptosis analysis of the BTTC nanoplatform of this invention are shown below; where Control is the blank group, BBT NPs is the BBT nanoplatform, CPT-CA4 is the pure drug nanoplatform, BTTC NPs is the BTTC nanoplatform, BBT NPs+L is the BBT nanoplatform under 1064 nm laser irradiation, and BTTC NPs+L is the BTTC nanoplatform under 1064 nm laser irradiation. Detailed Implementation

[0039] The accompanying drawings are for illustrative purposes only; the invention will be further described in detail below with reference to specific embodiments. However, this should not be construed as limiting the scope of the invention to the following embodiments; all technologies implemented based on the content of this invention fall within the scope of this invention.

[0040] It should be understood that, unless otherwise specified, all chemical reagents and materials used in this invention are available for purchase through conventional commercial channels; and all testing methods can be implemented through conventional experimental means.

[0041] Example 1 A near-infrared two-window responsive photothermal / chemotherapy synergistic nanoplatform is formed by the self-assembly of the near-infrared two-zone organic photothermal molecule BBT (benzobisthiadiazole), the dual-drug prodrug CPT-ss-CA4, and the amphiphilic polymer DSPE-PEG-cRGD. The BBT has a donor-acceptor-donor (DAD) structure and has light absorption and photothermal conversion capabilities in the near-infrared II region; The CPT-ss-CA4 is a dual-drug prodrug that connects CPT and CA4 via disulfide bonds; The DSPE-PEG-cRGD is used to encapsulate hydrophobic components and provide targeting functionality.

[0042] like Figure 1 As shown, a method for preparing a near-infrared two-window responsive photothermal / chemotherapy synergistic nanoplatform includes the following steps: (1) The preparation of BBT is as follows: Under argon protection, 0.237 g of 2,5-bis(2-octyldodecyl)-3,6-bis(5-(trimethylstanyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrolo-1,4-dione, 0.172 g of 4,9-bis(5-bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazo[3,4-g]quinoxaline, and 15 mg of Pd(PPh3)4 were dissolved in anhydrous toluene (20 mL), and the reaction was carried out at 120 °C for 24 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was added to excess petroleum ether to precipitate a solid. After filtration and drying, a black solid BBT (0.289 g, yield 80%) was obtained.

[0043] (2) The preparation of CPT-ss-OH is as follows: Under argon protection, CPT (1.50 g, 4.31 mmol) was dissolved in anhydrous dichloromethane (110 mL), triphosgene (0.473 g, 1.59 mmol) was added, and DMAP (catalytic amount) was added. The reaction system was stirred at room temperature for 30 min to activate the CPT hydroxyl groups. Then, 2,2'-dithiodiethanol (6.64 g, 43.1 mmol) dissolved in anhydrous THF (15 mL) was added, and the reaction was carried out overnight at room temperature. After the reaction was completed, the mixture was washed successively with 0.1 M hydrochloric acid solution, saturated brine, and deionized water. The organic phase was dried over anhydrous MgSO4, concentrated under reduced pressure, and the product was purified by recrystallization to give CPT-ss-OH (yield 65%).

[0044] (3) The preparation of CPT-ss-CA4 is as follows: Under argon protection, CPT-ss-OH (30 mg) was dissolved in anhydrous dichloromethane (5 mL), and triphosgene (7 mg) and DMAP (catalytic amount) were added. The reaction was carried out at room temperature for 30 min to further activate the hydroxyl groups. Subsequently, CA4 (25 mg) dissolved in anhydrous THF was added, and the reaction was continued at room temperature for 24 h. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography (ethyl acetate / methanol = 10:1) to obtain a light yellow solid CPT-ss-CA4.

[0045] (4) The specific steps for preparing the nanoplatform are as follows: BBT (1 mg) and CPT-ss-CA4 (0.017 mg) were dissolved in tetrahydrofuran (2 mL), and then rapidly injected under ultrasonic conditions into a deionized aqueous solution containing DSPE-PEG-cRGD (20 mg) to induce self-assembly and form a nanoparticle dispersion. The mixture was further sonicated, and the organic solvent was removed by nitrogen purging or vacuum evaporation. The resulting solution was filtered through a 0.22 μm microporous membrane to remove unloaded components, yielding stable BTTC nanoparticles (BTTCNPs).

[0046] Performance evaluation: a. To evaluate the physicochemical properties of the prepared BTTC nanoplatform, its particle size, potential, and optical properties were characterized. For example... Figure 2 As shown, the dynamic light scattering (DLS) test results indicate that the BTTC nanoplatform has good dispersibility in aqueous solution, with an average hydrated particle size of about 105 nm and a relatively uniform particle size distribution, indicating that the prepared nanoparticles have good stability and size controllability.

[0047] like Figure 3 As shown, the UV-Vis absorption spectroscopy results indicate that the BTTC nanoplatform exhibits significant absorption characteristics in the near-infrared II region, with its maximum absorption peak located near 971 nm. This demonstrates that BBT molecules retain good near-infrared absorption capabilities even after nano-assembly. This absorption characteristic is beneficial for improving the material's utilization efficiency of 1064 nm laser light, providing a foundation for subsequent photothermal therapy.

[0048] The above results demonstrate that the BTTC nanoplatform constructed in this invention possesses suitable nanoscale size and excellent near-infrared II absorption characteristics, which can meet the needs of subsequent biomedical applications.

[0049] b. To investigate the responsive drug release behavior of the BTTC nanoplatform, the effects of temperature, glutathione (GSH), and 1064 nm laser irradiation on drug release were studied.

[0050] like Figure 4 As shown in (AC), under conditions of 20 ℃, 37 ℃, and 55 ℃, the amount of drug released from the nanoplatform gradually increased with the extension of incubation time; at the same time, the higher the temperature, the higher the drug release efficiency. The release effect was most significant at 55 ℃, indicating that heating can promote the relaxation of the nanoplatform structure and drug diffusion, thereby improving the drug release efficiency.

[0051] like Figure 4As shown in Figure (DE), the fluorescence signal of the BTTC nanoplatform was significantly enhanced in the presence of 5 mM GSH, indicating that the responsive linkages in the prodrug structure were broken, thereby promoting drug release. Compared with the condition without GSH, the drug release rate was significantly increased in the presence of GSH, indicating that the nanoplatform has good reducing microenvironment response characteristics.

[0052] like Figure 4 As shown in Figure (F), under periodic irradiation with a 1064 nm laser, the drug release rate continuously increases with the number of irradiations. Each laser irradiation promotes further drug release, while the release rate tends to level off after irradiation stops, indicating that this nanoplatform has good photothermal controlled release capability.

[0053] The above results indicate that the BTTC nanoplatform constructed in this invention simultaneously possesses temperature response, GSH response, and laser-induced release characteristics, enabling controlled drug release.

[0054] c. To evaluate the photothermal performance of the BTTC nanoplatform, the effects of material concentration and laser power on its temperature rise were investigated.

[0055] comprehensive Figure 5 , 6 As shown, under the same 1064 nm laser power irradiation conditions, the system temperature continuously increases with the gradual increase of BTTC nanoplatform concentration, indicating that the higher the material concentration, the stronger its ability to absorb and convert light energy, exhibiting superior photothermal performance. Under the same material concentration conditions, the system temperature rise increases significantly with the gradual increase of laser power density, indicating that the BTTC nanoplatform can effectively respond to 1064 nm laser irradiation and rapidly convert the absorbed light energy into heat energy, with a photothermal conversion efficiency of 56.7%.

[0056] Further comparison of the photothermal properties of BBT NPs and BTTC NPs revealed that both exhibited similar temperature rise trends and photothermal conversion capabilities under the same experimental conditions, indicating that the introduction of the CPT-CA4 prodrug did not significantly affect the photothermal properties of the BBT core.

[0057] The above results indicate that the BTTC nanoplatform constructed in this invention has excellent near-infrared II photothermal response capability and can be used as an effective photothermal therapeutic agent for tumor treatment.

[0058] d. To evaluate the biosafety and in vitro antitumor activity of the BTTC nanoplatform, the CCK-8 assay, Calcein-AM / PI live / dead cell staining, and Annexin V-FITC / PI apoptosis detection were used for analysis.

[0059] like Figure 7As shown, under conditions without laser irradiation, the cell survival rate remained high after treatment with different concentrations of BTTC nanoplatform, indicating that the material has low dark toxicity and good biocompatibility.

[0060] like Figure 7 As shown, under 1064 nm laser irradiation, the cell survival rate gradually decreased with the increase of BTTC nanoplatform concentration, indicating that the material can effectively inhibit tumor cell activity under laser excitation and exhibits obvious concentration dependence.

[0061] like Figure 8 As shown, the Annexin V-FITC / PI flow cytometry results further confirmed that the proportion of apoptosis increased significantly after laser irradiation, and further increased with the increase of material concentration, indicating that the BTTC nanoplatform can effectively induce programmed cell death in tumor cells.

[0062] comprehensive Figures 7-8 The experimental results show that the BTTC nanoplatform constructed in this invention has good biocompatibility and exhibits low cytotoxicity under light-free conditions; while under 1064 nm laser irradiation, it can significantly inhibit tumor cell activity and induce apoptosis, showing good potential for in vitro anti-tumor applications.

[0063] Experimental results show that the BTTC nanoplatform constructed in this invention exhibits good nanodispersion with an average particle size of approximately 105 nm and significant absorption at 971 nm. This nanoplatform possesses excellent near-infrared II photothermal properties, with its photothermal effect increasing with material concentration and laser power, while also exhibiting good photothermal stability. Drug release experiments demonstrate that glutathione, high-temperature environments, and 1064 nm laser irradiation all promote drug release, achieving responsive and controllable release. Cell experiments show that this nanoplatform exhibits low cytotoxicity under light-free conditions, while significantly inhibiting tumor cell activity under laser irradiation. These results indicate that the BTTC nanoplatform constructed in this invention combines near-infrared II photothermal therapy and controllable drug release functions, demonstrating promising biomedical application prospects.

[0064] Example 2 A method for preparing a near-infrared two-window responsive photothermal / chemotherapy synergistic nanoplatform includes the following steps: (1) Preparation of BBT.

[0065] Under argon protection, 0.258 g of 2,5-bis(2-octyldodecyl)-3,6-bis(5-(trimethylstanyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrolo-1,4-dione, 0.172 g of 4,9-bis(5-bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazo[3,4-g]quinoxaline, and 20 mg of Pd(PPh3)4 were dissolved in 20 mL of anhydrous toluene. The reaction mixture was stirred at 110 °C for 20 h. After the reaction was completed, the mixture was cooled to room temperature, and the reaction solution was slowly added dropwise to 200 mL of n-hexane to precipitate a solid. The crude product was collected by filtration. The crude product was purified by column chromatography (eluent: petroleum ether / dichloromethane = 2:1, v / v) to obtain the target product BBT in 72% yield.

[0066] (2) Preparation of CPT-ss-CA4.

[0067] Synthesis of CPT-ss-OH: CPT (1.045 g, 3.0 mmol) and triphosgene (0.297 g, 1 mmol) were dissolved in 90 mL of anhydrous dichloromethane, and DMAP (catalytic amount) was added. The mixture was stirred at room temperature for 20 min to activate the hydroxyl groups of CPT. Subsequently, 2,2'-dithiodiethanol (30.0 mmol) dissolved in anhydrous THF (12 mL) was added, and the reaction was carried out overnight at room temperature (the reaction time is generally about 12 hours). After the reaction was completed, the mixture was washed successively with 0.1 M hydrochloric acid solution, saturated brine, and deionized water. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed by rotary evaporation. The crude product was purified by column chromatography (eluent: dichloromethane / methanol = 20:1, v / v) to obtain CPT-ss-OH in 62% yield.

[0068] Synthesis of CPT-ss-CA4: CPT-ss-OH (25 mg) was dissolved in anhydrous dichloromethane (4 mL), and triphosgene (5 mg) and DMAP (catalytic amount) were added. The reaction was carried out at room temperature for 20 min to further activate the hydroxyl groups. Then, CA4 (20 mg) dissolved in anhydrous THF was added, and the reaction was continued at room temperature for 20 h. After the reaction was completed, insoluble matter was removed by filtration, and the filtrate was concentrated and purified by column chromatography (eluent: dichloromethane / methanol = 20:1, v / v) to obtain the target product CPT-ss-CA4 in 58% yield.

[0069] (3) Assemble the BTTC nanoplatform.

[0070] BBT (1 mg) and CPT-ss-CA4 (0.018 mg) were dissolved in 2 mL of tetrahydrofuran. Under ultrasonic conditions (100 W), the solution was rapidly injected into 10 mL of an aqueous solution containing DSPE-PEG-cRGD (15 mg), and ultrasonic treatment was continued for 5 min. The mixture was then placed in a fume hood and magnetically stirred overnight to allow for complete evaporation of the tetrahydrofuran. Subsequently, the nano-suspension was filtered through a 0.22 μm aqueous filter membrane to obtain the BTTC nanoplatform solution. The average hydrated particle size of the obtained nanoparticles was 105 ± 8 nm, and UV-Vis-NIR absorption spectroscopy showed that the nanoplatform had a maximum absorption peak at 971 nm.

[0071] Comparative Example 1: A nanoplatform without CPT-ss-CA4 dual-drug prodrug.

[0072] According to the method of this invention, only BBT and DSPE-PEG-cRGD are self-assembled into nanoparticles (denoted as BT NPs) via nanoprecipitation, without adding the CPT-ss-CA4 prodrug. All other preparation conditions and operating steps are the same as in Example 2 of this invention.

[0073] Experimental results: BT NPs exhibited a good photothermal heating effect under 1064 nm laser irradiation, with a particle size of approximately 90 nm, and a photothermal conversion efficiency comparable to the BTTC nanoplatform of this invention. However, under no laser irradiation conditions, BT NPs showed no significant killing activity against tumor cells; under laser irradiation conditions, although BT NPs could inhibit cell activity through the photothermal effect, tumor cells showed significant regeneration after the laser was stopped. Drug release experiments showed that BT NPs did not possess any chemotherapy drug release capability and relied entirely on a single photothermal mode for treatment.

[0074] Comparative Conclusion: Compared with the BTTC nanoplatform of this invention, BT NPs without CPT-ss-CA4 can only achieve single photothermal therapy and cannot exert the synergistic effect of chemotherapy-photothermal therapy. This indicates that the introduction of CPT-ss-CA4 is a key component for achieving synergistic therapy and preventing tumor recurrence. The cRGD@C12T-BBT nanomedicine reported in the literature also uses a single BBT derivative for loading. Although it can achieve NIR-II imaging-guided photothermal therapy, its treatment mode is also limited to the synergy of photothermal effect and immune response, lacking the controllable release function of chemotherapeutic drugs.

[0075] Comparative Example 2: The system of the present invention in which CPT and CA4 are physically mixed rather than linked as prodrugs.

[0076] Free CPT (camptothecin) and CA4 (cobustatin A4) were co-assembled with BBT and DSPE-PEG-cRGD in a 1:1 molar ratio to form nanoparticles (denoted as BT+CPT / CA4 NPs), instead of using a CPT-ss-CA4 dual-drug prodrug linked by disulfide bonds. All other preparation conditions and procedures were the same as in the embodiments of this invention.

[0077] Experimental results: The BT+CPT / CA4 NPs have a particle size of approximately 110 nm, and their photothermal properties are basically consistent with the BTTC nanoplatform. However, drug release experiments showed that CPT and CA4 leaked significantly even without GSH stimulation, with cumulative release rates reaching 45% and 52% respectively after 48 h, leading to severe premature drug release and off-target toxicity. In the presence of GSH, the release of free drug did not differ significantly (release rates only increased to 52% and 58% respectively), indicating that this system lacks tumor microenvironment-responsive controlled release capabilities. Cytotoxicity experiments showed that under light-free conditions, BT+CPT / CA4 NPs also exhibited high toxicity to normal cells (cell viability approximately 55%), indicating poor selectivity.

[0078] Comparative Conclusion: Compared to the BTTC nanoplatform of this invention, physically mixed free drug systems lack GSH-responsive controlled-release capabilities, resulting in severe premature drug leakage and poor biosafety. This invention links CPT and CA4 into a single prodrug molecule via disulfide bonds, achieving specific release only within the high-GSH tumor microenvironment, significantly improving therapeutic selectivity and safety. Related studies also indicate that prodrug design based on disulfide bond linkages is an effective strategy for achieving specific drug release from the tumor microenvironment.

[0079] Of course, the above description is not intended to limit the present invention, and the present invention is not limited to the examples given above. Any changes, modifications, additions or substitutions made by those skilled in the art within the scope of the present invention should also fall within the protection scope of the present invention.

Claims

1. A near-infrared two-window responsive photothermal / chemotherapy synergistic nanoplatform, characterized in that, The nanoplatform is formed by the self-assembly of the near-infrared II organic photothermal molecule BBT, the dual-drug prodrug CPT-ss-CA4, and the amphiphilic polymer DSPE-PEG-cRGD. The BBT has a donor-acceptor-donor structure and has light absorption and photothermal conversion capabilities in the near-infrared II region; The CPT-ss-CA4 is a dual-drug prodrug that connects CPT and A4 via disulfide bonds; The DSPE-PEG-cRGD is used to encapsulate hydrophobic components and provide targeting functionality.

2. The near-infrared II-region responsive photothermal / chemotherapy synergistic nanoplatform according to claim 1, characterized in that, The average hydrated particle size of the nanoplatform is 80~150 nm, preferably 100~110 nm; The nanoplatform has a maximum absorption peak at 971 nm; The nanoplatform exhibits a photothermal heating effect under 1064 nm laser irradiation, and its photothermal conversion efficiency is not less than 40%.

3. The near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform according to claim 1, characterized in that, The nanoplatform exhibits glutathione-responsive drug release properties, where the disulfide bond in CPT-ss-CA4 breaks in the presence of glutathione, releasing CPT and CA4.

4. The near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform according to claim 1, characterized in that, The mass ratio of BBT to CPT-ss-CA4 in the nanoplatform is 50~65:1; The mass ratio of the amphiphilic polymer DSPE-PEG-cRGD to BBT is 15~25:

1.

5. The method for preparing a near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform as described in any one of claims 1-4, characterized in that, Includes the following steps: (1) Preparation of BBT: Under inert gas protection, the corresponding organotin compound and the brominated acceptor unit were subjected to a Stille coupling reaction in anhydrous toluene catalyzed by Pd(PPh3)4. After the reaction, BBT was obtained by separation and purification. The structural formula of BBT is as follows: ； (2) Preparation of CPT-ss-CA4: Using the hydroxyl activation strategy of triphosgene, CPT was reacted with dithiodiethanol to obtain the intermediate CPT-ss-OH, which was then esterified with CA4 to obtain CPT-ss-CA4; (3) Preparation of nanoplatform: BBT and CPT-ss-CA4 are dissolved in an organic solvent, and the resulting solution is injected into an aqueous solution containing DSPE-PEG-cRGD under ultrasonic conditions to form nanoparticles; ultrasonic treatment is continued, and the organic solvent is removed by nitrogen purging or vacuum evaporation, and then the nanoplatform is obtained by post-treatment.

6. The method for preparing a near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform according to claim 5, characterized in that, The Stille coupling reaction temperature in step (1) is 100~140 ℃, and the reaction time is 12~48 h; The separation and purification process includes adding the reaction solution to a non-polar solvent to precipitate a solid, followed by purification by column chromatography or recrystallization; wherein the non-polar solvent is selected from any one of isohexane, pentane, petroleum ether, and n-hexane.

7. The method for preparing a near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform according to claim 5, characterized in that, The organotin compound is 2,5-bis(2-octyldodecyl)-3,6-bis(5-(trimethylstanyl)thiophen-2-yl)-2,5-dihydropyrrolo[3,4-c]pyrrolo-1,4-dione, and the bromoacceptor unit is 4,9-bis(5-bromothiophen-2-yl)-6,7-bis(4-(hexyloxy)phenyl)-[1,2,5]thiadiazo[3,4-g]quinoxaline.

8. The method for preparing a near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform according to claim 5, characterized in that, The mass ratio of organotin compound to brominated acceptor unit is 1.0~1.5:

1.

9. The method for preparing a near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform according to claim 5, characterized in that, In step (2), CPT is first dissolved in anhydrous dichloromethane, then triphosgene is added, and DMAP is added as a catalyst. The reaction system is stirred at room temperature for 20-40 min to activate the hydroxyl groups of CPT. Then, 2,2'-dithiodiethanol dissolved in anhydrous THF is added, and the reaction is carried out overnight at room temperature. After the reaction is completed, the mixture is washed with 0.1 M hydrochloric acid solution, saturated brine and deionized water in sequence. The organic phase is dried with anhydrous MgSO4, concentrated under reduced pressure, and the product is purified by recrystallization to obtain CPT-ss-OH. The mass ratio of CPT, triphosgene and 2,2'-dithiodiethanol is 3-4:1:12-15. Under inert gas protection, the prepared CPT-ss-OH was dissolved in anhydrous dichloromethane, and triphosgene and DMAP catalyst were added. The reaction was carried out at room temperature for 20-40 min to further activate the hydroxyl groups. Then, CA4 dissolved in anhydrous THF was added, and the reaction was continued at room temperature for 20-30 h. After the reaction was completed, the solvent was removed under reduced pressure, and the crude product was purified by silica gel column chromatography to obtain a light yellow solid CPT-ss-CA4. The mass ratio of CPT-ss-OH, triphosgene and CA4 was 4-5:1:3-4.

10. The method for preparing a near-infrared II-responsive photothermal / chemotherapy synergistic nanoplatform according to claim 1, characterized in that, The organic solvent in step (3) is selected from one or more of tetrahydrofuran, acetone, and N,N-dimethylformamide; the post-treatment includes filtration and / or centrifugation.

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