A squid ink copper oxide-based nanocomposite biomaterial for treating tumor cells

Abstract

The application provides a kind of inkfish juice copper oxide-based nanocomposite, in this paper, inkfish juice (M) is as core, a layer of CuO is grown outside it, and M@CuO composite multifunctional nanomaterial is successfully constructed.M@CuO composite material is spherical, and the nanometer particle size is 128.2 nm, and it has good photo-thermal conversion performance (η T =47.6%) under near infrared light (NIR) irradiation conditions.In addition, M@CuO shows good Fenton effect at room temperature, and the Fenton reaction rate can be further improved by photo-thermal effect, and the Fenton reaction rate at 45 DEG C is more than twice that at 25 DEG C under the same conditions.In vitro cell experiments, M@CuO shows good biological safety, and under the effect of photo-thermal-chemical kinetics combined therapy, it can efficiently kill tumor cells, and provides an effective strategy for developing multifunctional nanomaterials with PTT-CDT combined therapy.

Description

Technical Field

[0001] This invention relates to a nanocomposite biomaterial, specifically a squid ink@copper oxide nanocomposite biomaterial, and its use in treating tumor cells or degrading dyes. Background Technology

[0002] Cancer, with its high incidence and mortality rates, seriously threatens people's lives and health. To effectively treat cancer, various novel treatment strategies have been developed in recent years. These treatments mainly include photodynamic therapy (PDT), photothermal therapy (PTT), sonodynamic therapy (SDT), and chemodynamic therapy (CDT). However, while each treatment method has its unique advantages, it also has some limitations. For example, PTT mainly uses near-infrared (NIR) light to generate heat and induce cancer cell death. However, NIR light has weak penetration into biological tissues, only treating superficial cancer cells. Furthermore, the PTT effect ceases immediately when the excitation light stops, making it unable to suppress tumors long-term, and tumor recurrence is common. In addition, the complexity and diversity of tumors further limit the effectiveness of single-therapy based on nanomedicines, leading to a current trend in clinical cancer research from single-therapy to comprehensive synergistic therapy to improve overall efficacy.

[0003] To overcome the limitations of single photothermal therapy, our research group recently developed a cuttlefish ink-protoporphyrin nanocomposite (CIPs) that can effectively perform PTT-SDT combined therapy on tumor cells. In SDT, ultrasound has a greater tissue penetration depth (>10 cm), effectively treating deep tumors; however, it also suffers from the problem of the therapeutic effect ceasing immediately after ultrasound stimulation stops, leading to tumor recurrence. CDT utilizes the reaction of nanomaterials with H2O2 in the tumor microenvironment to generate highly toxic hydroxyl radicals (•OH) to kill tumor cells. This treatment method can not only treat deep tumor cells but also produce a sustained chemokinetic effect over a long period; however, the rate of •OH generation via the Fenton reaction in CDT is slow, resulting in less than ideal efficacy. Combining PTT and CDT not only compensates for their respective shortcomings but also enhances the therapeutic effect of CDT by increasing the temperature and accelerating the •OH generation rate via the Fenton reaction through PTT. The key to PTT-CDT combined therapy is the development of multifunctional nanomaterials with significant photothermal conversion properties and chemokinetic effects. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a nanomaterial with both photothermal-chemokinetic therapy (PTT-CDT) functionality. Using squid ink (M) as the core, a layer of CuO is grown on its exterior, successfully constructing an M@CuO composite multifunctional nanomaterial. The M@CuO composite material exhibits nanosphere-like shapes and demonstrates excellent photothermal conversion performance (η) under near-infrared (NIR) irradiation. T =47% or more). Furthermore, M@CuO exhibits a good Fenton effect at room temperature (25℃), and the Fenton reaction rate can be further increased by the photothermal effect; under the same conditions, the Fenton reaction rate at 45℃ is more than twice that at 25℃. In in vitro cell experiments, M@CuO demonstrates good biocompatibility and can efficiently kill tumor cells under photothermal-chemokinetic combined therapy, providing an effective strategy for developing multifunctional nanomaterials with PTT-CDT combined therapy.

[0005] The technical solution of the present invention provides a nanocomposite biomaterial based on cuttlefish ink@copper oxide. The composite biomaterial is a multifunctional M@CuO composite nanomaterial with cuttlefish ink as the core and a CuO layer grown on its outside. The M@CuO composite material is spherical with a nanoparticle size of 100-130 nm and a surface potential of -8 to -10 mV.

[0006] Another aspect of the present invention provides a method for preparing a nanocomposite biomaterial based on cuttlefish ink@copper oxide, which includes the following steps: adjusting the pH of the ink sac of fresh cuttlefish to 12-13, stirring for 12-20 h and centrifuging; adding the obtained nanoparticles after centrifugation to CuSO4 solution, magnetically stirring for 3-5 min, filtering, drying, and calcining to obtain black particles, which are cuttlefish ink@copper oxide composite nanomaterials (M@CuO).

[0007] In the method, the pH value is adjusted to 12-13 using any one of sodium hydroxide, potassium hydroxide, sodium methoxide, or potassium methoxide solution.

[0008] The concentration of the CuSO4 solution is 0.01-0.2 mol·L⁻¹. -1 The concentration of the CuSO4 solution is any value within its range.

[0009] The calcination temperature is 350-500℃, and the calcination time is 3-5 hours. The calcination temperature and calcination time are any values ​​within these ranges.

[0010] Another technical solution of the present invention is to provide a photothermal material and / or a material with a specific Fenton effect, wherein the photothermal material and / or the material with a specific Fenton effect is the aforementioned cuttlefish ink@copper oxide nanocomposite biomaterial or the cuttlefish ink@copper oxide nanocomposite biomaterial prepared by the aforementioned method.

[0011] As a photothermal material, the aforementioned cuttlefish ink@copper oxide nanocomposite biomaterial achieves a temperature increase effect under near-infrared laser irradiation at 800-850 nm, and the concentration of the cuttlefish ink-porphyrin nanoconjugate is 0.025~0.4 mg / mL. -1 ;

[0012] And / or,

[0013] As a material that specifically exhibits the Fenton effect, it is a material that catalyzes the generation of •OH from H2O2 via the Fenton reaction.

[0014] Another technical solution of the present invention is the application of the aforementioned cuttlefish ink@copper oxide nanocomposite biomaterial or the cuttlefish ink@copper oxide nanocomposite biomaterial prepared by the method in the preparation of a drug for treating anti-tumor cells, wherein the tumor cells include cancer cells 4T1.

[0015] The aforementioned squid ink@copper oxide nanocomposite material is used to achieve in vitro photodynamic-photothermal combined therapy of tumor cells through tumor photothermal-chemokinetic combination.

[0016] The technical solution of the present invention also provides a photocatalytic reagent for degrading dyes, wherein the photocatalytic reagent is the aforementioned cuttlefish ink@copper oxide nanocomposite biomaterial or the cuttlefish ink@copper oxide nanocomposite material prepared by the method.

[0017] The dyes described in this invention include any one of MB, Rhodamine B, and Orange Red II.

[0018] In the technical solution of this invention, the amount of the photocatalytic reagent used to degrade the dye is 0.01-0.5 mg·mL. -1 Preferably, the dosage is 0.05-0.2 mg / mL. -1 Further preferred is 0.1 mg·mL -1 .

[0019] In the technical solution of this invention, cuttlefish juice is mainly composed of amorphous carbon with elements such as C, N, and O, and its surface contains abundant -OH, -C=O-, and -C=C- groups. Due to its natural biocompatibility and biodegradability, cuttlefish juice is considered an excellent photothermal reagent. Furthermore, copper-based nanomaterials have been proven to be effective Fenton reagents; copper-catalyzed Fenton reactions exhibit higher catalytic efficiency under weakly acidic and neutral conditions, reaching 160 times the rate of Fenton reactions mediated by iron-based nanomaterials. Therefore, this invention constructs a multifunctional cuttlefish juice@copper oxide composite nanomaterial (M@CuO) for cancer cell therapy. Research results show that M@CuO not only has good PTT and CDT effects, but its CDT performance can be further significantly enhanced through PTT effects, achieving a highly efficient PTT-CDT combined treatment effect. Figure 1 ).

[0020] This invention successfully prepared multifunctional M@CuO composite nanomaterials for photothermal-chemokinetic combined therapy (PTT-CDT) of tumors. The M@CuO exhibits a spherical structure with a particle size of 128.2 nm, slightly larger than the nanometer diameter of pure squid ink (105.6 nm). M@CuO still exhibits good near-infrared light absorption and good photothermal conversion efficiency (η) at 808 nm. T =47.6%). Furthermore, M@CuO exhibited Fenton reaction characteristics (25℃), and the reaction rate could be significantly increased (2.3-fold) by the photothermal effect (45℃). Co-incubation of the M@CuO dispersion with cells showed good biocompatibility, and under the combined photothermal-chemokinetic therapeutic effect, it could efficiently kill tumor cells. Attached Figure Description

[0021] Figure 1 Synthetic route of M@CuO and its application in combined photothermal-chemokinetic therapy of tumor cells.

[0022] Figure 2 (a,b) SEM images and particle size distribution of M. (c,d) SEM images and particle size distribution of M@CuO.

[0023] Figure 3 XRD pattern (a), X-ray photoelectron spectrum (b, c), and UV-Vis-NIR absorption spectrum (d) of M@CuO.

[0024] Figure 4(a) Temperature variation of M@CuO dispersions with different concentrations under 808 nm laser irradiation. (b) Photothermal cycling stability of M@CuO dispersions. (c) Temperature variation curves of M@CuO dispersions under laser on / off conditions. (d) Linear fitting of M@CuO linear time data to -ln(θ).

[0025] Figure 5 (a) Schematic diagram of photothermal-enhanced chemokinetics. (b, c) Time-dependent absorption spectra of MB mediated by M@CuO and H2O2 at 25℃ or 45℃. (d) Degradation rate of MB under different conditions.

[0026] Figure 6 (ac) Cell viability of HUVEC, CT26 and 4T1 cells after co-culturing with different concentrations of M@CuO for 24 h.

[0027] Figure 7 (a) Cell viability of PTT under different light exposure times. (b) Cell viability of CDT under different M@CuO concentrations.

[0028] Figure 8 Relative survival rate of 4T1 cells after different treatments (a) and fluorescence images (b).

[0029] Figure 9 This is a curve showing the relationship between temperature change (ΔT) and concentration.

[0030] Figure 10 The images show thermal images of M@CuO dispersions of different concentrations under 808 nm laser irradiation. Detailed Implementation

[0031] Experimental materials

[0032] Copper chloride dihydrate (CuCl2·2H2O), analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.; Sodium hydroxide (NaOH), analytical grade, Sinopharm Chemical Reagent Co., Ltd.; Polyvinylpyrrolidone (PVP-K30), analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.; Methylene blue (MB), analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.; Ethanol (CH3CH2OH), analytical grade, Shanghai Aladdin Biochemical Technology Co., Ltd.; 4',6-Diamidinyl-2-phenylindole (DAPI), analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.; Phosphate-buffered saline (PBS), analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.; 2′,7′-Dichlorofluorescein diacetate (DCFH-DA), analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.; Tetramethylazoazolium blue (MTT), analytical grade, Shanghai Maclean's Biochemical Technology Co., Ltd.

[0033] Example 1

[0034] Synthesis steps of squid ink@CuO

[0035] Cuttlefish ink (M) was obtained from the ink sac of fresh cuttlefish after washing and centrifugation. Then, 5 mg of M was added to a NaOH solution (5 mL, pH=12) and stirred for 12 h. Subsequently, the centrifuged nanoparticles were added to a CuSO4 solution (5 mL, 0.1 mol·L⁻¹). -1 The mixture was magnetically stirred for 5 minutes, filtered, dried, and finally calcined at 350℃ for 5 hours to obtain black particles, which are the squid ink@copper oxide composite nanomaterials (M@CuO).

[0036] Material characterization of M@CuO

[0037] First, the morphology and size of the composite material M@CuO were characterized. Scanning electron microscopy (SEM) images showed that the cuttlefish ink (M) extracted from the ink sac of fresh cuttlefish was in the form of uniformly sized spherical particles with a nanometer diameter of approximately 105.6 ± 2.3 nm. Figure 2 a and 2b). After forming the composite material M@CuO, it still exhibits a spherical structure with a particle size of 128.2 ± 1.9 nm, slightly larger than the nanometer diameter of pure squid ink. This may be due to the formation of CuO on the M surface increasing the nanometer diameter. Figure 2 c and 2d). Subsequently, the hydrodynamic dimensions and Zeta potential of M and M@CuO nanomaterials were further measured. The results showed that the hydrodynamic diameter of M was 259.7 nm and the surface potential was -17.6 mV; while the hydrodynamic diameter of M@CuO was 289.3 nm and the surface potential was -9.7 mV. This was also due to the formation of CuO on the M surface, which increased the nanometer diameter and decreased the surface potential. Figure 2 (e and 2f). In summary, the composite material M@CuO still exhibits a spherical shape, but its nanometer diameter is slightly increased compared to M.

[0038] Subsequently, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS) were used to further investigate the material composition and structure of the composite nanomaterial M@CuO. XRD results showed that M@CuO exhibited strong characteristic diffraction peaks at 35.4°, 38.5°, 48.7°, 66.4°, and 68.1°, consistent with the standard XRD card peaks of CuO (PDF 44-0706), indicating the successful growth of a CuO layer on the surface of squid ink. Figure 3 a). The XPS total spectrum showed strong signals from C, Cu, and O elements, where C originated from squid ink, and Cu and O originated from surface CuO (a). Figure 3b). The Cu 2p spectrum shows two peaks near 955.4 and 935.3 eV, respectively, which correspond to Cu... 2+ The Cu 2p1 / 2 and Cu 2p3 / 2 states. Furthermore, the three peaks near 963.2, 944.5, and 940.5 eV are characteristic peaks of +2 valence Cu salts. Figure 3 c). Therefore, the Cu element in the M@CuO sample has a +2 valence, consistent with the corresponding chemical formula result in the XRD. Finally, the optical properties of M and M@CuO were measured by UV-Vis-NIR spectroscopy. The results show that M@CuO exhibits strong light absorption in the 400–1000 nm range. Notably, compared with pure M, the light absorption of M@CuO in the near-infrared region (800–1000 nm) is the same as that of M, indicating that the formation of the composite material M@CuO did not affect the near-infrared light absorption effect of M. Figure 3 d). Therefore, we subsequently used an 808 nm laser to study its photothermal properties.

[0039] Example 2

[0040] Photothermal performance test

[0041] Photothermal conversion performance test: First, different concentrations (0, 0.05, 0.1, 0.2, 0.4 mg·mL) were prepared. -1 The M@CuO aqueous dispersion prepared in Example 1 was then subjected to an 808 nm laser (1.0 W·cm⁻¹). -2 The temperature changes of M@CuO dispersions of different concentrations were monitored using a thermal imaging camera.

[0042] Photostability test: Irradiation was performed using an 808 nm laser, switching it on and off four times (20 min / time), at a concentration of 0.1 mg·mL. -1 The temperature changes of the M@CuO dispersion were monitored over four cycles using a thermal imaging camera.

[0043] Photothermal conversion performance and photothermal enhancement of chemokinetics of M@CuO

[0044] First, the effects of different concentrations of M@CuO dispersions (0, 0.05, 0.1, 0.2, 0.4 mg·mL⁻¹) on the overall performance were investigated. -1 Irradiation with 808 nm laser (1.0 W·cm) -2 The photothermal conversion performance at that time. From Figure 4 As shown in Figure a, the heating effect of the M@CuO dispersion under light irradiation is concentration-dependent. Pure water only increases the temperature by 3.9°C within 10 min, while M@CuO (0.4 mg·mL⁻¹) increases the temperature by 10 min. -1The temperature increased to 62.5°C within 10 min, indicating that the M@CuO dispersion exhibits good photothermal conversion performance under 808 nm laser irradiation. Figure 9 ,10). In addition, a concentration of 0.1 mg·mL -1 The M@CuO dispersion was subjected to different laser powers (0.2-1 W·cm⁻¹). -2 The temperature change graph was monitored. The results showed that the heating effect of the M@CuO dispersion became more pronounced with increasing power, ΔT increasing from 0.2 W·cm⁻¹. -2 The temperature increased from 3.4°C to 1 W·cm -2 The equilibrium temperature of the M@CuO dispersion was 38.3°C, indicating that the M@CuO dispersion exhibits not only concentration-dependent photothermal properties but also laser power-dependent photothermal properties. Furthermore, the equilibrium temperature of the M@CuO dispersion did not change significantly after four 808 nm laser on / off cycles, demonstrating excellent photothermal cycling stability. Figure 4 b). Finally, by performing linear fitting on the data points of the heating / cooling process of the M@CuO dispersion, the photothermal conversion efficiency of M@CuO was studied ( Figure 4 c and 4d). M@CuO dispersion (0.1 mg·mL) -1 ) Exposed to 808 nm laser (1 W·cm -2 It reaches equilibrium temperature (T) in 600 s. max =55.8°C), then the laser was turned off, and the temperature dropped to the initial temperature (T). sur =27.6°C). The photothermal conversion efficiency (η) of M@CuO was calculated. T The percentage was 47.6%. In summary, M@CuO possesses excellent photothermal conversion properties and can be considered a potential photothermal material for tumor treatment.

[0045] Example 3

[0046] Detection of hydroxyl radicals (•OH)

[0047] Prepare 90 mL of solution with a concentration of 0.005 mg / mL. -1The aqueous solution of methylene blue (MB) and the M@CuO aqueous dispersion prepared in Example 1 (0.1 mg·mL⁻¹) were mixed and stirred in the dark for 30 min to reach adsorption-desorption equilibrium. Then, the mixed solution (M@CuO + MB) was evenly divided into three equal portions for three test groups: (1) M@CuO + MB (25℃); (2) M@CuO + MB + H₂O₂ (25℃); (3) M@CuO + MB + H₂O₂ (45℃). In (2) and (3), equal amounts of H₂O₂ were added in batches, with each addition occurring at 5-min intervals. The UV-Vis absorption spectra at 650 nm were measured and recorded using a UV-Vis spectrophotometer.

[0048] As is well known, Cu can catalyze the formation of highly reactive •OH from H₂O₂ via the Fenton reaction. Increasing the temperature increases the proportion of active H₂O₂ molecules, thereby increasing the Fenton reaction rate and producing more •OH (…). Figure 5 a). This paper uses the methylene blue (MB) colorimetric method to study the photothermal enhancement Fenton reaction rate of M@CuO. First, M@CuO (100 μg·mL⁻¹) was added to the mixture. -1 ) and MB (0.005 mg·mL) -1 The mixed solution of M@CuO and MB was stirred continuously in the dark for 30 min to reach adsorption-desorption equilibrium. Then, H2O2 was added to the mixture (M@CuO + MB) after adsorption-desorption equilibrium to investigate its Fenton reaction rate under different conditions. Without the addition of H2O2, the characteristic absorption peak of MB in the mixture (adsorption-desorption equilibrium) did not change, indicating that pure M@CuO does not produce •OH. However, when H2O2 was added at room temperature (25℃), the absorbance of the characteristic absorption peak of MB in the mixture slowly decreased from 1.64 to 1.17 within 30 min. Figure 5 (b) indicates that M@CuO can effectively catalyze the formation of •OH from H2O2 at room temperature. For comparison, the Fenton reaction with the same concentration / reaction time was performed under 45℃ water bath conditions (simulating photothermal heating effect). The results showed that the absorbance of the characteristic absorption peak of MB in the mixture rapidly decreased from 1.64 to 0.55 within 30 min. Figure 5 c) indicates that M@CuO can continuously and efficiently catalyze the formation of •OH from H2O2 at 45℃. Calculations show that, under the same conditions, the efficiency of the mixture M@CuO + MB + H2O2 in decomposing MB at 45℃ is 66.5%, which is 2.3 times that at 25℃ (28.7%). Figure 5 d). Therefore, M@CuO can effectively catalyze the generation of •OH from H2O2, and this effect can be further enhanced through photothermal effects.

[0049] Example 4

[0050] Cytotoxicity test

[0051] Human umbilical vein endothelial cells (HUVEC), mouse colon cancer cells (CT26), and mouse breast cancer cells (4T1) were cultured under standard conditions at a concentration of 1×10⁻⁶. 4 / wells were seeded in 96-well plates and incubated for 12 h. The old medium was then replaced with fresh medium containing a series of different concentrations of M@CuO NPs prepared in Example 1. After incubation for 24 or 48 h, the cells were washed with phosphate-buffered saline (PBS) and cell viability was assessed using the standard MTT assay.

[0052] In vitro cell experiments

[0053] Good biocompatibility is a prerequisite for the application of bionanomaterials. Therefore, this study compared human umbilical vein endothelial cells (HUVECs), mouse colon cancer cells (CT26), and mouse breast cancer cells (4T1) with different concentrations of M@CuO (0-400 μg·mL⁻¹). -1 Co-incubated to assess its cell compatibility ( Figure 6 (a, 6b, and 6c). The results showed that after 24 hours of co-incubation, non-cancer cells HUVEC and cancer cells CT26 and 4T1 cultured with M@CuO all exhibited high cell viability (>85%), indicating that M@CuO has good biocompatibility with cells in vitro.

[0054] Example 5: In Vitro Therapy

[0055] Photothermal cell therapy (PTT): First, cultured cells in a 96-well plate are placed with a solution containing 0.2 mg / mL... -1 The M@CuO prepared in Example 1 was co-incubated with fresh culture medium for 4 h. Then, the cells were irradiated with NIR light (808 nm) for different durations to perform photothermal therapy on tumor cells. Finally, the cell viability after photothermal therapy was assessed using the standard MTT assay, and the treated cells were co-stained using the Calcine-AM / PI staining method, and cell fluorescence images were captured.

[0056] Cytokinetic therapy (CDT): Discard the old culture medium from two 96-well plates containing cultured cells and wash once with PBS. Use fresh culture medium containing different concentrations of M@CuO (0-0.2 mg / mL) for each plate. -1 Replace with 150 μL). Another plate was prepared using fresh culture medium containing different concentrations of M@CuO (0-0.2 mg·mL⁻¹). -1The culture medium was replaced with 150 μL of H2O2 (100 μM). Two 96-well plates were placed together in a cell culture incubator and incubated for 12 h. The old culture medium was then discarded, and cell viability was assessed using the MTT assay. Furthermore, cells were co-stained using the Calcine-AM / PI staining method, and fluorescence images were captured.

[0057] 4T1 cells were then mixed with M@CuO (200 μg·mL⁻¹). -1 The cells were incubated for 12 h, and then the PTT effect under light conditions was tested using a cell viability assay kit (MTT). Under 808 nm laser irradiation (1.0 W·cm⁻¹), the cells were incubated for 12 h. -2 After 3 min, the cells still showed a high survival rate (87.4%), but with increasing light exposure time, the cell survival rate gradually decreased to 41.3% (6 min) and 16.1% (9 min), indicating that M@CuO has a good PTT effect under NIR light irradiation and the target effect can be achieved by adjusting the light exposure time (Figure 7a). Furthermore, the anticancer effect of different concentrations of M@CuO in in vitro chemokinetic therapy was studied using the MTT assay. Clearly, when 4T1 cells were co-incubated with M@CuO for 12 h at given concentrations (0-200 μg·mL⁻¹), the cell survival rate was significantly higher. -1 Within the specified range, cell viability exceeded 85%. Conversely, after co-incubation with M@CuO + H2O2 for 12 h, cell viability decreased from 0 μg / mL. -1 The concentration decreased from 100% to 200 μg·mL. -1 The efficacy rate was 51.7%, indicating that the combination of M@CuO and H2O2 has a good CDT effect, and the CDT effect is dose-dependent. Figure 7 b).

[0058] In vitro cell therapy: 4T1 cells were seeded into multiple 96-well plates (1×10⁻⁶ cells per well). 4 Cells were cultured for 12 h per well. The cultured cells were then randomly divided into 7 groups: (1) Control group, (2) M@CuO group, (3) Near-infrared (NIR) irradiation group, (4) H2O2 group, (5) M@CuO + NIR, (6) M@CuO + H2O2, and (7) M@CuO + H2O2 + NIR. After different treatments, the cells were washed with PBS, and cell viability was assessed using the standard MTT assay. Cells were also co-stained with Calcein-AM / PI and then fluorescent images were captured using a fluorescence microscope.

[0059] To evaluate the efficacy of in vitro photothermal-chemokinetic combined therapy, a mixture containing M@CuO (200 μg·mL⁻¹) was used. -14T1 cells were incubated in DMEM medium for 12 h, with some groups receiving 808 nm laser irradiation (1 W·cm⁻¹). −2 , 6 min) and / or add H2O2 (100 μM). These cells were divided into 7 groups, including: (1) Control group, (2) M@CuO group, (3) Near-infrared light (NIR) irradiation group, (4) H2O2 group, (5) M@CuO + NIR group, (6) M@CuO + H2O2 group, (7) M@CuO + H2O2 + NIR group. Cell viability in these groups was detected by MTT assay and cell fluorescence staining was observed by fluorescence microscopy ( ). Figure 8 In groups (1-4), cell survival rates all exceeded 85%, indicating that M@CuO, NIR, or H2O2 alone had no killing effect on 4T1 cancer cells. However, the cell survival rate in group (5) was 41.3%, indicating that M@CuO had a good PTT effect under NIR light irradiation. In addition, the cell survival rate in group (6) decreased to 52.8%, confirming a good CDT effect. More importantly, the cell survival rate in group (7) was the lowest, at only 19.2%, which was attributed to the good synergistic PTT-CDT therapeutic effect. Figure 8 a). Simultaneously, fluorescence imaging showed that almost all cells in groups (1-4) exhibited green fluorescence (live cells). Compared to groups (1-4), the green fluorescence area decreased in groups (5-7), while the red fluorescence area (dead cells) rapidly increased, indicating a decreased cancer cell survival rate. In particular, the M@CuO +H2O2+ NIR group (7) showed red fluorescence in most dead cells (…). Figure 8 (b) In summary, the combination of M@CuO with NIR light and H2O2 exhibits excellent photothermal-chemokinetic combined therapeutic efficacy, which can efficiently kill tumor cells.

Claims

1. A method for preparing a nanocomposite biomaterial based on cuttlefish ink@copper oxide, characterized in that, The process includes the following steps: adjusting the pH of fresh cuttlefish squid juice to 12-13, stirring for 12-20 hours, centrifuging, and then adding the resulting nanoparticles to a CuSO4 solution with a concentration of 0.01-0.2 mol·L⁻¹. -1 Stir magnetically for 3-5 minutes, filter, dry, and calcine. The calcination temperature is 350-500℃ and the calcination time is 3-5 hours. The resulting black particles are cuttlefish ink@copper oxide composite nanomaterials (M@CuO).

2. The method for preparing the cuttlefish ink@copper oxide nanocomposite material according to claim 1, characterized in that, Adjust the pH value to 12-13 using any one of the following solutions: sodium hydroxide, potassium hydroxide, sodium methoxide, or potassium methoxide.

3. The method for preparing the cuttlefish ink@copper oxide nanocomposite material according to claim 1, characterized in that, The composite material is a multifunctional M@CuO composite nanomaterial with a CuO layer grown on the outside of the squid ink core. The M@CuO composite material is spherical with a nanoparticle size of 100-130 nm and a surface potential of -8 to -10 mV.

4. The application of the squid ink@copper oxide-based nanocomposite material prepared by the preparation method according to any one of claims 1-3 in the preparation of materials for photothermal materials and / or the Fenton effect, characterized in that, The photothermal material and / or the material with a specific Fenton effect is a squid ink@copper oxide nanocomposite material prepared by the method described in any one of claims 1-3.

5. The application according to claim 4, characterized in that, As a photothermal material, the squid ink@copper oxide nanocomposite material achieves a temperature increase effect under near-infrared laser irradiation at 800-850 nm, and the concentration of the squid ink@copper oxide nanocomposite material is 0.4 mg / mL. -1 ; And / or, As a material for the Fenton effect, it is a material that catalyzes the generation of •OH from H2O2 via the Fenton reaction.

6. The use of the cuttlefish ink@copper oxide nanocomposite material prepared according to any one of claims 1-3 in the preparation of a drug for treating anti-tumor cells, characterized in that, The tumor cells were 4T1.

7. The application according to claim 6, characterized in that, The aforementioned squid ink@copper oxide nanocomposite biomaterial is used to achieve in vitro photodynamic-photothermal combined therapy of tumor cells through tumor photothermal-chemokinetic combination.

8. The application of the squid ink@copper oxide nanocomposite material prepared by the method according to any one of claims 1-3 in the photocatalytic degradation of dyes.

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