Water-soluble fibroin-novel indole blue-green nanoparticles as well as preparation method and application thereof

Abstract

The invention discloses a water-soluble fibroin-novel indole cyan green nano particle and a preparation method and application thereof, biocompatible silk fibroin and small photo-thermal molecules of novel indole cyan green dye are integrated together, and the water-soluble fibroin-novel indole cyan green nano particle expressed as SF (at) IR820 is obtained. The SF (at) IR820 prepared by the invention can go deep into tumor cells. The SF (at) IR820 can effectively eliminate 4T1 tumor cells in vitro and inhibit tumor growth in vivo when exposed to 808nm laser radiation, a promising strategy is provided for developing an efficient photothermal agent for cancer treatment, and the SF (at) IR820 has certain reference significance and application value.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of photothermal imaging and tumor treatment, in particular to a water-soluble silk fibroin-new indocyanine green nanoparticle and a preparation method and application thereof. BACKGROUND Cancer is the number one disease affecting human health, and has a serious negative impact on individual human beings and the entire society. Due to the high similarity between tumor cells and normal cells and their strong vitality, traditional tumor treatment often has limited effect and is accompanied by serious adverse reactions. Early clinical studies have shown that due to the good targeted delivery of nano-carriers, the intracellular uptake rate of nanoparticles by cancer cells in the tumor microenvironment is faster, so that nanoparticle-based treatment can show higher efficacy by enhancing the EPR effect, while minimizing toxic side effects. Therefore, nano-technology delivery systems have attracted the attention of researchers in recent years. So far, Doxil®, irinotecan hydrochloride liposome and more than a dozen nano-drugs have been approved for clinical treatment. This emerging drug delivery method can enhance the selectivity and effective delivery of drugs to target cells in the tumor microenvironment. It has achieved excellent therapeutic effect in many types of malignant tumors. In addition, nano-drugs not only can inhibit tumor growth, but also can reduce systemic toxicity by clearly defining drug dosage and administration route, providing new ideas for tumor treatment. Therefore, nano-technology-based treatment is becoming a new type of tumor treatment method with practical value.

[0002] Exogenous means of regulating treatment has the characteristics of non-invasiveness and adjustable treatment effect, and is the research hotspot of current cancer treatment technology. Photothermal therapy (PTT) is a new method of tumor treatment. In the whole process of PTT, the design of nano-materials, anticancer drugs, targeted carriers, nano-materials and other materials has high flexibility, and different combinations can be used according to the type of tumor to produce good therapeutic effect, so PTT is also a promising technology. In addition, for photothermal therapy, the main factor affecting photothermal therapy is the selection of photosensitizer, and near-infrared dye indocyanine green (IR820) has attracted widespread attention due to its excellent fluorescence stability and photothermal effect in the near-infrared range (NIR-II). Previously, Feng et al. reported that the formation of IR820 and albumin complex can greatly enhance the fluorescence effect of IR820, and the interaction between IR820 and albumin can prevent the formation of aggregates in aqueous solution, thereby reducing the quenching effect caused by aggregates. At the same time, the combination of the geometric shape of IR820 and the hydrophobic albumin carrier helps to form a rigid structure, thereby making the physical strength of IR820 more stable. These reasons all make the combination of IR820 and albumin significantly improve its fluorescence brightness and effectively improve its light stability.

[0003] Silk is a natural protein derived from cocoon, which plays an important role in biomedical science. As a main component, the structure of multi-component amino acids endows silk fibroin (SF) modifiers with good biocompatibility and degradability, negligible allergenicity and multiple bioactivities. SUMMARY

[0004] The present application integrates biocompatible silk fibroin and small photothermal molecules of a novel indocyanine green dye together to obtain a water-soluble silk fibroin-novel indocyanine green nanoparticle represented as SF@IR820. Dynamic structure design endows SF@IR820 with nanoscale, excellent biocompatibility, deep tumor penetration and high photothermal imaging and therapeutic effect. Under 808 nm laser irradiation, SF@IR820 can effectively kill 4T1 tumor cells in vitro and inhibit tumor growth in vivo through photothermal imaging monitoring, providing a promising strategy for constructing efficient photothermal reagents for cancer.

[0005] In view of this, the technical scheme adopted by the present application is: The cocoon is degummed by 0.3-0.7% (w / v) Na2CO3 solution at 50-100℃ for 45-90 minutes to obtain purified silk fibroin; The silk fibroin is dissolved in a ternary solvent composed of CaCl2, ethanol and water, and is dissolved by stirring at 85-95℃. The obtained solution is filtered by a 0.22μm filter membrane to remove impurities. The filtrate is dialyzed and concentrated to obtain a silk fibroin aqueous solution; The IR820 dye is first dissolved in dimethyl sulfoxide, and then mixed with acetone at a volume ratio of 1:5-1:10 to prepare an IR820 organic phase solution with a concentration of 100-200μg / mL; The obtained silk fibroin aqueous solution and the IR820 organic phase solution are mixed at a volume ratio of 1:8-1:12 under a buffer with pH 6.5-7.5, and are treated by 50-100 W ultrasonic waves with intermittent pulses for 5-15 minutes to induce self-assembly of nanoparticles; The self-assembled mixed solution is centrifuged at a high speed of 14000-16000 rpm to obtain water-soluble silk fibroin-novel indocyanine green nanoparticles.

[0006] Further, CaCl2, ethanol and water are mixed at a molar ratio of 1:2:8-1:3:10.

[0007] Specifically, the specific method of concentration is as follows: the dialyzed silk fibroin aqueous solution is mixed with a 0.8% (w / v) polyethylene glycol solution at a volume ratio of 1:1, and is concentrated to 1 / 3-1 / 2 of the original volume at 4℃. Then, the concentrated solution is treated at 4℃ under centrifugation conditions of 1000-1500 rpm for 10 minutes, and the supernatant is taken to obtain the silk fibroin aqueous solution with a concentration of 10-20 mg / mL.

[0008] The application further provides a water-soluble silk fibroin-new indocyanine green nanoparticle, which is prepared by the method.

[0009] The application of the nanoparticle in the application includes application of the water-soluble silk fibroin-new indocyanine green nanoparticle in preparation of a photothermal imaging material.

[0010] The application of the water-soluble silk fibroin-new indocyanine green nanoparticle in preparation of a drug for inhibiting growth of tumor cells.

[0011] The water-soluble silk fibroin-new indocyanine green nanoparticle can efficiently penetrate into the inside of a tumor spheroid.

[0012] The application adopts natural silk fibroin nanoprotein with better biocompatibility, controllable biodegradability and non-immunogenicity as a carrier, and loads IR820 to achieve better photothermal treatment effect. The application firstly evaluates SF@IR820, and the obtained experimental data show that SF is spherical, and can effectively load model drug IR820 into the SF nanosphere. In addition, the cell results show that the unloaded SF has acceptable low toxicity to tumor cells, indicating that it has good carrier low toxicity. At the same time, the nanoscale size allows the SF nanoparticle to penetrate into tumor cells. Under exposure to 808 nm laser radiation, SF@IR820 proves the ability of effectively eliminating 4T1 tumor cells in vitro and inhibiting tumor growth in vivo, and provides a promising strategy for developing high-efficiency photothermal agents for cancer treatment, and has certain reference significance and application value. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 To characterize SF@IR820 nanoparticles; (A) particle size SF@IR820, (B) TEM results SF@IR820, (C) UV fluorescence spectrum SF@IR820 free IR820, (D) with PBS (negative control), 0.1% Triton X-100 (positive control) and different concentrations of SF@IR820, (E) hemolysis analysis and hemolysis picture after incubation in different concentrations of SF-IR820, (F) temperature rise curve of SF@IR820 at different concentrations, (G) thermal cycle curve of SF@IR820 free IR820, (H) optical photo of temperature change in various processes; Figure 2 Thermal cycle photos of free IR820 and SF@IR820; Figure 3 Photothermal images (A) and temperature (B) of tumors in mice 4 hours after drug treatment; Figure 4Cell experiment for SF@IR820; (A) CLSM images of IR820 internalization in cells SF@IR820 nanoparticles were treated with 4T1 cells at different time points, (B) SF@IR820 was analyzed by flow cytometry at different times, (C) toxicity evaluation of SF@IR820 and free IR820 to 4T1 cells under different concentration conditions; Figure 5 (A) cell live and dead staining fluorescence pictures and (B) the proportion of live and dead cells; Figure 6 Tumor spheroid penetration experiment; (A) CLSM images of multicellular spheroids treated at different times. (B) Quantification of fluorescence intensity of each layer; Figure 7 Anti-tumor treatment experiment; (A) schematic diagram of tumor inhibition experiment, (B) individual and tumor volume growth curves of mice within 15 days of different treatments, (C) tumor inhibition rate, (D) tumor weight after treatment, (E) body weight change of different control groups during treatment, (F) quantitative results of CD31 immunofluorescence analysis, (G) CD31 and Ki67 staining pictures; Figure 8 Blood compatibility test results, all data are expressed as mean standard deviation (n=3); Figure 9 H&E section imaging of tumors in different treatment groups; Figure 10 Optical photos of various tissues in mice after treatment, including heart, liver, spleen, lung and kidney; Figure 11 Body weight changes of mice injected with normal saline, free IR820 and SF@IR820 nanoparticles within 15 days. DETAILED DESCRIPTION

[0014] The application is further illustrated in conjunction with the specific embodiments.

[0015] Take 2-3 g of cocoon, wash with warm deionized water for three times, and dry in a 40 °C oven for 24 hours. Cut the dried cocoon into small pieces of about 0.5 cm², and put them in 100 mL of 0.5% (w / v) sodium carbonate (Na2CO3) solution, and heat in a boiling water bath for 1 hour to completely remove sericin. Take out the degummed silk fibroin fibers, rinse them thoroughly with deionized water until neutral, and dry at 35 °C for more than 24 hours. Dissolve the dried pure silk fibroin fibers in 20 mL of ternary solvent (CaCl2: ethanol: water = 1:2:8 molar ratio) at 90 °C with magnetic stirring for 2 hours until the silk fibroin fibers are completely dissolved. After the solution is cooled to room temperature, perform preliminary filtration with gauze to remove insoluble substances. Put the filtrate into a dialysis bag (molecular weight cut-off 3.5 kDa), and dialyze against deionized water for 3 days, changing the water 3 times a day to completely remove calcium ions and ethanol. Mix the dialyzed silk fibroin aqueous solution with 0.8% (w / v) polyethylene glycol solution at a volume ratio of 1:1, and concentrate at 4 °C. Finally, centrifuge the concentrated solution at 1000 rpm for 10 minutes (4 °C), and take the supernatant to obtain a silk fibroin aqueous solution with a concentration of 15 mg / mL, which is stored at 4 °C for later use.

[0016] Accurately weigh 1.5 mg of IR820 dye, dissolve it in 100 μL of dimethyl sulfoxide (DMSO), and vortex until completely dissolved to obtain an IR820 DMSO stock solution. Slowly add the stock solution to 9 mL of acetone, and magnetically stir for 30 minutes to obtain a uniformly dispersed IR820 / acetone solution. Measure 1 mL of the obtained silk fibroin aqueous solution (15 mg / mL), mix and dilute it with 1 mL of pH 7.4 phosphate buffered saline (PBS) to ensure the pH environment of the subsequent mixed system, and slowly add it to the above-mentioned IR820 / acetone solution (water phase to organic phase volume ratio of 1:9) under continuous stirring (600 rpm). After the addition is completed, a mixed solution is obtained, which is placed in an ice water bath, and an ultrasonic cell disruptor is used to induce self-assembly of the nanoparticles. The ultrasonic treatment parameters are set as follows: power 80 W, intermittent pulse mode for 5 seconds, intermittent 2 seconds, and treatment time 10 minutes. The use of intermittent pulse mode combined with ice bath temperature control can maximize the promotion of silk fibroin coating IR820 and the formation of uniform nanoparticles while minimizing the destruction of the structure of the photothermal agent molecules by ultrasonic energy, thereby ensuring the photothermal activity After ultrasonic treatment, the mixture is moved to a fume hood, and left to stand for 6-8 hours to allow the acetone to evaporate naturally.

[0017] The suspension formed after evaporation is transferred to a centrifuge tube, centrifuged at 13000 rpm for 10 minutes at 4, the supernatant is discarded, and the precipitate is collected, which is the water-soluble silk-fibroin-indocyanine green nanoparticle of the present application.

[0018] For subsequent characterization and experiments, the obtained nanoparticle precipitate was re-dispersed in 1 mL of phosphate buffered saline (PBS, pH 7.4) and sterilized by filtration through a 0.22 pm pore size filter, resulting in a homogeneous aqueous stock dispersion of SF@IR820 NPs.

[0019] Photothermal properties of SF@IR820 nanoparticles Photothermal effect The SF@IR820 NPs were irradiated with 808 nm laser at different concentrations (0-50 pg / mL), and the thermal camera tracked the temperature change every 20 seconds. To investigate the photothermal stability, the SF@IR820 NPs (IR820 concentration: 50 pg / mL) were irradiated with 808 nm laser, followed by a 10-minute non-artificial cooling process. This heating-cooling cycle was repeated five times, and the photothermal camera recorded the solution temperature.

[0020] In vitro hemolysis test We used a classic red blood cell (RBC) assay to evaluate the SF@IR820 NPs. Specifically, red blood cells diluted to about 2% were transferred to a centrifuge tube. This SF@IR820 NPs (IR820 concentration: 0, 1, 5, 25, and 50 pg / mL) in the test tube were incubated at 37 °C for one hour, and after centrifugation, the ultraviolet absorption of the supernatant was measured, while the positive control group (1% Triton X-100) and the negative control group of PBS were detected.

[0021] In vitro cytotoxicity To evaluate the in vitro cytotoxicity, 4T1 cells were plated in 96-well plates (1.0 x 104 cells / well) and allowed to adhere overnight. Different concentrations of SF@IR820 NPs (IR820 concentration: 0, 0.1, 0.5, 1, 2, and 5 pg / mL) were placed in the wells. The irradiation group was irradiated with an 808 laser (1.0 W / cm2, 3 minutes). Next, after 24 hours of drug culture, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) was used to detect drug toxicity.

[0022] Penetrability of SF@IR820 NPs Multicellular tumor spheroids (MCTS) based on 4T1 cells were prepared according to previous reports. First, 50 pL of agarose and 4T1 (2 x 103 cells / well) were added to a 96-well plate and cultured for 3-4 days. After formation, SF@IR820 NPs (IR820 concentration: 20 pg / mL) were added to the wells. The SF@IR820 NPs addition time points were 2, 4, 6, 8, 12, and 24 hours, and the penetrability of the NPs in the MCTS was measured using a confocal laser scanning microscope (CLSM, Zeiss 800).

[0023] In vitro cellular uptake To evaluate the cellular uptake, 4T1 cells were plated in 12-well plates (5.0 x 104cells / well) and allowed to adhere for about 12 h. Then SF@IR820 NPs (IR820 concentration: 20 pg / mL) were added to the wells. The time points for SF@IR820 NPs addition were 0.1, 0.5, 1, 2, and 4 h, respectively, and the cellular uptake trend of the drug was observed by CLSM.

[0024] For flow cytometry (FCM), 4T1 cells were plated in 12-well plates (1.0 x 105cells / well) and allowed to adhere for about 12 h. Then SF@IR820 NPs (IR820 concentration: 5 pg / mL) were added to the wells. The time points for SF@IR820 NPs addition were 0.1, 0.5, 1, 2, and 4 h, respectively, and the cellular uptake trend of the drug was observed by FCM.

[0025] In vitro photothermal imaging 4T1 tumor-bearing mice were established and divided into three groups (three mice per group), and when the tumor volume grew to ~120 mm 3 , SF@IR820 was injected, respectively. After 4-6 h of injection, the mice were treated with 808 nm laser (1.0 W / cm 2 , 3 min), and the tumor temperature was monitored every minute by a photothermal imager.

[0026] In vitro therapeutic effect 4T1 Balb / c female mice were provided by Chongqing Tengxin Biotechnology Co., Ltd. for in vivo experiments and were strictly in accordance with the legal and institutional guidelines of Southwest University and were approved by the Ethics Committee of Southwest University in Chongqing, China. The mice were implanted with tumor cells and waited for the tumor volume to grow to ~100 mm 3 , SF@IR820 IR820+L, SF@IR820+L groups (n=5 per group). Next, the mice were administered with PBS, IR820, and SF@IR820 Tail vein injection was used on the first, fourth, and seventh days. Then, in the following days, the subcutaneous tumor site of IR820+L was irradiated with a laser (808 nm, 1.0 W / cm 2 ) for 3 min for the SF@IR820+L group. Finally, the body weight and tumor volume of all mice were recorded daily. When the 15-day treatment period ended, the mice were sacrificed, and the tumor and the required organs, including the heart, liver, spleen, lung, and kidney, were obtained for further histopathological experiments.

[0027] Statistical analysis All values are expressed as mean ± standard deviation (S.D.). Statistical significance was determined as *p <0.05, **p<0.01 and ***p <0.001.

[0028] Results and Discussion Synthesis and Characterization of SF@IR820 Nanoparticles Silk fibroin (SF) and photosensitizer IR820 form stable nanoparticles SF@IR820 through self-assembly. In order to characterize the morphology and distribution of SF@IR820 nanoparticles, nanoparticle, dynamic light scattering (DLS) and transmission electron microscopy (TEM) were used in this experiment to characterize the nanoparticles. As shown in FIG. 2B, SF@IR820 particles exhibit almost spherical morphology in TEM images, with an average size of about 70 nm. As shown in FIG. 2A, the PDI of SF@IR820 in aqueous phase is about 72.85 nm, and the distribution index detected by DLS is very narrow, with a relatively narrow PDI of 0.187. The diameter obtained from TEM images and DLS data is similar. The three-dimensional nanoeffect of nanoparticles can significantly prolong the half-life of blood circulation, preventing the rapid elimination of drugs from circulating blood. At the same time, the penetration ability of drug particles to tumor tissue has also been enhanced. Figure 1 Figure 1

[0029] In terms of optical properties, SF@IR820 nanoparticles as shown in FIG. 3C. In the ultraviolet (UV) visible spectrum, IR820 dispersed in the aqueous phase of SF@IR820 appears at about 835.6 nm, which is basically consistent with the characteristic peak of free IR820, indicating that the IR820 phototherapy drug has been successfully loaded into the silk nanoparticles SF@IR820 nanoparticles have been successfully obtained. Figure 1

[0030] In addition, considering that the blood compatibility of nanoparticles is a key influencing factor for later in vivo treatment, we continue to evaluate SF@IR820 by hemolysis experiment. In order to evaluate the content of leaked hemoglobin, the supernatant of each group was separated experimentally to detect their absorption intensity at about 540 nm and 570 nm, which is the characteristic peak of hemoglobin (FIG. ID). Then calculate the hemolysis rate under different concentrations of nanomedicine groups, as shown in FIG. IE. In addition, FIG. IE clearly reflects that all concentrations of SF@IR820 are less than 5%. The small amount of hemoglobin exuded in the supernatant indicates that SF@IR820 has good blood compatibility.

[0031] In order to obtain SF@IR820 we performed thermal cycling experiments to verify whether they have good photothermal effect and thermal stability. As shown in FIG. 4A, the temperature of the SF@IR820 solution increased rapidly after irradiation, and the temperature reached a maximum of about 50°C after 10 min of irradiation. The temperature of the SF@IR820 solution decreased rapidly after irradiation was stopped, and the temperature returned to room temperature after 10 min. The results show that SF@IR820 has good photothermal effect and thermal stability. Figure 1 ​​​As shown in Figure F, different concentrations of SF@IR820 exhibited good photothermal effects under near-infrared laser irradiation at a wavelength of 808 nm. With increasing drug concentration, when the concentration reached 30 μg / ml or higher, the drug temperature could reach above 40°C within 5 minutes, which is sufficient to achieve the therapeutic goal of inhibiting tumor cell growth. Furthermore, as... Figure 1 G and Figure 2 As shown, SF@IR820 and free IR820 under the same concentration conditions were compared. The study found that SF@IR820 exhibited better thermal stability than free IR820 under the same laser power, concentration, and ambient temperature conditions, maintaining good photothermal performance even after five thermal cycles. This indicates that the silk fibroin coating effectively enhances the photostability of IR820, laying the foundation for better tumor suppression. Simultaneously, time-varying photothermal images of various treatments were recorded using an infrared thermal imager. Figure 1 This clearly shows that temperature changes are closely related to concentration and radiation time (H), which clearly demonstrates that temperature changes are closely related to concentration and radiation time.

[0032] Photothermal reagents can provide the characteristics of real-time imaging in a photothermal mode, further facilitating convenient, rapid, and reliable site identification. Given the excellent photothermal properties of IR820 in vitro, we further investigated the in vivo performance of IR820 and SF@IR820 nanoparticles (…). Figure 3 Three groups of mice carrying 4T1 tumors were injected with PBS, IR820, and SF@IR820 via the tail vein, respectively. Four hours after injection, the mouse tumors were irradiated with an 808nm laser (1W / cm², 3 minutes), and photothermal images (Fig. S3A) and temperature changes (Fig. S3B) of the tumor site were recorded every minute using a thermal imaging camera. Within 5 minutes after irradiation, SF@IR820 reached temperatures above 40°C, exhibiting a faster heating rate and larger thermal radiation area than free IR820, while the tumor center temperature injected with PBS only rose below 35°C. This indicates that IR820 prepared by coating IR820 with silk fibroin can increase the cellular uptake of the photosensitizer IR820 by tumor tissue and further improve the photothermal performance of SF@IR820 compared to IR820. The photothermal imaging performance reveals the improved photothermal capabilities of SF@IR820 in vivo and also provides a simple in-situ spatiotemporal assessment for phototherapy applications.

[0033] Cell phagocytosis assay SF@IR820 Furthermore, CLSM technology was used to study the intracellular transport of SF@IR820 nanoparticles, revealing that the drug can be distributed throughout the cell. Figure 4A). 4T1 cells were incubated with SF@IR820 nanoparticles for 0.1 h, 0.5 h, 1 h, 2 h and 4 h, respectively, and the nuclei and cytoskeleton were stained with DAPI and FITC, respectively. Compared with the results of 0.1 h, the IR820 fluorescence in the cells was stronger at 4 h, which was due to the fact that more nanoparticles entered the cells as the incubation time increased. Interestingly, IR820 eventually accumulated in the cytoskeleton in the cells over time and played an anti-tumor role when exposed to laser irradiation. To verify SF@IR820 through tumor cells, we analyzed SF@IR820 nanoparticles using flow cytometry. In general, in principle, SF@IR820 tumor cells will increase over time as the incubation time of tumor cells increases, showing a time-dependent intracellular process. SF@IR820 nanoparticles, tumor cells were incubated for 4 h under the following conditions and detected using flow cytometry. As shown in Figure 4 B, SF@IR820 tumor cells can reach more than 90% within 4 h, while the uptake rate after 30 min of incubation is about 25%. This experimental result further confirms the time-dependent uptake of nanoparticles by tumor cells and demonstrates the high uptake rate of SF@IR820 tumor cells.

[0034] In vitro cytotoxicity verification of SF@IR820 To evaluate the ability of SF@IR820 nanoparticles to inhibit cancer cells, we performed in vitro cytotoxicity experiments and cell viability staining experiments. As shown in Figure 4 C, based on the MTT method, the toxicity of SF@IR820 with and without infrared laser irradiation was similar to that of free IR820 without infrared laser irradiation, both of which had good biocompatibility. At the same time, the SF@IR820 group under 808 nm infrared laser irradiation was significantly higher than the free IR820 group, indicating that SF@IR820 had good photothermal effects. It can activate the photothermal effect of IR820 under the trigger of exogenous near-infrared light, and has a strong killing effect on tumor cells. This indicates that SF@IR820 has a good inhibitory effect on tumor cell growth.

[0035] To verify the inhibitory effect of SF@IR820 on tumor cells from multiple angles, we further evaluated its toxicity using cell viability staining experiments. As shown in Figure 5 A, compared with the control group, the free IR820 group and the SF@IR820 group had similar fluorescence intensity (FDA represents green fluorescence-labeled live cells, and PI represents red-labeled dead cells), indicating that SF@IR820 had no photothermal effect under normal conditions (without light).

[0036] The most obvious difference is that when SF@IR820 was irradiated with a laser, the red fluorescence was significantly increased compared to the free IR820 group. Figure 5 (B) indicates that SF@IR820 nanoparticles have a more significant inhibitory effect on tumor cells under infrared laser excitation.

[0037] Tumor sphere penetration assay SF@IR820 This invention investigates the effect of SF@IR820 on tumor invasion using a multicellular spheroid model of 4T1 cells. SF@IR820 was used in vitro. After treatment, SF@IR820 (IR820 concentration: 20 μg / ml) was administered at different time points. Figure 6 A and Figure 6 As shown in Figure B, the SF@IR820 nanoparticles exhibited significant enhancement within tumor cell spheroids and continuously penetrated into the tumor spheroids, indicating excellent tumor penetration. These results clearly demonstrate that high fluorescence intensity is associated with good biocompatibility and tumor penetration, laying a solid foundation for future therapeutic applications.

[0038] Anti-tumor therapy SF@IR820 To conduct in vivo therapeutic experiments, we established a 4T1 tumor mouse model to evaluate the anticancer ability of the nanoparticles. Specifically, we established an anti-tumor model and measured the mice's body weight and tumor volume daily during treatment to assess the tumor-suppressive effect. Figure 7 A). 15 days after the end of treatment, if Figure 5 As shown in Figure B, there were significant differences in tumor volume changes among the groups, but no significant fluctuations in body weight among the groups. Figure 7 E). Among them, the saline group showed the fastest growth, with the largest tumor volume reaching ~800 mm. 3 Compared with the free IR820 group, the SF@IR820+L group showed the most significant therapeutic effect and the smallest tumor volume. Simultaneously, the nanoparticles also demonstrated good anti-tumor activity in terms of tumor mass and tumor inhibition rate. Figure 7 (D, 7C). The above results all confirm that the synthesized nanoparticles can effectively accumulate within tumors and have good tumor retention properties, further producing satisfactory PTT therapeutic effects in vivo.

[0039] We further investigated CD31 and Ki67 in the tumor. For example... Figure 7As shown in F-G, CD31 and Ki67 sections showed that the SF@IR820+L treatment group was the smallest, the tumor tissue damage was the most obvious, and the treatment effect was the best. CD31 sections further showed that the damage of SF@IR820+L was more serious than other groups, and green fluorescence was visible in the tumor blood vessels of the control PBS group. Ki67 sections showed that the SF@IR820+L treatment group showed obvious tumor damage, which was related to the severe atrophy of cells and the apoptosis characteristics of nuclear cohesion.

[0040] In vivo biological safety study Considering the balance requirement of high efficacy and low toxicity for ideal tumor treatment, we used blood compatibility test to evaluate the potential toxicity or adverse reactions of SF@IR820 nanoparticles on the organism, including granulocyte percentage (GRA), monocyte percentage (MON), lymphocyte percentage (LYM), mean cell volume (MCV), mean hemoglobin concentration (MCHC), and other factors. Red blood cell distribution width (RDW), red blood cell count (RBC), mean red blood cell content (MCH), platelet distribution width (PDW), and other parameters. As shown in Figure 8 Compared with the control group, these main parameters of the experimental group had no significant difference, all within the normal range. These results showed that the nanoparticles did not cause hemolytic anemia and coagulation dysfunction SF@IR820 nanoparticles had good blood compatibility, consistent with the results of in vitro experiments. After 15 days of treatment, the main organs (spleen, lung, heart, liver and kidney) of the mice were taken for hematoxylin-eosin (H&E) staining and histological section analysis, and no pathological changes were observed in any group ( Figure 9 ). At the same time, we took and recorded the photos of different organs (including heart, liver, spleen, lung, kidney) of mice after treatment ( Figure 10 ).

[0041] By weighing various tissues, it can be seen that the weight differences of heart, lung and kidney are not obvious, while the weight of spleen is roughly the same trend as the weight of tumor mass. We hypothesize that the splenomegaly is caused by the inflammatory response caused by the tumor.

[0042] In addition, blood compatibility SF@IR820 further studied the nanoparticles through blood routine test. In routine blood test, there was no significant change in the body weight of mice in each group after 15 days of SF@IR820 nanoparticle placement after injection of normal saline and free IR820 ( Figure 11 ), which also proved the good biological safety of SF@IR820 nanoparticles.

Claims

1. A method for preparing water-soluble silk fibroin-novel indocyanine green nanoparticles, characterized in that, Includes the following steps: S1: Silkworm cocoons are degummed by 0.3-0.7% (w / v) Na2CO3 solution at 50-100℃ for 45-90 minutes to obtain purified silk fibroin fibers; S2: Dissolve the silk fibroin fiber in a ternary solvent composed of CaCl2, ethanol and water, stir and dissolve at 85-95°C, filter the resulting solution through a 0.22μm filter membrane to remove impurities, and then dialyze and concentrate the filtrate to obtain an aqueous solution of silk fibroin protein. S3: Dissolve IR820 dye in dimethyl sulfoxide first, then mix it with acetone at a volume ratio of 1:5 to 1:10 to prepare an IR820 organic phase solution with a concentration of 100 to 200 μg / mL; S4: Under a pH buffer of 6.5–7.5, the obtained silk fibroin aqueous solution was mixed with the IR820 organic phase solution at a volume ratio of 1:8–1:12, and the mixture was treated with intermittent pulses of 50–100 W ultrasound for 5–15 minutes to induce the self-assembly of nanoparticles. S5: The self-assembled mixture was centrifuged at 14,000 to 16,000 rpm to obtain water-soluble silk fibroin-novel indocyanine green nanoparticles.

2. The method for preparing water-soluble silk fibroin-novel indocyanine green nanoparticles according to claim 1, characterized in that: The molar ratio of CaCl2, ethanol, and water is 1:2:8 to 1:3:

10.

3. The method for preparing water-soluble silk fibroin-novel indocyanine green nanoparticles according to claim 1, characterized in that, Ultrasound operates with intermittent pulses of 5 seconds followed by a 2-second pause, with a cycle count of 10 to 30 times.

4. The method for preparing water-soluble silk fibroin-novel indocyanine green nanoparticles according to claim 1, characterized in that, The silk fibroin obtained in S1 is washed three times with warm deionized water and dried at 30-40°C for 24 hours.

5. The method for preparing water-soluble silk fibroin-novel indocyanine green nanoparticles according to claim 1, characterized in that, The specific method for concentration in S2 is as follows: the dialyzed silk fibroin aqueous solution is mixed with 0.8% (w / v) polyethylene glycol solution at a volume ratio of 1:1, and the mixture is allowed to stand at 4°C to concentrate to 1 / 3 to 1 / 2 of the original volume; then the concentrate is centrifuged at 4°C and 1000 to 1500 rpm for 10 minutes, and the supernatant is taken to obtain the silk fibroin aqueous solution with a concentration of 10 to 20 mg / mL.

6. Water-soluble silk fibroin-novel indocyanine green nanoparticles, characterized in that: It is prepared by the method described in any one of claims 1-5.

7. Application of water-soluble silk fibroin-novel indocyanine green nanoparticles in the preparation of photothermal imaging materials.

8. Application of water-soluble silk fibroin-novel indocyanine green nanoparticles in the preparation of drugs that inhibit tumor cell growth.

9. The application according to claim 8, characterized in that: Water-soluble silk fibroin-novel indocyanine green nanoparticles efficiently penetrate into the tumor spheroid.

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