Preparation method of carrier-free nano-drug delivery system CSCN and application of carrier-free nano-drug delivery system CSCN in breast cancer treatment

Abstract

The invention discloses a preparation method of a carrier-free nano-drug delivery system CSCN and an application of the carrier-free nano-drug delivery system CSCN in breast cancer treatment. According to the system, a common photosensitizer Ce6 is chemically modified and reacts with 2-hydroxyethyl disulfide to obtain a new photosensitizer Ce6-SS, and Ce6-SS and Cel are self-assembled to form the carrier-free nano-particle CSCN. The nanoparticles are of a spherical structure with good dispersity, and the system has a relatively strong cell inhibition effect under near-infrared light, can be effectively internalized into 4T1 cells and presents time dependence. The system can also generate lysosome escape, and can significantly reduce the mitochondrial membrane potential of 4T1 cells and damage the mitochondrial function under near-infrared illumination, thereby inhibiting mitochondrial respiration, relieving tumor hypoxia and enhancing the PDT curative effect. The results prove that the nano system can play a good synergistic PDT treatment effect, and has a good application prospect in the anti-tumor field.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano drug delivery, and particularly relates to a preparation method of a carrier-free nano drug delivery system CSCN and its application in the treatment of breast cancer. Background Art

[0002] At present, nano drug delivery technology shows great potential in targeted therapy, sustained and controlled release, etc., but there are still some key problems to be solved. First is the biocompatibility and toxicity problems. Some nano materials (such as certain metal nano particles, carbon nanotubes) may trigger immune responses, cytotoxicity or long-term cumulative toxicity. Non-degradable nano carriers (such as inorganic nano particles) may remain in the body for a long time, leading to organ burden. Due to immune responses, nano particles may be recognized as foreign bodies by the immune system, triggering complement activation or inflammatory responses. Secondly is the problem of insufficient targeting efficiency. For example, the surface-modified targeting molecules may lose specificity due to the "protein corona" effect (adsorption of blood proteins), or cause off-target due to target heterogeneity. Physiological barriers such as vascular endothelium, blood-brain barrier, and high tumor interstitial pressure will also hinder the deep penetration of nano particles. Drug loading and release control is another difficult problem faced by nano drug delivery. In the nano drug delivery system, the low drug loading rate, imbalance between burst release and controlled release, and stability problems lead to the uncontrollability and failure of a large number of nano preparations. Therefore, nano drug delivery technology still needs to solve these key problems to realize its clinical potential.

[0003] Breast cancer is the most diagnosed malignant tumor in women and the second leading cause of cancer death. Breast cancer can be divided into three major subtypes: HR-positive, HER2-positive, and triple-negative breast cancer (TNBC). Triple-negative breast cancer accounts for 15-20% of all breast cancers and is named because it lacks estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). Its metastasis rate, recurrence rate, and mortality rate are higher than any other subtype, resulting in a lower overall survival rate. It is the most malignant subtype of breast cancer. The current treatment options mainly include surgery, radiotherapy, and adjuvant chemotherapy.

[0004] Photodynamic therapy (PDT) is a non-invasive cancer treatment method. It uses non-toxic components, namely photosensitizers, light of a specific wavelength, and oxygen dissolved in cells (ROS). There are mainly two mechanisms in photodynamic therapy PDT. The type I mechanism is characterized by the transfer of a hydrogen or an electron between the photosensitizer and the cancer tissue (substrate) in the excited state T1, forming free radicals and anion free radicals. The electrons interact with oxygen molecules, and the oxygen molecules maintain their ground energy state. This process leads to the initial generation of ROS in the form of superoxide anion radicals (O2·-), and further ROS are generated inside the cells. The oxidative stress caused by this cascade reaction can damage cancer cells. The type II mechanism is characterized by the direct release of energy to O2. That is, the photosensitizer transitions to the excited state, and the energy is directly transferred to the oxygen molecules in the ground energy state (ground triplet state), thus generating excited oxygen particles, namely so-called singlet oxygen, which is characterized by extremely strong oxidizing properties. Commonly used photosensitizers include: UV-vis activated photosensitizers, near-infrared light (NIR) activated photosensitizers, and photosensitizers activated by X-rays. Although photodynamic therapy has advantages such as minimally invasive and strong targeting, its application is still limited by problems such as limited light penetration depth, side effects of photosensitizers, and limitations of local treatment. It is necessary to develop combined strategies to optimize its efficacy and safety.

[0005] Celastrol (Cel) is a natural bioactive triterpenoid extracted from Tripterygium wilfordii, and it is one of the main bioactive components of Tripterygium wilfordii. The structural formula is as Figure 1 shown, and it has various biological properties such as anti-tumor, antioxidant, anti-obesity, neuroprotection, and immunosuppression. Modern pharmacological studies have shown that Cel exhibits significant broad-spectrum anti-cancer activity in the treatment of various cancers, including lung cancer, liver cancer, colorectal cancer, hematological malignancies, gastric cancer, prostate cancer, kidney cancer, breast cancer, bone tumors, brain tumors, cervical cancer, and ovarian cancer. Cel may cause various side effects, such as cardiotoxicity, hepatotoxicity, hematotoxicity, reproductive toxicity, and nephrotoxicity. In addition, its pharmacokinetic defects and clinical translation barriers limit its practical application, and the emergence of nanotechnology, such as exosomes, liposomes, polymeric micelles, nanoencapsulation, nanocarriers, and nanoparticles, can be used to improve the bioavailability of Cel, achieve its targeted delivery, and thus reduce its cytotoxicity to healthy cells.

[0006] Nanotherapies for treating TNBC are constantly evolving to overcome the limitations of traditional diagnostic and treatment methods. Nanocarriers based on nanotechnology have higher encapsulation efficiency, lower cytotoxicity, greater stability, and longer half-life than traditional therapies. The nanodrug delivery system improves pharmacokinetic and pharmacodynamic parameters due to its nanoscale size. Currently, various nanoplatforms are used for preclinical and clinical treatment of breast cancer, such as polymeric nanoparticles, micelles, nanobodies, magnetic nanoparticles, liposomes, gold nanoparticles, dendrimers, and carbon nanotubes. Nanoparticles based on nanodrug delivery systems (NDDS-based NPs) have changed the landscape of traditional cancer treatment.

[0007] Carrier-free nanodrugs are a new type of drug constructed by self-assembly of drug molecules without the participation of carriers. According to the different compositions of drug modules, they can be divided into three types: (1) simple drug nanoassemblies self-assembled from a single drug module; (2) dual-simple drug nanoassemblies co-assembled from two drugs; (3) multi-simple drug nanoassemblies co-assembled from more than two drugs. The assembly of drug modules is mainly driven by non-covalent interactions, including hydrophobic interactions, intermolecular π-π stacking, hydrogen bonds, and electrostatic forces. During the self-assembly process, balancing intermolecular interactions is more conducive to assembly. Carrier-free nanodrug delivery systems have advantages such as better cell permeability, high drug loading efficiency, low biotoxicity, and simple structure. Summary of the Invention

[0008] Object of the Invention: Aiming at the problems existing in the prior art, the present invention provides a preparation method of a carrier-free nanodrug delivery system CSCN and its application in the treatment of breast cancer. The carrier-free nanodrug delivery system of the present invention is prepared by chemically modifying the photosensitizer Ce6 with 2-hydroxyethyl disulfide to synthesize a new photosensitizer Ce6-SS, and self-assembling it with the natural chemical product Cel into carrier-free nanoparticles CSCN. In vitro experimental results show that CSCN has the effects of enhancing tumor killing and significantly inhibiting tumor growth.

[0009] Technical Solution: To achieve the above object, the preparation method of a carrier-free nanodrug delivery system CSCN described in the present invention includes the following steps:

[0010] (1) Preparation of Ce6-SS: Dissolve Ce6 (chlorin e6), 2-hydroxyethyl disulfide, DCC (1,3-dicyclohexylcarbodiimide), and DMAP (4-dimethylaminopyridine) in DMF, stir, extract, dry, dissolve, and purify to obtain Ce6-SS;

[0011] (2) Preparation of CSCN: Dissolve Ce6-SS and Cel (celastrol) in DMSO respectively. After mixing the obtained solutions, ultrasonic treatment, dialysis, and centrifugation are performed to obtain the nanoparticles CSCN.

[0012] Among them, in step (1), the molar ratio of Ce6, 2-hydroxyethyl disulfide, DCC, and DMAP is 0.3 - 0.6:0.3 - 0.7:1.5 - 2:1.5 - 2.0.

[0013] Preferably, dissolve 0.3 - 0.6 mmol of Ce6, 0.3 - 0.7 mmol of 2-hydroxyethyl disulfide, 1.5 - 2.0 mmol of DCC, and 1.5 - 2.0 mmol of DMAP in 1 - 5 mL of DMF.

[0014] Among them, in step (1), the stirring condition is to stir at room temperature for 8 - 12 h.

[0015] Preferably, in step (1), the extractant is any one of dichloromethane, ethyl acetate, petroleum ether, and methyl tert-butyl ether, and the desiccant is any one of anhydrous sodium sulfate, anhydrous magnesium sulfate, and anhydrous calcium chloride.

[0016] Among them, in step (2), the dissolution concentrations of Ce6-SS and Cel in DMSO are both 8 - 12 mg / mL.

[0017] Among them, in step (2), the volume ratio of mixing is 1.7:1 - 2.5:1.

[0018] The carrier-free nano-drug delivery system CSCN prepared by the preparation method of the carrier-free nano-drug delivery system CSCN described in the present invention.

[0019] Furthermore, an application of a carrier-free nano-drug delivery system CSCN in the preparation of a drug for treating breast cancer.

[0020] The drug for treating breast cancer described in the present invention comprises a carrier-free nano-drug delivery system CSCN and a pharmaceutically acceptable excipient or carrier.

[0021] Among them, the pharmaceutical preparation is an injection, a granule, a tablet, a capsule, a pill, or an oral liquid.

[0022] The photosensitizer described in the present invention has the following structure:

[0023]

[0024] In the present invention, a disulfide bond is introduced into Ce6 to obtain a new photosensitizer Ce6-SS, and a carrier-free nanoparticle CSCN is self-assembled from the photosensitizer Ce6-SS and Cel.

[0025] In the present invention, a nano-delivery system was prepared to chemically modify the commonly used photosensitizer Ce6. It was reacted with 2-hydroxyethyl disulfide to obtain a new photosensitizer Ce6-SS. The chemical structure of Ce6-SS was verified by liquid chromatography-mass spectrometry (LC-MS) and nuclear magnetic resonance hydrogen spectrometer (1H-NMR). It was self-assembled with Cel by the nanoprecipitation method to form a carrier-free nanoparticle CSCN. It was observed by TEM that the nanoparticle was a spherical structure with good dispersibility. In vitro pharmacodynamic studies showed that this system had a strong inhibitory effect on cells under near-infrared light. The uptake study of 4T1 cells on the nanoparticles showed that this system could be effectively internalized into 4T1 cells and was time-dependent. This system could also escape from lysosomes, significantly reduce the mitochondrial membrane potential of 4T1 cells under near-infrared light, damage mitochondrial function, thereby inhibiting mitochondrial respiration, alleviating tumor hypoxia, and enhancing the efficacy of PDT. The above results proved that this nano-system could exert a good synergistic PDT therapeutic effect and had good application prospects in the field of anti-tumor.

[0026] In the present invention, a photosensitizer Ce6-SS with a brand-new structure was designed and synthesized. Ce6-SS of the present invention could not only exert the PDT efficacy of Ce6 but also reduce the GSH concentration in tumor cells, and could exert a dual anti-tumor effect. The CSCN constructed in the present invention synergistically treated TNBC through PDT, chemotherapy, and ferroptosis, and showed a powerful inhibitory effect on tumor growth both in vitro and in vivo.

[0027] The carrier-free nano-drug delivery system constructed in the present invention avoided the potential biotoxicity problems caused by inert carriers compared with the carrier-based delivery system; the aggregated nano-scale drugs had a longer blood circulation time and better cell permeability in the organism, and could accumulate in the tumor site better through the EPR effect.

[0028] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0029] In the present invention, a carrier-free nanoparticle - CSCN self-assembled from Ce6-SS and Cel was prepared. Its morphology was a spherical structure with uniform size and had good dispersibility. When stored at 4 °C for 5 days, the particle size and PDI remained stable. The acting force of this nanoparticle was hydrophobic interaction and intermolecular electrostatic interaction; in this study, celastrol was self-assembled with the self-made photosensitizer Ce6-SS into a carrier-free nanoparticle, which could target and deliver drugs to the tumor site, avoid liver and kidney toxicity, and improve the therapeutic effect. The nanoparticles of the present invention could combine the chemotherapeutic effect of celastrol, the photodynamic therapy (PDT) of Ce6, and the effect of disulfide bonds consuming glutathione (GSH), thereby producing a synergistic effect and significantly amplifying the therapeutic effect. Description of the Drawings

[0030] Figure 1 is the structural formula of celastrol;

[0031] Figure 2 is the synthetic route of Ce6-SS;

[0032] Figure 3 is the transmission electron microscopy image of Ce6-SS;

[0033] Figure 4 is the LC-MS image of Ce6-SS (A) and the mass spectrometry image of Ce6-SS (B);

[0034] Figure 5 is the 1H NMR spectrum of Ce6-SS;

[0035] Figure 6 shows the killing effect of different concentration groups on 4T1 cells detected by CCK8 method (A), and the killing effect of different concentration groups on 4T1 cells under phototherapy B, where L refers to light therapy (B)

[0036] Figure 7 is the CLSM image of cell uptake (A) Scale bar: 50 μm, **p < 0.01, fluorescence semi-quantitative analysis (B);

[0037] Figure 8 is the CLSM image of 4T1 cells after CSCN treatment for 1, 2, 4 h and stained with Lyso Tracker Green, Scale bar: 50 μm;

[0038] Figure 9 is the CLSM image of 4T1 cells after 4 h under different treatments (A), stained with JC-1, Scale bar: 50 μm, **p < 0.01, and the change in green fluorescence intensity of 4T1 cells after 4 h under different treatments (B). Detailed implementation mode

[0039] The present invention will be further described below in conjunction with specific embodiments and the accompanying drawings.

[0040] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all purchased from conventional biochemical reagent companies unless otherwise specified.

[0041] Example 1

[0042] Preparation and characterization of CSCN

[0043] (1) Preparation of Ce6-SS

[0044] The synthetic route is as Figure 2As shown, Ce6 (200 mg, 0.3 mmol), 2-hydroxyethyl disulfide (62 mg, 0.4 mmol), DCC (345.05 mg, 1.7 mmol) and DMAP (204.35 mg, 1.7 mmol) were dissolved in 1.5 mL of DMF. After stirring overnight at room temperature, 5 mL of water was added to the reaction mixture, and the reaction mixture was extracted three times with dichloromethane and dried over anhydrous sodium sulfate. Then the crude product was dissolved in methanol and purified by reverse-phase preparative high-performance liquid chromatography with the mobile phase of DCM:MeOH = 50:1 to 5:1. Finally, a black powder Ce6-SS with a yield of 50% was obtained.

[0045] (2) Preparation of CSCN

[0046] The carrier-free nanoparticles Ce6-SS / CelNPs (CSCN) were prepared by the nanoprecipitation method. Ce6-SS and Cel were respectively dissolved in DMSO to obtain stock solutions with a final concentration of 10 mg / mL each. Then the Ce6-SS stock solution and the Cel stock solution were mixed at a volume ratio of 1.7:1, and the total volume was 75 μL. Subsequently, the mixture was dispersed into 1.5 mL of pure water, and a nano-suspension was formed under ultrasonic treatment. It was transferred into a dialysis bag (MCOW: 8000 Da) and dialyzed with distilled water for 4 h to remove organic solvents and unassembled nanoparticles, and the water was changed every 2 h. After dialysis, the solution in the dialysis bag was transferred into a centrifuge tube, and the nanoparticle CSCN solution was stored at 4 °C. Its transmission electron microscopy image is as Figure 3 shown. It can be seen from the figure that CSCN is spherical particles with an average particle size of about 100 nm, with a consistent morphology and uniform size.

[0047] (3) Characterization of CSCN

[0048] The prepared Ce6-SS was dissolved in methanol to prepare a 50 μg / mL solution. After filtering through a membrane, it was loaded into a sample vial and directly injected onto an LC-MS. LC-MS was used to verify the molecular weight of Ce6-SS. The results are as Figure 4 shown. When the absorption wavelength was set at 398 nm, the liquid phase had a relatively pure peak at 13.6 min, and the corresponding mass spectrometry molecular weight was 715.35. In addition, 1H-NMR was also used to 1 verify the structure of Ce6-SS. As Figure 5 shown, the characteristic peaks of Ce6 were 9.7, 9.6 and 9.1 ppm (=CH-in the porphyrin ring) and 8.2 ppm (-CH=CH2), and the characteristic peak of 2-hydroxyethyl disulfide (-CH2-CH2-) was observed at 2.1 - 2.4 ppm. These experimental results indicate the successful synthesis of Ce6-SS.

[0049] Example 2

[0050] In vitro anti-tumor activity of nanoparticles

[0051] Cell culture: The murine breast cancer cell line 4T1 was obtained from the National Centre for Cell Science (NCCS) and cultured in RPMI-1640 medium containing 10% fetal bovine serum. The cells were grown in an environment of 37 °C and 5% CO2 and passaged every 2 days.

[0052] Cell resuscitation procedure: Take out the cryopreservation tube from liquid nitrogen, quickly put it into a water bath preheated to 37 °C, and gently shake until the liquid is completely melted. Subsequently, in a laminar flow hood, transfer the cell suspension to a sterile centrifuge tube containing 10 mL of medium, and centrifuge at 1000 rpm for 5 min. After discarding the supernatant, resuspend the cells with 1 mL of medium, then aspirate it into a cell culture flask containing 10 mL of medium, pipette evenly and place it in the incubator for culture.

[0053] Cell passage: When the confluence of cells in the culture flask reaches 80%-90%, passage operation should be carried out immediately. First, pour out the original medium in the culture flask, add an appropriate amount of PBS for washing, and then pour out the PBS. Next, add an appropriate amount of trypsin and place the culture flask in the incubator for digestion for 1 min. When the cells become round, quickly add a double volume of serum-containing medium to terminate the digestion. Then, gently pipette the cells to disperse them. Collect the cell suspension into a sterile centrifuge tube and centrifuge at 1000 rpm for 5 min. After discarding the supernatant, resuspend the cells with medium and aliquot them into new culture flasks for continued culture.

[0054] Cell cryopreservation: Prepare a cryopreservation medium containing 7% DMSO, select cells in the logarithmic growth phase, remove the old medium and wash with PBS, then add trypsin to digest the cells into single-cell state. After digestion is completed, add serum-containing medium to terminate the digestion process. Then, gently mix the cell suspension and count, adjust the cell concentration to 5×10 6 -1×10 7 cell / mL. Then, aliquot the cell suspension into cryopreservation tubes, 1.0 - 1.5 mL per tube, and label the cryopreservation tubes with cell name, cryopreservation date and operator information. Place the cryopreservation tubes in a -20 °C refrigerator for 2 h first, and then transfer them to an -80 °C refrigerator overnight. The next day, transfer the cryopreservation tubes to a liquid nitrogen container for long-term storage.

[0055] Using 4T1 cells as a research model, the in vitro anti-tumor activity of CSCN was determined by the CCK8 method. Select 4T1 cells in the logarithmic growth phase, and seed the cells at 4×10 3Cells were seeded at a density of [cells per well] into 96-well plates and incubated in an incubator for 24 h until the cells adhered. Then, the cells were treated with Ce6-SS, Ce6-SS+L, Cel, Ce6-SS+Cel, Ce6-SS+Cel+L, CSCN, and CSCN+L prepared in Example 1 for 24 h, and the drug concentrations in each group are shown in Table 1. Among them, for the light treatment group: after the drug was incubated with the cells for 4 h, it was irradiated with near-infrared light at 660 nm (20 mW / cm 2 ) for 10 min, and the incubation was continued until 24 h. Finally, 10 μL of CCK8 solution was added to each well in the 96-well plate and incubated for another 1 h. The absorbance at 450 nm was measured using a microplate reader, and the cell viability was calculated. The formula for calculating cell viability: Cell viability (%) = (OD of experimental group - OD of blank group) / (OD of control group - OD of blank group) × 100%.

[0056] Table 1 Drug administration concentrations of different groups

[0057]

[0058] The results are as Figure 6 (A) and Table 2 show that the IC 50 of Cel is above 0.7 μg / mL, and the IC 50 of Ce6-SS is above 1.8 μg / mL. However, when they are directly physically mixed together, the IC 50 decreases to 1.35 μg / mL (Ce6-SS: 0.9 μg / mL, Cel: 0.45 μg / mL), which means that a lower concentration of Ce6-SS+Cel can synergistically enhance the anti-tumor effect. Similarly, when receiving light therapy ( Figure 6 (B)), Ce6-SS+Cel+L also shows a synergistically enhanced anti-tumor effect. Compared with the single-drug treatment groups (Ce6-SS+L or Cel), the IC 50 is less than 0.9 μg / mL. Further, the anti-tumor effect of the nanoparticles CSCN prepared in the present invention is significantly superior to other groups and significantly exceeds the physical mixture of equal doses of Ce6-SS+CEL. The above results indicate that CSCN can significantly enhance the inhibitory effect on tumor proliferation. The IC 50 of CSCN+L is 0.3 μg / mL, and the IC 50 is significantly lower than other treatments, indicating that CSCN has a strong inhibitory effect on cells under near-infrared light.

[0059] Table 2 IC 50 values of 4T1 cells under different treatments

[0060]

[0061] Example 3

[0062] Study on the Uptake of Nanoparticles by 4T1 Cells

[0063] 4T1 cells in the logarithmic growth phase were seeded on confocal culture dishes at a density of 1.2×10 4 cells / ml and cultured for 24 h until they adhered. Cells were treated with Ce6-SS and CSCN prepared in Example 1 for 1 h, 2 h, and 4 h, respectively (the administration concentrations were 0.2 μg / mL and 0.3 μg / mL, respectively). Then, the cells were fixed with 4% paraformaldehyde for 20 min, washed with PBS, stained with DAPI for 30 min to stain the cell nuclei, and washed with PBS. Ce6 has its own red fluorescence. The cellular uptake of Ce6-SS and CSCN was observed by CLSM.

[0064] 4T1 cells were co-cultured with Ce6-SS and CSCN for different times to observe the cellular uptake behavior. As Figure 7 (A) shows, after 1 h of treatment, the red fluorescence in the CSCN group was significantly stronger than that in the Ce6-SS group. After continuous incubation for 4 h, the red fluorescence intensity of both groups of cells increased, and the fluorescence intensity of the CSCN group was significantly higher than that of the Ce6-SS group, mainly aggregating around the cells. A significant increase in the red fluorescence of CSCN was also observed in the semi-quantitative fluorescence results ( Figure 7 B). These results indicate that CSCN can be effectively internalized into 4T1 cells in a time-dependent manner.

[0065] Example 4

[0066] Lysosomal Escape Behavior of Nanoparticles

[0067] 4T1 cells in the logarithmic growth phase were seeded on confocal culture dishes at a density of 1.2×10 4 cells / ml and cultured for 24 h until they adhered. After co-culturing 4T1 cells with CSCN+L prepared in Example 1 for 1 h, 2 h, and 4 h (concentration: 0.3 μg / mL), they were irradiated with near-infrared light at 660 nm for 10 min (20 mW / cm 2 ), the cells were washed with PBS, stained with Lyso Tracker Green for 30 min, washed with PBS again, fixed with 4% paraformaldehyde for 20 min, and washed 3 times with PBS to evaluate the lysosomal escape. Finally, the fluorescence of 4T1 cells was observed by CLSM.

[0068] The lysosomes of 4T1 cells were stained with a lysosomal green fluorescence probe (Lyso-Tracker Green) to study the lysosomal escape behavior of nanoparticles in cells ( Figure 8)。After incubation with CSCN+L for 1 h, the red fluorescence of CSCN merged with the green of the lysosome tracer, indicating that the nanoparticles were mainly localized in cellular lysosomes. As the incubation time increased, separation of the red fluorescence of CSCN from the green fluorescence of the lysosome tracer was observed at 4 h. These results suggest that CSCN can escape from lysosomes to avoid drug degradation by lysosomes.

[0069] Example 5

[0070] Study on the cell membrane potential of nanoparticles

[0071] To determine the change in mitochondrial membrane potential (MMP, ΔΨm), 4T1 cells in the logarithmic growth phase were seeded on confocal culture dishes at a density of 1.2×10 4 cells / ml and cultured for 24 h until they adhered. The cells were treated with PBS, Cel, Ce6-SS+L, Ce6-SS+Cel+L, CSCN, and CSCN+L prepared in Example 1 for 12 h (at concentrations of 0.1 μg / mL, 0.2 μg / mL, 0.3 μg / mL, 0.3 μg / mL, and 0.3 μg / mL respectively, except for the PBS group). Treatment for the light irradiation group: After the drugs were incubated with the cells for 4 h, they were irradiated with 660 nm near-infrared light for 10 min (20 mW / cm 2 ), and incubation was continued until 12 h. Subsequently, the cells were incubated with the mitochondrial membrane potential fluorescent probe JC-1 for 30 min and washed with PBS. The fluorescence change of JC-1 was observed by CLSM to analyze the change in mitochondrial membrane potential.

[0072] JC-1 is an ideal fluorescent probe widely used to measure the mitochondrial membrane potential ΔΨm. Under normal conditions, the mitochondrial membrane potential in cells is relatively high, and JC-1 accumulates in the mitochondrial matrix and forms polymers, which can produce red fluorescence. When the mitochondria are damaged and the membrane potential decreases, JC-1 cannot aggregate in the mitochondrial matrix, and JC-1 can produce green fluorescence in the mitochondrial matrix. Therefore, the change in mitochondrial membrane potential can be detected by the fluorescence color to reflect the degree of mitochondrial damage. As Figure 9 (A) shows, for the cells treated with light irradiation (Ce6-SS+L, Ce6-SS+Cel+L, CSCN+L), the red fluorescence decreased, and when treated with CSCN+L, the red fluorescence almost disappeared, and the green fluorescence intensity of JC-1 monomers increased significantly. This indicates that after treatment with this method, the cell membrane potential of 4T1 cells decreased significantly. Fluorescence quantification ( Figure 9 B) further confirmed this result. Therefore, CSCN+L can significantly reduce the mitochondrial membrane potential of 4T1 cells and damage mitochondrial function. By inhibiting mitochondrial respiration, tumor hypoxia is alleviated, and the efficacy of PDT is enhanced.

[0073] Example 6 According to the method of Example 1, when preparing CSCN, the volume ratio of the Ce6-SS stock solution and the Cel stock solution was adjusted. The nanoparticles obtained by mixing the Ce6-SS stock solution and the Cel stock solution in a volume ratio of 1.7:1 had the most stable particle size. The significant particle size differences at different ratios are shown in Table 3 below.

[0074] Table 3

[0075]

Claims

1. A preparation method of a carrier-free nano-drug delivery system CSCN, characterized in that, It includes the following steps: (1) Preparation of Ce6-SS: Dissolve Ce6, 2-hydroxyethyl disulfide, DCC and DMAP in an organic solvent, stir, extract, dry, redissolve and purify to obtain Ce6-SS; (2) Preparation of CSCN: Dissolve Ce6-SS and Cel in an organic solvent respectively, mix the obtained solutions, sonicate, dialyze and centrifuge to obtain the nanoparticles CSCN.

2. The preparation method of the nano-drug delivery system CSCN according to claim 1, characterized in that In step (1), the molar ratio of Ce6, 2-hydroxyethyl disulfide, DCC and DMAP is preferably 0.3 - 0.6: 0.3 - 0.7: 1.5 - 2: 1.5 - 2.

0.

3. The preparation method of the nano-drug delivery system CSCN according to claim 1, characterized in that, In step (1), the stirring condition is stirring at room temperature for 8 - 12 h.

4. The preparation method of the nano-drug delivery system CSCN according to claim 1, wherein In step (1), the extractant is any one of dichloromethane, ethyl acetate, petroleum ether and methyl tert-butyl ether, and the desiccant is any one of anhydrous sodium sulfate, anhydrous magnesium sulfate and anhydrous calcium chloride.

5. The preparation method of the nano-drug delivery system CSCN according to claim 1, characterized in that, In step (2), the dissolution concentrations of Ce6-SS and Cel in the organic solvent are both 8 - 12 mg / mL.

6. The preparation method of the nano-drug delivery system CSCN according to claim 1, wherein In step (2), the volume ratio of the solutions of Ce6-SS and Cel mixed is 1.3:1 - 2.5:

1.

7. A carrier-free nano-drug delivery system CSCN prepared by the preparation method of the carrier-free nano-drug delivery system CSCN described in claim 1.

8. Use of the carrier-free nano-drug delivery system CSCN described in claim 7 in the preparation of a drug for treating breast cancer.

9. A drug for treating breast cancer, characterized in that, It includes the carrier-free nano-drug delivery system CSCN described in claim 7 and a pharmaceutically acceptable excipient or carrier.

10. A photosensitizer, characterized in that, Its structure is as follows:

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