A targeted laryngeal cancer nanoparticle based on ferroptosis and photodynamic synergistic therapy and preparation and application thereof

Abstract

The application discloses a kind of based on ferroptosis and photodynamic cooperative treatment targeted laryngeal cancer nanoparticles and its preparation method and application.The nanoparticle is with polylactic acid-glycolic acid copolymer as carrier, co-loading photosensitizer indocyanine green and ferroptosis inducer Erastin, and realizes active targeting by cRGD peptide modification.Under laser irradiation, ICG produces reactive oxygen species and kills tumor cells, and Erastin induces ferroptosis by inhibiting GPX4 pathway, and the synergistic effect significantly enhances the anti-tumor effect.The application solves the problem of PDT hypoxic restriction and drug targeting deficiency, and PDT is combined with ferroptosis through nano-drug delivery platform, and the two promote each other, showing stronger anticancer effect, with high efficiency, stability and safety, suitable for targeted therapy of laryngeal squamous cell carcinoma.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a targeted nanoparticle for physical treatment of laryngeal cancer and a preparation method thereof, in particular, a nanoparticle drug delivery system based on the synergistic effect of ferroptosis and photodynamic therapy (PDT). BACKGROUND

[0002] Cancer is a major disease that threatens human health and life. The global burden of cancer continues to increase, bringing huge physical, psychological and economic burdens to individuals, families, communities and health systems. Although a series of treatment methods for tumors, including surgical intervention, chemotherapy, radiotherapy and gene therapy, have been significantly optimized and improved in recent years, these conventional therapies still cannot fully cure many patients, especially those who are diagnosed at a late stage. Therefore, tumor recurrence and metastasis are still a serious challenge faced by the contemporary field of oncology research.

[0003] Laryngeal cancer is a common malignant tumor of the head and neck, mainly originating from abnormal proliferation of laryngeal epithelial cells. In histopathological classification, laryngeal squamous cell carcinoma (LSCC) as the most common subtype not only has a high incidence, but also shows significant clinical invasive characteristics. LSCC, as the second most common squamous epithelial-derived malignant tumor of the head and neck, is one of the important types of respiratory system malignant tumors. With the aggravation of environmental pollution and tobacco exposure and other risk factors, the incidence of this disease shows a significant upward trend.

[0004] Due to its high incidence and mortality characteristics, the molecular mechanism and treatment strategy of LSCC have become a hot field of oncology research. The current clinical intervention scheme covers various means such as chemotherapy, radiotherapy, targeted therapy, immunotherapy and multi-modal surgical operation, but the overall prognosis and quality of life of patients are still at a low level. Such clinical difficulties are mainly due to high postoperative metastasis rate and high recurrence risk, resulting in a significant decrease in 5-year survival rate of patients. With the progress of medical technology, although the diagnosis and treatment technology of LSCC has made significant progress, there is still a lack of highly specific molecular markers. Therefore, postoperative recurrence and metastatic spread are still a serious clinical challenge faced by modern oncology research and head and neck surgeons, and the biological characteristics of tumors and treatment resistance mechanisms need to be further analyzed. The discovery of new markers has crucial clinical significance for improving early diagnosis, optimizing clinical treatment strategies and improving patient survival prognosis.

[0005] Photodynamic therapy (PDT) is a new method of tumor treatment. Compared with traditional surgery, radiotherapy and chemotherapy, PDT has the advantages of minimal invasion, simple operation, less side effects and multiple treatments. PDT has shown a broad application prospect in the treatment of skin cancer, breast cancer, esophageal cancer and other tumors. According to the mechanism, PDT is divided into type I and type II. Type II is the main reaction in PDT. Oxygen is necessary for the production of reactive oxygen species (ROS) in type II PDT. Therefore, the O2 content in the tumor microenvironment directly affects the ROS yield. Type II PDT needs to be carried out in a fully oxygenated environment. However, the rapidly growing tumor cells in the larynx need to consume a large amount of O2, and the tumor site often lacks oxygen supply. In addition, O2 needs to be continuously consumed during PDT, which further exacerbates the hypoxia in the tumor cell microenvironment, becoming one of the main reasons hindering the efficacy of PDT. In addition, photosensitizers are an important part of the PDT system. Due to the poor targeting of photosensitizers, low efficiency of selective enrichment in tumor tissue, and poor water solubility, the application of PDT in the treatment of laryngeal cancer is also seriously hindered. The photosensitizer approved by the US Food and Drug Administration (FDA) for clinical use in superficial tumors (such as ALA, Ce6, and Temoporfin) has a spectral absorption range below 700 nm in the visible light region. The light in this region can only penetrate the skin to a thickness of millimeters, limiting the efficacy of PDT for deep lesions such as laryngeal cancer. On the other hand, photodynamic killing of tumor cells is mainly achieved by generating toxic ROS under appropriate light irradiation. However, the ROS produced in tumor cells may be consumed by endogenous antioxidants-glutathione (GSH), affecting the ROS content in tumor cells and further weakening the therapeutic effect of PDT.

[0006] Ferroptosis is a non-apoptotic form of programmed cell death discovered by Stockwell's team in 2012. Its core feature is iron-dependent abnormal accumulation of lipid peroxides. This mode of cell death is significantly different from other types of cell death in terms of molecular mechanisms and cell morphology. Its occurrence is accompanied by characteristic morphological changes, including cell volume reduction, plasma membrane integrity destruction, and abnormal chromatin condensation. Although traditional treatment methods such as chemotherapy, radiotherapy, and immunotherapy mainly induce apoptosis to exert anti-tumor effects, tumor cells can escape apoptosis regulation through acquired drug resistance mechanisms or treatment resistance, ultimately leading to treatment failure. Based on the non-apoptotic characteristics of the ferroptosis pathway, targeting the ferroptosis pathway can effectively avoid the above treatment defects. In addition, tumor cells with apoptosis resistance phenotype are significantly susceptible to the ferroptosis pathway, which provides a new direction for breaking through the bottleneck of tumor treatment resistance.

[0007] Although photodynamic and ferroptosis therapy as a novel cancer therapy has shown significant potential for tumor treatment, there are still limitations that restrict their anticancer efficacy. First, photodynamic therapy relies on sufficient oxygen resources, but the lack of oxygen supply in the hypoxic tumor microenvironment seriously hinders the efficacy of PDT. In addition, the excessive expression of antioxidant glutathione (GSH) in malignant tumors can consume a large amount of reactive oxygen species (ROS), weakening the therapeutic effect of PDT. The effective initiation of ferroptosis therapy requires a high concentration of inducers, which not only poses a significant threat to the safety of normal tissues, but also may cause off-target toxicity due to the non-specific biological diffusion and drug efficacy persistence of some ferroptosis inducers. To overcome the limitations of current single therapy, combined therapy strategies of various ways have attracted increasing attention. This model can significantly improve the efficacy while minimizing damage to healthy cells or tissues by integrating the advantages of various anticancer treatments. Currently, the combination of ferroptosis therapy with chemotherapy, radiotherapy, and immunotherapy has shown significant therapeutic advantages, but the above combined antitumor therapies have many side effects, limiting their application in clinical work: such as activated immune cells in immunotherapy, which may damage normal cells and cause temporary autoimmune diseases; chemotherapy can cause damage to normal tissues throughout the body, manifested as alopecia, bone marrow suppression, and gastrointestinal reactions; and radiotherapy can cause skin ulcers in the irradiated area. Compared with the above combined therapies, the synergistic effect of photodynamic and ferroptosis provides another way for anticancer therapy. The ferroptosis process can convert the excess hydrogen peroxide (H2O2) produced by cancer cells into oxygen, thereby improving the hypoxic microenvironment of photodynamic therapy. In addition, the reactive oxygen species (ROS) produced by photodynamic therapy can effectively promote ferroptosis, and the local high heat induced by PDT enhances the permeability of the cancer cell membrane, thereby improving the cellular uptake efficiency of therapeutic drugs, further accelerating the Fenton reaction in cells, leading to the generation of excess reactive oxygen species to promote cell ferroptosis. The application of this therapy in multimodal combination therapy significantly enhances the synergistic effect of photodynamic-ferroptosis therapy, further promotes more efficient synergistic therapeutic effects, and significantly improves the lethality of cancer cells. In addition, combined with the specific binding properties of cRGD peptide and integrin αvβ3 on the surface of laryngeal cancer cells, we successfully coupled exogenous cRGD peptide with nanoparticles to construct a targeted nanodrug, which can significantly improve the active targeting of nanodrugs. Combined with the passive targeting of nanoparticles, the effector molecules can be accurately introduced into the tumor site, effectively exerting the antitumor effect and reducing the damage to normal tissues and cells during treatment.

[0008] Iron death inducer such as Erastin can regulate iron metabolism by inhibiting cystine / glutamate antiporter (System xc-), induce lipid peroxide accumulation, and selectively kill tumor cells. The high expression of SLC7A11 protein (a key component of System xc-) in laryngeal squamous cell carcinoma makes tumor cells more sensitive to iron death induction. However, single iron death treatment has problems such as insufficient efficiency and systemic toxicity. Indocyanine green (ICG) is the only near-infrared fluorescent dye approved by FDA, which has an absorption characteristic of 795-845 nm, suitable for deep tissue treatment. However, the instability of ICG in physiological environment and the extremely short half-life limit its application in the field of clinical photodynamic therapy. Therefore, constructing a transport carrier that can increase the stability of ICG, effectively target tumor cells and has a slow-release function is the key to solving this problem. Based on this, the application provides a targeted nanoparticle based on iron death and ICG, using PLGA as a nano-carrier, loading photosensitizer ICG and iron death inducer Erastin which has good absorption in the near-infrared region, and modifying the nano-complex with cRGD peptide, finally constructing cRGD / PLGA@ICG&Era nanoparticles. On the PLGA nano-platform, we integrate iron death and photodynamic therapy together, which better plays the role of combined therapy and synergistic anti-tumor therapy on the same nano-platform, and lays a foundation for realizing its multi-modal combined targeted physical therapy in vivo. SUMMARY

[0009] The purpose of the application is a preparation and application of a targeted laryngeal cancer nanoparticle based on iron death and photodynamic synergistic therapy.

[0010] The preparation method of the targeted laryngeal cancer nanoparticle based on iron death and photodynamic synergistic therapy provided by the application comprises the following steps:

[0011] (1) Dissolve polylactic acid-glycolic acid copolymer (PLGA), DLinDMA, maleimide-polyethylene glycol-phospholipid (DSPE-PEG-Mal) and iron death inducer Erastin in chloroform, and rotary evaporate at 40-50 DEG C until the solvent is completely volatilized;

[0012] The mass ratio of PLGA, DLinDMA, DSPE-PEG-Mal and Erastin is (4-6):(0.8-1.2):(3-5):(1.5-2.5), preferably 5:1:4:2.

[0013] (2) Add indocyanine green (ICG) solution to the rotary evaporation product, and place it at 35-40 DEG C, and ultrasonic treat for 5-10 min to form PLGA@ICG&Era nanoparticles;

[0014] The mass ratio of ICG to PLGA is 1: (8-12), preferably 1:10. The ultrasonic power is 120-140 W, and the frequency is 15-25 kHz.

[0015] (3) The cRGD-SH is added to the PLGA@ICG&Era nanoparticles and mixed uniformly, and then dialysis purification is carried out after standing at room temperature for 1-2 h, to obtain cRGD / PLGA@ICG&Era nanoparticles.

[0016] The mass ratio of cRGD-SH to ICG is 1: (400-600), preferably 1:500; the concentration of the indocyanine green (ICG) solution is 0.05-0.15 mg / mL. The dialysis bag has a molecular weight cut-off of 3500 Da.

[0017] The nanoparticles prepared by the above method are loaded with the iron death inducer Erastin and the photosensitizer indocyanine green (ICG) by the polylactic acid-glycolic acid copolymer (PLGA) carrier, and are modified by the cRGD peptide to realize active targeting. The nanoparticles produce a synergistic anti-tumor effect under 808 nm laser irradiation through Erastin-induced ferroptosis and ICG-mediated photodynamic therapy. The particle size of the nanoparticles is 80-100 nm, the ICG encapsulation rate is ≥94%, and the Erastin encapsulation rate is ≥89%.

[0018] The application provides an application of the nanoparticles in the preparation of a laryngeal cancer targeted treatment drug. The nanoparticles can induce an intracellular reactive oxygen species (ROS) burst through a nano photosensitizer under 808 nm laser irradiation, and can synergistically inhibit the expression of GPX4 and SLC7A11 proteins and the activity of FSP-1 protein, effectively promote ferroptosis, and convert hydrogen peroxide (H2O2) generated in excess by cancer cells into O2, thereby improving the tumor hypoxic microenvironment of photodynamic therapy and further enhancing the photodynamic effect. Therefore, ferroptosis and photodynamic therapy are mutually synergistic and promote each other, and a more efficient killing effect on tumor cells is realized.

[0019] Compared with the prior art, the application has the following beneficial effects:

[0020] 1. Synergistic treatment mechanism

[0021] By simultaneously loading the iron death inducer (Erastin) and the photosensitizer (ICG) by the PLGA nano carrier, under 808 nm laser irradiation, ICG produces reactive oxygen species (ROS) to destroy the cell redox balance, and Erastin induces ferroptosis by inhibiting the protein expression of GPX4 and SLC7A11, and the mutual synergistic effect significantly enhances the killing effect of laryngeal cancer TU686 cells.

[0022] The synergistic effect is as follows:

[0023] Oxygen self-supply effect: During the ferroptosis process induced by Erastin, Fenton reaction generates O2, which alleviates the hypoxic microenvironment of tumors and enhances the efficacy of PDT (solving the problem of limited PDT due to hypoxia). ROS multiplication effect: The ROS generated by ICG and the lipid peroxides accumulated by ferroptosis form an oxidative stress storm, significantly improving the efficiency of killing tumor cells and reducing the survival rate of tumor cells to 18.3%, far exceeding the effect of single therapy (experimental data show that Figures 10-15 ), the ROS level, LDH release amount and apoptosis rate of the combination therapy group are significantly higher than those of the single therapy group, confirming the synergistic effect).

[0024] Overcoming drug resistance of traditional treatment: Laryngeal cancer cells are prone to develop resistance to the apoptosis pathway, while ferroptosis as a non-apoptotic death mode can effectively avoid this problem. The physical killing mechanism of PDT further reduces the risk of drug resistance, providing a new strategy for recurrent or refractory laryngeal cancer.

[0025] 2. Optimization of targeting and stability

[0026] Active targeting design: By modifying nanoparticles with cRGD peptide (cRGD / PLGA@ICG&Era), the effect molecule can be specifically introduced into the tumor site by specifically binding to the high expression of ανβ3 integrin receptor on laryngeal cancer TU686 cells, significantly improving drug enrichment in the tumor site (laser confocal microscopy shows that the ICG fluorescence signal of the targeted group is enhanced, Figure 9 ) effectively plays a tumor inhibition role, and reduces damage to normal tissues and cells during treatment.

[0027] Advantages of nanocarriers:

[0028] High encapsulation efficiency and stability: PLGA carriers achieve high loading of ICG (encapsulation efficiency ≥ 94%) and Erastin (≥ 89%), solving the problem of short half-life of ICG. Dynamic light scattering (DLS) shows that the particle size of nanoparticles does not change significantly within 7 days ( Figure 7 ). By adjusting the ratio of PLGA / DLinDMA / DSPE-PEG-Mal, monodisperse nanoparticles (PDI ≈ 0.3) are obtained, and the Zeta potential changes from negative to positive ( Figure 3 ), enhancing the efficiency of cell uptake.

[0029] 3. Deep treatment and safety improvement

[0030] Near-infrared light penetration: The absorption peak of ICG is located in the near-infrared region of 795-845 nm, and 808 nm laser can penetrate deep tissues, overcoming the limitation of traditional photosensitizers (such as ALA) which are only suitable for superficial tumors.

[0031] Reducing toxic side effects: successful coupling of exogenous cRGD peptide with nanoparticles can significantly improve the active targeting of nanomedicines. Combined with the passive targeting of nanoparticles, the effector molecules can be accurately introduced into the tumor site, effectively exerting antitumor effects and reducing damage to normal tissues and cells during treatment.

[0032] 4. Multimodal treatment and diagnosis potential integration of diagnosis and treatment

[0033] The near-infrared fluorescence properties of ICG can be used for real-time imaging to locate tumors, and as a therapeutic agent to achieve "visual treatment". The molecular mechanism is clear, and by inhibiting GPX4 and SCL7A11 protein expression ( Figure 14 ), and PDT synergistic ferroptosis increases lactate dehydrogenase release ( Figure 13 ), etc. indicators, further verify the synergistic pathway of ferroptosis and PDT, and lay a theoretical foundation for clinical translation. BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 is a transmission electron micrograph of cRGD / PLGA@ICG&Era nanoparticles;

[0035] Figure 2 is a particle size analysis diagram of cRGD / PLGA@ICG&Era nanoparticles;

[0036] Figure 3 is a Zeta potential diagram of cRGD / PLGA@ICG&Era nanoparticles;

[0037] Figure 4 is a UV-visible spectrum diagram of cRGD / PLGA@ICG&Era nanoparticles;

[0038] Figure 5 is a UV standard curve diagram of ICG in cRGD / PLGA@ICG&Era nanoparticles;

[0039] Figure 6 is a UV standard curve diagram of Erastin in cRGD / PLGA@ICG&Era nanoparticles;

[0040] Figure 7 is a particle size stability test diagram of cRGD / PLGA@ICG&Era nanoparticles;

[0041] Figure 8 is a TU686 cell culture diagram;

[0042] Figure 9 is a cell uptake analysis diagram of cRGD / PLGA@ICG&Era nanoparticles under laser confocal microscope;

[0043] Figure 10 Figure for the killing effect of cRGD / PLGA@ICG&Era nanoparticles on laryngeal cancer cells at different concentration gradients;

[0044] Figure 11 Figure for the ICG / Era concentration value of different drug groups to achieve IC50;

[0045] Figure 12 Figure for the ROS production of different treatment groups in laryngeal cancer cells observed by laser confocal microscope;

[0046] Figure 13 Figure for the release activity of lactate dehydrogenase in laryngeal cancer cells;

[0047] Figure 14 Figure for the expression of iron death related proteins GPX4 and SCL7A11 detected by Western blot;

[0048] Figure 15 Figure for the apoptosis of TU686 detected by Annexin-V / PI double staining method. DETAILED DESCRIPTION

[0049] The application will be further described below through specific embodiments.

[0050] Example 1, preparation of GD / PLGA@ICG&Era nanoparticles:

[0051] (1) Accurately weigh 5 mg of PLGA, 1 mg of DLinDMA, 4 mg of DSPE-PEG-Mal (2000 da) and 2 mg of Erastin in turn, and dissolve them in 2 mL of chloroform. Then, transfer the solution to a chicken heart bottle and perform rotary evaporation at 45°C until the solvent is completely volatilized. The obtained product is ready for use;

[0052] (2) Add 5 mL of ICG (indocyanine green) solution with a concentration of 0.1 mg / mL to the product after rotary evaporation, and place it in a 37°C environment. Use an ultrasonic instrument to ultrasonically treat the mixture at a frequency of 20 kHz and a power of 130 W for 8 minutes, thereby obtaining PLGA@ICG&Era nanoparticles;

[0053] (3) To the above PLGA@ICG&Era nanoparticle solution, 1 μg of thiol-modified cRGD peptide (thiol-cRGD) was added and mixed uniformly by stirring or shaking, etc. Then it was placed at room temperature for 1 h. After that, the solution was dialyzed using a dialysis bag with a molecular weight cutoff of 3500 Da to remove the free cRGD peptide. Finally, a dark green cRGD / PLGA@ICG&Era aqueous solution was obtained, which was stored at 4°C in the dark for later use.

[0054] The structural formula of Erastin is:

[0055]

[0056] The structural formula of cRGD-SH is:

[0057]

[0058] The structural formula of indocyanine green (ICG) is:

[0059]

[0060] Example II. Structural characterization and identification of cRGD / PLGA@ICG&Era nanoparticles

[0061] 1. Surface morphology characterization of nanoparticles

[0062] Transmission electron microscopy (TEM, model: JEM F-200) was used to characterize the size and morphology of cRGD / PLGA@ICG&Era nanoparticles. Through TEM, we can directly observe the size and morphology of the synthesized cRGD / PLGA@ICG&Era nanoparticles. Figure 1 is the transmission electron micrograph of cRGD / PLGA@ICG&Era. It can be seen that the cRGD / PLGA@ICG&Era nanoparticles are round or spherical in shape, showing a monodisperse state, regular morphology, uniform size, and a particle size distribution of about 90 nm.

[0063] 2. Particle size distribution and Zeta potential

[0064] PLGA@ICG&Era and cRGD / PLGA@ICG&Era nanoparticles were measured by dynamic light scattering (DLS) method on Malvern Zetasizer Nano ZS. The average size and polydispersity index (PDI) of the nanoparticles were analyzed. The Zeta potential of the nanoparticles was also measured on Malvern Zetasizer. After opening the test software Zetasizer Software, the test parameters were set, and the potential test of the nanoparticles was carried out.

[0065] The particle size of the nanoparticles in aqueous solution was further measured by dynamic light scattering (DLS), as shown in Figure 2 The particle size of the aqueous dispersion phase of PLGA@ICG&Era and cRGD / PLGA@ICG&Era nanoparticles was 56 nm (PDI: 0.3) and 88±5.20 nm (PDI: 0.356), respectively, and the polydispersity index (PDI) was 0.3 and 0.356, respectively, as shown in Figure 3 The potential value of PLGA@ICG&Era was negative, and after coupling with the targeting group cRGD, the potential of cRGD / PLGA@ICG&Era became positive. The change of Zeta potential was due to the positive charge of cRGD offsetting part of the negative charge of PIE.

[0066] 3. Ultraviolet-visible spectroscopy of nanoparticles

[0067] Different concentrations of ICG solution, Erastin solution and nanoparticle solution were detected by ultraviolet spectrophotometer (wavelength range: 200-1000 nm). The results were recorded by scanning with ultraviolet spectrophotometer (model: UV-2700).

[0068] As shown in Figure 4 , the characteristic peaks of free Era drug molecules and ICG in aqueous solution were at 295 nm and 795 nm, respectively, while the synthesized nanoparticles cRGD / PLGA@ICG&Era had obvious Era and ICG absorption peaks in the range of 295 nm and 795 nm, which exactly matched the characteristic peaks of Era and ICG.

[0069] 4. Determination of encapsulation efficiency and drug loading of particles

[0070] The ultraviolet absorbance and corresponding solution concentration were recorded by ultraviolet spectrophotometer, and the standard curve of ICG solution and Erastin solution was drawn Figure 5 , 6). The absorbance of ICG and Erastin in the nanoparticles was substituted into the standard curve, respectively, to calculate the encapsulation efficiency and drug loading of ICG and Erastin.

[0071] The drug loading of Era and ICG in cRGD / PLGA@ICG&Era was 14.2%, 3.8%, respectively, and the encapsulation efficiency was 89%, 94%, respectively, as determined by the UV standard curve method.

[0072] 5. In vitro stability detection of GD / PLGA@ICG&Era nanoparticles

[0073] The particle size change of the nanoparticles cultured in vitro for 1 week was detected by DLS method on a Malvern nanoparticle size analyzer. The nanoparticles were placed in an aqueous solution (room temperature 25°C), and the particle size was detected by DLS after 1, 3, 5, and 7 days, respectively, and no significant change was found (P > 0.05), indicating that the drug-loaded nanoparticle platform constructed in this study had good stability. Figure 7

[0074] Example Three, in vitro anti-tumor activity of GD / PLGA@ICG&Era nanoparticles

[0075] 1. Culture

[0076] The TU686 cell strain was placed in a 37°C, 5% CO2 cell incubator and cultured with DMEM medium containing 10% FBS and 1% penicillin-streptomycin antibiotics. The cell growth was observed under an optical microscope, and when the cell density reached 80-90%, the subculture operation was performed. Figure 8

[0077] 2. Cell uptake experiment

[0078] Laser confocal microscope detection of cRGD / PLGA@ICG&Era nanoparticle cell uptake: under the laser confocal microscope, the uptake of ICG / Era mixed solution, PLGA@ICG&Era and cRGD / PLGA@ICG&Era nanoparticles was observed after 4 hours of incubation, and the distribution in the cells was recorded by taking pictures.

[0079] The laser confocal fluorescence image showed that (P < 0.05) Figure 9 The nucleus was stained blue with Hochest, and the red fluorescence signal of ICG could be clearly observed in the cytoplasm. Compared with the ICG / Era mixed drug group and the PLGA@ICG&Era nanoparticle drug group, the cRGD / PLGA@ICG&Era with surface modification of cRGD showed obvious targeting ability to TU686 cells, with obvious increase and uniform distribution of ICG signal in the cytoplasm. ​​

[0080] 3. Cell viability and nanoparticle toxicity assay

[0081] The cell survival rate of different concentrations of cRGD / PLGA@ICG&Era nanoparticles (0, 4, 7, 15, 30, 55 μg / mL) was evaluated by CCK-8 experiment method (cell survival rate (%) = experimental group absorbance value / control group absorbance value x 100%).

[0082] We co-incubated TU686 cells with different concentrations of cRGD / PLGA@ICG&Era nanoparticles, and evaluated the effect of cRGD / PLGA@ICG&Era on the cell viability of cancer cells by CCK-8 assay kit ( Figure 10 ). Under non-laser irradiation conditions, low concentrations of cRGD / PLGA@ICG&Era had little effect on laryngeal cancer TU686 cells; as the concentration of RPIEs gradually increased, the survival rate of TU686 cells gradually decreased, and when the concentration of RPIEs reached 55㎍ / ml, the activity of laryngeal cancer TU686 cells under non-laser irradiation conditions decreased to 66.9%, and after irradiation with 808nm laser (1W / cm 2 ) for 5min, the activity of laryngeal cancer TU686 cells was as low as 18.3%.

[0083] In addition, we co-incubated TU686 cells with different drug groups (simple ICG group, simple Erastin group, ICG / Era mixed group, RPIE nanoparticle group), and evaluated the effect of each group on the cell viability of cancer cells by CCK-8 assay kit ( Figure 11 ). As shown in Figure 6, the cell survival rate of the simple ICG group was 100%, the cell survival rate of the simple Erastin group was 62.3%, the cell survival rate of the ICG / Era mixed group was 73.6%, and the cell survival rate of the RPIE nanoparticle group was 66.9%. Figure 11As shown, at higher drug concentrations, free ICG and Erastin only caused limited cell viability decrease, with IC50 values of 5.73 μg / mL and 8.56 μg / mL, respectively. Compared with the free drug group, the ICG / Era mixed group (808 nm, 1 W / cm2, 5 min) and the RPIE nanoparticle group (808 nm, 1 W / cm2, 5 min) showed significantly enhanced cytotoxicity to TU686 cells, with IC50 values of ICG 2.74 μg / mL / Era 5.05 μg / mL and ICG 0.48 μg / mL / Era 1.81 μg / mL, respectively. This indicates that the combined use of ICG and Era can synergistically inhibit the proliferation of TU686 cells. Notably, the RPIE nanoparticle group showed strong cytotoxicity at very low concentrations, with tumor cell killing efficiency significantly stronger than that of the ICG / Era physical mixed group. This effect is mainly due to the active targeting ability of the cRGD peptide on the surface of the RPIE nanoparticles to TU686 cells, as well as the further enhancement of the solubility of the nanomedicine by the PLGA nanoparticle, which forms a higher local drug concentration around the tumor cells, thereby significantly inhibiting the proliferation of TU686 cells.

[0084] 4. Intracellular reactive oxygen species (ROS) assay

[0085] Reactive oxygen species (ROS) mainly include reactive superoxide anion (O2· - ), hydroxyl radical (·OH) and singlet oxygen ( 1 O2), which play an important role as signal molecules and regulatory molecules at physiological levels, but when the concentration is elevated to abnormal levels, they can cause damage to cells. DCFH-DA, as a classic ROS fluorescent probe, does not itself produce fluorescence, but after being taken up by cells, it can be hydrolyzed to form DCFH by intracellular esterase. DCFH, on the one hand, cannot penetrate the cell membrane, and on the other hand, can be oxidized to form DCF with green fluorescence in the presence of ROS, so that the generation level of intracellular ROS can be judged by detecting the strength of DCF green fluorescence. We detected the content of reactive oxygen species in each group of cells by DCFH-DA probe, observed the production of ROS in cells by laser confocal microscope, and took the results.

[0086] As shown in Figure 12 : The cRGD / PLGA@ICG&Era laser treatment group (808 nm, 1 W cm 2, 5 min) showed significantly stronger ROS fluorescence intensity compared with other groups. This phenomenon revealed that cRGD / PLGA@ICG&Era nanoparticles could significantly improve the generation of intracellular ROS, thereby promoting cell death caused by the disruption of cellular redox balance. This fully demonstrated the therapeutic effect of the combination of photodynamic and ferroptosis through the PLGA nanoparticle platform in vitro and the mutual promotion of each other.

[0087] 5. Lactate dehydrogenase release experiment

[0088] The release amount of lactate dehydrogenase, an indirect metabolite of ferroptosis of cRGD / PLGA@ICG&Era nanoparticles, was determined by a lactate dehydrogenase detection kit. As shown in Figure 13 compared with other positive control groups, the release amount of lactate dehydrogenase in the light irradiation group of cRGD / PLGA@ICG&Era nanoparticles reached more than 80%, which was significantly higher than that in other groups.

[0089] 6. Detection of GPX4 and SLC7A11 protein expression

[0090] The expression changes of SLC7A11 in Erastin-induced ferroptosis in laryngeal cancer TU686 cells were detected by Western blot experiment, and the effects of ICG, Erastin and cRGD / PLGA@ICG&Era nanoparticles on the expression levels of SLC7A11 and GPX4 in TU686 cells without treatment were evaluated, to confirm the role of ferroptosis in cRGD / PLGA@ICG&Era-induced cell death and the expression regulation of the two proteins by nano-ferroptosis inducers. The Western blot results showed that Figure 14 compared with the control group, the expression of SLC7A11 protein in the free Erastin group was slightly down-regulated. In contrast, the expression of SLC7A11 in the cRGD / PLGA@ICG&Era nanoparticle group was significantly down-regulated. In the free Erastin group, the laser irradiation group and the nanoparticle group without laser irradiation, the expression of GPX4 was significantly inhibited. In addition, compared with the free Erastin group, the expression level of GPX4 in the cRGD / PLGA@ICG&Era nanoparticle group was significantly decreased. These data showed that cRGD / PLGA@ICG&Era nanoparticle drugs promoted the regulation of protein expression in the process of inducing cell ferroptosis and could enhance the sensitivity of tumor cells to ferroptosis.

[0091] 7. Apoptosis experiment

[0092] Annexin-V / PI double staining is a commonly used method for detecting cell apoptosis. This study investigated the effect of cRGD / PLGA@ICG&Era on apoptosis in TU686 tumor cells using Annexin-V / PI double staining. Figure 15 As shown, the percentages of apoptotic cells (calculated as the sum of early and late apoptosis) in the ICG-only group, Erastin-only group, ICG / Era physical mixture group, and cRGD / PLGA@ICG&Era group were 52.6%, 44.4%, 66.2%, and 91.1%, respectively. Compared with the other groups, the TU686 cell apoptosis rate was highest in the cRGD / PLGA@ICG&Era laser treatment group, which is consistent with the above-mentioned cytotoxicity CCK8 assay results. These results indicate that RPIE nanoparticle-mediated ferroptosis induction combined with PDT can significantly enhance the phototoxicity of tumor cells, promote apoptosis, and thus enhance the anti-tumor effect of photodynamic therapy.

Claims

1. A method for preparing a targeted laryngeal cancer nanoparticle based on ferroptosis and photodynamic synergistic therapy, characterized in that, Comprising the following steps: (1) Dissolve polylactic acid-glycolic acid copolymer (PLGA), DLinDMA, maleimide-polyethylene glycol-phospholipid DSPE-PEG-Mal and ferroptosis inducer Erastin in chloroform, and rotary evaporate at 40-50°C until the solvent is completely volatilized; (2) Add indocyanine green (ICG) solution to the rotary evaporation product, and place it at 35-40°C, and ultrasonically treat for 5-10 min to form PLGA@ICG&Era nanoparticles; (3) Add cRGD-SH to the PLGA@ICG&Era nanoparticles and mix well, and then dialyze and purify after standing at room temperature for 1-2 h to obtain cRGD / PLGA@ICG&Era nanoparticles.

2. The production method according to claim 1, characterized by, The mass ratio of PLGA, DLinDMA, DSPE-PEG-Mal and Erastin in step (1) is (4-6):(0.8-1.2):(3-5):(1.5-2.5).

3. The production method according to claim 1, characterized by, The mass ratio of ICG to PLGA in step (2) is 1:(8-12).

4. The method of claim 1, wherein, The ultrasonic power in step (2) is 120-140 W, and the frequency is 15-25 kHz.

5. The preparation method according to claim 1, characterized in that, The mass ratio of cRGD-SH to ICG in step (3) is 1:(400-600).

6. The method of claim 1, wherein, The molecular weight cut-off of the dialysis bag in step (3) is 3500 Da.

7. Nanoparticles prepared according to the method of any one of claims 1 to 6, characterized in that, The polylactic acid-glycolic acid copolymer (PLGA) carrier simultaneously loads the ferroptosis inducer Erastin and the photosensitizer indocyanine green (ICG), and is modified by cRGD peptide to achieve active targeting. The nanoparticles produce a synergistic anti-tumor effect under 808 nm laser irradiation through Erastin-induced ferroptosis and ICG-mediated photodynamic therapy.

8. The nanoparticle of claim 7, wherein, The nanoparticle has a particle size of 80-100 nm, an ICG encapsulation efficiency of ≥94%, and an Erastin encapsulation efficiency of ≥89%.

9. The nanoparticles of claim 7 for use in the preparation of a laryngeal cancer targeted therapy drug.

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