Preparation method and application of light-operated mitochondrial autophagy nano inducer based on thermosensitive liposome

Abstract

The invention discloses a preparation method and application of a light-operated mitochondrial autophagy nano inducer based on thermosensitive liposome, and the preparation method comprises the following steps: (1) dissolving ICG and TPP-Ser in DMSO, carrying out ultrasonic treatment, dropwise adding into ultrapure water, carrying out room temperature stirring reaction, and carrying out centrifugal collection and washing to obtain TSI; (2) dissolving DPPC, DSPC and DSPE-PEG2000-iRGD in a chloroform-methanol mixed solvent, and carrying out rotary evaporation to remove the solvent so as to form a lipid membrane; and (3) adding an aqueous solution of TSI into the lipid membrane, and carrying out hydration and ultrasonic treatment to obtain a suspension. The light-operated mitochondrial autophagy nano inducer prepared by the invention is applied to precise treatment of cancers, and drug release and mitochondrial autophagy regulation are realized through near-infrared laser irradiation. The preparation method has the advantages of strong space-time controllability, high tumor targeting property, multi-mechanism synergistic anti-tumor effect and diagnosis and treatment integration capability, stable preparation process and convenience in clinical transformation.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of nanodrug delivery system, and particularly relates to a preparation method and application of a light-controlled mitochondrial autophagy nano-inducer based on thermosensitive liposomes. BACKGROUND

[0002] Mitochondrial autophagy, as a key process in cellular homeostasis and metabolic regulation, plays a complex dual role in tumor occurrence and development. On the one hand, moderate mitochondrial autophagy can timely remove damaged or dysfunctional mitochondria, maintain energy metabolism balance and redox stability in cells, thereby supporting cell survival and inhibiting malignant transformation; on the other hand, under continuous severe stress conditions, excessive mitochondrial damage can significantly induce mitochondrial autophagy level to rise, break through the threshold of cell self-protection, and finally irreversibly initiate the apoptosis program. The controllability of this dynamic balance provides a new strategy for tumor treatment, and by specifically enhancing the mitochondrial autophagy level in tumor cells, the transformation from pro-survival to pro-death can be promoted, thereby achieving the goal of selectively inhibiting tumor proliferation. Therefore, developing an intervention means capable of precisely regulating mitochondrial autophagy has become an important direction in the field of cancer treatment.

[0003] In the prior art, triphenylphosphine (TPP) is a classic and efficient mitochondrial targeting molecule, which can utilize the negative membrane potential of up to-180 mV on the inner side of the mitochondrial membrane to drive the active enrichment of the coupled therapeutic drugs in the mitochondrial matrix, thereby providing a basis for realizing precise delivery at the subcellular organelle level. At the same time, indocyanine green (ICG) as a near-infrared fluorescent dye approved by the US FDA for clinical application not only has excellent optical imaging capability, but also can realize real-time and non-invasive tumor positioning and tracking, and also shows good photothermal conversion efficiency (PTCE) and photodynamic effect (PDT), which makes it play an important role in photothermal therapy and photodynamic killing of tumors. The application of these components in tumor treatment lays a material foundation for developing new treatment strategies.

[0004] In order to effectively deliver the above components and realize on-demand release, the design of the carrier system is particularly crucial. Liposomes, especially thermosensitive liposomes, are a kind of drug delivery platform with great application prospect. Under mild heat stimulation slightly higher than the physiological temperature, the phospholipid bilayer structure of this kind of carrier can rapidly change from gel state to liquid crystal state, resulting in a sharp increase in membrane permeability, thereby realizing the rapid and local release of the encapsulated drugs. This property, combined with external photothermal stimulation, can construct a new type of nanodrug delivery system, providing a powerful tool for realizing precise treatment in the tumor microenvironment. In the prior art, a common strategy is to mix the mitochondrial targeting drug and ICG by simple physical mixing or co-encapsulate them in conventional liposomes. This way, to some extent, utilizes the encapsulation ability of liposomes to realize the preliminary protection and delivery of drugs in vivo.

[0005] For example, in some existing solutions, researchers co-load TPP-modified drug molecules and ICG in liposome carriers, aiming to combine mitochondrial targeting and photothermal effects for tumor treatment. This integrated application attempts to leverage the advantages of each component, such as the targeting drive of TPP, the photothermal conversion of ICG, and the delivery function of liposomes, to enhance treatment effectiveness. Specifically, TPP can guide drugs into mitochondria, interfere with their function, and induce autophagy; ICG generates a thermal effect under near-infrared light irradiation, which is used to directly kill tumor cells; and liposomes serve as carriers, providing drug encapsulation and preliminary tumor enrichment. These solutions show certain anti-tumor activity in in vitro experiments and achieve tumor monitoring through fluorescence imaging.

[0006] However, despite the unique advantages of mitochondrial-targeted drugs, ICG, and liposomes in tumor treatment, existing technical solutions still have a series of obvious defects in integrated applications. The current common strategy is to co-load mitochondrial-targeted drugs and ICG in conventional liposomes through simple physical mixing or co-loading, which lacks controlled release capability, leading to premature leakage of drugs during circulation, causing systemic toxicity and off-target effects. Specifically, in the blood circulation, conventional liposomes cannot effectively prevent drug molecules from escaping from the carrier, which not only reduces the effective concentration of drugs at the tumor site, but also can cause non-specific distribution of drugs in normal tissues, thereby causing systemic side effects, such as damage to healthy cell mitochondria.

[0007] On the other hand, ordinary liposomes mainly rely on the enhanced permeability and retention (EPR) effect to achieve passive targeting of tumors, which has limited targeting efficiency, significant individual differences, and slow and incomplete drug release, making it difficult to achieve effective intratumoral drug exposure. The EPR effect relies on the abnormal structure of tumor blood vessels, but there are significant differences in vascular permeability among different patients or tumor types, resulting in low reliability of targeted enrichment. In addition, the release process lacks external regulation, and the release rate and timing cannot be adjusted according to treatment needs, which further limits the optimization of therapeutic efficacy.

[0008] More fundamentally, existing research has largely failed to achieve the organic synergy of photothermal therapy and drug controlled release and its downstream biological effects. ICG, as a highly efficient photothermal agent, its thermal effect is usually used to directly kill tumor cells, and has not been systematically used to trigger intelligent drug release behavior, failing to fully exploit its multifunctional integration potential. For example, in some solutions, the photothermal effect of ICG is independent of drug release and only serves as an auxiliary killing means, which results in a lack of linkage between photothermal and autophagy induction mechanisms, and fails to form a synergistic amplification effect, thereby limiting the improvement of overall anti-tumor effect.

[0009] In addition, ICG itself is prone to aggregation and fluorescence quenching in aqueous environments, which seriously affects its optical stability and treatment reliability. In the carrier system, if ICG is not properly pre-assembled or protected, its aggregation tendency will reduce the photothermal conversion efficiency and weaken the accuracy of fluorescence imaging, which may lead to inaccurate tumor positioning or unstable treatment response in clinical applications.

[0010] The mitochondrial autophagy regulation strategy also has important limitations: existing methods mostly rely on irreversible autophagy inhibition or static "on / off" induction, lacking temporal and spatial precision and dynamic adjustability. This "all-or-nothing" regulation mode is difficult to adapt to the high heterogeneity of tumor cells, and cannot achieve fine control of mitochondrial autophagy levels, resulting in a narrow treatment window. Insufficient dosage cannot break through protective autophagy, leading to treatment failure; excessive dosage damages normal tissues, with significant side effects, which seriously restricts treatment safety and clinical translation potential. Specifically, existing autophagy inducers often act on mitochondria through a single mechanism, such as directly inhibiting the respiratory chain or inducing oxidative stress, but these effects cannot be adjusted according to real-time changes in the tumor microenvironment, resulting in poor applicability in different tumor stages or types. At the same time, due to the lack of external triggering mechanism, the autophagy induction process is difficult to activate at a specific time and location, which further increases the off-target risk.

[0011] Overall, existing nanodrug delivery systems have not effectively addressed the issues of drug leakage, low targeting efficiency, uncontrollable release, and insufficient multi-mechanism synergy when integrating mitochondrial targeting, photothermal effect, and autophagy regulation. These defects make the treatment strategy difficult to meet clinical needs in terms of precision and safety, limiting the progress from the laboratory to the clinic. To overcome the above problems, a new nanodrug delivery system that can induce mitochondrial autophagy in a spatiotemporal controllable manner, integrate multiple treatment mechanisms, and achieve synergistic effect is needed to improve the anti-tumor effect and improve treatment safety. SUMMARY

[0012] The present application aims to overcome the defects of the prior art and provide a preparation method of a light-controlled mitochondrial autophagy nanoinductor based on thermosensitive liposomes.

[0013] Another object of the present application is to provide the application of the light-controlled mitochondrial autophagy nanoinductor prepared by the above preparation method.

[0014] The technical solution of the present application is as follows:

[0015] A preparation method of a light-controlled mitochondrial autophagy nanoinductor based on thermosensitive liposomes, comprising the following steps:

[0016] (1) Dissolve ICG and TPP-Ser in DMSO, after ultrasonic treatment, drop them into ultrapure water, stir at room temperature, then collect and wash by centrifugation to obtain TSI;

[0017] (2) Dissolve DPPC, DSPC and DSPE-PEG 2000 -iRGD is dissolved in chloroform-methanol mixed solvent at a molar ratio of 80:15:5, rotary evaporation is carried out to form a lipid film, and then ventilation is carried out to remove excess chloroform-methanol mixed solvent;

[0018] (3) Add the aqueous solution of TSI prepared in step (1) to the lipid film obtained in step (2) to carry out hydration and ultrasonic treatment to obtain a suspension.

[0019] In a preferred embodiment of the present application, in step (1), the mass ratio of ICG and TPP-Ser is 1:4.

[0020] In a preferred embodiment of the present application, in step (2), the volume ratio of chloroform and methanol in the chloroform-methanol mixed solvent is 2:1.

[0021] Further preferably, the temperature of rotary evaporation is 45℃.

[0022] In a preferred embodiment of the present application, in step (3), the hydration and ultrasonic treatment temperature is 45℃, and the time is 2h.

[0023] In a preferred embodiment of the present application, the phase transition temperature of the light-controlled mitochondrial autophagy nano-inducer is 42-43℃.

[0024] The light-controlled mitochondrial autophagy nano-inducer prepared by the above preparation method is used for preparing a cancer treatment composition.

[0025] A cancer treatment composition, wherein the effective component comprises the light-controlled mitochondrial autophagy nano-inducer prepared by the above preparation method.

[0026] The beneficial effects of the present application are:

[0027] 1. The present application adopts a secondary structure of self-assembled nano-core and functionalized lipid shell, rather than simple co-encapsulation. The pre-assembly of the inner core improves the drug loading efficiency and ICG stability; the outer shell provides targeting, protection and intelligent release functions. The mitochondrial autophagy inducer (TPP-Ser), photothermal agent (ICG), heat-sensitive lipid material (DPPC, DSPC and DSPE-PEG2000-iRGD) and targeting peptide (iRGD) are combined in a specific ratio and process to form a new product with functions superior to the simple addition of each component, which improves the overall stability and multifunctional integration potential of the system.

[0028] 2、The prepared light-controlled mitochondrial autophagy nano-inducer liposome phospholipid bilayer effectively protects the active ingredients in the inner core from degradation and premature leakage in blood circulation. The polyethylene glycol modification on the surface provides "stealth" function, greatly reducing the speed of recognition and removal by the mononuclear phagocyte system, significantly prolonging the blood half-life. At the same time, the iRGD targeting peptide coupled to the surface of the liposome can specifically recognize and bind to the highly expressed ανβ3 / 5 integrin in tumor tissues, achieving enhancement from passive enrichment to active targeting, ensuring efficient aggregation and deep penetration of the nano-drug in the tumor site, and improving the efficiency and specificity of drug delivery.

[0029] 3、The prepared light-controlled mitochondrial autophagy nano-inducer lipid membrane adopts a precisely designed heat-sensitive formula, with the phase transition temperature regulated at 42-43 ℃ in the therapeutic window. When the nanoparticles are enriched in the tumor site, external near-infrared (NIR, 808 nm) laser irradiation is applied, and the ICG in the inner core rapidly generates a photothermal effect, causing the local microenvironment temperature to instantaneously rise and exceed the phase transition temperature of the liposome, resulting in a change in the lipid membrane structure from an ordered gel state to a disordered liquid crystal state, forming pores and rapidly and controllably releasing the internal wrapped mitochondrial autophagy inducer TPP-Ser. This process achieves spatiotemporal precise control of drug activity, avoiding ineffective release of the drug in non-targeted areas.

[0030] 4、The released TPP-Ser in the prepared light-controlled mitochondrial autophagy nano-inducer can efficiently enrich in mitochondria using its TPP targeting group. By precisely controlling the irradiation time of the NIR laser, mitochondrial damage can be quantitatively induced and the activation level of mitochondrial autophagy can be dynamically regulated. This external light control capability allows the autophagy intensity to be precisely elevated from the basal level to above the lethal threshold of overactivation, thereby converting mitochondrial autophagy from a protective function to inducing irreversible apoptosis of tumor cells, improving the precision and adjustability of treatment.

[0031] 5、The prepared light-controlled mitochondrial autophagy nano-inducer produces a high-temperature killing effect through ICG-mediated photothermal therapy and overactive mitochondrial autophagy induced by the light-controlled release of TPP-Ser, synergistically disrupting the energy metabolism and survival signals of tumor cells. This synergistic effect of photothermal therapy and regulated biological therapy significantly enhances the anti-tumor effect and effectively overcomes the limitations and drug resistance of single therapy. Through the cascade amplification of the photothermal effect, a powerful synergistic anti-tumor mechanism is formed, which is expected to play a more excellent role in complex tumor environments.

[0032] 6. The photocontrolled mitophagy nano-inducer based on thermosensitive liposomes prepared in this invention exhibits extremely high spatiotemporal controllability and therapeutic precision. Drug release is precisely controlled by an external laser, achieving precise regulation of mitophagy activity, minimizing off-target effects and damage to normal tissues, and significantly improving treatment safety. Simultaneously, ICG endows the system with real-time fluorescence imaging capabilities, providing a basis for integrated diagnosis and treatment and personalized therapy. The preparation process is stable and reproducible. The self-assembly combined with thin-film hydration-ultrasound method employed is a mature and controllable technology in nanomedicine preparation, which is beneficial for future clinical translation and large-scale production. Attached Figure Description

[0033] Figure 1 The figure shows the experimental results of Embodiment 1 of the present invention.

[0034] Figure 2 The figure shows the experimental results of Embodiment 2 of the present invention.

[0035] Figure 3 The figure shows the experimental results of Embodiment 3 of the present invention.

[0036] Figure 4 The figure shows the experimental results of Embodiment 4 of the present invention.

[0037] Figure 5 The figure shows the experimental results of Embodiment 5 of the present invention.

[0038] Figure 6 The figure shows the experimental results of Embodiment 6 of the present invention.

[0039] Figure 7 The figure shows the experimental results of Embodiment 7 of the present invention.

[0040] Figure 8 The figure shows the experimental results of Embodiment 8 of the present invention. Detailed Implementation

[0041] The technical solution of the present invention will be further explained and described below with reference to specific embodiments and accompanying drawings.

[0042] Example 1

[0043] The preparation steps in this embodiment are as follows: Figure 1 As shown, the details are as follows:

[0044] (1) Preparation of self-assembled nanoparticle TPP-Ser-ICG (TSI): 1 mg ICG (photothermal agent) and 4 mg TPP-Ser (mitophagy inducer) were dissolved in 200 μL DMSO and ultrasonically treated for 15 min. The resulting solution was quickly added to 3.8 mL ultrapure water, and after stirring at room temperature for 12 h, TSI was collected by centrifugation (13000 r / min, 5 min) and washed with deionized water three times to remove excess molecules, providing a uniform size and morphology of the load for subsequent liposome encapsulation.

[0045] (2) Preparation of thermosensitive phospholipid film layer: 20 mg DPPC (Beijing Solabio), 3.8 mg DSPC (Beijing Solabio) and 1.3 mg DSPE-PEG 2000 -iRGD (Xi'an Ruihi), dissolved in 6 mL chloroform-methanol (2:1, by volume). After complete dissolution, it was transferred to a round-bottom flask and rotary evaporated at 45 °C for 1 h to form a lipid film, followed by overnight in a fume hood to remove excess solvent;

[0046] (3) Preparation of light-controlled mitophagy nanoparticle inducer TPP-Ser-ICG@TSL based on thermosensitive liposomes: prepared by the thin film hydration method, 5 mL of TSI aqueous solution (ICG concentration, 1 mg / mL) prepared in step (1) was added to the lipid film prepared in step (2), and water hydration and ultrasonic treatment were carried out at 45 °C for 2 h to obtain a uniform, opaque green suspension, namely TPP-Ser-ICG@TSL (hereinafter referred to as TSL). This morphology is the result of the combined effect of the milky white opaque effect of the scattering of light by the lipid bilayer and the green color produced by the absorption of light of a specific wavelength by ICG. This unique visual performance is one of the intuitive evidence for the successful construction of TPP-Ser-ICG@TSL.

[0047] The resulting TSL was stored at 4 °C for later use.

[0048] Example 2 Phase transition temperature of TSL:

[0049] The phase transition behavior of the TSL prepared in Example 1 was characterized by differential scanning calorimetry (DSC). Before testing, an accurate amount of the TSL aqueous dispersion and an equal volume of phosphate buffered saline (PBS) were respectively loaded into the sample cell and the reference cell. Under nitrogen protection, a constant heating rate of 1.0 ℃ / min was used to scan in the temperature range of 4 ℃ to 90 ℃, and the heat flow curve was obtained; in order to eliminate the historical thermal effect and ensure the accuracy of the data, a pre-scanning cycle from 20 ℃ to 90 ℃ was first performed, and after the system was cooled and balanced, the second formal data acquisition cycle was performed, and the phase transition temperature (Tm) was determined by analyzing the peak value point or extrapolating the starting point of the endothermic peak on the heat flow curve through the instrument matching software. The specific results of this example are shown in Figure 2 The analysis of the DSC spectrum shows that the phase transition temperature is about 42.48 ℃, and reaching this temperature will trigger the rupture of the bilayer and the release of drug loading, and this temperature range is suitable for triggering the heat therapy induced drug release in the physiological environment.

[0050] Example 3 In vitro photothermal performance of TSL

[0051] In this example, the in vitro photothermal properties of the TSL prepared in Example 1 were characterized by infrared thermal imaging method. Before testing, 0.5 mL of TSL solution (ICG concentration of 100 μg / mL) was taken in a 2 mL centrifuge tube and irradiated with 1 W / cm 2 of near-infrared laser for a total of 10 min, and the temperature rise images were recorded every 30 s. The specific results of this example are shown in Figure 3 Under laser irradiation, TSL showed time-dependent temperature rise, with a maximum temperature rise of about 60 ℃, indicating that TSL has good in vitro photothermal properties.

[0052] Example 4 Drug release of TSL:

[0053] Drug release kinetics study: 1 mL of TSL prepared in Example 1 was dispersed in HEPES buffer at pH 6.5 (ICG concentration, 0.05 mg / mL), and placed in a constant temperature shaker. Incubation was carried out at 37 ℃ (simulating normal body temperature, as a negative control) and 43 ℃ (higher than the lipid phase transition temperature, triggering drug release) respectively, and the shaking speed was set to 100 rpm. Then at the preset time point, high-speed centrifugation (13,000 rpm, 4 ℃, 5 min) was carried out under pre-cooling conditions to precipitate the un-released liposomes. 100 μL of supernatant was carefully aspirated, and the drug concentration of ICG in the supernatant after centrifugation was measured by ultraviolet-visible spectrophotometer, so as to calculate the cumulative drug release efficiency.

[0054] Near-infrared light (NIR) induced drug release study: TSL dispersion (condition as above) was incubated in a 37 ℃ constant temperature shaker. At specific time points, the sample tube was irradiated with a near-infrared laser with a wavelength of 808 nm at a power density of 1 W / cm². The irradiation time was set to 3, 5 and 10 min, respectively. Finally, the concentration of free drug in the supernatant after centrifugation was measured by UV-Vis spectrophotometer, and the cumulative drug release efficiency was calculated. The specific results of this example are shown in Figure 4 At physiological temperature (37 ℃), the amount of drug release is very small (<10%), which confirms that the drug loading is stable in the thermosensitive bilayer structure under normal conditions. In contrast, at the phase transition temperature (43 ℃), the drug release is significantly increased. On this basis, after laser irradiation for 3, 5 or 10 min, an explosive release of the drug was observed. The cumulative drug release showed a clear irradiation time dependence, and the longer the irradiation time, the more the total drug loading was released, proving the controllable release mediated by near-infrared laser.

[0055] Example 5 In vitro cell autophagy level regulation effect

[0056] Human A549 cells were seeded in a 6-well plate at a density of 2×10 5 cells / well, and after 24 h of culture at 37 ℃, 5% CO2 to about 70-80% confluence, the experimental groups were treated; except for the control group, the experimental groups were pretreated with the culture medium containing the TSL prepared in Example 1, and the TSL+NIR group was irradiated with an 808 nm near-infrared laser (power density 1 W / cm²) for 3, 5 and 10 min, respectively; after the treatment, total protein was extracted with pre-cooled RIPA lysis buffer (containing protease inhibitors) on ice, and after quantification by BCA method, the protein samples were subjected to SDS-PAGE electrophoresis and transferred to PVDF membrane, then blocked with 5% skim milk for 1 h, and then incubated with specific autophagy-related protein primary antibodies (LC3B, p62, etc.) (4 ℃ overnight) and corresponding HRP-labeled secondary antibodies (room temperature for 1 h), finally, ECL chemiluminescence substrate was used for exposure under the imaging system; after the image was collected, the gray values of the target band and the internal reference β-actin band were analyzed using ImageJ software, and the relative expression was calculated as the integral optical density ratio of the target protein and the internal reference, and the effect of different treatments on the degree of cell autophagy was statistically analyzed, so as to evaluate the influence of different treatments on the degree of cell autophagy. The specific results of this example are shown in Figure 5As shown in FIG. 6B, with the extension of irradiation time, the ratio of LC3B-II / LC3B-I increased and the level of p62 decreased. Compared with the control group, 10 min of irradiation increased the ratio of LC3B-II / LC3B-I by about 4.2 times and decreased the level of p62 by about 8.1 times. These results showed that the laser-stimulated autophagy inducer TSL could induce autophagy activation and achieve programmed regulation of autophagy flux.

[0057] Example 6 In vitro regulation of tumor cell proliferation inhibition

[0058] Human A549 cells were seeded in a 6-well plate at a density of 2 x 10 3 After 24 h of incubation at 37 °C, 5% CO2, the experimental groups were treated; except for the control group, the experimental groups were pretreated with the TSL medium prepared in Example 1, and the TSL+NIR group was irradiated with an 808 nm near-infrared laser (power density 1 W / cm²) for 3, 5, and 10 min, respectively; after treatment, the drug-containing medium was discarded, the cells were gently washed twice with pre-warmed PBS, and fresh complete medium was added; thereafter, fresh medium was replaced every two days to maintain nutrition supply, and the cells were cultured for 14 days until obvious cell colonies were visible; after the culture ended, the medium was discarded, 4% paraformaldehyde solution was added to each well to fix the cells for 20 min, and after PBS rinsing, 0.1% crystal violet solution was used for staining for 20 min; after five times of PBS rinsing to remove background staining, the whole 6-well plate was photographed, and the inhibitory effect of each treatment group on tumor cell proliferation ability was evaluated. The specific results of this example are shown in FIG. 6A. With the extension of irradiation time, the number of colonies decreased significantly, and the proliferation ability of cells was significantly inhibited, which showed that the laser-stimulated autophagy inducer TSL could inhibit the proliferation ability of cells. Figure 6

[0059] Example 7 Bidirectional regulation of light-controlled mitochondrial autophagy function

[0060] Human A549 cells were seeded in a 6-well plate at a density of 5 x 10 4 ​The cells were seeded in 24-well plates at a density of 1 cell / well, and cultured at 37 °C, 5% CO2 for 24 h to about 70-80% confluence using culture medium containing 10% fetal bovine serum. Then, the cells were divided into groups for treatment, and were pretreated with 10 μM of autophagy inhibitor CQ and 2 mM of autophagy inhibitor 3-MA for 2 h to inhibit the intracellular autophagy level. Except for the control group, the experimental groups were pretreated with the culture medium containing TSL prepared in Example 1 (containing autophagy inhibitors), and then were irradiated with 808 nm near-infrared laser (power density 1 W / cm2) for 3, 5 and 10 min, respectively. After treatment, the cells in each group were stained using Calcein / PI Cell Viability Kit (Shanghai Biyun Tian), so as to evaluate the toxicity of different treatments on tumor cells. The specific results of this example are shown in Table 1. Figure 7 Under 3 min laser irradiation, the green fluorescence of the group with added autophagy inhibitors was weakened, the red fluorescence was enhanced, and the cell viability was less than that of the group without added autophagy inhibitors, indicating that the autophagy induced by 3 min laser irradiation played a protective function. Under 5 min and 10 min laser irradiation, the green fluorescence of the group with added autophagy inhibitors was enhanced, the red fluorescence was weakened, and the cell viability was greater than that of the group without added autophagy inhibitors, indicating that the autophagy induced by 5 min and 10 min laser irradiation played a death-promoting function. The above results show that precise regulation of the laser irradiation time can achieve precise regulation of the level of mitochondrial autophagy, so as to achieve the purpose of regulating the bidirectional function of autophagy.

[0061] Example 8 Tumor treatment

[0062] Balb / c female nude mice (4-6 weeks old) were subcutaneously injected with 5 x 10 6A549 cells were suspended in 0.1 mL of Corning Matrigel to establish a tumor model, and when the tumor volume grew to about 100-150 mm3, the tumor-bearing mice were randomly divided into six groups (n=5 each), including a saline control group, a simple laser irradiation group, a free drug group, a TSL group, and a TSL combined with different time near-infrared laser irradiation group; the mice in the treatment groups were injected with TSL prepared in Example 1 (2 mg / kg of drug dose) via the tail vein, and 24 h later, the mice in the laser irradiation groups were anesthetized and fixed, and an 808 nm near-infrared laser (the spot diameter covered the entire tumor area, and the power density was 1.0 W / cm2) was used to irradiate the tumor site for 3, 5 and 10 min, respectively, and an infrared thermal imager was used to monitor the local temperature changes of the tumor during irradiation; thereafter, the tumor volume was measured every three days using a vernier caliper (V = length × width2 / 2) and the body weight of the mice was recorded to evaluate systemic toxicity, and the treatment period lasted for 15 days; after the experiment, the mice were euthanized by cervical dislocation, and the tumor tissue and main organs such as the heart, liver, spleen, lung and kidney were completely stripped, and part of the tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and then subjected to hematoxylin-eosin staining and immunohistochemical analysis to comprehensively evaluate the treatment effect and biodistribution. The specific results of this example are shown in Figure 8 As shown in the results, TSL has good retention effect at the tumor site, and compared with the control group, the tumor growth volume is significantly reduced as the irradiation time is prolonged, and further histological section results show that as the laser irradiation time is prolonged, the expression level of autophagy protein LC3B is up-regulated, the expression level of autophagy substrate protein p62 is down-regulated, and the apoptosis signal is enhanced. These results show that the laser-stimulated autophagy inducer TSL can effectively achieve programmed regulation of the autophagy flux of the tumor and achieve tumor growth inhibition.

[0063] The above is only a preferred embodiment of the present application, and therefore cannot limit the scope of the present application, i.e., equivalent changes and modifications made in accordance with the scope and content of the present patent should still be within the scope of the present application.

Claims

1. A method for preparing a light-controlled mitophagy nano-inducer based on thermosensitive liposomes, characterized in that: The method comprises the following steps: (1) dissolving ICG and TPP-Ser in DMSO, adding the mixture into ultrapure water after ultrasonic treatment, stirring at room temperature to react, collecting and washing by centrifugation to obtain TSI; (2) DPPC, DSPC and DSPE-PEG 2000 -iRGD was dissolved in chloroform-methanol mixed solvent at a molar ratio of 80:15:5, rotary evaporation to form a lipid film, and ventilation to remove excess chloroform-methanol mixed solvent; (3) adding the aqueous solution of TSI prepared in step (1) into the lipid film obtained in step (2) to perform hydration and ultrasonic treatment to obtain a suspension.

2. The production method according to claim 1, characterized by: In step (1), the mass ratio of ICG and TPP-Ser is 1:

4.

3. The production method according to claim 1, wherein: In step (2), the volume ratio of chloroform and methanol in the chloroform-methanol mixed solvent is 2:

1.

4. The production method according to claim 3, characterized by: The temperature of the rotary evaporation is 45℃.

5. The production method according to claim 1, wherein: In step (3), the temperature of the hydration and ultrasonic treatment is 45℃, and the time is 2 hours.

6. The production method according to any one of claims 1 to 5, characterized by: The phase transition temperature of the light-controlled mitochondrial autophagy nano-inducer is 42-43℃.

7. The light-controlled mitochondrial autophagy nano-inducer prepared by the preparation method in any one of claims 1 to 6 is used in the preparation of a cancer treatment composition.

8. A cancer treatment composition, characterized by: The effective component of the composition comprises the light-controlled mitochondrial autophagy nano-inducer prepared by the preparation method in any one of claims 1 to 6.

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